Christian Boucher

Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus

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 YopJ/AvrRxv 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 YopJ/AvrRxv 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.

PopA1, a protein which induces a hypersensitivity-like response on specific Petunia genotypes, is secreted via the Hrp pathway of Pseudomonas solanacearum

This paper describes the identification of a new class of extracellular bacterial proteins, typified by PopA1 and its derivative PopA3, which act as specific hypersensitive response (HR) elicitors. These two heat‐stable proteins, with HR‐like elicitor activities on tobacco (non‐host plant) but without activity on tomato (host plant), have been characterized from the supernatant of the plant pathogenic bacterium Pseudomonas solanacearum strain GMI1000. These two proteins induced the same pattern of response on Petunia, as a function of the genotypes tested. popA, the structural gene for PopA1, maps outside of the hrp gene cluster but belongs to the hrp regulon. The amino acid sequence of PopA1 does not show homology to any characterized proteins. Its secretion is dependent on hrp genes and is followed by stepwise removal of the 93 amino‐terminal amino acids, producing the protein PopA3. Petunia lines responsive to PopA3 and its precursors were resistant to infection by strain GMI1000, whereas non‐responsive lines were sensitive, suggesting that popA could be an avirulence gene. A popA mutant remained fully pathogenic on sensitive plants, indicating that this gene is not essential for pathogenicity. While lacking PopA1, this mutant, which remained avirulent on tobacco and on resistant Petunia lines, still produced additional extracellular necrogenic compounds. On the basis of both their structural features and the biological properties of the popA mutant, PopA1 and PopA3 clearly differ from hairpins characterized in other plant pathogenic bacteria.

Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria

Genes of plant-pathogenic bacteria controlling hypersensitive response (HR) elicitation and pathogenesis were designated ‘hrp’ by Lindgren et al. in 1986 (J Bacteriol 168: 512–522). hrp genes have been characterized in several species of the four major genera of Gramnegative plant pathogens, Erwinia, Pseudomonas, Ralstonia (a new proposed genus including Pseudomonas solanacearum) and Xanthomonas. To date, hrp genes have been found mainly in large clusters, and they have been shown to be conserved physically and, in many cases, functionally among different bacteria. Hybridization studies and genetic analyses have revealed the presence of functional hrp genes even in species that are not typically observed to elicit an HR, such as Erwinia chrysanthemi and Erwinia stewartii, suggesting that hrp genes may be common to all Gram-negative plant pathogens, possibly excluding Agrobacterium spp. Current knowledge of hrp genes has been reviewed by Bonas (1994, Curr Top Microbiol Immunol 192: 79–98) and by Van Gijsegem et al. (1995, In Pathogenesis and Host–Parasite Specificity in Plant Diseases: Histopathological, Biochemical, Genetic and Molecular Basis. Volume 1. (Kohmoto et al., eds); Oxford: Pergamon Press, pp. 273–292). The nucleotide sequences of four hrp gene clusters, those of Ralstonia solanacearum (previously P. solanacearum) (Genin et al., 1992, Mol Microbiol 6: 3065–3076; Gough et al., 1992, Mol Plant–Microbe Interact 5: 384–389; Gough et al., 1993, Mol Gen Genet 239: 378–392; Van Gijsegem et al., 1995, Mol Microbiol 15: 1095–1114), Erwinia amylovora (Bogdanove et al., 1996, J Bacteriol 178: 1720– 1730; Wei and Beer, 1993, J Bacteriol 175: 7958–7967; Wei and Beer, 1995, J Bacteriol 177: 6201–6210; Wei et al., 1992, Science 257: 85–88; S. V. Beer, unpublished), Pseudomonas syringae pv. syringae (Huang et al., 1992, J Bacteriol 174: 6878–6885; Huang et al., 1993, Mol Plant–Microbe Interact 6: 515–520; Huang et al., 1995, Mol Plant–Microbe Interact 8: 733–746; Lidell and Hutcheson, 1994, Mol Plant–Microbe Interact 7: 488–497; Preston et al., 1995, Mol Plant–Microbe Interact 8: 717–732; Xiao et al., 1994, J Bacteriol 176: 1025–1036), and Xanthomonas campestris pv. vesicatoria (Fenselau et al., 1992, Mol Plant–Microbe Interact 5: 390–396; Fenselau and Bonas, 1995, Mol Plant–Microbe Interact 8: 845–854; U. Bonas, unpublished), have been largely determined. These clusters each contain more than twenty genes, many of which encode components of a novel proteinsecretion pathway designated ‘type III’. It has been shown directly that various extracellular proteins involved in pathogenesis and defence elicitation by plantpathogenic bacteria utilize this pathway (Arlat et al., 1994, EMBO J 13: 543–553; He et al., 1993, Cell 73: 1255–1266; Wei and Beer, 1993, ibid.), and the pathway is known to function in the export of virulence factors from the animal pathogens Salmonella typhimurium, Shigella flexneri, and Yersinia entercolitica, Yersinia pestis, and Yersinia pseudotuberculosis (for reviews, see Salmond and Reeves, 1993, Trends Biochem Sci 18: 7–12; and Van Gijsegem et al., 1993, Trends Microbiol 1: 175– 180). Nine type III secretion genes are conserved among all four of the plant pathogens listed above and among the animal pathogens. Based on sequence analysis and some experimental evidence, they are believed to encode one outer-membrane protein, one outer-membrane-associated lipoprotein, five inner-membrane proteins, and two cytoplasmic proteins, one of which is a putative ATPase. All of the predicted gene products, except the outer-membrane protein, show significant similarity to components of the flagellar biogenesis complex (for reviews see Blair, 1995, Annu Rev Microbiol 49: 489–522; and Bischoff and Ordal, 1992, Mol Microbiol 6: 23–28). We herein refer to the hrp-encoded type III pathway as the ‘Hrp pathway’. Because hrp genes have been characterized independently in diverse plant-pathogenic bacteria, hrp gene nomenclature differs in different species, and it is not always consistent even within the same organism. Different designations are used for homologous genes, and, even worse, the same designation is used for different genes in different organisms. For example, hrpI of E. amylovora is homologous with hrpC2 of X. campestris pv. vesicatoria and hrpO of R. solanacearum, and the homologue in P. syringae pv. syringae appears in the literature both as hrpI and as hrpJ2. Also, ‘hrpN ’ in R. solanacearum designates a secretion-pathway gene, whereas in E. amylovora, ‘hrpN ’ designates the gene encoding the elicitor harpin. Furthermore, in many bacteria the number of known hrp genes approaches 26. In anticipation of exhausting the alphabet, some authors chose to designate hrp genes with a letter and a number, creating the potential for confusion of distinct genes with alleles of the same gene. For hrp gene researchers, the current nomenclature is at best inconvenient; for other scientists, it is bewildering. Another problem exists: accumulation of knowledge about the structure of hrp loci has outpaced the accumulation of Molecular Microbiology (1996) 20(3), 681–683

Transposon Mutagenesis of Pseudomonas solanacearum: Isolation of Tn5-Induced Avirulent Mutants

Summary: Transposon mutagenesis in a tomato isolate of Pseudomonas solanacearum (strain Kourou) is reported, using Tn7 and Tn5 inserted in suicide conjugative plasmids. Whereas Tn7 integrates at high frequency in a particular site of the the genome, Tn5 appears to transpose much more randomly, allowing isolation of auxotrophic mutants with a frequency of 035%. The mutants showed a wide range of nutritional requirements. Following Tn5 mutagenesis, screening of 8250 clones on axenic tomato seedlings led to the isolation of 12 avirulent mutants. Southern blot analysis revealed that, for avirulent mutants, insertion of Tn5 occurred in at least 10 different EcoRI restriction fragments. Additional independent insertions of IS50 were also detected in four of these mutants. For each mutant, transformation experiments demonstrated that the Tn5-encoded kanamycin resistance and the avirulent phenotype are linked. Based on their ability or inability to induce a collapse of tobacco leaf parenchyma, and on the timing of reaction of the plant, avirulent mutants have been divided in to two and possibly three groups.

