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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: [email protected]

Abstract 1 INTRODUCTION Nb is a single dominant gene in potato that confers Genetic control of resistance to pathogens in plants is hypersensitive resistance to potato virus X group 1 and often determined by simple gene-for-gene interactions. group 2 (strains ROTH1 and CP2, respectively). Genetic The resistance response is induced only if the pathogen and molecular analysis showed that Nb is located on the encodes a strain-specific avirulence (avr) gene and the upper arm of chromosome V and form part of a cluster of plant carries the corresponding disease resistance (R) resistance genes encoding specificities to many different gene [1,2]. In many cases this recognition is manifested pathogens. Part of the strategy for cloning Nb was the as a hypersensitive response (HR) which is associated construction of a high-resolution genetic map around the with a programmed cell death (PCD) at the initial site of Nb locus using amplified fragment length polymorphism infection [3]. Two types of resistance to PVX have been (AFLP) technology in conjunction with a bulked identified in potato: hypersensitive resistance that is segregant approach. We describe the genetical localisation controlled by the genes Nb and Nx and extreme resistance of molecular markers tightly linked to the Nb locus and the (ER) that is conferred by the Rx1 and Rx2 genes. Rx1 and development PCR-based markers suitable for isolation of Rx2 are located on chromosome XII and V, respectively the Nb resistance gene by positional cloning. To [4,5]. Nx maps to chromosome IX [6]. Previous studies characterise the viral elicitor of Nb-mediated resistance have shown that two different features of the PVX coat we introduced modifications into the genome of the protein gene are involved in Rx and Nx interactions [7,8]. avirulent PVX strain ROTH1 and the virulent PVX strain In this paper, we have focused on the construction of a UK3. We show that the Nb avirulence determinant high resolution genetic map around the Nb locus and on corresponds to the 25 kDa PVX movement protein and the characterisation of its viral elicitor to study the that the isoleucine residue at position 6 in this protein is molecular basis of the Nb-PVX interaction. required for the activation of the Nb response. To study cellular events associated with the Nb response, the 25 2 MATERIALS AND METHODS kDa proteins of both viral strains were tagged with the green fluorescent protein (GFP). Using laser scanning 2.1 Plant material, virus strains, PVX cDNA confocal microscopy we showed that the Nb-mediated clones, expression vectors and resistance response is associated with degradation of subcellular assays structures. The tetraploid potato cv Pentland Ivory carrying Nb in the Keywords: Nb resistance gene, Solanum tuberosum, PVX, simplex condition (Nb nb nb nb) was self-pollinated to avirulence gene, map-based cloning produce an S1 population. PVX strains ROTH1, CP2, CP4 and UK3 were described previously [7,9,10]. María Rosa Marano and Isabelle Malcuit contributed ROTH1 and CP2 induce the Nb-mediated HR whereas equally to this work. CP4 and UK3 can infect Nb plants systemically. Plants were tested for resistance to PVX avirulent strains by Present address of María Rosa Marano: Instituto de graft inoculation [11] and/or particle bombardment of Biología Molecular y Celular (CONICET), Departamento detached leaves [10,12]. Plants were considered resistant Microbiología, Facultad de Ciencias Bioquímicas y if they displayed HR (necrotic lesions) on leaves Farmacéuticas, Universidad Nacional de Rosario. inoculated with ROTH1 or CP2 avirulent strains and if no Suipacha 531, 2000 Rosario, Argentina. 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. primers derived from GP21 and SPUD237 markers as ROTH1/UK3 hybrid viral genomes and 25K-GFP fusion described previously [11]. Twenty-six individual plants constructs have been described previously [10]. were identified with recombination events in the interval between markers GP21 and SPUD237. After screening of the recombinant plants the two markers SPUD839 and 2.2 PCR-based screening of the mapping TG432, initially found to cosegregate with GP21 and population and AFLP analysis SPUD237 respectively [11], could be separated from GP21 and SPUD237 by 3 and 2 recombination events Genomic DNA for AFLP and PCR-based analyses was (Fig. 1A). isolated according to [5]. Primers, PCR conditions and restriction enzymes used for the markers GP21, SPUD839, TG432 and SPUD237 have been described by Figure 1: High-resolution genetic map of the Nb locus. [11]. To isolate DNA for the resistant (R) and susceptible (S) bulked segregant pools, equivalent amounts of leaf A material from the individuals in each class were pooled Plant Number: 1 3 1 3 3 8 3 3 1 Markers before extraction [13]. Template DNA from the R and S pools as well as from each individual recombinant plant Distances R S A B C D E F G H I (cM) was prepared for AFLP as described previously [14,15], GP21-11 AFLPs 0.23 (3) using the restriction enzymes PstI and MseI. For selective SPUD839 - amplification, 741 primer combinations were analysed: 2 AFLPs 13 PstI/+2 primers (2 selective nucleotides) and 57 MseI/ 0.85 (11) +3 primers (3 selective nucleotides). AFLP reactions were GM339 performed as described by [14]. Bands of interest were 0.30 (4) cut out of the gel with a scalpel and incubated in 150 µl of Nb TE (10 mM Tris pH 7.5, 1 mM EDTA pH 8.0) overnight 0.46 (6) at 37˚C. AFLP fragments were recovered by PCR using TG432-GM637- 2 AFLPs the same conditions as the initial amplification. PCR 0.16 (2) M SPUD237 - products were cloned into the pGEM-T vector (Promega), B 26 AFLPs sequenced and converted to PCR-based markers. The sequences and PCR conditions used for AFLP-derived GM339 PCR markers can be obtained on request. 330 bp

