The Cyst Nematode SPRYSEC Protein RBP-1 Elicits Gpa2- and RanGAP2-Dependent Plant Cell Death

Plant NB-LRR proteins confer robust protection against microbes and metazoan parasites by recognizing pathogen-derived avirulence (Avr) proteins that are delivered to the host cytoplasm. Microbial Avr proteins usually function as virulence factors in compatible interactions; however, little is known about the types of metazoan proteins recognized by NB-LRR proteins and their relationship with virulence. In this report, we demonstrate that the secreted protein RBP-1 from the potato cyst nematode Globodera pallida elicits defense responses, including cell death typical of a hypersensitive response (HR), through the NB-LRR protein Gpa2. Gp-Rbp-1 variants from G. pallida populations both virulent and avirulent to Gpa2 demonstrated a high degree of polymorphism, with positive selection detected at numerous sites. All Gp-RBP-1 protein variants from an avirulent population were recognized by Gpa2, whereas virulent populations possessed Gp-RBP-1 protein variants both recognized and non-recognized by Gpa2. Recognition of Gp-RBP-1 by Gpa2 correlated to a single amino acid polymorphism at position 187 in the Gp-RBP-1 SPRY domain. Gp-RBP-1 expressed from Potato virus X elicited Gpa2-mediated defenses that required Ran GTPase-activating protein 2 (RanGAP2), a protein known to interact with the Gpa2 N terminus. Tethering RanGAP2 and Gp-RBP-1 variants via fusion proteins resulted in an enhancement of Gpa2-mediated responses. However, activation of Gpa2 was still dependent on the recognition specificity conferred by amino acid 187 and the Gpa2 LRR domain. These results suggest a two-tiered process wherein RanGAP2 mediates an initial interaction with pathogen-delivered Gp-RBP-1 proteins but where the Gpa2 LRR determines which of these interactions will be productive.     

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.     

Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode

The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.     

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.     

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.     

Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa2

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.Resistance (R) proteins play a central role in the recognition-based immune system of plants. Unlike vertebrates, plants lack an adaptive immune system with highly specialized immune cells. Instead, they rely on an innate immune system in which each cell is autonomous. Two types of immune receptors can be distinguished in plants, pathogen-associated molecular patterns recognition receptors that detect conserved molecular patterns in plant pathogens and intracellular R proteins that recognize specific effectors employed by pathogens as modifiers of host metabolism or defense mechanisms (Jones and Dangl, 2006). Effector-triggered activation of R proteins leads to an array of protective responses, often culminating in programmed cell death at the site of infection (Greenberg and Yao, 2004), thereby preventing further ingress of the pathogen. Pathogens have evolved mechanisms to evade recognition by R proteins and to regain their virulence (Dodds and Rathjen, 2010). This continuous coevolutionary process between host and pathogen has resulted in a reservoir of highly diverse R proteins in plants, enabling them to counteract a wide range of pathogens and pests.The most common class of R proteins consists of nucleotide-binding (NB)-leucine-rich repeat (LRR) proteins with a tripartite domain architecture, which roughly corresponds to an N-terminal response domain (a coiled coil [CC] or Toll/Interleukin-1 receptor [TIR] domain) involved in downstream signaling, a central molecular switch domain (the NB domain present in the mammalian apoptosis regulator Apaf1, plant R proteins, and the Caenorhabditis elegans apoptosis regulator CED4 [NB-ARC]), and a C-terminal sensor domain (the LRR domain). The NB-ARC domain is an extended nucleotide-binding domain that plant immune receptors share with metazoan apoptosis regulators and immune receptors such as Apaf1, CED4, and nucleotide-binding oligomerization domain (NOD-like) receptors (NLRs) and belongs to the STAND (signal transduction ATPases with numerous domains) family of nucleoside triphosphatase domains (van der Biezen and Jones, 1998; Leipe et al., 2004; Albrecht and Takken, 2006; Maekawa et al., 2011b). The overall modular architecture of metazoan STAND nucleoside triphosphatase is similar to that of NB-LRR plant immune receptors, but the domains flanking the NB-ARC domain often differ. In NLRs, for example, several N-terminal domains can be found, including caspase-recruiting domains and Pyrin domains (Proell et al., 2008). In the mammalian protein Apaf1, the sensor involved in cytochrome c detection consists of C-terminal WD40 repeats (Zou et al., 1997).In plant NB-LRR resistance proteins, the recognition of a pathogen effector via the LRR domain is thought to switch the conformation of the protein from a closed, autoinhibited “off” state into an open, active “on” state (Lukasik and Takken, 2009). The activation of NB-LRR proteins is most likely a multistep process in which the NB-ARC domain plays a central role. The three subdomains of the NB-ARC, the NB, ARC1, and ARC2, collectively form a nucleotide-binding pocket that adopts different conformations depending on the bound nucleotide. This mechanism seems to be conserved between proteins from organisms as distant as bacteria, metazoans, and plants (Rairdan and Moffett, 2007; Danot et al., 2009; Takken and Tameling, 2009). The conformational change coincides with the exchange of bound ADP for ATP in the NB-ARC, probably stabilizing the active conformation (Tameling et al., 2006; Ade et al., 2007). Hydrolysis of the bound ATP is hypothesized to return the domains to their inactive state. The exact mechanism by which elicitor recognition via the LRR leads to a conformational change of the NB-ARC and the subsequent activation of immune signaling pathways is not clear.Previous studies have shown that the CC/TIR, NB-ARC, and LRR domains in plant immune receptors interact and cooperate with each other in an interdependent manner (Moffett et al., 2002; Leister et al., 2005; Ade et al., 2007; Rairdan et al., 2008). From these data, a picture emerges in which the LRR domain is not only involved in pathogen recognition, but also plays a role in maintaining an autoinhibited resting state in the absence of pathogens via its interactions with the other domains (Bendahmane et al., 2002; Hwang and Williamson, 2003; Ade et al., 2007; Qi et al., 2012). A similar role as regulatory domain has been found for the sensor domains of other NLRs, such as the mammalian Apaf1 (Hu et al., 1998). For the potato (Solanum tuberosum) immune receptor Rx1, a model plant NB-LRR protein, it has been shown that the LRR cooperates with the ARC subdomains in retaining the inactive state of the protein. The deletion of the ARC and LRR domains leads to a constitutive activity of the NB (Bendahmane et al., 2002; Rairdan et al., 2008). In addition, it was demonstrated that the elicitor, the Potato virus X (PVX) coat protein, modifies the interdomain interactions in Rx1 (Moffett et al., 2002; Rairdan et al., 2008). Sequence exchanges between Rx1 and the highly homologous nematode resistance protein Gpa2 (88% amino acid identity) resulted in incompatibilities between the domains that give rise to inappropriate activation of cell death responses (Rairdan and Moffett, 2006), indicating that the cooperation between the sensor and switch domains depends on an interaction fine tuned by intramolecular coevolution. In this light, it is interesting to note that a functional ortholog of Rx1, Rx2 from Solanum acaule, is almost identical to Rx1 in its LRR region but displays a higher similarity to Gpa2 in stretches of its CC-NB-ARC sequence (Bendahmane et al., 2000).The aim of our study was to pinpoint the molecular determinants controlling the switch between the resting and activation state of NB-LRR proteins. The incompatibility between the ARC and LRR domains of Rx1 and Gpa2 was used as a guideline to dissect the molecular and structural determinants involved in the cooperation between the switch (NB-ARC) and sensor (LRR) domain. An extensive exchange of polymorphic residues between these two homologous NB-LRR proteins resulted in the identification of a minimal fragment of 68 amino acid residues in the ARC2 domain and the first LRR repeats as being crucial for proper activation of Gpa2 and Rx1. Within this minimal region, we identified two amino acids that, despite their proximity in the amino acid sequence, differentiate between elicitor-dependent (position 401) and independent activation (position 403). However, structural modeling of the domains shows that the residue at position 403 operates at the interface of the ARC2 and N-terminal part of the LRR domain, while residue 401 mapped at the interface between the ARC2 and NB domain. Furthermore, an acidic loop region in the ARC2 domain and complementary-charged basic patches in the N-terminal half of the LRR domain are shown to be required for the physical interaction between these domains. We demonstrate that the binding between the CC- NB-ARC and LRR domains is disrupted upon elicitor-dependent activation and that the complementary-charged residues are predicted to facilitate reassociation. Two independent docking simulations of the NB-ARC and LRR domain indicate that the LRR domain binds to the NB-ARC domain at the surface formed by the interaction of the ARC2 and NB subdomains. We present a mechanistic model in which the first repeats of the LRR, the ARC2 subdomain, and the NB form a clamp, which governs the shuttling between a closed, autoinhibited “off” state and an open, active “on” state of the resistance protein. Finally, we discuss the consequences of the functional constraints imposed by the interface of the NB, ARC2, and LRR domain for the generation of novel resistance specificities via evolutionary processes and genetic engineering.     

