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.

The virulence of Pseudomonas syringae and many other proteobacterial pathogens is dependent on complex repertoires of effector proteins injected into host cells by type III secretion systems. The 28 well-expressed effector genes in the repertoire of the model pathogen P. syringae pv. tomato DC3000 were deleted to produce polymutant DC3000D28E. Growth of DC3000D28E in Nicotiana benthamiana was symptomless and 4 logs lower than that of DC3000Δ hopQ1-1 , which causes disease in this model plant. DC3000D28E seemed functionally effectorless but otherwise WT in diagnostic phenotypes relevant to plant interactions (for example, ability to inject the AvrPto-Cya reporter into N. benthamiana ). Various effector genes were integrated by homologous recombination into native loci or by a programmable or random in vivo assembly shuttle (PRIVAS) system into the exchangeable effector locus in the Hrp pathogenicity island of DC3000D28E. The latter method exploited dual adapters and recombination in yeast for efficient assembly of PCR products into programmed or random combinations of multiple effector genes. Native and PRIVAS-mediated integrations were combined to identify a minimal functional repertoire of eight effector genes that restored much of the virulence of DC3000Δ hopQ1-1 in N. benthamiana , revealing a hierarchy in effector function: AvrPtoB acts with priority in suppressing immunity, enabling other effectors to promote further growth (HopM1 and HopE1), chlorosis (HopG1), lesion formation (HopAM1-1), and near full growth and symptom production (AvrE, HopAA1-1, and/or HopN1 functioning synergistically with the previous effectors). DC3000D28E, the PRIVAS method, and minimal functional repertoires provide new resources for probing the plant immune system.

Summary: The 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 hrpB‐independent 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.

Summary: As a key virulence strategy to cause bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo) injects into the plant cell DNA‐binding proteins called transcription activator‐like effectors (TALEs) that bind to effector‐binding elements (EBEs) in a sequence‐specific manner, resulting in host gene induction. TALEs AvrXa7, PthXo3, TalC and Tal5, found in geographically distant Xoo strains, all target OsSWEET14, thus considered as a pivotal TALE target acting as major susceptibility factor during rice–Xoo interactions. Here, we report the generation of an allele library of the OsSWEET14 promoter through stable expression of TALE‐nuclease (TALEN) constructs in rice. The susceptibility level of lines carrying mutations in AvrXa7, Tal5 or TalC EBEs was assessed. Plants edited in AvrXa7 or Tal5 EBEs were resistant to bacterial strains relying on the corresponding TALE. Surprisingly, although indels within TalC EBE prevented OsSWEET14 induction in response to BAI3 wild‐type bacteria relying on TalC, loss of TalC responsiveness failed to confer resistance to this strain. The TalC EBE mutant line was, however, resistant to a strain expressing an artificial SWEET14‐inducing TALE whose EBE was also edited in this line. This work offers the first set of alleles edited in TalC EBE and uncovers a distinct, broader range of activities for TalC compared to AvrXa7 or Tal5. We propose the existence of additional targets for TalC beyond SWEET14, suggesting that TALE‐mediated plant susceptibility may result from induction of several, genetically redundant, host susceptibility genes by a single effector.