MOLECULAR ANALYSIS OF THE INTERACTION BETWEEN POTATO
VIRUS X AND THE RESISTANCE GENE NB IN SOLANUM TUBEROSUM
María Rosa Marano, Isabelle Malcuit and David C. Baulcombe
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK.
Contact details of submitting author (name underlined):
Tel. 54-341-4370008, Fax. 54-341-4390465
E-mail: marano@ciudad.com.ar
Abstract
Nb is a single dominant gene in potato that confers
hypersensitive resistance to potato virus X group 1 and
group 2 (strains ROTH1 and CP2, respectively). Genetic
and molecular analysis showed that Nb is located on the
upper arm of chromosome V and form part of a cluster of
resistance genes encoding specificities to many different
pathogens. Part of the strategy for cloning Nb was the
construction of a high-resolution genetic map around the
Nb locus using amplified fragment length polymorphism
(AFLP) technology in conjunction with a bulked
segregant approach. We describe the genetical localisation
of molecular markers tightly linked to the Nb locus and the
development PCR-based markers suitable for isolation of
the Nb resistance gene by positional cloning. To
characterise the viral elicitor of Nb-mediated resistance
we introduced modifications into the genome of the
avirulent PVX strain ROTH1 and the virulent PVX strain
UK3. We show that the Nb avirulence determinant
corresponds to the 25 kDa PVX movement protein and
that the isoleucine residue at position 6 in this protein is
required for the activation of the Nb response. To study
cellular events associated with the Nb response, the 25
kDa proteins of both viral strains were tagged with the
green fluorescent protein (GFP). Using laser scanning
confocal microscopy we showed that the Nb-mediated
response is associated with degradation of subcellular
structures.
Keywords: Nb resistance gene, Solanum tuberosum, PVX,
avirulence gene, map-based cloning
María Rosa Marano and Isabelle Malcuit contributed
equally to this work.
Present address of María Rosa Marano: Instituto de
Biología Molecular y Celular (CONICET), Departamento
Microbiología, Facultad de Ciencias Bioquímicas y
Farmacéuticas, Universidad Nacional de Rosario.
Suipacha 531, 2000 Rosario, Argentina.
1 INTRODUCTION
Genetic control of resistance to pathogens in plants is
often determined by simple gene-for-gene interactions.
The resistance response is induced only if the pathogen
encodes a strain-specific avirulence (avr) gene and the
plant carries the corresponding disease resistance (R) gene
[1,2]. In many cases this recognition is manifested as a
hypersensitive response (HR) which is associated with a
programmed cell death (PCD) at the initial site of
infection [3]. Two types of resistance to PVX have been
identified in potato: hypersensitive resistance that is
controlled by the genes Nb and Nx and extreme resistance
(ER) that is conferred by the Rx1 and Rx2 genes. Rx1 and
Rx2 are located on chromosome XII and V, respectively
[4,5]. Nx maps to chromosome IX [6]. Previous studies
have shown that two different features of the PVX coat
protein gene are involved in Rx and Nx interactions [7,8].
In this paper, we have focused on the construction of a
high resolution genetic map around the Nb locus and on
the characterisation of its viral elicitor to study the
molecular basis of the Nb-PVX interaction.
2 MATERIALS AND METHODS
2.1 Plant material, virus strains, PVX cDNA
clones, expression vectors and resistance assays
The tetraploid potato cv Pentland Ivory carrying Nb in the
simplex condition (Nb nb nb nb) was self-pollinated to
produce an S1 population. PVX strains ROTH1, CP2,
CP4 and UK3 were described previously [7,9,10].
ROTH1 and CP2 induce the Nb-mediated HR whereas
CP4 and UK3 can infect Nb plants systemically. Plants
were tested for resistance to PVX avirulent strains by
graft inoculation [11] and/or particle bombardment of
detached leaves [10,12]. Plants were considered resistant
if they displayed HR (necrotic lesions) on leaves
inoculated with ROTH1 or CP2 avirulent strains and if no
virus was detected on non-inoculated leaves. Plants were
considered susceptible if after challenging with ROTH1
or CP2 the plants became systemically infected with the
virus and it could be detected in non-inoculated leaves.