2.3 Bombardment of detached potato leaves and GM637 confocal microscopy experiments 220 bp

The methods employed for transient expression using A. Map position of the Nb locus based on the screening of 741 AFLP particle bombardment have been described previously primer combinations for AFLP markers linked to the Nb locus and 1300 [10,12]. Imaging of GFP fluorescence was performed segregant plants for recombination events in the interval GP21- SPUD237. The genetic distance (in cM) corresponds to the percent of with a LEICA TCS-NT confocal laser scanning recombination between molecular markers or molecular markers and Nb microscope equipped with a 20X or 40X dry objective. in a population of 1300 plants. B. Shows the analysis of individuals with Laser illuminations at 488 and 568 nm (Argon ion laser) recombination events in the GP21-SPUD237 interval using the GM339 and GM637 markers. The resistant pool and susceptible pools are were recorded through a 530 nm or 600 to 630 nm indicated as R and S respectively. Arrows show the sizes of the markers bandpass filter.

3 RESULTS Each recombinant plant was then tested for resistance to PVX avirulent strains CP2 or ROTH1 and Nb was 3.1 Selection of recombinants in the GP21- mapped between SPUD839 and TG432 at 1.15 cM and SPUD237 interval 0.46 cM, respectively (Fig. 1A). 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 resolution map around the Nb locus. This S1 population was screened for recombination events using PCR analysis of DNA samples from individual plants with 3.2 High-resolution genetic map around the Nb containing the 25K gene from the virulent strain UK3, or locus with the wild-type virulent control pUK3 (Fig. 2). To identify DNA markers in the region delimited by the A B . markers SPUD839 and TG432, containing Nb, AFLP technology [15] was employed in conjunction with a ROTH1 Ile6 bulked segregant approach [13]. A total of 741 random Replicase 25K 8K CP selected PstI/MseI primer combinations were analysed and 69 of those revealed polymorphisms between the R 12K and S pools. To map these new polymorphic markers UK3 Ser6 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 co- segregated with GP21, 2 AFLP markers (GM715 and REP31 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 REP13 remaining 26 markers were mapped outside the GP21- SPUD237 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, FA313 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 FA131 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 UK3-Ile6 Ile6 analysis were in accordance with the map presented in Fig. 1.

3.3 Mapping of the Nb avirulence determinant in the PVX genome To identify the PVX avirulence determinant that is Figure 2: Analysis of ROTH1/UK3 hybrids and UK3-Ile6 involved in the activation of Nb-mediated HR in potato, mutant by particle bombardment of detached Nb potato two viral strains were selected, ROTH1 [10] and UK3 [7]. leaves. To map the Nb avirulence determinant in the PVX A. Schematical representation of the wild type and modified PVX genome, a set of hybrid viruses was constructed by genomes. REP31, REP13, FA313 and FA131 were generated by exchanging cDNA fragments between the PVX avirulent strain ROTH1 exchanging cDNA sequences between the avirulent and the Nb resistance-breaking strain UK3 [10]. UK3-Ile6 is a viral strains ROTH1 and the Nb resistance-breaking strain mutant derived from UK3 in which the serine residue at position 6 in the UK3 (Fig. 2A). The interaction between Nb and the 25 kDa protein is replaced by isoleucine [10]. B. Responses induced on Nb leaves three days after bombardment with the hybrid and mutant ROTH1/UK3 viral hybrids was analysed by particle cDNA constructs. HR is manifested by the appearance of necrotic bombardment of these chimeric genomes on potato lesions (black spots) at the site of impact. 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 All of these PVX chimeras induced the formation of avirulent control pROTH1 led to the formation of necrotic symptoms in susceptible (nb) potato plants and lesions 3 days after bombardment, indicative of an accumulated to the same level as the wild-type strains, as interaction between Nb and the corresponding PVX measured by ELISA detection of the viral coat protein avirulence determinant (Fig. 2). In contrast, there were no [10]. Therefore, induction of the Nb-mediated resistance hypersensitive lesions with REP13 and FA131, in potato is dependent upon either the PVX 25 kDa movement protein or its mRNA. Comparative sequence analysis of the 25 kDa proteins of fluorescence reflect degradation of subcellular structures ROTH1, UK3, CP2, and CP4 identified 32 amino acid specifically associated with Nb-mediated HR. 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 A (Ile6) in the 25 kDa protein of ROTH1 and CP2 is p25K1GFP 25K1 GFP 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 Nb- Nb mediated necrosis (Fig. 2), whereas UK3 produced systemic chlorotic symptoms in graft-inoculated Nb plants (Fig. 2). These results indicate that the presence of Ile6 in the 25 kDa protein is required for activation of the Nb response.

