Philippe Remigi

Phytophthora species in forest streams in Oregon and Alaska

Eighteen Phytophthora species and one species of Halophytophthora were identified in 113 forest streams in Alaska, western Oregon and southwestern Oregon that were sampled by baiting or filtration of stream water with isolation on selective media. Species were identified by morphology and DNA characterization with single strand conformational polymorphism, COX spacer sequence and ITS sequence. ITS Clade 6 species were most abundant overall, but only four species, P. gonapodyides (37% of all isolates), P. taxon Salixsoil, P. taxon Oaksoil and P. pseudosyringae, were found in all three regions. The species assemblages were similar in the two Oregon regions, but P. taxon Pgchlamydo was absent in Alaska and one new species present in Alaska was absent in Oregon streams. The number of Phytophthora propagules in Oregon streams varied by season and in SW Oregon, where sampling continued year round, P. taxon Salixsoil, P. nemorosa and P. siskiyouensis were recovered only in some seasons.

Symbiosis within Symbiosis: Evolving Nitrogen-Fixing Legume Symbionts.

Bacterial accessory genes are genomic symbionts with an evolutionary history and future that is different from that of their hosts. Packages of accessory genes move from strain to strain and confer important adaptations, such as interaction with eukaryotes. The ability to fix nitrogen with legumes is a remarkable example of a complex trait spread by horizontal transfer of a few key symbiotic genes, converting soil bacteria into legume symbionts. Rhizobia belong to hundreds of species restricted to a dozen genera of the Alphaproteobacteria and Betaproteobacteria, suggesting infrequent successful transfer between genera but frequent successful transfer within genera. Here we review the genetic and environmental conditions and selective forces that have shaped evolution of this complex symbiotic trait.

Functional diversification of the GALA type III effector family contributes to Ralstonia solanacearum adaptation on different plant hosts

Type III effectors from phytopathogenic bacteria exhibit a high degree of functional redundancy, hampering the evaluation of their precise contribution to pathogenicity. This is illustrated by the GALA type III effectors from Ralstonia solanacearum, which have been shown to be collectively, but not individually, required for disease on Arabidopsis thaliana and tomato. We investigated evolution, redundancy and diversification of this family in order to understand the individual contribution of the GALA effectors to pathogenicity. From sequences available, we reconstructed GALA phylogeny and performed selection studies. We then focused on the GALAs from the reference strain GMI1000 to examine their ability to suppress plant defense responses and contribution to pathogenicity on three different host plants: A. thaliana, tomato (Lycopersicum esculentum) and eggplant (Solanum melongena). The GALA family is well conserved within R. solanacearum species. Patterns of selection detected on some GALA family members, together with experimental results, show that GALAs underwent functional diversification. We conclude that functional divergence of the GALA family likely accounts for its remarkable conservation during R. solanacearum evolution and could contribute to R. solanacearum’s adaptation on several host plants.

The Exotic Legume Tree Species Acacia holosericea Alters Microbial Soil Functionalities and the Structure of the Arbuscular Mycorrhizal Community

ABSTRACT The response of microbial functional diversity as well as its resistance to stress or disturbances caused by the introduction of an exotic tree species, Acacia holosericea, ectomycorrhized or not with Pisolithus albus, was examined. The results show that this ectomycorrhizal fungus promotes drastically the growth of this fast-growing tree species in field conditions after 7 years of plantation. Compared to the crop soil surrounding the A. holosericea plantation, this exotic tree species, associated or not with the ectomycorrhizal symbiont, induced strong modifications in soil microbial functionalities (assessed by measuring the patterns of in situ catabolic potential of microbial communities) and reduced soil resistance in response to increasing stress or disturbance (salinity, temperature, and freeze-thaw and wet-dry cycles). In addition, A. holosericea strongly modified the structure of arbuscular mycorrhizal fungus communities. These results show clearly that exotic plants may be responsible for important changes in soil microbiota affecting the structure and functions of microbial communities.

Transient hypermutagenesis accelerates the evolution of legume endosymbionts following horizontal gene transfer.

Stress-responsive error-prone DNA polymerase genes transferred along with key symbiotic genes ease the evolution of a soil bacterium into a legume endosymbiont by accelerating adaptation of the recipient bacterial genome to its new plant host.

Bistability in a metabolic network underpins the de novo evolution of colony switching in Pseudomonas fluorescens

Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation of a number of underlying molecular mechanisms. However, little is known about how phenotypic switches arise and function in their early evolutionary stages. The first opportunity to provide empirical insight was delivered by an experiment in which populations of the bacterium Pseudomonas fluorescens SBW25 evolved, de novo, the ability to switch between two colony phenotypes. Here we unravel the molecular mechanism behind colony switching, revealing how a single nucleotide change in a gene enmeshed in central metabolism (carB) generates such a striking phenotype. We show that colony switching is underpinned by ON/OFF expression of capsules consisting of a colanic acid-like polymer. We use molecular genetics, biochemical analyses, and experimental evolution to establish that capsule switching results from perturbation of the pyrimidine biosynthetic pathway. Of central importance is a bifurcation point at which uracil triphosphate is partitioned towards either nucleotide metabolism or polymer production. This bifurcation marks a cell-fate decision point whereby cells with relatively high pyrimidine levels favour nucleotide metabolism (capsule OFF), while cells with lower pyrimidine levels divert resources towards polymer biosynthesis (capsule ON). This decision point is present and functional in the wild-type strain. Finally, we present a simple mathematical model demonstrating that the molecular components of the decision point are capable of producing switching. Despite its simple mutational cause, the connection between genotype and phenotype is complex and multidimensional, offering a rare glimpse of how noise in regulatory networks can provide opportunity for evolution.