The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex

Five transcription units of the Pseudomonas solanacearum hrp gene cluster are required for the secretion of the HR‐inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions., Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant‐pathogenic bacteria include proteins encoded by hrp genes. For Hrp‐related proteins of animal pathogens, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solanacearum, include proteins encoded by fli and fli genes of S. typhimurium, Bacillus subtils and Escherichia coli and by mop genes of Erwinia carotovora. P. solanacearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity.

A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain

The family of PulD proteins, which has been characterized in a wide variety of microorganisms, comprises several membrane-associated proteins essential for the transport of macromolecules across bacterial membranes. These proteins are involved in the transport of complex structures (such as phage particles, DNA) or various proteins (such as extracellular enzymes and pathogenicity determinants). Amino acid sequence analysis revealed a possible modular organisation of proteins of this superfamily, with highly conserved C-terminal domains and dissimilar N-terminal domains. In the C-terminal domain, four highly conserved regions have been found, one of them containing a remarkable common motif: (V, I)PXL(S, G)XIPXXGXLF. Structural comparisons between the N-terminal domains indicate that proteins of this superfamily can be divided into at least two subgroups, probably reflecting the existence of distinct secretion mechanisms. This implies that members of the superfamily of PulD-related proteins are independently involved in (1) the general secretory pathway, (2) a new signal-peptide-independent secretion pathway found in several bacterial pathogens, and possibly in (3) the translocation of bacteriophage particles through the bacterial cell envelope.

Conservation of secretion pathways for pathogenicity determinants of plant and animal bacteria

Extracellular proteins of plant and animal bacteria are important in virulence. Many of these are secreted through the type I sec-independent and the type II sec-dependent pathways. Recently, a third distinct pathway, involved in secretion of Yops, has been discovered in Yersinia. This pathway has homology with pathways in plant pathogenic bacteria that are putatively involved in the secretion of proteins active on plant cells, such as harpin and possibly some avr gene products

Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identification of novel effector proteins translocated to plant host cells through the type III secretion system

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.

Evidence that the hrpB gene encodes a positive regulator of pathogenicity genes from Pseudomonas solanacearum

The hrp gene cluster of Pseudomonas solanacearum GMI1000 strain encodes functions that are essential for pathogenicity on tomato and for the elicitation of the hypersensitive response on tobacco. In this study, we present the nucleotide sequence of one of the hrp genes (hrpB) located at the left‐hand end of the cluster and we show that hrpB encodes a positive regulator controlling the expression of hrp genes. hrpB has a coding capacity for a 477‐amino‐acid polypeptide, which shows significant similarity to several prokaryotic transcriptional activators including the AraC protein of Escherichia coli, the XylS protein of Pseudomonas putida and the VirF protein of Yersinia enterocolitica. The predicted hrpB gene product belongs to a family of bacterial regulators different from the previously described HrpS protein of the hrp gene cluster of Pseudomonas syringae pv. phaseolicola. Genetic evidence demonstrates that the hrpB gene product acts as a positive regulator of the expression in minimal medium of all but one of the putative transcription units of the hrp gene cluster and also controls the expression of genes located outside this cluster. We also show in this paper that the transcription of hrpB is induced in minimal medium and is partly autoregulated.

A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes

The hrp genes of the plant pathogen Ralstonia solanacearum are key pathogenicity determinants; they encode a type III protein secretion machinery involved in the secretion of mediators of the bacterium–plant interaction. These hrp genes are under the genetic control of the hrpB regulatory gene, expression of which is induced when bacteria are co‐cultivated with plant cell suspensions. In this study, we used hrp–gfp transcriptional fusions to demonstrate that the expression of the hrpB and type III secretion genes is specifically induced in response to the bacterium–plant cell contact. This contact‐dependent induction of hrpB gene expression requires the outer membrane protein PrhA, but not a functional type III secretion apparatus. Genetic evidence indicates that PrhA constitutes the first example of a bacterial receptor for a non‐diffusible signal present in the plant cell wall and which triggers the transcriptional activation of bacterial virulence genes.