Rôle de la protéine CLV1 dans la sensibilité d'Arabidopsis thaliana à la bactérie phytopathogène Ralstonia solanacearum

Les mécanismes moléculaires associés au développement de la maladie causée par la bactérie phytopathogène R. solanacearum sont relativement peu connus à ce jour. La recherche de mutants d'Arabidopsis incapables de développer des symptômes de flétrissement associée à la maladie a mené à l'identification de gènes dit de sensibilité. Les produits de ces gènes peuvent être des cibles de facteurs de virulence aussi bien que des composantes végétales requises pour la " fitness " de la bactérie. Le gène CLAVATA1 (CLV1), très étudié dans le contexte du développement, code pour une protéine appartenant à la famille des récepteurs kinase possédant un domaine extracellulaire riche en leucine. Cette protéine joue un rôle crucial dans le maintien d'une population de cellules souches au niveau du méristème caulinaire donnant naissance à toute la partie aérienne de la plante. La mutation clv1 entraîne une résistance accrue à R. solanacearum associée à une réduction de la croissance bactérienne in planta. Mes travaux de thèse ont consisté à élucider les mécanismes sous-tendant la résistance accrue conférée par la mutation du gène CLV1 en utilisant différents types d'approche (génétique, moléculaire et transcriptomique). Nous avons été capables de démontrer l'implication de facteurs de transcription de la famille de gènes NF-YA, eux-mêmes contrôlés par les microARNs miR169, dans la résistance accrue des mutants clv1. Ces résultats démontrent que la protéine CLV1 est une composante requise pour l'établissement de la maladie causée par R. solanacearum. Mes travaux de thèse mettent en lumière une nouvelle fonction de cette protéine et illustrent la grande diversité des rôles biologiques de protéines de type récepteur-kinase.The molecular mechanisms associated to disease development caused by the phytopathogenic bacteria Ralstonia solanacearum are poorly understood. Search for mutants altered in their response to the pathogen led to the identification of some susceptibility genes including targets of virulence factors as well as plant components required for pathogen fitness. The CLAVATA1 (CLV1) gene, extensively studied for its role in plant development, encodes a receptor-like kinase with a leucin-rich repeat extracellular domain. This protein plays indeed a key role in maintaining a pool of stem cell within the shoot apical meristem. The clv1 mutation leads to an increased resistance to R. solanacearum, associated with a decrease of in planta bacterial growth. The aim of my PhD work was the understanding of the mechanisms underlying the increased resistance conferred by the clv1 mutation in using different approach (molecular, genetic and transcriptomic). We have been able to demonstrate the implication of the NF-YA transcription factor family, controlled by microRNA miR169, in the increased resistance of these mutants. These results demonstrate that the CLV1 protein is a required component for the establishment of the disease caused by R. solanacearum. and illustrate the wide diversity of functions fulfilled by receptors kinases

Identification of transcription factors controlling floral morphology in wild Petunia species with contrasting pollination syndromes

Adaptation to different pollinators is an important driver of speciation in the angiosperms. Genetic approaches such as QTL mapping have been successfully used to identify the underlying speciation genes. However, these methods are often limited by widespread suppression of recombination due to divergence between species. While the mutations that caused the interspecific differences in floral color and scent have been elucidated in a variety of plant genera, the genes that are responsible for morphological differences remain mostly unknown. Differences in floral organ length determine the pollination efficiency of hawkmoths and hummingbirds, and therefore the genes that control these differences are potential speciation genes. Identifying such genes is challenging, especially in non‐model species and when studying complex traits for which little prior genetic and biochemical knowledge is available. Here we combine transcriptomics with detailed growth analysis to identify candidate transcription factors underlying interspecific variation in the styles of Petunia flowers. Starting from a set of 2284 genes, stepwise filtering for expression in styles, differential expression between species, correlation with growth‐related traits, allele‐specific expression in interspecific hybrids, and/or high‐impact polymorphisms resulted in a set of 43 candidate speciation genes. Validation by virus‐induced gene silencing identified two MYB transcription factors, EOBI and EOBII, that were previously shown to regulate floral scent emission, a trait associated with pollination by hawkmoths

Arabidopsis CLAVATA1 and CLAVATA2 receptors contribute to Ralstonia solanacearum pathogenicity through a miR169-dependent pathway

Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial plant diseases. Although many molecular determinants involved in R. solanacearum adaptation to hosts and pathogenesis have been described, host components required for disease establishment remain poorly characterized. Phenotypical analysis of Arabidopsis mutants for leucine-rich repeat (LRR)-receptor-like proteins revealed that mutations in the CLAVATA1 (CLV1) and CLAVATA2 (CLV2) genes confer enhanced disease resistance to bacterial wilt. We further investigated the underlying mechanisms using genetic, transcriptomic and molecular approaches. The enhanced resistance of both clv1 and clv2 mutants to the bacteria did not require the well characterized CLV signalling modules involved in shoot meristem homeostasis, and was conditioned by neither salicylic acid nor ethylene defence-related hormones. Gene expression microarray analysis performed on clv1 and clv2 revealed deregulation of genes encoding nuclear transcription factor Y subunit alpha (NF-YA) transcription factors whose post-transcriptional regulation is known to involve microRNAs from the miR169 family. Both clv mutants showed a defect in miR169 accumulation. Conversely, overexpression of miR169 abrogated the resistance phenotype of clv mutants. We propose that CLV1 and CLV2, two receptors involved in CLV3 perception during plant development, contribute to bacterial wilt through a signalling pathway involving the miR169/NF-YA module

PERKing up our understanding of the proline‐rich extensin‐like receptor kinases, a forgotten plant receptor kinase family

International audienceProline-rich extensin-like receptor kinases (PERKs) are an important class of receptor-like kinases (RLKs) containing an extracellular proline-rich domain. While they are thought to be putative sensors of the cell wall integrity, there are very few reports on their biological functions in the plant, as compared with other RLKs. Several studies support a role for PERKs in plant growth and development, but their effect on the cell wall composition to regulate cell expansion is still lacking. Gene expression data suggest that they may intervene in response to environmental changes, in agreement with their subcellular localization. And there is growing evidence for PERKs as novel sensors of environmental stresses such as insects and viruses. However, little is known about their precise role in plant immunity and in the extracellular network of RLKs, as no PERK-interacting RLK or any coreceptor has been identified as yet. Similarly, their signaling activities and downstream signaling components are just beginning to be deciphered, including Ca 2+ fluxes, reactive oxygen species accumulation and phosphorylation events. Here we outline emerging roles for PERKs as novel sensors of environmental stresses, and we discuss how to better understand this overlooked class of receptor kinases via several avenues of research

Hrp mutant bacteria as biocontrol agents: toward a sustainable approach in the fight against plant pathogenic bacteria.

Sustainable agriculture necessitates development of environmentally safe methods to protect plants against pathogens. Among these methods, application of biocontrol agents has been efficiently used to minimize disease development. Here we review current understanding of mechanisms involved in biocontrol of the main Gram-phytopathogenic bacteria-induced diseases by plant inoculation with strains mutated in hrp (hypersensitive response and pathogenicity) genes. These mutants are able to penetrate plant tissues and to stimulate basal resistance of plants. Novel protection mechanisms involving the phytohormone abscisic acid appear to play key roles in the biocontrol of wilt disease induced by Ralstonia solanacearum in Arabidopsis thaliana. Fully understanding these mechanisms and extending the studies to other pathosystems are still required to evaluate their importance in disease protection

New strategies and tools in quantitative genetics: how to go from the phenotype to the genotype

ISBN : 978-0-8243-0668-7Quantitative genetics has a long history in plants: It has been used to study specific biological processes, identify the factors important for trait evolution, and breed new crop varieties. These classical approaches to quantitative trait locus mapping have naturally improved with technology. In this review, we show how quantitative genetics has evolved recently in plants and how new developments in phenotyping, population generation, sequencing, gene manipulation, and statistics are rejuvenating both the classical linkage mapping approaches (for example, through nested association mapping) as well as the more recently developed genome-wide association studies. These strategies are complementary in most instances, and indeed, one is often used to confirm the results of the other. Despite significant advances, an emerging trend is that the outcome and efficiency of the different approaches depend greatly on the genetic architecture of the trait in the genetic material under study

Natural variation at FLM splicing has pleiotropic effects modulating ecological strategies in Arabidopsis thaliana

International audienceInvestigating the evolution of complex phenotypes and the underlying molecular bases of their variation is critical to understand how organisms adapt to their environment. Applying classical quantitative genetics on a segregating population derived from a Can-0xCol-0 cross, we identify the MADS-box transcription factor FLOWERING LOCUS M (FLM) as a player of the phenotypic variation in plant growth and color. We show that allelic variation at FLM modulates plant growth strategy along the leaf economics spectrum, a trade-off between resource acquisition and resource conservation, observable across thousands of plant species. Functional differences at FLM rely on a single intronic substitution, disturbing transcript splicing and leading to the accumulation of non-functional FLM transcripts. Associations between this substitution and phenotypic and climatic data across Arabidopsis natural populations, show how noncoding genetic variation at a single gene might be adaptive through pleiotropic effects

Tight genetic linkage of genes causing hybrid necrosis and pollinator isolation between young species.

