Matthieu Arlat

Genome sequence of the plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. Itxa0is 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.

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.

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.

PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells

The Ralstonia solanacearum hrp gene cluster is organized in five transcriptional units. Expression of transcriptional units 2, 3 and 4 is induced in minimal medium and depends on the hrp regulatory gene hrpB, which belongs to unit 1. This regulatory gene also controls the expression of genes, such as popAlocated to the left of the hrp cluster. Here, we show that, upon co‐culture with Arabidopsis thaliana and tomato cell suspensions, the expression of the hrp transcriptional units 1, 2, 3 and 4 is induced 10‐ to 20‐fold more than in minimal medium. This induction is not triggered by diffusible signals but requires the presence of plant cells. Moreover, we show that this specific plant cell induction of hrp genes is controlled by a gene, called prhA (plant regulator of hrp genes), located next to popA. This gene codes for a putative protein of 770 amino acids, which shows similarities with TonB‐dependent outer membrane siderophore receptors. Expression of prhA and hrp genes is not regulated by iron status, and we postulate that iron is not the signal sensed by PrhA. In prhA mutants, the induction of hrpB and other hrp genes is abolished in co‐culture with Arabidopsis cells, partially reduced in co‐culture with tomato cells and not modified in minimal medium. prhA mutants are hypoaggressive on Arabidopsis (accessions Col‐0 and Col‐5) but remain fully pathogenic on tomato plants, suggesting that the co‐culture assays mimic the in planta conditions. A model suggesting that PrhA is a receptor for plant specific signals at the top of a novel hrp regulatory pathway is discussed.

Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins

Xylem plays a major role in plant development and is considered part of the apoplast. Here, we studied the proteome of Brassica oleracea cv Bartolo and compared it to the plant cell wall proteome of another Brassicaceae, the model plant Arabidopsis thaliana. B. oleracea was chosen because it is technically difficult to harvest enough A. thaliana xylem sap for proteomic analysis. We studied the whole proteome and an N‐glycoproteome obtained after Concanavalin A affinity chromatography. Altogether, 189 proteins were identified by LC‐MS/MS using Brassica EST and cDNA sequences. A predicted signal peptide was found in 164 proteins suggesting that most proteins of the xylem sap are secreted. Eighty‐one proteins were identified in the N‐glycoproteome, with 25 of them specific of this fraction, suggesting that they were concentrated during the chromatography step. All the protein families identified in this study were found in the cell wall proteomes. However, proteases and oxido‐reductases were more numerous in the xylem sap proteome, whereas enzyme inhibitors were rare. The origin of xylem sap proteins is discussed. All the experimental data including the MS/MS data were made available in the WallProtDB cell wall proteomic database.

AvrACXcc8004, a Type III Effector with a Leucine-Rich Repeat Domain from Xanthomonas campestris Pathovar campestris Confers Avirulence in Vascular Tissues of Arabidopsis thaliana Ecotype Col-0

Xanthomonas campestris pathovar campestris causes black rot, a vascular disease on cruciferous plants, including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC(Xcc8004), functions as an avirulence gene whose product seems to be recognized in vascular tissues.

The Decoy Substrate of a Pathogen Effector and a Pseudokinase Specify Pathogen-Induced Modified-Self Recognition and Immunity in Plants

In plants, host response to pathogenic microbes is driven both by microbial perception and detection of modified-self. The Xanthomonas campestris effector protein AvrAC/XopAC uridylylates the Arabidopsis BIK1 kinase to dampen basal resistance andxa0thereby promotes bacterial virulence. Here we show that PBL2, a paralog of BIK1, is similarly uridylylated by AvrAC. However, in contrast to BIK1, PBL2 uridylylation is specifically required for host recognition of AvrAC to trigger immunity, but not AvrAC virulence. PBL2 thus acts as a decoy and enables AvrAC detection. AvrAC recognition also requires the RKS1 pseudokinase of the ZRK family and the NOD-like receptor ZAR1, which is known to recognize the Pseudomonas syringae effector HopZ1a. ZAR1 forms a stable complex with RKS1, which specifically recruits PBL2 when the latter is uridylylated by AvrAC, triggering ZAR1-mediated immunity. The results illustrate how decoy substrates and pseudokinases can specify and expand the capacity of the plant immune system.

Host plant-dependent phenotypic reversion of #Ralstonia solanacearum# from non-pathogenic to pathogenic forms via alterations in the phcA gene

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild‐type pathogenic to a non‐pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild‐type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64u2003bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.

PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family

Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads

BackgroundXanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences.ResultsComparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations.ConclusionsThis work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.