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PROJECT TITLE
Molecular basis of pathogenicity and Cucurbit host specificity in the Erwinia
tracheiphila genome
PROJECT INVESTIGATOR
5Siti Izera
1. PROJECT SUMMARY
Erwinia tracheiphila infects cultivated cucurbit crops and causes bacterial wilt diseases.
These bacterial wilts can cause major financial losses for cucurbit growers in the United
States of more than $13 million every year (Latin et al. 1993). The bacterial wilt of
cucurbits is therefore an important disease problem for growers in the United States.
E. tracheiphila is transmitted by striped and spotted cucumber beetles. These bacteria
remain in the gut of cucumber beetles during the winter. During a growing season,
bacteria residing in the infested feces enter plants through wounds or flower nectaries.
These bacteria colonize the xylem; multiply within the host cells and synthesis
exocellular polysaccharide (EPS) production and plant cell wall degrading enzymes. The
bacteria block xylem vessels to cause wilting of plant hosts. Preliminary cross inoculation
studies at Iowa State University showed that E. tracheiphila strains isolated from
Cucumis species cause slower wilting symptoms on Cucurbita plant species. This result
strongly suggests that Erwinia tracheiphila strains have host specificity to cause disease.
We hypothesize that there is a key genes of Erwinia tracheiphila that is responsible for
host specificity on Cucumis and Cucurbita plants. Many gram-negative bacteria use a
type three-secretion system (T3SS) to colonize their hosts. T3SS is needed to transport
effector proteins into the eukaryotic host cells. T3SS are encoded by hrp genes, and these
genes are require to cause disease in susceptible plants and the hypersensitive response in
resistant plants (Lingren et al.1986). Hrp genes have been identified in most plant
pathogenic bacteria such as Pseudomonas syringae (foliar spots and blight), Ralstonia
solanecearum (wilt of solanaceous plants) and Erwinia amylovora (fire blight of apple
and pear) (Buttner and Bonas, 2006). We hypothesize T3SS is also present in E.
tracheiphila strains. The overall goal for this project is to gain better understanding the
genetic mechanisms for virulence and host specificity in Erwinia tracheiphila. Our
specific objectives of proposed research are first to compare genome sequences of
Erwinia tracheiphila strains isolated from 2 different plant species, Cucumis spp
(muskmelon) and Cucurbita spp (summer squash); second, to identify new candidate
virulence genes involved in pathogenicity of bacterial wilt diseases; and third, to
characterize the function of new candidate genes and type three secretion system in hostspecificity. To begin to test these hypotheses, it is first essential to collect and compare
genome sequence of two E. tracheiphila strains isolated from Cucumis and Cucurbita
hosts. For this E. tracheiphila, the genome of this plant pathogenic bacterium is not
available. Therefore, the time is right to explore the molecular genetics involved in hostspecificity in E.tracheiphila strain. We will also conduct approaches such as genome
sequencing and assembly, bioinformatics analysis and molecular technique to answer
objective 1 and objective 2. Another objective of this proposed research is to create T3SS
mutant construct of E. tracheiphila subspecies Cucumis and Cucurbita and then
transform into its host in order to determine T3SS is present in E. tracheiphila strain. We
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hope by comparing the genome sequence of E. tracheiphila can help us to better
understand the function of virulence genes that are involved in pathogenicity of bacterial
wilt and develop new strategies to manage bacterial wilt diseases.
II PROJECT DESCRIPTION
A. INTRODUCTION
Bacterial wilt damages cucurbit crops throughout the eastern half of the united sates.
According to the U.S national agricultural statistics service, more than 10,000 cucurbit
growers in this region produce a value of fresh market cucurbit crops (muskmelon,
summer squash, cucumber and pumpkins) of about $400 million in 2009 (USDA-NASS
2009). Bacteial wilt can cut yield by >80% (Latin 1993). Our project focus muskmelon
and summer squash crops because they are widely grown in the Midwest and are at high
risk from cucumber beetles and bacteria wilt.
