Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: 
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you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Snakin genes from potato: overexpression confers
blackleg disease resistance
____________________
A thesis
submitted in partial fulfilment
of the requirements for the Degree of
Doctor of Philosophy (PhD) in Plant biotechnology
at Lincoln University, New Zealand
By
Sara Mohan
__________________
Lincoln University
2011
Abstract of thesis submitted in partial fulfilment of the requirements for the
Degree of Doctor of Philosophy
Snakin genes from potato: overexpression confers blackleg
disease resistance
by
Sara Mohan
Antimicrobial peptides (host defense peptides) are an evolutionarily conserved component of
the innate immune response, and have been shown to kill or inhibit the growth of microbes
such as bacteria, fungi, viruses, or parasites. Snakin-1 (SN1) and Snakin-2 (SN2) are lowmolecular weight antimicrobial peptides produced in potato tubers. They share common
structural features with more than 500 antimicrobial peptides discovered in plants and
animals, such as an N-terminal putative signal sequence, a highly divergent intermediate
region and a conserved cysteine-rich 60 amino-acid carboxyl-terminal domain. These proteins
are thought to play important roles in the innate defense against invading microbes.
SN1 and SN2 genes were isolated and sequenced from a range of potato cultivars/clones
(Solanum tuberosum), as well as a diverse range of other Solanaceous species. The sequences
were aligned with reference sequences of the StSN1 gene (AJ320185) and the StSN2 gene
(AJ312424) using Vector NTI Advance 10 software. Allelic variation both within and
between genotypes and/or species was observed for both SN1 (four alleles) and SN2 (six
alleles). Most of the variation occurred within introns, with relatively few nucleotide
substitutions in exons. The predicted amino acid sequence was highly conserved; identical for
all SN1 alleles, with only minor amino acid substitutions observed in SN2 alleles present in
different Solanum species.
Overexpression of the SN genes in potato was achieved using Agrobacterium-mediated gene
transfer. Plant expression vectors with both sense and antisense orientations of SN1 (a1 allele)
and SN2 (b1 allele) genes were constructed under the regulatory controls of the potato light
inducible Lhca3 gene. Potato plants, cultivar Iwa, were transformed via Agrobacterium
tumefaciens harboring binary vector containing the intragenic SN cassettes. The resulting
i
plants were confirmed as being transgenic by PCR, then analysed at the molecular level for
expression of the transgenes. Quantitative Reverse Transcriptase-PCR (qRT-PCR) analysis
demonstrated that the majority of the transgenic potato lines overexpressed the SN genes at
RNA level. Based on qRT-PCR of each SN gene, seven and eight lines respectively were
selected from each of the Lhca3-SN1 and Lhca3-SN2 transgenic plant populations for further
characterisation. Overexpression of the SN genes in leaves, stems, roots and tubers was
determined by qRT-PCR analysis and these selected transgenic lines were evaluated in
bioassays for resistance to Pectobacterium atrosepticum (formally Erwinia carotovora subsp.
atroseptica) causal agent of blackleg in potatoes. Results from the pathogenicity bioassays
showed that overexpression of either the SN1 gene or the SN2 gene from the intragenic
cassettes conferred resistance to P. atrosepticum blackleg disease. This result has established
the functional role of SN genes in plant defense against pathogens in potato. Antisense
transformants were difficult to recover and failed to regenerate plants. This indicates that
these highly conserved Snakin proteins may also play an important role in cellular
metabolism other than just defense against invading microorganism.
Key words: potato, Snakin-1, Snakin-2, alleles, Agrobacterium-mediated transformation,
Pectobacterium atrosepticum, Erwinia carotovora subsp. atroseptica, qRT-PCR, Blackleg,
Lhca3 promoter.
ii
Table of Contents
Abstract
i
Contents
iii
List of Tables
vii
List of Figures
viii
Abbreviations
x
Chapter 1 Literature Review
1
1.1
Introduction
1
1.2
Blackleg of potato
3
1.2.1 Pathogen biology
3
1.2.2 Symptoms
4
1.2.3 Epidemiology
4
1.2.4 Disease management
8
1.2.4.1 Cultural Control
8
1.2.4.2 Chemical Control
8
1.2.4.3 Biological control
9
1.2.4.4 Traditional breeding
9
1.3
Plant Genetic Engineering
10
1.4
Selectable marker genes
12
1.5
Regulation of transgene expression
12
1.6
Antimicrobial peptides
14
1.6.1 Properties of antimicrobial peptides
14
1.6.2 Antimicrobial peptides for engineering disease-resistant plants
15
1.6.3 Snakin-1 and Snakin-2 antimicrobial peptides
20
Aims and objectives of this thesis
21
1.7
Chapter 2 Highly conserved alleles of Snakin-1 and Snakin-2 genes in Solanaceous
species
22
2.1
Introduction
22
2.2
Materials and Methods
24
2.2.1 Plant Material
24
iii
2.3
2.2.2 Genomic DNA isolation
25
2.2.3 PCR amplification of the SN1 and SN2 genes
25
2.2.4 Cloning of PCR fragments
26
2.2.5 E. coli transformation and plasmid isolation
26
2.2.6 Sequencing of the SN1 and SN2 genes
27
2.2.7 Phylogenetic analysis
28
Results
28
2.3.1 PCR isolation of the SN1 and SN2 genes
28
2.3.2 Identifying alleles of the SN genes in Solanaceous species.
29
2.3.3 Allele diversity in the exons (coding regions) of SN1 and SN2 genes
34
2.3.4 Allele diversity in the introns (non-coding regions) of SN1 and SN2 genes
34
2.3.5 Predicted amino acid sequences of SN alleles
36
2.3.6 Phylogenetic relationships between the various SN alleles
38
2.3.7 Comparative analysis of SN protein identity with other species
39
2.3.8 Comparative study of highly conserved C-terminal region in different plant
families
2.4
Discussion
45
49
Chapter 3 Overexpression of Snakin-1 and Snakin-2 genes under a potato light
inducible Lhca3 promoter in transgenic potatoes
52
3.1
Introduction
52
3.2
Materials and methods
54
3.2.1 Construct development
54
3.2.2 Agrobacterium transformation
57
3.2.3 Plant material
57
3.2.4 Potato transformation
59
3.2.5 Regeneration and selection of transgenic potato plants
59
3.2.6 Molecular characterisation of transformed potato.
59
3.2.7 Phenotypic evaluation of transgenic lines in glasshouse
60
3.2.8 RNA isolation and cDNA synthesis
60
3.2.9 Overexpression of specific gene in leaves using qRT-PCR
61
Results
62
3.3.1 Vector Construction
62
3.3.2 Potato transformation
64
3.3
iv
3.4
3.3.3 DNA isolation and PCR based screening of regenerated lines
66
3.3.4 Phenotypic evaluation
68
3.3.5 Endogenous expression levels of SN1 and SN2
70
3.3.6 Quantitative RT-PCR analysis of transgenic lines
71
Discussion
74
Chapter 4 Resistance of transgenic potato plants overexpressing SN1 and SN2 to
Pectobacterium atrosepticum
78
4.1
Introduction
78
4.2
Material and methods
79
4.2.1 Plant material
79
4.2.2 Expression of SN gene in different tissues of selected lines using qRT-PCR
80
4.2.3 Pectobacterium atrosepticum pathogenicity assays
80
4.2.3.1 Experiment 1
82
4.2.3.2 Experiment 2
82
4.2.3.3 Experiment 3
83
4.2.4 Disease Assessment
4.3
84
4.2.4.1 Assessment of disease resistance
84
4.2.4.2 PCR analysis of Pca from lesions
85
4.2.4.3 Dilution plating of Pca from lesions
85
4.2.4.4 Quantitative PCR analysis
86
4.2.4.5 Statistical Analysis
86
Results
87
4.3.1 Quantification of the transcript levels of the SN genes in different tissues
using qRT-PCR
87
4.3.1.1 SN1 transcript levels in Lhca3-SN1 transgenic lines
87
4.3.1.2 SN2 transcript levels in Lhca3-SN2 transgenic lines
87
4.3.2 Black leg assays in transgenic potato lines overexpressing SN1 and SN2 genes 90
4.3.2.1 Experiment 1
90
4.3.2.2 Experiment 2
96
4.3.2.3 Experiment 3
100
4.3.3 PCR screening of the pathogen using the genomic DNA of the infected stem. 103
4.3.4 Viable bacterial colony count
103
4.3.5 Quantitative PCR analysis of the infected stems
103
v
4.4
Discussion
107
Chapter 5 General Discussion
115
5.1
Contributions of thesis findings to research
118
5.2
Directions of further research
118
Acknowledgments
122
References
124
Appendix A Media and Solutions
152
Appendix B Protocols
157
Appendix C Sequences from GenBank
159
Appendix D Sequencing the coding regions of StSN genes
161
Appendix E Analysis of SN1 and SN2 function using an antisense approach
165
E.1 Introduction
165
E.2 Materials and methods
166
E.2.1 Vector Construction
166
E.2.2 Agrobacterium transformation
167
E.2.3 Plant material
167
E.2.4 Potato transformation
167
E.2.5 DNA isolation and PCR based analysis of transgenic colonies
167
E.3 Results
168
E.3.1 Vector Construction
168
E.3.2 Potato transformation
168
E.3.3 DNA isolation and PCR based analysis of transgenic colonies
172
E.4 Discussion
172
Appendix F ANOVA tables
176
F.1
176
Chapter 4 ANOVA tables
Appendix G List of publications, presentations and awards
181
vi
List of Tables
Table 1.1 Hybrids with wild species used in traditional breeding
11
Table 1.2 Major antimicrobial peptides showing toxicity to bacterial pathogens
17
Table 1.3 Transgenic potato plants resistance to Erwinia inducing diseases
19
Table 2.1 Primers used to isolate SN genes
26
Table 2.2 SN1 and SN2 alleles in various Solanaceous species and genotypes
31
Table 2.3 Nucleotide variations in different alleles of SN1 and SN2 genes
34
Table 2.4A SNPs in the SN1 alleles with reference to StSN1 coding sequence (exons)
35
Table 2.4B SNPs in the SN2 alleles with reference to StSN2 coding sequence (exons)
35
Table 2.5 Amino acid changes in the SN2 alleles
38
Table 3.1 Details of the primers used in this chapter
58
Table 3.2 Details of the primers designed for qRT-PCR
62
Table 3.3 Efficiency of A. tumefaciens transformation
65
Table 4.1 Potato lines selected for use in pathogen assay study
81
Table 4.2 Bioassay of Lhca3-SN1 transgenic lines against Pca (Expt 1)
92
Table 4.3 Bioassay of Lhca3-SN2 transgenic lines against Pca (Expt 1)
94
Table 4.4 Bioassay of Lhca3-SN1 transgenic lines against Pca (Expt 2)
97
Table 4.5 Bioassay of Lhca3-SN2 transgenic lines against Pca (Expt 2)
98
Table 4.6 Bioassay of Lhca3-SN1 transgenic lines against Pca (Expt 3)
105
Table 4.7 Bioassay of Lhca3-SN2 transgenic lines against Pca (Expt 3)
106
Table 4.8 Summary of the status of transgenic lines across the three different Pca assays 108
vii
List of Figures
Figure 1.1 Different stages of black leg infection on potato
5
Figure 1.2 Potato stem showing early blackleg symptoms with decayed internal tissue
5
Figure 1.3 Potato tubers infected with the blackleg-inducing bacterium
6
Figure 1.4 Tuber decay due to blackleg disease
6
Figure 1.5 Disease cycle of potato blackleg caused by Pca
7
Figure 2.1A PCR amplified SN1 gene from various Solanaceous species and genotypes
30
Figure 2.1B PCR amplified SN2 gene from various Solanaceous species and genotypes
30
Figure 2.2 Alignment of SN1 sequences
32
Figure 2.3 Alignment of SN2 sequences
33
Figure 2.4 Multiple amino acid sequence alignment between reference sequence and alleles37
Figure 2.5 Phylogenetic tree indicating the relationship of SN1 alleles and SN2 alleles
39
Figure 2.6 Amino acid alignment of SN1 and its homologues
41
Figure 2.7 A phylogenetic tree based on amino acid sequences of SN1 and its homologues 42
Figure 2.8 Amino acid alignment of SN2 and its homologues
43
Figure 2.9 A phylogenetic tree based on amino acid sequences of SN2 and its homologues 44
Figure 2.10 C-terminal regions alignment of the SN family and other related families
46
Figure 2.11 An unrooted phylogenetic tree constructed based in Figure 2.10
48
Figure 3.1 An intragenic expression cassette based on the potato StLhca3 gene
56
Figure 3.2 Schematic representation of the Lhca3 cassette with specific genes
62
Figure 3.3 Schematic diagrams of the whole binary vectors
63
Figure 3.4 PCR analysis of representative Lhca3-SN1 regenerated lines
67
Figure 3.5 PCR analysis of representative Lhca3-SN2 regenerated lines
67
Figure 3.6 Randomly arranged transgenic potato lines grown in bags in the greenhouse
68
Figure 3.7 Off-type transgenic potato lines grown in the containment greenhouse
69
Figure 3.8 Endogenous SN expression levels in non-transgenic Iwa
70
Figure 3.9 Quantitative PCR amplification of the SN1 transcripts in the leaves
72
Figure 3.10 Quantitative PCR amplification of the SN2 transcripts in the leaves
73
Figure 4.1 Expt 1 set up showing Pca inoculated potato plants in the inoculation chamber 83
Figure 4.2 Expt 3 set up showing inoculated potato plants in the artificially made tent
84
Figure 4.3 SN1 transcript levels in different tissues of the selected lines
88
Figure 4.4 SN2 transcripts levels in different tissues of the selected lines
89
Figure 4.5 Disease severities in control plants after inoculating Pca
91
viii
Figure 4.6 Disease appearance observed in Lhca3-SN2 transgenic lines
95
Figure 4.7 Internal necrotic lesions observed in Lhca3-SN1 transgenic lines
99
Figure 4.8 Internal necrotic lesions observed in Lhca3-SN2 transgenic lines
99
Figure 4.9 Disease appearance observed in Lhca3-SN1 transgenic lines
102
ix
Abbreviations
°C
Degrees Celsius
µg
microgram
µL
microlitre
BAP
benzylaminopurine
bp
base pair
dATP
deoxyadenosine triphosphate
DEPC
diethyl pyrocarbonate
DNA
deoxyribonucleic acid
EDTA
ethylene diaminetetraacetic acid
g
gram
GA3
gibberellic acid
h
hour
IPTG
Isopropyl-β-D-thiogalactopyranoside
kb
kilobase
L
litre
mg
milligram
min
minutes
mL
millilitre
mm
millimeter
mM
millimolar
mRNA
messenger ribonucleic acid
NAA
napthaleneacetic acid
ng
nanogram
nm
nanometer
nM
nanomolar
Pca
Pectobacterium atrosepticum
PCR
polymerase chain reaction
RNA
ribonucleic acid
qRT-PCR
quantitative reverse transcriptase PCR
qPCR
quantitative PCR
s
second
T-DNA
transfer DNA
V
volts
X-Gal
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
x
Chapter 1
Literature Review
1.1 Introduction
Potato (Solanum tuberosum L.) is a crop of worldwide importance. It is an integral part of the
diet of a large proportion of the world’s population (Hawkes, 1990) and one of the world’s
main food crops, differing from others in that the edible part of the plant is a tuber (i.e. the
swollen end of an underground stem). The failure of cereal surpluses to meet international
demand together with rapidly increasing food prices has increased the requirements for potato
as an easily grown and nutritious food source. Reflecting its importance as a food source, the
year 2008 was designated “The International Year of the Potato” by the Food and Agricultural
Organization of the United Nations (UN information service, Vienna).
Solanum tuberosum is an annual crop of the Solanaceae family. Potato plants have a lowgrowing habit and bear white to purple flowers with yellow stamens (Logan, 1983). The most
important cultivated potatoes are tetraploids (2n=4x=48) (Gebhardt and Valkonen, 2001).
Any potato cultivar can be propagated vegetatively by planting pieces of existing tubers, cut
to include at least one or two eyes. Potatoes are the world's most widely grown tuber crop,
and the third main food crop in terms of both area cultivated and total production (after rice
and wheat) (Gebhardt and Valkonen, 2001). According to the Food and Agriculture
Organization, the worldwide production of potatoes in 2010 was 314.1 million metric tonnes
from 18.1 million hectares (FAOSTAT, 2010). Major potato producing countries are China,
Russian Federation, India, USA, Germany, Ukraine, United Kingdom and Poland. Potato
production in developed countries has increased rapidly. Annual potato production grew from
30 million tonnes in the early 1960s to 165 million tonnes in 2007. Developing countries are
expecting a 2.8% increase in the production of potato each year (Ghislain et al., 1997).
The potato is one of the major vegetable crops grown in New Zealand for fresh use and for
processing (Petrie and Bezar, 1998). In New Zealand nearly 12,600 hectares were harvested
in 2009 yielding 490,000 metric tonnes (FAOSTAT, 2009). The adaptability of potato enables
it to be grown in most parts of New Zealand in a wide variety of soils and climates (Logan,
1983). Most of the potatoes grown in New Zealand are consumed in the domestic market
(Petrie and Bezar, 1998).
Potatoes are very versatile and can be used fresh or processed into a variety of products, such
as French fries, crisps, mashed potatoes, flour, alcohol, starch and its derivatives (Hawkes,
1990). Nutritionally, potatoes are best known for their carbohydrate content. Starch is the
1
predominant form of carbohydrate found in potatoes. A small but significant portion of the
starch in potatoes is resistant to enzymatic digestion in the stomach and small intestine and
reaches the large intestine essentially intact. This resistant starch is considered to have similar
physiological effects and health benefits as fibre by providing bulk, offering protection
against colon cancer, improving glucose tolerance and insulin sensitivity, lowering plasma
cholesterol and triglyceride concentrations, increasing satiety, and possibly even reducing fat
storage (Cummings et al., 1996; Hylla et al., 1998; Raben et al., 1994). Potatoes also contain
an assortment of phytochemicals, such as carotenoids and polyphenols and a number of
important vitamins and minerals. Jacques Diouf, Director-General of the Food and
Agriculture Organisation of the United Nations (FAO) stated that “The potato is on the
frontline in the fight against world hunger and poverty” (Caliskan and Struik, 2010).
Potatoes are a popular and reliable crop. Unfortunately, commercial cultivars are particularly
susceptible to diseases. Potato production is threatened by a range of bacterial, fungal,
nematode, oomycete, and viral pathogens. The yield loss caused by disease and pests is
estimated at 22% per year (Ross, 1986). Most of the commercial cultivars in Europe
originated from a limited number of potato clones imported from South America in the
beginning of 16th century. Consequently, many commercially grown potato cultivars lack
adequate genetic resistance to continually evolving pathogens, which currently constrain
potato production worldwide (Düring, 1996; Nielsen, 1954).
Potato late blight, caused by Phytophthora infestans, is an important disease of potato, and
although one of the most intensively researched plant diseases in the world, it is still a major
threat to crop production in both western industrial nations and in developing countries
(Duncan, 1999; Garelik, 2002). Rhizoctonia solani causes black scurf on tubers and stem
canker on underground stems and stolons, and occurs wherever potatoes are grown causing
economically significant damage in cool wet soils (James and Mckenzie, 1972). Another
disease with worldwide importance is powdery scab caused by the pathogen Spongospora
subterranea (Harrison et al., 1997). However, of all the agents known to cause plant diseases,
bacteria are especially difficult to control with limited pesticide options available (Mourgues
et al., 1998). Many antimicrobials used for controlling pathogenic bacteria also eliminate
beneficial natural microbes in soil. Because of the loss of the natural beneficial microbes in
soil plants may be more vulnerable to future disease attacks due to the lack of competition
against the pathogenic bacteria. Although no accurate figures are available for losses
sustained on a worldwide basis, Pérombelon and Kelman (1980) estimated that
2
Pectobacterium diseases alone may be responsible for US$50-100 million in crop damage
annually.
1.2 Blackleg of potato
Blackleg is one of the important bacterial diseases capable of causing serious damage to
potato in temperate regions (Hightower et al, 1994; Düring, 1996). The most common
bacterial species causing blackleg are in the genus Pectobacterium, and of these, isolates
belonging to the Carotovora group are usually referred to as the blackleg bacteria. These
include Pectobacterium carotovora subsp. carotovora (Pcc) (former name Erwinia
carotovora subsp. carotovora; Dye, 1969; Gardan et al., 2003), Pectobacterium atrosepticum
(Pca) (former name Erwinia carotovora subsp. atroseptica; Dye, 1969; Gardan et al., 2003),
and Dickeya spp. (former name Erwinia chrysanthemi; Burkholder, 1953; Samson et al.,
2005). Pca is restricted only to potato, and mainly causes blackleg in temperate regions, such
as found in New Zealand (Pérombelon and Salmond, 1995). It can cause severe economic
losses to the potato crop. To ensure potato production under New Zealand climatic conditions,
research on resistance to Pca is essential.
1.2.1 Pathogen biology
Pectobacterium carotovora subsp. atroseptica is a gram-negative, rod-shaped, non sporeforming bacterium (Rowe et al., 1995). The pathogen can grow both aerobically and
anaerobically and is motile with numerous peritrichous flagella. Pca is host specific and
mostly associated with potatoes. These bacteria produce a variety of cell-wall-degrading
enzymes such as polygalacturonase, pectin methyl esterase, pectin lyase and isomers of
pectate lyase that allow infiltration and maceration of plant tissues on which they feed. Some
of the enzymes are secreted while others remain in the periplasm. Pectate lyase is the most
abundantly produced and is believed to be the most important in pathogenesis (Pérombelon
and Kelman, 1980). This bacterium can be isolated on a selective medium such as crystal
violet pectate (CVP) medium (Cuppels and Kelman, 1974). The bacterium develops pits or
craters in the medium due to the excretion of pectolytic enzymes that liquefy the pectate
(McMillan et al., 1994). Isolates of this bacterium do not survive well in soil after a year,
unless they are contained within diseased tubers or other potato plant debris.
SCRI 1043 (American Type Culture Collection BAA-672) is a well studied isolate of Pca
isolated from a potato stem with blackleg disease symptoms in 1985 in Perthshire, Scotland.
This strain has been used in epidemiological and molecular studies for many years and is
amenable to genetic manipulation (Hinton et al., 1985; Toth et al., 1997). It is a well
3
characterized strain with a fully sequenced genome of 5.06 million bp containing
approximately 4614 predicted genes (GenBank accession NC-004547) (Bell et al., 2004).
1.2.2 Symptoms
The bacterium Pca affects the stems and tubers of potato plants. Blackleg begins from
contaminated seed tubers, but symptoms can occur at several stages of plant development
(Pérombelon, 1974). Blackleg disease can sometimes develop early in the growing season
soon after the plants emerge. In this case, the first blackleg symptoms appear as a black
discoloration of previously healthy stems, followed by a rapid wilting (Figure 1.1 A) (De
Boer, 2004). Stems of infected plants typically have inky black symptoms and may extend up
the entire length of the stem. The stem pith can decay above the black discoloration and the
vascular tissue can be discoloured (Figure 1.1 B and Figure 1.2). Leaves turn yellow and
leaflets tend to roll upwards at the margins. The plant normally dies within 15 days of the first
appearance of symptoms (Pérombelon, 1992) (Figure 1.1 C).
Tubers with blackleg disease generally first become decayed at the stolon attachment site
where the tuber tissue becomes blackened and soft (Figure 1.3) (Czajkowski et al., 2011). As
the disease progresses, the entire tuber may decay or the rot may remain partially restricted to
the inner perimedullary (or parenchymal) tissue, the tissue inside the vascular ring (Figure
1.4) (Helias et al., 2000; De Boer, 2004).
1.2.3 Epidemiology
There are many ways by which Pca may reach the progeny tubers produced on the potato
plant. The primary blackleg inoculum is latently infected seed tubers (Pérombelon, 1974;
Hooker, 1981; Czajkowski et al., 2011). After being planted, seed pieces rot and release large
numbers of bacteria into the soil (Figure 1.5). The bacteria may move some distance in the
soil water and contaminate adjacent plants. The secondary blackleg inoculum is contaminated
soil. These bacteria can enter the plant system through wounds to cause stem rot. Although
the pathogen is usually present on or in the potato plants, disease occurs only when the
environmental conditions are favorable for growth of the pathogen (Pérombelon and Kelman,
1980).
4
Figure 1.1 Different stages of blackleg infection on potato. A. Early blackleg symptoms with rapid wilting
and yellowing of the leaves. B. Stems becoming slimy and pale with a light to dark discolouration of the vascular
system. C. Severely infected plants collapsed and desiccated.
Figure 1.2 Potato stem showing early blackleg symptoms with decayed internal tissue. The dark
discoloration of the lower stem is typical of blackleg disease. Decay has moved up into the pith beyond the area
with external symptoms (reproduced with permission from De Boer, 2004).
5
Figure 1.3 Potato tubers infected with the blackleg-inducing bacterium. Decay has spread outward from the
stolon attachment site (reproduced with permission from Benlioğlu et al., 1991).
Figure 1.4 Tuber decay due to blackleg disease (reproduced with permission from De Boer, 2004).
6
Environmental factors that favor infection in the field include cool, wet conditions and poor
aeration of the soil. Pca tends to predominate in rotting seed tubers and in diseased stems at
temperatures lower than 25ºC. Bacteria normally survive in soil for a short time. Bacterial
cells enter lenticels of the developing tubers and either become inactive, or when conditions
are favorable, initiate decay (Pérombelon, 1992). Bacteria can also enter the tubers during
harvesting and handling through wounds via mechanical flailing (Pérombelon and van der
Wolf, 2002).
Figure 1.5 Disease cycle of potato blackleg caused by Pectobacterium atrosepticum (reproduced with
permission from Benlioğlu et al., 1991 and De Boer, 2004).
7
1.2.4 Disease management
1.2.4.1
Cultural Control
Cultural control practices are the first line of defence against bacterial diseases. Blackleg can
controlled by: (1) using clean, certified, well-healed seed pieces; the use of healthy tissueculture plantlets to initiate seed potato stocks is also an important method to reduce infection;
(2) using clean planting equipment; (3) good field hygiene and crop rotations; (4) planting
into well drained soil; (5) avoiding excessive irrigation; (6) removal of diseased plant material
to help reduce the survival and spread of the pathogen (Toth et al., 2003); (7) preventing the
wounding of seed pieces when planting; and (8) delaying harvest until the skin has matured to
reduce tuber injuries and the entry points for the pathogen.
Calcium ions and nitrogen compounds improve the structure and integrity of the plant cell
wall components and help to minimise the incidence of blackleg. Soil naturally low in these
nutrients can be amended by adding CaSO4 (gypsum) and nitrogen fertilizers to decrease
disease development (Bain et al; 1996; Graham and Harper, 1966).
Thermotherapy of tubers prior to storage has been shown to reduce blackleg incidence.
Dashwood et al. (1991) demonstrated that when small batches of tubers were sequentially
dipped in water at 55ºC for five minutes before being dried in high speed airflow, blackleg
incidence was reduced. However, this system was considered unlikely to be widely adopted
for use because of the high cost of the treatment when used with large numbers of tubers.
1.2.4.2
Chemical Control
Control of bacterial diseases relies on prevention of infection as once disease has been
initiated chemical control is ineffective since bacteria increase rapidly and are protected
within host tissue. The other main problem is the limited number of registered pesticides
available for disease control, and those that can be used are expensive and potentially
damaging to the environment and human health. A small range of chemicals have been tested
to reduce infection on or in tubers. Antibiotics such as streptomycin (Robinson et al., 1960),
oxytetracycline (Bonde and de Souza, 1954) and tetracycline have been used to treat tubers
and shown to reduce blackleg infection. However, their use is restricted due to risks of
introducing antibiotic resistance in human and animal pathogens. Copper-containing
compounds or Bordeaux mixture should be used before development of bacterial disease
(Mills et al., 2006). Another major problem associated with the use of pesticide is the risk of
pesticide resistance developing in the pathogen, i.e., copper resistance has developed in some
bacterial plant pathogens (Cooksey and Azad, 1992). Treatment with mercuric chloride has
8
proven highly effective in preventing blackleg (Hoyman, 1957), but is no longer used due to
deregistration of mercuric compounds as pesticides. Additionally, the overall effect of this
chemical use was found to be a delay in blackleg initiation, rather than prevention, and was
therefore ineffective. The realization of the risks of using pesticides in potato production have
resulted in an increase awareness of serious problems such as development of bacteria
resistant to pesticides, persistence of residues in tubers for consumption, destruction of
beneficial organisms, human intoxication, and contamination of the environment.
1.2.4.3
Biological control
Although a number of microbes such as fluorescent Pseudomonas spp. (Kastelein et al., 1999)
and Lactobacillus spp. (Trias et al., 2008) have shown potential to control blackleg in
laboratory assays and small scale disease trials, none have been developed commercially.
1.2.4.4
Traditional breeding
Use of resistant cultivars is the most important bacterial disease control procedure. Although
potato cultivars vary in disease susceptibility, none are known to have immunity to blackleg
disease (Nielsen, 1954). However, there are some cultivars such as Karaka (Anderson et al.,
1993) and Dawn (Genet et al., 2001) with partial resistance to the related Pcc pathogen
causing bacterial soft rot. High levels of bacterial resistance can be observed in many wild
potato species (most of them present in Central and South America). Through traditional
hybridization crosses with these diploid species and tetraploid species, some of these hybrids
display resistance to Pectobacterium spp. and Dickeya spp., although they also suffer some
major drawbacks (Table 1.1). Clonal crops such as potatoes are highly heterozygous and
usually suffer from severe inbreeding depression and instant loss of their genetic integrity
upon selfing or out crossing (Conner and Christey, 1994). Therefore, it is impossible to
maintain the genetic integrity of a cultivar whilst combining traits through sexual
hybridization. Furthermore, sexual breeding is complicated in potatoes by tetrasomic
segregation patterns and incomplete fertility in many tetraploids commercial cultivars (Ebora
and Sticklen, 1994).
In summary, traditional breeding has not succeeded in producing potato cultivars resistant to
blackleg because of the genetic complexity of the potato. None of the previously mentioned
interspecific hybrid lines (Table 1.1) have been commercialized to date. Other than that, the
breeding of resistant crops is time consuming, and has to be a continuous process to counter
crops becoming susceptible due to new races of the pathogen evolving.
9
Throughout the world a combination of all the above discussed management methods are used
to control blackleg disease of potato. Despite this, control of this disease remains a major
problem. To make maximum use of the potential of potato production and potato utilization,
research, development, and innovation play a crucial role. For all these reasons many research
groups are trying to develop strategies to improve potato plant resistance to bacterial diseases
through genetic engineering (Mourgues et al., 1998; Liu, 2006).
1.3 Plant Genetic Engineering
Since 1983, genetic engineering has been accomplished in many plant species, either by
biolistic methods or via Agrobacterium-mediated transformation. This approach facilitates the
transfer and expression of genes of non-plant origin in plants and greatly expands the
potential sources of disease resistance genes available for introduction into crop plants
(Conner and Jacobs, 1999). The major goal of plant biotechnology is genetic engineering of
disease and insect resistance in crops (Rao, 1995).
The major advantages of genetic engineering (GE) over traditional plant breeding were
described by Conner and Jacobs (1999) as:
-
in traditional breeding, genetic information can only transfer among individuals from
the same or closely related species, but in GE approaches DNA can be transferred into
plant from any organism;
-
in GE, only well-characterized genes are transferred; in contrast, traditional breeding
may transfer traits from wild germplasm into a crop plant, without knowing the genes
responsible for this trait, and
-
the breeding process can be considerably faster in GE because a gene is transferred in
a single step, while other characteristics of the recipient variety remains unaffected in
theory and the amount of backcrossing needed can be considerably reduced.
There has been enormous progress in the past decade in providing a number of options and
strategies which can be, and have been, used to make transgenic plants resistant to pathogens.
10
Table 1.1 Hybrids with wild species used in traditional breeding.
Wild species/Hybrids
Resistance
Drawback
Reference
S. canasense x S. Tuberosum
Pectobacterium carotovora subsp. atroseptica
Dickeya spp.
Diploid
Carputo et al., 1997
S. tarijense x S. Tuberosum
Pectobacterium carotovora subsp. atroseptica
Dickeya spp.
Diploid
Carputo et al., 1997
S. tuberosum subsp. andogena x S.
tuberosum
Dickeya spp.
Diploid
Hidalgo and Echandi, 1982
S. phureja x S. tuberosum
Pectobacterium carotovora subsp. atroseptica
S. commeronnii x S. tuberosum
Ralstonia solanacearum
Less tuber yield and temperature sensitive
resistance
Chance of genetic imbalance and somatic
incompatibility
Rousselle- Bourgeois and
Priou, 1995
Laferriere et al., 1999
S. stenotomum x S. tuberosum
Ralstonia solanacearum
Chance of genetic imbalance and somatic
incompatibility
Fock et al., 2001
S. chacoense x S. tuberosum
Pectobacterium carotovora subsp. atroseptica
Pectobacterium carotovora subsp. carotovora
High glycoalkaloid content-toxic to
humans and animals.
Carputo et al., 1997
S. sparsipillum x S. tuberosum
Pectobacterium carotovora subsp. atroseptica
Pectobacterium carotovora subsp. carotovora
Pectobacterium carotovora subsp. atroseptica
Pectobacterium carotovora subsp. carotovora
Pectobacterium carotovora subsp. atroseptica
Pectobacterium carotovora subsp. carotovora
High glycoalkaloid content-toxic to
humans and animals.
High glycoalkaloid content-toxic to
humans and animals.
Wide range of variation in plant
morphology
Carputo et al., 1997
S. multidissectum x S. tuberosum
S. brevidens + S. tuberosum
Carputo et al., 1997
Austin et al., 1986; Austin et
al., 1988; ZimnochGuzowska et al., 1999
11
1.4 Selectable marker genes
Transformation of potato via Agrobacterium-mediated techniques has become a common
practice and provides the chance to genetically engineer new disease resistance into potato
like crop plants (Zupan et al., 2000). In the event of Agrobacterium transformation, only a
small proportion of plant cells are recipients of the gene insertion event. Therefore a method
is required to recognize the transformed cells. The standard/current approach is the cointroduction of a selectable marker gene that confers resistance to phytotoxic chemicals such
as antibiotics or herbicides. These genes are frequently used because of their extensive utility,
efficiency as reliable selection markers, ease of use, and availability (Sunder and Sakthivel,
2008). This will allow the growth, division and development of only those plant cells
receiving such selectable marker genes to become complete plants on culture medium
supplemented with the appropriate selective agent (Draper and Scott, 1991). The most widely
using selectable marker gene for plant transformation is neomycin phosphotransferase II
(nptII) gene, from the bacterial transposon Tn5 (Flavell et al., 1999). The expression of this
gene in plant cells gives resistance to kanamycin and its derivative antibiotics as a selection
agent in media through detoxification by phosphorylation (Conner et al., 1991; Webb and
Morris, 1992). Kanamycin resistance has been commonly used as the marker system for the
recovery of transgenic potato plants and enables the selection of transformed cells for
subsequent regeneration into complete plants (Conner et al., 1991).
Initially, kanamycin tends to bleach tissues and then inhibits further growth of nontransformed plant tissue without causing rapid necrosis (Draper and Scott, 1991). The use of
the selection agent at a optimum threshold concentration is very important since too low
concentrations fails to eliminate the non-transformed cells and higher concentrations leads to
recovery of transgenic lines with higher expression due to integration of multiple copies of the
transgenes, which may result in subsequent problems due to homology induced gene silencing
(Barrell et al., 2002).
Barrell et al. (2002) and Barrell and Conner (2006) investigated a range of alternative
selectable markers and ranked them in terms of their relative efficiency of recovering
transgenic lines in potato transformation as kanamycin (nptII) > hygromycin (hpt) >
methotrexate (dhfr) > phosphinothricin (bar) > phleomycin (ble).
1.5 Regulation of transgene expression
Expression of heterologous genes requires the delivery of a construct containing a gene of
interest fused to a regulatory sequence. Regulatory elements ensure stable gene expression
12
and enable the expression to be switched on and off depending on the environmental
conditions, tissue type and developmental stage. A promoter is a regulatory region of DNA
located upstream (the 5’ region) of a gene, providing a control point for regulated gene
transcription. The promoter contains specific DNA sequences that are recognized by proteins
known as transcription factors. These factors bind to the promoter sequences, recruiting RNA
polymerase, the enzyme that synthesizes the mRNA from the coding region of the gene
(Webb and Morris, 1992; Hartl and Jones, 1998).