The mechanisms of reproductive isolation that cause phenotypic diversification and eventually speciation are a major topic of evolutionary research. Hybrid necrosis is a post-zygotic isolation mechanism in which cell death develops in the absence of pathogens. It is often due to the incompatibility between proteins from two parents. Here we describe a unique case of hybrid necrosis due to an incompatibility between loci on chromosomes 2 and 7 between two pollinator-isolated Petunia species. Typical immune responses as well as endoplasmic reticulum stress responses are induced in the necrotic line. The locus on chromosome 2 encodes ChiA1, a bifunctional GH18 chitinase/lysozyme. The enzymatic activity of ChiA1 is dispensable for the development of necrosis. We propose that the extremely high expression of ChiA1 involves a positive feedback loop between the loci on chromosomes 2 and 7. ChiA1 is tightly linked to major genes involved in the adaptation to different pollinators, a form of pre-zygotic isolation. This linkage of pre- and post-zygotic barriers strengthens reproductive isolation and probably contributes to rapid diversification and speciation

The complex genetic architecture of shoot growth natural variation in <em>Arabidopsis thaliana</em>

International audienceOne of the main outcomes of quantitative genetics approaches to natural variation is to reveal the genetic architecture underlying the phenotypic space. Complex genetic architectures are described as including numerous loci (or alleles) with small-effect and/or low-frequency in the populations, interactions with the genetic background, environment or age. Linkage or association mapping strategies will be more or less sensitive to this complexity, so that we still have an unclear picture of its extent. By combining high-throughput phenotyping under two environmental conditions with classical QTL mapping approaches in multiple Arabidopsis thaliana segregating populations as well as advanced near isogenic lines construction and survey, we have attempted to improve our understanding of quantitative phenotypic variation. Integrative traits such as those related to vegetative growth used in this work (highlighting either cumulative growth, growth rate or morphology) all showed complex and dynamic genetic architecture with respect to the segregating population and condition. The more resolutive our mapping approach, the more complexity we uncover, with several instances of QTLs visible in near isogenic lines but not detected with the initial QTL mapping, indicating that our phenotyping accuracy was less limiting than the mapping resolution with respect to the underlying genetic architecture. In an ultimate approach to resolve this complexity, we intensified our phenotyping effort to target specifically a 3Mb-region known to segregate for a major quantitative trait gene, using a series of selected lines recombined every 100kb. We discovered that at least 3 other independent QTLs had remained hidden in this region, some with trait- or condition-specific effects, or opposite allelic effects. If we were to extrapolate the figures obtained on this specific region in this particular cross to the genome- and species-scale, we would predict hundreds of causative loci of detectable phenotypic effect controlling these growth-related phenotypes. Author summary The question of the complexity of the genetic variants underlying diversity in plant size and shape is central in evolutionary biology to better understand the impacts of selection and adaptation. In this work, we have combined the high resolution of a robotized platform designed to grow Arabidopsis plants under strictly-controlled conditions and the power of quantitative genetics approaches to map the individual genetic components (the 'QTLs') controlling diverse phenotypes, and hence reveal the so-called 'genetic architecture' of these traits. We show that the more we increase our resolution to map QTLs, the more complex of a genetic architecture we reveal. For instance, by focusing all of our mapping power on a small region representing 2.5% of the genome in an unprecedented phenotyping effort, we reveal that several independent QTLs had remained hidden in this region beyond a major-effect QTL that is always clearly visible. If this region is representative of the genome, this means that our current understanding misses potentially hundreds of variants finely controlling traits of evolutionary or agronomical interest

Deletion of <i>EWR1</i> enhances Arabidopsis susceptibility to Verticillium wilt.

<p>(A) Typical symptoms of <i>V. dahliae</i> on the wild-type (Col-0) and <i>EWR1</i> knock out (<i>ewr1</i>) plants. Picture was taken at 21 days post inoculation (dpi) and a representative of three independent experimental replicates is shown. (B) Disease severity score for the wild-type (Col-0) and <i>ewr1</i> at 14 (white bar) and 21 (grey bar) days post inoculation (dpi). The total number of rosette leaves and the number of rosette leaves that showed Verticillium symptoms were counted at least from eight plants and percentage of the diseased leaves were calculated as an indication of disease severity. The bars represent averages of three independent experiments with standard deviation and asterisks indicate significance differences (Dunnett t-test at <i>P = 0.05</i>). (C) Relative quantification (RQ) by real-time PCR of Verticillium colonization by comparing levels of the <i>V. dahliae</i> internal transcribed spacer (ITS) region of the ribosomal DNA (as measure for fungal biomass) relative to levels of the large subunit of the Arabidopsis <i>RubisCo</i> gene (for equilibration) at 21 dpi. Bars represent averages with standard deviation of four technical replicates. A representative of three independent experiments is shown.</p