Erwinia tracheiphila, the causal agents of bacterial wilt of cucurbits, is transmitted when
cucumber beetles carrying these bacteria feed on cucurbit plants. The infested feces from
its vector are deposited on cucurbit plants and enter plant cells through fresh wounded
leaf or flower nectaries. E. tracheiphila is vectored by two species of cucumber beetles,
Acalymma vittatum F. (striped cucumber bettles) and Diabrotica undecimpunctata
howardii (spotted cucumber beetles). Bacteria enter the vascular system through these
openings, multiply within intercellular and xylem tissues, and produce exopolysaccarides,
which plug the water flow and eventually kill cucurbit plants. Many gram-negative
bacteria regulate EPS production. This feature is important for phytopathogenic bacteria
to protect them from being recognized by plant defense mechanisms (Leigh & Coplin
1992). Diiferent structure of EPS molecule is thought to be one of the reasons for
phytopathogenic bacteria to have their own preferred hosts (Maes et al. 2001). Insecticide
is common strategy to control cucumber beetles in the growing season. Unfortunately,
this situation creates more problems to the ecosystem as it suppresses the population of
honeybees (Apis mellifera) and other pollinator species (Hodges et al. 2007). Research
community urgently needs to find ways to protect bees more effectively. Also, there are
no genetic resistant cultivars commercially available at this moment.
E. amylovora requires the type three secretion system (hereafter, T3SS) to infect its hosts
and allows bacteria to inject virulence proteins into plant host cells (S. Y. He et al. 2004).
In phytopathogenic bacteria, genes of the T3SS are also known as hrp genes, are needed
for hypersensitive response (HR) induction. In E. amylovora, hrp genes encode the
component of T3SS system and their expression is linked to virulence (C.-S. Oh &
Steven V Beer 2005). The molecular switch for hrp system is HrpL which can bind to the
hrp-box promoter regions in all hrp genes and dsp genes (Ry 2005) (Wei & S V Beer
1995). Erwinia amylovora secrete key pathogenicity factor known as DspA/E for
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developing fire blight disease. Mutants of DspA/E were unable to produce bacteria ooze
and no longer pathogenic in apple plant cells. DspA/E is a type III effector protein
secreted through the hrp T3SS (Bogdanove et al., 1998). Erwinia amylovora also carry
host-specificity gene called eop1 gene that was isolated from wilted tissues of Rubus spp.
infected by E. amylovora (Asselin et al. 2011).
More recently, complete genomes of four Erwinia species which are E. amylovora, E.
pyrifoliae, E. tasmaniensis and E. biliangiae have been sequenced and published (Kube
et al. 2010)(Kube et al. 2008)(Smits et al. 2010). Pairwise 16S rDNA comparison showed
that E. tracheiphila is closely related to E. amylovora and E. pyrifoliae (Kado 2006). E.
tracheiphila also share similar features of E. amylovora such as production of
exopolysaccharide which cause plugging of vessel elements in the host plant. The early
symptoms of infected plants are water soaking then vascular wilting and eventually dies.
The availability of complete genome of E. amylovora offers great opportunities for
characterizing virulence factors of E. tracheiphila. The comparisons of different Erwinia
genomes permit a direct assessment of changes in gene structure and sequence that have
arisen during the evolution. Such comparisons also refine the identification of specific
and conserved protein coding genes within a given genomes. For example, E. amylovora
need a functional hypersensitive response and pathogenicity factor T3SS and formation
of EPS for iniatiating disease on host plant in Rosaecous family. One of striking findings
of comparative genome is existing 5 effectors gene including eop1, eop3, avrRpt2Ea,
dspA/E and hopC1 which regulates HrpL (Zhao et al. 2006)(Ry 2007). Mutants also have
been created for some of those effectors by transposon and site-directed mutagenesis,
which lost of virulence (Bugert & Geider 1995)(W. Kim et al. 2002).