Certain sequences from A. tumefaciens and A. rhizogenes have been used as promoters, such
as those from nos (nopaline synthase), ocs (octopine synthase) and mas (mannopine synthase)
genes (Webb and Morris, 1992). Promoters are classified according to the gene expression
they control. Gene expression induced by environmental stimuli such as heat-shock promoters
(Moore et al., 1998), light sensitive promoters (Nap et al., 1993), chemically induced
promoters (Cao et al., 2001) and wound-induced promoters (Peferoen et al., 1990) are also
available. Phloem-specific promoters (Liu, 2006) and seed-specific promoters (Liu, 2006) are
classified as controlling tissue-specific gene expression. In eukaryotes, in addition to the
promoter sequences, there are also other DNA sequences called enhancers that interact with
the promoter to determine the level of transcription (Hartl and Jones, 1998).
Cauliflower mosaic virus (CaMV) 35S promoter is the most widely used in transgenic crops
to transcriptionally control foreign genes in plants. It is classified as a constitutive promoter to
drive gene expression in the majority of developed potato transgenic plants (Conner et al.,
1991; Douches et al., 1998 and 2002; Davidson et al., 2002 and 2004). It is important to
identify alternative promoters for potato transformation that restrict transgene expression to
foliar and stem plant parts. The most relevant approach to achieving this involves the isolation
of a promoter from potatoes. The promoter from the potato Lhca3St1 gene encoding
apoprotein 2 of the light-harvesting complex of Photosystem 1 is transcriptionally regulated
by light (Nap et al., 1993). This promoter is active in leaves, stems and other green parts of
potato, while less expression has been observed in roots, tubers and underground stolons. The
Lhca3St1 promoter gives twice the expression of the CaMV 35S promoter (Nap et al., 1993;
Annadana et al., 2001).
The recent improvements in plant-transformation techniques and progress in the
understanding of plant-pathogen interactions enables the use of genetic engineering for the
development of disease-resistant plants. Potato is an ideal crop for the introduction of disease
resistance technology due to its susceptibility to a number of bacterial, fungal, oomycetes and
viral diseases and the relative economic importance of these diseases.
13
Genetic engineering approaches to increase resistance to pathogens have proven to be
successful in a number of agriculturally important crops, including potato. The approaches
that have been taken by researchers can be grouped into five general categories (Mourgues et
al., 1998):
(1) The expression of gene products that destroy or neutralize a component of the pathogen
arsenal such as polygalacturonase, oxalic acid, and lipase.
(2) The expression of gene products that can potentially enhance the structural defences in the
plant. These include elevated levels of peroxidase and lignin.
(3) The expression of gene products releasing signals that can regulate plant defences. This
includes the production of specific elicitors, hydrogen peroxide (H2O2), salicylic acid (SA),
and ethylene (C2H4).
(4) The expression of resistance gene (R) products involved in the hypersensitive response
(HR) and in interactions with avirulence (Avr) factors.
(5) The expression of gene products that are directly toxic to pathogens or that reduce their
growth. These include pathogenesis-related proteins (PR proteins) such as hydrolytic enzymes
(chitinases, glucanases), antifungal proteins (osmotin and thaumatin-like), antimicrobial
peptides (thionins, defensins), ribosome inactivating proteins (RIP), and phytoalexins.
Of these approaches, genetic engineering with antimicrobial proteins conferring resistance to
bacteria offers a valuable opportunity for the development of bacteria-resistant transgenic
plants.
1.6 Antimicrobial peptides
1.6.1 Properties of antimicrobial peptides
Antimicrobial peptides (AMPs), also known as host defence peptides, are an evolutionarily
preserved component of the native immune response in plants. These peptides are produced to
kill or inhibit the progression of microbes such as bacteria, fungi, viruses, or parasites that are
ubiquitous in the plant and animal kingdoms (Hancock, 2001; Yeaman and Yount, 2003). The
majority of these AMPs are Cys-rich and have a globular structure which is stabilized by
disulfide bridges (Papagianni, 2003; Durr et al., 2006). The peptides are normally encoded by
multigenic families in which some genes are developmentally regulated (Does et al., 1999).
These peptides are potent, broad-spectrum antibiotics which demonstrate potential as novel
therapeutic agents (Bohlmann, 1994). Their mode of action varies in different pathogens and
includes disrupting membranes, interfering with metabolism, and targeting cytoplasmic
14
components (Rao, 1995). In general the antimicrobial activity of these peptides is determined
by measuring the minimal inhibitory concentration. Cysteine-rich peptides are the major class
of antimicrobial proteins found in plants. These peptides are categorized together, and all
contain even numbers of cysteine residues, which connect pairwise to stabilise the protein
structure.
The examples of antimicrobial peptide discovered in animals include: cecropin peptide from
Hyalophora cecropia moth (Hultmark et al., 1980), attacins from Hyalophora cecropia pupae
(Hultmark et al., 1980), sarcotoxin1 from flesh-fly (Okada and Natori, 1984), magainin from
the skin of the African clawed frog (Zasloff, 1987), tachyplesin from Southeast Asian
horseshoe crab (Nakamura et al., 1988), cecropin P1 from pig intestine (Lee et al., 1989),
defensin from insect Phormia terranovae (Dimarcq et al., 1990), caerin 1 from Australian tree
frog (Stone et al., 1992), moricin from silkworm Bombyx mori (Hara and Yamakawa, 1995),
paradaxin from Moses sole fish (Oren and Shai, 1996), parasin 1 from catfish (Park et al.,
1998) and Penaeidin4-1 from shrimp (Cüthbertson et al., 2006). Antimicrobial peptides have
also been isolated from plants, for example, thionine (Bohlman and Apel, 1987), antifungal
proteins from radish (Terras et al., 1992) and Snakin-1 and Snakin-2 from potato (Segura et
al., 1999; Berrocal-Lobo et al., 2002). The genes encoding these antimicrobial proteins are
now considered as a potential source of resistance genes to pathogens in plants (Evans and
Greenland, 1998).
1.6.2 Antimicrobial peptides for engineering disease-resistant plants
The expression of genes encoding antimicrobial peptides like magainin II (Barrell and
Conner, 2009), cecropin (Nordeen et al., 1992; Huang et al.,1997b; Wilson et al., 1998; Arce
et al., 1999, Zakharchenko et al., 2005; Jan et al., 2010), attacin (Arce et al., 1999; Norelli et
al.,1994, Reynoird et al.,1999, Ko et al.,2002), tachyplesin I (Allefs et al., 1996), lysozymes
(Ko et al., 2002), thionine (Molina and García-Olmedo, 1993; Epple et al., 1997), a heveinlike peptide (Koo et al., 2002), osmotin-like proteins (Zhu et al., 1996), pathogenesis-related
protein 1a (Alexander et al., 1993), non-specific lipid transfer proteins (Molina and GarcíaOlmedo, 1997), small cysteine-rich plant defensins (Bi et al., 1999; Gao et al., 2000), Snakins
(Almasia et al., 2008; Balaji and Smart, 2011), Pendeidin4-1 (Zhou et al., 2011) and snakindefensin fusion proteins (Kovalskaya et al., 2011) have been shown to enhance resistance to
many pathogens in various plant species.
As described above, a wide range of defence antimicrobial proteins have been identified and
are being utilized in attempts to provide disease protection in transgenic crops (Evans and
Greenland, 1998). To date, less effort has been devoted to the creation of transgenic plants
15
that are resistant to bacteria. A number of antimicrobial peptides are known to exhibit
bacterial toxicity in vitro; examples are listed in Table 1.2. Using genes for these
antimicrobial peptides offers opportunities to develop transgenic potato plants resistant to
blackleg disease.
The major antimicrobial peptides expressed in transgenic potato plants and investigated for
their improved resistance to Pectobacterium diseases are listed in Table 1.3. Many of these
antimicrobial peptides originated from animals or microorganisms. This contributes to some
of the public concerns over the deployment of GM crops in agriculture, especially ethical
issues associated with the transfer of DNA across wide taxonomic boundaries (Conner and
Jacobs, 2006). Therefore it is important that research focuses on the overexpression of genes
responsible for plant-derived antimicrobial peptides that can intensify or trigger the
expression of existing defence mechanisms. To date the two main plant-derived antimicrobial
peptide groups known to have antimicrobial activity are thionins and snakins.
Thionins are plant antimicrobial proteins which are able to inhibit a broad range of pathogenic
bacteria in vitro (Molina and García-Olmedo, 1993). Carmona et al. (1993) reported the
expression of alpha-thionin gene from barley in transgenic tobacco confers enhanced
resistance to two pathogens of P. syringae. Unfortunately, most thionins can be toxic to
animal and plant cells and thus may not be ideal for developing transgenic plants (ReimannPhilipp et al., 1989). Antimicrobial peptides Snakin-1 (SN1) and Snakin-2 (SN2) isolated
from Solanum tuberosum cv. Desiree have been found to be active against a number of
important plant pathogens in vitro (Segura et al., 1999; Berrocal-Lobo et al., 2002).
16
Table 1.2 Major antimicrobial peptides showing toxicity to bacterial pathogens.
Antimicrobial Source
peptide
Magainin
Cecropins
and its
analogs
Attacins
Penaeidin4-1
Skin of the
african clawed
frog (Xenopus
laevis)
Hemolymph of
Hyalophora
cecropia, the
giant silk moth
Action
Toxicity to
Reference
These peptides potentially kill
microbes inside the intercellular
space prior to its entry into the
cytoplasm.
These peptides interact with the
outer phospholopid membranes of
both Gram-negative and Grampositive bacteria and modify them
by forming a large number of
transient ion channels
Erwinia carotovora subsp. atroseptica, Streptomyces
scabies, Streptomyces sp., Erwinia carotovora
Wilson and Conner, 1995
Pseudomonas syringae pv. tabaci
Huang et al., 1997b
Erwinia carotovora subsp. atroseptica (Pca)
Erwinia amylovora
Erwinia carotovora subsp. atroseptica, Streptomyces
scabies, Streptomyces sp., Erwinia carotovora
Clavibacter michiganensis subsp. michiganensis,
Erwinia carotovora subsp. betavasculorum, Erwinia
carotovora, Pseudomonas corrugate, Pseudomonas
syringae pv. tabaci, Pseudomonas syringae pv.
glycinea, Pseudomonas solanacearum,
Xanthomomas campestris pv. vesicatoria.
Erwinia carotovora subsp. atroseptica (Pca)
Arce et al., 1999
Norelli et al.,1994
Wilson and Conner, 1995
Erwinia amylovora
Norelli et al., 1994;
Reynoird et al., 1999;
Ko et al., 2002
Botrytis cinerea, Penicillium crustosum, Fusarium
oxysporum Micrococcus luteus, Streptococcus.
viridans, Cryptococcus neoforman (Steroform A,
Steroform B, Steroform C, Steroform D) and
Candida spp. (Candida lipolytica, Candida
inconspicua, Candida krusei, Candida lusitaniae and
Candida glabrata)
Cüthbertson et al., 2004;
Cüthbertson et al., 2006
Hyalophora
cecropia pupae
The mechanisms of antibacterial
activity of this protein are to inhibit
the synthesis of the certain outer
membrane protein in gram negative
bacteria.
Atlantic White
Shrimp
Upon pathogen challenge to the
host, the peptides are released from
granular haemocytes to the plasma
and attached to cuticles fighting
microbial infection
Nordeen et al., 1992
Arce et al., 1999
17
Table 1.2 continued.....
Antimicrobial
peptide
Source
Lysozymes
Toxicity to
Reference
T4
The lysozyme attacks the murein layer
bacteriophage of bacterial peptidoglycan resulting in
and chicken
cell wall weakening and eventually
leading to lysis of both Gram-negative
and Gram-positive bacteria.
Horseshoe
Enhance the production of proteins and
crab,
other organic molecules produced prior
to infection or during pathogen attack
Erwinia carotovora
During et al., 1994
Erwinia amylovora
Ko et al., 2002
Erwinia carotovora
Allefs et al., 1996
Thionins
Plant
antimicrobial
proteins
Not yet defined
Molina and GarcíaOlmedo, 1993;
Florack et al., 1993
Snakin-1
Potato tubers
Not yet defined
Pseudomonas syringae pv. tabaci,Clavibacter
michiganensis subsp. michiganensis,Clavibacter
michiganensis subsp. sepedonicus,
Xanthomonas campestris pv. vesicatoria.
Clavibacter michiganensis subsp. sepedonicus ,
Fusarium solani, Fusarium culmorum, Bipolaris
maydis and Botrytis cinerea
Snakin-2
Potato tubers
Not yet defined
Ralstonia solanacearum and Erwinia
chrysanthemi
Berrocal-Lobo et al., 2002
Fusion Protein
(SN1+PTH1)
Potato
Not yet defined
Clavibacter michiganensis, Pseudomonas
syringae pv. tabaci, Pseudomonas syringae pv.
syringae
Kovalskaya et al., 2011
Dermaseptins
Arboreal frog
Not yet defined
Pseudomonas aeruginosa, Staphylococcus
aureus, Aspergillus flavus, A. fumigatus, A.
niger, Fusarium noniliforme and F oxysporum
De Lucca et al., 1998;
Nanon-Venezia et al., 2002
CaAMP1
Pepper
Not yet defined
Botrytis cinerea, Cladosporium cucumerinum,
Phytophthora capsici, Candida albicans,
Saccharomyces cerevisiae, and Bacillus subtilis
Lee et al., 2008
Tachyplesin I
Action
Segura et al., 1999
18
Table 1.3 Transgenic potato plants resistance to Erwinia (now known as Pectobacterium) inducing diseases.
Protein
Origin
Transgenic
Resistance to
species
Level of
Referance
resistance
Lyzozyme
T4 bacteriophage
Potato
Erwinia carotovora
Partial
During et al., 1994
Tachyplesin
Horse shoe crab
Potato
Erwinia carotovora
Partial
Allefs et al., 1996
Pectate lyase
Erwinia carotovora
Potato
Erwinia carotovora
Partial
Wegener et al., 1996
Glucose oxidase
Aspergillus niger
Potato
Erwinia carotovora
Partial
Wu et al., 1995
Cecropin analogs
Hyalophora cecropia pupae
Potato
Erwinia carotovora subsp. atroseptica
Partial
Arce et al., 1999
Attacin
Hyalophora cecropia pupae
Potato
Erwinia carotovora subsp. atroseptica
Total
Arce et al., 1999
Magainin
African clawed frog
Potato
Erwinia carotovora subsp. atroseptica
Total
Barrell and Conner, 2009
Dermaseptin
Arboreal frog
Potato
Erwinia carotovora
Partial
Osusky et al., 2005
Snakin-1
Potato
Potato
Erwinia carotovora subsp. atroseptica
Total
Almasia et al., 2008
19
1.6.3 Snakin-1 and Snakin-2 antimicrobial peptides
Snakin-1 (SN1) and Snakin-2 (SN2) are cysteine-rich antimicrobial peptides isolated from
potato that show broad spectrum activity against a range of bacterial and fungal pathogens
(Segura et al., 1999; Berrocal-Lobo et al., 2002). SNI and SN2 share common structural
features with more than 500 antimicrobial peptides discovered in plants and animals, such as
an N-terminal putative signal sequence, a highly divergent intermediate region and a
conserved, cysteine-rich carboxyl-terminal domain. Based on the homology to other genes,
these proteins have also been hypothesized to be involved in diverse biological processes,
including cell division, cell elongation, cell growth, transition to flowering, and signaling
pathways (Ben-Nissan et al., 2004; Nahirñak et al., 2012).
The SN1 peptide has 63 amino acid residues, 12 of which are highly conserved cysteines. The
amino acid sequence has some motifs in common with kistrin and other snake venoms, but
lacks the RGD motif responsible for disintegrin activity (Segura et al., 1999). The
antimicrobial activity of the purified SN1 protein was tested in vitro against bacterial and
fungal species. The SN1 protein (molecular mass of 6,922 D) showed toxicity to many
bacterial pathogens such as Clavibacter michiganensis subsp. sepedonicus and fungal
pathogens such as Fusarium solani, Fusarium culmorum, Bipolaris maydis and Botrytis
cinerea (Segura et al., 1999). SN1 expression was detected in tubers, stems, auxiliary buds,
and young floral buds, as well as in sepals, petals, stamens, and carpels from fully developed
flowers. Expression was not detected in roots, stolons or leaves, and not induced by either
abiotic or biotic stress, or chemical treatments such as jasmonic acid, salicylic acid,
isonicotinic acid, abscisic acid, gibberellic acid, and indoleacetic acid. This suggests that SN1
is a component of the constitutive defence barrier, especially of the storage and reproductive
organs (Segura et al. 1999). Overexpression of the SN1 gene was shown to confer enhanced
resistance in transgenic potato plants to Rhizoctonia solani and Erwinia caratovora (Almasia
et al., 2008).
The SN2 peptide has 66 amino acid residues, 15 of which are acidic residues and 12 of which
are cysteines. SN2 (molecular mass of 7,025.14 D) has been reported to be active against
many bacterial and fungal species (Berrocal-Lobo et al., 2002). SN2 is developmentally
expressed in tubers, petals and carpels, with expression also in stems, shoot apices, leaves,
flower buds and stamens, but not in roots, stolons and sepals (Berrocal-Lobo et al., 2002). The
SN2 gene was locally up-regulated in leaves by wounding and abscisic acid treatments,
responded weakly to salinity stress, but drought stress or treatments with gibberellic acid,
chitosan, jasmonic acid, ethylene, benzothiadiazole-7-carbothioic acid or S-methyl ester had
20
no effect. Expression of this SN2 gene is up-regulated after infection of potato tubers with the
fungus Botrytis cinerea and down-regulated by the virulent bacteria Ralstonia solanacearum
and Erwinia chrysanthemi. Overexpression of the tomato (Solanum lycopersicum)
(throughout this thesis tomato is referred to as Solanum lycopersicum) SN2 gene was shown
to confer enhanced resistance in transgenic tomato plants to Clavibacter michiganensis subsp.
michiganensis (Balaji and Smart, 2011).
SN1 and SN2 amino acid sequence alignments show similarity with members of the GAST
family from tomato and GASA family from Arabidopsis thaliana (Berrocal-Lobo et al.,
2002). Homologous genes have been also identified in a wide range of species such as GASA2
and GASA3 from A. thaliana, GAST1 from S. lycopersicum and Os0951 from Oryza sativa
(Ben-Nissan and Weiss, 1996; Kotilainen et al., 1999; Shi et al., 1992).
1.7 Aims and objectives of this thesis
A clear challenge for the biotechnology industry is to develop technologies, such as smallscale, rapid screens in vivo, to identify target gene products for each crop and disease
situation. Also, as success will depend not only upon the characteristics of the antimicrobial
protein, but also upon the regulatory elements utilised in their expression, the focus should not
only be on expression levels and timing, but also on targeting of the product to the correct
location in the crop plant. This study investigates the development of broad-spectrum diseaseresistant potato cultivars, by exploiting the antimicrobial nature of the endogenous SN1 and
SN2 genes. Using blackleg disease induced by Pca as a model, the overall aim of this thesis
was to test the hypothesis that overexpression of Snakin genes confers disease resistance in
plants. To achieve this aim, three objectives were established:
Objective 1: To determine the allele diversity of the Snakin-1 and Snakin-2 genes in potato
and related species (Chapter 2);
Objective 2: To overexpress the Snakin-1 and Snakin-2 genes under a potato light inducible
Lhca3 promoter in transgenic potatoes (Chapter 3); and
Objective 3: To determine the susceptibility of the transgenic potato lines to Pectobacterium
carotovora subsp. atroseptica (Chapter 4).
21
Chapter 2
Highly conserved alleles of Snakin-1 and Snakin-2 genes in
Solanaceous species
2.1 Introduction
An allele is one of two or more alternative forms of a gene with a unique nucleotide sequence
(Ayala and Kiger, 1980). Naturally occurring allelic diversity in plants accounts for the
genetic component of phenotypic variation in natural populations (Doebley and Lukens, 1998;
Buckler and Thornsberry, 2002). These allelic variations may play a role in gene regulation
and gene function. Identification of allelic variations that influence the plant phenotype is of
utmost importance for the utilization of genetic resources, such as in the genetic improvement
of plants. The transfer of novel alleles from wild relatives of crop plants into elite cultivars
(deVicente and Tanksley, 1993; Xiao et al., 1996; 1998; McCouch et al., 2007; Kaur et al.,
2008a) has clearly demonstrated that allele mining or allele diversity studies are set to play
important roles in future plant breeding research. Allele diversity studies also enable direct
access to key alleles conferring biotic and abiotic stresses, greater nutrient use efficiency,
enhanced yield and improved quality. In addition, such studies can provide information on the
rate of evolution of alleles, allelic similarity/dissimilarity of candidate genes, and allelic
synteny with other members of the gene family. Allele mining may facilitate molecular
discrimination among related species, development of molecular markers and marker assisted
selective breeding (Kumar et al., 2010).
In recognition of the potential for allele mining to explore the plant genetic resources, many
research institutes started maintaining crop germplasms and initiated allele diversity studies in
candidate gene of interest in various crop plants. Examples of loci investigated include: Mal d
3 in apple (Gao et al., 2005), Amy32b, rps2, VRN-HI, VRN-H2, genes coding GAMYB
transcription factor in barley (Polakova et al., 2005; Kubo et al., 2005; Cockram et al., 2007;
Haseneyer et al., 2008), Badh2 in rice (Sun et al., 2008), Alt3 in ryegrass (Miftahudin et al.,
2005), DGAT in soybean (Wang et al., 2006), Wx-A1 and SSIIa in wheat (Saito and
Nakamura, 2005; Shimbata et al., 2005). Investigating allele diversity facilitates the discovery
of novel variants of disease resistance genes that can be used in breeding programs.
Transferring beneficial alleles conferring disease resistance to elite genetic backgrounds has
resulted in increased trait performance in rice (Xiao et al., 1996; 1998; McCouch et al., 2007)
and tomato (deVicente and Tanksley, 1993). Other major disease resistant genes identified by
allele mining research are Pi ta and Pikh from rice that confer rice blast resistance (Bryan et
22
al., 2000; Huang et al., 2008; Wang et al., 2008a) Pto from tomato for bacterial speck
resistance (Rose et al., 2005), and Pm3 from wheat with powdery mildew resistance
(Yahiaoui et al., 2006; Kaur et al., 2008b).
In the case of potato, various types of molecular markers have been used for studies on
genetic variation of allelic diversity. Many studies produced RFLP marker based linkage
maps for diploid potato using gDNA and cDNA RFLP probes of potato (Bonierbale et al.,
1988; Gebhardt et al., 1989; 1991; Tanksley et al., 1992; Jacobs et al., 1995) and such linkage
maps will increase the efficiency and accuracy of plant breeding programmes. van Eck et al.
(1993; 1994a; 1994b) identified and mapped multiple alleles for flower colour, tuber shape,
anthocyanin pigmentation and tuber skin colour in potato using RFLPs. van Eck et al. (1995)
constructed an AFLP map consisting of 264 AFLP markers and the length of 1170cM in noninbred potato offspring. van Os et al. (2006) also developed an ultra-dense genetic linkage
map consisting > 10,000 AFLP loci in 136 lines of a diploid potato mapping population.
Milbourne et al. (1997) conducted a comparative analysis of allelic diversity at different loci
amongst 16 tetraploid potato cultivars using different PCR based marker systems like SSRs,
AFLP and RFLP. They subsequently developed 112 SSR markers of which 98 markers
exhibited high level of allelic polymorphism (Milbourne et al., 1998). Ghislain et al. (2004)
identified 931 potato germplasm assessions using 48 SSR markers. Likewise Feingold et al.
(2005) characterized 30 potato cultivars using 94 SSR markers. All these studies enable
qualitative and quantitative trait identification; marker assisted breeding and map-based gene
cloning in potato.
One of the first reported allele diversity studies involved the actin gene in Solanum tuberosum
where two allelic variants were reported (Drouin and Dover, 1990). Menendez et al. (2002)
and Li et al. (2005) identified the potential candidate genes involvement in tuber traits. These
included functional variants of orthologous invertase genes responsible for the chip quality
(invGE-f and invGF-d alleles) and tuber starch content (invGF-b allele). More recently, eight
allelic variants of the apoplastic invertase inhibitor gene from potato, hypothesised to
contribute to the genetic variation in cold-induced sweetening response, were also reported
(Datir et al., 2012). Wang et al (2008b) studied the allelic frequency and variation of Rpi-blb1
and Rpi-blb2 in a large number of tuber-bearing Solanum species and discovered highly
conserved late blight resistant RGA1-blb homologues in all tested Solanum species. Finding
new alleles of disease resistance genes in potato will benefit the production of disease
resistant potato germplasm in the future. The initial analysis of the potato genome sequencing
23
(The Potato Genome Sequencing Consortium, 2011) identified more than 800 putative disease
resistance genes, all with potential allelic variants among potato germplasm resources.
Antimicrobial peptides are the effector molecules of innate immunity and are considered to be
the hidden weapons of microbial destruction in plant genomes (Sitaram and Nagaraj, 2002).
Snakin-1 (SN1) and Snakin-2 (SN2) are low-molecular weight, small cysteine-rich
antimicrobial peptides isolated from potato tubers and found to be active against bacterial and
fungal plant pathogens (Segura et al., 1999; Berrocal-Lobo et al., 2002). The StSN1 gene is
763 bp containing two exons encoding a protein of 88 amino acids (Segura et al., 1999). The
StSN2 gene is slightly smaller; 727 bp containing three exons encoding a protein of 104
amino acids (Berrocal-Lobo et al., 2002). Almasia et al. (2008) compared the sequences of the
SN1 gene in three different wild Solanum species (S. bulbocastanum, S. commersonii and two
genotypes of S. chacoense) and one potato cultivar (S. tuberosum cv. Kennebec) and revealed
the conserved nature of the SN1 gene. Snakin proteins share common structural features with
more than 500 antimicrobial peptides discovered in plants and animals, such as an N-terminal
putative signal sequence, a highly divergent intermediate region and a conserved, cysteinerich, 60 amino-acid carboxyl-terminal domain (Berrocal-Lobo et al., 2002). Orthologues of
Snakin genes have been detected in other plants, such as tomato (GAST1; Shi et al., 1992),
arabidopsis (GASA1 and GASA4; Herzog et al., 1995), strawberry (GAST; De la Fuente et
al., 2006), Petunia hybrida (GIP1; Ben-Nissan et al., 2004), Gerbera hybrida (GEG;
Kotilainen et al., 1999). Based on the homology to other genes, these proteins have also been
hypothesised to be involved in diverse biological processes, including cell division, cell
elongation, cell growth, transition to flowering, and signalling pathways (Nahirñak et al.,
2012). The main focus of this study was to identify the allele diversity among SN1 and SN2
genes from a wide collection of Solanaceous species. The motivation for this work was to find
alternative useful alleles with the potential to contribute a different spectrum of resistance to
potato diseases.
2.2 Materials and Methods
2.2.1 Plant Material
SN1 and SN2 genes were isolated from potato (S. tuberosum) cultivars Iwa, Desiree and the
diploid potato clones ‘SH83-92-488’ and ‘RH89-039-16’ (here after referred to as ‘SH’ and
‘RH’ respectively), as well as a range of other Solanaceous species such as S. bulbocastanum
Donal (PT29), S. chacoense Bitter (Lincoln population, Herbarium No. CHR571411), S.
nigrum L. (Lincoln population), S. lycopersicum L. (cv. Moneymaker), S. palustre Poepp. (PI
218228; US Potato Genebank, Sturgeon Bay, WI), Petunia hybrida Jess. (cv. Mitchell),
24
Nicotiana tabacum L. (cv. Petit Havana SR1) and Capsicum annuum L. (cv. Californian
Wonder). Plants were grown by sowing seeds or planting tubers in PB5 polyethylene bags
containing potting mix (Conner et al., 1994). The greenhouse conditions included heating
below 15ºC and ventilation above 21ºC. Day length was supplemented to 16 h when needed
with 500 W metal halide vapor bulbs. For genomic DNA extraction, leaves from 4 week-old
plant were harvested. DNA from ‘SH83-92-488’ and RH89-039-16’ was provided by Dr
Jeanne Jacobs (Plant & Food Research, New Zealand), and DNA of S. bulbocastanum was
provided by Dr James Bradeen (University of Minnesota, USA).
2.2.2 Genomic DNA isolation
Genomic DNA was isolated from the leaves of greenhouse-grown plants based on the method
described by Bernatzky and Tanksley (1986) (Appendix B). The quality and quantity of the
extracted DNA was confirmed by gel-electrophoresis in a 1% agarose gel in 1xTAE buffer at
5.5 V/cm for 80 min and visualized under UV light after staining with ethidium bromide (5
mg/L) for 15 min, and by measuring the absorbance ratio at 260nm and 280nm using a
NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA).
Pure DNA preparations have expected A260/280 ratios of 1.7-1.9.
2.2.3 PCR amplification of the SN1 and SN2 genes
Primers (Table 2.1) were designed based on the reference gene sequences for the StSN1 gene
(Genbank accession AJ320185) and the StSN2 gene (Genbank accession AJ312424) using
Primer 3 software (http://frodo.wi.mit.edu/primer3/; Rozen and Skaletsky, 2000). The primers
used to isolate the SN1 gene were SN1-F1 and SN1-R5 which produce a predicted product of
883 bp. For the SN2 gene, the primers SN2-F2 and SN2-R3 were used which produce a
predicted product of 1009 bp.
PCR Extender kit (5 PRIME, Gaithersburg, MD, US) was used for all the PCR reactions.
PCR reactions were performed using Extender enzyme mix to isolate the SN genes from all
sources of DNA.
Each PCR amplification was carried out in a total volume of 50 µL containing ~100 ng
genomic DNA, 200 nM of each primer, 0.2 U Extender enzyme mix (5 U/µL), 200 µM of
each dNTP, 1x HighFidelity Reaction Buffer (5 PRIME). PCR reactions were performed in a
Mastercycler (Eppendorf, Hamburg, Germany). The conditions for PCR for the SN1 gene
were: 1 cycle at 94C for 2min, 34 cycles of (20 s 94C, 15 s 47C, 90 s 72C), followed by 6
min extension at 72C. The SN2 PCR was performed using the same PCR conditions with
25
49C annealing temperature. Amplified products were separated by gel-electrophoresis and
visualised as described above.
Table 2.1 Primers used to isolate SN genes.
Target gene
SN1
SN2
Primer pairs
Primer sequence (5’ to 3’)
SN1-F1
CATCAAATTTTCAGCTTCG
SN1-R5
TTTTCTATTTCAATATGTATAAC
SN2-F2
GCTTATCAATTTATAGAAAAATA
SN2-R3
ACTCGAACTTCATTTATTCCTC
Product size (bp)
883
1009
2.2.4 Cloning of PCR fragments
Fragments of the expected size, 883 bp for SN1 and 1009 bp for SN2, were excised from the
ethidium bromide stained agarose gels using a scalpel and the DNA products were extracted
using a QIAquick gel extraction kit (QIAGEN, GmbH, Germany) according to the
manufacturer’s instructions. The quality and concentration of the purified products was
confirmed by gel-electrophoresis in a 1% agarose gel in 1xTAE buffer at 5.5V/cm for 45 min
and visualized under UV light after staining with ethidium bromide (5mg/L) for 15 min, and
by measuring the absorbance ratio at 260nm wavelength using a NanoDrop® ND-1000
spectrophotometer. The purified PCR products were ligated into pGEM®-T Easy vector
(Promega, Mannheim, Germany). Ligation was performed according to the manufacturer’s
instructions.
2.2.5 E. coli transformation and plasmid isolation
Ligated DNA was used for transformation into Escherichia coli. Subcloning EfficiencyTM
DH5αTM Competent Cells (Invitrogen, Carlsbad, CA, USA) were transformed with DNA from
ligation reactions according to the manufacturer’s instructions. An aliquot of 200 µL from
each transformation was spread onto Luria Bertani (LB) agar media (Appendix A) containing
ampicillin (100µg/mL), 0.5 mM IPTG (isopropyl β-D-thiogalactopyranoside) and 80µg/mL
X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside), then incubated at 37°C overnight for
colony formation. Transformed colonies were identified by blue-white colony screening. The
white colonies were further screened using PCR with specific SN gene primers to identify
whether they contained inserts of SN genes. Individual white colonies were picked using a
sterile pipette tip and resuspended in 10 µL of PCR mix. Each 10 µL PCR mix contained
1xThermoPol Reaction Buffer [20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM
26
MgSO4, 0.1% Triton X-100, pH 8.8 at 25°C], 0.2 mM of each dNTP, 0.2 µM of each primer
and, 0.4 U of Taq DNA polymerase (New England BioLabs, Massachusetts, USA). PCR
conditions as described in Section 2.2.3 except the initial denaturing was performed at 94ºC
for 4 min. Amplified products were separated by gel-electrophoresis and visualised as
described above. White colonies used for PCR screening were also streaked on LB agar plates
containing 100µg/mL ampicillin and the Petri dishes were incubated at 37°C overnight.
Isolated single white colonies growing on LB plates, confirmed as PCR-positive for SN genes
were inoculated individually into 2 mL liquid LB media (Appendix A) containing 100 µg/mL
ampicillin. These were then incubated at 37ºC overnight at 1000 rpm in a Thermomixer®
(Eppendorf). Plasmids were isolated from the overnight cultures using the High Pure Plasmid
Isolation Kit (Roche Applied Science, Indianapolis, USA) according to the manufacturer’s
instructions.
The
plasmid
DNA
was
quantified
using
NanoDrop®
ND-
1000
spectrophotometer and gel electrophoresis. To confirm the presence of positive intact clones,
restriction enzyme digestion of plasmid DNA was also carried out with NotI restriction
enzyme at 37ºC for 3 h. Each 10 µL digestion mix contained 1X manufacturer supplied
buffer, 0.5 µL BSA (2 mg/ml), 0.1 µL of NotI (10 U/µL) (New England Biolabs,
Massachusetts, USA) and 4 µL of plasmid DNA. Digested fragments were separated by gelelectrophoresis and visualized as described in Section 2.2.2. A maximum of sixteen plasmids
with intact clones were selected for sequencing.
2.2.6 Sequencing of the SN1 and SN2 genes
Both strands of each fragment were sequenced by Sanger sequenced using M13 primers. Each
10 µL sequencing reaction contained 1.75 µL 5x Sequencing buffer, 0.5 µL Big Dye (Applied
Biosystems, Warrington, UK), 0.16 µL of primer (M13 forward or M13 reverse at 20 µM)
and 150-300 ng of plasmid DNA. A 96-well microtitre plate was used for sequencing
reactions. The conditions for the sequencing reactions were: 1 cycle at 96C for 1 min at 50
cycles of [10 s 96C, 5 s 50C, 4 min 60C], then ramped to 4C. To collect the samples in
the bottom of the plate, the plate was briefly spun (Eppendorf 5510R bench top centrifuge),
templates were precipitated by the addition of 2.5 µL 125 mM EDTA (pH 8.0) and 25 µL
100% ethanol to each well. The plate was then sealed with aluminium film and the contents
were mixed by inverting the plate at least 4 times. The plate was left at room temperature for
15 min, and then centrifuged at 3,200 x g for 30 min at 4C. This step was immediately
followed by the inversion of the plate on paper towels and centrifugation at 200 x g
(Eppendorf 5510R bench top centrifuge) for 30 s to remove the supernatants without
disturbing the DNA pellets. To each well 35 µL 70% ethanol was then added and the plate
27
was centrifuged again for 15 min at 3000 x g at 4C. The removal of ethanol, without
disturbing the DNA pellets, was immediately carried out as described above. The plate was
then air dried in an incubator at 37C for 10 min, after which 10 µL of Hi-Di formamide
buffer (Applied Biosystems) was added to each well. The samples were denatured at 95C for
5 min using Mastercycler (Eppendorf, Hamburg, Germany) before being placed on ice for 5
min. Finally, the plate was briefly centrifuged to collect samples in the bottom of each well.
The sequencing reactions were analysed using an Applied Biosystems 3130xl genetic analyser
(Applied Biosystems).
Vector NTI Advance 10 software package (Invitrogen) was used to analyse the sequences and
assembled into contigs. Homology of the contigs sequences from the different Solanaceous
species and genotypes were aligned with the reference sequence obtained from the GenBank
database. The aligned sequences were verified for their homology and variability. The protein
sequences were determined using DNAMAN (Version 3.2, Lynnon Biosoft, Canada) to
identify any amino acid polymorphisms. Searches for sequence similarities were carried out
using BLASTn (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi).
