Temperature-induced changes in the wheat phosphoproteome reveal temperature-regulated interconversion of phosphoforms

Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower, and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental, and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term and mild increase in temperature on the wheat leaf and spikelet phosphoproteome. In total, 3822 (containing 5178 phosphosites) and 5581 phosphopeptides (containing 7023 phosphosites) were identified in leaf and spikelet samples, respectively. Following statistical analysis, the resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature. This community resource on the high temperature-mediated wheat phosphoproteome will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which probably impacts protein activity

Molecular analysis of unreduced microgametes formation in Rosa spp

Dans l’histoire évolutive des végétaux, la polyploïdisation a été un phénomène récurrent qui a façonné les génomes, aurait contribué à l’avènement de grandes étapes évolutives et aurait favorisé la survie de nombreuses lignées lors de crises écologiques majeures. Le principal mécanisme d’apparition d’espèces polyploïdes est la polyploïdisation sexuelle, qui implique la formation de gamètes 2n résultant de modifications de la division méiotique. Récemment plusieurs mutants produisant des taux élevés de gamètes 2n ont été identifiés chez A. thaliana. Chez cette espèce, la perte de fonction du gène AtPS1 conduit à la mise en place de fuseaux parallèles en méiose II et celle du gène AtCYCA1;2/TAM à l’omission de la seconde division méiotique. L’objectif de cette thèse a été de déterminer des facteurs et mécanismes responsables de la formation de gamètes 2n, en utilisant le rosier comme modèle végétal. Ces travaux ont permis : (i) d’identifier un facteur abiotique, la température élevée, comme inducteur de production de forts taux de gamètes 2n, (ii) de montrer que la fenêtre de sensibilité à ce facteur est restreinte à la méiose et (iii) de révéler que ces gamètes 2n produits sont principalement issus de la mise en place de fuseaux parallèles en méiose II. Afin de déterminer les mécanismes moléculaires à l’origine de leur formation, deux gènes candidats, RhPS1 et RhCYCA1 ont été identifiés chez Rosa. L’analyse de leur expression a révélé : (i) en condition non inductible, leur forte expression dans les étamines au stade méiose et (ii) la répression rapide de leurs niveaux de transcrits en condition d’induction de gamètes 2n. La fonction méiotique du gène RhPS1 a été validée par complémentation du mutant atps1-1 d’A. thaliana et par l’obtention d’une lignée rosier transgénique p35S::ARNi-RhPS1. Compte tenu de ces résultats, l’étude de la polyploïdisation et de ses mécanismes peut désormais être replacée dans le contexte actuel de changement climatique.In the evolutionary history of plants, polyploidization has been a recurring phenomenon that has shaped the genomes, might have contributed to the occurrence of major evolutionary step and might have facilitated the survival of many plant families during major ecological crises. The main mechanism of polyploidization is sexual polyploidization, which involves the formation of 2n gametes resulting from meiotic division changes. Recently, mutants highly producing 2n gametes have been isolated in A. thaliana. Loss of AtPS1 gene function leads to parallel spindles orientation in meiosis II and loss of AtCYCA1;2/TAM gene function leads to the omission of the second meiotic division. The aim of this PhD project was to identify factors and mechanisms responsible for the 2n gametes formation, using Rosa as a model. This work permitted to: (i) discover an abiotic factor, high temperature, that can induce a high production of 2n gametes, (ii) show that the sensitivity window to this factor is narrow and restricted to meiosis and (iii) reveal that 2n gamete production in inductive condition, results from parallel spindle orientation in meiosis II. To determine molecular mechanisms responsible for their formation, two candidate genes, RhPS1 and RhCYCA1 were identified in Rosa. Analysis of their expression revealed: (i) their high expression level in stamens at meiosis stage in non-inductive condition and (ii) the rapid repression of their transcript levels under inductive condition. Meiotic gene function of RhPS1 was validated by complementation of atps1-1 mutant and by generating a rose transgenic line p35S:: RNAi-RhPS1. According to these results, polyploidization and its mechanisms can now be replaced in the context of the current climate

Evolutionary genomics of Ralstonia solanacearum emergence and host range expansion

International audienceMembers of the Ralstonia solanacearum species complex (RSSC) are naturally competent soil-borne vascular pathogens that causes severe outbreaks in staple carbohydrate and high-value crops like banana (Musa spp.), potato (Solanum tuberosum), and tomato (S. lycopersicum). Phylotypes I and II have been documented in Martinique since the 1960s, however in 1999 a novel lineage was identified, prompting a large-scale sampling effort. This new lineage, referred to as 4NPB, can infect cucurbits and Heliconia spp. and is more aggressive on solanaceous crops than previous variants. We sequenced 487 RSSC isolates sampled across Martinique and French Guiana during the emergence of this novel lineage. Pangenome and phylogenetic analyses were performed to identify genomic changes linked with the emergence of the new lineage. This analysis reveals the 4NPB population likely emerged from an ancestral mainland population infecting Musa sp. The emergence of the 4NPB lineage is associated with the exchange of Type 3 secreted effectors and multiple genes with predicted catalytic activity. Recombination hotspots found between 4NPB and close relatives include various two partner secretion systems potentially involved in intra-species competition and signaling. These changes may underlie the altered pathogenicity and host-range profile of 4NPB, contributing to the dramatic expansion of this lineage across Martinique

Evidence for increased fitness of a plant pathogen conferred by epigenetic variation

