Large-Scale Structure –Function Analysis of the Arabidopsis RPM1 Disease Resistance Protein

Abstract

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...

Molecular Genetic Evidence for the Role of SGT1 in the Intramolecular Complementation of Bs2 Protein Activity in Nicotiana benthamiana

“…This is in contrast with barley RAR1-dependent MLA6, which does not interact with SGT1 (Bieri et al, 2004). The Bs2 RAR1 independence may simply be the result of high Bs2 expression, similar to the RAR1 independence that results for high RPS2 expression in Arabidopsis (Tornero et al, 2002). SGT1 is a highly conserved eukaryotic protein involved in such diverse pathways as the regulation of cell division, SCF (Skip1p/ Cdc53p-Cullin-F-box)-mediated ubiquitination of proteins, and the regulation of the cAMP pathway in yeast (Kitagawa et al, 1999;Schadick et al, 2002).…”

A Gain-of-Function Mutation in a Plant Disease Resistance Gene Leads to Constitutive Activation of Downstream Signal Transduction Pathways in suppressor of npr1-1, constitutive 1

“…A large number of mutations in both the NB and LRR domains have been found to inactivate the R proteins (Warren et al, 1998;Dinesh-Kumar et al, 2000;Tao et al, 2000, Axtell et al, 2001Tornero et al, 2002), implying that the mutated residues are important for R protein function. Although negative regulation of this group of R proteins also may play a crucial role in R gene-mediated resistance, as suggested by the data that RPS2 can be activated constitutively by the elimination of its negative regulator RIN4 (Axtell and Staskawicz, 2003;Mackey et al, 2003), it is not known which regions of the R genes are important for this negative regulation.…”

Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation

“…Both took advantage of sensitized genetic backgrounds. The first was a modification of a previous screen (9), using a ␤-estradiol-inducible copy of the avrRpm1 bacterial type III effector gene whose product is recognized in Arabidopsis by the RPM1 NB-LRR protein (Fig. S1 A).…”

“…S1 A). Given the very high recovery ratio of rpm1 alleles compared with second-site loci isolated previously (9), we modified the screen by crossing into this background a well-characterized transgenic, myc-epitope tagged copy of RPM1 expressed from the native promoter ( Fig. S1 A and ref.…”