The type VI secretion system of Stenotrophomonas rhizophila CFBP13503 limits the transmission of Xanthomonas campestris pv. campestris 8004 from radish seeds to seedlings.

Stenotrophomonas rhizophila CFBP13503 is a seedborne commensal bacterial strain, which is efficiently transmitted to seedlings and can outcompete the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc8004). The type VI secretion system (T6SS), an interference contact-dependent mechanism, is a critical component of interbacterial competition. The involvement of the T6SS of S. rhizophila CFBP13503 in the inhibition of Xcc8004 growth and seed-to-seedling transmission was assessed. The T6SS cluster of S. rhizophila CFBP13503 and nine putative effectors were identified. Deletion of two T6SS structural genes, hcp and tssB, abolished the competitive advantage of S. rhizophila against Xcc8004 in vitro. The population sizes of these two bacterial species were monitored in seedlings after inoculation of radish seeds with mixtures of Xcc8004 and either S. rhizophila wild-type (wt) strain or isogenic hcp mutant. A significant decrease in the population size of Xcc8004 was observed during confrontation with the S. rhizophila wt in comparison with T6SS-deletion mutants in germinated seeds and seedlings. We found that the T6SS distribution among 835 genomes of the Stenotrophomonas genus is scarce. In contrast, in all available S. rhizophila genomes, T6SS clusters are widespread and mainly belong to the T6SS group i4. In conclusion, the T6SS of S. rhizophila CFBP13503 is involved in the antibiosis against Xcc8004 and reduces seedling transmission of Xcc8004 in radish. The distribution of this T6SS cluster in the S. rhizophila complex could make it possible to exploit these strains as biocontrol agents against X. campestris pv. campestris.

Genome mining reveals the genus Xanthomonas to be a promising reservoir for new bioactive non-ribosomally synthesized peptides.

BACKGROUND: Various bacteria can use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. Conserved features within the NRPS machinery allow the type, and sometimes even the structure, of the synthesized polypeptide to be predicted. Thus, bacterial genome mining via in silico analyses of NRPS genes offers an attractive opportunity to uncover new bioactive non-ribosomally synthesized peptides. Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant species. To date, the only known small molecule synthesized by NRPS in this genus is albicidin produced by Xanthomonas albilineans. This study aims to estimate the biosynthetic potential of Xanthomonas spp. by in silico analyses of NRPS genes with unknown function recently identified in the sequenced genomes of X. albilineans and related species of Xanthomonas. RESULTS: We performed in silico analyses of NRPS genes present in all published genome sequences of Xanthomonas spp., as well as in unpublished draft genome sequences of Xanthomonas oryzae pv. oryzae strain BAI3 and Xanthomonas spp. strain XaS3. These two latter strains, together with X. albilineans strain GPE PC73 and X. oryzae pv. oryzae strains X8-1A and X11-5A, possess novel NRPS gene clusters and share related NRPS-associated genes such as those required for the biosynthesis of non-proteinogenic amino acids or the secretion of peptides. In silico prediction of peptide structures according to NRPS architecture suggests eight different peptides, each specific to its producing strain. Interestingly, these eight peptides cannot be assigned to any known gene cluster or related to known compounds from natural product databases. PCR screening of a collection of 94 plant pathogenic bacteria indicates that these novel NRPS gene clusters are specific to the genus Xanthomonas and are also present in Xanthomonas translucens and X. oryzae pv. oryzicola. Further genome mining revealed other novel NRPS genes specific to X. oryzae pv. oryzicola or Xanthomonas sacchari. CONCLUSIONS: This study revealed the significant potential of the genus Xanthomonas to produce new non-ribosomally synthesized peptides. Interestingly, this biosynthetic potential seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement of non-ribosomally synthesized peptides in plant-bacteria interactions.

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

BACKGROUND: Xanthomonads 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. RESULTS: Comparative 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. CONCLUSIONS: This 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.

Genomic and evolutionary features of the SPI-1 type III secretion system that is present in Xanthomonas albilineans but is not essential for xylem colonization and symptom development of sugarcane leaf scald.

Xanthomonas albilineans is the causal agent of sugarcane leaf scald. Interestingly, this bacterium, which is not known to be insect or animal associated, possesses a type III secretion system (T3SS) belonging to the injectisome family Salmonella pathogenicity island 1 (SPI-1). The T3SS SPI-1 of X. albilineans shares only low similarity with other available T3SS SPI-1 sequences. Screening of a collection of 128 plant-pathogenic bacteria revealed that this T3SS SPI-1 is present in only two species of Xanthomonas: X. albilineans and X. axonopodis pv. phaseoli. Inoculation of sugarcane with knockout mutants showed that this system is not required by X. albilineans to spread within xylem vessels and to cause disease symptoms. This result was confirmed by the absence of this T3SS SPI-1 in an X. albilineans strain isolated from diseased sugarcane. To investigate the importance of the T3SS SPI-1 during the life cycle of X. albilineans, we analyzed T3SS SPI-1 sequences from 11 strains spanning the genetic diversity of this species. No nonsense mutations or frameshifting indels were observed in any of these strains, suggesting that the T3SS SPI-1 system is maintained within the species X. albilineans. Evolutionary features of T3SS SPI-1 based on phylogenetic, recombination, and selection analyses are discussed in the context of the possible functional importance of T3SS SPI-1 in the ecology of X. albilineans.