For the past 30 years, DNA sequencing relied on the capillary electrophoresis (CE)-based
Sanger sequencing by using Applied Biosystems. However, this tehnology has some
limitations such as low speed, low sequence coverage and the cost data production is
expensive. More recently, a new sequencing technology called Next-Generation
Sequencing (NGS) has transformed biological enterprise and sequencing whole genome
only take a short amount of time. For example, researchers can now sequence 6 human
genomes in a single run, producing data in about one week for cost data production of
less than $5000 per genome. By using CE-based Sanger sequencing, the first human
genome took about 13 years to complete entire genome sequencing. In other words, NGS
sequencing offers sequencing technology that can be completed in months and it costs for
about thousand of dollars. Also, NGS technology is highly scalable. For sequencing
bacterial or viral genome, a researcher can choose Multiplexing for a large number of
samples. Multiplexing enables to sequence more than individual of bacterial genome
accurately at the same time in a single run by applying specific Barcode to each sample.
For this project, we choose to do conduct the whole genomic analysis of NGS-generated
sequence data as it allows one-shot sequencing of the entire Erwinia’s genome. As a
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result, it is faster, easier and cheaper for highly increased coverage that can be obtained in
a single sequencing run.
In preliminary study at Iowa State University, strains of E. tracheiphila can be grouped
into two-groups based on host range. We will refer isolates from different hosts as
subspecies of E. tracheiphila. i) Strains isolated from muskmelon, Cucumis spp.
(hereafter, E. tracheiphila subsp. muskmelon) ii) Strains isolated from summer squash,
Cucurbita spp. (hereafter E. tracheiphila subsp. squash). Cross-inoculation of Cucumis
strains into Cucurbita seedlings developed slower wilting symptom, but Cucumis
seedlings caused very rapidly wilting symptom. These observations clearly showed that
E. tracheiphila strains have host-specificity factor or pathogenicity determinant. In order
to understand how E. tracheiphila subsp. muskmelon and E. tracheiphila subsp. squash
strains differ, preliminary study revealed that they differ in host symptom development.
Further work is needed to demonstrate whether these differences affect the host ranges of
E. tracheiphila subsp. muskmelon and E. tracheiphila subsp. squash. Now, we proposed
to compare genome sequences of E. tracheiphila subsp. muskmelon and E. tracheiphila
subsp. squash with other pathogenic E. amylovora strains with different host ranges
might suggest potential candidate host-specificity factors. We also will do genome
comparison with non-pathogenic Erwinia, which is E. bilingiae. This non-pathogenic
bacterium is epiphytic Erwinia and plays a role as antagonist for biocontrol of fire blight
disease. Also, E. bilingiae lacks any T3SS in contrast to E. amylovora (Kube et al. 2010)
The bacterium E. tracheiphila will have a complete genome sequence and assembly. It is
a logical choice since it has a small genome size and share similar features of other
pathogenic Erwinia strains. E. tracheiphila resembles some properties of E. amylovora,
thus fulfill the requirements for phytopathogenic bacteria associated their interactions
with plants because, i) it damages high-value cucurbit crops; ii) it survives in the gut of
insect beetles in winter time; iii) spread E. tracheiphila by feeding wound and iv) it
regulates EPS production and plant cell degrading enzymes and colonizes the xylem
tissues.
HYPOTHESIS AND OBJECTIVES.
Our overall research goal is to understand its host-specificity and virulence mechanisms
of bacterial wilt disease of Cucurbitaceae caused by E. tracheiphila. We expect the
results of whole genome sequencing and functional genomics will provide key genetic
basis of host-specificity in E. tracheiphila strains. We will identify key genes with
potential impact to virulence of pathogenic erwinias for each of two recently discovered
subspecies of E. tracheiphila. Then, we will investigate the role of strain-specific genes
and the function of type three-secretion system (T3SS, hrp genes) in host-specificity of E.
tracheiphila. Successful completion of this project will enable us for the first time to fill a
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gap in the knowledge of molecular genetics of E. tracheiphila. Our specific objectives
are:
Objective 1: To obtain draft whole genome sequences of E. tracheiphila strains isolated
from muskmelon and summer squash plants.
Objective 2: To compare two-draft genome sequences to recently sequenced genome of
Erwinia amylovora ATCC 49946 and non-pathogenic Erwinia strain, which is Erwinia
biliangiae Eb661. Our working hypothesis for this objective is that Erwinia tracheiphila
strains have sub-species-specific genes with potential virulence and pathogenicity
mechanism in analyzed draft genomes.
