The positive effects of root-colonizing bacteria cooperating with plants lead to improved growth and/or health of their eukaryotic hosts. Some of these Plant Growth-Promoting Rhizobacteria (PGPR) display several plant-beneficial properties, suggesting that the accumulation of the corresponding genes could have been selected in these bacteria. Here, this issue was targeted using 23 genes contributing directly or indirectly to established PGPR effects, based on genome sequence analysis of 304 contrasted Alpha- Beta- and Gammaproteobacteria. Most of the 23 genes studied were also found in non-PGPR Proteobacteria and none of them were common to all 25 PGPR genomes studied. However, ancestral character reconstruction indicated that gene transfers -predominantly ancient- resulted in characteristic gene combinations according to taxonomic subgroups of PGPR strains. This suggests that the PGPR-plant cooperation could have established separately in various taxa, yielding PGPR strains that use different gene assortments. The number of genes contributing to plant-beneficial functions increased along the continuum -animal pathogens, phytopathogens, saprophytes, endophytes/symbionts, PGPR- indicating that the accumulation of these genes (and possibly of different plant-beneficial traits) might be an intrinsic PGPR feature. This work uncovered preferential associations occurring between certain genes contributing to phytobeneficial traits and provides new insights into the emergence of PGPR bacteria.

Vibrios are frequently associated with oyster mortality; however whether they are the primary causative agent or secondary opportunistic colonizers is not well understood. Here we combine analysis of natural infection dynamics, population genomics and molecular genetics to ask (i) to what extent oysters are passively colonized by Vibrio population present in the surrounding water, (ii) how populations turn over during pathogenicity events and (iii) what genetic factors are responsible for pathogenicity. We identified several populations of Vibrio preferentially associated with oyster tissues. Among these, Vibrio crassostreae is particularly abundant in diseased animals while nearly absent in the surrounding water, and its pathogenicity is correlated with the presence of a large mobilizable plasmid. We further demonstrate that the plasmid is essential for killing but not necessary for survival in tissues of oysters. Our results suggest that V. crassostreae first differentiated into a benign oyster colonizer that was secondarily turned into a pathogen by introgression of a virulence plasmid into the population, possibly facilitated by elevated host density in farming areas.

Successive disease outbreaks in oyster (Crassostrea gigas) beds in France have resulted in dramatic losses in production, and subsequent decline in the oyster-farming industry. Deaths of juvenile oysters have been associated with the presence of a herpes virus (OsHV-1 μvar) and bacterial populations of the genus Vibrio. Although the pathogenicity of OsHV-1 μvar, as well as several strains of Vibrio has been demonstrated by experimental infections, our understanding of the complexity of infections occurring in the natural environment remains limited. In the present study, we use specific-pathogen-free (SPF) oysters infected in an estuarine environment to study the diversity and dynamics of cultured microbial populations during disease expression. We observe that rapid Vibrio colonization followed by viral replication precedes oyster death. No correlation was found between the vibrio concentration and viral load in co-infected animals. We show that the quantity of viral DNA is a predictor of mortality, however, in the absence of bacteria, a high load of herpes virus is not sufficient to induce the full expression of the disease. In addition, we demonstrate that juvenile mortalities can occur in the absence of herpes virus, indicating that the herpes virus appears neither essential nor sufficient to cause juvenile deaths; whereas bacteria are necessary for the disease. Finally, we demonstrate that oysters are a reservoir of putative pathogens, and that the geographic origin, age, and cultivation method of oysters influence disease expression.

Vibriospecies cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. HowVibriosubverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulentVibriospecies in an ecologically relevant host model, oyster, to study interactions with marineVibriospecies. AllVibriostrains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together withVibriogene knock-outs, we discovered thatVibrio crassostreaeandVibrio tasmaniensisuse distinct mechanisms to cause hemocyte lysis. WhereasV. crassostreaecytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function,r5.7,V. tasmaniensiscytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies onVibriospecies-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