2.2.7 Phylogenetic analysis
The evolutionary relationship within the SN gene family was examined by constructing a
phylogenetic tree based on multiple sequence alignments of the DNA sequences of the SN1
and SN2 alleles from a wide range of potato cultivars/clones, as well as a wide range of
Solanum species using the neighbour-joining method implemented in the Geneious Software
(Drummond et al., 2011). The length of the branch indicates the amount of divergence
between two nodes in a phylogenetic tree.
2.3 Results
2.3.1 PCR isolation of the SN1 and SN2 genes
The SN gene products were successfully amplified from all species using the two primer sets,
suggesting the presence of intact SN genes in all genotypes. The size of the PCR product
using the SN1-F1 and SN1-R5 primer set was of the expected size of ~883 bp (Figure 2.1A)
and the product using the SN2-F2 and SN2-R3 primer set was the expected size of ~1009 bp
(Figure 2.1 B).
Fragments of the expected size for the SN genes were successfully purified from the gel using
a gel extraction kit (QIAGEN), and cloned into the pGEM®-T Easy vector and transformed
into E. coli strain DH5. Intact clones were identified via PCR directly from E. coli colonies.
28
Plasmids were recovered from PCR-positive white colonies and the NotI restriction digestion
of plasmid DNA, isolated from PCR-positive white colonies, released the expected ~883 bp
SN1 and ~1009 bp SN2 fragment and ~3 kb vector fragment.
2.3.2 Identifying alleles of the SN genes in Solanaceous species.
A total of 154 clones were sequenced for identifying alleles of the SN1 and SN2 genes from
four potato genotypes, as well as eight other Solanaceous species. Sixteen positive clones
from each of the tetraploid potato cultivars and 6 clones each from the diploid species, N.
tabacum and S. nigrum were selected and sequenced for both genes to ensure >95% chance of
detecting all alleles for each species (Simko, 2004).
The sequences were aligned with reference to the StSN1 gene (AJ320185) and the StSN2 gene
(AJ312424) using Vector NTI Advance Software. Reference sequences were obtained from
the NCBI Nucleotide sequence database (Appendix C). This study has revealed several allelic
variants within and between genotypes/species for both the SN1 and SN2 genes (Table 2.2).
Sequence analysis led to the discovery of four SN1 alleles (designated a1-a4) and six SN2
alleles (designated b1-b6). Alleles a1, a2, a3 and a4 showed 99.7%, 96.1%, 93.6% and 96.5%
nucleotide identity to the reference StSN1 (AJ320185) sequence, respectively. Alleles b1, b2,
b3, b4 , b5 and b6 showed 99.7%, 93.6%, 76.7%, 78.2% ,78.6% and 93.4% nucleotide
identity to the reference StSN2 (AJ312424) sequence, respectively. The a1 and b1 alleles are
almost identical to the GenBank reference sequences for the SN1 gene (AJ320185) and the
SN2 gene (AJ312424), respectively. The boundaries between exons and introns regions were
confirmed by sequencing cDNA sequences of the StSN genes (Appendix D; provided by Dr.
S. Meiyalaghan, Plant and Food Research, Lincoln).
The SN1 gene has two exons (82 and 185 nucleotides) and one large intron (496 nucleotides)
(Figure 2.2). The SN2 gene has 3 exons (87, 46 and 182 nucleotides respectively) and two
introns (249 and 163 nucleotides) (Figure 2.3). Alignment of the allele sequences in Figure
2.2 and Figure 2.3 reveals all the nucleotide variations between the alleles and the reference
sequences. Both the coding regions and non-coding regions of the genes are highly conserved
throughout the wide range of Solanaceous species and genotypes studied.
A comparison of the variation in the different SN1 and SN2 alleles relative to the reference
sequences is provided in Table 2.3. For both the SN1 and SN2 genes, the majority of the
allelic variation occurs within the intron regions, with relatively few nucleotide substitutions
in the exon regions.
29
Figure 2.1 A. PCR amplified SN1 gene from various Solanaceous species and genotypes. Lane 1,
HyperLadderTM II (Bioline, UK). Lanes 2-13 PCR products using the primer set SN1-F1/SN1-R5 producing a
product of ~883 bp. Lane 2, S. chacoense; lane 3, S. nigrum; lane 4, S. lycopersicum; lane 5, Petunia hybrida;
lane 6, Capsicum annuum; lane 7, Nicotiana tabacum; lane 8, S. tuberosum ‘RH’; lane 9, S. tuberosum ‘Iwa’;
lane 10, S. tuberosum ‘Desiree’; lane 11, S. bulbocastanum; lane 12, S. palustre; and lane 13, S. tuberosum ‘SH’.
Figure 2.1 B. PCR amplified SN2 gene from various Solanaceous species and genotypes. Lanes 1 and 10,
HyperLadderTM II (Bioline). Lanes 2-9 and 11-14 PCR products using the primer set SN2-F2/SN2-R3
producing a product of ~1009 bp. Lane 2, S. chacoense; lane 3, S. nigrum; lane 4, S. lycopersicum; lane 5, S
tuberosum ‘SH’; lane 6, S. tuberosum ‘RH’; lane 7, Petunia hybrida; lane 8, Capsicum annuum; lane 9,
Nicotiana tabacum; lane 11, S. tuberosum ‘Iwa’; lane 12, S. tuberosum ‘Desiree’; lane 13, S. bulbocastanum;
and lane 14, S. palustre.
30
Table 2.2 SN1 and SN2 alleles in various Solanaceous species and genotypes. [presence (√) absence (X)]
SN1
SN2
Solanaceous species/genotypes
a1
a2
a3
a4
b1
b2
b3
b4
b5
b6
S. tuberosum ‘Iwa’ (4n)
√
√
X
X
√
√
X
X
X
X
S. tuberosum ‘Desiree’ (4n)
√
√
√
X
√
X
X
X
X
X
S. tuberosum ‘SH83-92-488’ (2n)
X
√
X
X
√
√
X
X
X
X
S. tuberosum ‘RH 89-039-16’ (2n)
X
√
√
X
√
√
X
X
X
X
S. palustre (2n)
X
√
X
X
X
X
√
X
X
X
S. bulbocastanum (2n)
X
√
√
X
X
X
X
√
X
X
S. lycopersicum (2n)
√
X
X
X
X
X
X
X
√
X
S. chacoense (2n)
X
X
X
√
√
X
X
X
X
√
S. nigrum (6n)
√
X
X
√
√
X
X
X
√
√
Petunia hybrida (2n)
√
X
X
X
√
X
X
X
X
X
Capsicum annuum (2n)
√
X
X
X
√
X
X
X
X
X
Nicotiana tabacum (4n)
√
X
X
X
√
X
X
X
X
X
31
Ref.StSN1
Allele a1
Allele a2
Allele a3
Allele a4
1
100
ATGAAGTTATTTCTATTAACTCTGCTTTTGGTCACTCTTGTTATTACCCCTTCTCTCATTCAAACTACCATGGCTGGTTCAAGTAAGTACCAATTCTTCC
ATGAAGTTATTTCTATTAACTCTGCTTTTGGTCACTCTTGTTATTACCCCTTCTCTCATTCAAACTACCATGGCTGGTTCAAGTAAGTACCAATTCTTCC
ATGAAGTTATTTCTATTAACTCTGCTTTTGGTCACTCTTGTCATTACCCCTTCTCTCATTCAAACTACTATGGCTGGTTCAAGTAAGTACCAATTCTTCC
ATGAAGTTATTTCTATTAACTCTGCTTTTGGTCACTCTTGTTATTACCCCTTCTCTCATTCAAACTACTATGGCTGGTTCAAGTAAGTACCAATTCTTCC
ATGAAGTTATTTCTATTAACTCTGCTTTTGGTCACTCTTGTTATTACCCCTTCTCTCATTCAAACTACTATGGCTGGTTCAAGTAAGTACCAATTCTTCC
Ref.StSN1
Allele a1
Allele a2
Allele a3
Allele a4
101
200
ATCATCTTTAAAATTTGCTACTAGTCTTCTGGAAACATCCACCAGGTAGTAGTAAGGTTTTCGTATACTTTGCTTTTTTCATACCTTATCAAGTGAAATT
ATCATCTTTAAAATTTGCTACTAGTCTTCTGGAAACATCCACCAGGTAGTAGTAAGGTTTTCGTATACTCTGCTTTTTTCATACCTTATCAAGTGAAATT
ATCATCTTTAAAATTTGCTACTAGTCTTCTGGAAACATTCACGAGGTAGTAGTAAGGTTTTCGTATACTCTGCTTTTTTCATACCTTACCAAGTGAAATT
ATCATCTTTAAAATTTGCTACTAGTCTTCTGGAAACAT-CATGAGATAGTGGTAAGGTTTTCGTATACTCTACTTTTTTCATACCTTATCAAGTGAAATT
ATCATCTTTAAAATTTGCTACTAGTCTTCTGGAAACATCCACCAGATAGTAGTAAGATTTTCGTATACTTTGCTTTTTTCATACCTTATCAAGTGAAATT
Ref.StSN1
Allele a1
Allele a2
Allele a3
Allele a4
201
300
TGAATATGTACTTGTCCTC-GGGCATAGCTATAGCCATTCCAGGGTATCCAATTATATGTCAAATT-TACATTTAAAGAATATATATTTAAAA-TTGAAT
TGAATATGTACTTGTCCTC-GGGCATAGCTATAGCCATTCCAGGGTATCCAATTATATGTCAAATT-TACATTTAAAGAATATATATTTAAAA-TTGAAT
TGAATATGTACTTGTTCTCGGGGCATAGCTATAGCCACTCCAGGGTATCCAATTATACGTCAAATTCTACATTTAAAGAATATATATTTAAAA-TTGAAT
TGAATATGTACTTGTCCTTGGGGCATAGCTATAGCCACTCCAGGGTATCCAATTATATGTCAAATTTTACATTTAAAGAATATATATTTAAAAATTGAAT
TGAATATGTACTTGTCCTC-GGGCATAGCTATAGCCACTCCAGGGTGTCCAATTATATCTCAAATTCTACATTTAAAGAATATATATTTAAAA-TTGAAT
Ref.StSN1
Allele a1
Allele a2
Allele a3
Allele a4
301
400
ACACTTGACAAAAGTTATGTCTTTAGCTAGTGGTAATGAGTTTTCATTTGAAC-------TTTTGGGTTTGAACCC-----------------CAAATAG
ACACTTGACAAAAGTTATGTCTTTAGCTAGTGGTAATGAGTTTTCATTTGAAC-------TTTTGGGTTTGAACCC-----------------CAAATAG
ACACTTGACAAAAGTTATGCCTTTAGCTAGTGGTAATGAGTTTTCATTTGAATGAAAGAACTTTGGGTTTGAACCC-----------------CAAATAG
ACACTTGACAAAAGTTATGTCTTTAGCTAGTGGTAATGAGTTTTCATTTGAAC-AAAGAATTTTGGGTTTGAACCCCAAATAGTACAATATCCCAAATAG
ACATTTGACAAAAGTTATGTCTTTAGCTAGTGGTAATGAGTTTTCATTTGAAC–AAAGAATTTTTGGGTTGAACCC-----------------CAAATAG
Ref.StSN1
Allele a1
Allele a2
Allele a3
Allele a4
401
500
TACAATATTTTTTTGTTTTATTTTGACAGCCACACATCATTATTTTTAGACACCTTTAATAAAATTTCTAGCTTAACCACTGATTGTTCTAATATTTTCA
TACAATATTTTTTTGTTTTATTTTGACAGCCACACATCATTATTTTTAGACACCTTTAATAAAATTTCTAGCTTAACCACTGATTGTTCTAATATTTTCA
AACAATATTTTTTTGTTTTATTTTGACAGCCACACACCATTATTTTTAGACACTTTTAATAAAATTTCTGGCTTAACCACTGATTGTTCTAATATTTTCA
TACAATATTTTTTTGTTTTATTTTGACAGCCACAC--CATTATTTTTAGACATCTTTAATAAAATTTCTGGCTTAACCATTGATTGTTCTAATATTTTCA
TACAATATTTTTTTGTTTTATTTTGAGAGTCACACACCATTATTTTTAGAGACCTTTAATAAAATTTCTGACTTAACCACTGATTGTTCTAATATTTTCA
Ref.StSN1
Allele a1
Allele a2
Allele a3
Allele a4
501
600
CTACTATCCACTAACACAACACATATATAAGATAATTTTGTCCACTAATTTTTTTTTTTAAA--GTTATA-TGTTCTAATTATGAGTTTTTTTTTT-AAT
CTACTATCCACTAACACAACACATATATAAGATAATTTTGTCCACTAATTTTTTTTTTTAAA--GTTATA-TGTTCTAATTATGAGTTTTTTTTTTTAAT
CTACTATCCACTAACACAACACATATATAAGATAATTTTGTCCAATAATTTTTTTTTTAAAA--GTTATA-TGTTCTAATTATGAGTTTTTTTTTTT--T
CTACTATACACTAACACAACACATATATAAGATAATTTTGTCCACTAATTTTTTTTTAAAAAAAGTTATAATGTTCTAATTATGAGTTTTTTTTTT---T
CTACTATCCACTAACGCAACACATATATAAGATAATTTTGTCCACTAATTTTTTTTTTAAAAA-GTTATA-TGTTCTAATTATAAGTTTTT--------T
Ref.StSN1
Allele a1
Allele a2
Allele a3
Allele a4
601
700
TTTTTAAAGATTTTTGTGATTCAAAGTGCAAGCTGAGATGTTCAAAGGCAGGACTTGCAGACAGATGCTTAAAGTACTGTGGAATTTGTTGTGAAGAATG
TTTTTAAAGATTTTTGTGATTCAAAGTGCAAGCTGAGATGTTCAAAGGCAGGACTTGCAGACAGATGCTTAAAGTACTGTGGAATTTGTTGTGAAGAATG
TTTTTAAAGATTTTTGTGATTCAAAGTGCAAGCTGAGATGTTCAAAGGCAGGACTTGCAGACAGATGCTTAAAGTACTGTGGAATTTGTTGTGAAGAATG
TTTTTAAAGATTTTTGTGATTCAAAGTGCAAGCTGAGATGCTCAAAGGCAGGACTTGCAGACAGATGCTTAAAGTACTGTGGAATTTGTTGTGAAGAATG
TTTTTAAAGATTTTTGTGATTCAAAGTGCAAGCTGAGATGTTCAAAGGCAGGACTTGCAGACAGATGCTTAAAGTACTGTGGAATTTGTTGTGAAGAATG
Ref.StSN1
Allele a1
Allele a2
Allele a3
Allele a4
701
794
CAAATGTGTGCCTTCTGGAACTTATGGTAACAAACATGAATGTCCTTGTTATAGGGACAAGAAGAACTCTAAGGGCAAGTCTAAATGCCCTTGA
CAAATGTGTGCCTTCTGGAACTTATGGTAACAAACATGAATGTCCTTGTTATAGGGACAAGAAGAACTCTAAGGGCAAGTCTAAATGCCCTTGA
CAAATGTGTGCCTTCTGGAACTTATGGTAACAAACATGAATGTCCTTGTTATAGGGACAAGAAGAACTCTAAGGGCAAATCTAAATGCCCTTGA
CAAATGTGTGCCTTCTGGAACTTATGGTAACAAACATGAATGTCCTTGTTATAGGGACAAGAAGAACTCTAAGGGAAAGTCTAAATGCCCTTGA
CAAATGTGTGCCTTCTGGAACTTATGGTAACAAACATGAATGTCCTTGTTATAGGGACAAGAAGAACTCTAAGGGCAAGTCTAAATGCCCTTGA
Figure 2.2 Alignment of SN1 sequences. The reference StSN1 gene (AJ320185) aligned with the sequences of
the SN1 alleles from this study. Features shown are exons (yellow) with start and stop codons (grey),
substitutions (blue), insertions (green) and deletions (red).
32
Ref.StSN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
1
100
ATGGCCATTTCGAAAGCTCTCTTTGCTTCATTACTTCTCTCCTTGCTCCTTCTCGAGCAAGTCCAATCTATTCAGACCGATCAAGTGGT-GAGTTATTTT
ATGGCCATTTCGAAAGCTCTCTTTGCTTCATTACTTCTCTCCTTGCTCCTTCTCGAGCAAGTCCAATCTATTCAGACCGATCAAGTGGT-GAGTTATTTT
ATGGCCATTTCGAAAGCTCTCTTTGCTTCATTACTTCTCTCCTTGCTCCTTCTCGAGCAAGTCCAATCTATTCAGACCGATCAAGTGGT-GAGTTACTTT
ATGGCCATCTCTAAAGCTCTCTTTGCTTCATTACTTCTCTCCTTGCTCCTTCTCGAGCAAGTTCAATCTATTCAGACCGATCAAGTGGTAAAGTTATTTT
ATGGCCATTTCTAAAGCTCTCTTTGCTTCATTACTTCTCTCCTTGCTCCTTCTCGAGCAAGTCCAATCTATTCAGACCGATCAAGCGGT-AAGTTATTCT
ATGGCCATTTCTAAAGCTCTCTTTGCTTCATTACTTCTCTCCTTGCTCCTTCTCGAGCAAGTCCAATCTATTCAGACTGATCAAGTGGT-GAGTTATTTT
ATGGCCATTTCGAAAGCTCTCTTTGCTTCATTACTTCTCTCCTTGCTCCTTCTCGAGCAAGTCCAATCTATTCAGACCGATCAAGTGGT-GAGTTATTTT
Ref.StSN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
101
200
ATTTG--TTCCTTAGCAATTTACTTAAAAATTA-TATCTATGCTACGTA-----------TATTTCTTCGGTTCTAAA-ATAGAAGAACTCATGATAAAT
ATTTG--TTCCTTAGCAATTTACTTAAAAATTA-TATCTATGCTACGTA-----------TATTTCTTCGGTTCTAAA-ATAGAAGAACTCATGATAAAT
ATTTG--TTCCTTAGCAA-TTACTTAAAATTTT-TATCTATGCTACGTA-----------TATTTCTTCGGTTCTAAA-ATAGAAGAACTCATGATAAAT
ATTTAGCTTACTCAAAAATCTA--GAAAGTTTACTACCCATGTTACGTACGTATGTATACTACTTTTTATGTTCTAAAAAGAGAAGAATTC---ATAAAT
ATTTAGCTTTCTCAAAAATCTA—-GGAAGTTTACTACGCATGT-----------------TACTTTTTATCTTCTAAAAAGAGAAGAATTC---ATAAAT
ATTTA--TTCCTTAGC---TTACACAAAAATTA-TATTTATCCTAG--------------TATATTTT-----------------GAACTCGTAATAAAC
ATTTG--TTTCTTAGCAA-TTACTTAAAAATT-CTATCTATGCTACGTA-----------TATTTCTTCGGTTCTAAA-ATAGAAGAACTCATGATAAAT
Ref.StSN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
201
300
AGTCTAAGAG------GTTTGAATTACCGTTTCATCATGAATTTATGT----------------------GGAAAAAAAAAATCTAGT-------TGACT
AGTCTAAGAG------GTTTGAATTACCGTTTCATCATGAATTTATGT----------------------GGAAAAAAAAAATCTAGT-------TGACT
AGT-AAAGAG------GTTTGAATTACCGTTTCATCATGAATTTATGTTTCAA--CGTTT-TTTACTACATGAAAAAAAAATTCTAGT-------TGACT
AGTC-CAGAGGTTATAATTTGAATTATTATTTCATCATGAGTTCAAGTTTCAT--CAGT-CTTTACTGCATG--AAAAAAAATCTATTAATTATTTGAGT
AGTC-CATAGGTTATAATTTGAATTATTATTTCATCATGGGTTCAAGTTTCAT--CAGT-CTTTACTGCATGAAAAAAAAAATCTATTAATTATGTGAGT
AGTCAAAAAGATTATAATTTGGATTAGTGTTTCATCATAAAATTATGTTTCAAACGTTTTCTTTACTACATG--AAAAAAAAT-TAGT--------GACT
AGT-AAAGAG------GTTTGAATTACCGTTTCATCATGAATTTATGTTTCAA---CGTTTTTTACTACATG-AAAAAAAATTCTAGT-------TGACT
Ref.StSN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
301
400
GTCTAAATATATAGTTAAGAATTTAATCATACCCCTTAA--TTTAAAGTTTAATTT-ATTCACCCGTGATATATGTTTTGACTTTTGCAGACCAGCAATG
GTCTAAATATATAGTTAAGAATTTAATCATACTCCTTAA--TTTAAAGTTTAATTT-ATTCACTCGTGATATATGTTTTGACTTTTGCAGACCAGCAATG
GTCTAAATATATAGTTAAGAATTTAATCATACTCCTTAA--TTTAAAGTTTAATTT-ATTCACTCGTGATATATGTTATGACTTTTGCAGACCAGCAATG
GTCTAAATTTATAATTAAGTATTTAATTATATTCC--------------TTATTTTCATTCACTCATGATATATGTT-TGACTTTTGCAGACCAGCAATG
GTCTAAATTTATAATTAAGTATTTAATTATATTCCTTAA--TTTCAAGTTTAATTTCATTCACTCATGATATATGTT-TGACGTTTGCAGACCAGCAATG
ATCTAAGTATATATAGAAAAATTTAATCATATTCCTTAATTTTTCAAGATTAATTT-ATTCACTGGTGATATATGTT-TGACTTTTGCAGAGCAGCAATG
GTCTAAATATATAGTTAAGAATTT-----------TTAA--TTTCAAGTTTAATTT-ATTCACTCGTGATATATGTTATGACTTTTGCAGACCAGCAATG
Ref.StSN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
401
500
CTATTTCTGAAGCCGCTTATTCCTACAAGAAAATTGGTATGTTGTA-ATT-----------TCT-----------------------ATACAACATTTTG
CTATTTCTGAAGCCGCTTATTCCTACAAGAAAATTGGTATGTTGTA-ATT-----------TCT-----------------------ATACAACATTTTG
CTATTTCTGAAGCCGCTTATTCCTACAAGAAAATTGGTATGTTCTA-ATT---T--------CT-----------------------ATACAACATTTTG
CTATTTCTGAAGCCGCTAATTTCTACAAGAAAATTAGTATGTTCTA-ATT--=--------TCTGA------A-----------GCCATACAACATTTTG
CTATTTCTGAAGCAGCTTATTCCTACAAGAAAATTGGTATGCTCTATATGCTTTACCACCGTTTAATTACTCTCCCGCTAG-ATGCCATGCAGCATTTTG
CTATTTCTGAAGGCGCTGATTCCTACAAGAAAATTGGTATTTTCCA-ATT-----------TCTTATTACTCTCCCGCTAGAATGCCATACAACATTTTG
CTATTTCTGAAGCCGCTTATTCCAACAAGAAAATTGGTATGTTGTA-ATT-----------TCT-----------------------ATACAACATTTTG
Ref.StSN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
501
600
TCACCTTTTTCAAT-TATAAAT----ATTTATTCCCCTATGTCCTGTATCAATTATAAACAATACACTTTTT----ATATAAATATATATTAAACCTGGA
TCACCTTTTTCAAT-TATAAAT----ATTTATTCCCCTATGTCCTGTATCAATTATAAACAATACACTTTTT----ATATAAATATATATTAAACCTGGA
TCACCTTTTTCAAT-TATAAAT----ATTTATTCCCCTATGTCCTGTATCAATTATAAACAATACACTTTTTATATATATATATATATATTAAACCTGGA
TCACCTTTTTCAAT-TATAAATATTTATTTATTCCCCTCTGTCATGTAT----TATCAATTATACTGTGTTT-----------TATAGATTAAAAGT--T
TCACGTTTTTCAAT-TATAAAT----GTTTATTCCCCTATGTCATATATCAATTATAAACAATACCCTTTTT----------ATATATATTAAACCT-GA
TCACCTTTTTCAATTTATAAAT----ATCTATTCCCCCTATGTCTGTATCAATTATAAATAATACGCTTCT------------TATATATTAAACCTCGA
TCACCTTTTTCAAT-TATAAAT----ATTTATTCCCCTATGTCCTGTATCAGTTATAAACAATACACTTTT—TATATATATATATATATTAAACCTGGA
601
700
Ref.StSN2 ACATCCTT-AGTGAAAT-------------------------TTCTT------TGTGTTGTTTATATATGTACAGACTGTGGGGGAGCTTGTGCAGCAAG
Allele b1 ACATCCTT-AGTGAAAT-------------------------TTCTT------TGTGTTGTTTATATATGTACAGACTGTGGGGGAGCTTGTGCAGCAAG
Allele b2 ACATCCTT-AGCGAAATA----------------------AATTTCT------TGTGTTGTTTATATATGTACAGACTGTGGGGGAGCTTGTGCAGCAAG
Allele b3 ACTTTTTT-ATATAAATT--A----ATAAACCT---C--GAATTTCT------TGTGTTGTTAAT----GTACAGATTGTGGGGGAGCTTGTGCTGCAAG
Allele b4 ACATCCTT-AGCGAAATTTGC----CACGGCATATATATTAATTTCT------TGTGTTGTTTAT----GTACAGACTGTGGGGGAGCTTGTGCAGCAAG
Allele b5 ACATTCTTAAGCGAAATTCGAGCATATATAACTAATTATTAATTTCTCATATATATGTTGTTTAC----GTACAGACTGTGGGGGAGCTTGTGCAGCAAG
Allele b6 ACATCCTT-AGCGAAAT-------------------------TTCGA------TGTGTTGTTTATATATGTACAGACTGTGGGGGAGCTTGTGCAGCAAG
701
800
Ref.StSN2 GTGCCGATTATCATCAAGGCCAAGATTGTGTAATAGGGCATGTGGAACTTGTTGTGCTAGATGCAACTGTGTTCCTCCTGGTACTTCTGGCAACACTGAG
Allele b1 GTGCCGATTATCATCAAGGCCAAGATTGTGTAATAGGGCATGTGGAACTTGTTGTGCTAGATGCAACTGTGTTCCTCCTGGTACTTCTGGCAACACTGAG
Allele b2 GTGCCGATTATCATCAAGGCCAAGATTGTGTAATAGAGCATGTGGAACTTGCTGTGCTAGATGCAATTGTGTTCCTCCTGGTACTTCTGGCAACACTGAG
Allele b3 GTGCCGATTATCATCGAGACCAAGATTGTGTAATAGGGCATGTGGAACTTGTTGTGCTAGATGCAACTGTGTTCCTCCTGGTACTTCTGGTAACACTGAG
Allele b4 GTGCCGATTATCATCGAGGCCAAGATTGTGTAATAGGGCATGTGGAACTTGTTGTGCTAGATGCAACTGTGTTCCTCCTGGTACTTCTGGCAACACTCAG
Allele b5 GTGCCGATTATCATCAAGGCCAAGATTGTGTCATAGGGCATGTGGAACTTGTTGTGCTAGATGCAACTGTGTTCCTCCTGGTACTTCTGGCAACACTGAG
Allele b6 GTGCCGATTATCATCAAGGCCAAGATTGTGTAATAGGGCATGTGGAACTTGTTGTGCTAGATGCAACTGTGTTCCTCCTGGTACTTCTGGCAACACTGAG
Ref.StSN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
801
857
ACTTGCCCTTGCTATGCCAGTTTGACTACTCATGGCAACAAACGTAAATGCCCTTAA
ACTTGCCCTTGCTATGCCAGTTTGACTACTCATGGCAACAAACGTAAATGCCCTTAA
ACTTGCCCTTGCTATGCCAGTTTGACTACTCATGGAAACAAACGTAAATGCCCTTAA
ACTTGCCCTTGCTATGCCAATTTGACTACTCACGGCAACAAACGCAAATGCCCTTAA
ACTTGCCCTTGCTATGCCAATTTGACTACTCACGGCAACAAACGCAAATGCCCTTAA
ACTTGCCCTTGCTATGCCAGTTTGACTACTCATGGCAACAAACGTAAATGCCCTTAA
ACTTGCCCTTGCTATGCCAGTTTGACTACTCATGGCAACAAACGTAAATGCCCTTAA
Figure 2.3 Alignment of SN2 sequences. The reference StSN2 gene (AJ312424) aligned with the sequences of
the SN2 alleles from this study. Features shown are exons (yellow) with start and stop codons (grey),
substitutions (blue), insertions (green) and deletions (red).
33
Table 2.3 Nucleotide variations in different alleles of SN1 and SN2 genes.
Gene Allele
SN1
SN2
Exons (coding region)
Substitutions Insertions Deletions
Introns (non-coding region)
Substitutions Insertions Deletions
a1
0
0
0
1
1
0
a2
3
0
0
16
4
1
a3
3
0
0
14
7
3
a4
1
0
0
17
3
1
b1
0
0
0
2
0
0
b2
4
0
0
12
4
2
b3
14
0
0
59
12
9
b4
8
0
0
61
11
6
b5
6
0
0
46
7
9
b6
1
0
0
7
3
6
2.3.3 Allele diversity in the exons (coding regions) of SN1 and SN2 genes
For the SN1 gene, the coding sequence of the a1 allele was identical to the reference coding
sequence (AJ320185). Alleles a2 and a3 differed by three single nucleotide substitutions from
the reference sequence and a4 differs by single nucleotide substitution (Figure 2.2, Table 2.3).
For the SN2 gene, the coding sequence of the b1 allele was homologous to the reference
coding sequence (AJ312424). The b2, b3, b4, b5 and b6 alleles differed by 4, 14, 8, 6 and 1
nucleotide substitutions, respectively, from the reference sequence (Figure 2.3, Table 2.3).
2.3.4 Allele diversity in the introns (non-coding regions) of SN1 and SN2 genes
The a1, a2, a3 and a4 alleles of the SN1 gene showed 1, 16, 14 and 17 single nucleotide
substitutions, respectively, relative to the reference sequence (AJ320185). The intron
sequence of allele a1 showed a single nucleotide insertion compared to the reference SNI
sequence.
34
Table 2.4 A. SNPs in the SN1 alleles with reference to StSN1 coding sequence (exons).
SNP
SNP
SNP
SNP
42
69
641
776
779
Ref.SN1
T
C
T
C
G
a1
--
--
--
--
--
a2
C
T
--
--
A
a3
--
T
C
A
--
a4
--
T
--
--
--
Alleles
SNP
Table 2.4 B. SNPs in the SN2 alleles with reference to StSN2 coding sequence (exons).
Alleles
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
SNP
9
12
63
78
86
392
413
414
418
422
424
436
677
695
716
719
732
737
752
767
791
799
819
835
836
845
Ref.SN2
T
G
C
C
T
C
C
C
T
C
T
G
C
A
A
G
A
G
T
C
C
G
G
T
C
T
b1
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
b2
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
A
C
T
--
--
--
--
A
--
b3
C
T
T
--
--
--
--
--
A
T
--
A
T
T
G
A
--
--
--
--
T
--
A
C
--
C
b4
--
T
--
--
C
--
--
A
--
--
--
--
--
--
G
--
--
--
--
--
--
C
A
C
--
C
b5
--
T
--
T
--
G
G
--
G
--
--
--
--
--
--
--
C
--
--
--
--
--
--
--
--
--
b6
--
--
--
--
--
--
--
--
--
--
A
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
35
Allele a2 differs from the SN1 reference by three single nucleotide insertions and a seven
nucleotide insertion; allele a3 has four single nucleotide insertions and three insertions of 1, 7
and 17 nucleotides respectively. Allele a4 has two single nucleotide insertions and a six
nucleotide insertion (Figure 2.2, Table 2.3). The a1, a2, a3 and a4 alleles of the SN1 gene
showed 0, 1, 3 and 1 nucleotide deletions, respectively, relative to the reference sequence.
In SN2, the sequence of the introns of the b1, b2, b3, b4, b5 and b6 alleles exhibit 2, 12, 59,
61, 46 and 7 single nucleotide substitutions respectively compared to the reference sequence
(AJ312424). An allele b1 intron sequence was identical to the reference non-coding sequences
except two substitutions. Allele b2 had two single nucleotide deletions and four insertions
sized eighteen, one, four and three nucleotides respectively. Allele b3 showed four single
nucleotide insertions and eight insertions of 2-17 nucleotides. In alleles b4 and b5 single
nucleotide insertions and deletions were scattered throughout the introns. Allele b4 showed
four single nucleotide insertions and seven insertions sized 2-20 nucleotides. Allele b4 also
showed two single nucleotide deletions and four deletions of 2-7 nucleotides, whereas allele
b5 showed one single nucleotide insertion and six insertions of 2-26 nucleotides. It also
showed four single nucleotide deletions and five 2-18 nucleotide deletions. Allele b6 showed
five single deletions, three insertions and one 11 nucleotide deletion compared to the
reference sequence (Figure 2.3, Table 2.2).
2.3.5 Predicted amino acid sequences of SN alleles
Nucleotide sequence variation can potentially alter the amino acid sequence in proteins and
result in functional phenotypic changes. Therefore an analysis of the predicted amino acid
sequence was carried out using DNAMAN software for all SN1 and SN2 alleles. The 88
predicted amino acids from the SN1 protein (Figure 2.4A) were identical for the a1, a2, a3
and a4 alleles, despite some point mutations in the nucleotide sequence (Table 2.4A). The 104
predicted amino acids from the SN2 protein were also was very highly conserved; identical
for two SN2 alleles (b1 and b2) (Figure 2.4B). The alleles b3, b4, b5 and b6 of the SN2 gene
showed 3, 3, 4 and 1 amino acid substitutions respectively (Table 2.5). All these alleles show
more than 96% identity to the reference amino acid sequence. The high degree of sequence
similarity among the alleles suggests that the proteins produced by the different alleles might
be functionally and structurally equivalent.
36
(A)
Ref SN1
Allele a1
Allele a2
Allele a3
Allele a4
1
44
MKLFLLTLLLVTLVITPSLIQTTMAGSNFCDSKCKLRCSKAGLA
MKLFLLTLLLVTLVITPSLIQTTMAGSNFCDSKCKLRCSKAGLA
MKLFLLTLLLVTLVITPSLIQTTMAGSNFCDSKCKLRCSKAGLA
MKLFLLTLLLVTLVITPSLIQTTMAGSNFCDSKCKLRCSKAGLA
MKLFLLTLLLVTLVITPSLIQTTMAGSNFCDSKCKLRCSKAGLA
Ref SN1
Allele a1
Allele a2
Allele a3
Allele a4
45
88
DRCLKYCGICCEECKCVPSGTYGNKHECPCYRDKKNSKGKSKCP
DRCLKYCGICCEECKCVPSGTYGNKHECPCYRDKKNSKGKSKCP
DRCLKYCGICCEECKCVPSGTYGNKHECPCYRDKKNSKGKSKCP
DRCLKYCGICCEECKCVPSGTYGNKHECPCYRDKKNSKGKSKCP
DRCLKYCGICCEECKCVPSGTYGNKHECPCYRDKKNSKGKSKCP
(B)
Ref SN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
1
52
MAISKALFASLLLSLLLLEQVQSIQTDQVTSNAISEAAYSYKKIDCGGACAA
MAISKALFASLLLSLLLLEQVQSIQTDQVTSNAISEAAYSYKKIDCGGACAA
MAISKALFASLLLSLLLLEQVQSIQTDQVTSNAISEAAYSYKKIDCGGACAA
MAISKALFASLLLSLLLLEQVQSIQTDQVTSNAISEAANFYKKIDCGGACAA
MAISKALFASLLLSLLLLEQVQSIQTDQATSNAISEAAYSYKKIDCGGACAA
MAISKALFASLLLSLLLLEQVQSIQTDQVSSNAISEGADSYKKIDCGGACAA
MAISKALFASLLLSLLLLEQVQSIQTDQVTSNAISEAAYSNKKIDCGGACAA
Ref SN2
Allele b1
Allele b2
Allele b3
Allele b4
Allele b5
Allele b6
53
104
RCRLSSRPRLCNRACGTCCARCNCVPPGTSGNTETCPCYASLTTHGNKRKCP
RCRLSSRPRLCNRACGTCCARCNCVPPGTSGNTETCPCYASLTTHGNKRKCP
RCRLSSRPRLCNRACGTCCARCNCVPPGTSGNTETCPCYASLTTHGNKRKCP
RCRLSSRPRLCNRACGTCCARCNCVPPGTSGNTETCPCYANLTTHGNKRKCP
RCRLSSRPRLCNRACGTCCARCNCVPPGTSGNTQTCPCYANLTTHGNKRKCP
RCRLSSRPRLCHRACGTCCARCNCVPPGTSGNTETCPCYASLTTHGNKRKCP
RCRLSSRPRLCNRACGTCCARCNCVPPGTSGNTETCPCYASLTTHGNKRKCP
Figure 2.4 Multiple amino acid sequence alignment between reference sequence and alleles. (A) Reference
StSN1 and SN1 alleles. (B) Reference StSN2 and SN2 alleles. Analysis of predicted aminoacid sequences was
carried out using DNAMAN software. The changes in amino acids are shown in red.