Abstract Adaptation is usually explained by adaptive genetic mutations that are transmitted from parents to offspring and become fixed in the adapted population. However, more and more studies show that genetic mutation analysis alone is not sufficient to fully explain the processes of adaptive evolution and report the existence of non-genetic (or epigenetic) inheritance and its significant role in the generation of adapted phenotypes. In the present work, we tested the hypothesis of the role of DNA methylation, a form of epigenetic modification, in adaptation of the plant pathogen Ralstonia solanacearum to the host plant during an experimental evolution. Using SMRT-seq technology, we analyzed the methylomes of 31 experimentally evolved clones that were obtained after serial passages on a given host plant during 300 generations, either on susceptible or tolerant hosts. Comparison with the methylome of the ancestral clone revealed between 12 and 21 differential methylated sites (DMSs) at the GTWWAC motif in the evolved clones. Gene expression analysis of the 39 genes targeted by these DMSs revealed limited correlation between differential methylation and differential gene expression. Only one gene showed a correlation, the RSp0338 gene encoding the EpsR regulator protein. The MSRE-qPCR (Methylation Sensitive Restriction Enzyme - qPCR) technology was used as an alternative approach to assess the methylation state of the DMSs found by SMRT-seq between the ancestral and evolved clones. This approach also found the two DMSs upstream of RSp0338. Using site-directed mutagenesis, we demonstrated the contribution of these two DMSs in host adaptation. As these DMSs appeared very quickly in the experimental evolution, we hypothesize that such fast epigenetic changes can allow rapid adaptation to the plant stem environment. To our knowledge, this is the first study showing a link between epigenetic variation and evolutionary adaptation to new environment

Changes in DNA methylation contribute to rapid adaptation in bacterial plant pathogen evolution

International audienceAdaptation is usually explained by beneficial genetic mutations that are transmitted from parents to offspring and become fixed in the adapted population. However, genetic mutation analysis alone is not sufficient to fully explain the adaptive processes, and several studies report the existence of nongenetic (or epigenetic) inheritance that can enable adaptation to new environments. In the present work, we tested the hypothesis of the role of DNA methylation, a form of epigenetic modification, in adaptation of the plant pathogen Ralstonia pseudosolanacearum to the host during experimental evolution. Using SMRT-seq technology, we analyzed the methylomes of 31 experimentally evolved clones obtained after serial passages on 5 different plant species during 300 generations. Comparison with the methylome of the ancestral clone revealed a list of 50 differential methylated sites (DMSs) at the GTWWAC motif. Gene expression analysis of the 39 genes targeted by these DMSs revealed limited correlation between differential methylation and differential expression of the corresponding genes. Only 1 gene showed a correlation, the RSp0338 gene encoding the EpsR regulator protein. The MSRE-qPCR technology, used as an alternative approach for DNA methylation analysis, also found the 2 DMSs upstream RSp0338. Using site-directed mutagenesis, we demonstrated the contribution of these 2 DMSs in host adaptation. As these DMSs appeared very early in the experimental evolution, we hypothesize that such fast epigenetic changes can allow rapid adaptation to the plant stem environment. In addition, we found that the change in DNA methylation upstream RSp0338 remains stable at least for 100 generations outside the host and thus can contribute to long-term adaptation to the host plant. To our knowledge, this is the first study showing a direct link between bacterial epigenetic variation and adaptation to a new environment

INDICANTS project : INnovative DIagnostiCs for bANana paThogens Surveillance

International audienceBanana is a major staple in developing countries and the most eaten fruit in Europe. Global banana production is constrained by several diseases responsible for yield losses and low productivity which may compromise food security. Four vascular diseases are of particular concern: Fusarium wilt disease of banana caused by the fungus Fusarium oxysporum f.sp. cubense (Foc), Moko and banana blood diseases, caused by Ralstonia solanacearum and R. syzygii subsp. celebesensis, respectively) and Xanthomonas wilt of banana caused by Xanthomonas vasicola pv. musacearum. Efficiency of surveillance and plant disease management requires the availability of Point of care (POC) diagnostics that can be operated directly on site. This poster describes the main objectives and early results of the INDICANTS project (INnovative DIagnostiCs for bANana paThogens Surveillance): (i) the development of low-cost multi-pathogens LAMP (Loop-mediated isothermal amplification) assays and the production of ready-to use kits by a private company (ii) the comparison of different simplified DNA extraction methods adapted to field. (iii) the validation of the LAMP protocols via an interlaboratory test and field surveys. (iiii) the evaluation of an emergent technology (CRISPR/Cas12a) for the diagnosis of Foc

Genomic approach to study floral development genes in Rosa sp.

International audienceCultivated for centuries, the varieties of rose have been selected based on a number of flower traits. Understanding the genetic and molecular basis that contributes to these traits will impact on future improvements for this economically important ornamental plant. In this study, we used scanning electron microscopy and sections of meristems and flowers to establish a precise morphological calendar from early rose flower development stages to senescing flowers. Global gene expression was investigated from floral meristem initiation up to flower senescence in three rose genotypes exhibiting contrasted floral traits including continuous versus once flowering and simple versus double flower architecture, using a newly developed Affymetrix microarray (Rosa1_Affyarray) tool containing sequences representing 4765 unigenes expressed during flower development. Data analyses permitted the identification of genes associated with floral transition, floral organs initiation up to flower senescence. Quantitative real time PCR analyses validated the mRNA accumulation changes observed in microarray hybridizations for a selection of 24 genes expressed at either high or low levels. Our data describe the early flower development stages in Rosa sp, the production of a rose microarray and demonstrate its usefulness and reliability to study gene expression during extensive development phases, from the vegetative meristem to the senescent flower