High-Quality Draft Genome Sequences of Two Xanthomonas Pathotype Strains Infecting Aroid Plants.

We present here the draft genome sequences of bacterial pathogens of the Araceae family, Xanthomonas axonopodis pv. dieffenbachiae LMG 695 and Xanthomonas campestris pv. syngonii LMG 9055, differing in host range. A comparison between genome sequences will help understand the mechanisms involved in tissue specificity and adaptation to host plants.

Draft Genome Sequence of the Flagellated Xanthomonas fuscans subsp. fuscans Strain CFBP 4884.

We report the draft genome sequence of the flagellated strain CFBP 4884 of Xanthomonas fuscans subsp. fuscans, which was isolated in an outbreak of common bacterial blight of beans along with non-flagellated strains. Comparative genomics will allow one to decipher the genomic diversity of strains cohabiting in epidemics.

Draft Genome Sequence of Xanthomonas axonopodis pv. allii Strain CFBP 6369.

We report here the draft genome sequence of Xanthomonas axonopodis pv. allii strain CFBP 6369, the causal agent of bacterial blight of onion. The draft genome has a size of 5,425,942 bp and a G+C content of 64.4%.

High-Quality Draft Genome Sequence of Xanthomonas alfalfae subsp. alfalfae Strain CFBP 3836.

We report the high-quality draft genome sequence of Xanthomonas alfalfae subsp. alfalfae strain CFBP 3836, the causal agent of bacterial leaf and stem spot in lucerne (Medicago sativa). Comparative genomics will help to decipher the mechanisms provoking disease and triggering the defense responses of this pathogen of the model legume Medicago truncatula.

High-Quality Draft Genome Sequences of Xanthomonas axonopodis pv. glycines Strains CFBP 2526 and CFBP 7119.

We report here the high-quality draft genome sequences of two strains of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule on soybeans. Comparison of these genomes with those of phylogenetically closely related pathovars of Xanthomonas spp. will help to understand the mechanisms involved in host specificity and adaptation to host plants.

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. 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.

[Current concepts on the pathogenicity of phytopathogenic bacteria]. / Concepts actuels sur la pathogénie chez les bactéries phytopathogènes.

What are the molecular determinants that make a bacterium a plant pathogen? In the last 10-20 years, important progress has been made in answering this question. In the early 20th century soon after the discovery of infectious diseases, the first studies of pathogenicity were undertaken. These early studies relied mostly on biochemistry and led to the discovery of several major pathogenicity determinants, such as toxins and hydrolytic enzymes which govern the production of major disease symptoms. From these pioneering studies, a simplistic view of pathogenicity arose. It was thought that only a few functions were sufficient to transform a bacterium into a pathogen. This view rapidly changed when modern techniques of molecular genetics were applied to analyse pathogenicity. Modern analyses of pathogenicity determinants took advantage of the relatively simple organization of the haploid genome of pathogenic bacteria. By creating non-pathogenic mutants, a large number of genes governing bacterium-host interactions were identified. These genes are required either for host colonization or for the production of symptoms. Even though the role of motility and chemotaxis in these processes is still unclear, it is clear that a strong attachment of Agrobacterium to plant cells is a prerequisite for efficient plant transformation and disease. Other important pathogenicity factors identified with a molecular genetic approach include hydrolytic enzymes such as pectinases and cellulases which not only provide nutrients to the bacteria but also facilitate pathogen invasion into host tissues. The precise role of exopolysaccharide in pathogenicity is still under discussion, however it is has been established that it is crucial for the induction of wilt symptoms caused by Ralstonia solanacearum. Trafficking of effector proteins from the invading bacterium into the host cell emerged recently as a new central concept. In plant pathogenic bacteria, protein translocation takes place through the so-called 'type II secretion machinery' encoded by hrp genes in the bacterium. These genes are present in representatives of all the major groups of Gram negative plant pathogenic bacteria except Agrobacterium. Most of these genes have counterparts in pathogens of mammals (including those of human) and they also play a central role in pathogenicity. Additionally, recent evidence suggests that a 'type IV secretion machinery' injects bacterial proteins into host cells. This machinery, originally found to be involved in the transfer of t-DNA from Agrobacterium into plant cells, was recently shown to translocate pathogenicity proteins in pathogens of mammals such as Helicobacter pylori and Brucella. Discovery of the trafficking of proteins from the pathogen into host cells revolutionized our conception of pathogenicity. First, it rather unexpectedly established the conservation of basic pathogenicity strategies in plant and animal pathogens. Second, this discovery changes our ideas about the overall strategy (or mechanism) of pathogenicity, although we still think the end result is exploitation of host cell nutritive components. Rather than killing the host cell from outside, we envision a more subtle approach in which pathogens inject effector proteins into the host cell to effect a change in host cell biology advantageous to the pathogen. Identification of the effector proteins, of their function and of the corresponding molecular targets in the host is a new challenge which will contribute to the conception of new strategies to control diseases.

Two novel proteins, PopB, which has functional nuclear localization signals, and PopC, which has a large leucine-rich repeat domain, are secreted through the hrp-secretion apparatus of Ralstonia solanacearum.