37
Table 2.5 Amino acid changes in the SN2 alleles
Alleles
b3
b4
b5
b6
Amino acid
position
39
Amino acid changes
40
Serine to Phenylalanine
93
Serine to Asparagine
29
Valine to Alanine
86
Glutamic acid to Glutamine
93
Serine to Asparagine
30
Threonine to Serine
37
Alanine to Glycine
39
Tyrosine to Aspartic acid
64
Asparagine to Histidine
41
Tyrosine to Asparagine
Tyrosine to Asparagine
2.3.6 Phylogenetic relationships between the various SN alleles
A phylogenetic tree was constructed by the neighbour-joining method based on the full length
nucleotide sequences of different alleles of SN1 and SN2 genes from this study. Grouping and
cluster analysis of the data generated revealed the phylogenetic relationship between different
alleles (Figure 2.5). Sequences with high similarity were clustered together. In the tree, SN1
alleles and SN2 alleles clearly made their own separate clusters. The branch connection
between the clusters reveals the homology between the alleles. The branch lengths are very
short within each allele, illustrating these alleles are highly conserved. One cluster contains
four closely related SN1 alleles (a1, a2, a3, a4) and the reference sequence. The second cluster
is formed by three closely related alleles of SN2 (b1, b2 and b6) and the reference sequence.
The b3 and b4 made their own clusters near to the main SN2 cluster. Out of all six alleles, b5
allele has more divergent sequence compared to other alleles. The connection between these
clusters denotes the homology between SN1 and SN2 genes.
38
Figure 2.5 Phylogenetic tree indicating the relationship between SN1 alleles and SN2 alleles. The
relationships between SN1 and SN2 genes based on the sequence analysis of the whole gene sequence. The scale
bar represents substitutions per nucleotide site.
2.3.7 Comparative analysis of SN protein identity with other species.
A comparative study was also conducted on the protein sequences of different species in the
NCBI database using the reference protein of StSN1 and StSN2 as query sequence. For the
SN1 protein, all the Solanaceous species showed high conservation throughout the amino acid
sequences.
The SN1 amino acid sequence showed high similarity with amino acid sequences from some
other highly divergent species such as Vitis vinifera (XP-002271730.1), Ricinus communis
39
(XP-002510126.1), Populus trichocarpa (XP-002327192.1), Vigna radiata (AAY51975.1),
Medicago truncatula (ABD33278.1), Brassica napus (ACJ68116.1), Arabidopsis thaliana
(NP-568914.1), Zea mays (NP-001149639), Sorghum bicolor (XP-002463849.1), Oryza
sativa (NP-001051348.1), Gymnadenia conopsea (ABN12953.1), and Glycine max
(B1470705). All these genes encode small polypeptides sharing a highly divergent N-terminal
domain, cysteine conserved intermediate region and highly conserved carboxyl-terminal
domain containing 12 conserved cysteine residues (Figure 2.6). All the monocotyledonous
species showed three amino acid insertions at the 98th amino acid. The unrooted phylogenetic
tree reveals the SN1 amino acid similarity among those species (Figure 2.7).
The SN2 amino acid sequence also showed high similarity with amino acid sequences from
some other highly divergent species such as Vitis vinifera (XP-002275694.1), Ricinus
communis (XP-002526823.1), Populus trichocarpa (XP-002300706.1), Arabidopsis thaliana
(ABK22434.1), Sorghum bicolor (XP-002448761.1), Oryza sativa (NP-001055630.1), Litchi
chinensis (ABK21133.1), Glycine max (ACU14458.1), Gerbera hybrida (AJ005206),
Lavatera thuringiaca (AF007784), Elaeis guineensis (ACF21739.1) and Jatropha curcas
(ACV70139.1) (Figure 2.8). The unrooted phylogenic tree reveals the SN2 amino acid
similarity among those species (Figure 2.9).
40
Figure 2.6 Amino acid alignment of SN1 and its homologues. A combined multiple sequence alignment of the SN1 amino acid sequence in different species from this
study and the comparative results of GenBank query of homologues of SN1 amino acid sequences. All twelve highly conserved cysteine are are shown in red colour and other
residues are shown in same colour.
41
Figure 2.7 A phylogenetic tree based on amino acid sequences of SN1 and its homologues. An unrooted phylogenetic tree constructed using neighbour-joining method
based on the multiple sequence alignment of the SN2 amino acid sequence in different species from this study and the comparative results of a GenBank query of homologues
of SN2 amino acid sequences. All Solanaceae sequences other than Solanum commersonii (ABQ42628) and Solanum tuberosum cv Desiree (CAC44032.1) in this tree were
derived from this study; * indicates identical sequences reported by Segura et al. (1998) and Almasia et al. (2008). The scale bar represents substitutions per amino acid site.
42
Figure 2.8 Amino acid alignment of SN2 and its homologues. A combined multiple sequence alignment of the SN2 amino acid sequence in different species from this
study and the comparative results of a GenBank query of homologues of SN2 amino acid sequences. All twelve highly conserved cysteine are are shown in red colour and
43
other residues are shown in same colour.
Figure 2.9. A phylogenetic tree based on amino acid sequences of SN2 and its homologues. An unrooted phylogenetic tree constructed using neighbour-joining method
based on the multiple sequence alignment of the SN2 amino acid sequence in different species from this study and the comparative results of a GenBank query of homologues
of SN2 amino acid sequences. All Solanaceae other than the Solanum tuberosum cv. Jaerla (CAC44011.1) are derived from this study.The scale bar represents substitutions
per amino acid site.
44
2.3.8 Comparative study of highly conserved C-terminal region in different
plant families
Amino acid sequence alignments showed that the C-terminal region of the SN proteins and
the GASA proteins share common cysteine conservation (Figure 2.10). The unrooted
phylogenic tree (Figure 2.11) reveals conservation of cysteine residues also occurs in:
GASA1-14 (Roxrud et al., 2007) and GAST1 (AK229086) from Arabidopsis thaliana;
GAST1, GASAI, GASAII (Shi et al., 1992) and RSI-1 from tomato (Taylor and Scheuring,
1994); GEG from Gerbera hybrida (Kotilainen et al., 1999); GAST from strawberry (De la
Fuente et al., 2006); SNIII from potato (BG597515); 153 from Ricinus communis
(EMBLT24153); GIP1, GIP4 and GIP5 from Petunia hybrida (Ben-Nissan et al., 2004);
GSL8 (Os06g0729400); GASR1 from rice (Furukawa et al., 2006); and GASL1 from maize
(Zimmermann et al., 2009). All Solanaceae sequences derived from this study showed the
same cysteine conservation. All 12 characteristic cysteine residues are highly conserved
throughout all these peptides. Among the 70 C-terminal amino acids, there are 22 identical
residues of which 12 are cysteines.
45
46
Figure 2.10 caption on next page
Figure 2.10 C-terminal regions alignment of the SN family and other related families. A comparative study of the conserved C-terminal regions of GASA genes, SN
genes and other related genes using the Geneious software. Multiple sequence alignment of the conserved C-terminal domain of the 14 putative GASA genes (Roxrud et al.,
2007), and GAST1 (AK229086) from Arabidopsis, SN1 (Segura et al., 1999), SN2 (Berrocal-Lobo et al., 2002), SNIII from potato (cited in Berrocal-Lobo et al., 2002),
GAST1, GASAI, GASAII, RSI-1 from tomato, GEG from Gerbera hybrida, GAST from Fragaria ananassa (De la Fuente et al., 2006), 153 from Ricinus communis, GIP2 and
GIP4 from Petunia hybrida (Ben-Nissan et al., 2004), GASRI (Furukawa et al., 2006), GSL8 (cited in Zimmermann et al., 2009), GSL1 (Zimmermann et al., 2009) and GAST
from Picea marina (AF051227). All twelve highly conserved cysteine are are shown in red colour and other residues are shown in same colour.
47
Figure 2.11 An unrooted phylogenetic tree constructed based in Figure 2.10. The scale bar represents substitutions per amino acid site. * indicates the sequences from this
study.
48
2.4 Discussion
This study has revealed that the sequences of SN1 and SN2 genes are highly conserved in a
range of potato genotypes, as well as a wide range of other Solanaceous species. Allelic
variation both within and between genotypes and/or species was observed for both the SN1
gene (four alleles) and the SN2 gene (six alleles). Most of the sequence variation in the alleles
of the SN1 and SN2 genes occurs in introns. A few point mutations occur in exons, but are
usually silent, suggesting that the SN1 and SN2 proteins may play an important role in
cellular metabolism beyond the defence against invading microorganisms. The phylogenetic
tree shown in Figure 2.5 gives a good indication of the relationship between the various
alleles as well as the conserved homology between the SN1 and SN2 genes.
For the allele diversity study the number of colonies selected for sequencing is important.
Therefore, for tetraploid potato, sixteen clones for each SN gene were sequenced in both
directions to improve the chances of detecting all alleles (Simko, 2004). At least six clones for
each SN gene per cultivar or species were sequenced from all diploid species, N. tabacum and
S. nigrum. Almasia et al. (2008) conducted a comparative sequencing study of the SN1 gene
in three different wild Solanum species (S. bulbocastanum, S. commersonii and two genotypes
of S. chacoense) and a commercially cultivated S. tuberosum cv. Kennebec. They produced a
consensus sequence from five independent clones of each species for comparative
bioinformatic analysis that revealed the highly conserved nature of the nucleotide sequences
in the SN1 gene in the four Solanum species. However, the derivation of a consensus sequence
for each species disguises the possibility of allele diversity within each species. In this chapter
the specific gene fragment corresponding to full length SN genes was cloned prior to
sequencing. This demonstrates the value of sequencing multiple clones to ensure the recovery
of all alleles present in heterozygous and/or polyploidy genotypes, which provides a more
informative approach on genetic diversity than assembling a consensus sequence for a
species.
Sequence analysis in various Solanaceous species and potato genotypes revealed four alleles
of SN1 and six alleles of SN2 found throughout all species analysed. The out-groups b3, b4,
b5 showed more divergence from the other SN2 alleles. The specific effect of these alleles on
disease resistance to specific pathogens remains to be established and further evaluation of
functional significance of these allelic differences is important. This can be evaluated by
transforming potato with the different alleles, thereby providing a strategy to examine the role
of each allele in a common genetic background.
49
According to the protein BLAST results, the amino acid sequences for the StSN1 gene and
StSN2 genes also show high homology with proteins of highly divergent species (Figure 2.6
and Figure 2.8). Based on the NCBI database the majority of these proteins from different
species are named as hypothetical proteins; proteins whose existence has been predicted, but
for which there is no experimental evidence of their function in vivo. Interestingly, all those
proteins show >70% similarity in the amino acid residues and 100% identity in the positions
of cysteine residues. This high conservation of 12 characteristic cysteine residues at eleven
positions in the C-terminus in all studied Solanaceous species as well as other species (Figure
2.6, Figure 2.8 and Figure 2.10), indicates that the cysteine residues may play an important
role in their physiological function, presumably to maintain globular conformation through
disulfide bridges (Rietsch and Beckwith, 1998).
Due to the absence of structural and biochemical data, the functional roles of most of these
conserved genes are unknown, although some are involved in cell division, cell elongation or
cell elongation arrest. Expression data and mutant anlysis in Petunia hybrida suggests that the
GIP1 gene is involved in the elongation of the stem and corolla (Ben-Nissan et al., 1996).
GAST1 from tomato is also associated with stem elongation (Shi et al., 1992). In contrast,
GEG from Gerbera hybrida (Kotilainen et al., 1999) and FaGAST in strawberry (De la Fuente
et al., 2006) lead to the arrest of cell elongation. Petunia GIP4/GIP5 (Ben-Nissan et al., 1996)
and Arabidopsis GASA4 (Aubert et al., 1998) were suggested to act in cell division. Also in
Arabidopsis GASA4 controls seed development and regulates flowering (Roxrud et al., 2007),
and is identified as a downstream target of the DELLA protein, a key component of the GA
signalling pathway (Zhang and Wang, 2008). In rice it has been suggested that OsGASR1 and
OsGASR2 are involved in panicle differentiation (Furukawa et al., 2006). All of these might
reflect involvement of the SN proteins in similar processes. The highly conserved nature of
these genes within and outside the Solanaceous species adds support to the hypothesis of the
vital role of the SN1 and SN2 proteins in cellular metabolism beyond the defence against
invading microorganisms (Segura et al., 1999; Berrocal-Lobo et al., 2002).
The mechanism of action of Snakin proteins remains unknown, but they could play a role in
the control of the pathogens in vivo. Snakin peptides show sequence similarity with cysteinerich domains of proteins from the hemotoxic snake venome disintegrin from agkistrodon
halysblomhoffii snake (Gloydius blomhoffii; P21858), kistrin from the Malayan pit viper
(Adler et al. 1991), and mammalian peptides such as HsMuc (Q9ESP3), HsvWF (X04F385),
HsMDC, and HsMDC2a (Sagane et al., 1998) from humans and BtvWF from Bos taurus
(P80012). Most of these proteins mainly play protective roles in those species against
50
invading infectious agents. It is therefore assumed that snakin proteins provide a similar
protective role.
The gibberellic acid signaling pathway regulates growth and influences various
developmental processes, including stem elongation, germination, dormancy, flowering, sex
expression, enzyme induction, and leaf and fruit senescence (Daviere et al., 2008). On the
other hand, ABA-mediated signaling plays an important part in plant responses to
environmental stress and plant pathogens. DELLA proteins are the key components of these
signalling pathways and play important roles in the progress of plant growth (Fleet et al.,
2005). Recently Zhang and Wang (2008) found that GA regulates the expression of four
GASA genes (GASA1, GASA4, GASA6 and GASA9) through DELLA proteins. GASA is
upregulated in the absence of DELLA protein. The expression of GASA1 is inhibited by GA3
(Zhang and Wang, 2008). SN1 and SN2 show some homology with GASA genes at the
nucleotide and amino acid level (Segura et al., 1999; Berrocal-Lobo et al., 2002; Broekaert et
al., 1997; García-Olmedo et al., 1998). The highly conserved C domain in SN and GASA
suggests a conserved mode of action at the protein level.
This study focused on two SN genes, SN1 and SN2, originally derived from Solanum
tuberosum. Highly conserved SN1 and SN2 homologues were discovered in all tuber-bearing
and non-tuber-bearing Solanum species, suggesting that both SN1 and SN2 genes were present
before the divergence of tuberisation in Solanum species. Wang et al. (2008b) also came with
the similar evolutionary concept from their allele mining study in the Rpi-blb1 gene in
Solanum species.
Evaluation of the allele diversity in a specific gene among different species is valuable to
identify new alleles with potential agronomical relevance. Exploiting wild relatives as a
source of novel alleles is challenging, but a well worthwhile endeavor. To date, this has
provided notable success for crop improvement; for example, the case of the RB gene from S.
bulbocastanum conferring broad spectrum resistance to potato late blight (Song et al., 2003).
An important objective of characterizing potato germplasm is the discovery of potentially
superior alleles and their utilization in potato improvement. This is not only important for the
identification of disease resistance genes, but it is also useful for identifying the alleles with
resistance to other biotic stresses, tolerance to abiotic stresses, as well as for improving
nutrition, quality and yield.
51
Chapter 3
Overexpression of Snakin-1 and Snakin-2 genes under a potato
light inducible Lhca3 promoter in transgenic potatoes.
3.1 Introduction
Potatoes (Solanum tuberosum L.) are the most important non-cereal food crop in the world.
Potatoes have the potential to substantially contribute to the future food needs of developing
countries. Worldwide, diseases are the most important threat to potato production and
strategies for managing potato diseases remain unsustainable and costly. Environmental and
human health concerns have prompted much research on ecologically safe, non-chemical
methods of disease control, including breeding of pathogen-resistant cultivars. Potato
breeding for disease-resistant cultivars is not easy because of the genetic complexity of this
crop. Genetic engineering approaches involving the overexpression of genes associated with
the natural defence mechanisms used by plants to protect themselves against pathogens, offers
new opportunities to produce disease-resistant cultivars. The most important class of genes
that has been used by breeders for disease control is the plant resistance (R) genes (Bent,
1996; Tai et al., 1999; Kim et al., 2002; Mysore et al., 2003). However, most R-genes are
non-durable and are rapidly overcome by pathogens when introduced into cultivars (Nelson,
1978). A transgenic approach involving the overexpression of genes encoding antimicrobial
peptides offers an alternative to R genes for the improvement of disease resistance (Gao et al.,
2000). Antimicrobial peptides are low molecular weight cysteine-rich peptides which play an
important role in innate defence against invading microbes (López-Solanilla et al., 2003;
Pelegrini et al., 2011).
A wide range of plant defence antimicrobial proteins have been identified and are being
utilized in attempts to provide protection in transgenic crops (Evan and Greenland, 1998). The
overexpression of genes encoding chitinase (Brogue et al., 1991; Vellicce et al., 2006; Jayaraj
and Punja, 2007), osmotin-like proteins (Zhu et al., 1996), thionin (Florack et al., 1993; Epple
et al., 1997), non-specific lipid transfer proteins (Molina and Gacia-Olmedo, 1997), small
cysteine-rich plant defensins (Bi et al., 1999; Gao et al., 2000), magainin II (Barrell and
Conner, 2009), cecropin (Nordeen et al., 1992; Arce et al., 1999, Zakharchenko et al., 2005;
Jan et al., 2010), attacin (Arce et al., 1999; Norelli et al., 1994, Reynoird et al., 1999, Ko et
al., 2002), tachyplesin I (Allefs et al., 1996), lysozymes (Ko et al., 2002) and a hevein-like
peptide (Koo et al., 2002), have been shown to enhance resistance to pathogenic fungi and
bacteria in various plant species. All the above examples involve the transfer of genes across
52
divergent taxonomic boundaries, a concept that struggles to gain acceptance from society
(Barrell et al., 2010). To help circumvent this problem, the approach taken in this study is to
overexpress endogenous genes responsible for plant-derived antimicrobial peptides with the
expectation that they will intensify or trigger the expression of existing defence mechanisms.
The antimicrobial peptides SN1 and SN2 have been isolated from potato tubers and found to
be active against bacterial and fungal plant pathogens (Segura et al., 1999; Berrocal-Lobo et
al., 2002). Although StSN1 and StSN2 only show 38% conserved amino acid residues, they
have highly conserved cysteine residues as well as a similar antimicrobial spectrum. There is
a growing body of literature reporting that these and related proteins may be involved in
diverse biological processes, such as cell division, cell elongation, cell growth, transition to
flowering, and signaling pathways other than disease resistance (Aubert et al., 1998; BenNissan et al., 2004; Berrocal-Lobo et al., 1999; Roxrud et al., 2007; Segura et al., 1999). The
overexpression of the SN1 gene in potato plants has been reported to confer enhanced
resistance to both Rhizoctonia solani and Pectobacterium carotovora (syn Erwinia
carotovora) (Almasia et al., 2008). The overexpression of the SN2 gene in tomato plants has
been
reported
to
confer
enhanced
resistance
to
Clavibacter
michiganensis
subsp.michiganensis (Balaji and Smart, 2011).
SN1 proteins cause a rapid in vitro aggregation of both gram-positive and gram-negative
bacteria. This protein is also reported to be active in vitro against bacterial species such as
Clavibacter michiganensis subsp. sepedonicus and fungal species such as Fusarium solani,
Fusarium culmorum, Bipolaris maydis and Botrytis cinerea (Segura et al., 1999; Almasia et
al., 2008). The expression profile of the StSN1 gene showed no response to gibberellic acid
(GA3) or biotic and abiotic treatments (Segura et al., 1999). Like SN1, SN2 proteins also
cause a rapid aggregation of both gram-negative and gram-positive bacteria. The expression
of this gene is up-regulated after infection of potato tubers with the compatible fungus
Botrytis cinerea and down-regulated by the virulent bacteria Ralstonia solanacearum and
Pectobacterium chrysanthemii (syn. Erwinia chrysanthemum). The expression of the SN2
gene is also up-regulated by wounding and by abscisic acid treatment (Berrocal-Lobo et al.,
2002). In Chapter 2, the sequences of SN1 and SN2 genes were found to be highly conserved
within and between Solanaceous species, with the predicted amino acid sequence being very
highly conserved. Based on the homology to other genes, these proteins have also been
hypothesised to be involved in functional roles other than defence (Nahirñak et al., 2012).
The main aim of this chapter is to obtain potato plants over expressing StSN1 and StSN2
genes. The intention is to develop broad-spectrum, a durable disease-resistant potato cultivar
53
using single genes and without altering cultivar identity. For that, potato plants (S. tuberosum
cv. Iwa) were transformed via Agrobacterium tumefaciens with a construct encoding the
potato cv. Iwa SN1 gene or SN2 gene under the regulation of a light inducible potato cv. Iwa
Lhca3 promoter. In contrast to other recent studies using the SN1 gene (Almasia et al., 2008)
and SN2 gene (Balaji and Smart, 2011), a potato-derived expression cassette using a light
inducible promoter was constructed to limit gene overexpression to the non-consumed organs
of the potato plant and thereby overcome some of the biosafety and food safety concerns from
the public toward GMO products (Conner and Jacobs, 1999).
3.2 Materials and methods
3.2.1 Construct development
Allele a1 of StSN1 and allele b1 of StSN2 genes were isolated from plasmid DNA of pGEMTEasy harboring specific SN alleles (see Chapter 2 for cloning of SN alleles into pGEMT-Easy
vectors) by PCR using the SN primers (SN1-F2 and SN1-R2 primer set for SN1 and SN2-F
and SN2-R primer set for SN2) described in the Table 3.1. Each PCR amplification was
carried out in a total volume of 50 µL containing 1x ThermoPol Reaction Buffer [20 mM
Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8 at
25°C], ~1 ng plasmid DNA, 0.2 mM of each dNTP, 0.4 µM of each primer and 0.6 U of
VentR® DNA polymerase (New England BioLabs, Massachusetts, USA). PCR reactions were
performed in a Mastercycler (Eppendorf, Hamburg, Germany). The conditions for PCR were:
1 cycle at 93C for 1min, 34 cycles of (30 s 92C, 35 s 58C, 90 s 72C), followed by 7 min
extension at 72C.
Amplified products were separated by electrophoresis in 1% agarose gel in TAE buffer at
5.5V/cm for 80 min and visualized under UV light after staining with ethidium bromide
(5mg/L) for 15 min. PCR products of the expected size (813 bp for the StSN1 gene, allele a1
and 953 bp for the StSN2 gene, allele b1) were gel-purified using QIAquick Gel Extraction
Kit (QIAGEN, Venlo, The Netherlands) and 5’-phosphorylated for efficient blunt-end ligation
using Quick Blunting Kit (NEBioLabs, Massachusetts, USA) according to the manufactures’
instructions. These DNA fragments were then ligated into the PsiI site of Lhca3 expression
cassette (Meiyalaghan et al., 2009; Figure 3.1) using T4 DNA Ligase (NEBioLabs,
Massachusetts, USA) according to manufacturer’s recommendation.
One Shot® TOP10 ElectrocompTM E. coli cells (Invitrogen, Carlsbad, CA, USA) were
transformed with DNA from blunt-end ligation reactions according to the manufacturer’s
instructions. Aliquots of 50 µL, 100 µL and 150 µL from each transformation were spread
54
individually onto LB agar media (Appendix A) containing ampicillin (100µg/mL) and
incubated at 37°C overnight. Colonies with transformed cells were identified by PCR (full
details are given in Chapter 2) with specific SN gene primers (Table 3.1). PCR conditions as
described for SN primers except the initial denaturing at 94ºC for 4 min. Plasmid DNA from
clones selected by PCR was isolated using the QIAprep Spin Miniprep kit (QIAGEN, Venlo,
Netherlands) according to the manufacturer’s instructions. The plasmid DNA was sequenced
to confirm the orientation of expression cassette. The primers Cab-Fa and Cab-Ra (Table 3.1)
were used individually as sequencing primers in each sequencing reaction. The full details of
Sanger sequencing using Big Dye (Applied Biosystems, Warrington, UK) are given in
Chapter 2.
Plasmids containing expression cassettes with Lhca3 promoter directed towards the correct
end of the StSN genes (sense expression cassettes) and those with inserts in the opposite
orientation (anti-sense expression cassettes) were identified by sequencing. Plasmids of sense
expression cassettes containing SN1 and SN2 genes were named as pStLhca3cas-SN1 and
pStLhca3cas-SN2 respectively.
These two resulting plasmids were digested with HindIII, the 1631 bp Lhca3-SN1 and the
1770 bp Lhca3-SN2 fragments were blunt-ended using Quick Blunting Kit (NEBioLabs,
Massachusetts, USA) and were ligated individually into the blunt-ended NotI site of the
binary vector pMOA33 (Barrell and Conner, 2006) using T4 DNA Ligase (NEBiolabs,
Massachusetts, USA) according to the manufacturer’s instructions, to produce pMOA33Lhca3-SN1 and pMOA33-Lhca3-SN2 binary vectors. Ligation products were transformed into
MAX Efficiency® DH5αTM Competent Cells (Invitrogen, Carlsbad, CA, USA) according to
the manufacture’s recommendation. After transformation the bacterial cells were plated on
LB agar plates containing 100 mg L-1 spectinomycin and incubated at 37ºC overnight.
55
Figure 3.1 An intragenic expression cassette based on the potato StLhca3 gene. The 5’ promoter region
consists of nucleotides 1-600 from NCBI accession number EU234502 and the 3’terminator region consists of
nucleotides 101- 487 from NCBI accession number EU293853. Theses sequences were synthesized as a single
988bp fragment (Genscript Corporation, Piscataway, NJ, USA) and cloned into pUC57 to produce pStLhca3cas.
The sequence across the junction of the promoter and terminator regions creates a unique PsiI restriction site for
the insertion of the coding regions from other potato genes (Provided by Dr S. Meiyalaghan, Plant & Food
Research).
Plasmid DNA was isolated from putatively transformed E. coli colonies using the QIAprep
Spin Miniprep kit (QIAGEN, Venlo, Netherlands) according to the manufacturer’s
instructions, and the insertion and orientation of the chimeric Lhca3-SN-Lhca3 genes into the
T-DNA of the binary vector was verified by restriction analysis using EcoRV and Sca1 for the
Lhca3-SN1 insert and EcoRV and XhoI for the Lhca3-SN2 insert.
Colonies containing recombinant plasmids were identified by PCR (full details are given in
Chapter 2) with Cab-Fa and Cab-Ra primers (Table 3.1). PCR conditions as described except
the initial denaturing at 94ºC for 4 min. Plasmid DNA from clones selected by PCR was
isolated using the QIAprep Spin Miniprep kit (QIAGEN) according to the manufacturer’s
instructions. The orientation of the chimeric Lhca3-SN-Lhca3 genes within the T-DNA in
both binary vectors, pMOA33-Lhca3-SN1 and pMOA33-Lhca3-SN2, was tested by restriction
analysis to select the binary vector that contains the StLhca3 promoter adjacent to the right
border within the T-DNA. Restriction analysis using EcoRV and Sca1 for pMOA33-Lhca3SN1, with the Lhca3 promoter adjacent to the right border, produced 1253 bp, 4800 bp and
56
5600 bp fragments. The plasmid, pMOA33-Lhca3-SN2 with the Lhca3 promoter adjacent to
the right border, produced 678 bp, 4800 bp and 6315 bp fragments when cut with EcoRV and
XhoI.
3.2.2 Agrobacterium transformation
The resulting binary vectors, pMOA33-Lhca3-SN1 and pMOA33-Lhca3-SN2 were
individually transferred to the disarmed Agrobacterium tumefaciens strain EHA105 (Hood et
al., 1993) using the freeze-thaw method (Hofgen and Willmitzer, 1988). After transformation
Agrobacterium cells were plated on LB agar plates containing 100 mg L-1 spectinomycin and
100 mg L-1 streptomycin. Plates were then incubated at 28ºC and transformed colonies
appeared within 2-3 days.
Single colonies were picked and screened using colony PCR with specific SN primers and
virG-F and virG-R primers (Table 3.1) to confirm their authenticity. The conditions for PCR
with specific SN primers as described except the initial denaturing at 94ºC for 5 min. The
virG PCR was performed using the same PCR conditions with 50C annealing temperature. A
single positive colony, for each recombinant plasmid, was selected and inoculated
individually in a 50 mL of LB broth supplemented with 300 mg L-1 spectinomycin and 100
mg L-1 streptomycin). After overnight incubation at 28ºC with shaking 300 rpm, cells were
centrifuged for 5 min at 4000 g, resuspended in 5 mL of LB containing 10% glycerol as
cryoprotectant and stored at -80ºC as 250 µL aliquots. Prior to use for co-cultivation with
potato leaf explants, the Agrobacterium cultures from -80ºC storage were cultured overnight
on a shaking table at 28ºC in 50mL LB broth supplemented with 300 mg/L spectinomycin.
3.2.3 Plant material
Virus-free plants of the cultivar Iwa were multiplied in vitro on multiplication medium
(Appendix A). The agar was added after the pH was adjusted to 5.8 with 0.1 M KOH, and
then the media was autoclaved at 121°C for 15 min. Aliquots of 50 mL were dispensed into
pre-sterilised plastic containers (80 mm diameter x 50 mm high; Vertex Plastics, Hamilton,
New Zealand). Plants were routinely subcultured as two to three node segments every 3-4
weeks and incubated at 26°C under cool white fluorescent lamps (80-100 μmol m-2s-1; 16h
photoperiod).
57
Table 3.1 Details of the primers used in this chapter
Target
gene
SN1
Primer pairs
Primer sequence (5’ to 3’)
SN1-F2
AAATGAAGTTATTTCTATTAACTCTGC
SN1-R2
TGTGAAGACGCAAATATAACCAC
SN2-F
AAATATTTCAAATTCCAATGGC
SN2-R
CAATACAATGCAAACCAGAACAA
VirG-F
GCGTCAAAGAAATA
VirG-R
GCGGTAGCCGACAG
Lhca3SN1
Cab-Fa
TTCTAGTGGAGCTAAGTGTTCA
SN1-R4
ATAATTGGATACCCTGGAATGG
Lhca3SN2
SN2-bF4
TCATCAAGGCCAAGATTGTG
Cab-Ra
TGTTACATTACACATAAGAGAAGG
Actin-F
GATGGCAGAAGGCGAAGATA
Actin-R
GAGCTGGTCTTTGAAGTCTCG
Npt-II a
ATGACTGGGCACAACAGACAATTCGGCTGCT
Npt-II b
CGGGTAGCCAACGCTATGTCCTGATAGCGG
813
955
SN2
virG
Product size
(bp)
599
330
461
1069
Actin
612
nptII
58
3.2.4 Potato transformation
Fully expanded leaves from in vitro micro-propagated virus-free plants of potato cultivar Iwa
were excised, cut in half across midribs while submerged in the liquid Agrobacterium culture.
After about 30 s, the leaf segments were blotted dry on Whatman 3MM sterile filter paper to
eliminate excess bacterial cell suspension. The leaf segments were then cultured on callus
induction medium (multiplication medium supplemented with 0.2 mg L-1 napthaleneactic acid
(NAA) and 2 mg L-1 benzylaminopurine (BAP) in standard plastic Petri dishes under reduced
light intensity, incubated in a controlled environment chamber as described above. After two
days, the leaf segments were transferred to callus induction medium supplemented with 200
mg L-1 Timentin™ to prevent Agrobacterium overgrowth and to allow the formation of
transformed cell colonies. After 5 days, transformed cell colonies were selected by
transferring the explants to the same medium supplemented with 100 mg L-1 kanamycin. The
initial callus was observed at the site of wounding on the explants.
3.2.5 Regeneration and selection of transgenic potato plants
The individual cell colonies were transferred to regeneration medium (potato multiplication
medium with sucrose reduced to 5 g L-1, plus 1.0 mg L-1 zeatin and 5 mg L-1 GA3, both filter
sterilised and added after autoclaving) supplemented with 200 mg L-1 Timentin™ and 100 mg
L-1 kanamycin. These were also incubated under low intensity light until regenerated shoots
were recovered. When the green shoots reached a length of 1 cm, they were transferred to a
selective multiplication medium (Appendix A) containing 50 mg L-1 kanamycin and 200 mg
L-1 Timentin™. Plantlets with well-developed roots were propagated on potato multiplication
medium supplemented with 200 mg L-1 Timentin™ for further molecular analysis.
3.2.6 Molecular characterisation of transformed potato.
Genomic DNA was extracted from 0.2 g of leaf material from putative independent transgenic
plants and control plants using a modified CTAB method (Sosinski and Douches, 1996). PCR
reactions were performed from the transgenic lines to confirm the presence of transgene, nptII
and endogenous potato actin gene by using Taq DNA polymerase (New England BioLabs).
All PCRs were carried out in a Mastercycler (Eppendorf, Hamburg, Germany). Initially, to
verify the quality of the genomic DNA template, PCRs were conducted to amplify the
endogenous actin gene using Actin-F and Actin-R primers (Table 3.1). The PCR reaction was
denatured at 93C for 1 min, followed by 40 cycles of (30 s at 92C, 30 s at 58C, and 90 s at
72C), then 6 min extension at 72C. Each 10 µL PCR mix contained 1x ThermoPol Reaction
Buffer [20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1 % Triton X59
100, pH 8.8 at 25°C], 0.2 mM of each dNTP, 0.2 µM of each primer and 0.4U of Taq DNA
Polymerase (New England BioLabs). The Npt-IIa and Npt-IIb primers (Table 3.1) were then
used to amplify the nptII gene from the samples. Each 10 µL reaction mix and the thermal
cycling profile were as described previously, except for an annealing step of 20 s at 60C and
extension step of 45 s at 72C. Finally, to amplify the transgene from genomic DNA samples,
a SN gene specific primer and Lhca3 promoter specific primer were used (to avoid
endogenous gene amplification). The conditions for PCR for the Lhca3-SN1 gene using the
primers Cab-Fa and SN1-R4 (Table 3.1) were: denatured at 94C for 2 min, followed by 34
cycles of (15 s 93C ,30 s 55C, 80 s 72C), followed by a 3 min extension at 72C. The
conditions for PCR for the Lhca3-SN2 using the primers SN2-bF4 and Cab-Ra (Table 3.1)
were as follows: denatured at 94C for 1 min, followed by 34 cycles of (20 s 93C, 20 s 55C,
80 s 72C), then 3 min extension at 72C. All these amplified products were separated by
electrophoresis in a 1% agarose gel in 1xTAE buffer at 5.5V/cm for 80 min and visualized
under UV light after staining with ethidium bromide (5mg/L) for 15 min.
3.2.7 Phenotypic evaluation of transgenic lines in glasshouse
Selected putatively transformed lines with well-defined shoots and roots were transferred to
the containment greenhouse using the method described in Conner et al., 1994. The transgenic
lines were initially transferred to a 6-cell plastic tray (5 cm x 5 cm x 10 cm) and kept in low
light under a greenhouse bench for acclimatization. For each line, two plants were established
in each of three PB5 bags (15 cm x 15 cm x 15 cm black polythene) containing potato potting
mix (Conner et al., 1994), with each PB5 bag treated as a replicate, and the bags placed in the
greenhouse in a randomised block design (one replicate plant per bench). The greenhouse
conditions provided heating below 15ºC and ventilation above 22ºC. Day length was
supplemented to 16 h when needed with 500 W metal halide vapor bulbs, and relative
humidity was maintained above 60%.
After 6-8 weeks in the greenhouse, the appearance of the foliage from each line was recorded
using the several categories: phenotypically normal, marginal leaf curl, leaf wrinkling,
reduced vigor, and/or stunted plants (Conner et al., 1994). Tubers were also evaluated based
on their size and appearance at the time of harvest, 16 weeks after planting in the greenhouse.
3.2.8 RNA isolation and cDNA synthesis
One gram of leaf tissues (approximately 10 discs of 8 mm diameter) were collected from the
youngest, fully expanded leaves from 2 month old glasshouse-grown plants and immediately
frozen in liquid nitrogen and stored at -80ºC until use. Total RNA was extracted with Illustra
60
RNAspin Mini Isolation kit (GE healthcare, Buckinghamshire, UK), including DNase
treatment according to the manufacturer’s instructions after grinding the frozen tissue in
liquid nitrogen. The quality of the RNA was assessed by agarose gel electrophoresis and
quantity was determined with a NanoVueTM Spectrophotometer (GE healthcare).
The total RNA was stored at -80ºC until used for cDNA synthesis. First-strand cDNA was
synthesised using the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen, Carlsbad, USA)
according to the manufacturer’s instructions. VILO™ Reaction Mix contains random primers,
MgCl2 and dNTPs in a buffer formulation. Synthesized cDNA was stored at -20ºC for further
use.