ROTH1/UK3 hybrid viral genomes and 25K-GFP fusion
constructs have been described previously [10].
2.2 PCR-based screening of the mapping
population and AFLP analysis
Genomic DNA for AFLP and PCR-based analyses was
isolated according to [5]. Primers, PCR conditions and
restriction enzymes used for the markers GP21,
SPUD839, TG432 and SPUD237 have been described by
[11]. To isolate DNA for the resistant (R) and susceptible
(S) bulked segregant pools, equivalent amounts of leaf
material from the individuals in each class were pooled
before extraction [13]. Template DNA from the R and S
pools as well as from each individual recombinant plant
was prepared for AFLP as described previously [14,15],
using the restriction enzymes PstI and MseI. For selective
amplification, 741 primer combinations were analysed: 13
PstI/+2 primers (2 selective nucleotides) and 57 MseI/+3
primers (3 selective nucleotides). AFLP reactions were
performed as described by [14]. Bands of interest were
cut out of the gel with a scalpel and incubated in 150 µl of
TE (10 mM Tris pH 7.5, 1 mM EDTA pH 8.0) overnight
at 37˚C. AFLP fragments were recovered by PCR using
the same conditions as the initial amplification. PCR
products were cloned into the pGEM-T vector (Promega),
sequenced and converted to PCR-based markers. The
sequences and PCR conditions used for AFLP-derived
PCR markers can be obtained on request.
resolution map around the Nb locus. This S1 population
was screened for recombination events using PCR
analysis of DNA samples from individual plants with
primers derived from GP21 and SPUD237 markers as
described previously [11]. Twenty-six individual plants
were identified with recombination events in the interval
between markers GP21 and SPUD237. After screening of
the recombinant plants the two markers SPUD839 and
TG432, initially found to cosegregate with GP21 and
SPUD237 respectively [11], could be separated from
GP21 and SPUD237 by 3 and 2 recombination events
(Fig. 1A).
A
Plant Number: 1
3
1
3
3
8
3
Distances R S A B C
(cM)
D
E F G
3
1
Markers
H I
GP21-11 AFLPs
0.23 (3)
SPUD839 2 AFLPs
0.85 (11)
GM339
0.30 (4)
Nb
0.46 (6)
0.16 (2)
B
M
TG432-GM6372 AFLPs
SPUD237 26 AFLPs
GM339
330 bp
2.3 Bombardment of detached potato leaves and
confocal microscopy experiments
The methods employed for transient expression using
particle bombardment have been described previously
[10,12]. Imaging of GFP fluorescence was performed
with a LEICA TCS-NT confocal laser scanning
microscope equipped with a 20X or 40X dry objective.
Laser illuminations at 488 and 568 nm (Argon ion laser)
were recorded through a 530 nm or 600 to 630 nm
bandpass filter.
3 RESULTS
3.1 Selection of recombinants in the GP21SPUD237 interval
Nb was previously mapped on chromosome V in a 3.3
centimorgan (cM) interval delimited by markers
GP21/SPUD839 and TG432/SPUD237 [11]. We
extended the original mapping population to a total of
1300 S1 progeny to allow the construction of a high
220 bp
GM637
Figure 1: High-resolution genetic map of the Nb locus.
A. Map position of the Nb locus based on the screening of 741 AFLP
primer combinations for AFLP markers linked to the Nb locus and 1300
segregant plants for recombination events in the interval GP21SPUD237. The genetic distance (in cM) corresponds to the percent of
recombination between molecular markers or molecular markers and Nb
in a population of 1300 plants. B. Shows the analysis of individuals
with recombination events in the GP21-SPUD237 interval using the
GM339 and GM637 markers. The resistant pool and susceptible pools
are indicated as R and S respectively. Arrows show the sizes of the
markers
Each recombinant plant was then tested for resistance to
PVX avirulent strains CP2 or ROTH1 and Nb was
mapped between SPUD839 and TG432 at 1.15 cM and
0.46 cM, respectively (Fig. 1A).