Objective 3: To determine the function of strain-specific genes and type three secretion
systems (hrp) in host-specificity of E. tracheiphila strains. Our working hypothesis for
this objective is E. tracheiphila have T3SS that can export effector protein in the bacterial
cytoplasm to their site of action in the host.
C. RATIONALE AND SIGNIFICANCE.
The rationale of this project is that E. tracheiphila is essentially plant-associated
pathogenic Enterobacteria, which damages cucurbit crops, bacterial wilt complex.
Cucumber beetles efficiently vector these bacteria. Lack of knowledge of E. tracheiphila
biology and its interaction with their host plants has hampered efforts to develop effective
control strategies against bacteria wilt. One of the biggest challenges associated with E.
tracheiphila is that these bacteria are difficult to isolate from wilted plant tissue (Rojas et
al. 2012) As a result, the genetics of E. tracheiphila remain unexplored. This project is
significant because the knowledge of new key genetic loci for host-specificity pathogens
of E. tracheiphila can contribute to novel control strategy especially for disease
resistance, which has been delayed due to insufficient information of E. tracheiphila.
Study by Rand in (1915) clearly showed that E. tracheiphila survived in the gut of E.
tracheiphila and developed bacterial wilt symptoms in cucurbit field in the spring of
1914. Unfortunately, the research community has neglected this study and still no
researchers have ever reported about the genetics of E. tracheiphila. The results of this
project will facilitate developing control strategy if we could find out the molecular
underpinnings of host-specificity and pathogenicity in the E. tracheiphila’s genome.
Spraying insecticides for reducing bacterial wilt incidence is the only management
option. In early summer, cucurbit growers rely mainly on neonicotinoid insecticides to
suppress cucumber bettles carrying E. tracheiphila during the critical period of
infestation (Ricky, Foster 2012). The downside using this insecticide is it kills the bees
that pollinate cucurbit crops. Growers need environmentally safer and more effective
ways to manage cucumber beetles and cucumber wilt. In the near future, the outcomes of
this project have potential to reduce the use of insecticides in agriculture for manage wilt
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diseases and sustains the health of environment and people. It will give multiple benefits
to U.S agriculture, environment and society. We hope to develop and release cucurbit
cultivars that are resistance to disease and this can improve crop yield and enhance
profitability of U.S cucurbit growers.
Also, next-generation sequencing is faster and less expensive than the CE-based Sanger
sequencing. To obtain complete genome of 2 strains of Erwinia, it will take less time to
assemble DNA sequences of the bacterial chromosome and will generate powerful
results. The whole genome sequence data will be published and help other researchers
use the genome sequence data for future studies.
D. EXPERIMENTAL APPROACH
PROJECT INTEGRATION PLAN
This project has one principal investigator (PI), which is responsible to manage the
project and supervise the graduate student. We will have one Ph.D student and one
postdoctoral research to complete this project. Post-doctoral researcher will have a strong
role in setting the whole genome sequencing analysis, including coordinate of all the
aspects of the next-generation sequencing and bioinformatics data analysis. The graduate
student will create various mutants and testing the mutants in host plants.
OBJECTIVE 1: Obtain genome sequences of Erwinia tracheiphila strains isolated
from 2 different plant species, Cucumis spp (muskmelon) and Cucurbita spp
(summer squash).
Generation of genome sequencing
Two Erwinia tracheiphila strains collection, including one strain for subspecies
muskmelon and summer squash will be sequenced at the Iowa State University DNA
Sequencing and Synthesis Facility. These strains have been isolated from the wilting
muskmelon and summer squash plants in Iowa in 2009 (Rojas et al. 2012). E.tracheiphila
strains will be cultured on nutrient agar peptone (NAP) plates containing 75 ug/ml of
rifampicin antibiotic and verify the biological identity of these phytopathogenic bacteria
by using 16S rRNA gene sequence and BIOLOG plates for determining carbon source
utilization. The white colony morphology bacteria colonies that resemble features of the
E.tracheiphila will be confirmed positive by PCR using specific primers ETC1 and ETC2
(Rojas et al. 2012).
