Patrick Barberis

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.

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.

Transcriptional organization and expression of the large hrp gene cluster of Pseudomonas solanacearum.

Cloning and localized mutagenesis of the larger cluster of hrp genes of Pseudomonas solanacearum strain GMI1000 allowed the definition of the borders of this cluster, which now extends about 2 kb to the left of the insert of the previously described plasmid pVir2 (Boucher et al. 1987, J. Bacteriol. 169:5626-5632). The size of the cluster has also been expanded 3 kb to the right to include a region previously described as dsp; our present data demonstrate that insertions occurring in these 3 kb lead to leaky mutations affecting both pathogenicity on tomato and ability to induce the hypersensitive response (HR) on tobacco. Therefore, the size of the entire hrp gene cluster is estimated to be about 22 kb. The use of transposon Tn5-B20, which promotes transcriptional gene fusions, allowed us to demonstrate that the hrp gene cluster is organized in a minimum of six transcriptional units, which are transcribed when the culture is grown in minimal medium but are repressed during growth in rich medium or in the presence of peptone or Casamino Acids. The level of expression in minimal medium is modulated by the carbon source provided; pyruvate is the best inducer. Under these conditions the level of expression observed in vitro appears to be representative of the actual expression observed in planta.

prhJ and hrpG, two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum

hrp gene expression in the phytopathogenic bacterium Ralstonia solanacearum GMI1000 is induced through the HrpB regulator in minimal medium and upon co‐culture with plant cell suspensions. The putative outer membrane protein PrhA is specifically involved in hrp gene activation in the presence of plant cells and has been proposed to be a receptor of a plant‐dependent signal transduction pathway. Here, we report on the identification of two regulatory genes, hrpG and prhJlocated at the right‐hand end of the hrp gene cluster, that are required for full pathogenicity. HrpG belongs to the OmpR subclass of two‐component response regulators and is homologous to HrpG, the activator of hrp genes in Xanthomonas campestris pv. vesicatoria. PrhJ is a novel hrp regulatory protein, sharing homology with the LuxR/UhpA family of transcriptional activators. As for HrpG of X. c. pv. vesicatoria, HrpG is required for hrp gene expression in minimal medium, but, in addition, we show that it also controls hrpB gene activation upon co‐culture with Arabidopsis thaliana and tomato cell suspensions. In contrast, PrhJ is specifically involved in hrp gene expression in the presence of plant cells. hrpG and prhJ gene transcription is plant cell inducible through the PrhA‐dependent pathway. From these results, we propose a regulatory cascade in which plant cell signal(s) sensed by PrhA are transduced to the prhJ gene, whose predicted product controls hrpG gene expression. HrpG then activates the hrpB regulatory gene, and, subsequently, the remaining hrp transcriptional units in all known inducing conditions.

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.

A Signal Transfer System Through Three Compartments Transduces the Plant Cell Contact-Dependent Signal Controlling Ralstonia solanacearum hrp Genes

Ralstonia solanacearum hrp genes encode a type III secretion system required for disease development in host plants and for hypersensitive response elicitation on non-hosts. hrp genes are expressed in the presence of plant cells through the HrpB regulator. This activation, which requires physical interaction between the bacteria and the plant cell, is sensed by the outer membrane receptor PrhA. PrhA transduces the plant cell contact-dependent signal through a complex regulatory cascade integrated by the PrhJ, HrpG, and HrpB regulators. In this study, we have identified two genes, named prhI and prhR, that belong to the hrp gene cluster and whose predicted products show homology with extracytoplasmic function sigma factors and transmembrane proteins, respectively. Strains carrying a mutation in prhIR show a delayed pathogenic phenotype toward host plants. PrhIR control the plant cell contact-dependent activation of hrp genes. prhIR gene expression is induced by a signal present in the plant cell coculture that is not PrhA-dependent. Genetic evidence shows that PrhIR act upstream of PrhJ in the regulatory cascade, likely transducing the signal sensed by PrhA through the periplasm as described for signal transfer systems through three compartments. This is the first report of such a surface signaling mechanism activating pathogenicity determinants in response to a nondiffusible plant cell wall signal.

Two Type III Secretion System Effectors from Ralstonia solanacearum GMI1000 Determine Host-Range Specificity on Tobacco

The model pathogen Ralstonia solanacearum GMI1000 is the causal agent of the bacterial wilt disease that attacks many solanaceous plants and other hosts but not tobacco (Nicotiana spp.). We found that two type III secretion system effector genes, avrA and popP1, are limiting the host range of strain GMI1000 on at least three tobacco species (N. tabacum, N. benthamiana, and N. glutinosa). Both effectors elicit the hypersensitive response (HR) on these tobacco species, although in different manners; AvrA is the major determinant recognized by N. tabacum and N. benthamiana, while PopP1 appears to be the major HR elicitor on N. glutinosa. Only the double inactivation of the avrA and popP1 genes allowed GMI1000 to wilt tobacco plants, thus showing that GMI1000 intrinsically possesses the functions necessary to wilt tobacco plants. A focused analysis on AvrA revealed that the first 58 N-terminal amino acids are sufficient to direct its injection into plant cells. We identified a hypervariable region in avrA, which contains variable numbers of tandem repeats (VNTR), each composed of 12 base pairs. We show that an 18-amino acid region in which the VNTR insertion occurs is an important domain involved in HR elicitation on N. benthamiana. avrA appears to be the target of various DNA insertions or mobile elements that probably allow R. solanacearum to evade the recognition and defense responses of tobacco.

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

SummaryA class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acrr), harbour a deletion of over 85 kb in their genome. This deletion affects, a≤1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acrr mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.

A Competitive Index Assay Identifies Several Ralstonia solanacearum Type III Effector Mutant Strains with Reduced Fitness in Host Plants

Ralstonia solanacearum, the causal agent of bacterial wilt, is a soil bacterium which can naturally infect a wide range of host plants through the root system. Pathogenicity relies on a type III secretion system which delivers a large set of approximately 75 type III effectors (T3E) into plant cells. On several plants, pathogenicity assays based on quantification of wilting symptoms failed to detect a significant contribution of R. solanacearum T3E in this process, thus revealing the collective effect of T3E in pathogenesis. We developed a mixed infection-based method with R. solanacearum to monitor bacterial fitness in plant leaf tissues as a virulence assay. This accurate and sensitive assay provides evidence that growth defects can be detected for T3E mutants: we identified 12 genes contributing to bacterial fitness in eggplant leaves and 3 of them were also implicated in bacterial fitness on two other hosts, tomato and bean. Contribution to fitness of several T3E appears to be host specific, and we show that some known avirulence determinants such as popP2 or avrA do provide competitive advantages on some susceptible host plants. In addition, this assay revealed that the efe gene, which directs the production of ethylene by bacteria in plant tissues, and hdfB, involved in the biosynthesis of the secondary metabolite 3-hydroxy-oxindole, are also required for optimal growth in plant leaf tissues.