contributed equally to this workThe Arabidopsis protein RPM1 activates disease resistance in response to Pseudomonas syringae proteins targeted to the inside of the host cell via the bacterial type III delivery system. We demonstrate that speci®c mutations in the ATP-binding domain of a single Arabidopsis cytosolic HSP90 isoform compromise RPM1 function. These mutations do not affect the function of related disease resistance proteins. RPM1 associates with HSP90 in plant cells. The Arabidopsis proteins RAR1 and SGT1 are required for the action of many R proteins, and display some structural similarity to HSP90 co-chaperones. Each associates with HSP90 in plant cells. Our data suggest that (i) RPM1 is an HSP90 client protein; and (ii) RAR1 and SGT1 may function independently as HSP90 cofactors. Dynamic interactions among these proteins can regulate RPM1 stability and function, perhaps similarly to the formation and regulation of animal steroid receptor complexes.

Plant disease resistance ( R ) genes mediate specific pathogen recognition, leading to a successful immune response. Downstream responses include ion fluxes, an oxidative burst, transcriptional reprogramming, and, in many cases, hypersensitive cell death at the infection site. We used a transgenic Arabidopsis line carrying the bacterial avirulence gene avrRpm1 under the control of a steroid-inducible promoter to select for mutations in genes required for RPM1 -mediated recognition and signal transduction. We identified an allelic series of eight mutants that also were allelic to the previously identified pbs2 mutation. Positional cloning revealed this gene to be AtRAR1 , the Arabidopsis ortholog of barley RAR1 , a known mediator of R function. AtRAR1 is required for both full hypersensitive cell death and complete disease resistance mediated by many, but not all, tested R genes. Double mutant analysis of Atrar1 in combination with the R signal intermediate ndr1 suggests that AtRAR1 and NDR1 can operate in both linear and parallel signaling events, depending on the R gene function triggered. In Atrar1 null plants, the levels of RPM1-myc are reduced severely, suggesting that AtRAR1 may regulate R protein stability or accumulation. INTRODUCTIONPlant recognition of pathogens is mediated by large families of highly polymorphic disease resistance ( R ) genes (Dangl and Jones, 2001; Jones, 2001). The products of these genes function to recognize, directly or indirectly, the products of pathogen-encoded avirulence ( Avr ) genes (Nimchuk et al., 2001). Recognition stimulates a signal transduction cascade leading to the activation of multiple defense responses, including, in many cases, hypersensitive plant cell death (HR) at the site of infection (reviewed by Heath, 2000). Most R products contain a central nucleotide binding site and C-terminal Leucine-rich repeat domains (NB-LRR). There are ‫ف‬ 150 NB-LRR proteins encoded in the complete Arabidopsis genome (Arabidopsis Genome Initiative, 2000). The N termini of these proteins contain either potential coiledcoil (CC) or Toll-Interleukin 1 receptor homology (TIR) domains.Genetic screens in Arabidopsis have defined several loci required for R function. There is evidence from these studies that the NB-LRR class of R proteins trigger multiple signaling pathways. Many, but not all, CC-NB-LRR proteins require NDR1, a protein of undefined biochemical function (Century et al., 1995(Century et al., , 1997. In contrast, all tested members of the TIR class require the EDS1 protein (Parker et al., 1996). EDS1 encodes a protein of unknown function, although it has homology with lipases (Falk et al., 1999). Whether or not these pathways converge into a simple linear signal transduction cascade is unknown, but it is unlikely given the fact that no locus defined by mutant phenotype is required for the function of all NB-LRR proteins.RPM1 conditions resistance to Pseudomonas syringae strains expressing either avrRpm1 or the sequence-unrelated avrB (Bisgrove et al., 1994; Grant et al....

The Arabidopsis RPM1 gene confers resistance against Pseudomonas syringae expressing either the AvrRpm1 or the AvrB type III effector protein. We present an exhaustive genetic screen for mutants that no longer recognize avrRpm1 . Using an inducible avrRpm1 expression system, we identified 110 independent mutations. These mutations represent six complementation groups. None discriminates between avrRpm1 and avrB recognition. We identified 95 rpm1 alleles and present a detailed structure-function analysis of the RPM1 protein. Several rpm1 mutants retain partial function, and we deduce that their residual activity is dependent on the level of avrRpm1 signal. In these mutants, the hypersensitive response remains activated if the signal goes above a certain threshold. Missense mutations in rpm1 are highly enriched in the nucleotide binding domain, suggesting that this region plays a key role either in the hypersensitive response associated with RPM1 activation or in RPM1 stability. Cluster analysis of rpm1 alleles defines functionally important residues that are highly conserved between nucleotide binding site leucine-rich repeat R proteins and those that are unique to RPM1 . Regions of RPM1 to which no loss-of-function alleles map may represent domains in which variation is tolerated and may contribute to the evolution of new R gene specificities. INTRODUCTIONPlants can recognize certain products produced by pathogens and mount an appropriate disease resistance response. Soon after the rediscovery of Gregor Mendel's work, it became clear that single loci could confer resistance to otherwise susceptible lines if transferred by introgression (Biffin, 1905). Flor (1971) condensed this idea by postulating the existence of genes in the plant, called resistance ( R ) genes, that are able to interact genetically with genes of the pathogen, called avirulence ( Avr ) genes. If, and only if, both genes are present, a resistance response is triggered. Frequently, the resistance phenotype is accompanied by a programmed cell death called the hypersensitive response (HR) (reviewed by Morel and Dangl, 1997;Heath, 2000). It is not clear if HR is the cause or the consequence of the resistance response.We work with Pseudomonas syringae pv tomato ( Pst ), a bacterial pathogen of Arabidopsis. This pathogen is extracellular and causes disease by "delivering" protein disease effectors into the host cell interior by means of an evolutionarily conserved type III secretion apparatus (Nimchuk et al., 2001). If the plant genotype infected carries an appropriate R gene, conditioning the recognition of one of the type III effectors, then disease resistance occurs. If recognition does not occur, colonization and disease ensue. We study the recognition of avrRpm1 and avrB by Arabidopsis. The RPM1 protein recognizes the presence of either of these two sequence-unrelated type III effectors (Dangl et al., 1992;Bisgrove et al., 1994). The deduced RPM1 sequence predicts three main protein domains (Grant et al., 1995). The N terminus features a predict...