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BGPI :équipe 5; The Botrytis genus encompasses more than 30 species, most of them being able to infect a restricted range of host plants. Nevertheless, B. cinerea is considered a generalist pathogen, recently found to infect 586 plant genera. This makes B. cinerea an interesting candidate to study ecological specialization. This was first approached while describing population structure in France, from strains collected on greenhouse tomato, grapevine, blackberry, strawberry and hydrangea. Understanding the causes of population subdivision is of fundamental importance, as studying barriers to gene flow between populations may reveal key aspects of the process of adaptive divergence and, for pathogens, may help forecasting disease emergence and implementing sound management strategies. Population genetics analyses revealed a weak association between population structure and geography, but a clear differentiation according to the host plant of origin, and especially for greenhouse tomato and grapevine. Host specialization was validated by cross-inoculation experiments, carried out in vitro on detached leaves. Genomes from strains contrasting for their specialization on tomato, grapevine, blackberry and hydrangea were Illumina-sequenced and genomic phylogeny analysis confirmed the similarities between genomes from strains collected on the same host. Population genomics analysis used to compare variation between and within lines of strains provided promising sets of genes that may induce host specialization. In addition to coding sequences, analysis of small RNAs from strains collected either on tomato or grapevine was initiated. Preliminary sequencing data suggest that these two populations have contrasted repertories of small RNAs. Further analysis and additional genomes and small RNA sequencing are required to definitely identify the determinants of host specialization. Our findings open up new perspectives for disease control by managing Botrytis hosts in agricultural landscapes, and reinforcing prophylactic measures.

National audience; Botrytis cinerea is a necrotrophic plant pathogen that exhibits prominent light responses including the formation of the reproduction structures (photomorphogenesis), secondary metabolites/ pigments, and antioxidant enzymes. A complex regulatory network of photoreceptors, transcription factors (TFs) and chromatin modulates is supposed to initiate, transmit, and fine-tune the responses to different wavelengths of light on the transcriptional level that finally leads to the observable phenotypes. As the formation of the reproduction structures is strictly regulated by light in this fungus - conidia are formed in the light, sclerotia in the dark - the output can be easily monitored. The GATA-type TFs BcLTF1 and BcWCL1 are important regulators as their deletions resulted in conidiation in light and dark («always conidia») (Schumacher et al. 2014; PLoS Genet 10:e1004040, Canessa et al. 2013; PLoS One 8:e84223). Study of gene expression in both deletion mutants by microarray analyses highlighted the role of the TFs in mediating the transcriptional responses of the majority of lightresponsive genes including a number of other transcriptional regulators. As the group of lightresponsive genes also contained genes that are induced by oxidative and osmotic stress in a BcSAK1-dependent manner (Heller et al. 2012; MPMI 25:802-816), their expression levels were studied in the delta-Bcsak1 mutant. Indeed, light induction does not occur in the mutant background indicating that the MAP kinase cascade is another functional unit of the light regulatory network. More detailed expression analyses of chosen LTF-encoding genes showed that three major transcriptional profiles exist: genes that exhibit maximal expression after 15 min (early), 60 min (moderate) or 120 min (late) of light treatment. Expression levels of the majority of genes decrease after prolonged light exposure (photoadaptation); however, this is not observed for bcltf1. Eight LTFs have been functionally characterized by deletion and overexpression analyses so far: While BcLTF1 has various functions (growth in light, ROS homoeostasis, virulence, differentiation), other LTFs specifically regulate certain developmental stages such as the initiation of conidiophore development or conidiogenesis.

National audience; Botrytis cinerea is responsible for the gray mold disease on more than 200 plant species. Among the virulence factors identified in this Ascomycete are two non-host specific toxins (and their derivatives): botrydial (BOT, a sesquiterpene) and botcinic acid (BOA, a polyketide). The genes responsible for their biosynthesis are clustered in AT-rich subtelomeric regions of chromosome 1 (BOA genes) and chromosome 12 (BOT genes). Previous studies pointed out their co-regulation by conserved signalling pathways as well as by global transcription factors (TFs) like the calcineurin-dependent BcCrz1. A strong link between secondary metabolism gene expression and light-dependent development has been showed in B. cinerea, notably by the characterization of Velvet complex members BcVel1 and BcLae1. Global regulation data are available for BcBOT and BcBOA genes, but their direct regulators have not been characterized so far. A TF encoding gene present within a secondary metabolism cluster usually specifically regulates genes of this cluster. Amongst BcBOA genes, BcBOA13 is predicted to encode a Zn(II)2Cys6 TF. In addition, a new version of B. cinerea genome annotation allowed adding new putative BcBOT genes to this cluster, including a gene predicted to encode a Zn(II)2Cys6 TF (BcBOT6). In our study, we functionally characterized BcBOT6 and BcBOA13. Our hypothesis is that these TFs are specific regulators of their respective cluster. The data accumulated so far through the generation of deletion mutants and the analysis of gene expression by RT-qPCR support this hypothesis. In order to investigate the putative direct interaction between BcBot6 and BcBoa13 with promoters of genes from BcBOT and BcBOA culsters, a Yeast One Hybrid strategy was carried out. We searched for regulators of BcBOT6 and BcBOA13 to make the link between these TFs and known regulatory pathways. In parallel, we tested the role of epigenetic modifications in the transcriptional control of these clusterss. We generated deletion mutants of the orthologs of chromatin modifiers known to affect secondary metabolism gens expression in fungi (Hp1, Dim-5 and Kmt6). The expression profiles of BcBOT and BcBOA genes will be assessed in these mutants. Altogether, these results give a more precise view of the regulatory network controlling the expression of BcBOT and BcBOA gene clusters in B. cinerea.

