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Plant disease resistance genes function is highly specific pathogen recognition pathways. PRS2 is a resistance gene of Arabidopsis thaliana that confers resistance against Pseudomonas syringae bacteria that express avirulence gene avrRpt2. RPS2 was isolated by the use of a positional cloning strategy. The derived amino acid sequence of RPS2 contains leucine-rich repeat, membrane-spanning, leucine zipper, and P loop domains. The function of the RPS2 gene product in defense signal transduction is postulated to involve nucleotide triphosphate binding and protein-protein interactions and may also involve the reception of an elicitor produced by the avirulent pathogen.

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasm'id includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine 1'iosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf)' and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix-nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.A great many natural isolates of rhizobia, agrobacteria, and pseudomonads have been shown to carry a variety of large plasmids (for examples, see references 8, 21, and 38). Some of these are megaplasmids, with molecular masses over 450 megadaltons (38). Genes for a few functions have been localized to these large plasmids, notably including pathogenicity gepes for the Ti and Ri plasmids of agrobacteria (6, 21) and symbiotic nodulation (nod) and nitrogen fixation (nif) genes for the Sym megaplasmids of the fast-growing rhizobia (3,8,28,33,37,38). Nevertheless, the significance of this genomic organization remains obscure, although the fact that these are all plant-associated soil bacteria does suggest an underlying evolutionary cause.Rhizobial Sym megaplasmids are being characterized extensively with regard to symbiosis. In addition, Rhizobium meliloti 41 has recently been shown to carry a second megaplasmid, with a molecular weight very nearly that of pSym, on which a region for surface exclusion (although none for symbiotic functions) has tentatively been identified (2). A second megaplasmid has also been identified in R.

SummaryA new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauli¯ower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These ®ndings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.

SUMMARY Successful pathogen infection likely involves the suppression of general antimicrobial host defences. One Pseudomonas syringae virulence factor proposed to act in this manner is coronatine (COR), a phytotoxin believed to function as an analogue of one or more jasmonates, a family of plant growth regulators. COR biosynthetic (COR(-)) mutants of P. syringae pv. tomato strain DC3000 exhibit reduced virulence on Arabidopsis thaliana and tomato. In the present study, three genetically and biochemically defined COR(-) mutants of DC3000 were used to explore potential effects of COR and its precursors, coronafacic acid (CFA) and coronamic acid (CMA), on defence signalling pathways in A. thaliana. Inoculation with wild-type DC3000 resulted in the accumulation of several jasmonate-responsive transcripts, whereas infection with a mutant strain that accumulates CFA, which is structurally similar to methyl jasmonate (MeJA), did not. Thus, COR, but not CFA, stimulates jasmonate signalling during P. syringae infection of A. thaliana. The ability of the COR(-) mutants to grow to high levels in planta was fully restored in A. thaliana lines deficient for salicylic acid (SA) accumulation. Although the COR(-) mutants grew to high levels in SA-deficient plants, disease symptoms were reduced in these plants. Collectively, these results indicate that COR is required both for overcoming or suppressing SA-dependent defences during growth in plant tissue and for normal disease symptom development in A. thaliana.

The Pseudomonas syringae type III effector AvrRpt2 promotes bacterial virulence on Arabidopsis thaliana plants lacking a functional RPS2 gene (rps2 mutant plants). To investigate the mechanisms underlying the virulence activity of AvrRpt2, we examined the phenotypes of transgenic A. thaliana rps2 seedlings constitutively expressing AvrRpt2. These seedlings exhibited phenotypes reminiscent of A. thaliana mutants with altered auxin physiology, including longer primary roots, increased number of lateral roots, and increased sensitivity to exogenous auxin. They also had increased levels of free indole acetic acid (IAA). The presence of AvrRpt2 also was correlated with a further increase in free IAA levels during infection with P. syringae pv. tomato strain DC3000 (PstDC3000). These results indicate that AvrRpt2 alters A. thaliana auxin physiology. Application of the auxin analog 1-naphthaleneacetic acid promoted disease symptom development in PstDC3000-infected plants, suggesting that elevated auxin levels within host tissue promote PstDC3000 virulence. Thus, AvrRpt2 may be among the virulence factors of P. syringae that modulate host auxin physiology to promote disease.disease ͉ indole acetic acid ͉ pathogen ͉ virulence ͉ host physiology

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.

Plant pathogen resistance is mediated by a large repertoire of resistance (R) genes, which are often clustered in the genome and show a high degree of genetic variation. Here, we show that an Arabidopsis thaliana R-gene cluster is also subject to epigenetic variation. We describe a heritable but metastable epigenetic variant bal that overexpresses the R-like gene At4g16890 from a gene cluster on Chromosome 4. The bal variant and Arabidopsis transgenics overexpressing the At4g16890 gene are dwarfed and constitutively activate the salicylic acid (SA)-dependent defense response pathway. Overexpression of a related R-like gene also occurs in the ssi1 (suppressor of SA insensitivity 1) background, suggesting that ssi1 is mechanistically related to bal.