Structural Basis for the Interaction between the Potato Virus X Resistance Protein (Rx) and Its Cofactor Ran GTPase-activating Protein 2 (RanGAP2)

The potato (Solanum tuberosum) disease resistance protein Rx has a modular arrangement that contains coiled-coil (CC), nucleotide-binding (NB), and leucine-rich repeat (LRR) domains and mediates resistance to potato virus X. The Rx N-terminal CC domain undergoes an intramolecular interaction with the Rx NB-LRR region and an intermolecular interaction with the Rx cofactor RanGAP2 (Ran GTPase-activating protein 2). Here, we report the crystal structure of the Rx CC domain in complex with the Trp-Pro-Pro (WPP) domain of RanGAP2. The structure reveals that the Rx CC domain forms a heterodimer with RanGAP2, in striking contrast to the homodimeric structure of the CC domain of the barley disease resistance protein MLA10. Structure-based mutagenesis identified residues from both the Rx CC domain and the RanGAP2 WPP domain that are crucial for their interaction and function in vitro and in vivo. Our results reveal the molecular mechanism underlying the interaction of Rx with RanGAP2 and identify the distinct surfaces of the Rx CC domain that are involved in intramolecular and intermolecular interactions.     

Membrane microdomain may be a platform for immune signaling

Arabidopsis RPS2 is a typical disease resistance (R) protein with nucleotide-binding leucine-rich repeats (NB-LRR). Previously, we reported that RPS2 is physically associated with some Arabidopsis hypersensitive induced reaction (AtHIR) proteins, which are enriched in membrane microdomains. Biochemical and genetic analyses suggested that members of the AtHIR gene family have a function in RPS2-mediated immune signaling. Here, we provide evidence that the pattern recognition receptor (PRR) FLS2 is also physically associated with AtHIR2 in a N. benthamiana transient expression system. We thus speculate that PM microdomains provide a platform for both types of immune receptors, R proteins and PRRs, and that the activation of the receptors is facilitated by AtHIR proteins.     

Distinct domains in the ARC region of the potato resistance protein Rx mediate LRR binding and inhibition of activation

Plant nucleotide binding and leucine-rich repeat (NB-LRR) proteins contain a region of homology known as the ARC domain located between the NB and LRR domains. Structural modeling suggests that the ARC region can be subdivided into ARC1 and ARC2 domains. We have used the potato (Solanum tuberosum) Rx protein, which confers resistance to Potato virus X (PVX), to investigate the function of the ARC region. We demonstrate that the ARC1 domain is required for binding of the Rx N terminus to the LRR domain. Domain-swap experiments with Rx and a homologous disease resistance gene, Gpa2, showed that PVX recognition localized to the C-terminal half of the LRR domain. However, inappropriate pairings of LRR and ARC2 domains resulted in autoactive molecules. Thus, the ARC2 domain is required to condition an autoinhibited state in the absence of elicitor as well as for the subsequent elicitor-induced activation. Our data suggest that the ARC region, through its interaction with the LRR, translates elicitor-induced modulations of the C terminus into a signal initiation event. Furthermore, we demonstrate that physical disruption of the LRR-ARC interaction is not required for signal initiation. We propose instead that this activity can lead to multiple rounds of elicitor recognition, providing a means of signal amplification.     

