Search

We report here a new application, CustomProteinSearch (CusProSe), whose purpose is to help users to search for proteins of interest based on their domain composition. The application is customizable. It consists of two independent tools, IterHMMBuild and ProSeCDA. IterHMMBuild allows the iterative construction of Hidden Markov Model (HMM) profiles for conserved domains of selected protein sequences, while ProSeCDA scans a proteome of interest against an HMM profile database, and annotates identified proteins using user-defined rules. CusProSe was successfully used to identify, in fungal genomes, genes encoding key enzyme families involved in secondary metabolism, such as polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), hybrid PKS-NRPS and dimethylallyl tryptophan synthases (DMATS), as well as to characterize distinct terpene synthases (TS) sub-families. The highly configurable characteristics of this application makes it a generic tool, which allows the user to refine the function of predicted proteins, to extend detection to new enzymes families, and may also be applied to biological systems other than fungi and to other proteins than those involved in secondary metabolism.

National audience

International audience

National audience

National audience

Botrytis cinerea is a 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 modifiers 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 BcWCL1 (as part of the White Collar complex (WCC)) and BcLTF1 are important regulators as their deletions result in light-independent conidiation (“always conidia”) due to the deregulation of BcLTF2 (Schumacher et al. 2014; Canessa et al. 2013; Cohrs et al. 2016). Study of light-induced gene (LIG) expression in both deletion mutants highlighted the role of the TFs in activating EARLY and in repressing LATE LIG expression, including bcltf2 encoding the master regulator of conidiation and further LTFs that may function downstream or in parallel with BcLTF2 in regulating the conidiation process. As the group of LIGs also contained genes that are induced by various stresses in a BcSAK1-dependent manner, the phosphorylation status of the stress-activated MAPK BcSAK1 was studied and shown to increase after exposure to light. Deletion of bcsak1 impairs LIG expression suggesting that BcSAK1 functions as a co-activator of the WCC in inducing EARLY genes, and as a component releasing the LATE promoters (e.g. bcltf2) from repression by BcLTF1 and the WCC resulting in the expression of the conidiation genes. This preliminary model is in agreement with the observations that the deletion of BcSAK1 results in a “never conidia” phenotype (Segmüller et al. 2007), and that the exposure of the wild type to heat and osmotic stress bypasses the requirement of light for conidiation.

Unlike plants that use light also as source of energy, filamentous fungi exclusively use light as signal; here, light may trigger morphogenesis, pigment formation and circadian rhythms. Light plays an important role in the biology of the gray mold fungus by determining the mode of asexual reproduction (conidia in the light, sclerotia in the dark) and by triggering the differentiation of the apothecia. Field populations of B. cinerea are highly diverse with regard to different phenotypic traits and can be generally divided into two groups; strains that undergo photomorphogenesis (considered as the initial situation) and strains that produce the same reproductive structures under different light conditions (considered as loss of light control). However, the inability to form certain reproductive structures such as conidia is expected to decrease the fitness of the pathogen, and thus, it is questionable why “blind” strains are abundant in field populations, especially as this phenotype may be associated with attenuated virulence as shown for the “always conidia” phenotype caused by SNPs in bcvel1. Therefore, we aim to understand the fundamental mechanisms of photoperception and the involved signal transduction pathways. Though overlaps with the well-studied fungal systems i.e. Neurospora crassa and Aspergillus nidulans exist, the light signaling machinery in B. cinerea appears more sophisticated. Thus, the fungus possesses more photoreceptors than saprophytic fungi and responds to different wavelengths of light (near-UV, blue, green, red, far-red). More than 200 genes are responsive to white light, including 24 transcription factor (TF)-encoding genes. BcLTF2 has an outstanding function as its expression is necessary and sufficient to induce conidiation and simultaneously to suppress sclerotial development. Its tight regulation by several signaling pathways governs the balance between vegetative growth that allows for the colonization of the substrate and entering sclerotial development, and reproduction via conidia in the light.

