Analyses transcriptomiques du dimorphisme levure-mycélium chez le champignon phytopathogène Ophiostoma novo-ulmi

L’analyse de données de transcriptomique par le biais du séquençage d’ARN messagers (RNAseq) offre une perspective globale de la régulation de l’expression génique au cours d’un évènement biologique. Dans cette thèse, nous avons exploité cette technique dans le but de comprendre les mécanismes moléculaires qui régulent la transition morphologique réversible levure-mycélium qui est une caractéristique souvent liée au pouvoir pathogène chez les champignons. Dans un premier temps, par le biais de comparaisons de données de transcriptomique entre sept espèces fongiques dimorphiques, nous avons observé une certaine conservation des processus biologiques associés au changement de morphologie chez des champignons issus de branches très éloignées de l’arbre phylogénétique fongique. Dans un second temps, nous nous sommes concentrée sur notre modèle d’étude principal, Ophiostoma novo-ulmi, le champignon pathogène responsable de la maladie hollandaise de l’orme. Par l’analyse comparée des gènes exprimés en phases levure et mycélienne, nous avons défini les facteurs moléculaires qui sont spécifiques à chacune des phases chez O. novo-ulmi et établi une distinction claire entre les deux phases d’un point de vue du contenu en gènes exprimés. Par la suite, nous avons affiné notre étude en nous focalisant sur l’évènement de transition levure-mycélium afin déterminer les gènes dont l’expression était modulée au cours du temps dans le processus de changement morphologique. Nous avons mis en évidence plusieurs facteurs potentiellement impliqués dans la transition, notamment des gènes liés à la cascade de phosphorylation des MAPKs, connues pour jouer un rôle clé dans le dimorphisme chez plusieurs espèces fongiques. Finalement, dans le but d’évaluer plus précisément le niveau de conservation des processus biologiques liés au dimorphisme chez des espèces non modèles éloignées, nous avons comparé la régulation de l’expression génique au niveau des gènes orthologues entre O. novo-ulmi et l’espèce basidiomycète, Pseudozyma flocculosa. Nous nous sommes concentrée sur les gènes qui étaient différentiellement exprimés entre les phases de germination et de filamentation. Dans l’ensemble, les processus associés aux gènes pour lesquels la régulation de l’expression est conservée chez les deux espèces portent sur des fonctions essentielles du développement fongique. Ainsi, cette comparaison a permis de définir ce qui semble constituer la « base minimale » génique commune nécessaire à la transition asexuée levure-mycélium chez des espèces phylogénétiquement éloignées.Large-scale transcriptomic analyses via messenger RNA sequencing (RNAseq) give access to the information on expression regulation of all the genes present in a sample at a given time and in a given experimental condition. In this thesis, we took advantage of this technology in order to investigate the molecular mechanisms that regulate the reversible yeast-to-hypha morphological switch which is a characteristic often linked to virulence in fungal pathogens. To begin with, we compared transcriptomic data among seven dimorphic fungi and found conserved biological processes associated with the morphological switch among species from very distant branches of the fungal phylogenetic tree. Later, we focused on our model species, Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. We first compared the gene expression levels in yeast and mycelium growth phases. We defined the molecular factors that are specific to each growth phase and highlighted a clear molecular distinction between the two phases in terms of expressed gene contents. We further narrowed down our analysis by focusing on the yeast-to-hypha transition in a time course experiment. We determined the set of genes for which the expression was regulated during the morphological switch, thus potentially involved in the yeast-to-hypha transition. In particular, we identified genes that could be related to the MAPK cascade, known to play a crucial role in the dimorphic switch in many fungal species. Finally, in order to address the level of conservation in the biological processes linked to dimorphism in highly divergent non-model species, we compared the gene expression regulation of the orthologous genes between O. novo-ulmi and the basidiomycete Pseudozyma flocculosa. We focused on the genes that were differentially expressed between the germination and the filamentation phases. We identified several factors for which the regulation of expression seems conserved during the switch from germinating spore to filamentous growth. Overall, these genes are associated with biological processes that play essential roles in fungal development. Hence, our comparison here highlighted core components necessary for the yeast-to-hypha transition in phylogenetically distant species