Mapping of the cyst nematode resistance locus Gpa2 in potato using a strategy based on comigrating AFLP markers

 The nematode resistance locus Gpa2 was mapped on chromosome 12 of potato using information on the genomic positions of 733 known AFLP markers. The minimum number of AFLP primer combinations required to map Gpa2 was three. This demonstrates that a reference collection of potato AFLP markers may be a valuable tool for mapping studies in potato. By use of RFLP probes, Gpa2 was more precisely mapped at the distal end of chromosome 12. Gpa2 confers resistance to a distinct group of populations of the potato cyst nematode Globodera pallida and originates from the same potato accession as locus H1, conferring resistance to pathotype Ro₁ of G. rostochiensis. This study shows that these two nematode resistance loci are unlinked and that Gpa2 is linked to the Rx1 locus conferring resistance to potato virus X. The efficiency of AFLPs for genetic mapping of a highly heterozygous crop like potato is discussed and compared with the RFLP technique. Received: 24 February 1997/Accepted: 2 May 1997     

Molecular mechanisms of generation for nitric oxide and reactive oxygen species, and role of the radical burst in plant immunity

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and H₂O₂ accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.     

The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana

Background

The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance.

Principal Findings

We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs.

Conclusions/Significance

We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.     

Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato

Rx2 confers resistance against potato virus X (PVX). To clone Rx2, we developed a system based on Agrobacterium-mediated transient expression of candidate R genes in transgenic tobacco leaves expressing the PVX coat protein elicitor of Rx2-mediated resistance. Using this system, a potato gene eliciting HR specifically in the presence of the elicitor was identified. Based on genetical and functional analysis, it is concluded that the cloned gene is Rx2. The transient expression system is potentially adaptable to cloning of any other resistance gene. The Rx2 locus is on chromosome V of potato and the encoded protein is highly similar to the products of Rx1 and Rxh1 encoded on potato chromosome XII. Rxh1 has been shown elsewhere to encode a potato cyst nematode resistance gene Gpa2. All three proteins are in the leucine zipper-nucleotide binding site-leucine rich repeat class of resistance gene products. Rx1 and Rx2 are functionally identical and are almost identical in the C terminal region consistent with a role of the leucine rich repeats in recognition of the PVX coat protein. In the N terminal, half there are some regions where the Rx1 and Rx2 proteins are more similar to each other than to the Rxh1 protein. However, in other regions these proteins are more similar to Rxh1 than to each other. Based on this mosaic pattern of sequence similarity, we conclude that sequence exchange occurs repeatedly between genetically unlinked disease resistance genes through a process of gene conversion.     

The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR.     

Genetic and physical mapping of homologues of the virus resistance gene <Emphasis Type="Italic">Rx1</Emphasis> and the cyst nematode resistance gene <Emphasis Type="Italic">Gpa2</Emphasis> in potato

Nine resistance gene homologues (RGHs) were identified in two diploid potato clones (SH and RH), with a specific primer pair based on conserved motifs in the LRR domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene Rx1. A modified AFLP method was used to facilitate the genetic mapping of the RGHs in the four haplotypes under investigation. All nine RGHs appeared to be located in the Gpa2/ Rx1 cluster on chromosome XII. Construction of a physical map using bacterial artificial chromosome (BAC) clones for both the Solanum tuberosum ssp. tuberosum and the S. tuberosum ssp. andigena haplotype of SH showed that the RGHs are located within a stretch of less than 200 kb. Sequence analysis of the RGHs revealed that they are highly similar (93 to 95%) to Gpa2 and Rx1. The sequence identities among all RGHs range from 85 to 100%. Two pairs of RGHs are identical, or nearly so (100 and 99.9%), with each member located in a different genotype. Southern-blot analysis on genomic DNA revealed no evidence for additional homologues outside the Gpa2/ Rx1 cluster on chromosome XII.     

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.     

Rice BiP3 regulates immunity mediated by the PRRs XA3 and XA21 but not immunity mediated by the NB-LRR protein,Pi5

Plant innate immunity is mediated by pattern recognition receptors (PRRs) and intracellular NB-LRR (nucleotide-binding domain and leucine-rich repeat) proteins. Overexpression of the endoplasmic reticulum (ER) chaperone, luminal-binding protein 3 (BiP3) compromises resistance to Xanthomonas oryzae pv. oryzae (Xoo) mediated by the rice PRR XA21 [12]. Here we show that BiP3 overexpression also compromises resistance mediated by rice XA3, a PRR that provides broad-spectrum resistance to Xoo. In contrast, BiP3 overexpression has no effect on resistance mediated by rice Pi5, an NB-LRR protein that confers resistance to the fungal pathogen Magnaporthe oryzae (M. oryzae). Our results suggest that rice BiP3 regulates membrane-resident PRR-mediated immunity.