Fluorescent pseudomonads protecting plant roots from phytopathogens by producing 2,4-diacetylphloroglucinol (DAPG) are considered to form a monophyletic lineage comprised of DAPG+ Pseudomonas strains in the “P. corrugata” and “P. protegens” subgroups of the “Pseudomonas fluorescens” group. However, DAPG production ability has not been investigated for many species of these two subgroups, and whether or not the DAPG+ Pseudomonas are truly monophyletic remained to be verified. Thus, the distribution of the DAPG biosynthetic operon (phlACBD genes) in the Pseudomonas spp. was investigated in sequenced genomes and type strains. Results showed that the DAPG+ Pseudomonas include species of the “P. fluorescens” group, i.e., P. protegens, P. brassicacearum, P. kilonensis, and P. thivervalensis, as expected, as well as P. gingeri in which it had not been documented. Surprisingly, they also include bacteria outside the “P. fluorescens” group, as exemplified by Pseudomonas sp. OT69, and even two Betaproteobacteria genera. The phl operon-based phylogenetic tree was substantially congruent with the one inferred from concatenated housekeeping genes rpoB, gyrB, and rrs. Contrariwise to current supposition, ancestral character reconstructions favored multiple independent acquisitions rather that one ancestral event followed by vertical inheritance. Indeed, based on synteny analyses, these acquisitions appeared to vary according to the Pseudomonas subgroup and even the phylogenetic groups within the subgroups. In conclusion, our study shows that the phl+ Pseudomonas populations form a polyphyletic group and suggests that DAPG biosynthesis might not be restricted to this genus. This is important to consider when assessing the ecological significance of phl+ bacterial populations in rhizosphere ecosystems.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a five month time-series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting cooccurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences are underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Diseases of marine animals caused by bacteria of the genus Vibrio are on the rise worldwide. Understanding the eco-evolutionary dynamics of these infectious agents is important for predicting and managing these diseases. Yet, compared to Vibrio infecting humans, knowledge of their role as animal pathogens is scarce. Here we ask how widespread is virulence among ecologically differentiated Vibrio populations, and what is the nature and frequency of virulence genes within these populations? We use a combination of population genomics and molecular genetics to assay hundreds of Vibrio strains for their virulence in the oyster Crassostrea gigas, a unique animal model that allows high-throughput infection assays. We show that within the diverse Splendidus clade, virulence represents an ancestral trait but has been lost from several populations. Two loci are necessary for virulence, the first being widely distributed across the Splendidus clade and consisting of an exported conserved protein (R5.7). The second is a MARTX toxin cluster, which only occurs within V. splendidus and is for the first time associated with virulence in marine invertebrates. Varying frequencies of both loci among populations indicate different selective pressures and alternative ecological roles, based on which we suggest strategies for epidemiological surveys.

24 ORIGINALITY-SIGNIFICANCE STATEMENT 25A recent study highlighted the role of a herpes virus as primary etiological agent of Pacific oyster 26 mortality syndrome (POMS), which affects juveniles of the oyster Crassostrea gigas. We show 27 here that the selection of virulent bacteria in oyster farms is also an important piece of the POMS 28 puzzle. This bacteria taxonomically assigned to Vibrio crassostreae species, carries a plasmid 29 that encodes a Type 6 Secretion System (T6SS), which increases its ability to kill the major 30 cellular players of oyster immunity, the hemocytes. This T6SS was identified in two additional 31 species that infect mollusks, suggesting a parallel evolution of these pathogens. Finally, our 32 results indicate that broad range of pathogens kill their hosts via hemocyte cytotoxicity. 33 34 ABSTRACT 35Pacific oyster mortality syndrome affects juveniles of Crassostrea gigas oysters and threatens the 36 sustainability of commercial and natural stocks of this species. Vibrio crassostreae has been 37 repeatedly isolated from diseased animals and the majority of the strains have been demonstrated 38 to be virulent for oysters. In this study we showed that oyster farms exhibited a high prevalence 39 of a virulence plasmid carried by V. crassostreae while oysters, at an adult stage, were reservoirs 40 of this virulent population. The pathogenicity of V. crassostreae depends on a novel 41 transcriptional regulator, which activates the bidirectional promoter of a Type 6 Secretion System 42 (T6SS) genes cluster. Both the T6SS and a second chromosomal virulence factor, r5.7, are 43 necessary for virulence but act independently to cause to hemocyte (oyster immune cell) 44 cytotoxicity. A phylogenetically closely related T6SS was identified in V. aestuarianus and V. 45 tapetis, which infect adult oysters and clams, respectively. We propose that hemocyte 46 cytotoxicity, is a lethality trait shared by a broad range of mollusk pathogens and we speculate 47 107 108 RESULTS 110The virulence plasmid is widespread in V. crassostreae population occurring in oyster farms 111We previously hypothesized that the introgression of the virulence plasmid pGV into 112 V. crassostreae might have been favored by elevated host density in farming areas (Bruto et al., 113 2017). However, wild oyster beds can also reach high densities, as exemplified by the recent 114 invasion of C. gigas oysters into the Wadden sea (North Sea) (Reise et al., 2017). To date, no 115Vibrio-associated mass mortalities have been observed in this area, in contrast to observations in 116 heavily farmed areas. We thus investigated the presence and frequency of the pGV plasmid in 117 V. crassostreae strains sampled from Sylt. For this, 910 Vibrio strains were isolated from 118 seawater fractions and oysters from Sylt, genotyped by partial hsp60 gene sequencing and 119 assigned to Vibrio populations as described previously ( Figure S1). Multi Locus Sequencing 120Typing (MLST) further confirmed the taxonomic assignment of 47 V. crassostreae str...