3.2 High-resolution genetic map around the Nb
locus
To identify DNA markers in the region delimited by the
markers SPUD839 and TG432, containing Nb, AFLP
technology [15] was employed in conjunction with a
bulked segregant approach [13]. A total of 741 random
selected PstI/MseI primer combinations were analysed
and 69 of those revealed polymorphisms between the R
and S pools. To map these new polymorphic markers
relative to SPUD839 and TG432, the AFLP analysis was
repeated using the recombinants detected in the GP21 and
SPUD237 interval. Of these, 11 AFLP markers cosegregated with GP21, 2 AFLP markers (GM715 and
GM1350, Fig. 1A) co-segregated with SPUD839, 1 AFLP
marker (GM339, Fig. 1A) was mapped between
SPUD839 and Nb, 3 AFLP markers (GM637, GM1216
and GM1219, Fig. 1A) co-segregated with TG432, 26
AFLP markers co-segregated with SPUD237 and the
remaining 26 markers were mapped outside the GP21SPUD237 interval (Fig. 1A). The result of this analysis
places Nb in an interval of 0.76 cM, flanked by the marker
GM339 and the cosegregating markers TG432, GM637,
GM1216 and GM1219 (Fig. 1A). To develop a fast
screening method, the AFLP markers closest to Nb and
bigger in length, GM339 and GM637, were converted to
PCR markers. GM339-derived primers amplified a 330 bp
fragment and GM637-derived primers a 220 bp fragment
(Fig. 1B). The GM339 and GM637 primers amplified
only the resistant allele and were used to analyse the 26
plants with recombination events between GP21 and
SPUD237. The results obtained from this analysis were in
accordance with the map presented in Fig. 1.
virulent strain UK3, or with the wild-type virulent control
pUK3 (Fig. 2).
A
B
ROTH1
Replicase
.
Ile6
25K 8K
CP
12K
UK3
Ser6
REP31
REP13
FA313
FA131
UK3-Ile6
Ile6
3.3 Mapping of the Nb avirulence determinant
in the PVX genome
To identify the PVX avirulence determinant that is
involved in the activation of Nb-mediated HR in potato,
two viral strains were selected, ROTH1 [10] and UK3 [7].
To map the Nb avirulence determinant in the PVX
genome, a set of hybrid viruses was constructed by
exchanging cDNA sequences between the avirulent strains
ROTH1 and the Nb resistance-breaking strain UK3 (Fig.
2A). The interaction between Nb and the ROTH1/UK3
viral hybrids was analysed by particle bombardment of
these chimeric genomes on potato plants. Bombardment
of detached resistant (Nb) potato leaves with viral cDNA
hybrids carrying the 25K gene from ROTH1 (REP31 and
FA313) or the wild-type avirulent control pROTH1 led to
the formation of necrotic lesions 3 days after
bombardment, indicative of an interaction between Nb
and the corresponding PVX avirulence determinant (Fig.
2). In contrast, there were no hypersensitive lesions with
REP13 and FA131, containing the 25K gene from the
Figure 2: Analysis of ROTH1/UK3 hybrids and UK3-Ile6
mutant by particle bombardment of detached Nb potato
leaves.
A. Schematical representation of the wild type and modified PVX
genomes. REP31, REP13, FA313 and FA131 were generated by
exchanging cDNA fragments between the PVX avirulent strain ROTH1
and the Nb resistance-breaking strain UK3 [10]. UK3-Ile6 is a viral
mutant derived from UK3 in which the serine residue at position 6 in
the 25 kDa protein is replaced by isoleucine [10]. B. Responses induced
on Nb leaves three days after bombardment with the hybrid and mutant
cDNA constructs. HR is manifested by the appearance of necrotic
lesions (black spots) at the site of impact.