Functional Annotation of the Ophiostoma novo-ulmi Genome: Insights into the Phytopathogenicity of the Fungal Agent of Dutch Elm Disease

International audienceThe ascomycete fungus Ophiostoma novo-ulmi is responsible for the pandemic of Dutch elm disease that has been ravaging Europe and North America for 50 years. We proceeded to annotate the genome of the O. novo-ulmi strain H327 that was sequenced in 2012. The 31.784-Mb nuclear genome (50.1% GC) is organized into 8 chromosomes containing a total of 8,640 protein-coding genes that we validated with RNA sequencing analysis. Approximately 53% of these genes have their closest match to Grosmannia clavigera kw1407, followed by 36% in other close Sordariomycetes, 5% in other Pezizomycotina, and surprisingly few (5%) orphans. A relatively small portion (~3.4%) of the genome is occupied by repeat sequences; however, the mechanism of repeat-induced point mutation appears active in this genome. Approximately 76% of the proteins could be assigned functions using Gene Ontology analysis; we identified 311 carbohydrate-active enzymes, 48 cytochrome P450s, and 1,731 proteins potentially involved in pathogen– host interaction, along with 7 clusters of fungal secondary metabolites. Complementary mating-type locus sequencing, mating tests, and culturing in the presence of elm terpenes were conducted. Our analysis identified a specific genetic arsenal impacting the sexual and vegetative growth, phytopathogenicity, and signaling/plant–defense–degradation relationship between O. novo-ulmi and its elm host and insect vectors. Introduction During the last centuries, increased movements of people and goods across countries and continents have favored the emergence and global spread of plant pathogens, insect pests, and invasive weeds which have substantially altered the landscape of several parts of the world. One well-documented example is Dutch elm disease (DED), the most destructive disease of elms. It has been estimated that over 1 billion mature elms were killed by two successive pandemics since the early 1900s (Paoletti et al. 2005). The first pandemic, which prompted initial investigations by Dutch scientists shortly after the First World War (Holmes and Heybroek 1990), was caused by the ascomycete fungus Ophiostoma ulmi (Buisman) Nannf. As it spread relentlessly over Western Europe and, a few decade

Comparative Analysis of Transcriptomes of Ophiostoma novo-ulmi ssp. americana Colonizing Resistant or Sensitive Genotypes of American Elm

The Ascomycete Ophiostoma novo-ulmi threatens elm populations worldwide. The molecular mechanisms underlying its pathogenicity and virulence are still largely uncharacterized. As part of a collaborative study of the O. novo-ulmi-elm interactome, we analyzed the O. novo-ulmi ssp. americana transcriptomes obtained by deep sequencing of messenger RNAs recovered from Ulmus americana saplings from one resistant (Valley Forge, VF) and one susceptible (S) elm genotypes at 0 and 96 h post-inoculation (hpi). Transcripts were identified for 6424 of the 8640 protein-coding genes annotated in the O. novo-ulmi nuclear genome. A total of 1439 genes expressed in planta had orthologs in the PHI-base curated database of genes involved in host-pathogen interactions, whereas 472 genes were considered differentially expressed (DEG) in S elms (370 genes) and VF elms (102 genes) at 96 hpi. Gene ontology (GO) terms for processes and activities associated with transport and transmembrane transport accounted for half (27/55) of GO terms that were significantly enriched in fungal genes upregulated in S elms, whereas the 22 GO terms enriched in genes overexpressed in VF elms included nine GO terms associated with metabolism, catabolism and transport of carbohydrates. Weighted gene co-expression network analysis identified three modules that were significantly associated with higher gene expression in S elms. The three modules accounted for 727 genes expressed in planta and included 103 DEGs upregulated in S elms. Knockdown- and knockout mutants were obtained for eight O. novo-ulmi genes. Although mutants remained virulent towards U. americana saplings, we identified a large repertoire of additional candidate O. novo-ulmi pathogenicity genes for functional validation by loss-of-function approaches