A Computation Method Based on the Combination of Chlorophyll Fluorescence Parameters to Improve the Discrimination of Visually Similar Phenotypes Induced by Bacterial Virulence Factors.

Phenotyping biotic stresses in plant-pathogen interactions studies is often hindered by phenotypes that can hardly be discriminated by visual assessment. Particularly, single gene mutants in virulence factors could lack visible phenotypes. Chlorophyll fluorescence (CF) imaging is a valuable tool to monitor plant-pathogen interactions. However, while numerous CF parameters can be measured, studies on plant-pathogen interactions often focus on a restricted number of parameters. It could result in limited abilities to discriminate visually similar phenotypes. In this study, we assess the ability of the combination of multiple CF parameters to improve the discrimination of such phenotypes. Such an approach could be of interest for screening and discriminating the impact of bacterial virulence factors without prior knowledge. A computation method was developed, based on the combination of multiple CF parameters, without any parameter selection. It involves histogram Bhattacharyya distance calculations and hierarchical clustering, with a normalization approach to take into account the inter-leaves and intra-phenotypes heterogeneities. To assess the efficiency of the method, two datasets were analyzed the same way. The first dataset featured single gene mutants of a Xanthomonas strain which differed only by their abilities to secrete bacterial virulence proteins. This dataset displayed expected phenotypes at 6 days post-inoculation and was used as ground truth dataset to setup the method. The efficiency of the computation method was demonstrated by the relevant discrimination of phenotypes at 3 days post-inoculation. A second dataset was composed of transient expression (agrotransformation) of Type 3 Effectors. This second dataset displayed phenotypes that cannot be discriminated by visual assessment and no prior knowledge can be made on the respective impact of each Type 3 Effectors on leaf tissues. Using the computation method resulted in clustering the leaf samples according to the Type 3 Effectors, thereby demonstrating an improvement of the discrimination of the visually similar phenotypes. The relevant discrimination of visually similar phenotypes induced by bacterial strains differing only by one virulence factor illustrated the importance of using a combination of CF parameters to monitor plant-pathogen interactions. It opens a perspective for the identification of specific signatures of biotic stresses.

Role of the acquisition of a type 3 secretion system in the emergence of novel pathogenic strains of Xanthomonas.

Cases of emergence of novel plant-pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non-pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non-pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild-type, non-pathogenic strains induced a hypersensitive response (HR)-like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non-pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non-pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter-selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.

Assessment of the genetic diversity of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans as a basis to identify putative pathogenicity genes and a type III secretion system of the SPI-1 family by multiple suppression subtractive hybridizations.

Fluorescent amplified fragment length polymorphism revealed that strains of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans are genetically distinct and can be grouped into four genetic lineages. Four suppression subtractive hybridizations were then performed to isolate DNA fragments present in these bean pathogens and absent from closely related xanthomonads. Virulence gene candidates were identified such as homologs of hemagglutinins, TonB-dependent receptors, zinc-dependent metalloproteases, type III effectors, and type IV secretion system components. Unexpectedly, homologs of the type III secretion apparatus components (SPI-1 family), usually reported in animal pathogens and insect symbionts, were also detected.

Recombination-prone bacterial strains form a reservoir from which epidemic clones emerge in agroecosystems.

The acquisition of virulence-related genes through horizontal gene transfer can modify the pathogenic profiles of strains and lead to the emergence of new diseases. Xanthomonas arboricola is a bacterial species largely known for the damage it causes to stone and nut fruit trees worldwide. In addition to these host-specific populations called pathovars, many nonpathogenic strains have been identified in this species. Their evolutionary significance in the context of pathogen emergence is unknown. We looked at seven housekeeping genes amplified from 187 pathogenic and nonpathogenic strains isolated from various plants worldwide to analyze population genetics and recombination dynamics. We also examined the dynamics of the gains and losses of genes associated with life history traits (LHTs) during X. arboricola evolution. We discovered that X. arboricola presents an epidemic population structure. Successful pathovars of trees (i.e. pruni, corylina and juglandis) are epidemic clones whose emergence appears to be linked to the acquisition of eight genes coding for Type III effectors. The other strains of this species are part of a recombinant network, within which LHT-associated genes might have been lost. We suggest that nonpathogenic strains, because of their high genetic diversity and propensity for recombination, may promote the emergence of pathogenic strains.