3.2.9 Overexpression of specific gene in leaves using qRT-PCR
The transgene expression and the relative expression levels of SN1 and SN2 transcripts in
various plant tissues were examined using qRT-PCR. This is a highly sensitive method for the
detection of low abundance mRNAs (Fleige and Pfaffl, 2006). Constitutively expressed
reference genes are commonly used as internal controls for qRT-PCR. In this study Ef1α
(elongation factor 1 alpha) was used as an internal standard (Nicot et al., 2005) to normalize
the small difference in template amounts thus avoiding bias. Primers were designed to
amplify the target SN gene and the internal reference gene Ef1α. The design of primers was
performed to avoid primer-dimers (heterodimer) or self-priming (homodimer) formation using
Primer express 3.0 software (Applied Biosystems, Foster City, CA, USA) (Table 3.2). The
cDNA was used as the template in the qRT-PCR experiments. Quantitative RT-PCRs were
carried out in the StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA,
USA) with EXPRESS SYBR® GreenER™ qPCR Supermix with Premixed ROX (Invitrogen,
Carlsbad, CA, USA).
Every reaction was performed in triplicate per sample and triplicate biological replicates were
carried out for each transgenic line, results for gene expression for each line were averaged.
PCR reactions were in a total volume of 20 µL containing 0.2 µM of each primer (forward
and reverse), 1X EXPRESS SYBR® GreenER™ qPCR Supermix with premixed ROX
reference dye (500 nM) (Invitrogen) and 1µg cDNA template. PCR conditions were as
follows: 2 min at 50ºC, 2 min at 95ºC followed by 40 cycles of 15 s at 95ºC, 60 s at 60ºC,
finally dissociation curve analysis was carried out by cycling for 15 s at 95ºC, 60 s at 60 ºC,
15 min at 95ºC. The relative expression levels were determined using the StepOne™ software
v2.1. Quantification was based on the comparative Ct method (∆∆Ct). Cycle threshold (C T)
was calculated for each gene from three replicates. This is the PCR cycle number when the
reaction exceeds a threshold value that is arbitrarily set in the exponential phase of reaction
61
fluorescence. Expression levels of the target gene were determined relative to the expression
of the Eflα reference gene. ∆Ct between target gene and Ef1α was calculated for each sample.
Comparative ∆∆Ct method was then used to determine the expression levels. The specificity
of the primers was validated by the dissociation curve analysis after qRT-PCR.
Table 3.2 Details of the primers designed for qRT-PCR.
Target gene
SN1
SN2
Eflα
Primer pairs
Primer sequence (5’ to 3’)
SN1-exF1
TGGCTGGTTCAAGTTTTTGTGA
SN1-exR1b
GGGCATTTAGACTTGCCCTTA
SN2-exF2
CAAGTGACCAGCAATGCTATTTCT
SN2-exR2b
AGGGCATTTACGTTTGTTGC
Eflα F
ATTGGAAACGGATATGCTCCA
Eflα R
TCCTTACCTGAACGCCTGTCA
Product size (bp)
193
231
101
3.3 Results
3.3.1 Vector Construction
To overexpress the SN1 and SN2 genes in non-consumed organs of potato plants, the strong
Lhca3 light inducible promoter was ligated to the coding regions of the a1 allele of SN1 and
the b1 allele of SN2 (Figure 3.2). The pMOA33-Lhca3-SN1 (Figure 3.3A) and pMOA33Lhca3-SN2 (Figure 3.3B) constructs containing either StSN1 or StSN2 gene in a sense
orientation were used to generate transgenic potato plants.
Figure 3.2 Schematic representation of the Lhca3 cassette with specific genes. (A) Lhca3 cassette containing
the SN1 gene (a1 allele). (B) Lhca3cassette containing the SN2 gene (b1 allele).
62
(A)
(B)
Figure 3.3 Schematic diagrams of the whole binary vectors. (A) pMOA33-Lhca3-SN1 and (B) pMOA33Lhca3-SN2 used for Agrobacterium transformation.
63
3.3.2 Potato transformation
Following co-cultivation of potato leaf discs with A. tumefaciens possessing either of the two
binary vectors, cell colonies developed along the cut leaf margins and leaf surfaces after 5-6
weeks on callus induction medium with kanamycin. Most of the cell colonies were green and
grew as a hard, compact callus while some were pale-green and friable. Following transfer to
regeneration medium 90% of the hard green cell colonies produced shoots. Only 10% of the
friable cell colonies produced shoots. On average, more than 80% of the total cell colonies
transferred onto the regeneration medium produced shoots after two to three weeks.
Regenerated shoots were recovered over a period of three to four months following cocultivation. Although multiple shoots were regenerated from each cell colony, only a single
shoot was chosen per colony for further analysis. Healthy single shoots were excised from
regenerating clumps and transferred into multiplication medium with kanamycin. More than
95% of shoots produced roots within one week, while no rooting occurred in control plants on
the selective multiplication medium. In total, 299 independently derived kanamycin-resistant
cell colonies for the Lhca3-SN1 and Lhca3-SN2 transgenic potato lines were regenerated
from 435 cell colonies over three separate transformation experiments. From these, 108 lines
(45 from Lhca3-SN1 and 63 from Lhca3-SN2) were selected from those that regenerated
plants on the basis of healthy and phenotypically normal appearance and were used for the
remaining experiments. Regeneration and transformation efficiencies are summarised in
Table 3.3.
64
Table 3.3 Efficiency of A. tumefaciens transformation. The results of three transformation experiments are presented.
Specific Transformation Number
gene
experiment
of
explants
Lhca3SN1
Lhca3SN2
Number
of cell
colonies
Number of
Root
colonies
growth in
regenerating kanamycin
plants
1
120
81
63
63
2
80
59
41
41
3
90
55
40
40
1
122
89
60
60
2
88
64
42
42
3
101
87
53
53
Number PCR
of plants positive
screened for
Actin
PCR
positive
for
nptII
PCR
positive
for
Lhca3-SN
Putative Transformation
transgenic efficiency (%)*
lines
63
63
44
42
42
67
45
45
41
38
38
84
* Transformation efficiency = (Total number of putative transgenic lines / Total number of plants screened) x 100.
65
3.3.3 DNA isolation and PCR based screening of regenerated lines
The presence of the actin gene, nptII genes and Lhca3-SN1 and Lhca3-SN2 chimeric genes, in
regenerated lines were confirmed using independent PCR reactions (Figure 3.4 and Figure
3.5). Two of the transgenic plants from the Lhca3-SN1 transformations and three from the
Lhca3-SN2 transformations contained the nptII gene conferring kanamycin resistance but
lacked the chimeric Lhca3-StSN genes. The lines which contained both the Lhca3-SN gene
and the nptII gene were used for further analysis (Figures 3.4 and 3.5). The results of
molecular analyses indicate that 67% of screened Lhca3-SN1 lines and 84% of Lhca3-SN2
lines contained both the nptII and Lhca3-SN genes (Table 3.3).
From the PCR results 33 Lhca3-SN1 lines and 32 Lhca3-SN2 lines were selected for further
analysis based on the growth vigor and appearance of the plants resembling the nontransgenic controls. For convenience, the Lhca3-SN1 and Lhca3-SN2 transgenic lines were
labeled from 101 to 133 and 201 to 232, respectively. Each line originated from independent
explants following A. tumefaciens transformation. All 65 selected transgenic plants were
multiplied in vitro and transferred to the glasshouse (Figure 3.6).
66
Figure 3.4 PCR analysis of representative Lhca3-SN1 regenerated lines. Products of DNA amplification
after polymerase chain reaction using DNA from transgenic lines as the template and using the primers specific
to Lhca3-SN1, neomycin phosphotransferase (nptII), and Actin genes. Lane 1- GeneRulerTM DNA ladder mix
(Fermentas); lanes 2- 7 putative transformed lines 101-106, lane 8- non-transgenic Iwa, lane 9- pMOA33-Lhca3SN1 plasmid (+), lane 9- no template control.
Figure 3.5 PCR analysis of representative Lhca3-SN2 regenerated lines. Products of DNA amplification
after polymerase chain reaction using DNA from transgenic lines as the template and using the primers specific
to Lhca3-SN2, neomycin phosphotransferase (nptII) and Actin genes. Lane 1- GeneRulerTM DNA ladder mix
(Fermentas); lanes 2- 7 putative transformed lines 201-206, lane 8- non-transgenic Iwa, lane 9- pMOA33-Lhca3SN2 plasmid (+), lane 9- no template control.
67
Figure 3.6 Randomly arranged transgenic potato lines grown in bags in the greenhouse.
3.3.4 Phenotypic evaluation
The majority of the transformants developed normally and exhibited a similar phenotypic
appearance to the untransformed control plants in the greenhouse. These plants were also
normal during subsequent vegetative propagation. Out of 33 transgenic Lhca3-SN1 lines, 26
were classified as phenotypically normal. For the Lhca3-SN2 lines, out of 32 lines, 30 were
phenotypically normal. Seven lines with the Lhca3-SN1 gene (#111, #112, #120, #123, #125,
#129 and #133) and two lines with the Lhca3-SN2 gene (#204 and #215) showed a range of
abnormal characteristics such as marginal leaf curl, leaf wrinkling, reduced vigor, stunted
plant with small tubers, or a combination of all these traits (Figure 3.7).
68
Figure 3.7 Off-type transgenic potato lines grown in the containment greenhouse. (A) Phenotypically
normal transgenic plant (right) compared with a variant stunted plant (left). (B) Phenotypically normal tubers
(right) compared with phenotypically abnormal ‘peanut tubers’ from the stunted plant (left).
69
3.3.5 Endogenous expression levels of SN1 and SN2
To confirm the natural variability in expression of StSN1 and StSN2 genes in the leaves of
untransformed Iwa plants a qRT-PCR was carried out. Expression of the StSN1 and StSN2
genes was seen to vary between five independent plants.
StSN1 expression in untransformed Iwa
5
4
∆CT
3
2
1
0
Iwa 1
Iwa 2
Iwa 3
Iwa 4
Iwa 5
StSN2 expression in untransformed Iwa
5
∆CT
4
3
2
1
0
Iwa 1
Iwa 2
Iwa 3
Iwa 4
Iwa 5
Figure 3.8 Endogenous SN expression levels in non-transgenic Iwa. StSN1 expression levels and StSN2
expression levels in leaves of different Iwa control plants. The 95% confidence limits for each mean is
represented by the vertical line (n=3).
70
3.3.6 Quantitative RT-PCR analysis of transgenic lines
The transcript levels of the SN genes in leaves of all transgenic lines were determined by
qRT-PCR analysis relative to the endogenous SN levels in non-transgenic Iwa plants. In
comparison to the wild type, Lhca3-SN1 expression levels varied in the different transgenic
lines. Some lines had similar expression levels to the wild type, whilst in others an increase in
expression by up to 12-fold was observed (Figure 3.9). Of the 33 Lhca3-SN1 transgenic lines,
117, 121 and 131 showed high levels of SN1 mRNA expression and 108, 116 and 128 showed
moderate levels of SN1 mRNA expression. The transgenic lines 106 and 107 showed low
levels of SN1 expression similar to the untransformed Iwa plant. In total, out of the 33 SN1
transgenic lines, seven Lhca3-SN1 lines (107, 108, 116, 117, 121, 128 and 131) with different
levels of SN1 expression were selected for pathogen assays.
Similar results were seen for the Lhca3-SN2 lines, with the highest SN2 expression level
being 15 fold higher than in the corresponding wild type (Figure 3.10). Of the 32 lines in the
Lhca3-SN2 series, 3 lines (202, 218 and 219) showed more than 10 fold increase in
expression and 7 lines (203, 205, 206, 207, 223, 224 and 232) showed more than 5 fold
increases in SN2 expression compared to the wild type. Two lines (209 and 228) showed low
expression in leaves, similar to the wild type. From these, eight Lhca3-SN2 lines (202, 203,
205, 211, 218, 219, 223 and 228) with different level of SN2 expression were selected for
pathogen assay.
71
10
9
Relative Expression
8
7
6
5
4
3
2
1
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
Iwa
0
SN1 transgenic lines
Figure 3.9 Quantitative PCR amplification of the SN1 transcripts in leaves. The qRT-PCR data for each sample was normalized to the amount of Ef1α transcript using the
same amplification conditions. The relative abundance of the SN1 gene transcript in transgenic plants was determined using the ΔΔCT method relative to the non-transgenic
Iwa control. The 95% confidence limits for each mean is represented by the vertical line (n=3). The relative expression value of the non-transgenic control is set at 1. The
lines selected for further analysis are shown in red.
72
18
Le
16
Relative Expression
14
12
10
8
6
4
2
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
Iwa
0
SN2 transgenic lines
Figure 3.10 Quantitative PCR amplification of the SN2 transcripts in leaves. The qRT-PCR data for each sample was normalized to the amount of Ef1α transcript using
the same amplification conditions. The relative abundance of the SN2 gene transcript in transgenic plants was determined using the ΔΔCT method relative to the nontransgenic Iwa control. The 95% confidence limits for each mean is represented by the vertical line (n=3). The relative expression value of the non-transgenic control is set at
1. The lines selected for further analysis are shown in red.
73
3.4 Discussion
Since domestication of plants for human use began, diseases have caused major yield losses
and have impacted the well-being of humans worldwide (Agrios, 1997). The potential
application of transgenic technology offers a powerful strategy to generate agriculturally
valuable transgenic germplasm with disease resistance. However, the application of genetic
engineering too many food crops, including potato, has raised public concerns (Conner and
Jacobs, 1999). This could be overcome by modifying crop plants using only genes and
promoters from the same species (Barrell et al., 2010).
The research in this chapter investigated the transfer and overexpression of the potato SN1
and SN2 genes in the potato cultivar Iwa. This cultivar was selected because it is known to
have high efficiency for plant transformation (Conner et al., 1991) and it has a good field
resistance to both late blight and early blight and it is resistant to viruses PVX and PVY
(Genet, 1985). However, Iwa is known to be highly susceptible to soft rot (Barrell and
Conner, 2009).
The production and evaluation of genetically modified (GM) crops is an active area of
research, but the access of growers to this technology in many countries is currently restricted
primarily because of political and bioethical issues. Development of intragenic vectors and
marker-free transgenic plants may help to ease some of the political concerns about GM
technologies (Conner et al., 2007). To help develop the concept of intragenics the goal in this
study was to design an intragenic expression cassette completely derived from potato
sequences. Potato promoter and terminators as part of intragenic cassettes to target the desired
expression of transferred genes are vital components. The potato-derived Lhca3 promoter was
already known to be a good transcriptional control of gene expression in transgenic potato
plants (Nap et al., 1993; Shang et al., 2000; Meiyalaghan et al., 2006). This study used the
fragment homologous to this promoter, as well as the Lhca3 terminator sequence, isolated
from potato, as a potato expression cassette into which coding regions of other potato genes
can be cloned (Meiyalaghan et al., 2009).
The success in the development of cultivars with improved resistance to pathogens involving
transgenic expression of antimicrobial genes mainly depends on the nature of the transferred
gene. The potato derived SN1 and SN2 proteins are known to confer broad spectrum activity
against wide range of bacterial and fungal pathogens (Segura et al., 1999; Berrocal-Lobo et
al., 2002). Here SN1 and SN2 genes isolated from potato cultivar Iwa as candidate genes for
the intragenic cassette construction. To avoid the inadvertent inclusions of foreign nucleotides
74
in the expression cassette, high fidelity polymerase enzymes were used in all PCRs. Blunt-end
ligations were often required to avoid creating a duplication of cohesive ends at restriction
sites. Therefore, VentR®DNA polymerase was used to isolate blunt-end fragments from the a1
allele of SN1 and the b1 allele of SN2. An intragenic chimeric gene construct was successfully
built and based solely on Iwa sequences for transfer back into Iwa. This chimeric potato
intragene has been tested using the transgenic binary vector pMOA33 (Barrell and Conner,
2006) for ease of gene transfer. In this study the SN1 and SN2 genes were overexpressed and
established a group of transgenic potato plants using an intragenic cassette to determine if
these approaches would be relevant to improving the disease resistance at the crop level.
In most overexpression studies in plants (Brogue et al., 1991; Epple et al., 1997; Winicov and
Bastola, 1999; Koo et al., 2002; Hu et al., 2006; Jayaraj and Punja., 2007; Chen et al., 2009;
Kang et al., 2010) a constitutive promoter, such as CaMV 35S is utilized for transcriptional
control. Almasia et al (2008) and Balaji and Smart (2011) also used this promoter for the
constitutive overexpression of the SN1 and SN2 genes respectively. In some instances, where
gene overexpression is only required in specific tissues, e.g. leaves in case of foliar diseases
or stems in stem-based diseases, constitutive expression of the transferred gene is
unnecessary. Meiyalaghan et al. (2006) reported that the expression of transgenes, under the
transcriptional control of the Iwa-derived light-inducible Lhca3 promoter, was higher in
foliage coupled with minimal expression or no expression in tuber. For avoiding the public
concerns regarding the food safety of transgenic potatoes, the Iwa-derived light-inducible
Lhca3 promoter was used in this study.
In course of six independent transformation experiments, SN1 and SN2 genes under
transcriptional control of Lhca3 promoter were transferred to Iwa potato with the nptII
selectable marker gene conferring resistance to kanamycin. Approximately 600 leaf explants
of potato cultivar Iwa were co-cultivated with disarmed Agrobacterium strain EHA105
containing either the binary vector pMOA33-Lhca3-SN1 or pMOA33-Lhca3-SN2. More than
80% (Section 3.3.2) of the selected cell colonies produced regenerated shoots. A total of 144
Lhca3-SN1 and 155 Lhca3-SN2 putative transgenic potato lines produced roots in the
kanamycin selection medium (Table 3.3). The PCR screening of selected lines established
more than 60% transformation frequency from both binary vectors. This confirms the high
rate of success for the Agrobacterium-mediated gene transfer system of potato using
kanamycin resistance as a selectable marker (Barrell et al., 2002).
Analysis of putative transgenic lines using independent PCR (Figure 3.3 and Figure 3.4)
established the presence of nptII gene and either Lhca3-SN1 or Lhca3-SN2 genes using
75
precise primers in selected regenerated lines. Actin gene PCR was also conducted to
determine the genomic DNA quality. Out of 108 selected transgenic lines, two lines in the
Lhca3-SN1 series and three lines in the Lhca3-SN2 series contained the nptII gene, but not
the target Lhca3-SN gene. This may be because of the partial integration of the T-DNA
construct in the host plant. Davidson et al. (2002) also reported similar partial integration in
their studies. For screening the presence of the Lhca3-SN-Lhca3 potato intragenes, care was
taken to use one promoter specific primer and one SN gene specific primer to prevent the
amplification of the endogenous genes.
Ninety percent of the putatively transgenic lines were phenotypically normal when grown in
the greenhouse. A low frequency (10%) of the transgenic lines showed plants exhibited some
form of off-type appearance, with phenotypes such as marginal leaf curl, leaf wrinkling,
reduced vigor, and stunted plants with small tubers (Figure 3.6). These off-type phenotypes
may be the result of somoclonal variation or some epigenetic effect during the tissue culture
phase of the transformation process (Conner and Christey, 1994). The growth, development
and tuber yield of the remaining transgenic plants were not different compared to the wild
control type. Therefore, the results suggest that overexpression of the SN genes is not
influencing the physiology of the plants and their overall phenotypic appearance. These plants
were devoid of the negative traits such as dwarfism observed for magnesium transport gene
(Deng et al., 2006) and maize homeo box (KNOTTED-1) gene (Sinha et al., 1993), or high
branching from a gibberellin 20-oxidase gene (Niki et al., 2001) and arabinogalactan protein
gene (Sun et al., 2004).
To examine the variation of endogenous StSN1 and StSN2 expression in untransformed Iwa
plants qRT-PCR was carried out using cDNA samples from different untransformed Iwa. The
results revealed slight variations of StSN1 and StSN2 expressions between different
untransformed plants (Figure 3.7).
The qRT-PCR results for RNA levels in transformed plants leaves revealed the
overexpression of the SN genes at the mRNA level. Eflα reference gene was chosen as an
endogenous control based on studies by Nicot et al. (2005) who reported Eflα was the most
stable among the seven reference genes they tested in potato. SN overexpression was observed
in 90% lines in Lhca3-SN2 series. In comparison the overall expression was less in Lhca3SN1 series. Expression of transferred genes can be highly variable between different plant
lines transformed with the same transgene (Conner and Christey, 1994). There are several
potential reasons for this variation in expression. The effect of the position of the transgene
within the potato genome may result in varied levels of expression (position effect) (Conner
76
and Christey, 1994). The transformed lines 117, 118, 121 and 131 from Lhca3-SN1 series and
202, 218 and 219 from Lhca3-SN2 series had the highest level of expression of the SN gene
(Figures 3.8 and 3.9) Two lines in each series, 106 and 107 among the Lhca3-SN1 lines
(Figure 3.8) and 209 and 228 among the Lhca3-SN2 lines (Figure 3.9), showed lower
expression level of the SN genes comparable with the control Iwa plant. Co-suppression may
be responsible for such low mRNA levels in those transgenic plants (Taylor, 1997; Palauqui
et al., 1997). The mechanism of co-suppression is not understood. According to a threshold
model, when RNA transcripts of a gene accumulate beyond a critical, threshold level, they are
selectively degraded by RNases. Gene silencing sometimes results from aberrant RNA
transcripts of the transgene as well as the endogenous gene, which can lead to a drastic
reduction in the level of expression of the transgene and endogenous gene (Wassenegger and
Pelissier, 1998).
In summary, this chapter set out to construct a potato intragene for overexpression of the
potato SN genes in potato. Binary vectors for Agrobacterium-mediated gene transfer were
successfully constructed with the StSN1 and StSN2 coding regions under transcriptional
control of the Lhca3 promoter. Over 30 independently derived transgenic lines for both
Lhca3-SN1 and Lhca3-SN2 were recovered and mRNA expression levels were assessed using
qRT-PCR. This study has therefore established two new sources of potato germplasm with
overexpressed SN1 and SN2 genes. These results suggest that the genetic manipulation
technique used in the present study is effective means of producing phenotypically normal
potato plants with an enhanced SN gene expression. Based on the results obtained from the
expression analysis (Figures 3.8 and Figure 3.9), seven Lhca3-SN1 transgenic lines (3 high
expression lines, 3 medium expression lines and 1 low expression) and eight Lhca3-SN2 lines
(3 high expression lines, 4 medium expression lines and 1 low expression) transgenic lines
were selected as a valuable resource for assessing the phenotypic role of these genes in potato,
especially for assays on disease resistance.
77
Chapter 4
Resistance of transgenic potato plants overexpressing SN1 and
SN2 to Pectobacterium atrosepticum
4.1 Introduction
Blackleg of potato is one of the most important destructive diseases affecting potato
production throughout the world (Pérombelon, 1992; De Boer, 2004), because the disease not
only affects yield, but also affect the quality of the crop. This disease was first reported in
Amsterdam in 1902 (Fredricks and Metcalf, 1970). Although average losses are generally
small, the wide spread occurrence of the pathogen and the possibility of outbreaks reaching
epidemic proportions means blackleg is potentially one of the most economically serious
diseases affecting potatoes (Pérombelon, 1992). Pectobacterium atrosepticum (Pca) (former
name Erwinia carotovora subsp. atroseptica; Dye, 1969; Gardan et al., 2003),
Pectobacterium carotovorum subsp. carotovorum (Pcc) (former name Erwinia carotovora
subsp. carotovora) (Dye, 1969; Gardan et al., 2003) and Dickeya spp. (former name Erwinia
chrysanthemi; Burkholder, 1953; Samson et al., 2005) are the main bacteria responsible for
blackleg disease. Temperature has the greatest effect on the ability of these bacteria to infect
potatoes (Pérombelon and Kelman, 1980; Pérombelon, 1992). Pectobacterium atrosepticum is
found under cool temperate climates (<25ºC) and where the soil temperatures are lower
(between 15ºC and 20ºC) (Smadja et al., 2004) causes blackleg only in potatoes. Because of
these reasons Pca can cause severe damage to potatoes under New Zealand climatic
conditions.
Control of bacterial disease relies largely on prevention of infection. Once disease has been
initiated, chemical control is ineffective since bacteria increase rapidly and are protected
within host tissue. Due to the lack of effective chemical treatments and cultural practices to
reduce the impact of both blackleg and soft rot, the use of resistant cultivars is considered the
most effective, economical and environmentally friendly approach to control this disease
(Czajkowski et al., 2011). To date traditional potato breeding for resistant cultivars has not
been very successful, mainly because of the genetic complexity of this crop and the lack of
suitable germplasm with resistance to blackleg (De Boer, 2004). An area of increasing interest
is to genetically modify plants to enhance resistance to plant pathogens.
Enormous progress has been made over the past decade in our understanding of the highly
complex molecular events that occur in plant-pathogen interactions. This knowledge in turn
78
has provided a number of options and strategies which can be, and have been, used to make
plants resistant to pathogens (Hammond-Kosack et al., 2004; Dong, 1998). Plants have
evolved many different mechanisms to cope with the constant threat of attack by
phytopathogenic microorganisms. In the last few decades, more than 30 resistance genes
which confer resistance against a wide range of pathogens, including viruses, bacteria, fungi,
oomycetes and nematode have been identified (Johal and Briggs, 1992; Martin et al., 1993;
Song et al., 1995; Bent et al., 1994; Grant et al., 1995; Salmeron et al., 1994; Whitham et al.,
1996; Lawrence et al., 1995; Jones et al., 1994; Dixon et al., 1996; Mysore et al., 2003;
Huang et al., 2005).
One of the most significant developments in plant molecular biology and biotechnology has
been the use of gene isolation and genetic transformation techniques to develop diseaseresistant transgenic plants. One group of genes which have been shown to have potential are
those encoding antimicrobial proteins (Selitrennikoff, 2001; Zasloff, 2002; Sitaram and
Nagaraj, 2002; Yeaman and Yount, 2003). There is evidence that the overexpression of these
transgenes may confer protection against pathogen attack (Brogue et al., 1991; Nordeen et al.,
1992; Molina and García-Olmedo, 1993; Zhu et al., 1996; Allefs et al., 1996; Bi et al., 1999;
Gao et al., 2000; Koo et al., 2002; Vellicce et al., 2006; Zakharchenko et al., 2005; Jayaraj
and Punja, 2007; Barrell and Conner, 2009; Jan et al., 2010).
As outlined in Chapter 1 antimicrobial peptides Snakin-1 (SN1) and Snakin-2 (SN2) isolated
from Solanum tuberosum have been found to be active against a number of important plant
pathogens in vitro (Segura et al., 1999; Berrocal-Lobo et al., 2002). The SN genes were found
to be highly conserved throughout Solanaceous species as reported in Chapter 2. To elucidate
the role of these SN genes and to potentially develop new disease-resistant germplasm,
transgenic potato plants that overexpress SN1 or SN2 genes were regenerated (Chapter 3). In
this chapter, selected transgenic lines were further characterized for transgene expression and
assayed for resistance to Pectobacterium atrosepticum (causing blackleg disease) of potato.
4.2 Material and methods
4.2.1 Plant material
Based on the results obtained from Chapter 3, phenotypically normal potato lines of cultivar
Iwa, transgenic for varying levels of SN gene expression in leaves (Figure 3.9 and 3.10) were
selected for more detailed assessment and pathogen bioassays. These included six Lhca3-SN1
lines (108, 116, 117, 121, 128 and 131) and seven Lhca3-SN2 lines (202, 203, 205, 211, 218,
219 and 223) (Table 4.1) with medium-high transcriptional expression and two low
79
expressing lines respectively. An untransformed Iwa also included in each sets as a control.
For the first two pathogenicity experiments plants were micro-propagated in tissue culture and
transferred to pots as described in Chapter 3. In the third experiment plants were grown from
tubers which had been stored for 4 months at 6ºC.
4.2.2 Expression of SN gene in different tissues of selected lines using qPCR
Total RNA was extracted from leaves, stems and tubers of Lhca3-SN1 selected lines and
leaves, stems, roots and tubers of Lhca3-SN2 selected lines
using the RNAspin Mini
Isolation Kit (GE Healthcare, UK) following the manufacturer’s instructions as described in
Chapter 3 (Section 3.2.8) after the plants had grown for 2.5 months in PB5 bags in the
greenhouse (Section 3.2.7). Complementary DNAs were synthesized from 1µg of total RNA
using SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s
protocol. Quantitative RT-PCRs were carried out in an Applied Biosystems StepOne RealTime PCR System with Express Sybr GreenER qPCR Supermix (Invitrogen) An endogenous
housekeeping gene eflα as an internal control to provide a baseline for standardizing gene
expression between transgenic lines.
Each 20 µL reaction mix and PCR profile was as described in Chapter 3 (Section 3.2.9). The
relative expression levels were determined using StepOneTM software v2.1 as described in the
Chapter 3.
4.2.3 Pectobacterium atrosepticum pathogenicity assays
The selected transgenic plants and control plants were assayed for their susceptibility to
Pectobacterium atrosepticum in three separate pot experiments under different conditions.
Pectobacterium atrosepticum isolate SCRI 1043 (American Type Culture Collection BAA672) was isolated from a potato stem with blackleg disease symptoms in 1985 in Perthshire,
Scotland. This strain, obtained from Dr Andrew Pitman (Plant & Food Research), has been
used in epidemiological and molecular studies for many years (Hinton et al., 1985; Toth et al.,
1997) and is a well characterised strain with a full genome sequence (GenBank accession NC004547) (Bell et al., 2004). The isolate was stored in 8% glycerol at -80ºC and cultured onto
Luria-Bertani (LB) agar at 28ºC as required.
80
Table 4.1 Potato lines selected for use in pathogen assay study (based on its transcriptional
expression in leaves). (A) Lhca3-SN1-Lhca3 transgenic lines, and (B) Lhca3-SN2-Lhca3 transgenic
lines. The origin of all lines is outlined in Chapter 3.
(A)
Line
Engineered gene construct
Transcriptional expression level in leaves
107
Lhca3-SN1-Lhca3
Low expression
108
Lhca3-SN1-Lhca3
Medium expression
116
Lhca3-SN1-Lhca3
Medium expression
117
Lhca3-SN1-Lhca3
High expression
121
Lhca3-SN1-Lhca3
High expression
128
Lhca3-SN1-Lhca3
Medium expression
131
Lhca3- SN1-Lhca3
High expression
Line
Engineered gene construct
Transcriptional expression level in leaves
202
Lhca3-SN2-Lhca3
High expression.
203
Lhca3-SN2-Lhca3
Medium expression
205
Lhca3-SN2-Lhca3
Medium expression
211
Lhca3-SN2-Lhca3
Medium expression
218
Lhca3-SN2-Lhca3
High expression
219
Lhca3-SN2-Lhca3
High expression
223
Lhca3-SN2-Lhca3
Medium expression
228
Lhca3-SN2-Lhca3
Low expression
(B)
81
To produce inoculum for the pathogenicity assays, 20 mL of (LB) broth (Appendix A) was
inoculated with one colony picked off a newly cultured LB agar plate and incubated overnight
in a shaking incubator at 28ºC and 200 rpm. The cells were harvested by centrifugation at
4,000 x g for 10 min. The supernatant was discarded and the cells were washed in 10 mM
MgCl2, and resuspended (OD 1.2 at 600 nm) in 20 mL of 10 mM MgCl2.
In all experiments four week old plants from either micro-propagation in vitro or tubers were
established in pots (6 cm x 6 cm x 7 cm deep) containing potting mix (Conner et al., 1994)
and transferred to the inoculation chamber one week before bacterial inoculation for
acclimatisation. For each transgenic line and the Iwa control, up to 14 replicate plants were
inoculated with Pca. The stem under the second fully expanded leaf was punctured using the
tip of a micropipette tip and inoculated with 10 µL of the bacterial suspension. Up to 14
plants were mock-inoculated with 10 mM MgCl2 and same numbers of plants were left
uninoculated under the same conditions. The inoculation site was then sealed with vaseline to
avoid desiccation.
4.2.3.1
Experiment 1
Lhca3-SN1 experiment was conducted in the inoculation chamber facility at Plant & Food
Research, Lincoln and Lhca3-SN2 experiment was conducted in the containment greenhouse
facility at Plant & Food Research, Lincoln using in vitro micro-propagated plants. In both set
the plants were arranged in a completely randomised design in the growth cabinet
(CONTHERM, CAT610/620 Biosyn. Growth Chamber (Figure 4.1)/ artificially made tent
and incubated at ~ 22ºC under 8/16 hour dark/light cycle (500 W metal halide vapor bulbs
(light intensity maximum 1150µmol m-2 s-1) and 80-90% humidity for 14 days respectively.
4.2.3.2 Experiment 2
The second experiment was conducted in the Biotron containment facility at Lincoln
University
(http://bioprotection.org.nz/the-new-zealand-biotron)
using
plants
micro-
propagated in vitro. The conditions provided heating between 18ºC-22ºC. Day length was set
at 16 h with light provided by 400 W metal halide vapor bulbs and 100W incandescent bulbs
(light intensity maximum 1100µmol.m2.s1). A tent was made with plastic sheets under a metal
frame (1.8 m x 0.8 m x 0.6 m deep) to maintain high humidity with three humidity controller
probes (Onset, HOBO Pro series data loggers) placed inside the tent to monitor the humidity
level. The inoculated plants were placed in this tent, randomly arranged in trays. Humidity
was maintained at over 80% inside the tent throughout the experiment, by adding water to the
trays as required.
82
Figure 4.1 Experiment 1 set up showing inoculated potato plants in the inoculation chamber.
4.2.3.2
Experiment 3
This experiment was performed in the containment greenhouse facility at Plant & Food
Research, Palmerston North. A humidity tent was made under the greenhouse bench using
plastic sheets (Figure 4.2). The inoculated plants were randomly arranged in this tent with
heating providing a temperature of 22-25ºC and incubated for 10 days. Relative humidity was
maintained above 80% by maintaining the water level in the trays. Disease was assessed after
83
10 days and pathogen inoculated stem samples were also collected for PCR analysis, qPCR
and dilution plating from this experiment.
4.2.4 Disease Assessment
4.2.4.1
Assessment of disease resistance
The disease severity on each replicate plant was scored by observing symptoms such as
chlorosis, necrosis, stem wilting, wilted and rolled leaves, and measuring the length of lesion
that developed at each inoculation site. The first experiment and second experiment were
recorded daily for 14 and 11 days respectively. The experiment 3 in Palmerston North, which
was assessed only after ten days. Longitudinal sections of the infected stems were also
assessed in the experiment 2. In the third experiment, PCR analysis, qPCR and dilution
plating were also conducted to determine the persistence and invasion of Pca from the
inoculation sites.
Figure 4.2 Experiment 3 set up showing inoculated potato plants in the artificially made tent.
84
4.2.4.2
PCR analysis of Pca from lesions.
To confirm the presence or absence of Pca in the lesions, PCR was conducted on transgenic
and control potato plant samples. A 3cm long stem section was taken from just below the
visible symptoms of the lesion 10 days after inoculation. For each transgenic line and control,
genomic DNA was isolated from stems of three randomly selected pathogen-inoculated
replicate plants and the uninoculated MgCl2 controls using the CTAB method (Doyle and
Doyle, 1990). Negative controls (no template DNA) and positive controls (DNA of Pac) were
also included. Primers specific for the Pca515 gene present in Pca was used for the PCR
amplification. PCR reactions were carried out in a Mastercycler (Eppendorf, Hamburg,
Germany). The Pca515 Forward (5’ATGTCAGAACTGACCGCCTT3’) and Pca515 Reverse
(5’ATTAAGAACACGGCCACCTG3’) primer set (designed by Bhanupratap Vanga, Plant &
Food Research) was used for the PCR reaction and amplifies a fragment with an expected size
of 215 bp. Each 25 µL PCR mix contained 10 x PCR buffer, 0.2 mM of each dNTP, 0.5 µM
of forward primer, 0.5 µM of reverse primer and 0.4 U of Taq DNA Polymerase (New
England Biolabs). The conditions for PCR were: denaturation at 94C for 5 min, followed by
40 cycles of (30 s 94C, 30 s 60C, 40 s 72C), then by 7 min extension at 72C. Amplified
products were separated by electrophoresis in a 1% agarose gel in 1xTAE buffer at 5.5V/cm
for 80 min and visualized under UV light after staining with ethidium bromide (1 mg/L) for
15 min.
4.2.4.3
Dilution plating of Pca from lesions
Dilution plating was also carried out to determine the number of viable Pca in the sample.