Many endophytic bacteria colonize host tissues internally without causing damage or eliciting disease symptoms. Some of them also promote plant growth. Previously, we obtained a collection of bacterial endophytes from leaves of field-grown tomato and identified strains capable to systemically protect this plant against B. cinerea infection. In the present work we used the model plant A. thaliana to analyze the mechanisms involved in the induction of systemic resistance to B. cinerea by one of these strains, Pantoea eucalypti NT6 (PeNT6). In order to evaluate if PeNT6 triggers induced systemic resistance (ISR) in A. thaliana, plants were soil-inoculated with this bacterium and challenged by leaf inoculation with B. cinerea four weeks later. PeNT6 inoculation reduced (30-60%) the size of necrotic lesions caused by B. cinerea. Inoculation with PeNT6 had no effect on the expression of the defense-related genes PR1, PDF1.2, MPK3 and WRKY33. However, plants inoculated with PeNT6 and further challenged with B. cinerea exhibited higher levels of MPK3 and WRKY33 expression than those plants challenged with B. cinerea without previous inoculation with the bacterium. NT6 was unable to trigger ISR in A. thaliana mutants affected in defense signaling pathways mediated by jasmonic acid (coi1), ethylene (ein2) and salicylic acid (sid2). However, the npr1 mutant showed an ISR similar to the wild type. Mutants in MPK3 and MPK6 showed a differential response. While the mpk3 mutant showed no induction of resistance, the mpk6 showed a similar level of ISR to wild type plants. ISR triggered by PeNT6 also involved priming of callose deposition upon Botrytis infection. Finally, the use of a GFP-labeled B. cinerea strain allowed us to determine that in planta germination of fungal conidia was retarded in NT6-inoculated plants. Results hereby presented demonstrate that P. eucalypti triggers ISR to B. cinerea by priming specific components of the defense machinery, such as the kinase MPK3 and the transcription factor WRKY33. ISR triggered by NT6 was also found to depend on jasmonic acid, ethylene and salicylic acid signaling pathways, but did not depend on the regulatory protein NPR1. The kinase MPK3 was also found to play a key role in the onset of ISR by PeNT6. These findings contribute to understand the mechanisms underlying ISR to B. cinerea provided by plant inoculation with beneficial bacteria with the potential to be used as biological control agents.

Botrytis cinerea is a well-known phytopathogenic fungus that infects more than two hundreds plants species. It shows a wide variety of mechanisms for infecting host plants, as for example the secretion of broad range of cell wall-degrading enzymes, toxins and many other secondary metabolites including i) non ribosomal peptides and amino acid-derived compounds, (ii) polyketides and fatty acid-derived compounds and (iii) terpenes. Botrytis produces three series of sesquiterpenes, botrydial (1) and its derivatives, a new family of eremophilenols (2) and the phytohormone abscisic acid (ABA) (3). Botrydial is one of the main toxins involved in the infection mechanism and its biosynthetic pathway has recently been completed. A sesquiterpene family with eremophilenol skeleton has been characterized and its role in the Botrytis cycle of life reported. The role of these sesquiterpenes in the interaction plant-pathogen will be discussed in this communication. On the other hand, studies on ABA biosynthesis had identified a gene cluster of at least four genes responsible for this pathway in B. cinerea. However, until now, the sesquiterpene cyclase (STC) involved in the ABA biosynthesis had not yet been identified and it should be located in another part of the genome of B. cinerea. The metabolomic study of the strains ATCC G3, an overproducer of ABA, its null mutants ΔBcSTC5 and mutants Δbcaba1-4, have allowed to identify the sesquiterpene cyclase involved in the ABA biosynthesis and to present an updated ABA biosynthetic pathway.