Evolutionary history of the plant pathogenic bacterium Xanthomonas axonopodis.

Deciphering mechanisms shaping bacterial diversity should help to build tools to predict the emergence of infectious diseases. Xanthomonads are plant pathogenic bacteria found worldwide. Xanthomonas axonopodis is a genetically heterogeneous species clustering, into six groups, strains that are collectively pathogenic on a large number of plants. However, each strain displays a narrow host range. We address the question of the nature of the evolutionary processes--geographical and ecological speciation--that shaped this diversity. We assembled a large collection of X. axonopodis strains that were isolated over a long period, over continents, and from various hosts. Based on the sequence analysis of seven housekeeping genes, we found that recombination occurred as frequently as point mutation in the evolutionary history of X. axonopodis. However, the impact of recombination was about three times greater than the impact of mutation on the diversity observed in the whole dataset. We then reconstructed the clonal genealogy of the strains using coalescent and genealogy approaches and we studied the diversification of the pathogen using a model of divergence with migration. The suggested scenario involves a first step of generalist diversification that spanned over the last 25,000 years. A second step of ecology-driven specialization occurred during the past two centuries. Eventually, secondary contacts between host-specialized strains probably occurred as a result of agricultural development and intensification, allowing genetic exchanges of virulence-associated genes. These transfers may have favored the emergence of novel pathotypes. Finally, we argue that the largest ecological entity within X. axonopodis is the pathovar.

Multilocus sequence analysis and type III effector repertoire mining provide new insights into the evolutionary history and virulence of Xanthomonas oryzae.

Multilocus sequence analysis (MLSA) and type III effector (T3E) repertoire mining were performed to gain new insights into the genetic relatedness of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), two major bacterial pathogens of rice. Based on a collection of 45 African and Asian strains, we first sequenced and analysed three housekeeping genes by MLSA, Bayesian clustering and a median-joining network approach. Second, we investigated the distribution of 32 T3E genes, which are known to be major virulence factors of plant pathogenic bacteria, in all selected strains, by polymerase chain reaction and dot-blot hybridization methods. The diversity observed within housekeeping genes, as well as within T3E repertoires, clearly showed that both pathogens belong to closely related, but distinct, phylogenetic groups. Interestingly, these evolutionary groups are differentiated according to the geographical origin of the strains, suggesting that populations of Xoo and Xoc might be endemic in Africa and Asia, and thus have evolved separately. We further revealed that T3E gene repertoires of both pathogens comprise core and variable gene suites that probably have distinct roles in pathogenicity and different evolutionary histories. In this study, we carried out a functional analysis of xopO, a differential T3E gene between Xoo and Xoc, to determine the involvement of this gene in tissue specificity. Altogether, our data contribute to a better understanding of the evolutionary history of Xoo and Xoc in Africa and Asia, and provide clues for functional studies aiming to understand the virulence, host and tissue specificity of both rice pathogens.

A "repertoire for repertoire" hypothesis: repertoires of type three effectors are candidate determinants of host specificity in Xanthomonas.

BACKGROUND: The genetic basis of host specificity for animal and plant pathogenic bacteria remains poorly understood. For plant pathogenic bacteria, host range is restricted to one or a few host plant species reflecting a tight adaptation to specific hosts. METHODOLOGY/PRINCIPAL FINDINGS: Two hypotheses can be formulated to explain host specificity: either it can be explained by the phylogenetic position of the strains, or by the association of virulence genes enabling a pathological convergence of phylogenically distant strains. In this latter hypothesis, host specificity would result from the interaction between repertoires of bacterial virulence genes and repertoires of genes involved in host defences. To challenge these two hypotheses, we selected 132 Xanthomonas axonopodis strains representative of 18 different pathovars which display different host range. First, the phylogenetic position of each strain was determined by sequencing the housekeeping gene rpoD. This study showed that many pathovars of Xanthomonas axonopodis are polyphyletic. Second, we investigated the distribution of 35 type III effector genes (T3Es) in these strains by both PCR and hybridization methods. Indeed, for pathogenic bacteria T3Es were shown to trigger and to subvert host defences. Our study revealed that T3E repertoires comprise core and variable gene suites that likely have distinct roles in pathogenicity and different evolutionary histories. Our results showed a correspondence between composition of T3E repertoires and pathovars of Xanthomonas axonopodis. For polyphyletic pathovars, this suggests that T3E genes might explain a pathological convergence of phylogenetically distant strains. We also identified several DNA rearrangements within T3E genes, some of which correlate with host specificity of strains. CONCLUSIONS/SIGNIFICANCE: These data provide insight into the potential role played by T3E genes for pathogenic bacteria and support a "repertoire for repertoire" hypothesis that may explain host specificity. Our work provides resources for functional and evolutionary studies aiming at understanding host specificity of pathogenic bacteria, functional redundancy between T3Es and the driving forces shaping T3E repertoires.