Diversity in yeast-mycelium dimorphism response of the Dutch elm disease pathogens: the inoculum size effect

Dutch elm disease (DED) is caused by the dimorphic fungi Ophiostoma ulmi, O. novo-ulmi and O. himal-ulmi. A cell population density-dependent phenomenon related to quorum sensing was previously shown to affect the reversible transition from yeast-like- to mycelial growth in liquid shake cultures of O. novo-ulmi strain NRRL 6404. Since the response to external stimuli often varies among DED fungal strains, we evaluated the effect of inoculum size on eight strains of the three species of DED agents by determining the proportion of yeast and mycelium produced at different spore inoculum concentrations in defined liquid shake medium. Results show that not all DED fungi strains respond similarly to inoculum size effect since variations were observed among strains. It is thus possible that the different strains belonging to phylogenetically close species use different signalling molecules or molecular signalling pathways to regulate their growth mode via quorum sensing mechanisms.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Deciphering the Genome-Wide Transcriptomic Changes during Interactions of Resistant and Susceptible Genotypes of American Elm with Ophiostoma novo-ulmi

Dutch elm disease (DED), caused by Ophiostoma novo-ulmi (Onu), is a destructive disease of American elm (Ulmus americana L.). The molecular mechanisms of resistance and susceptibility against DED in American elm are still largely uncharacterized. In the present study, we performed a de novo transcriptome (RNA-sequencing; RNA-Seq) assembly of U. americana and compared the gene expression in a resistant genotype, ’Valley Forge’, and a susceptible (S) elm genotype at 0 and 96 h post-inoculation of Onu. A total of 85,863 non-redundant unigenes were identified. Compared to the previously characterized U. minor transcriptome, U. americana has 35,290 similar and 55,499 unique genes. The transcriptomic variations between ‘Valley Forge’ and ‘S’ were found primarily in the photosynthesis and primary metabolism, which were highly upregulated in the susceptible genotype irrespective of the Onu inoculation. The resistance to DED was associated with the activation of RPM1-mediated effector-triggered immunity that was demonstrated by the upregulation of genes involved in the phenylpropanoids biosynthesis and PR genes. The most significantly enriched gene ontology (GO) terms in response to Onu were response to stimulus (GO:0006950), response to stress (GO:0050896), and secondary metabolic process (GO:0008152) in both genotypes. However, only in the resistant genotype, the defense response (GO:0006952) was among the topmost significantly enriched GO terms. Our findings revealed the molecular regulations of DED resistance and susceptibility and provide a platform for marker-assisted breeding of resistant American elm genotypes

Systemic control of nodule formation by plant nitrogen demand requires autoregulation-dependent and independent mechanisms

International audienceIn legumes interacting with rhizobia, the formation of symbiotic organs involved in the acquisition of atmospheric nitrogen gas (N2) is dependent on the plant nitrogen (N) demand. We used Medicago truncatula plants cultivated in split-root systems to discriminate between responses to local and systemic N signaling. We evidenced a strong control of nodule formation by systemic N signaling but obtained no clear evidence of a local control by mineral nitrogen. Systemic signaling of the plant N demand controls numerous transcripts involved in root transcriptome reprogramming associated with early rhizobia interaction and nodule formation. SUPER NUMERIC NODULES (SUNN) has an important role in this control, but we found that major systemic N signaling responses remained active in the sunn mutant. Genes involved in the activation of nitrogen fixation are regulated by systemic N signaling in the mutant, explaining why its hypernodulation phenotype is not associated with higher nitrogen fixation of the whole plant. We show that the control of transcriptome reprogramming of nodule formation by systemic N signaling requires other pathway(s) that parallel the SUNN/CLE (CLAVATA3/EMBRYO SURROUNDING REGION-LIKE PEPTIDES) pathway

Autoregulation dependent and independent mechanisms are responsible for the systemic control of nodule formation by the plant N demand

