Physical association of the NB-LRR resistance protein Rx with a Ran GTPase-activating protein is required for extreme resistance to Potato virus X

Nucleotide binding leucine-rich repeat (NB-LRR) proteins play an important role in plant and mammalian innate immunity. In plants, these resistance proteins recognize specific pathogen-derived effector proteins. Recognition subsequently triggers a rapid and efficient defense response often associated with the hypersensitive response and other poorly understood processes that suppress the pathogen. To investigate mechanisms associated with the activation of disease resistance responses, we investigated proteins binding to the potato (Solanum tuberosum) NB-LRR protein Rx that confers extreme resistance to Potato virus X (PVX) in potato and Nicotiana benthamiana. By affinity purification experiments, we identified an endogenous N. benthamiana Ran GTPase-Activating Protein2 (RanGAP2) as an Rx-associated protein in vivo. Further characterization confirmed the specificity of this interaction and showed that the association occurs through their N-terminal domains. By specific virus-induced gene silencing of RanGAP2 in N. benthamiana carrying Rx, we demonstrated that this interaction is required for extreme resistance to PVX and suggest that RanGAP2 is part of the Rx signaling complex. These results implicate RanGAP-mediated cellular mechanisms, including nucleocytoplasmic trafficking, in the activation of disease resistance.     

Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death

Many plant disease resistance (R) genes encode proteins predicted to have an N-terminal coiled-coil (CC) domain, a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. These CC-NBS-LRR proteins recognize specific pathogen-derived products and initiate a resistance response that often includes a type of cell death known as the hypersensitive response (HR). Co-expression of the potato CC-NBS-LRR protein Rx and its elicitor, the PVX coat protein (CP), results in a rapid HR. Surprisingly, co-expression of the LRR and CC-NBS as separate domains also resulted in a CP-dependent HR. Likewise, the CC domain complemented a version of Rx lacking this domain (NBS- LRR). Correspondingly, the LRR domain interacted physically in planta with the CC-NBS, as did CC with NBS-LRR. Both interactions were disrupted in the presence of CP. However, the interaction between CC and NBS-LRR was dependent on a wild-type P-loop motif, whereas the interaction between CC-NBS and LRR was not. We propose that activation of Rx entails sequential disruption of at least two intramolecular interactions.     

Perturbation of nuclear–cytosolic shuttling of Rx1 compromises extreme resistance and translational arrest of potato virus X transcripts

Many plant intracellular immune receptors mount a hypersensitive response (HR) upon pathogen perception. The concomitant localized cell death is proposed to trap pathogens, such as viruses, inside infected cells, thereby preventing their spread. Notably, extreme resistance (ER) conferred by the potato immune receptor Rx1 to potato virus X (PVX) does not involve the death of infected cells. It is unknown what defines ER and how it differs from HR-based resistance. Interestingly, Rx1 can trigger an HR, but only upon artificial (over)expression of PVX or its avirulence coat protein (CP). Rx1 has a nucleocytoplasmic distribution and both pools are required for HR upon transient expression of a PVX-GFP amplicon. It is unknown whether mislocalized Rx1 variants can induce ER upon natural PVX infection. Here, we generated transgenic Nicotiana benthamiana producing nuclear- or cytosol-restricted Rx1 variants. We found that these variants can still mount an HR. However, nuclear- or cytosol-restricted Rx1 variants can no longer trigger ER or restricts viral infection. Interestingly, unlike the mislocalized Rx1 variants, wild-type Rx1 was found to compromise CP protein accumulation. We show that the lack of CP accumulation does not result from its degradation but is likely to be linked with translational arrest of its mRNA. Together, our findings suggest that translational arrest of viral genes is a major component of ER and, unlike the HR, is required for resistance to PVX.     