The Ralstonia solanacearum hrp gene cluster codes for components of a type III secretion pathway necessary for the secretion of PopA1, a hypersensitive response-like elicitor protein. In the present study, we show that several other Hrp-secreted proteins can be detected by growing wild-type bacteria in minimal medium in the presence of Congo red. Two of these proteins, PopB and PopC, are encoded by genes located downstream of popA and constitute an operon with popA. popABC mutants retain the wild-type ability to cause disease in hosts and to elicit the hypersensitive response on non-hosts. Expression of the popABC operon is controlled by the hrpB regulatory gene and is induced upon co-culture with Arabidopsis cell suspensions. This plant cell-specific induction depends on PrhA, a putative receptor for plant specific signal(s). The transcription of the popABC operon is not modified by the addition of Congo red to the growth medium and the intracellular pools of PopB and PopC are very similar in the absence or presence of Congo red. Preliminary data suggest that Congo red stabilizes secreted proteins in the extracellular medium. PopB is a 173-amino-acid-basic protein that contains a functional bipartite nuclear localization signal. PopC is a 1024-amino-acid protein that carries 22 tandem leucine-rich repeats (LRR). The LRR domain of this protein forms a consensus that perfectly matches the predicted eukaryotic cytoplasmic LRR consensus. We propose that PopB and PopC may be translocated into plant cells via the Hrp pathway.

Genetic characterization of RRS1, a recessive locus in Arabidopsis thaliana that confers resistance to the bacterial soilborne pathogen Ralstonia solanacearum.

The soilborne, vascular pathogen Ralstonia solanacearum, the causative agent of bacterial wilt, was shown to infect a range of Arabidopsis thaliana accessions. The pathogen was capable of infecting the Col-5 accession in an hrp-dependent manner, following root inoculation. Elevated bacterial population levels were found in leaves of Col-5, 4 to 5 days after root inoculation by the GMI1000 strain. Bacteria were found predominantly in the xylem vessels and spread systematically throughout the plant. The Nd-1 accession of A. thaliana was resistant to the GMI1000 strain of R. solanacearum. Bacterial concentrations detected in leaves of Nd-1, inoculated with an hrp+ strain of R. solanacearum, were only slightly higher than those detected in the susceptible accession, Col-5, following inoculation with a strain whose hrp gene cluster was deleted. Leaf inoculation of the GMI1000 strain on the resistant accession Nd-1 induced the formation of lesions in the older leaves of the rosette whereas the same strain of R. solanacearum provoked complete wilting of Col-5. Resistance to strain GMI1000 of R. solanacearum segregated as a simply inherited recessive trait in a genetic cross between Col-5 and Nd-1. F9 recombinant inbred lines generated between these two accessions were used to map a locus, RRS1, that was the major determinant of resistance between restriction fragment length polymorphism markers mi83 and mi61 on chromosome V. This region of the A. thaliana genome is known to contain many other pathogen recognition capabilities.

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 popA, located 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 hypo-aggressive 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.

Restriction Fragment Length Polymorphism Analyses of Iranian Strains of Xanthomonas campestris from Cereals and Grasses.

Restriction fragment length polymorphism (RFLP) analyses of the genomic DNA of 45 Xanthomonas campestris strains from cereals and grasses in Iran, and of 17 reference strains, were performed using two probes originating from X. campestris and including hrp genes. The Iranian strains studied belonged to three clearly distinct RFLP groups related to the grouping previously established on the basis of biochemical and physiological characters and host range. RFLP group 1 encompassed all the strains pathogenic to barley but not to the other plants tested (i.e., wheat, rye, Bromus inermis, Lolium multiflorum, Agropyron elongatum, and oat). RFLP group 2 contained strains that are pathogenic to all the above mentioned plants except oat. One strain, which has the same host range as group 2, was classified as RFLP group 3. Reference strains were distributed over these three groups, independently of their geographical origin. Strains in groups 1 and 3 had highly conserved RFLP patterns. In contrast, group 2 strains were easily split into two RFLP subgroups, although they did not differ significantly for other characters. The data suggest that RFLP analysis is a useful tool to distinguish among X. campestris strains causing bacterial leaf streak of cereals.

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. solancearum, include proteins encoded by fli and flh genes of S. typhimurium, Bacillus subtilis 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.

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.

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.

Xanthomonas campestris contains a cluster of hrp genes related to the larger hrp cluster of Pseudomonas solanacearum.

All Xanthomonas campestris pathovars tested contain DNA which hybridizes to the large hrp gene cluster of Pseudomonas solanacearum (C.A. Boucher, F. Van Gijsegem, P.A. Barberis, M. Arlat, and C. Zischek, J. Bacteriol. 169:5626-5632, 1987). Clones carrying these sequences were isolated from genomic libraries of X. campestris pvs. campestris and vitians. Mutagenesis of the corresponding genomic regions of both pathovars gave strains defective in both pathogenicity and hypersensitive response induction. X. c. pv. campestris contained a hrp gene cluster covering about 25 kb, which was homologous and colinear over a continuous 19-kb DNA region with the P. solanacearum hrp cluster. Cross-complementation showed that X. c. pv. vitians and X. c. pv. campestris hrp sequences are functionally interchangeable, but the source of the hrp genes did not determine the compatibility-incompatibility of the host-pathogen interaction. One X. c. pv. campestris Hrp- mutant was "complemented" by specific subclones of the P. solanacearum hrp cluster, suggesting the existence of some functional homology between the clusters of the two species. Expression of hrp genes (studied by lacZ fusions) was repressed in rich medium, and in minimal medium the level of expression depended on the carbon source supplied to the cells. Transcription of hrp genes was not regulated by genes that control the synthesis of extracellular enzymes, which are required for pathogenicity. In addition X. campestris Hrp- mutants produced wild-type levels of these extracellular enzyme activities. These results suggest the existence of two independent sets of pathogenicity genes that are regulated differently.

Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered.

A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.