Ten days after inoculation, three replicate stem (a 3cm long stem section was taken from just
below the visible lesion symptoms) from each line were sampled. Stem segments were
washed in running tap water, surface-sterilized in 1% sodium hypochlorite (commercial
bleach) for 5 min, washed once with sterile water and subsequently sterilized with 70%
ethanol for 5 min. After sterilization, the stem samples were washed twice with sterile water
and the surface blotted dry with sterile tissue paper. Collected stem sections (40 mg each)
were crushed with a mortar and pestle with 1 mL 10 mM MgCl2. Serial dilutions (100 to 10-6)
of stem extracts were made in 10 mM MgCl2 and 100 µL of each dilution were spread-plated
on three crystal violet pectate agar (CVP) plates. After incubation for 72 h at 28C the
numbers of colonies were counted.
85
4.2.4.4
Quantitative PCR analysis
Quantitative PCR was also carried out to estimate the total amount of pathogen in the
transgenic and control potato samples using the genomic DNA (Section 4.2.4.2) as a template
(triplicate) and the pure pathogen genomic DNA as control. The pair of primers described in
Section 4.2.4.2 was used for these qPCRs. PCR reactions were in a total volume of 20 µL
containing 0.5 µM of forward primer and 3 µM of reverse primer, 1X EXPRESS SYBR®
GreenER™ qPCR Supermix with premixed ROX reference dye (500 nM) (Invitrogen) and
DNA template. The PCR conditions were as follows; 2 min at 50ºC, 2 min at 95ºC followed
by 40 cycles (15 s at 95ºC , 60s at 60ºC), finally dissociation curve analysis was carried out by
cycling for 15s at 95ºC, 60s at 60ºC, 15 min at 95ºC. Each plate always contained known Pca
DNA samples that were used to develop the standard curve, as well as DNA samples from
uninoculated potato plants and a negative control reaction (no template DNA).
4.2.4.5
Statistical Analysis
Bioassays were conducted three times with a selected transgenic line with same number of
replicates per treatment and included appropriate controls. The results were represented as
two key parameters: the percentage of plants showing symptoms and the length of lesions on
the plants. The necrotic lesion was recorded daily for 14 days in first experiment. Analysis of
variance was performed on key parameters from each experiment using GENSTAT (GenStat
Committee, 2010 and Payne et al., 2009), and least significant differences (LSDs) were
derived from the residual mean square of the analysis of variance just done for day 9 or day
11 or day 10 depending on the experiment. For the qPCR data obtained from Experiment 3, a
standard curve was developed by plotting the logarithm of known concentrations (10-fold
dilution series) of Pca DNA against the Ct values. Ct being the cycle number at which the
fluorescence emission of the PCR product is statistically significant from the background. Ct
value is inversely related to the log of the initial concentration, so that the lower the Ct value
the higher the initial DNA concentration. Theoretically, Ct values should decrease by one unit
as the number of DNA molecules in the reaction double (i.e. 100% efficiency of the
amplification reaction). The log10 of the estimated dilutions for these samples were analyzed
using analysis of variance. Data for the calibrations, non template control and any plants for
which all values were undetermined (MgCl2 inoculated plants) were excluded. The analysis of
variance included factors for the individual replicate plants, and for the triplicate sampling
from each plant. Differences between the lines and comparisons with the Pca positive (pure
Pca culture) were made within the analysis of variance, using F-tests at P=0.05. For
translating the estimated dilution into DNA/CFU content, the known log10 dilutions were
86
converted into log10CFU or log10DNA concentrations on the basis of the known CFU for 100
dilution. CFU values for dilution plating were log10 transformed prior to analysis by analysis
of variance.
4.3 Results
4.3.1 Quantification of the transcript levels of the SN genes in different tissues
using qRT-PCR
The expression of the SN1 gene in leaves, stem and tubers and SN2 gene in leaves, stems, root
and tubers of selected transgenic lines was determined by qRT-PCR analysis.
4.3.1.1
SN1 transcript levels in Lhca3-SN1 transgenic lines
Three SN1 lines (117, 121 and 128) exhibited SN1 expression ranging from a 4- 6.5 fold
increase over the Iwa control (Figure 4.3). Two lines (108, 131) showed medium level of
expression ranging from 1.8- 2.5 fold increase over the control, while the selected low
expression line (107) and line 116 showed no change in SN1 expression compared with the
control plant.
None of the lines showed any increase in SN1 expression in stems and tubers compared with
the control plant (Figure 4.3). The low expressing line (107) showed almost similar SN1
expression to the control Iwa in all tissues analyzed.
4.3.1.2
SN2 transcript levels in Lhca3-SN2 transgenic lines
The SN2 transcript level in the leaf tissues exhibited similar trends among lines to the similar
to that observed in the earlier experiment (Figure 3.9, Chapter 3); seven lines retained a 2.5 to
5 fold increase in transcription of SN2 over the control, while the selected low expressing line
(228) retained similar expression to the non-transgenic Iwa control. Stems of the selected
Lhca3-SN2 trandgenic lines showed considerably higher expression of SN2 transcripts in
most transgenic lines, except for the line 228 which also showed low expression in the leaves.
The transgenic lines 202, 203 and 218 showed a 55 to 130-fold increase in SN2 transcripts
over the Iwa control, whereas lines 205, 219 and 223 showed a 15 to 30-fold increase in
expression. Root tissue of all lines showed a 1.5 to 4.5-fold increase in SN2 expression,
except line 202 which showed no significant change in SN2 expression. In the tuber tissue
most of the transgenic lines showed no change in SN2 expression when compared to the Iwa
control plants. However, line 203 showed more than a two-fold increase in SN2 transcripts in
the tuber tissue relative to the Iwa control (Figure 4.4).
87
Figure 4.3 SN1 transcript levels in the different tissues of the selected lines. The qRT-PCR data for each
sample was normalized to the amount of Ef1α transcript using the same amplification conditions. The relative
abundance of SN1 gene transcript in transgenic plants was determined using the ΔΔC T method relative to the
non-transgenic ‘Iwa’ control for three biological replicates. The relative expression value of the non-transgenic
control is set at 1. Analysis of variance established highly significant differences between lines for SN1
expression in leaves (Fs (7, 16) = 7.79, P<0.001), stem (Fs (7, 16) = 1.04, P<0.442) and tuber (Fs (7, 16) = 2.34,
P<0.075). Vertical bars represent the least significant difference (LSD) at the 0.05 probababilty level.
88
Figure 4.4 SN2 transcript levels in the different tissues of the selected lines. The qRT-PCR data for each
sample was normalized to the amount of Ef1α transcript using the same amplification conditions. The relative
abundance of SN2 gene transcript in transgenic plants was determined using the ΔΔC T method relative to the
non-transgenic ‘Iwa’ control for three biological replicates. The relative expression value of the non-transgenic
control is set at 1. Analysis of variance established highly significant differences between lines for GSL1
expression in leaves (Fs (8, 18) =6.85, P<0.001), stem (Fs (8, 18) = 68.61, P<0.001), roots (Fs (7, 16) =373, P<0.001) and
tuber (Fs (7, 16) =10.81, P<0.001). Vertical bars represent the least significant difference (LSD) at the 0.05
probability level.
89
4.3.2 Black leg assays in transgenic potato lines overexpressing SN1 and SN2
genes
Selected lines from the two populations of independently derived transgenic lines were
challenged with Pca. The assays were performed three times under different experimental
conditions to confirm the results, with similar trends observed in all experiments. Initial
disease symptoms such as chlorosis, necrosis of leaves and stem wilting, wilting and rolling
of leaves, as well as more severe symptoms such as extent of necrotic lesion development and
stem breakdown were observed in control plants and some susceptible transgenic lines 9-12
days after inoculation with Pca.
4.3.2.1
Experiment 1
In the first Lhca3-SN1 experiment in the inoculation chamber, characteristic disease
symptoms were observed on the stems of all the Iwa control plants indicating that the
bioassay was successful at inducing blackleg disease in potato (Figure 4.5). The control Iwa
and low expressing line (107) were very susceptible to Pca (92% of stems with disease
symptoms and large lesion length). Two days after inoculation with the pathogen, the first
sign of disease was observed on the non-transgenic control plants with the inoculation site
becoming dark and swollen (Figure 4.5). On the fourth day, virtually all the leaves of the
control plants and susceptible transgenic lines had wilted and noticeable extension of the stem
lesions was observed (Figure 4.5). The resistant lines 128 and 131 also showed slow lesion
extension from fourth day onwards. At 9 dpi onwards, stems of the control and other
susceptible lines apart from lines 128 and 131, had collapsed, leading to plant death. Four
Lhca3-SN1 transgenic lines (108, 116, 117 and 121) showed comparatively reduced lesion
length and two lines (128 and 131) exhibited significantly reduced lesion length compared to
the non-transgenic control. Out of seven Lhca3-SN1 transgenic lines inoculated with Pca, two
(128 and 131) could be classified as resistant, three as partial resistant (108, 116 and 121) and
one as susceptible (107, low expressing line) (Table 4.2). Line 107 with no transcriptional
overexpression of SN1 was susceptible to P. atrpsepticum. Interestingly, line 117 also proved
to be susceptible in this bioassay. MgCl2 inoculated control plants were devoid of all disease
symptoms (Table 4.2).
90
Figure 4.5 Disease severities in control plants after inoculating Pca at different assessment times. The arrows indicate the inoculation site
91
Table 4.2 Bioassay of Lhca3-SN1 transgenic lines against Pectobacterium atrosepticum (Experiment 1).
Summary of disease parameters from twelve replicates of each transgenic line, non-transgenic control Iwa and
MgCl2 inoculated plant.
Plant line
Incidence of
Frequency of
Mean blackleg lesion
blackleg
collapsed plants
length (cm at 10 dpi)
(% at 14 dpi)
(% at 14 dpi)
Statusa
Iwa control
92
92
4.6
Susceptible
107
92
92
4.6
Susceptible
108
75
42
2.6
Partial resistant
116
92
42
1.7
Partial resistant
117
92
75
4.0
Susceptible
121
75
42
2.5
Partial resistant
128
58
8
0.9
Resistant
131
42
0
0.4
Resistant
MgCl2 inoculated plants
0
0
-
-
Fsb(df)
11.54*** (7,88)
LSD (5%)
a
1.34
= Status was calculated on the basis of an arbitrary scale: resistant (mean lesion length <1 cm), partially
resistant (1-3 cm) and susceptible (>3 cm).
b
= F value from analysis of variance, *** represents significant
difference between means at the 0.1% probability level, LSD = least significant differences between two means
at the 5% probability level. c = Not Applicable.
92
In the pathogen assay with the Lhca3-SN2 lines, the control Iwa and low expressing line
(228) exhibited severe disease symptoms (Table 4.3) Disease symptoms were first observed
on the Iwa control plants at day 2 after pathogen inoculation. Rapid disease progression was
observed resulting in completely rotten stems with folding and curling of leaves after 5 days,
followed by stem collapse and death of all plants by 12 dpi. All lines showed a small swelling
around the lesion (wound response) within 4 days, which developed into necrotic lesions in
susceptible lines. For transgenic lines 202, 203, 211 and 219, some necrosis developed in a
few plants, from which most recovered by 9 dpi. These four Lhca3-SN2 lines (202, 203, 211
and 219) were healthy and showed no symptoms of blackleg 14 dpi and were considered to be
resistant to blackleg disease caused by P. atrosepticum (Table 4.3, Figure 4.6). Two lines
(205 and 223) showed significantly reduced lesion length compared to the non-transgenic
control and were given the status of partially resistant (Table 4.3). Line 228 with no
measurable transcriptional overexpression of SN2 was susceptible to P. atrosepticum (Table
4.3, Figure 4.6). Surprisingly, line 218 also proved to be susceptible in this bioassay. The
MgCl2 inoculated control and non-inoculated plants did not develop any disease symptoms or
necrotic lesions (Table 4.3).
93
Table 4.3 Bioassay of Lhca3-SN2 transgenic lines against Pectobacterium atrosepticum (Experiment 1).
Summary of disease parameters from fourteen replicates of each transgenic line, non-transgenic control Iwa and
MgCl2 inoculated plant.
Plant line
Statusa
Incidence of
Frequency of
Mean blackleg lesion
blackleg
collapsed plants
length (cm at 9 dpi)
(% at 14 dpi)
(% at 14 dpi)
Iwa control
93
71
3.5
Susceptible
202
0
0
0
Resistant
203
0
0
0
Resistant
205
71
14
0.8
Partially resistant
211
0
0
0.1
Resistant
218
100
100
3.6
Susceptible
219
0
0
0.2
Resistant
223
93
0
0.8
Partially resistant
228
93
86
3.5
Susceptible
MgCl2 inoculated plants
0
0
-
-
Non-inoculated plants
0
0
-
-
Fsb(df)
35.83*** (8,117)
LSD (5%)
a
0.77
Status was calculated on the basis of an arbitrary scale: resistant (mean lesion length <0.5 cm), partially
resistant (0.5-2 cm) and susceptible (>2 cm).
b
F value from analysis of variance, *** represents significant
difference between means at the 0.001 probability level, LSD = least significant differences between two means
at the 0.05 probability level.
94
Figure 4.6 Disease appearance observed in Lhca3-SN2 transgenic lines (Experiment 1). Variation in the
disease response following pathogen challenge among the Lhca3-SN2 transgenic lines compared with nontransgenic control (Iwa). From left to right each row represents a line that has 14 replicates plants. (A) Control
Iwa, (B) line 228, (C) line 211, (D) line 205 and (E) line 202 (F) line 219. The photograph was taken 14 days
after inoculation.
95
4.3.2.2
Experiment 2
The growth environment of the potato plants in the Biotron facility during Experiment 2 had a
major influence on the physiology of the plants, resulting in the plants having ‘woody’ stems.
As a consequence the external disease symptoms were not visible. Nevertheless, an
association between disease resistance and the overexpression of the SN gene was visible in
longitudinal sections through the inoculated stems (Figure 4.7 and Figure 4.8). The internal
lesion length and the percentage of plants with symptoms in this experiment supported the
results of Experiment 1.
A high percentage of Iwa control plants had symptoms and significantly larger internal lesions
than most of the transgenic lines. Analysis of variance established that the there was a highly
significant difference in the lesion lengths caused by Pca inoculations between the transgenic
lines and the Iwa control for both Lhca3-SN1 and Lhca3-SN2 transgenic lines (Tables 4.4 and
4.5). Two Lhca3-SN1 lines (128 and 131) showed substantial reduction in the frequency of
plants with symptoms and internal lesion length, with three lines (108, 116 and 121) also
showing a significantly reduced lesion length compared to the non-transgenic control (Table
4.4 and Figure 4.7). Four Lhca3-SN2 lines (202, 203, 211 and 219) showed highly reduced
disease symptoms as noted for both the frequency of stems with symptoms and the internal
lesion length, whereas three other lines (205, 218 and 223) showed significantly reduced
internal lesion lengths and one line (228, low expressing line) was very marginal in exhibiting
a difference from the non-transgenic control (Table 4.5 and Figure 4.8).
In both experiments the MgCl2 inoculated control plants did not develop any disease
symptoms or necrotic lesions (Tables 4.4 and 4.5, Figures 4.7 and 4.8).
96
Table 4.4 Bioassay of Lhca3-SN1 transgenic lines against Pectobacterium atrosepticum (Experiment 2).
Summary of disease parameters from three replicates of each transgenic line, non-transgenic control Iwa and
MgCl2 inoculated plant.
Plant line
Incidence of blackleg
Mean internal blackleg
(% at 11 dpi)
lesion length (cm at 11dpi )
Statusa
Iwa control
100
3.2
Susceptible
107
100
2.6
Susceptible
108
100
1.3
Partially resistant
116
67
1.4
Partially resistant
117
100
2.5
Susceptible
121
100
2.0
Partially resistant
128
67
0.8
Resistant
131
33
0.2
Resistant
MgCl2 inoculated plants
0
-
-
Non-inoculated plants
0
-
-
Fsb
-
8.78***(7,16)
(df)
LSD (5%)
a
1.0
Status was calculated on the basis of an arbitrary scale: resistant (mean internal lesion length <1 cm), partially
resistant (1-2 cm) and susceptible (>2 cm).
b
F value from analysis of variance, *** represents significant
differences between means at the 0.001 probability level, LSD = least significant difference between two means
at the 0.05 probability level.
97
Table 4.5 Bioassay of Lhca3-SN2 transgenic lines against Pectobacterium atrosepticum (Experiment 2).
Summary of disease parameters from fourteen replicates of each transgenic line, non-transgenic control Iwa and
MgCl2 inoculated plant.
Plant line
Incidence of
Mean internal blackleg
blackleg
lesion length
(% at 11 dpi)
(cm at 11 dpi )
Statusa
Iwa
100
2.8
Susceptible
202
7
0.2
Resistant
203
0
0.0
Resistant
205
79
1.4
Parially resistant
211
14
0.3
Resistant
218
86
1.3
Partially resistant
219
14
0.6
Resistant
223
86
1.3
Partially resistant
228
93
2.2
Susceptible
MgCl2 inoculated plants
0
-
-
Non-inoculated plants
0
-
-
Fsb
-
44.28 ***(8,117)
(df)
LSD (5%)
a
0.46
Status was calculated on the basis of an arbitrary scale: resistant (mean internal lesion length <1 cm), partially
resistant (1-2 cm) and susceptible (>2 cm).
b
F value from analysis of variance, *** represents significant
differences between means at the 0.001 probability level, LSD = least significant difference between two means
at the 0.05 probability level.
98
Figure 4.7 Internal necrotic lesions observed in Lhca3-SN1 transgenic lines response to Pectobacterium
atrosepticum (Experiment 2). Variation in the internal necrotic lesion following pathogen challenge among
Lhca3-SN1 transgenic lines compared with the non-transgenic control (Iwa) and the MgCl2 inoculated negative
control. (A) MgCl2 inoculated non transgenic plant, (B) line 131, (C) line 128, (D) line 107 and (E) control Iwa
The photograph was taken 11 dpi.
Figure 4.8 Internal necrotic lesions observed in Lhca3-SN2 transgenic lines response to Pectobacterium
atrosepticum (Experiment 2). Variation in the internal necrotic lesion following pathogen challenge among
Lhca3-SN2 transgenic lines compared with the non-transgenic control (Iwa) and the MgCl2 inoculated negative
control. (A) MgCl2 inoculated non transgenic plant, (B) line 202, (C) line 211, (D) line 228 and (E) control Iwa
The photograph was taken 11 dpi.
99
4.3.2.3
Experiment 3
The third experiment was conducted in Palmerston North greenhouse, where rapid disease
progression in non-transgenic control plants was also observed with stem collapse and death
of plants at 10 dpi (Table 4.6 and 4.7).
A high frequency of plants for five Lhca3-SN1 transgenic lines (107, 108, 116, 117 and 121)
and control Iwa plant exhibited symptoms (70-86%). The frequency of plants with symptoms
was low (38-58%) for lines 128 and 131. The percentage of collapsed plants was also
analyzed as the secondary disease parameter, except lines. The percentage of collapsed plants
was only 8% for 128, with no plants seen to collapse for 131. Other than that all the other
lines including Iwa showed a range of collapsed stems. Mean lesion lengths on stems of lines
108, 116, 128 and 131 were significantly reduced compared with the Iwa control. Lines 107,
117 and 121 performed similar to the non transgenic control Iwa. No disease symptoms were
seen on MgCl2 inoculated plants and non-inoculated plants (Table 4.6).
Overall, Lhca3-SN1 transgenic lines 128 and 131 showed noticeable disease resistance at 10
days after inoculation with Pca (Figure 4.9; Table 4.6) with all disease parameters measured
being substantially lower than the inoculated Iwa control plants. Transgenic lines 108 and 116
showed reduced level for some of the disease parameters, whereas lines 107, 117 and 121
performed similar to the non transgenic control Iwa. The susceptibility status of the transgenic
plants in this experiment was analyzed on the basis of the three disease parameters assessed.
Out of seven transgenic lines inoculated with Pca, two (128 and 131) were classified as
resistant, two (108 and 116) as partially resistant, and three as susceptible (107, 117 and 121)
(Table 4.6).
Three Lhca3-SN2 lines (205, 218 and 223) and control Iwa plants showed high frequency of
plants with symptoms (79-86%). Four lines (202, 203, 211 and 219) showed substantial
reduction (29-43%) in the frequency of plants with symptoms compared with control Iwa
(Table 4.7). A high percentage of plants for the Iwa control and the low expressing line 228
collapsed 83% and 50%, respectively. Less than 30% of plants for transgenic lines 205 and,
218 collapsed. One plant each was collapsed from line 211 and 223. No plants from lines 202,
203 and 219 collapsed. Apart from lines 218 and 228, lesion lengths which developed on the
stems of the transformed lines were significantly shorter than those which developed on the
control Iwa plants. Lesion lengths on the stem of the transgenic lines 202, 203 and 219 (less
than 1.5 cm) were significantly shorter than the lesion lengths compared with all other lines
(Table 4.7).
100
The status of the transgenic plants in this experiment was analyzed on the basis of these three
disease parameters.In summary, lines 202, 203, 211 and 219 exhibited resistance to P.
atrosepticum with no (or minimal) plant collapse observed and significantly reduced lesion
length compared with the Iwa control (Table 4.7), reinforcing the results from the previous
experiments. Lines 205 and 223 all exhibited partial resistance with a low frequency of plant
collapse and significantly reduced lesion length. As expected, line 228 with no SN2 overexpression was susceptible and exhibited a similar response to the non-transgenic control.
Consistent with experiment 1, line 218 also proved to be susceptible in this bioassay. The
MgCl2 inoculated control and non-inoculated controls plants failed to develop any disease
symptoms or necrotic lesions (Table 4.7).
101
Figure 4.9 Disease appearance observed in Lhca3-SN1 transgenic lines (Experiment 3). Variation in the
disease response following pathogen challenge among the Lhca3-SN1 transgenic lines compared with nontransgenic control (Iwa). From left to right each row represents a line that has 14 replicates plants. (A) Line 131,
(B) line 128, (C) line 107 and (D) control Iwa. The photograph was taken 10 days after inoculation.
102
4.3.3 PCR screening of the pathogen using the genomic DNA of the infected
stem.
Species-specific PCR amplification was conducted to confirm the presence of Pca in three
replicate samples of stem tissue isolated from immediately below the lesion for each treatment
in experiment 3. Genomic DNA isolated from the stem tissue was used as the template. A 215
bp product was amplified from all replicates of all the Pca inoculated lines, but not from the
MgCl2 inoculated plants and non-inoculated plants (Tables 4.6 and 4.7), thereby indicating
the presence of the inoculated Pca bacterium.
4.3.4 Viable bacterial colony count
The numbers of viable Pca bacteria were estimated from counts of colony forming units
(CFU) on CVP agar plates from the stem region immediately below visible symptoms. The
results show a strong association between disease symptoms and viable Pca CFU counts
(Tables 4.6 and 4.7). Pca CFU counts for the high Lhca3-SN1 expressing lines (128 and 131)
were significantly lower, by 4 orders of magnitude, compared with the high counts from the
Iwa control and low expressing line (107). Similarly, Pca CFU counts for the high Lhca3-SN2
expressing lines (202, 203, 211 and 219) were significantly lower compared with the Iwa
control and low expressing line (228) by several orders of magnitude. No Pca colonies were
recovered from the MgCl2 inoculated plants.
4.3.5 Quantitative PCR analysis of the infected stems
To determine Pca density by qPCR, a standard curve analysis was determined using pure Pca
DNA as a control established by plotting the log of known Pca DNA concentrations against
Ct values. Calibration standards ranged from 10-2 to 10-7 dilutions of the Pca DNA (6
standards). The Ct regression calculated from the standard curve was 97.2 indicating the highefficiency of the qPCR assay in this study. Ct values resulting from assays with unknown
samples were plotted onto this curve and the inferred concentration of Pca DNA was
calculated.
For the Lhca3-SN1 transgenic lines there were large differences in the amount of amplified
DNA product between the lines (P<0.001 for overall line differences), with differences
between any pair of lines also significant (P<0.05 or smaller). From these DNA values the
CFU/g was estimated on the basis of the known CFU for 100 dilution (8.1×107 cfu/mL) (Table
4.6). The greatest CFU count was isolated from stem tissue of the low expressing line 107,
which was significantly greater than for the Iwa control and line 128. The CFU count
103
estimated from the qPCR results for line 131 was significantly lower than all of the other lines
and the Iwa control. No Pca DNA was detected in the MgCl2 inoculated stems.
For the Lhca3-SN2 transgenic lines the calibration DNA standards ranged from 10-2 to 10-6 (5
standards). The Ct regression calculated from the standard curve was 99.3, indicating the
high-efficiency of the PCR assay in this study. A standard curve was established and Pca
DNA was calculated as described. There were also large difference in amount of amplified
DNA between lines (P<0.001 for overall line differences), with differences between any pair
of lines also significant (P< 0.05). The CFU/g was estimated as described above and the
results are shown in Table 4.7. The greatest CFU count was isolated from stem tissue of Iwa
control, which was significantly greater than any of the transgenic lines. Among the
transgenic lines, there was significantly higher Pca CFU/g from low expressing line 228
compared with all other lines, followed by lines 219 and 211. The CFU estimated from the qPCR results for lines 202 and 203 were significantly lower than for all of the other lines. No
Pca DNA was detected in the MgCl2 inoculated stem.
104
Table 4.6 Bioassays of Lhca3-SN1 transgenic lines against Pectobacterium atrosepticum (Experiment 3). Summary of disease parameters and pathogen density from
fourteen replicates of each transgenic line, non-transgenic control Iwa and MgCl2 inoculated plant.
Plant lines
Control Iwa
Frequency of collapsed
Mean blackleg
P. atrosepticum count
P. atrosepticum density
blackleg
plants
lesion length
log10 CFU/g plant stem
qPCR log CFU/g plant stem
(% at 10 dpi)
(% at 10 dpi)
(cm at 10 dpi)
((back transformed mean)
(back transformed mean)
86
29
9.1
5.
5
5.15 (1.4×10 )
5
5.72 (5.2×105)
7
Statusa
Susceptible
107
79
43
9.0
5.96 (9.3×10 )
7.00 (1.0×10 )
Susceptible
108
71
14
4.7
NDd
ND
Partially resistant
116
79
43
6.5
ND
ND
Partially resistant
117
71
57
9.3
ND
ND
Susceptible
121
86
50
9.5
ND
128
36
7
1.6
ND
1
1.84 (7.7×10 )
Susceptible
4
Resistant
3
4.79 (6.2×10 )
131
57
0
1.4
0
3.07 (1.2×10 )
Resistant
MgCl2 inoculated plant
0
0
-
0
0
-
Fsc
(df)
LSD (5%)
a
Incidance of
5.4*** (7,104)
4.1
10*** (4,10)
0.22
9.0*** (4,40)
0.50
Status was calculated on the basis of an arbitrary scale: resistant ( mean lesion length <2 cm), partially resistant (2-7 cm) and susceptible (>7 cm). b F value from analysis of
variance, *** represents significant differences between means at the 0.001 probability level, LSD = least significant difference between two means at the 0.5 probability
level. d ND = Not determined.
105
6
Table 4.7 Bioassays of Lhca3-SN2 transgenic lines against Pectobacterium atrosepticum (Experiment 3). Summary of disease parameters and pathogen density from
fourteen replicates of each transgenic line, non-transgenic control Iwa and MgCl2 inoculated plant.
Plant line
Incidence of
Frequency of
Mean blackleg
P. atrosepticum count
P. atrosepticum qPCR
blackleg
collapsed plants
lesion length
log10 CFU/g plant stem
log10 cells/g plant stem
(% at 10 dpi)
(% at 10 dpi)
(cm at 10 dpi)
(back transformed mean)
(back transformed mean)
Iwa control
202
203
205
29
29
86
79
0
0
14
9.4
0.6
0.8
5.2
6.80 (6.3×10 )
0
0.56 (3.7×10 )
0
0.30 (2×10 )
ND
d
1
6.99 (9.9x106 )
Susceptible
2
Resistant
2
2.15 (1.4x10 )
Resistant
ND
Partially resistant
2.93 (8.5x10 )
3
211
43
7
1.6
1.13 (1.7×10 )
3.18 (1.5x10 )
Resistant
218
86
29
8.4
ND
ND
Susceptible
1
4
219
50
0
1.5
1.01 (1.1×10 )
4.23 (1.7x10 )
Resistant
223
86
7
6.0
ND
ND
Partially resistant
4
6
228
79
50
10.1
4.56 (3.6×10 )
6.34 (2.2x10 )
MgCl2 inoculated plants
0
0
-
0
0
15.81*** (8,117)
14*** (6,12)
13***(6,48)
2.6
0.16
0.23
Fsb (df)
LSD
a
86
6
Statusa
Susceptible
Status was calculated on the basis of an arbitrary scale: resistant ( mean lesion length <2 cm), partially resistant (2-6 cm) and susceptible (>6 cm). b F value from analysis of
variance, *** represents significant differences between means at the 0.001 probability level, LSD = least significant difference between two means at the 0.5 probability
level. d ND = Not determined.
106
6
4.4 Discussion
A major goal for plant biotechnology is genetic engineering for disease and insect resistance
in crops. The development of disease resistant potatoes by genetic engineering to insert genes
resistance to bacteria could provide a significant advantage in reducing crop losses. The
overall goal of this project was to determine whether genetically engineered potato plants
overexpressing endogenous SN1 and SN2 gene would have improved resistance to bacterial
blackleg disease caused by Pectobacterium atrosepticum.
From both transformation series 299 transgenic lines were generated via Agrobacteriummediated transformations (six experiments). For convenience 65 healthy lines were selected
for further analysis (Chapter 3). Quantitative RT-PCR in 33 transgenic lines of Lhca3-SN1
and 32 transgenic lines of Lhca3-SN2 revealed the transcriptional expression of the SN
transgene in leaf tissue. Endogenous SN1 expression was previously detected in tubers, stems,
axillary buds and young floral buds by Northern blot assays (Segura et al., 1999). Endogenous
SN2 expression was previously detected in tubers, petals, stamen and fully developed flowers
(Berrocal-Lobo et al., 2002). In the present study SN mRNA was also detected in different
tissues of the selected transgenic lines. SN transcript accumulation levels in different tissues
varied between each transgenic line. There may be several reasons for why transgene
expression is highly variable between different plant lines transformed with same transgene.
The effect of the position of the transgene within the potato genome may result in varied
levels of expression of the peptide (positional effect) (Conner and Christey, 1994). The
variations suggest that the regions flanking the inserts could alter temporal or tissue-specific
expression patterns. In fact, insertion of multiple copies of a transgene into the genome of the
plant cell could increase the possibility of gene silencing and reduced gene expression (Stam
et al., 1997; Butaye et al., 2004), while a transgene inserted as a unique copy is
transcriptionally more active than those inserted in multiple copies (Hobbs et al., 1993; Bhat
and Srinivasan, 2002; Butaye et al., 2004) which may show post-transriptional gene silencing
phenomena.
Seven phenotypically normal Lhca3-SN1 lines and eight Lhca3-SN2 lines were selected in
this chapter for more detailed studies based on leaf expression data in Chapter 3. This chapter
included qRT-PCR to determine SN transcript abundance in different tissues, as well as
bioassays against Pca.
107
Table 4.8 Summary of the status of transgenic lines across the three different Pca assays
(A)
Plant lines
Status of Lhca3-SN1 lines in pathogen assay
Overall Status
Experiment 1
Experiment 2
Experiment 3
Control Iwa
Susceptible
Susceptible
Susceptible
Susceptible
107
Susceptible
Susceptible
Susceptible
Susceptible
108
Partial resistant
Partial resistant
Partial resistant
Partial resistant
116
Partial resistant
Partial resistant
Partial resistant
Partial resistant
117
Susceptible
Susceptible
Susceptible
Susceptible
121
Partial resistant
Partial resistant
Susceptible
Partial resistant
128
Resistant
Resistant
Resistant
Resistant
131
Resistant
Resistant
Resistant
Resistant
(B)
Plant lines
Status of Lhca3-SN2 lines in pathogen assay
Overall Status
Experiment 1
Experiment 2
Experiment 3
Control Iwa
Susceptible
Susceptible
Susceptible
Susceptible
202
Resistant
Resistant
Resistant
Resistant
203
Resistant
Resistant
Resistant
Resistant
205
Partial resistant
Partial resistant
Partial resistant
Partial resistant
211
Resistant
Resistant
Resistant
Resistant
218
Susceptible
Partial resistant
Susceptible
Susceptible
219
Resistant
Resistant
Resistant
Resistant
223
Partial resistant
Partial resistant
Partial resistant
Partial resistant
228
Susceptible
Susceptible
Susceptible
Susceptible
108
The important prerequisite for controlling the pathogen in the transgenic plant is the presence
of the antimicrobial peptide at the site of interaction with the pathogen. The previous SN
overexpression studies (Almasia et al., 2008; Balaji and Smart, 2011) a constitutive CaMV
35S was utilized for transcriptional control. This will raise the public concerns regading the
food safety of transgenic product. To accomplish this, an Iwa derived light inducible promoter
was used to overexpress the transgene expression in the stem and leaves in this study.
All those selected plant lines (Table 4.8A and 4.8B) were subjected to further analysis of
transcript levels in different tissues. All the Lhca3-SN1 lines showed SN1 transcript
abundance in leaves and comparatively less SN1 mRNA in tubers as expected. In contrast to
our expectation, SN1 transcript accumulation levels in stems of Lhca3-SN1 lines were only
marginally or no different from the non-transgenic control Iwa. All the Lhca3-SN2 lines
showed SN transcript abundance in leaves and stems and comparatively less mRNA in roots,
with one line exhibited overexpression in tubers. The limited over-expression in roots and
tubers is expected given the foliage- specific nature of the light-inducible Lhca3 gene
controlling the SN1 and SN2 overexpression (Nap et al., 1993; Meiyalaghan et al., 2006).
The selected lines in this study were chosen on the basis of transcriptional expression in
leaves. Despite having significant overexpression in leaves, some lines showed a significant
reduction in SN transcripts in stems; e.g. lines 117, 121, 128 and 131. This can be explained
by cosuppression. By engineering the overexpression or underexpression of genes of interest
in wild-type or mutant plants introduced genes can suppress the expression of related
endogenous genes and/or transgenes already present in the genome, a phenomenon termed
post-transcriptional homology-dependent gene silencing (i.e., cosuppression). Cosuppression
mainly occurs when transgene transcripts are abundant, and it is generally thought to be
triggered at the level of mRNA processing, localization, and/or degradation (Taylor, 1997). It
is interesting the observation of cosuppression associated with the SN genes has tissue
specificity.
The selected plants were challenged with Pca under various experimental conditions to
analyse the blackleg tolerance of these plants. All the overexpressed lines showed consistent
resistance levels across all repeated bioassays, except 117 from Lhca3-SN1 and 218 from
Lhca3-SN2 (Table 4.8). Overall, five of the Lhca3-SN1 lines and six of the Lhca3-SN2
transgenic lines exhibited significant reduction in disease symptoms compared to both their
respective low-expression lines and the non-transformed control plant under all environmental
conditions.
109
Of the seven selected Lhca3-SN1 transgenic lines, two lines (128 and 131) showed a high
level of resistance to Pca and three lines (108, 116 and 121) showed significantly reduced
disease symptoms compared with the non-transgenic control and low expressing line (107)
(Table 4.8A). However, as discussed earlier Lhca3-SN1 lines did not show significant
accumulation of SN1 transcript levels in the stems compared to the control Iwa plant. The
PCR analysis (Chapter 3) revealed that all the Lhca3-SN1 lines contained insertion of the
transgene, and apart with, apart from the low expressing line 107 and line 117, all lines
displaying significant resistance to Pca in the three pathogen assays. The previous SN1
overexpression studies (Almasia et al., 2008) did not attempt to associate the disease
resistance with the stem expression levels. There are two mechanisms that could account for
the contradiction between low expressions in the stems but high level of resistance in the
stems when inoculated with Pca. One mechanism could be due to the migration of the
proteins from the leaves to the stems described earlier (Chapter 1) SN1 and SN2 antimicrobial
peptides are small, basic globular antimicrobial peptides (Segura et al., 1999; Berrocal-Lobo
et al., 2002) and the small size of the protein could enhance its translocation around the plant.