Systemic acquired resistance is induced by R gene-mediated responses independent of cell death

On infection by pathogens, plants initiate defence responses that are able to curtail infection locally. These responses are mediated either by receptor-like proteins that recognize pathogen-associated molecular patterns or by the protein products of disease resistance ( R ) genes. At the same time, primary defence responses often result in the generation of signals that induce what is known as systemic acquired resistance (SAR), such that defence responses are enhanced on secondary pathogen challenge in distal tissues. R protein-mediated SAR induction is normally accompanied by a type of programmed cell death known as the hypersensitive response (HR) and, in some instances, cell death alone has been implicated in the induction of SAR. This has raised the question of whether R protein-mediated signalling per se induces SAR or whether SAR is an indirect result of the induction of HR. Using the Rx gene of potato, which confers resistance to Potato Virus X in the absence of cell death, we have shown that the HR is dispensable for R protein-mediated induction of SAR and that Rx-induced SAR is mediated by the same salicylate-dependent pathway induced by other R proteins.     

Constitutive gain-of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato

Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRR) that confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure-function relationships of NBS-LRR proteins we exploited the potential of Rx to mediate a cell death response. With wild-type Rx cell death is elicited only in the presence of the viral coat protein. However, following random mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat protein. Out of 2500 Rx clones tested there were seven constitutive gain-of-function mutants carrying eight independent mutations. The mutations encoded changes in the LRR or in conserved RNBS-D and MHD motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and LRR. The constitutive gain-of-function phenotypes would be due to deletion or modification of these inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not active unless the protein is overexpressed.     

High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance

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.     

Functionally Homologous Host Components Recognize Potato Virus X in Gomphrena globosa and Potato

All known isolates of potato virus X (PVX), with the exception of a South American isolate PVXHB, induce an extreme resistance response on potato carrying the Rx gene and elicit the production of necrotic lesions on Gomphrena globosa: PVXHB establishes systemic infection on Rx genotypes of potato and infects the inoculated leaf of G. globosa without lesion formation. Previously, we have shown that the Rx-mediated resistance is affected by a feature of the coat protein that depends on the presence of a threonine residue at position 121 in the coat protein of PVXCP4 and that the resistance is an induced response expressed in protoplasts of potato with the Rx genotype. In this study, we provide evidence, based on the analysis of PVXCP4/PVXHB hybrids, that the elicitation of lesions on G. globosa also requires the presence of a threonine residue at position 121 of the viral coat protein. The lesion-forming phenotype was not associated with the ability of the viral isolate to accumulate in the infected plant. We therefore propose that there is a homologous component of both potato carrying Rx and G. globosa that interacts with a feature of the PVX coat protein and, following the interaction, activates an induced response in the plant cell.     

Rds and Rih mediate hypersensitive cell death independent of gene-for-gene resistance to the oat crown rust pathogen Puccinia coronata f. sp. avenae.

The Pca crown rust resistance cluster in the diploid Avena genus confers gene-for-gene specificity to numerous isolates of Puccinia coronata f. sp. avenae. Recombination breakpoint analysis indicates that specificities conferred by the Pca cluster are controlled by at least five distinct genes, designated Pc81, Pc82, Pc83, Pc84, and Pc85. Avena plants with the appropriate genotype frequently respond to P. coronata by undergoing hypersensitive cell death at the sites of fungal infection. Autofluorescence of host cells in response to P. coronata occurs in plants that develop visible necrotic lesions but not in plants that lack this phenotype. Two newly described, non-Pc loci were shown to control hypersensitive cell death. Rds (resistance-dependent suppressor of cell death) suppresses the hypersensitive response (HR), but not the resistance, mediated by the Pc82 resistance gene. In contrast, Rih (resistance-independent hypersensitive cell death) confers HR in both resistant and susceptible plants. Linkage analysis indicates that Rds is unlinked to the Pca cluster, whereas Rih is tightly linked to it. These results indicate that multiple synchronous pathways affect the development of hypersensitive cell death and that HR is not essential for resistance to crown rust. Further characterization of these genes will clarify the relationship between plant disease resistance and localized hypersensitive cell death.     