The Illumina HiSeq 2000 operated by the DNA facility of the Iowa State University is
capable to generate a maximum number of 180 million short reads (50-100 bases
sequence per read) per lane simultaneously and sequencing of all sequence reads happen
at the same time. The Illumina sequencer is a direct step-by-step detection of each
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nucleotide base incorporated during sequencing reaction. Next-generation Illumina
sequencer offer paired end read capability for example sequences can be derived for both
ends of library fragments. For Illumina sequencing, hundred of millions of reactions will
be captured per run, which called ‘massively parallel sequencing’. ISU DNA facility also
provides library construction services for all Illumina application. For the genomic DNA
preparation, we will use TruSeq kit from Illumina. We will extract DNA genomic from
each of the 2 Erwinia strains and will be measured the quality of DNA by reading
absorbance at ratio of A260nm/280nm. Then, the DNA genomics will be sent to the DNA
facility of the Iowa State University Office of Biotechnology for library construction,
quality checking and sequencing reaction. The steps of library construction involve in
fragmentation the DNA genomic consisting of 300-500 base fragments, add on adaptors
or linker by ligation, amplification to multiple copies to make a ‘library’. For Illumina
Next generation sequencing, a whole genome sequenced at 30X coverage will generate
an average; 30 sequencing reads will cover each base in the genome. We will use 100cycle paired-end cluster generation and sequencing per lane of 2 strains of about
5,000,000 bp genome sizes of each genome is estimated to cost about $2375.
Assembly of the short sequence reads
The Genome Informatics Facility of Iowa State University will perform the genome
assembly and annotation of the draft genomes into contigs. The professional staff at this
facility will operate and assemble the short sequence reads into contigs. The short
sequence reads derived-contigs will be sent to the project investigator for further analysis.
Short-read sequencing by using Illumina sequencer has been successfully used for de
novo assembly of small bacteria genomes (2-5 Mbp) (Farrer et al. 2009). De novo
assembly using Illumina sequencing technology has been successfully used for bacteria
genome for example E. amylovora, the causal agent of fire blight and for fungal genomes
including Sordaria macrospora and Ventura inaequalis (Diguistini et al.
2009)(Nowrousian et al. 2010).
Analysis of the draft genome sequence
Post-doctoral researcher at Iowa State University will analyze the draft genome
sequences by using Iowa State University bioinformatics servers. The computing power
to analyze the genome sequence is estimated to cost about $2000. This centralized
bioinformatics server offers some advantages; powerful, web-based platform is open to
public, allowing the users to work remotely within different locations. Also, it can
analyze de novo assembly Sanger and high-throughput sequencing data. The draft
genome will be analyzed to identify genetic elements with potential impact to virulence
and the presence of key genes of E. tracheiphila. We also interested to determine the
chromosome size (bp), sequence coverage, G+C (%) content of the chromosome, number
of plasmid, number of protein coding (%), number of coding sequences, assigned
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function and sequence similarity between Erwinia subspecies muskmelon and summer
squash. Also, the analyzed genome will be compared with previously published genome
sequence of Erwinia species.
Recent genome sequencing of the pathogenic Erwinia strain identified T3SS system,
which were found to be important for delivery effector proteins into the host cytoplasm
(Hueck 1998; Coburn et al. 2007). In plant pathogenic bacteria, T3SS are encoded by hrp
genes which are found in most of gram-negative bacteria (Lavie et al. 2002). The T3SS in
Erwinia species is composed of the hrp/hrc-gene cluster and two flanking regions (hrp
elicitors and effectors (HEE) and hrp-associated enzymes involved in systemic virulence
(Kube et al. 2008); Ry 2005). For this project, we will focus on the key genetic players in
the pathogenesis of E. tracheiphila genomes with respect to T3SS, which will be focused
to secretion systems and effectors and production of exopolysacarides (EPS). We also
want to investigate the role of T3SS, hrp genes in host-specificity of E. tracheiphila
strains. We will obtain complete segments of hrp elicitor/effector clusters of the 2
E.tracheiphila strains. We will align the hrp clusters of 2 E.tracheiphila strains to
identify key genes that are specific to each of the subspecies. We also will look for any
differences in the amino acid level and the functional content of hrp clusters. The key
genes will be the targets for host-specificity studies.