National audience; Botrytis cinerea est l’agent pathogène responsable de la pourriture grise qui touche plus de 200 espèces végétales. Parmi les facteurs de virulence identifiés chez ce champignon ascomycète figurent deux toxines (et leurs dérivés respectifs) ayant un rôle redondant dans la nécrotrophie. Il s’agit du sesquiterpène botrydial (BOT) et du polycétide acide botcinique (BOA). Les gènes responsables de leur synthèse sont agencés en clusters au sein du génome comme dans la plupart des cas pour les gènes du métabolisme secondaire chez les champignons. Des études précédentes ont permis de mettre en évidence la régulation de ces clusters par des voies de transductions conservées de type MAP kinases ainsi que par des facteurs de transcription globaux comme le facteur de transcription calcineurine-dépendant BcCrz1. De plus, un fort lien entre l’expression de ces gènes du métabolisme secondaire et le développement en réponse à la lumière a été démontré chez l’agent de la pourriture grise, notamment par la caractérisation des membres du complexe Velvet BcVel1 et BcLae1. Des données de régulation globale sont donc disponibles pour les gènes Bcbot et Bcboa mais leurs régulateurs directs n’ont pas été caractérisés. D’après de nombreux exemples de la littérature, lorsqu’un gène codant un facteur de transcription se trouve au sein d’un cluster du métabolisme secondaire, la protéine produite régule spécifiquement les gènes du cluster. Parmi les gènes Bcboa, Bcboa13 est prédit pour coder un facteur de transcription de type Zn(II)2Cys6. D’autre part, une nouvelle version du génome de B. cinerea a permis de compléter la liste des gènes putatifs du cluster BOT et de mettre en évidence à proximité des gènes Bcbot1 à 5 un gène (Bcbot6) prédit pour coder également un FT de type Zn(II)2Cys6. Dans le cadre de notre étude, la caractérisation fonctionnelle de BcBot6 et BcBoa13 est réalisée. Notre hypothèse est que ces facteurs de transcription sont des régulateurs spécifiques de leur cluster. Les données acquises jusqu’ici via la création de mutants et des analyses d’expression par RT-qPCR confirment cette hypothèse. Afin d’étudier la capacité de BcBot6 et BcBoa13 d’intervenir dans des interactions directes avec les promoteurs Bcbot et Bcboa, une approche par simple hybride est actuellement réalisée. Finalement, la banque de facteurs de transcription dont nous disposons sera criblée pour identifier des régulateurs de Bcbot6 et Bcboa13 afin de faire le lien avec les cascades d’activation déjà̀ connues.

International audience; Botrytis cinerea is responsible of the gray mold disease on a wide range of host plants. During the infection process, it produces unspecific phytotoxins (botrydial and botcinic acid) and other secondary metabolites that play a significanT role in plant tissue colonization. Regulation of the biosynthesis of these bioactive compounds is therefore crucial for the outcome of fungus/plant interactions. Combining genetics and transcriptomic approaches, the role of different signaling cascades, transcription factors, and other regulators such as those belonging to the VELVET complex have recently been investigated. Overall, these studies suggest a strong link between the regulation of secondary metabolism and the light-dependent development in B.cinerea. Further knowledge of the complex regulatory network controlling secondary metabolism in B.cinera is crucial to understand the interaction between B.cinerea and its different host plants and may help to define new strategies to control this crop-devastating fungus.

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.