B. cinerea and other filamentous fungi colonize their substrates in the form of vegetative mycelia and produce asexual spores (macroconidia and microconidia) and sexual spores in high quantities for their distribution and survival. As both processes, vegetative growth and reproduction, are competing for the same limiting resources, they should not occur simultaneously and need therefore be tightly coordinated by genetic programming and environmental conditions. For instance, excessive conidiation (e.g. microcycle conidiation) is disadvantageous as it may prevent vegetative growth and entering the sexual cycle. In this context, light serves as an important cue for the gray mold fungus: It induces the formation of conidia and at the same time represses the formation of sclerotia that represent asexual survival structures and are required for sexual recombination. As conidiation is induced by light, we considered the 24 identified light-responsive transcription factors (LTFs) as possible regulators of the conidiation program in B. cinerea. Nine 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 (BcATF1, BcLTF2, BcLTF7) or conidiogenesis (BcLTF3, BcREG1), or do not have obvious functions (BcLTF4, BcLTF5, BcLTF6). The C2H2-TF BcLTF2 has an outstanding function as its expression is necessary and sufficient to induce conidiation and simultaneously to suppress sclerotial development. In contrast, expression of BcLTF7 promotes conidiation but is not required for the process. By expression profiling of the remaining LTFs in mutants exhibiting deregulated conidiation, we identified five further candidates (bcltf12, bcltf15, bcltf16, bcltf20, bcltf21) that may act downstream of the major regulator BcLTF2 in the network of LTFs governing the asexual differentiation programs in the gray mold fungus.

The sesquiterpene Botrydial (BOT) and the polyketide botcinic acid (BOA) are non-host specific phytotoxins produced by Botrytis cinerea. In addition, their botryanes and botcinines derivatives were shown to have biological activities against bacteria and fungi. Both BOT and BOA biosynthetic gene clusters were previously identified. Inactivation of the key enzymes-encoding genes, i.e. the sesquiterpene cyclase-encoding gene Bcbot2 and the polyketide synthase-encoding gene Bcboa6, revealed that they may have a redundant role in virulence. In order to understand how BOT, BOA and their derivatives are regulated during the life cycle of B. cinerea, we investigated the genomic environment of the BOT and BOA clusters and searched for putative transcription factor (TF) encoding genes. Thanks to the recent release of the gapless genome of B05.10 strain (J. van Kan, Univ. Wageningen, NL), the BOT and BOA clusters were localized in two A+T-rich regions, one being subtelomeric (BOA). Our bioinformatics analyses revealed that the A+T/G+C-equilibrated regions that contain the Bcbot and Bcboa genes alternate with A+T-rich regions (>85%) made of relics of transposable elements that have undergone repeat-induced point (RIP) mutations. The occurrence of RIP raises questions about possible chromatin-based regulation of BOT and BOA synthesis. Several chromatin modifiers (KMT1, KMT6, HP1) are under studies to test this hypothesis. Amongst the previously identified Bcboa genes, Bcboa13 was predicted to encode a Zn(II)2Cys6 TF. In addition, the gapless version of the genome allowed us to identify a similar gene (Bcbot6) flanking the previously identified BOT cluster. Gene inactivation demonstrated that BcBot6 and BcBoa13 are the major positive regulators of BOT and BOA biosynthesis, respectively. Interestingly, the functional fusion protein BcBot6-GFP and BcBoa13-GFP localize into nuclear foci. Identification of BcBot6 as the major regulators of BOT and BOA synthesis is the first step toward a comprehensive understanding of the complete regulation network of toxin synthesis in B. cinerea. We are currently using a Yeast-One-Hybrid approach to search the upstream TF(s) that are interacting with the promoters of Bcbot6 and Bcboa13 genes.