These structural features may enable these antimicrobial peptides to move towards the site of
infection. All Lhca3-SN1 lines, except the low expressing line (107), showed high level of
SN1 transcript in the leaves. The overexpressed protein migrating from leaves into the
adjacent stem may act to suppress the pathogen when inoculated in the stem thus making the
plant resistant. In the present study protein levels in the plant tissue was not determined; with
only mRNA levels measured using qRT-PCR. As previously stated, the cysteine residues play
an important role in maintaining the globular conformation of the SN proteins through
disulfide bridges and to provide stability to these proteins. It is possible that only small
amount of these stable proteins are sufficient to confer improved tolerance pathogen attack.
Interestingly line 117 showed high SN1 mRNA accumulation in leaves and exhibited P.
atrosepticum susceptibility. It is possible that the region flanking the inserts alter temporal or
tissue expression patterns in this line in a manner that limits disease resistance.
Of the eight selected Lhca3-SN2, transgenic lines, four lines (202, 203, 211 and 219) showed
a high level of resistance to Pca and two lines (205 and 223) showed significantly reduced
disease symptoms compared with the non-transgenic control and low expressing line (228)
(Table 4.8B). This response generally reflected the magnitude of transcriptional
overexpression. An exception was line 218 which had high transcriptional overexpression in
leaves and stems (Figure 4), but was susceptible to P. atrosepticum. Despite a high SN2
transcript level, line 218 may have compromised protein accumulation, as observed in rare
110
transgenic lines overexpressing genes encoding other antimicrobial peptides (Barrell and
Conner, 2009). Conversely, line 211 exhibits high resistance to P. atrosepticum, but did not
have significantly increased SN2 transcript levels in the stems. Since SN2 is a small protein, it
is possible that protein translocation from the high expression in leaves accounts for the
bacterial resistance following stem inoculation.
Almasia et al. (2008) and Balaji and Smart (2011) who showed that their SN overexpressing
plants were resistant to Rhizoctonia solani and Erwinia carotovora, and Clavibacter
michiganensis subsp. michiganensis, respectively in potatoes and tomato. However, unlike the
present study their findings were based on a single pathogen assay. In contrast, the results
from this study are more robust, being based on three bioassays under different experimental
conditions. To the best of my knowledge, this is the first report on the isolation of full-length
potato SN genes and development of transgenic potato lines with enhanced tolerance to Pca
using a potato derived cassette.
For the Lhca3-SN2 lines, four lines showed high level of resistance and and two lines showed
significant reduction in disease symptoms to Pca (Table 4.8B). According to the qRT-PCR
results, all of these Lhca3-SN2 transgenic lines, except the low expressing line, exhibited
significantly higher SN2 transcript levels in the stem tissue compared with the Iwa control
(Figure 4.4). Balaji and Smart (2011) analysed the overexpression of the SN2 gene in stem
tissue using semi-quantitative RT-PCR, but the level of expression between the lines was not
compared to that of control non-transgenic plants. Consequently no association of disease
resistance with the stem expression studies was not possible. However, all of their
overexpressed lines showed a distinguishable decrease in disease symptoms such as wilting
severity and wilting timing compared with control plants. Correspondingly, in this study most
of the transgenic lines with increased SN gene expression also showed an increase in plant
survival and significant reduction in disease symptoms, such as lesion extension and stem
collapse. Line 205 and 223 showed medium SN2 mRNA accumulation levels in stems, and
exhibited partial resistance to the pathogen. But line 218 exhibited high SN2 mRNA
accumulation, but exhibited susceptibility to the pathogen. It is possible that the region
flanking the inserts alter temporal or tissue expression patterns in these two lines in a manner
that limits disease resistance.
The results also suggest that SN1 and SN2 are interesting candidate genes for creating
genetically modified disease resistant plants, although the underlying molecular mechanisms
of antimicrobial activity of SN genes are not clearly understood. The hypothesis of a defense
role for SN gene was also supported by different reports on genes homologous to SN genes
111
which play protective roles in those species against invading infectious agents (Thornburg et
al., 2003; Trainotti et al., 2003; Bindschedler et al., 2006). The main example is the sequence
similarity with cysteine-rich domains of proteins from the hemotoxic snake venome disinterin
(mentioned in Chapter 2- Adler al., 1991) and some related proteins (mucins; Li et al., 1998,
MDC proteins; Sagane et al., 1998) have some defence protective roles. Balaji and Smart
(2011) postulated that SN proteins contain putative redox-active cysteines; these proteins
could be involved in redox regulation. It is well known that reactive oxygen species (ROS)
are involved in pathogenesis and wounding. Recently, it has been shown that GIP2 (another
GASA-like protein) from Petunia hybrida regulates ROS levels (Wigoda et al., 2006).
Consequently SN1 and SN2 as well as all members of this peptide family may also act as
antioxidants.
The observed antibacterial activity of these genes in vitro has been proposed to play a role in
vivo through the control of pathogen migration (Segura et al., 1999; Berrocal-Lobo et al.,
2002; Kovalskaya and Hammond, 2009). Overexpression of the SN1 gene in transgenic potato
plants has been previously reported to confer enhanced resistance to Rhizoctonia solani and
Erwinia carotovora (Almasia et al., 2008). Overexpression of the SN2 gene was shown to
confer enhanced resistance in transgenic tomato plants to Clavibacter michiganensis subsp.
michiganensis (Balaji and Smart, 2011). Results from the bioassays in the present study
identified potato lines with significantly improved resistance to blackleg compared with
controls lines. The highly conserved nature of these genes shown in the allele diversity studies
(Chapter 2) also support this and identified many homologues of SN1 and SN2 genes found
throughout the plant kingdom which may be involved in defence.
The qPCR studies revealed the high concentration of Pca in the stem tissue of the control
plant compared with that from the stems of two Lhca3-SN1 transgenic plants (128 and 131)
(Table 4.6 and Table 4.7). The highest bacterial concentration was observed in the low
expressing line (107). Similar result were found for the Lhca3-SN2 lines where the
concentration of the bacteria was found to be 103 times higher in the control plant stem tissue
compared with two Lhca3-SN2 lines (202 and 203) and significantly lower in other studied
Lhca3-SN2 lines except low expressing line (228). Re-isolation of the inoculating bacteria, in
this case Pca, and confirmation of its identity as was done in this study is crucial to
confirming its role as the casual agent and thereby validating Koch’s postulates (Agrios,
1997). Additionally, isolation of Pca from the infected tissue both by DNA methodology and
by dilution plating was also carried out to try and elucidate the mode of action these genes
against Pca infection and tissue colonisation.
112
The qPCR result measure the total amount of pathogen DNA in the sample which can
originate from live or dead cells; whereas the dilution plating method provides a viable
bacterial colony count similar to that carried out by Balaji and Smart (2011) with SN2
overexpressing tomato lines. Lower Clavibacter michiganensis subsp. michiganensis counts
were isolated from infected stem tissue in SN2 overexpressed lines than in control plants.
Similar trends of bacterial growth restriction were observed in Lhca3-SN1 and Lhca3-SN2
overexpressed transgenic lines in the present study. The live bacterial count (CFU) was found
to be 104 times higher from the control plant stems compared with that from the resistant
Lhca3-SN1 transgenic plant stems (Table 4.6) and 105 higher than that from Lhca3-SN2
transgenic plants (Table 4.7). Very low level bacteria were recovered from the transgenic
potato plants in this study, with none recovered from transgenic line 131. In summary, the
results presented show that the strategy for the design and construction of an overexpressing
SN gene was effective for producing plants with improved antimicrobial activity and disease
resistance.
The Pca viable count and Pca density determined by qPCR for the Lhca3-SN1 and Lhca3SN2 transgenic lines gave contrasting results. For the Lhca3-SN1 resistant plants although
low bacterial viable counts were recovered from the stems Pca DNA levels was high. In
contrast, both low Pca viable counts and low Pca DNA level was recovered from Lhca3-SN2
resistant plants. These differences in qPCR and dilution plating results may indicate different
modes of action for the SN1 and SN2 proteins against Pca. It is postulated that the
overexpression of the SN1 proteins prevent the migration of bacteria to new tissue and
thereby limiting the population of the bacterial pathogen in the plant, on the other hand,
overexpression of SN2 proteins may act by killing the live bacteria thereby reducing cell
viability.
The present investigation showed that the SN1 gene and SN2 gene overexpressed in potato
plants had no deleterious effects on the transgenic plants. Plant morphology, plant growth,
and tuber yields were similar to that seen with the Iwa control grown in the glasshouse. This
is similar to that observed by Almasia et al. (2008) and Balaji and Smart (2011) who reported
that potato lines overexpressing SN1 and tomato lines overexpressing SN2, respectively had
similar morphology to their respective untransformed control plants.
The blackleg bioassay used in this study was a very severe test. It is very rare that plants
grown under normal field conditions would encounter the inoculum intensity (equivalent to
106 cells per inoculation site) of Pca at one time as used in these experiments. In the field an
initial mild infection often leads to major infections. However, significant delays in infection
113
and substantial reductions in disease symptoms were found in majority of the Lhca3-SN1 and
Lhca3-SN2 transgenic lines other than those with only low transcriptional expression. The
similar type of delay in disease was observed in other overexpression studies with SN genes
(Almasia et al., 2008 and Balaji and Smart, 2011). The results showed that these transgenic
plants overcome the initial attack and prevent further disease development in the field.
In conclusion, resistance to Pca infection can be successfully engineered into potato through
the overexpression of a gene that encodes the antimicrobial protein SN1 and SN2. Pca related
bacteria (Pectobacterium carotovorum subsp. carotovorum and Dickeya spp.) can cause
disease in a wide range of hosts such as tomato, tobacco, eggplant (Czajkowski et al., 2011).
Identifying and overexpressing SN genes which confer resistance to these bacteria may also
be beneficial for other commodities in addition to potato. Furthermore, it is possible that these
lines may also confer resistance to a wide range of other pathogens which have not been
tested. Future research will show whether the use of the SN transgenes are useful for
engineering resistance to other important pathogens in a broad range of crops.
114
Chapter 5
General Discussion
The overall goal of this thesis was to determine whether genetically engineered potato plants
overexpressing SN genes would have improved resistance to blackleg disease caused by
Pectobacterium atrosepticum. The potato cultivar Iwa was used as a model system to test the
efficiency of this approach due the ease with which it can be transformed via Agrobacteriummediated transformation, and that it is highly susceptible to blackleg caused by Pca.
Identification and access to allelic variation that influences plant phenotype is important for
the utilization of genetic resources, such as in plant cultivar development. The strategy of
finding valuable, previously unknown, alleles at a known locus is referred to as ‘allele
mining’. This allows the molecular isolation of new alleles with potential agronomic
relevance. The main focus of the Chapter 2 was to analyse the allele diversity and variation of
SN1 and SN2 genes in diverse range of Solanaceous species. The SN1 and SN2 genes were
isolated and sequenced from several unrelated potato cultivar/clones, as well as a wide range
of other Solanaceous species. This revealed several allelic variants for both SN1 and SN2.
Four alleles of SN1 (a1, a2, a3 and a4) and six alleles of SN2 (b1, b2, b3, b4, b5 and b6) were
identified from this study (Table 2.2). Three alleles of SN1 (a1, a2 and a3) and two alleles of
SN2 (b1, b2) were present in a wide range of Solanaceous species. The slightly variable a4
allele was found only in S. nigrum and S. chacoense and the b3, b4 alleles were found only in
S. palustre and S. bulbocastanum respectively. Allele b5 was identified in S. lycopersicum
and S. nigrum. Allele b6 was identified in S. chacoense and S. nigrum. Virtually all the
variation in DNA sequence occurred within introns, with only rare neutral nucleotide
substitutions in exons (Figure 2.2 and Figure 2.3). The predicted amino acid sequence for all
SN1 and SN2 alleles was very highly conserved (Figure 2.4). More intriguing is the exact
identity of DNA sequence for the alleles in the different potato cultivars and divergent
Solanum species. Such very highly conserved sequence suggests that the SN1 and SN2 genes
may play an important role in plant metabolism in addition to defence against invading
microorganisms.
Diseases are a major problem of potato industry worldwide. An approach to the enhancement
of disease resistance has been the overexpression in plants of single genes whose products
have been shown to have in vitro activity against one or more plant pathogens (HammondKosack et al., 2004; Dong, 1998). Snakin peptides isolated from potato show significant in
115
vitro antimicrobial activity against diverse microbes (Segura et al., 1999; Berrocal-Lobo et al.,
2002). As the first step towards an intragenic solution to potato disease problems, endogenous
snakin genes were selected as candidates for overexpression in potato. Chapter 3 describes an
effective experimental approach to produce SN1 and SN2 overexpressing transgenic plants.
For implementing the intragenic approach, SN1 and SN2 genes were isolated from potato
cultivar Iwa and the coding regions of these genes were individually cloned into the potato
derived expression cassette harboring Lhca3 promoter and Lhca3 terminator, also from
cultivar Iwa. The resulting intragenic Lhca3’-SN-Lhca3’ chimeric genes were cloned into the
binary vector pMOA33 and used to transform potato cultivar Iwa via Agrobacteriummediated transformation (Figure 3.3). A total of 299 kanamycin-resistant lines were obtained
using the transformation protocol from separate transformation experiments with two binary
vectors (Table 3.3). Thirty-three and thirty-two independently derived transgenic lines of
potato cultivar Iwa were selected for the SN1 and SN2 genes respectively for further analysis.
PCR confirmed the presence of the nptII selectable marker gene and specific gene in all
regenerated lines (Figure 3.4 and Figure 3.5). All 65 lines transgenic lines were grown with
three replicates in the greenhouse to allow flowering and grown until natural senescence
(Figure 3.6).
Phenotypic analysis demonstrated 90% of the transgenic plants appeared normal and
exhibited no detrimental phenotype from the overexpression of the SN1 or SN2 genes. The
off-types observed in a few transgenic lines were attributed to somoclonal variation (Figure
3.7). The majority of lines overexpressed the SN genes at the RNA level as determined by
qRT-PCR from leaf tissue (Figure 3.9 and Figure 3.10). From these results eight Lhca3-SN1
and nine Lhca3-SN2 transgenic plants were selected with varying transcript levels and
phenotypically normal appearance for further analysis (Table 4.1).
The main objective of this thesis was fulfilled in Chapter 4 where the SN overexpressing lines
were assayed for disease resistance. In preparation for this, the expression of SN genes in
different plant tissues were ascertain by qRT-PCR (Figure 4.3 and Figure 4.4). The result
revealed the limited expression of transgene in tubers as expected for the foliage-specific
Lhca3 promoter (Nap et al., 1993). The final component of this study was to challenge the
selected transgenic lines with Pectobacterium atrosepticum, the causative agent of blackleg
disease in potato. Bioassays were conducted in three independent experiments times with 3-14
replicates per treatment and included appropriate controls. The control and low expressing
lines were highly susceptible to Pca in all the pathogen trials. All other Lhca3-SN1 and
Lhca3-SN2 transgenic potato plants exhibited varying levels of resistance to Pca. Two Lhca3116
SN1 lines (128 and 131) showed a significantly high level of resistance, and other three lines
(108, 116 and 121) were partially resistant compared with control plants (Figure 4.8A). In the
pathogen assays on Lhca3-SN2 lines, four lines (202, 203, 211 and 219) showed a high level
of resistance and other two lines (205 and 223) overexpressed lines exhibited minimal
resistance to the disease (Figure 4.8B). In all three pathogen challenge experiments, low
expressing lines were disease susceptible like parental line.
These final results clearly demonstrated that the overall goal of this thesis was achieved.
Genetically engineered potato plants overexpressing snakin genes had improved resistance to
bacterial blackleg disease caused by Pca. This is the first report on the isolation of full-length
potato SN1 or SN2 genes and development of transgenic potato lines with enhanced tolerance
to Pca using a complete intragenic cassette.
As part of this research, knockdown of SN expression was also attempted using antisense
constructs (Appendix E). All putative transformations failed to regenerate and died (Figure
E.3A). This suggests that expression of the SN gene family is essential for plant development.
Coupled with the highly conserved nature of the SN genes (Chapter 2) this result supports the
hypothesis of these proteins involvement in diverse biological process (including cell
division, cell elongation, cell growth, transition to flowering and signaling pathways), other
than just defense against invading microorganisms.
The hypothesis of a defense role for the Snakin genes is well supported by previous studies in
SN genes and its orthologous genes (Thornburg et al., 2003; Bindschedler et al., 2006;
Almasia et al., 2008; Kovalskaya and Hammond, 2009; Balaji and Smart, 2011). No
information is yet available regarding the mechanism of action of SN1 and SN2 peptides. The
recent studies (Segura et al., 1999; Berrocal-Lobo et al., 2002; Kovalskaya and Hammond,
2009) anticipated that the observed aggregation of both Gram-negative and Gram-positive
bacteria in vitro might play a role in vivo through the control of pathogen migration. Snakin
peptides show sequence similarity with cysteine-rich domains of proteins from animals which
are playing an important role in pathogen control in-vivo (Berrocal-Lobo et al., 2002).
Recently, a chitin binding proline-rich protein from French bean that has been previously
characterised through its involvement in plant-pathogen interaction was shown to be
composed of two components: a proline-rich polypeptide and a twelve cysteine peptide with
amino acid similarity to Snakin proteins (Bindschedler et al., 2006). Overexpression of the
SN1 gene was shown to confer enhanced resistance in transgenic potato plants to Rhizoctonia
solani and Pectobacterium caratovora (Almasia et al., 2008). Overexpression of the tomato
SN2 gene was shown to confer enhanced resistance in transgenic tomato plants to Clavibacter
117
michiganensis subsp. michiganensis (Balaji and Smart, 2011). From this study, Lhca3-SN1
and Lhca3-SN2 overexpressing transgenic lines exhibited good resistance when challenged
with Pca.
5.1 Contributions of thesis findings to research
The allele diversity study in the Chapter 2 established the highly conserved nature of Snakin
genes within and between Solanaceous species.
The lethality of antisense knockdown expression of SN1 and SN2 establishes a critical role of
this gene family for plant development (Appendix E).
The observed resistance to pathogens confirms the results of previous research by
overexpression SN1 in potato (Almasia et al., 2008) and extends these observations to SN2.
The use of a Iwa derived light inducible tissue specific Lhca3 promoter for transcriptional
control of Snakin-1 and Snakin-2 genes provided the successful use of an Iwa derived
intragenic expression cassette.
Two new sources - Lhca3-SN1 and Lhca3-SN2 - of Pca-resistant potato were produced using
an intragenic cassette.
5.2 Directions of further research
There are many ways to further analyse the genetic attributes of the existing transgenic lines.
Southern blot analysis will confirms the insertion of the gene of interest and the copy number.
Western blotting and ELISA (Enzyme-linked-immunosorbent assay) may useful for further
characterizing expression of these transgene at the protein level. Raising antibodies against
SN1 and SN2 peptides may be useful for evaluating the peptide levels in Lhca3-SN1 and
Lhca3-SN2 transgenic lines.
Chapter 4 results established that the SN overexpressing transgenic potato lines had
significant resistance against Pca, and with the broad specificity of Snakin peptides against a
wide range of microbes, these plant lines could be challenged with other common
phytopathogens of potato. Some of the transgenic plants exhibit overexpression in tubers as
well as roots these can be used for challenging against root and tuber disease of potato.
Appropriate targets could include Verticillium dahlia (fungal wilt), Phytophthora infestans
(late blight), Xanthomoas campestris, Pseudomonas solanacearum (bacterial wilt),
Streptomyces scabies (common scab), Pectobacterium carotovorum (soft rot), Spongospora
subterranean (powdery scab) and nematodes.
118
Prior to starting a breeding program with any gene, evaluation of the allele diversity for that
specific gene in different species will be valuable to identify new alleles with potential
agronomical relevance. Exploiting wild relatives as a source of novel alleles is challenging
but well worthwhile. So far it has provided notable successes in crop improvement such as the
case of the RB gene from S. bulbocastanum conferring broad spectrum resistance to potato
late blight (Song et al., 2003). An objective for characterization of potato germplasm involves
the discovery of potentially superior alleles and their utilization in potato improvement. An
allele diversity study is not only important in the identification of disease resistance genes; it
is also useful for identifying the alleles with resistance to other biotic stresses and tolerance to
abiotic stresses, as well as for improving nutrition, quality and yield.
Sequence analysis in various Solanaceous species and potato genotypes revealed four alleles
of SN1 and five alleles of SN2 throughout all those species. The out-group (b3, b4 and b5)
needs further evaluation to identify any functional differences of these specific alleles. A
further question relates to the significance of these alleles and whether their low frequency is
a reflection of inferior quality or superior quality. Moreover, the specific effect of these alleles
on disease resistance is also still obscure and needs further clarification. The functional
significance of these alleles can be evaluated by transforming potato independently with
specific alleles, or with transcriptional fusion of promoters and coding regions through the
introduction of full length DNA clones. This strategy might be used to generate transgenic
plants containing each allele in a common genetic background. The end products will reveal
the importance of each allele.
We have already established a PCR based strategy for the isolation, cloning, sequencing and
annotation of SN1 and SN2 genes. Since cultivated potato is an autotetraploid with high
heterozygosity, each genotype may contain up to four different alleles at each locus. To
determine allele diversity within and between cultivars it is therefore important to isolate and
sequence sufficient clones to ensure recovery of all alleles present in an individual genotype.
This approach will help further allelic variation analysis in wider range of Solanum species,
including crops such as eggplant, pepino as well as ornamental species and weeds. These
studies may allow the finding of new alleles in the future with predicted changes in amino
acid sequence that might confer changes in functionality.
A novel component of the research in this thesis is the use of a complete potato-derived
expression cassette for overexpressing the SN genes. The resultant intragenic cassettes with
chimeric intragenes were cloned into transgenic binary vector pMOA33 and this construct
was used to create a disease-resistant germplasm. The cloning these chimeric potato
119
intragenes into a suitable potato-derived vector can fulfill the concept of intragenic plant
transformation (Conner et al., 2007; Barrell et al., 2010). The use of such intragenic vectors
for the transfer of genes from within the gene pools of crops may help to alleviate some of the
public concerns over the deployment of GM crops in agriculture, especially ethical issues
associated with the transfer of DNA across wide taxonomic boundaries.
Here in this work we used potato cultivar Iwa as a model system for this experiment because
of its high transformation efficiency and its high susceptibility to disease. It is no longer used
as a commercially important cultivar. This approach used in this thesis provides a successful
method for transforming elite commercial potato cultivars to generate transformed lines with
disease resistance without the transfer of foreign DNA. This work also demonstrated that SN
like small peptides is effective in conferring disease resistance. Genes for other small
antimicrobial peptides may be of value for introducing disease resistance to crop species, and
transformed into crop plants using system described in this thesis. The Chapter 2 described
the presence of SN genes in tobacco, tomato, capsicum. The same intragenic approach is
applicable for these species too.
Multiple gene cloning or gene pyramiding is a better approach for increasing the disease
resistance in plants. Our studies revealed that single SN genes are highly effective to control
bacterial pathogen Pca when overexpressed in potato plants (Chapter 3 and Chapter 4).
Considerable strategies have been proposed for introducing and expressing multiple
transgenes in crops (Huang et al., 1997b; Singh et al., 2001; Narayanan et al., 2002;
Hittalmani et al., 2000; Kumaravadivel et al., 2006; Cox et al., 1993; Liu et al., 2000; Jackson
et al., 2003; Gahan et al., 2005; Schneider et al., 2002 and Werner et al., 2005). In contrast
with all these studies, it is difficult to pyramid transgenes in potato by sexual crossing of
different transgenic parental lines because potato is vegetatively propagated crop. Pyramiding
of genes in clonal crops that cannot be usually crossed sexually, presents new challenge for
deployment of gene strategies in agriculture. Therefore, experimental design of strategies for
pyramiding
in
potato
using
Agrobacterium-mediated
transformation
is
important
(Meiyalaghan et al., 2010). Therefore, developing potato plants pyramided with both these
genes using simultaneous transformation or re-transformation would improve the efficiency
of plant breeding leading to the development of genetic stocks and precise development of
broad-spectrum resistance capabilities. The success of gene pyramiding depends upon several
critical factors, including the number of genes to be transferred, the distance between the
target genes and flanking markers, the number of genotype selected in each breeding
generation, the nature of germplasm etc (Joshi and Nayak, 2010). The innovative tools such as
120
DNA chips, microarrays, SNPs like advance experimental techniques and the information’s
from the potato genome sequence published recently (The Potato Genome Sequencing
Consortium, 2011) can be used to develop SN-pyramided broad-spectrum durable disease
resistant potato plants.
The transgenic lines developed and described in this thesis were not evaluated for Pca
resistance under field conditions. The field testing of these transgenic lines is essential to
confirm that the overexpression of snakin genes retain performance under field conditions,
especially when the SN genes are under the control of light regulated Lhca3 promoter. This is
important since transgene expression can vary with environmental conditions (Broer, 1996).
Furthermore, off types due to somaclonal variation can sometimes only be exhibit under field
conditions (Conner et al., 1994). Sufficient quantities of tubers produced from numerous sites
and field trials are necessary for the complete evaluation of processing characteristics,
nutritional content, and assessment of glycoalkaloid level. It is also important that this elite
genetic material is incorporated into long term traditional breeding programmes as soon as
practically possible.
The black rot bioassay as described in this chapter was a very severe test. It is very rare that
plants grown under normal field conditions would encounter the billions of Pectobacterium at
one time as in the assay used in this experiment. In the field mild infection leads to the major
infections. However, significant delays of infection and substantial reductions of disease
symptoms were found on all Lhca3-SN1 and Lhca3-SN2 transgenic lines other than low
expressing line and control plant. Our results showed that these transgenic plants will
efficiently overcome the initial attack from excessive inoculation conditions and prevent the
further disease development. Therefore under the lower inoculation loads of natural infection
in the field, these lines may exhibit even higher tolerances to Pca infection.
For the past three decades, many transgenic plants with improved resistance to microbial
diseases have been reported (Johal and Briggs., 1992; Martin et al., 1993; Song et al., 1995;
Bent et al., 1994; Grant et al., 1995; Salmeron et al., 1996; Whitham et al., 1996). These
researches may be useful to control some of the economically important plant pathogens. The
experimental approaches developed in this study should be useful for producing diseaseresistant plants for applications in agriculture and thereby reduce the chemical load of
pesticide used in the environment.
121
Acknowledgments
Foremost, I would like to express my sincere gratitude to my principal supervisor Prof. Tony
Conner for the continuous support of my PhD study and research, for his patience,
motivation, enthusiasm, and immense knowledge. His guidance helped me in all the time of
research and writing of this thesis. I could not have imagined having a better advisor and
mentor for my PhD study. I consider myself lucky to have worked with him.
I was delighted to having Dr Eirian Jones as my associate supervisor. I very much appreciated
her enthusiasm, intensity and vast knowledge in pathology. She offered her advice and
suggestions whenever I need them.
Besides both, I would like to thank the rest of my thesis committee: Dr Jeanne Jacobs, and Dr
Sathiyamoorthy Meiyalaghan, Dr Hayley Ridgway for their encouragement, insightful
comments, and hard questions. In particular, I would like to extend my gratitude to Dr.
Sathiyamoorthy Meiyalaghan for his friendship, guidance and help in the past four years, and
for teaching me basics of molecular biology, cloning techniques, potato transformation and
offered advice at every stages of this research. I thank Dr Jeanne Jacobs for her assistance in
molecular aspects of this research and for thought provoking input into the writing phase of
this thesis.
I would like to thank the many people who have taught me science: my high school science
teachers (T.T.Leelamma and A.J.Elsamma (my mom)), and my graduate teachers (especially
Dr T.T.Mathew, Dr Dennis Thomas and Dr Sr. Jancy). In particular, I am grateful to Dr
T.T.Mathew for enlightening me the first glance of research.
Thanks also the team from the upstairs lab including Dr Philippa Barrell, Michelle Gatehouse,
Julie Latimer who inspired me in research and life through our interactions during the long
hours in the lab. Particularly Julie Latimer, who sets an example of a outstanding researcher
for her rigor and passion on research, her friendship and technical guidance are valued. A big
thanks to Julie Latimer for help with the qRT-PCR.
I would also like to acknowledge the Dr David Lewis, Ian King and Julie Ryan from
Palmerston North, Stuart Larsen from Lincoln Biotron Facility, Dr Andy Pitman and his team
(Bhanupratap Vanga, Sandi Keenan) for their help and assistance with Pectobacterium
122
bioassays. My grateful appreciation to Dr Ruth Butler for valuable advice and help with
statistical analysis.
I am grateful to Dianne Hall for her help with maintaining plants in the containment
greenhouse. Thanks to Robert Lamberts for help with photography.
My heartfelt thanks are due to Dr. Jessica Dohmen Vereijssen, Dr. Karen Armstrong from the
Entomology Group, Lincoln University and Heather Watson from Lincoln Hospitality Ltd
who provide me with part time jobs.
I thank my roommate, Dr Ning Zhang for the stimulating discussions, for the sleepless nights
we were working together before deadlines, and for all the fun we have had in the last one
year. Furthermore, I am grateful to come across several life-long friends at work Rashidi
Othman, Roopashree Revanna, Saira Wilson, Sagar Datir, Jayaseelan Balabhaskaran, Anju
George, Azmi Muda, and Anu Jose.
My deepest gratitude goes to my family for their unflagging love and support throughout my
life; this thesis is simply impossible without them. I wish to thank my parents, Mohan
Abraham and late Elsamma Mohan. They bore me, raised me, supported me, taught me, and
loved me. I would like to thank my husband’s family for all their love, encouragement
especially K.U.Kurian and Annakutty Kurian were particularly supportive. I wish to extend
my deepest gratitude to my brother, brother-in-laws, and sister-in-laws, and all my friends for
their support and encouragement.
And most of all for my loving, supportive, encouraging, and patient husband Sajan Joseph
Kurian whose faithful support during all stages of this PhD is so appreciated and his sweat
paid my tuition fees. He has been, always, my pillar, my joy and my guiding light, and I thank
him. This thesis is dedicated to Stephen and Anna, my lovely kids. They have lost a lot
because of mama’s PhD study.
Last but not least, thanks be to God for my life through all tests in the past four years. You
have made my life more bountiful. May your name be exalted, honoured, and glorified.
123
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Appendix A
Media and Solutions
Bacterial Culture Media:
SOC medium – Invitrogen Cat # 15544-034
Formulation per one litre:
2% tryptone
0.5% yeast extract
10 mM sodium chloride
2.5 mM potassium chloride
10 mM magnesium chloride
10 mM magnesium sulphate
20 mM glucose
Luria-Bertani (LB) Broth
Bacto-tryptone
10 g/L
Bacto-yeast extracts
NaCl
5 g/L
10 g/L
Adjust pH to 7.0 with NaOH and autoclave for 15 min at 121oC then autoclave to sterilise.
Luria-Bertani (LB) Agar
Bacto-tryptone
Bacto-yeast extracts
NaCl
10 g/L
5 g/L
10 g/L
Adjust pH to 7.0 with NaOH and add 15g/L bacto- agar then autoclave to sterilise.
CVP Medium based on Hyman et al, 2001
Five hundred millilitres of boiling distilled water was placed into blander. The blender was
switched onto a slow speed and the following added;
1.0 mL of a 0.075% (w/v) aqueous crystal violet solution
4.5 mL of 1 N NaOH (8 g NaOH/200 mL distilled water)
6.8 mL of 10% CaCl2.2H20 (fresh solution)
2.0 g Agar
1 g NaNO3
152
2.5 g of trisodium citrate dihydrate
0.5 g tryptone
The blender was then set at high speed for 15 s with the lid on. Bulmer’s sodium polypectate
(9 g) was slowly added while blending. After blending at high speed for 15 s the solution was
divided into two lots of 250 mL and placed into 800-1000 mL bottles and autoclaved for 25
min at 121oC. Plates need to be dried thoroughly before use.
Genomic DNA extraction reagents:
Nuclei-extraction buffer
63.77 g sorbitol, 100 mM Tris pH 7.5, 5 mM, Na2-EDTA, water to 1 L and autoclaved, just
prior to use add 3.8 g/L NaSO4.
Lysis buffer
0.2M Tris pH 7.5, 50b mM Na2-EDTA salt, 116.88 g NaCl, 20 g CTAB, sterile water up to 1
litre. Autoclave.
TE-4
10 mM Tris, 0.1 mM EDTA water to 100 mL
CTAB extraction buffer
20 mM EDTA pH 8.0, 20 mM Tris pH 8.0, 40.9g NaCl, dH20 to 500mL.Autoclave, allow to
cool then add 10 g CTAB (Cetyl-Trimethyl-Ammonium Bromide) and incubate at 60 oC until
dissolved.
Plant tissue culture media:
MS Macro 20X (Murashige and Skoog 1962)
Ammonium nitrate
33.0 g/L
Potassium nitrate
38.0 g/L
Potassium dihydrogen phosphate
8.8 g/L
Calcium chloride dihydrate
3.4 g/L
Magnesium sulphate heptahydrate
7.4 g/L
MS Micro 200X
Boric Acid
1.24 g/L
153
Manganese Sulfate Tetrahydrate
4.46 g/L
Zinc Sulfate Heptahydrate
1.72 g/L
Potassium Iodide
0.166 g/L
Sodium Molybdate Dihydrate
0.05 g/L
Copper Sulfate Pentahydrate
0.005 g/L
Cobalt Chloride Hexahydrate
0.005 g/L
MS Iron 200X
EDTA (FeNa Salt)
7.34 g/L
MS Vitamins 200X
Myo-inositol
20.0 g/L
Nicotinic Acid
0.10 g/L
Pyridoxine HCl
0.10 g/L
Thiamine HCl
0.02 g/L
Glycine
0.40 g/L
Potato Multiplication media (PM)
Stock Solution:
Stock Conc.
MS Macro
20x
50 mL/L
MS Micro
200x
5 mL/L
MS Iron
200x
5 mL/L
MS Vitamins
200x
5 mL/L
Ascorbic Acid
40 mg/mL
1 mL/L
Casein Hydrolysate
0.5 g/L
0.5 g/L
Sucrose (3%)
30 g/L
30 g/L
Adjust pH to 5.8
Agar (Coast biologicals
Cat # AST001)
10 g/L
Autoclave for 15 minutes
Potato Callusing media (PCM)
Stock Solution:
Stock Conc.
MS Macro
20x
50 mL/L
MS Micro
200x
5 mL/L
154
MS Iron
200x
5 mL/L
MS Vitamins
200x
5 mL/L
Ascorbic Acid
40 mg/mL
1 mL/L
Casein Hydrolysate
0.5 g/L
0.5 g/L
Benzylaminopurine (BAP)
2.0 mg/mL
1 mL
Sucrose (3%)
30 g/L
30 g/L
0.2 mg/mL
1 mL
Napthaleneactic
Acid
(NAA)
Adjust pH to 5.8
Agar (Coast biologicals
Cat # AST001)
10 g/L
Autoclave for 15 minutes
Potato Callusing media with Timentin (PCT)
Prepare PCM media
Autoclave for 15 minutes
Cool to 60°C and add 1 mL Timentin (200 mg/mL)
Potato Regeneration Media (PRM)
Stock Solution:
Stock
Conc.
MS Macro
20x
50 mL/L
MS Micro
200x
5 mL/L
MS Iron
200x
5 mL/L
MS Vitamins
200x
5 mL/L
Ascorbic Acid
40 mg/mL 1 mL/L
Casein Hydrolysate
0.5 g/L
0.5 g/L
Sucrose (0.5%)
5 g/L
5 g/L
Adjust pH to 5.8
Agar (Coast biologicals
Cat # AST001)
10 g/L
Autoclave for 15 minutes
Cool to 60OC and add 1 mL Timentin (200 mg/mL)
1 mL Kanamycin (100 mg/mL), 1ml Zeatin (1 mg/mL) and 1 ml Gibberelic Acid (GA3) (5
mg/mL)
155
Potato Callusing media with Timentin and Kanamycin (PTK)
Prepare PCM media
Autoclave for 15 minutes
Cool to 60°C and add 1 mL Timentin (200 mg/mL) and 1ml Kanamycin (100 mg/mL)
50X TAE Buffer
2M Tris base in 250 mL ddH2O, 1 M actic acid, 0.5 M EDTA (pH 8.0) and make up to
1000mL.
1X TAE Buffer
10mL 50X TAE buffer 490 mL ddH2O
Loading buffer
2 g Ficoll 400, 0.02 g Bromophenol blue, 0.02 g Xylene cyanol, 1 mL 10% SDS (w/v), add 7
mL distilled water and 2mL 0.5 M EDTA.