Abstract In legumes interacting with rhizobia the formation of symbiotic organs responsible for the acquisition of atmospheric nitrogen is depending of the plant nitrogen (N) demand. We discriminated between local and systemic impact of nitrogen on nodule formation using Medicago truncatula plants cultivated in split-root systems. We obtained evidence of the control of nodule formation by whole plant systemic N-satisfaction signaling but obtained little evidence of a local control by mineral nitrogen. We characterized the impact of systemic N signaling on the root transcriptome reprogramming associated to nodule formation. We identified, large genes clusters displaying common expression profiles in response to systemic N signaling enriched in particular fonctions required during these biological processes. We found evidence of a strong effect of SUNN in the control by systemic N signaling of many genes involved in the early interaction with rhizobium as well as organogenesis supporting a role of autoregulation pathway in systemic N signaling. However, we also found evidence that major SUNN independent systemic N signaling controls were maintained in the mutant. This study shed light on the unexpected high complexity of the control of nodule formation by systemic N signaling, that probably involves multiple pathways

Comparative Analysis of Transcriptomes of <i>Ophiostoma novo-ulmi</i> ssp. <i>americana</i> Colonizing Resistant or Sensitive Genotypes of American Elm

The Ascomycete Ophiostoma novo-ulmi threatens elm populations worldwide. The molecular mechanisms underlying its pathogenicity and virulence are still largely uncharacterized. As part of a collaborative study of the O. novo-ulmi-elm interactome, we analyzed the O. novo-ulmi ssp. americana transcriptomes obtained by deep sequencing of messenger RNAs recovered from Ulmus americana saplings from one resistant (Valley Forge, VF) and one susceptible (S) elm genotypes at 0 and 96 h post-inoculation (hpi). Transcripts were identified for 6424 of the 8640 protein-coding genes annotated in the O. novo-ulmi nuclear genome. A total of 1439 genes expressed in planta had orthologs in the PHI-base curated database of genes involved in host-pathogen interactions, whereas 472 genes were considered differentially expressed (DEG) in S elms (370 genes) and VF elms (102 genes) at 96 hpi. Gene ontology (GO) terms for processes and activities associated with transport and transmembrane transport accounted for half (27/55) of GO terms that were significantly enriched in fungal genes upregulated in S elms, whereas the 22 GO terms enriched in genes overexpressed in VF elms included nine GO terms associated with metabolism, catabolism and transport of carbohydrates. Weighted gene co-expression network analysis identified three modules that were significantly associated with higher gene expression in S elms. The three modules accounted for 727 genes expressed in planta and included 103 DEGs upregulated in S elms. Knockdown- and knockout mutants were obtained for eight O. novo-ulmi genes. Although mutants remained virulent towards U. americana saplings, we identified a large repertoire of additional candidate O. novo-ulmi pathogenicity genes for functional validation by loss-of-function approaches

Natural Wolbachia infections are common in the major malaria vectors in Central Africa

International audienceDuring the last decade, the endosymbiont bacterium Wolbachia has emerged as a biological tool for vector disease control. However, for long time, it was believed that Wolbachia was absent in natural populations of Anopheles. The recent discovery that species within the Anopheles gambiae complex host Wolbachia in natural conditions has opened new opportunities for malaria control research in Africa. Here, we investigated the prevalence and diversity of Wolbachia infection in 25 African Anopheles species in Gabon (Central Africa). Our results revealed the presence of Wolbachia in 16 of these species, including the major malaria vectors in this area. The infection prevalence varied greatly among species, confirming that sample size is a key factor to detect the infection. Moreover, our sequencing and phylogenetic analyses showed the important diversity of Wolbachia strains that infect Anopheles. Co‐evolutionary analysis unveiled patterns of Wolbachia transmission within some Anopheles species, suggesting that past independent acquisition events were followed by co‐cladogenesis. The large diversity of Wolbachia strains that infect natural populations of Anopheles offers a promising opportunity to select suitable phenotypes for suppressing Plasmodium transmission and/or manipulating Anopheles reproduction, which in turn could be used to reduce the malaria burden in Africa