[show abstract][hide abstract] ABSTRACT: To evaluate the role of salicylic acid (SA) in Nb-mediated hypersensitive resistance to Potato virus X (PVX) avirulent strain ROTH1 in Solanum tuberosum, we have constructed SA-deficient transgenic potato plant lines by overexpressing the bacterial enzyme salicylate hydroxylase (NahG), which degrades SA. Evaluation of these transgenic lines revealed hydrogen peroxide accumulation and spontaneous lesion formation in an age- and light-dependent manner. In concordance, NahG potato plants were more sensitive to treatment with methyl viologen, a reactive oxygen species-generating compound. In addition, when challenged with PVX ROTH1, NahG transgenic lines showed a decreased disease-resistance response to infection and were unable to induce systemic acquired resistance. However, the avirulent viral effector, the PVX 25-kDa protein, does induce expression of the pathogenesis-related gene PR-1a in NahG potato plants. Taken together, our data indicate that SA is involved in local and systemic defense responses mediated by the Nb gene in Solanum tuberosum. This is the first report to show that basal levels of SA correlate with hypersensitive resistance to PVX.
[show abstract][hide abstract] ABSTRACT: In animals and plants, innate immunity is regulated by nucleotide binding domain and leucine-rich repeat (NB-LRR) proteins that mediate pathogen recognition and that activate host-cell defense responses. Plant NB-LRR proteins, referred to as R proteins, have amino-terminal domains that contain a coiled coil (CC) or that share similarity with animal Toll and interleukin 1 receptors (TIR). To investigate R protein function, we are using the TIR-NB-LRR protein N that mediates resistance against tobacco mosaic virus (TMV) through recognition of the TMV p50 protein. Here, we describe N requirement gene 1 (NRG1), a novel N-resistance component that was identified by a virus-induced gene silencing (VIGS) screen of a cDNA library. Surprisingly, NRG1 encodes an NB-LRR type R protein that, in contrast to N, contains a CC rather than a TIR domain. Our findings support emerging evidence that many disease-resistance pathways each recruit more than a single NB-LRR protein. The results also indicate that, in addition to the previously recognized role in elicitor recognition, NB-LRR proteins may also function in downstream signaling pathways.
Current Biology 06/2005; 15(10):968-73. · 9.49 Impact Factor
[show abstract][hide abstract] ABSTRACT: Virus-induced gene silencing was used to assess the function of random Nicotiana benthamiana cDNAs in disease resistance. Out of 4992 cDNAs tested from a normalized library, there were 79 that suppressed a hypersensitive response (HR) associated with Pto-mediated resistance against Pseudomonas syringae. However, only six of these clones blocked the Pto-mediated suppression of P.syringae growth. The three clones giving the strongest loss of Pto resistance had inserts corresponding to HSP90 and also caused loss of Rx-mediated resistance against potato virus X and N-mediated tobacco mosaic virus resistance. The role of HSP90 as a cofactor of disease resistance is associated with stabilization of Rx protein levels and could be accounted for in part by SGT1 and other cofactors of disease resistance acting as co-chaperones. This approach illustrates the potential benefits and limitations of RNA silencing in forward screens of gene function in plants.
The EMBO Journal 12/2003; 22(21):5690-9. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying inserts derived from host genes the process can be additionally targeted against the corresponding mRNAs. VIGS has been used widely in plants for analysis of gene function and has been adapted for high-throughput functional genomics. Until now most applications of VIGS have been in Nicotiana benthamiana. However, new vector systems and methods are being developed that could be used in other plants, including Arabidopsis. Here we discuss practical and theoretical issues that are specific to VIGS rather than other gene "knock down" or "knockout" approaches to gene function. We also describe currently used protocols that have allowed us to apply VIGS to the identification of genes required for disease resistance in plants. These methods and the underlying general principles also apply when VIGS is used in the analysis of other aspects of plant biology.
[show abstract][hide abstract] ABSTRACT: Nb is a single dominant gene in potato that confers hypersensitive resistance to potato virus X (PVX) isolates from strain groups 1 and 2. Genetic and molecular analyses showed that Nb is located on the upper arm of chromosome V and forms part of a cluster of resistance genes encoding specificities to many different pathogens. 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. A bulked segregant approach was applied to identify polymorphic AFLP markers tightly linked to the Nb locus. These markers were mapped in a population of segregating S1 progeny (1,300 plants) from a self-pollinated potato cultivar, Pentland Ivory. From this analysis, Nb was placed in an interval of 0.76 cM, flanked by the AFLP markers GM339 and GM637. Recombinant PVX strains carrying different combinations of avirulence genes were used in biological assays to show that Nb was also present in potato cv. Cara but was masked by the extreme PVX resistance conferred by the Rx gene. PCR-based screening of a Cara genomic BAC library with markers closest to the Nb locus identified a new marker tightly linked to Nb.
Theoretical and Applied Genetics 09/2002; 105(2-3):192-200. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Homologues of the yeast ubiquitin ligase-associated protein SGT1 are required for disease resistance in plants mediated by nucleotide-binding site/leucine-rich repeat (NBS-LRR) proteins. Here, by silencing SGT1 in Nicotiana benthamiana, we extend these findings and demonstrate that SGT1 has an unexpectedly general role in disease resistance. It is required for resistance responses mediated by NBS-LRR and other R proteins in which pathogen-derived elicitors are recognized either inside or outside the host plant cell. A requirement also exists for SGT1 in nonhost resistance in which all known members of a host species are resistant against every characterized isolate of a pathogen. Our findings show that silencing SGT1 affects diverse types of disease resistance in plants and support the idea that R protein-mediated and nonhost resistance may involve similar mechanisms.
Proceedings of the National Academy of Sciences 09/2002; 99(16):10865-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Resistance to potato virus X (PVX) is determined by the product of a host resistance gene and a viral determinant specifying either virulence (resistance-breaking ability) or avirulence (resistance sensitivity). The viral coat protein is the determinant of resistance mediated by the host Nx gene while the 25 kDa movement protein is the determinant of Nb-mediated resistance. Group 1 and group 4 strains of PVX are avirulent or virulent respectively for both these determinants while group 2 and group 3 strains are virulent for one but avirulent for the other determinant. There are two alternative evolutionary mechanisms by which the various strain groups might have evolved: either by recombination between strains carrying virulence (or avirulence) determinants that evolved once only, or alternatively, by independent evolution of at least one virulence (or avirulence) determinant in distinct phylogenetic branches. These alternative hypotheses were investigated by (i) determining the complete genomic sequence of a group 1 and a group 4 strain and (ii) comparing the completely sequenced genomes of six isolates representative of the four strain groups. The analysis revealed the same phylogeny for all five PVX genes. Thus, there is no evidence that the PVX strain groups evolved by recombination.
[show abstract][hide abstract] 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.