OBJECTIVE 2: Identify subspecies-speficic genes and type three secretion system
T3SS in host specificity
Comparisons of genome sequences of E. tracheiphila strains isolated from Cucumis spp.
and Cucurbita spp. and non-pathogenic Erwinia strain such as Erwinia tasmaniensis and
Erwinia bilingiae (Kube et al. 2008) might suggest potential candidate host specificity
factors and pathogenicity determinants. We hope to identify these through genome
comparison of the E. tracheiphila subsp. muskmelon and E. tracheiphila subsp. summer
squash for strain-specific gene (Objective 1). We also hope to identify hrpL and dspA/E
gene representing T3SS system in E. tracheiphila draft genome. More recently, genome
sequence of the apple pathogen, Erwinia amylovora strain ATCC 49946, pear pathogen
Erwinia pyrifoliae strain Ep1/96 and the non-pathogenic Erwinia billingiae strain Eb661
have been characterized (Powney et al. 2011; Sebaihia et al. 2010; Smits et al. 2010;
Kube et al. 2008). We also will investigate the role of T3SS effector protein because the
T3SS effectors from plant pathogenic bacteria also act as host specificity factors and
pathogenicity (Gaudriault et al. 1997). Then, we will create mutants of strain-specific
genes and T3SS and will analyze the virulence of the mutants in Cucurbitaceae plants.
Construction of strain-specific mutants and T3SS mutants
We will determine if strain-specific genes and a cluster of hrp genes might contribute to
host specificity. To answer this hypothesis, we will mutate strain-specific genes. As an
alternative strategy, we will work with HrpL and dspA/E gene in E. tracheiphila genome.
These genes will be manipulated in the respective Erwinia tracheiphila strains. DNA
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sequences of E. tracheiphila will be used to design primers for PCR amplification of the
HrpL and dspA/E genes from E. tracheiphila. We will evaluate whether if T3SS is
present in E. tracheiphila.
The method called site-directed mutagenesis will be performed to create mutants.
Custom-designed nucleotides harboring mismatch to selected gene can be used to make a
directed mutation in the mutant contructs. Crossover PCR mutagenesis will be used to
make precise gene knockouts in selected genes of interest. Then, the deletion contructs
will be cloned into SmaI site of mobilizable suicide vector pJQ200uc1. This suicide
vector carries P15A origin of replication (Ori), SacB-based system (Bacillus subtilis),
gentamicin resistence marker (gtmR) and lacZ system. This vector only replicates in
enterobacteria (Quandt & Hynes 1993). We will introduce antibiotic marker into the
deletion constructs and will be transferred to E. tracheiphila by electroporation. E.
tracheiphila transformants will be selected on Luria Bertani (LB) media containing
appropriate antibiotic and 5% sucrose. The sucrose-resistant coloniesres will be tested by
PCR, using universal primer sets that produce amplicons of different sizes for wild-type
strain and mutated contructs. The E.tracheiphila subsp. muskmelon-specific genes will
be cloned for heterologous expression in E.tracheiphila subsp. summer squash. Total
RNA will be isolated from the mutant constructs, will be treated with DNAse enzyme,
and will be checked for quality by gel electrophoresis and A260nm/A280nm ratio. High
quality RNA transcripts will be used to reverse transcribed with reverse transcriptase
enzyme. We will conduct quantitative real-time PCR (qPCR) assay for E. amylovora
genes, including HrpL and dspA/E. Primers sequences will be obtained from available
sequence information in GenBank: HrpL (U36244) and dspA/E (Y13831.1). Specific
amplification of the targets will be verified by the presence of a specific product in cDNA
template.