The coiled-coil and nucleotide binding domains of the Potato Rx disease resistance protein function in pathogen recognition and signaling

Plant genomes encode large numbers of nucleotide binding and leucine-rich repeat (NB-LRR) proteins, some of which mediate the recognition of pathogen-encoded proteins. Following recognition, the initiation of a resistance response is thought to be mediated by the domains present at the N termini of NB-LRR proteins, either a Toll and Interleukin-1 Receptor or a coiled-coil (CC) domain. In order to understand the role of the CC domain in NB-LRR function, we have undertaken a systematic structure-function analysis of the CC domain of the potato (Solanum tuberosum) CC-NB-LRR protein Rx, which confers resistance to Potato virus X. We show that the highly conserved EDVID motif of the CC domain mediates an intramolecular interaction that is dependent on several domains within the rest of the Rx protein, including the NB and LRR domains. Other conserved and nonconserved regions of the CC domain mediate the interaction with the Ran GTPase-activating protein, RanGAP2, a protein required for Rx function. Furthermore, we show that the Rx NB domain is sufficient for inducing cell death typical of hypersensitive plant resistance responses. We describe a model of CC-NB-LRR function wherein the LRR and CC domains coregulate the signaling activity of the NB domain in a recognition-specific manner.     

A RanGAP protein physically interacts with the NB-LRR protein Rx, and is required for Rx-mediated viral resistance

Race-specific disease resistance in plants is mediated by the products of host disease resistance (R) genes. Plant genomes possess hundreds of R gene homologs encoding nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. NB-LRR proteins induce a disease resistance response following recognition of pathogen-encoded avirulence (Avr) proteins. However, little is known about the general mechanisms by which NB-LRR proteins recognize Avr proteins or how they subsequently induce defense responses. The Rx NB-LRR protein of potato confers resistance to potato virus X (PVX). Using a co-purification strategy, we have identified a Ran GTPase-activating protein (RanGAP2) as an Rx-interacting protein. We show by co-immunoprecipitation that this interaction is mediated in planta through the putative signaling domain at the Rx amino terminus. Overexpression of RanGAP2 results in activation of certain Rx derivatives. Likewise, knocking down RanGAP2 expression in Nicotiana benthamiana by virus-induced gene silencing compromises Rx-mediated resistance to PVX. Thus, we have demonstrated a novel role for a RanGAP in the function of a plant disease resistance response.     

High-resolution genetic map of Nb,a gene that confers hypersensitive resistance to potato virus X in Solanum tuberosum

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.     

Coordinated Activation of Programmed Cell Death and Defense Mechanisms in Transgenic Tobacco Plants Expressing a Bacterial Proton Pump

In plants, programmed cell death is thought to be activated during the hypersensitive response to certain avirulent pathogens and in the course of several differentiation processes. We describe a transgenic model system that mimics the activation of programmed cell death in higher plants. In this system, expression of a bacterial proton pump in transgenic tobacco plants activates a cell death pathway that may be similar to that triggered by recognition of an incompatible pathogen. Thus, spontaneous lesions that resemble hypersensitive response lesions are formed, multiple defense mechanisms are apparently activated, and systemic resistance is induced in the absence of a pathogen. Interestingly, mutation of a single amino acid in the putative channel of this proton pump renders it inactive with respect to lesion formation and induction of resistance to pathogen challenge. This transgenic model system may provide insights into the mechanisms involved in mediating cell death in higher plants. In addition, it may also be used as a general agronomic tool to enhance disease protection.     

A Potato Virus X Resistance Gene Mediates an Induced,Nonspecific Resistance in Protoplasts

The Rx locus in potato controls extreme resistance to most isolates of potato virus X (PVX). The resistance is expressed in whole plants and in protoplasts. Rx-mediated resistance in protoplasts causes reduced accumulation of all PVX RNA species, including the (-) strand RNA after a lag of 8 hr postinoculation. In work reported elsewhere, we have shown that the Rx-breaking property of PVXHB was associated with the coat protein gene of PVXUK3 and PVXCP4. Here, we describe how a frameshift mutation in the coat protein gene had no effect on Rx resistance breaking but compromised the Rx-mediated resistance to PVXCP4. We also describe how in coinoculation experiments, the Rx-mediated resistance could be induced to affect PVXHB or cucumber mosaic virus (CMV). In these experiments, PVXHB or CMV was coinoculated to protoplasts (Rx genotype) together with an isolate of PVX, which is affected by Rx. We interpret this data to indicate that Rx-mediated resistance is induced when the PVX coat protein is produced in the infected cells and that the induced resistance mechanism is effective against viruses unrelated to PVX.     