All of these PVX chimeras induced the formation of
symptoms in susceptible (nb) potato plants and
accumulated to the same level as the wild-type strains, as
measured by ELISA detection of the viral coat protein
[10]. Therefore, induction of the Nb-mediated resistance
in potato is dependent upon either the PVX 25 kDa
movement protein or its mRNA.
Comparative sequence analysis of the 25 kDa proteins of
ROTH1, UK3, CP2, and CP4 identified 32 amino acid
substitutions in total [16]. However, there is only one
amino acid difference when the 25 kDa protein sequences
of ROTH1 and CP2 were compared to that of UK3 and
CP4 respectively [10,16]. The isoleucine at position 6
(Ile6) in the 25 kDa protein of ROTH1 and CP2 is
replaced by a serine (Ser6) in UK3 or a threonine (Thr6) in
CP4 [10]. This conservation of Ile6 in the two avirulent
strains indicates that it is probably essential for activation
of the Nb response. To test this hypothesis, a mutant
transcription clone was generated, resulting in a single
substitution in the UK3 25 kDa protein sequence at
position 6 (Ser6Ile6: UK3-Ile6 mutant) [10]. When this
construct was tested on plants, UK3-Ile6 induced Nbmediated necrosis (Fig. 2), whereas UK3 produced
systemic chlorotic symptoms in graft-inoculated Nb plants
(Fig. 2). These results indicate that the presence of Ile 6 in
the 25 kDa protein is required for activation of the Nb
response.
of subcellular structures specifically associated with Nbmediated HR.
A
25K1
GFP
p25K3GFP
25K3
GFP
Nb
3.4 Dynamics of the subcellular distribution of
25KGFP fusion proteins in relation to the Nbmediated response
To investigate the cellular processes associated with
pathogen-induced HR, GFP was used as a marker [17] to
follow the localisation of the 25 kDa protein in potato
cells and monitor cellular changes specifically associated
with the activation of Nb-mediated HR. This gene was
fused to the 3’ end of the 25K gene of ROTH1
(p25K1GFP) and UK3 (p25K3GFP), (Fig. 3).
GFP fluorescence was detected 6 hours after
bombardment in epidermal cells for all the constructs
tested (Fig. 3). Free GFP expressed from the CaMV 35S
promoter (pGFP construct) accumulated in the nucleus
and was found in the cytoplasm, as described previously
[17] (Fig. 3C). In contrast, GFP fluorescence in potato
cells expressing the 25K1GFP and 25K3GFP fusion
proteins was initially localised in inclusions in the
cytoplasm of Nb and nb cells and also seemed to be
present in or associated with the nucleus (Fig. 3A,B).
High resolution imaging of 25KGFP inclusions revealed a
network of Y-shaped tubular structures (Fig. 3A).
Eventually, starting between 28 and 34 hours after
bombardment, these inclusion bodies fragmented into
progressively smaller pieces in Nb potato cells bombarded
with the avirulent construct p25K1GFP (Fig. 3A). After
48 hours, there was no remaining GFP fluorescence in Nb
cells bombarded with p25K1GFP, indicating that the cell
death process was complete (data not shown). In control
experiments involving bombardment of nb plants with
p25K1GFP and Nb plants with p25K3GFP, the green
fluorescence was still visible after 48 hours and the large
inclusions remained intact (Fig. 3B). These data suggest
that the changes in GFP fluorescence reflect degradation
p25K1GFP
nb
B
Nb
C
pGFP
GFP
Nb
Figure 3: Real time imaging of the subcellular distribution
of 25KGFP fusion proteins in Nb and nb potato cells.
GFP fluorescence was detected by laser scanning confocal microscopy.
Each picture shows the distribution of GFP fluorescence in different
epidermal cells at different times after bombardment of detached Nb
and nb potato leaves with p25K1GFP (A), p25K3GFP (B) and pGFP
(C). The CaMV 35S promoter and terminator sequences are indicated
by purple and brown boxes respectively. h: hours post-bombardment.
The position of the nucleus (n) is indicated by an arrow. A large
inclusion body (i) associated with the nucleus is composed of a network
of Y-shaped tubular structures.