3.4 Dynamics of the subcellular distribution of 25KGFP fusion proteins in relation to the Nb- nb mediated response To investigate the cellular processes associated with pathogen-induced HR, GFP was used as a marker [17] to B follow the localisation of the 25 kDa protein in potato p25K3GFP 25K3 GFP 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 Nb 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 C pGFP GFP [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). Nb High resolution imaging of 25KGFP inclusions revealed a network of Y-shaped tubular structures (Fig. 3A). Eventually, starting between 28 and 34 hours after Figure 3: Real time imaging of the subcellular distribution bombardment, these inclusion bodies fragmented into of 25KGFP fusion proteins in Nb and nb potato progressively smaller pieces in Nb potato cells cells. bombarded with the avirulent construct p25K1GFP (Fig. GFP fluorescence was detected by laser scanning confocal microscopy. 3A). After 48 hours, there was no remaining GFP Each picture shows the distribution of GFP fluorescence in different fluorescence in Nb cells bombarded with p25K1GFP, epidermal cells at different times after bombardment of detached Nb and indicating that the cell death process was complete (data nb potato leaves with p25K1GFP (A), p25K3GFP (B) and pGFP (C). not shown). In control experiments involving The CaMV 35S promoter and terminator sequences are indicated by purple and brown boxes respectively. h: hours post-bombardment. The bombardment of nb plants with p25K1GFP and Nb plants position of the nucleus (n) is indicated by an arrow. A large inclusion with p25K3GFP, the green fluorescence was still visible body (i) associated with the nucleus is composed of a network of Y- after 48 hours and the large inclusions remained intact shaped tubular structures. (Fig. 3B). These data suggest that the changes in GFP 5 DISCUSSION associated with cleavage of microfilament proteins by caspases [25]. Previous studies in plants have implicated In this report we have identified new molecular markers caspase-like enzymes in disease resistance [26]. tightly linked to the Nb locus and characterised the PVX Therefore, it is possible that the fragmentation of the elicitor of the Nb-mediated response. 25K1GFP inclusion bodies during the Nb-mediated In the high resolution genetical map described above, we response is due to the action of a caspase-like protease positioned the Nb locus in an interval of approximately and/or the disruption of actin microfilaments. 0.76 cM between the AFLP markers GM339 and GM637 The cloning and characterisation of the Nb gene might (Fig. 2A). Given that the average recombination reveal new domains involved in the recognition of frequency in potato is about 1000 kb.cM-1 [18], the pathogen elicitors and the explanation about the difference distance between the closest flanking markers should between HR provided by Nb and ER conferred by the Rx correspond to approximately 760 kb. However, previous gene to PVX, that are elicited by two different virus- studies in potato have shown that the relationship between encoded proteins, respectively the 25 kDa protein and the genetical and physical distances can vary considerably. coat protein [10,27]. In addition, Nb is particularly For instance, in the case of potato cultivar Cara, which important because of its genetic linkage to other loci was used to isolate the Rx1 gene, recombination conferring resistance against fungus, nematodes and other frequencies were found to vary from 180 kb.cM-1 to 2677 viruses [11,28-30]. Consequently, the isolation of Nb and kb.cM-1, estimated from the number of recombination the physical analysis of this cluster of R genes will provide events in individual BAC clones [19]. Therefore, the a good experimental system to study evolution of R genes physical distance in the genetical interval between in potato and the mechanisms by which plants generate markers GM339 and GM637 cannot be estimated new recognition specificities. 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 6 ACKNOWLEDGEMENTS very large. Future experiments towards the isolation of Nb We thank Gianinna Brigneti for critical reading of the will focus on the screening of a BAC library from potato manuscript. M.R.M. was supported by a Marie Curie cultivar Pentland Ivory with the new tightly linked Research Training Grant no: ERBFMBICT 961303 and is markers. a MCFA member, MN3207. Sainsbury Laboratory is As part of our strategy to analyse the Nb/PVX interaction supported by the Gatsby Charitable Foundation. 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 7 REFERENCES movement of the virus, probably by interacting with [1]H. H. Flor (1971). "Current status of the gene-for-gene concept". plasmodesmata [20,21]. At this stage we do not know Annu.Rev.Phytopathol. 9, 275-298. whether these two different functions are inter-related. It is also possible that Nb, like other resistance genes, is part [2]N. T. Keen (1990). "Gene-for-gene complementarity in plant- of a surveillance system for detection of foreign pathogen interactions". 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