Plants expressing the Pto disease resistance gene confer resistance to recombinant PVX containing the avirulence gene AvrPto

Elicitation of hypersensitive cell death and induction of plant disease resistance by Pseudomonas syringae pv. tomato (Pst) is dependent on activity of the Pst Hrp secretion system and the gene-for-gene interaction between the tomato resistance gene Pto and the bacterial avirulence gene avrPto. AvrPto was expressed transiently in resistant or susceptible plant lines via a potato virus X (PVX) vector. We found that while PVX is normally virulent on tomato, a PVX derivative expressing avrPto was only capable of infecting plants lacking a functional Pto resistance pathway. Mutations in either the Pto or Prf genes allowed systemic spread of the recombinant virus. These results indicate that recognition of AvrPto by Pto in resistant plant lines triggers a plant defense response that can confer resistance to a viral as well as a bacterial pathogen.     

Vascular associated death1, a novel GRAM domain-containing protein, is a regulator of cell death and defense responses in vascular tissues

The hypersensitive response (HR) is a programmed cell death that is commonly associated with plant disease resistance. A novel lesion mimic mutant, vad1 (for vascular associated death1), that exhibits light conditional appearance of propagative HR-like lesions along the vascular system was identified. Lesion formation is associated with expression of defense genes, production of high levels of salicylic acid (SA), and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato. Analyses of the progeny from crosses between vad1 plants and either nahG transgenic plants, sid1, nonexpressor of PR1 (npr1), enhanced disease susceptibility1 (eds1), or non-race specific disease resistance1 (ndr1) mutants, revealed the vad1 cell death phenotype to be dependent on SA biosynthesis but NPR1 independent; in addition, both EDS1 and NDR1 are necessary for the proper timing and amplification of cell death as well as for increased resistance to Pseudomonas strains. VAD1 encodes a novel putative membrane-associated protein containing a GRAM domain, a lipid or protein binding signaling domain, and is expressed in response to pathogen infection at the vicinity of the hypersensitive lesions. VAD1 might thus represent a new potential function in cell death control associated with cells in the vicinity of vascular bundles.     

RanGAP2 mediates nucleocytoplasmic partitioning of the NB-LRR immune receptor Rx in the Solanaceae, thereby dictating Rx function

The potato (Solanum tuberosum) nucleotide binding-leucine-rich repeat immune receptor Rx confers resistance to Potato virus X (PVX) and requires Ran GTPase-activating protein 2 (RanGAP2) for effective immune signaling. Although Rx does not contain a discernible nuclear localization signal, the protein localizes to both the cytoplasm and nucleus in Nicotiana benthamiana. Transient coexpression of Rx and cytoplasmically localized RanGAP2 sequesters Rx in the cytoplasm. This relocation of the immune receptor appeared to be mediated by the physical interaction between Rx and RanGAP2 and was independent of the concomitant increased GAP activity. Coexpression with RanGAP2 also potentiates Rx-mediated immune signaling, leading to a hypersensitive response (HR) and enhanced resistance to PVX. Besides sequestration, RanGAP2 also stabilizes Rx, a process that likely contributes to enhanced defense signaling. Strikingly, coexpression of Rx with the Rx-interacting WPP domain of RanGAP2 fused to a nuclear localization signal leads to hyperaccumulation of both the WPP domain and Rx in the nucleus. As a consequence, both Rx-mediated resistance to PVX and the HR induced by auto-active Rx mutants are significantly suppressed. These data show that a balanced nucleocytoplasmic partitioning of Rx is required for proper regulation of defense signaling. Furthermore, our data indicate that RanGAP2 regulates this partitioning by serving as a cytoplasmic retention factor for Rx.