5 DISCUSSION
In this report we have identified new molecular markers
tightly linked to the Nb locus and characterised the PVX
elicitor of the Nb-mediated response.
In the high resolution genetical map described above, we
positioned the Nb locus in an interval of approximately
0.76 cM between the AFLP markers GM339 and GM637
(Fig. 2A). Given that the average recombination
frequency in potato is about 1000 kb.cM-1 [18], the
distance between the closest flanking markers should
correspond to approximately 760 kb. However, previous
studies in potato have shown that the relationship between
genetical and physical distances can vary considerably.
For instance, in the case of potato cultivar Cara, which
was used to isolate the Rx1 gene, recombination
frequencies were found to vary from 180 kb.cM -1 to 2677
kb.cM-1, estimated from the number of recombination
events in individual BAC clones [19]. Therefore, the
physical distance in the genetical interval between
markers GM339 and GM637 cannot be estimated
accurately at this stage. However, the small number of
AFLP markers identified near the GP21 and SPUD237
interval and their resistant-allele specificity may indicate
that the physical distance in this genetic interval is not
very large. Future experiments towards the isolation of Nb
will focus on the screening of a BAC library from potato
cultivar Pentland Ivory with the new tightly linked markers.
As part of our strategy to analyse the Nb/PVX interaction
we have also shown that the PVX 25 kDa protein is the
elicitor of Nb-mediated HR. In compatible interactions the
role of this protein is to facilitate cell-to-cell movement of
the virus, probably by interacting with plasmodesmata
[20,21]. At this stage we do not know whether these two
different functions are inter-related. It is also possible that
Nb, like other resistance genes, is part of a surveillance
system for detection of foreign molecules in plant cells
[22]. Analysis of both resistance-inducing (ROTH1) and
resistance-breaking (UK3) strains showed that the elicitor
function involves the isoleucine at position 6 (Ile6) in the
25 kDa protein. We used 25KGFP fusions to study
subcellular distribution of the PVX 25 kDa protein in
living potato cells, and to monitor its fate and other
cellular events associated with Nb-mediated cell death.
The HR-eliciting property of the 25 kDa protein allowed
the visualisation by laser scanning confocal microscopy of
a cellular process specifically associated with the Nbmediated response. The large GFP-labelled perinuclear
bodies in the bombarded potato cells expressing the
25KGFP fusion may represent the lamellar inclusions
detected previously by immunocytochemistry and in
ultrastructural studies [23]. These inclusion bodies are
intimately associated with the endoplasmic reticulum and
with actin microfilaments [24]. As the Nb-elicited cells
died, these perinuclear structures fragmented and
disappeared. In animal cells, programmed cell death was
associated with cleavage of microfilament proteins by
caspases [25]. Previous studies in plants have implicated
caspase-like enzymes in disease resistance [26].
Therefore, it is possible that the fragmentation of the
25K1GFP inclusion bodies during the Nb-mediated
response is due to the action of a caspase-like protease
and/or the disruption of actin microfilaments.
The cloning and characterisation of the Nb gene might
reveal new domains involved in the recognition of pathogen
elicitors and the explanation about the difference between
HR provided by Nb and ER conferred by the Rx gene to
PVX, that are elicited by two different virus-encoded
proteins, respectively the 25 kDa protein and the coat
protein [10,27]. In addition, Nb is particularly important
because of its genetic linkage to other loci conferring
resistance against fungus, nematodes and other viruses
[11,28-30]. Consequently, the isolation of Nb and the
physical analysis of this cluster of R genes will provide a
good experimental system to study evolution of R genes in
potato and the mechanisms by which plants generate new
recognition specificities.
6
ACKNOWLEDGEMENTS
We thank Gianinna Brigneti for critical reading of the
manuscript. M.R.M. was supported by a Marie Curie
Research Training Grant no: ERBFMBICT 961303 and is
a MCFA member, MN3207. Sainsbury Laboratory is
supported by the Gatsby Charitable Foundation.
7
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