Botrytis cinerea est le champignon nécrotrophe responsable de la pourriture grise qui affecte plus de 200 espèces végétales. Cet agent phytopathogène possède un fort potentiel de synthèse de métabolites secondaires (MSs). Le rôle de plusieurs de ces métabolites, dont le polycétide acide botcinique, a été mis en évidence dans la mort des cellules de l’hôte et dans la colonisation de ses tissus. Les mécanismes moléculaires qui régulent l’expression des gènes codant ces MSs restent cependant jusqu’ici méconnus et leur étude présente donc un réel intérêt. Les gènes responsables de la synthèse de l’acide botcinique sont organisés en cluster (BOA) et l’un d’entre eux code un facteur de transcription (BOA13). L’analyse transcriptomique globale du mutant Δboa13 a permis de mettre en exergue le rôle régulateur attendu du facteur de transcription (FT) sur les gènes du cluster BOA mais aussi de façon inattendue sur des gènes impliqués dans d’autres aspects de la nécrotrophie (production d’enzymes de dégradation des parois végétales, protéases, transporteurs membranaires et réponse au stress oxydatif). Ce résultat est original puisqu’il révèle une régulation par BOA13 de gènes qui ne sont pas impliqués dans le métabolisme secondaire. Les travaux en cours visent (i) à identifier les motifs de fixation de BOA13 et (ii) à déterminer si ce FT interagit directement avec les promoteurs des gènes candidats. Pour cela, ils consistent dans un premier temps à réaliser une analyse en microscopie à fluorescence des promoteurs entiers et tronqués fusionnés à la GFP au sein de transformants de B. cinerea. Le niveau d’intensité du signal de fluorescence permettra de mettre en évidence l’implication des régions d’intérêt dans la fixation de FTs. Ensuite, l’outil simple-hybride développé au sein du laboratoire sera utilisé pour mettre en évidence l’interaction physique possible entre BOA13 et les motifs validés lors de l’étape de microscopie. L’ensemble des FTs de la banque simple-hybride sera aussi criblé afin de vérifier si certains d’entre eux interagissent avec les motifs étudiés. L’ensemble de ces résultats nous permettra d’approfondir les connaissances relatives à la régulation de la synthèse de toxines chez le champignon nécrotrophe modèle B. cinerea.

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POSTER P11 - p.133; absent

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Atf1-homologous basic region leucine zipper (bZIP) transcription factors are known to act downstream of the stress-activated mitogen-activated protein kinase (SAPK) cascade in mammals, as well as in several fungi; they regulate the transcription of genes involved in the general stress response. Functional analyses of BcAtf1 in Botrytis cinerea show that it is also connected to the SAPK BcSak1, as it shares several stress response target genes. However, Dbcatf1 mutants are not hypersensitive to osmotic or oxidative stress, as are Dbcsak1 mutants. Both BcSak1 and BcAtf1 are regulators of differentiation, but their roles in these processes are almost inverse as, in contrast with Dbcsak1, Dbcatf1 mutants are significantly impaired in conidia production and do not differentiate any sclerotia. They show extremely vigorous growth in axenic culture, with a thick layer of aerial hyphae and a marked increase in colonization efficiency on different host plants and tissues. In addition, the sensitivity to cell wall-interfering agents is increased strongly. Microarray analyses demonstrate that the loss of BcAtf1 leads to extensive transcriptional changes: apart from stress response genes, the expression of a broad set of genes, probably involved in primary metabolism, cell wall synthesis and development, is affected by BcAtf1. Unexpectedly, BcAtf1 also controls secondary metabolism: the mutant contains significantly elevated levels of phytotoxins. These data indicate that BcAtf1 controls a diversity of cellular processes and has broad regulatory functions.

Session 1 : Interactions Moléculaires. Poster n°1.2; absent

Session 3 : Post-Genome Functional Analysis : P3.6; absent

Session 3 : Post-Genome Functional Analysis : P3.4; absent

Session 3 : Post-Genome Functional Analysis : P3.3; absent

Session 3 : Post-Genome Functional Analysis : P3.2; absent

The grey mould fungus Botrytis cinerea produces two major phytotoxins, the sesquiterpene botrydial, for which the biosynthesis gene cluster has been characterized previously, and the polyketide botcinic acid. We have identified two polyketide synthase (PKS) encoding genes, BcPKS6 and BcPKS9, that are up-regulated during tomato leaf infection. Gene inactivation and analysis of the secondary metabolite spectra of several independent mutants demonstrated that both BcPKS6 and BcPKS9 are key enzymes for botcinic acid biosynthesis. We showed that BcPKS6 and BcPKS9 genes, renamed BcBOA6 and BcBO9 (for B. cinerea botcinic acid biosynthesis), are located at different genomic loci, each being adjacent to other putative botcinic acid biosynthetic genes, named BcBOA1 to BcBOA17. Putative orthologues of BcBOA genes are present in the closely related fungus Sclerotinia sclerotiorum, but the cluster organization is not conserved between the two species. As for the botrydial biosynthesis genes, the expression of BcBOA genes is co-regulated by the Gα subunit BCG1 during both in vitro and in planta growth. The loss of botcinic acid production does not affect virulence on bean and tomato leaves. However, double mutants that do not produce botcinic acid or botrydial (bcpks6Δbcbot2Δ) exhibit markedly reduced virulence. Hence, a redundant role of botrydial and botcinic acid in the virulence of B. cinerea has been demonstrated.

International audience

International audience