156
Appendix B
Protocols
Plant DNA isolation
-based on Bernatzky and Tanksley (1986) method
Ten discs from healthy leaves were collected in a 1.5 mL microcentrifuge from the glass
house grown plants. The tubes were immediately snap frozen in liquid nitrogen and stored at 80°C until use. DNA was extracted from stored leaf material by grinding to a fine powder,
using a pestle, under liquid nitrogen. One ml nuclei-extraction buffer was added to ground
tissue. The tubes were inverted to mix the tissues and then mixture was centrifuged at 4°C for
20 min at 7,828 x g and the supernatant was discarded. The remaining pellet was resuspended
in 200 µL of Lysis buffer and 80 µL (0.4 volume) 5 % (w/v) Sodium-N-Lauroylsarcosine
(sarcosyl). Samples were mixed by inversion 10 times then incubated at 65°C for 5 min,
mixed again by invertion 10 times and incubated then incubated for a further 10 min.
Chloroform/ isoamyl alcohol (24:1 iaa) solution (500 µL or 1 volume) was added and the
sample inverted 40 times. The samples were centrifuged at 4°C for 5 min at 14,857 x g. and
the top aqueous layer of each transferred to a sterile microcentrifuge tube. The chloroform/
isoamyl alcohol step was then repeated. The DNA was precipitated using 250 µL (2/3
volume) ice cold (-20°C) isopropanol. The tubes were inverted 15 times and the precipitated
DNA hooked out and transferred to a new microcentrifuge tube, using a pipette tip. The tube
containing the isopropanol and remaining sample DNA was stored at -20°C overnight. This
was then centrifuged at 14,857 x g for 20 min and the supernatant removed. The precipitated
DNA (either cloud or pelleted sample) was then washed using 70% ethanol. The samples
were centrifuged briefly and the ethanol drained off. The DNA was air dried at room
temperature and resuspended in 250 µL of TE-4 (10mM Tris, 0.1 mM EDTA water to 100
mL).
-using CTAB method based on Doyle and Doyle, 1990)
Approximately 0.2 g of young leaf tissue was harvested into 1.5 mL eppendorf tubes held in
racks suspended above liquid nitrogen. The frozen tissue was then crushed with glass beads
before addition of 500 µL, and samples mixed by shaking to combine the extraction buffer
and the ground leaf tissue. Samples were then placed in a 65°C water bath for 60 min. Tubes
were removed from the water bath and allowed to cool to room temperature of 500 µL of
chloroform: octan-2-ol (24:1) was added to each sample and samples were mixed by inversion
157
20 times. After mixing, tubes were centrifuged at 10,000 x g for 5 min and the supernatant
was removed with a pipette and placed into a new Eppendorf tube, where it was mixed with 1
volume of cold isopropanol and 1/10th volume 3M Sodium Acetate (pH5.2). The tubes were
inverted gently to precipitate the DNA, followed by another centrifugation at 10,000 x g for
10 min to pellet the DNA. The supernatant was discarded, and the pellet was washed with 800
µL of cold 70% (v/v) ethanol, precipitated by centrifugation and dried. The pellet was resuspended in 50 µL of TE buffer in a 50°C water bath.
Freeze-thaw method for transformation of Agrobacterium tumefaciens
Thaw 500 µL of chemically competent A.tumefaciens cells and 0.5-1.0 µg of plasmid DNA
on ice. Add the DNA to the competent cells and incubate on ice for 5 min. Immerse the cells
in liquid nitrogen for 5 min then thaw at 37°C for 5min. Add 1mL of liquid LB medium and
incubate at 28°C for 3 h shaking at 300 rpm (Thermomixer, Eppendorf) Spread aliquots of
200 µL onto LB medium containing the appropriate antibiotics in Petri dishes and incubate at
28°C for 2 days.
158
Appendix C
Sequences from GenBank
Reference Sequence SN1
DEFINITION Solanum tuberosum SN1 gene for snakin1.
ACCESSION
AJ320185
1 tcatcaaatt ttcagcttcg aaaaaatgaa gttatttcta ttaactctgc ttttggtcac
61 tcttgttatt accccttctc tcattcaaac taccatggct ggttcaagta agtaccaatt
121 cttccatcat ctttaaaatt tgctactagt cttctggaaa catccaccag gtagtagtaa
181 ggttttcgta tactttgctt ttttcatacc ttatcaagtg aaatttgaat atgtacttgt
241 cctcgggcat agctatagcc attccagggt atccaattat atgtcaaatt tacatttaaa
301 gaatatatat ttaaaattga atacacttga caaaagttat gtctttagct agtggtaatg
361 agttttcatt tgaacttttg ggtttgaacc ccaaatagta caatattttt ttgttttatt
421 ttgacagcca cacatcatta tttttagaca cctttaataa aatttctagc ttaaccactg
481 attgttctaa tattttcact actatccact aacacaacac atatataaga taattttgtc
541 cactaatttt tttttttaaa gttatatgtt ctaattatga gttttttttt taatttttta
601 aagatttttg tgattcaaag tgcaagctga gatgttcaaa ggcaggactt gcagacagat
661 gcttaaagta ctgtggaatt tgttgtgaag aatgcaaatg tgtgccttct ggaacttatg
721 gtaacaaaca tgaatgtcct tgttataggg acaagaagaa ctctaagggc aagtctaaat
781 gcccttgaat ttgatgtatt ccaaataatc taagtggtta tatttgcgtc ttcacatatt
841 aagctttcta ggtatgttaa agttatacat attgaaatag aaaaaaaaa
Reference Sequence SN2
DEFINITION Solanum tuberosum SN2 gene for snakin2.
ACCESSION
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
AJ312424
aagcttatca
tgcttcatta
agtggtgagt
tatatttctt
accgtttcat
ttaagaattt
ttgacttttg
tatgttgtaa
tatgtcctgt
catccttagt
cagcaaggtg
gtgctagatg
atgccagttt
tcccctacca
tcaatattca
taattttgag
ttgcattgta
aaa
atttatagaa
cttctctcct
tattttattt
cggttctaaa
catgaattta
aatcataccc
cagaccagca
tttctataca
atcaattata
gaaatttctt
ccgattatca
caactgtgtt
gactactcat
gtatattgtg
aaatccagta
gaaattgtat
ttgtattgta
aaatatttca
tgctccttct
gttccttagc
atagaagaac
tgtggaaaaa
cttaatttaa
atgctatttc
acattttgtc
aacaatacac
tgtgttgttt
tcaaggccaa
cctcctggta
ggcaacaaac
cgattactat
aaatgtcttt
tcactttgat
taataagatg
aattccaatg
cgagcaagtc
aatttactta
tcatgataaa
aaaaatctag
agtttaattt
tgaagccgct
acctttttca
tttttatata
atatatgtac
gattgtgtaa
cttctggcaa
gtaaatgccc
gaatatgtta
agcgtgtact
ttgtgtgttt
aggaataaat
gccatttcga
caatctattc
aaaattatat
tagtctaaga
ttgactgtct
attcacccgt
tattcctaca
attataaata
aatatatatt
agactgtggg
tagggcatgt
cactgagact
ttaacttttc
ttgttctgtc
tttttggtgg
ggtgtgatat
gaagttcgag
aagctctctt
agaccgatca
ctatgctacg
ggtttgaatt
aaatatatag
gatatatgtt
agaaaattgg
tttattcccc
aaacctggaa
ggagcttgtg
ggaacttgtt
tgcccttgct
ttctaaaata
atatattgtg
gttgtctttt
ttgttctggt
taaaaaaaaa
159
Sequences were deposited with GenBank from this work
The nucleotide sequence SN1 and SN2 alleles from this study were deposited in the GenBank:
the accession numbers are in Table C.1 and Table C.2 respectively. The Highlighted data of
Iwa and Desiree accessible to public and other datas are to be confidential until October 1,
2012.
Table C.1 SN1 allele nucleotide GenBank accession numbers
SN1
Solanaceous species/genotypes
a1
a2
S. tuberosum ‘Iwa’ (4n)
FJ195646
FJ195647
S. tuberosum ‘Desiree’ (4n)
FJ195648
FJ195649
X
NS
S. tuberosum ‘RH 89-039-16’ (2n)
X
NS
S. palustre (2n)
X
S. bulbocastanum (2n)
X
a4
X
X
X
FJ542040
a
S. tuberosum ‘SH83-92-488’ (2n)
a3
X
X
NS
X
JN809786
X
X
JN809785
JN809787
X
JN809784
X
X
X
X
X
X
JN797794
S. nigrum (6n)
JN809781
X
X
JN809788
Petunia hybrida (2n)
JN809782
X
X
X
Capsicum annuum (2n)
JN809780
X
X
X
Nicotiana tabacum (4n)
JN809783
X
X
X
S. lycopersicum (2n)
S. chacoense (2n)
a
= Not Submitted.
Table C.2 SN2 allele nucleotide GenBank accession numbers
SN2
Solanaceous species/genotypes
b1
S. tuberosum ‘Iwa’ (4n)
EU848497
S. tuberosum . ‘Desiree’ (4n)
EU848499
b2
EU848498
b3
b4
b5
b6
X
X
X
X
X
X
X
X
X
S. tuberosum . ‘SH (2n)
NSa
NS
X
X
X
X
S. tuberosum ‘RH (2n)
NS
NS
X
X
X
X
S. palustre (2n)
X
X
JN802275
X
X
X
S. bulbocastanum (2n)
X
X
X
JN802276
X
X
S. lycopersicum (2n)
X
X
X
X
JN802277
X
S. chacoense (2n)
JN809793
X
X
X
X
JN802278
S. nigrum (6n)
JN809790
X
X
X
JN809794
JN809795
Petunia hybrida (2n)
JN809791
X
X
X
X
X
Capsicum annuum (2n)
JN809789
X
X
X
X
X
Nicotiana tabacum (4n)
JN809792
X
X
X
X
X
a
= Not Submitted.
160
Appendix D
Sequencing the coding regions of StSN genes
(Provided by Dr S. Meiyalaghan, Plant & Food Research)
To determine the intron-exon boundaries in the StSN genes, cDNA of these genes was
sequenced and aligned with the genomic sequence for comparison.
Total RNA was isolated from the youngest, fully expanded leaves of 2 month old greenhousegrown Iwa potato plants using the Illustra RNAspin Mini Isolation Kit (GE healthcare,
Buckinghamshire, UK), including DNase treatment according to the manufacturer’s
instructions. The integrity of the total RNA was checked by electrophoresis in 1% agarose gel
in Tris-acetate-EDTA (TAE) buffer and quantity was determined with a NanoVueTM
Spectrophotometer (GE healthcare).
First-strand cDNA was synthesised using the SuperScript® VILO™ cDNA Synthesis Kit
(Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. VILO™ Reaction
Mix contains random primers, MgCl2 and dNTPs in a buffer formulation. Single-stranded
cDNA was then used as a template in the PCR reactions using the Expand High FidelityPLUS
PCR system (Roche Applied Science, Mannheim, Germany). Approximately 50 ng of cDNA,
corresponding to the amount of total RNA isolated from Iwa plants, was used as a template.
The PCRs were carried out in a C1000TM Thermal Cycler (Bio-Rad). The 50 µL PCR mix
contained 1x Expand High FidelityPLUS Reaction Buffer containing 1.5mM MgCl2, 0.2 mM of
each dNTP, 0.4 µM of each primer and, 2.5 U of Expand High Fidelity Taq polymerase
(Roche Applied Science). For the StSN1 coding region, the primers were 5′ATGAAGTTATTTCTATTAACTCTGCTTT-3′ and 5′- TCAAGGGCATTTAGACTTGC-3′.
For the StSN2 coding region, the nucleotide sequences of the primers were 5′ATGGCCATTTCGAAAGC-3′ and 5′- TTAAGGGCATTTACGTTTGTT-3′. The PCR
conditions for the StSN1 coding region were: 1 cycle at 94C for 2min, 34 cycles of (30 s
94C, 30 s 59C, 30 s 72C), followed by 7 min extension at 72C. The PCR for StSN2
coding region was performed using the same PCR conditions with 57C annealing
temperature. PCR products were separated by electrophoresis in a 2% agarose gel in TAE
buffer and visualized under UV light after staining with ethidium bromide. The primers for
StSN1 and StSN2 coding regions generated expected PCR products of 267 bp and 315 bp
respectively.
161
Figure D.1 An alignment of StSN1 coding sequences (GU137307) with StSN1 reference gene sequences
(FJ195646).
162
Figure D.2 An alignment of StSN2 coding sequences (JF683606) with StSN2 reference gene sequences
(EU848497).
PCR products were purified using the Illustra GFX™ PCR DNA and Gel Band Purification
Kit (GE Healthcare) according to the manufacturer’s recommendations and sequenced
directly using Applied Biosystems BigDye® Terminator v3.1 kit. Sequencing reactions were
analysed using an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, USA).
Each fragment was sequenced from both directions individually using 3.2 pmole of primer.
The primers described above were used individually as sequencing primer in each sequencing
reaction. Sequences of StSN1 and StSN2 coding regions were analysed and aligned with their
163
reference sequences by MUSCLE alignment method (Edgar 2004) using Geneious software
(Drummond et al., 2011).
Sequences analysis showed all the exon-intron boundaries do conform to the splice
donor/acceptor consensus sequences, GT and AG, respectively (Figure D.1 and D.2).
Reference
Drummond, A.J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., Duran, C., Field, M.,
Heled, J., Kearse, M., Markowitz, S., Moir, R., Stones-Havas, S., Sturrock, S., Thierer, T.
and Wilson, A. (2011) Geneious v5.4, Available from http://www.geneious.com/
Edgar, R.C. (2004) MUSCLE; multiple sequence alignment with high accuracy and high
throughput. Nuclic acid research, 32, 1792-1797
164
Appendix E
Analysis of SN1 and SN2 function using an antisense approach
E.1 Introduction
Functional genomics is a field of molecular biology that attempts to make use of wealth of
data generated by genomic projects to clarify gene and protein functions and interactions.
Snakin-1 (SN1) and Snakin-2 (SN2) are low-molecular weight cysteine-rich antimicrobial
peptides produced in potato tubers. Overexpression of the SN1 and SN2 genes is known to
provide broad spectrum activity against a broad series of bacterial and fungal pathogens in
potato (Segura et al., 1999; Berrocal-Lobo et al., 2002; Almasia et al., 2008). Chapter 2
results suggest that SN1 and SN2 proteins are highly conserved through all studied
Solanaceous species. Such very highly conserved nature of these proteins suggests that these
proteins may play an important role in plant metabolism in addition to defence against
invading microorganisms. Transgenic approaches involving reverse genetics such as antisense
suppression, and RNA interference can be employed to elucidate gene function and
complements biochemical approaches for understanding the function and role of specific
proteins. For example, antisense transformation of tobacco to produce β-1,3-glucanasedeficient plants showed that these plants had increased deposition of callose and showed more
resistance towards tobacco mosaic virus and tobacco necrosis virus (Beffa et al., 1996). Most
classes of cysteine-rich proteins (CRPs) in plants are involved in various defence or stresssignalling pathways, but some CRPs, such as the GASA family members, possess nondefence functions (Silverstein et al., 2007). The diverse functional roles of GASA genes in
cell division, cell elongation, and flower, root and fruit development have been previously
reported (Kotilainen et al.,1999; Ben-Nissan et al.,2004; De la Fuente et al., 2006: Furukawa
et al., 2006 and Roxrud et al., 2007). GIP2, one of the five GASA members in Petunia
hybrida, exhibits antioxidant activity and affects stomatal closure (Wigoda et al., 2006).
Chapter 2 of this thesis illustrates the highly conserved C-terminal regions of SN1 proteins
and GASA proteins (Section 2.3.8). SN1 and SN2 are often considered as defence proteins
involved in the innate defence against invading microorganisms (Segura et al., 1999;
Berrocal-Lobo et al., 2002; Mao et al., 2011). However, given the highly conserved nature of
SN proteins, they may also have additional roles of biological significance. This study
investigates the response to knockdown expression of SN1 and SN2 genes in potato plants
using antisense strategy.
165
E.2 Materials and methods
E.2.1 Vector Construction
Full details are given in Chapter 3 with the clones having the SN1 and SN2 genes in the
antisense orientation being utilised (Section 3.2.1). Plasmids with antisense expression
cassettes containing SN1 and SN2 genes were named as pStLhca3cas-antiSN1 and
pStLhca3cas-antiSN2 respectively.
These two resulting plasmids were digested with HindIII and the resulting fragments, the
1631 bp Lhca3-antiSN1 and the 1770 bp Lhca3-antiSN2 fragments were blunt-ended using
Quick Blunting Kit (NEBioLabs) and were ligated individually into the blunt-ended NotI site
of the binary vector pMOA33 using T4 DNA Ligase (NEBiolabs) according to the
manufacturer’s instructions. This produced pMOA33-Lhca3-antiSN1 and pMOA33-Lhca3antiSN2 binary vectors. Ligation products were transformed into MAX Efficiency® DH5αTM
Competent Cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
instructions. After transformation the bacterial cells were plated on LB agar plates containing
100 mg L-1 spectinomycin and incubated at 37ºC overnight.
Plasmid DNA was isolated from putatively transformed E. coli colonies using the QIAprep
Spin Miniprep kit (QIAGEN) according to the manufacturer’s instructions, and the insertion
and orientation of the chimeric Lhca3-antiSN-Lhca3 genes into the T-DNA of the binary
vector was verified by restriction analysis using EcoRV and ScaI for the Lhca3-antiSN1 insert
and EcoRV and XhoI for the Lhca3-antiSN2 insert.
Colonies containing recombinant plasmids were identified by PCR (full details are given in
Chapter 2) with Cab-Fa and Cab-Ra primers (Table E.1). PCR reactions were performed in a
Master cycler. The conditions for PCR were: 1 cycle at 94 ºC for 4 min, 34 cycles of (30 s
92ºC, 35 s 58ºC, 90 s 72ºC) followed by 7 min extension at 72ºC. Plasmid DNA from clones
selected by PCR was isolated using the QIAprep Spin Miniprep kit (QIAGEN, Venlo,
Netherlands) according to the manufacturer’s instructions. The orientation of the chimeric
Lhca3-SN-Lhca3 genes within the T-DNA in both binary vectors, pMOA33-Lhca3-antiSN1
and pMOA33-Lhca3-antiSN2, was tested by restriction analysis to select the binary vector
that contains the Lhca3 promoter adjacent to the right border within the T-DNA.
Restriction analysis using EcoRV and ScaI for pMOA33-Lhca3-antiSN1, with the Lhca3
promoter adjacent to the right border, produced 772 bp, 4800 bp and 6080 bp fragments. The
plasmid, pMOA33-Lhca3-antiSN2 with the Lhca3 promoter adjacent to the right border,
produced 1491 bp, 4800 bp and 5502 bp fragments when cut with EcoRV and XhoI.
166
Figure E.1 Schematic representation of the Lhca3 cassette used for the transformation with specific genes.
(A) Lhca3 cassette containing the SN1 gene (a1 allele) in reverse orientation. (B) Lhca3cassette containing the
SN2 gene (b1 allele) in reverse orientation.
E.2.2 Agrobacterium transformation
Full details are given in Chapter 3 (Section 3.2.2).
E.2.3 Plant material
Full details are given in Chapter 3 (Section 3.2.3).
E.2.4 Potato transformation
Full details are given in Chapter 3 (Section 3.2.4).
E.2.5 DNA isolation and PCR based analysis of transgenic colonies
Genomic DNA was extracted from the cell colonies using a modified CTAB method
(Appendix B). PCR reactions were performed using Taq DNA polymerase (BioLabs, New
England). Initially, to verify the quality and quantity of the genomic DNA template, PCRs
were conducted to amplify the endogenous actin gene using Actin-F and Actin-R primers as
described in Section 3.2.6.
To confirm the presence of the antisense-SN constructs in the cell colonies, genomic DNA of
these cell colonies were subjected to PCR analysis with primer specific to Lhca3-antiSN1 and
Lhca3-antiSN2 construct. The conditions for PCR for the Lhca3-antiSN1 gene using the
primers Cab-Fa and SN1-R4 (Table E.1) were: 1 cycle at 94C for 2 min, 34 cycles of (15 s
93C ,30 s 55C, 80 s 72C), followed by a 3 min extension at 72C. The conditions for PCR
for the Lhca3-antiSN2 using the primers SN2-bF4 and Cab-Ra (Table 3.1) were as follows: 1
cycle at 94C for 1 min, 34 cycles of (20 s 93C, 20 s 55C, 80 s 72C), followed by a 3 min
167
extension at 72C. Each 10 µL PCR mix contained 1x ThermoPol Reaction Buffer [20 mM
Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8 at
25°C], 0.2 mM of each dNTP, 0.2 µM of each primer and 0.4U of Taq DNA Polymerase
(New England Biolabs). All these amplified products were separated by electrophoresis in a
1% agarose gel in 1xTAE buffer at 5.5V/cm for 40 min and visualized under UV light after
staining with ethidium bromide (5 mg/L) for 15 min.
Table E.1 Details of the primers used to screen the colonies.
Target gene
Primer
pairs
SN1-R4
Primer sequences (5’ to 3’)
Cab-Fa
TTCTAGTGGAGCTAAGTGTTCA
SN2-bF4
TCATCAAGGCCAAGATTGTG
Cab-Ra
TGTTACATTACACATAAGAGAAGG
Actin-F
GATGGCAGAAGGCGAAGATA
Actin-R
GAGCTGGTCTTTGAAGTCTCG
Product size (bp)
ATAATTGGATACCCTGGAATGG
Lhca3-antiSN1-Lhca3
Lhca3-antiSN2-Lhca3
Actin
652
720
1069
E.3 Results
E.3.1 Vector Construction
The two binary vectors outlined in Figure 5.1 were successfully constructed (Figure E.2);
these constructs containing either SN1 or SN2 gene in an antisense orientation were used to
generate transgenic potato plants.
E.3.2 Potato transformation
The binary vectors were used for Agrobacterium-mediated gene transfer to potato cv. Iwa.
After 4-5 weeks of incubating Agrobacterium co-cultivated explants on callus induction
medium, only very few cell colonies were developed along the cut leaf margins and leaf
surfaces. The few green cell colonies were transferred into the regeneration medium
supplemented with 200 mg L-1 TimentinTM and 100 mg L-1 kanamycin, but these cell colonies
failed to regenerate. Antisense transformation experiments were conducted on three
independent occasions. In the third experiment, transformations using the ‘sense’ vectors
described in Chapter 3 were performed simultaneously.
168
(A)
(B)
Figure E.2 Schematic diagrams of the whole antisense binary vectors. (A) pMOA33-Lhca3antiSN1 and (B) pMOA33-Lhca3-antiSN2 used for Agrobacterium transformation
169
Transgenic plants overexpressing SN1 and SN2 genes in the sense orientation grew well
(Table E.2 and Figure E.3A). In contrast, the antisense transformations again failed to produce
shoots and all eventually died (Table E.2 and Figure E.3B).
Table E.2 Regeneration efficiency for A. tumefaciens transformation of Solanum tuberosum
cultivar Iwa with Lhca3-antiSN1 and Lhca3-antiSN2. The results of three transformation
experiments are presented.
Specific gene
Lhca3-antiSN1
Lhca3-SN1
Lhca3-antiSN2
Lhca3-SN2
Transformation
experiment
Number of
explants
Number of
Colonies
Colonies
regenerated
1
112
10
0
2
109
8
0
3
132
15
0
3
121
101
95
1
118
15
0
2
121
20
0
3
103
14
0
3
105
91
83
Regeneration
frequency (%)
0
79
0
79
170
(A)
(B)
Figure E.3 Cell colony growth in kanamycin-supplemented regeneration medium. (A) Healthy sense
colonies; and (B) senescent antisense colonies
171
E.3.3 DNA isolation and PCR based analysis of transgenic colonies
The presence of the actin gene and the Lhca3-antiSN1 and Lhca3-antiSN2 constructs in
recovered cell colonies was confirmed by independent PCR reactions.
Figure E.4 PCR analysis of representative Lhca3-antiSN1 cell colonies. Products of DNA amplification after
polymerase chain reaction using DNA from cell colonies as the template and using the primers specific to
Lhca3-antiSN1 and Actin genes. Lane 1- GeneRulerTM DNA ladder mix (Fermentas); lane 2- no template
control, lane 3- pMOA33- Lhca3-SN1 plasmid (+), lane 4- non-transgenic Iwa, lanes 5- 9 colonies A1- A5.
Figure E.5 PCR analysis of representative Lhca3-antiSN2 cell colonies. Products of DNA amplification after
polymerase chain reaction using DNA from cell colonies as the template and using the primers specific to
Lhca3-antiSN1 and Actin genes. Lane 1- GeneRulerTM DNA ladder mix (Fermentas); lane 2- no template
control, lane 3- pMOA33-Lhca3-SN2 plasmid (+), lane 4- non-transgenic Iwa, lanes 5- 9 colonies B1- B5.
E.4 Discussion
Potato leaf explants were transformed with antisense constructs containing Lhca3-antiSN1Lhca3 cassette or the Lhca3-antiSN2-Lhca3 cassette. Out of four antisense transformations
majority of the transformants fail to produce cell colonies and those that did failed to
regenerate in regeneration media. This very low frequency of recovery transformed potato cell
colonies and their failure to regenerate plants has never been reported in our laboratory
172
previously over the past 20 years, despite the transfer of many genes to potato using the same
standard protocol (Liew, 1994; Jaimes, 1999; Barrell et al., 2002, 2006 and 2009; Davidson et
al., 2002 and 2004; Chan, 2006; Meiyalaghan et al., 2006, 2009 and 2010; Jacob et al., 2009;
Vellasamy, 2010). Since transformed potato lines were recovered the SN sense constructs.
The third experiment attempting potato transformation with the antisense constructs also
included the sense constructs as a positive control. Given the successful transformation using
these sense constructs and the repeated failure using the antisense constructs, is highly
suggestive that the antisense knockdown expression of the SN genes is a lethal condition. This
is further substantiated by the confirmation that the rare cell colonies that were recovered, but
died before they regenerated, were PCR positive for the antisense construct.
All these results strongly indicated that knockdown of the SN1 and SN2 gene expression and
therefore the absence of the SN1 and SN2 proteins is deleterious to the growth and plant
development of potato plants. The lethality of antisense knockdown expression of SN1 and
SN2 establishes an essential role of this gene family for potato development in addition to
defense against invading microbes.
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175
Appendix F
ANOVA tables
F.1 Chapter 4 ANOVA tables
F.1.1 Analysis of variance (ANOVA) table for the SN1 transcripts in leaves (selected
lines) (Figure 4.3)
Source of
variation
Analysis
SN1 transcripts
in leaves
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
7
75.202
10.743
7.79
<0.001
Residual
16
22.078
1.380
Total
23
97.280
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.2 Analysis of variance (ANOVA) table for the SN1 transcripts in stems (selected
lines) (Figure 4.3)
Source of
variation
Analysis
SN1 transcripts
in stems
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
7
4.8295
0.6899
1.04
<0.442
Residual
16
10.6046
0.6628
Total
23
15.4342
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.3 Analysis of variance (ANOVA) table for the SN1 transcripts in tubers (selected
lines) (Figure 4.3)
Source of
variation
Analysis
SN1 transcripts
in tubers
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
7
6.3538
0.9077
2.34
<0.075
Residual
16
6.1958
0.3872
Total
23
12.5496
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.4 Analysis of variance (ANOVA) table for the SN2 transcripts in leaves (selected
lines) (Figure 4.4)
Analysis
SN2 transcripts
in leaves
Source of
variation
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
8
55.877
6.985
6.85
<0.001
Residual
18
18.365
1.020
Total
26
74.241
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
176
F.1.5 Analysis of variance (ANOVA) table for the SN2 transcripts in stems (selected
lines) (Figure 4.4)
Source of
variation
Analysis
SN2 transcripts
in stems
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
8
42566.37
5320.3
68.61
<0.001
Residual
18
1398.83
77.71
Total
26
43965.20
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.6 Analysis of variance (ANOVA) table for the SN2 transcripts in roots (selected
lines) (Figure 4.4)
Source of
variation
Analysis
SN2 transcripts
in roots
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
8
29.65541
3.70693
373.00
<0.001
Residual
18
0.18488
0.01027
Total
26
29.84029
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.7 Analysis of variance (ANOVA) table for the SN2 transcripts in tubers (selected
lines) (Figure 4.4)
Analysis
SN2 transcripts
in tubers
Source of
variation
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
8
11.08352
1.3857
10.81
<0.001
Residual
18
2.4808
0.1378
Total
26
13.5660
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.8 Analysis of variance (ANOVA) table for the Pathogen assay 1 (SNI selected lines)
(Table 4.2)
Analysis
Length of the
lesion
Source of
variation
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
7
221.289
31.613
11.54
<0.001
Residual
88
241.063
2.739
Total
95
462.352
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
177
F.1.9 Analysis of variance (ANOVA) table for the Pathogen assay 1 (SN2 selected lines)
(Table 4.3)
Analysis
Source of
variation
d.f.
s.s.
m.s.
v.r.
F pr.
8
286.629
35.829
35.83
<0.001
Residual
117
125.321
1.071
Total
123
411.950
Treatment
Length of the
lesion
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.10 Analysis of variance (ANOVA) table for the Pathogen assay 2 (SN1 selected lines)
(Table 4.4)
Analysis
Length of the
lesion
Source of
variation
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
7
21.9917
3.1417
8.78
<0.001
Residual
16
5.7267
0.3579
Total
23
27.7183
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.12 Analysis of variance (ANOVA) table for the Pathogen assay 2 (SN2 selected lines)
(Table 4.5)
Analysis
Source of
variation
d.f.
s.s.
m.s.
v.r.
F pr.
8
134.1814
16.7727
44.28
<0.001
Residual
117
44.3186
0.3788
Total
125
178.5000
Treatment
Length of the
lesion
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.13 Analysis of variance (ANOVA) table for the Pathogen assay 3 (SN1 selected lines)
(Table 4.6)
Analysis
Source of
variation
Treatment
Length of the
lesion
d.f.
s.s.
m.s.
v.r.
F pr.
5.44
<0.001
7
1158.3013
165.4716
Residual
104
3161.5535
30.3995
Total
111
4319.8549
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
178
F.1.14 Analysis of variance (ANOVA) table for the Pathogen assay 3 (SN2 selected lines)
(Table 4.7)
Source of
variation
Analysis
d.f.
s.s.
m.s.
v.r.
F pr.
8
1495.3845
186.9230
15.81
<0.001
Residual
117
1383.1071
11.8214
Total
125
2878.4916
Treatment
Length of the
lesion
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.15 Analysis of variance (ANOVA) table for P.atrosepticum count (SN1 selected lines)
(Table 4.7)
Source of
variation
Analysis
P.atrosepticum
count
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
4
28.55995
14.27998
10
<0.001
Residual
10
0.15277
0.02546
Total
14
28.71272
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.16 Analysis of variance (ANOVA) table for P.atrosepticum count (SN2 selected lines)
(Table 4.7)
Source of
variation
Analysis
P.atrosepticum
count
d.f.
s.s.
m.s.
v.r.
F pr.
Treatment
6
84.33346
16.86669
14
<0.001
Residual
Total
12
18
0.13028
84.46374
0.01086
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
F.1.17 Analysis of variance (ANOVA) table for P.atrosepticum density (SN1 selected
lines) (Table 4.7)
Analysis
P.atrosepticum
density
Source of
variation
d.f.
Treatment
4
s.s.
24.6271
Residual
40
1.0099
Total
44
25.6370
m.s.
8.2090
0.1262
v.r.
F pr.
9.0
<0.001
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
179
F.1.18 Analysis of variance (ANOVA) table for P.atrosepticum density (SN2 selected
lines) (Table 4.7)
Analysis
P.atrosepticum
density
Source of
variation
d.f.
Treatment
6
s.s.
53.75410
Residual
48
0.91694
Total
54
54.67104
m.s.
10.75082
0.07641
v.r.
13.0
F pr.
<0.001
NB: d.f = degrees of freedom; s.s =sums of squares; m.s = mean square; v.r = variance ratio; F pr. = F probability
180
Appendix G
List of publications, presentations and awards
Publications
Meiyalaghan, S., Mohan, S., Pringle, J.M., Baldwin, S.J., Jacobs, J.M.E., and Conner, A.J.
2009. Towards disease resistance in potatoes using intragenic approaches. Communications in
Agricultural and Applied Biological Sciences, 74, 667-679.
Seven NCBI Gene Bank Submissions
Accession Numbers
FJ195649, FJ195648, FJ195647, FJ195646, EU848499, EU848498, EU848497, JN809786,
JN809785, JN809787, JN809784, JN809781, JN809782, JN809780, JN809783, JN797794,
JN809788, JN802275, JN802276, JN802277, JN802278, JN809795, JN809794, JN809793,
JN809790, JN809791, JN809789 and JN809792.
Presentations
Meiyalaghan, S., Mohan, S., Jacobs, J.M.E., Barrell, P.J., Baldwin, S.J. and Conner, A.J.
2007. Sequence diversity in SN1 and SN2 genes from potato. Proceedings of the 17th
Queenstown Molecular Biology Meeting 2007, 28-31 August, Rydges Lakeland Resort,
Queenstown, abstract 91.
Meiyalaghan, S., Mohan, S., Jacobs, J.M.E. and Conner, A.J. 2007. Allele diversity for the
StSN1 and StSN2 genes encoding antimicrobial peptides. Abstracts of the 4th Solanaceae
Genome Workshop 2007, 9-13 September, Ramada Plaza Jeju, Jeju Island, Korea, p. 77.
Mohan, S., Meiyalaghan, S., Conner, A. and Jacobs, J. 2008. Highly conserved nature of
Snakin 1 and Snakin 2 proteins in Solanum. Abstracts of the 5th Solanaceae Genome
Workshop, 12-16 October, Cologne, Germany, p. 223.
Meiyalaghan, S., Mohan. S., Pringle, J.M., Jacobs, J.M.E. and Conner, A.J. 2009. Towards
disease resistance in potatoes using intragenic approaches. Conference Proceedings of the 18th
Biennial Meeting of the New Zealand Branch of the International Association of Plant
Biotechnology, 16-19 February, Napier, New Zealand, p. 23.
Mohan, S., Meiyalaghan, S., Jones, E., Jacobs, J.M.E. and Conner, A.J. 2009. Allele diversity
for Snakin-1 and Snakin-2 genes in Solanaceous species. Conference Proceedings of the 18th
Biennial Meeting of the New Zealand Branch of the International Association of Plant
Biotechnology, 16-19 February, Napier, New Zealand, p. 36.
181
Meiyalaghan, S., Mohan, S., Pringle, J.M., Jacobs, J.M.E. and Conner, A.J. 2009. Towards
disease resistance in potatoes using intragenic approaches. Abstracts of the 61st International
Symposium on Crop Protection, Gent, Belgium, p. 87
Mohan, S., Meiyalaghan, S., Jones, E., Jacobs, J.M.E. and Conner, A.J. 2009. Highly
conserved alleles of Snakin-1 and Snakin-2 genes in Solanaceous species. Abstracts of
COMBIO 2009, Christchurch Convention Centre, Christchurch 6-10 December, Proceedings
of the Australian Society for Biochemistry and Molecular Biology Volume 41, CD Rom
Meiyalaghan, S., Mohan, S., Pringle J.M., Jacobs J.M.E., and Conner, A.J. 2010. Exploring
intragenic approaches towards disease resistance in potatoes. Abstracts of the European
Association for Potato Research – Eucarpia Congress, Potato Breeding after the Completion
of the DNA Sequence of the Potato Genome, 27-30 June 2010, Wageningen, the Netherlands,
p. 45.
Mohan, S., Meiyalaghan, S., Pringle, J.M., Jones, E., Jacobs, J.M.E. and Conner, A.J. 2010.
Over expression of Snakin-1 and Snakin-2 genes under a potato light-inducible Lhca3
promoter in transgenic potatoes for broad-spectrum disease resistance. OzBio 2010 ‘The
Molecules of Life - from Discovery to Biotechnology’, 27 September - 1 October 2010,
Melbourne Convention and Exhibition Centre, Australia. p. 188.
Mohan, S., Meiyalaghan, S., Jones, E., Jacobs, J.M.E. and Conner, A. 2011. Overexpression
of endogenous snakin-1 and snakin-2 genes in potato confers resistance to Pectobacterium
atrosepticum. Abstracts of the 19th Biennial Meeting of the New Zealand Branch of the
International Association of Plant Biotechnology, 8-11 February, Hanmer Springs, New
Zealand, p. 32.
Awards

Best outgoing student award from Bharathidasan University (2004)

Bharathidasan University Postgraduate scholarship holder (2002-2004)

Student Award Winner (Oral Presentation) at the IAPB Conference, Napier (2009
February)

MacMillen Brown Agriculture Scholarship (2009)

Howard Bezer Scholarship (2009)

NZSBMB Travel Award (2010)
182