Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors.

RRS1-R confers broad-spectrum resistance to several strains of the causal agent of bacterial wilt, Ralstonia solanacearum. Although genetically defined as recessive, this R gene encodes a protein whose structure combines the TIR-NBS-LRR domains found in several R proteins and a WRKY motif characteristic of some plant transcriptional factors and behaves as a dominant gene in transgenic susceptible plants. Here we show that PopP2, a R. solanacearum type III effector, which belongs to the YopJ͞AvrRxv protein family, is the avirulence protein recognized by RRS1-R. Furthermore, an interaction between PopP2 and both RRS1-R and RRS1-S, present in the resistant Nd-1 and susceptible Col-5 Arabidopsis thaliana ecotypes, respectively, was detected by using the yeast split-ubiquitin two-hybrid system. This interaction, which required the full-length R protein, was not observed between the RRS1 proteins and PopP1, another member of the YopJ͞AvrRxv family present in strain GMI1000 and that confers avirulence in Petunia. We further demonstrate that both the Avr protein and the RRS1 proteins colocalize in the nucleus and that the nuclear localization of the RRS1 proteins are dependent on the presence of PopP2. P lants rely on an innate immune response for their survival after pathogen attack. Specific recognitions between pathogen Avr and plant R proteins are crucial for the onset of the resistance response and determine the issue of many plantpathogen interactions by triggering plant defense. Disease results from the inactivation or absence of one or both partners (1). It has been postulated that R gene products are receptors for pathogen-encoded Avr components (2). Despite the cloning of numerous R and Avr genes, only two plant R proteins, Pto and Pi-ta, were shown to interact physically with their pathogen Avr counterparts, Avr-Pto and Avr-Pita, respectively, by using the yeast two-hybrid system (3-5). The hypothesis that R proteins are part of protein complexes was recently substantiated by the identification of multiprotein complexes including R and Avr proteins (6-9). Additionally, whereas Avr bacterial proteins expressed in plants carrying the cognate R protein generally induce a cell death program (10), termed the hypersensitive response, closely linked to resistance, they can also cause disease-like symptoms when expressed in plants lacking the appropriate R protein. Bacterial pathogenicity effectors such as Avr proteins are injected into the host cell via a type III secretion system (TTSS) (11). According to the guard model (9, 12), such effectors can associate and induce modifications of plant targets functioning as negative regulators of basal defense responses leading to disease development in plants lacking the corresponding R protein. In a resistant host, the plant target that interacts with both R and Avr proteins is guarded by the R protein, preventing its manipulation by pathogen effectors. The recent characterization of RIN4, a negative regulator of plant defense, strengthens this model (13).Most resistance p...

SummaryThe ability of Ralstonia solanacearum strain GMI1000 to cause disease on a wide range of host plants (including most Solanaceae and Arabidopsis thaliana ) depends on genes activated by the regulatory gene hrpB . HrpB controls the expression of the type III secretion system (TTSS) and pathogenicity effectors transiting through this pathway. In order to establish the complete repertoire of TTSS-dependent effectors belonging to the Hrp regulon and to start their functional analysis, we developed a rapid method for insertional mutagenesis, which was used to monitor the expression of 71 candidate genes and disrupt 56 of them. This analysis yielded a total of 48 novel hrpB -regulated genes. Using the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter fusion system, we provide direct biochemical evidence that five R. solanacearum effector proteins are translocated into plant host cells through the TTSS. Among these novel TTSS effectors, RipA and RipG both belong to multigenic families, RipG defining a novel class of leucine-rich-repeats harbouring proteins. The members of these multigenic families are differentially regulated, being composed of genes expressed in either an hrpB -dependent or an hrpBindependent manner. Pathogenicity assays of the 56 mutant strains on two host plants indicate that, with two exceptions, mutations in individual effectors have no effect on virulence, a probable consequence of genetic and functional redundancy. This large repertoire of HrpB-regulated genes, which comprises > 20 probable TTSS effector genes with no counterparts in other bacterial species, represents an important step towards a full-genome understanding of R. solanacearum virulence.

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