Shaping Bacterial Symbiosis With Legumes by Experimental Evolution

Nitrogen-fixing symbionts of legumes have appeared after the emergence of legumes on earth, approximately 70 to 130 million years ago. Since then, symbiotic proficiency has spread to distant genera of α- and β-proteobacteria, via horizontal transfer of essential symbiotic genes and subsequent recipient genome remodeling under plant selection pressure. To tentatively replay rhizobium evolution in laboratory conditions, we previously transferred the symbiotic plasmid of the Mimosa symbiont Cupriavidus taiwanensis in the plant pathogen Ralstonia solanacearum, and selected spontaneous nodulating variants of the chimeric Ralstonia sp. using Mimosa pudica as a trap. Here, we pursued the evolution experiment by submitting two of the rhizobial drafts to serial ex planta-in planta (M. pudica) passages that may mimic alternating of saprophytic and symbiotic lives of rhizobia. Phenotyping 16 cycle-evolved clones showed strong and parallel evolution of several symbiotic traits (i.e., nodulation competitiveness, intracellular infection, and bacteroid persistence). Simultaneously, plant defense reactions decreased within nodules, suggesting that the expression of symbiotic competence requires the capacity to limit plant immunity. Nitrogen fixation was not acquired in the frame of this evolutionarily short experiment, likely due to the still poor persistence of final clones within nodules compared with the reference rhizobium C. taiwanensis. Our results highlight the potential of experimental evolution in improving symbiotic proficiency and for the elucidation of relationship between symbiotic capacities and elicitation of immune responses.

Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum

The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1-like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine-rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease-controlling T3E, and sheds light on the co-evolutionary arms race between the bacterium and its hosts.

Spatio-temporal control of mutualism in legumes helps spread symbiotic nitrogen fixation

Mutualism is of fundamental importance in ecosystems. Which factors help to keep the relationship mutually beneficial and evolutionarily successful is a central question. We addressed this issue for one of the most significant mutualistic interactions on Earth, which associates plants of the leguminosae family and hundreds of nitrogen (N2)-fixing bacterial species. Here we analyze the spatio-temporal dynamics of fixers and non-fixers along the symbiotic process in the Cupriavidus taiwanensis–Mimosa pudica system. N2-fixing symbionts progressively outcompete isogenic non-fixers within root nodules, where N2-fixation occurs, even when they share the same nodule. Numerical simulations, supported by experimental validation, predict that rare fixers will invade a population dominated by non-fixing bacteria during serial nodulation cycles with a probability that is function of initial inoculum, plant population size and nodulation cycle length. Our findings provide insights into the selective forces and ecological factors that may have driven the spread of the N2-fixation mutualistic trait.

Modeling and experimental determination of infection bottleneck and within-host dynamics of a soil-borne bacterial plant pathogen.

The soil is known to be a very microbe-rich environement. Plant roots are surrounded by a complex microbiota among which, in warm climates, the pathogenic bacteria belonging to the Ralstonia sp. species complex. We used a combination of mathematical modelling and experimental plant infection methods, mimicking the natural conditions, to define the key parameters describing the infection, colonization and wilting of the host plant (bacterial wilt disease). Importantly, our model takes into account the possibility for the orthologous re-infection of already infected plants. We showed that it is the case in our experimental setup and likely to also happen in natura. We were able to model and experimentaly measure the plant infection bottleneck, under these non-forced infection conditions. We then quantified to what extend the plant natural barriers can restrict (up to 50 times) this bottleneck size. We also measured the importance of the bacterial main virulence determinants (type III effectors) to allowing infection, as there is a reduction of the bottleneck by 70 times when the type III arsenal is absent. We further validated the model by predicting a strain caracteristics using only a few experimentally determined parameters. Finally the analysis of the global colonization dynamics allowed an accurate assessment of the in planta bacterial load triggering disease.In natura, many organisms face multiple infections by pathogens. The ability of a pathogen to reinfect an already-infected host affects the genetic makeup of the pathogen population at the end of the infectious cycle. Despite the likely prevalence of this situation, the population dynamics of pathogens during multiple infections over time is still poorly understood. Here we combined theoretical and empirical investigations of the founding population size, a critical driver of the evolution of pathogens, in a setting allowing for multiple and subsequent re-infections. Using the soil-borne bacterial pathogen Ralstonia solanacearum and tomato as its host, we first assessed the strength of the host infection bottleneck, and showed that both the host barrier and the immune system work additively to constrain the infection. Then, by increasing the temperature, we experimentally demonstrated that the increased pathogen proliferation within the host reduces the contribution of subsequent re-infection leading to a lower founding population size. Our study highlights the importance of within-host pathogen proliferation in determining founding population size – and thus bacterial genetic diversity during epidemics – for pathosystems where multiple re-infections occur. Under current global changes, our work notably predicts that an increased temperature provided this increase has a beneficial impact on pathogen growth, should decrease the founding population size and as a consequence potentially lower the diversity of the infecting and transmitted pathogen population. Significance Statement Founder population size is a major determinant of pathogen evolution, yet we still have limited insights into effective populations in natural settings. Most studies have considered infection as a single event, followed by pathogen growth in the host. But, in natura, organisms typically face multiple infections by several co-exisiting pathogen strains. Therefore, effective population size will depend on the timing and relative growth rate of the different infecting strains. In this work, we predict and experimentally show that both priority effects and within-host competition determines effective founding size, with an over-contribution of fast-growing and early infecting genotypes. This work sheds a new light on the ecological and evolutionary pressures affecting infection dynamics in realistic conditions.