Stéphane Poussier

The complete genome sequence of Xanthomonas albilineans provides new insights into the reductive genome evolution of the xylem-limited Xanthomonadaceae

BackgroundThe Xanthomonadaceae family contains two xylem-limited plant pathogenic bacterial species, Xanthomonas albilineans and Xylella fastidiosa. X. fastidiosa was the first completely sequenced plant pathogen. It is insect-vectored, has a reduced genome and does not possess hrp genes which encode a Type III secretion system found in most plant pathogenic bacteria. X. fastidiosa was excluded from the Xanthomonas group based on phylogenetic analyses with rRNA sequences.ResultsThe complete genome of X. albilineans was sequenced and annotated. X. albilineans, which is not known to be insect-vectored, also has a reduced genome and does not possess hrp genes. Phylogenetic analysis using X. albilineans genomic sequences showed that X. fastidiosa belongs to the Xanthomonas group. Order of divergence of the Xanthomonadaceae revealed that X. albilineans and X. fastidiosa experienced a convergent reductive genome evolution during their descent from the progenitor of the Xanthomonas genus. Reductive genome evolutions of the two xylem-limited Xanthomonadaceae were compared in light of their genome characteristics and those of obligate animal symbionts and pathogens.ConclusionThe two xylem-limited Xanthomonadaceae, during their descent from a common ancestral parent, experienced a convergent reductive genome evolution. Adaptation to the nutrient-poor xylem elements and to the cloistered environmental niche of xylem vessels probably favoured this convergent evolution. However, genome characteristics of X. albilineans differ from those of X. fastidiosa and obligate animal symbionts and pathogens, indicating that a distinctive process was responsible for the reductive genome evolution in this pathogen. The possible role in genome reduction of the unique toxin albicidin, produced by X. albilineans, is discussed.

Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region, AFLP and 16S rRNA sequence analysis, and identification of an African subdivision.

The genetic diversity among strains in a worldwide collection of Ralstonia solanacearum, causal agent of bacterial wilt, was assessed by using three different molecular methods. PCR-RFLP analysis of the hrp gene region was extended from previous studies to include additional strains and showed that five amplicons were produced not only with all R. solanacearum strains but also with strains of the closely related bacteria Pseudomonas syzygii and the blood disease bacterium (BDB). However, the three bacterial taxa could be discriminated by specific restriction profiles. The PCR-RFLP clustering, which agreed with the biovar classification and the geographical origin of strains, was confirmed by AFLP. Moreover, AFLP permitted very fine discrimination between different isolates and was able to differentiate strains that were not distinguishable by PCR-RFLP. AFLP and PCR-RFLP analyses confirmed the results of previous investigations which split the species into two divisions, but revealed a further subdivision. This observation was further supported by 16S rRNA sequence data, which grouped biovar 1 strains originating from the southern part of Africa.

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.

Contrasting recombination patterns and demographic histories of the plant pathogen Ralstonia solanacearum inferred from MLSA

We used multilocus sequence analysis (MLSA) on a worldwide collection of the plant pathogenic Ralstonia solanacearum (Betaproteobacteria) to retrace its complex evolutionary history. Using genetic imprints left during R. solanacearum evolution, we were able to delineate distinct evolutionary complex displaying contrasting dynamics. Among the phylotypes already described (I, IIA, IIB, III, IV), eight groups of strains with distinct evolutionary patterns, named clades, were identified. From our recombination analysis, we identified 21 recombination events that occurred within and across these lineages. Although appearing the most divergent and ancestral phylotype, phylotype IV was inferred as a gene donor for the majority of the recombination events that we detected. Whereas this phylotype apparently fuelled the species diversity, ongoing diversification was mainly detected within phylotype I, IIA and III. These three groups presented a recent expanding population structure, a high level of homologous recombination and evidences of long-distance migrations. Factors such as adaptation to a specific host or intense trading of infected crops may have promoted this diversification. Whether R. solanacearum lineages will eventually evolve in distinct species remains an open question. The intensification of cropping and increase of geographical dispersion may favour situations of phylotype sympatry and promote higher exchange of key factors for host adaptation from their common genetic pool.

Type Three Effector Gene Distribution and Sequence Analysis Provide New Insights into the Pathogenicity of Plant-Pathogenic Xanthomonas arboricola

ABSTRACT Xanthomonas arboricola is a complex bacterial species which mainly attacks fruit trees and is responsible for emerging diseases in Europe. It comprises seven pathovars (X. arboricola pv. pruni, X. arboricola pv. corylina, X. arboricola pv. juglandis, X. arboricola pv. populi, X. arboricola pv. poinsettiicola, X. arboricola pv. celebensis, and X. arboricola pv. fragariae), each exhibiting characteristic disease symptoms and distinct host specificities. To better understand the factors underlying this ecological trait, we first assessed the phylogenetic relationships among a worldwide collection of X. arboricola strains by sequencing the housekeeping gene rpoD. This analysis revealed that strains of X. arboricola pathovar populi are divergent from the main X. arboricola cluster formed by all other strains. Then, we investigated the distribution of 53 type III effector (T3E) genes in a collection of 57 X. arboricola strains that are representative of the main X. arboricola cluster. Our results showed that T3E repertoires vary greatly between X. arboricola pathovars in terms of size. Indeed, X. arboricola pathovars pruni, corylina, and juglandis, which are responsible for economically important stone fruit and nut diseases in Europe, harbored the largest T3E repertoires, whereas pathovars poinsettiicola, celebensis, and fragariae harbored the smallest. We also identified several differences in T3E gene content between X. arboricola pathovars pruni, corylina, and juglandis which may account for their differing host specificities. Further, we examined the allelic diversity of eight T3E genes from X. arboricola pathovars. This analysis revealed very limited allelic variations at the different loci. Altogether, the data presented here provide new insights into the evolution of pathogenicity and host range of X. arboricola and are discussed in terms of emergence of new diseases within this bacterial species.

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.

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.

Specific detection of biovars of Ralstonia solanacearum in plant tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 103 cfu ml−1, which corresponds to 1 cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.

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

ABSTRACT 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.

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.