OBJECTIVE 3: Characterize the function of subspecies-specific genes and type
three-secretion system (T3SS) in host specificity
Host plant inoculations, pathogenicity and HR assays
The mutants of each subspecies along with wild-type will be tested for pathogenesis in
the two cucurbit hosts, which are muskmelon and summer squash. The plants will be
raised in the greenhouse. We will maintain seedlings in growth chamber in greenhouse
set at 25 C with 14 hr light/10 hr dark. The trays containing seedlings will be spaced apart
from each other to prevent cross contamination. Three replicate plants per treatment will
be inoculated by infiltration with various mutant strains and wild-type strains. Mock
inoculation will be performed with buffer only. We will also inoculate on non-host plant,
which is tobacco plant (Nicotiana tabacum cv. Xanthi). Hypersensitive response (HR)
will be observed in tobacco plants. Photographs of the disease symptom will be taken
daily beginning 2 days post inoculation (dpi) and 4 days post inoculation. The population
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of the bacteria in the plant hosts will be measured by agar-plate bioassay. We expect to
see that the T3SS mutants will have an effect on the phenotypes, which inhibit
compatible and incompatible reactions. Mutants in effector genes may only affect
pathogenicity or HR on specific host species. The strain-specific genes will be expressed
in strains, which do not carry them to investigate the ability to develop host-specificity to
the expressing strain.
Plant gene expression analysis
Expression of the pathogen-related protein-1 (PR-1; GenBank: AF507974.1) will be
determined by qPCR as described in Milcevicova et al. (2010). Analysis of relative gene
expression will be calculated between inoculated and uninoculated plants.
B. FUTURE DIRECTIONS.
In summary, the whole genome sequencing of two species of E. tracheiphila will provide
key genetic loci for host-specificity and pathogenicity for the conserved regions. In the
near future, it can help us to correct the classification of Erwinia species and its
evolutionary divergence among other Erwinia species. We also can focus on ecological
study to better understand their modes of adaptation to different ecological niches. With
the advances of technologies, future work should be address question such as; (i) How E.
tracheiphila can survive in insect guts? (ii) How they can survive on host plants when
release in feces? Polysaccharides are important virulence factors in erwiniae. Another
future study, we can emphasize on the genetics of EPS by E. tracheiphila by looking at
gene cluster involved in EPS-synthesis. We also can try to identify genes that code for
proteins homologous to Avr proteins and investigate the role of these genes in
pathogenicity. We expect that there will be a lot of progress in the future in studying
bacterial wilts disease, which will lead to the development of environmentally safe
disease management strategies.
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F. PROJECT TIMELINE
Objectives
Objective 1:
1.1 Generation of genome sequencing for E.
tracheiphila
1.2 Assembly of the short sequence reads
1.3 Analysis if the draft genome sequence
Objective 2:
2.1 Identify new candidate virulence genes involved
in pathogenicity of bacterial wilt diseases
2.2 Construction of strain-specific mutants and
T3SS mutants
Objective 3:
3.1 Host plant inoculations
3.2 Plant gene expression analysis
Year 1
Year 2
Year 3
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III. REFERENCES
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exopolysaccharide synthesis of Erwinia amylovora. , 15, pp.917–933.
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and E . tasmaniensis with the pear pathogen E . pyrifoliae. , pp.1–15.
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IV. ESTIMATED BUDGET
First year
Second year
Third year
Total
1. Salaries and
social benefits
$48, 800
$50, 264
$51, 772
$150, 836
2. Non-expendable
equipment
$5000
$2000
0
$7000
3. Operating
expenses/supplies
$19, 263
$ 19, 717
$15, 709
$54, 689
4. Travel
0
$1000
$1000
$2000
Total Direct Costs
$73, 063
$72, 981
$68, 481
$214, 525
Indirect costs
$27, 264
$27, 967
$27, 971
$83, 201
Annual totals
$100, 327
$101, 428
$95, 492
$297, 247
Category
15
JUSTIFICATION OF THE BUDGET
Wages and salaries: Funds to support one postdoctoral researcher are requested
($40000/year). Funds to support one full time (100%) qualified Ph.D student are
requested ($22000/year).
Operating expenses: Funds are requested for whole-genome sequencing, DNA
manipulation, DNA primer synthesis, plant inoculations and grow plant in the growth
chamber at the greenhouse and costs for publication.
Travel: The estimated budget for principal investigator to travel (United States) is $1000
per year.