Advances in genomic studies, in combination with increased access to DNA sequencing, are providing a wealth of information about how natural products are assembled, mechanisms by which natural product gene clusters can be manipulated to yield new product diversity, and the genetic potential of individual organisms. Botrytis cinerea is a well-known pathogen of a number of commercial crops and produces many structurally diverse metabolites. Botrytis produces three series of sesquiterpenes, botrydial and its derivatives, a new family of eremophilenols and the phytohormone abscisic acid (ABA). Studies on ABA biosynthesis have identified a gene cluster of at least four genes responsible for this pathway in B. cinerea: two P450 monooxygenase-encoding genes (bcaba1 and bcaba2), an open reading frame (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Until now, the sesquiterpene cyclase (STC) involved in the ABA biosynthesis has not yet been identified and it should be located in another part of the genome of B. cinerea. The metabolomic study of the strains ATCC G3, an overproducer of ABA, and null mutants ΔBcSTC5 and Δbcaba1-4, together with studies on the level of expression of the STC-encoding genes, have shown the STC5 as the sesquiterpene synthase involved in the ABA biosynthesis. Additionally, the studies of the transformants Δbcaba1-4 have allowed to present an updated ABA biosynthetic pathway.

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.

Summary: D-galacturonic acid (GalA) is the most abundant monosaccharide component of pectin. Previous transcriptome analysis in the plant pathogenic fungus Botrytis cinerea identified eight GalA-inducible genes involved in pectin decomposition, GalA transport and utilization. Co-expression of these genes indicates that a specific regulatory mechanism occurs in B. cinerea. In this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. The Bclga1 promoter, containing all these motifs, was functionally analysed and the motif designated GalA Responsive Element (GARE) was identified as the crucial cis-regulatory element in regulation of GalA utilization in B. cinerea. Yeast one-hybrid screening with the GARE motif led to identification of a novel Zn2Cys6 transcription factor (TF), designated BcGaaR. Targeted knockout analysis revealed that BcGaaR is required for induction of GalA-inducible genes and growth of B. cinerea on GalA. A BcGaaR-GFP fusion protein was predominantly localized in nuclei in mycelium grown in GalA. Fluorescence in nuclei was much stronger in mycelium grown in GalA, as compared to fructose and glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa. Short Abstract: The transcriptional regulator that controls D-galacturonic acid (GalA) utilization in fungi was unknown. We identified in Botrytis cinerea a novel Zn2Cys6 transcription factor (TF), designated BcGaaR, which is required for the induction of GalA-utilization genes. The BcGaaR protein was predominantly localized in nuclei in cultures grown on GalA, but remained in the cytoplasm in cultures grown on glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa.

Just accepted; Botrytis cinerea is a plant pathogenic fungus known for its utilization of light as environmental cue to regulateasexual differentiation: conidia are formed in the light, while sclerotia are formed in the dark. As noorthologues of known regulators of conidiation (e.g.,Aspergillus nidulans BrlA, Neurospora crassa FL)exist in the Leotiomycetes, we initiated a de novo approach to identify the functional counterpart in B.cinerea. The search revealed the light-responsive C2H2 transcription factor BcLTF2 whose expression– usually restricted to light conditions – is necessary and sufficient to induce conidiation and simultaneouslyto suppress sclerotial development. Lightinduced expression of bcltf2 is mediated via a so far unknown pathway, and is attenuated in a (blue) lightdependent fashion by the White Collar complex, BcLTF1 and the VELVET complex. Mutation of either component leads to increased bcltf2 expression and causes light-independent conidiation (always conidia phenotype). Hence, the tight regulation of bcltf2 governs the balance between vegetative growth that allows for the colonization of the substrate and subsequent reproduction via conidia in the light. The orthologue ssltf2 in the closely related species Sclerotinia sclerotiorum is not significantly expressed suggesting that its deregulation may cause the lack of the conidiation program in this fungus.

International audience

International audience; Botrytis cinerea is the causal agent of gray mold diseases in a range of dicotyledonous plant species. The fungus can reproduce asexually by forming macroconidia for dispersal and sclerotia for survival; the latter also participate in sexual reproduction by bearing the apothecia after fertilization by microconidia. Light induces the differentiation of conidia and apothecia, while sclerotia are exclusively formed in the absence of light. The relevance of light for virulence of the fungus is not obvious; infections are observed under natural illumination as well as in constant darkness. By a random mutagenesis approach, we identified a novel virulence-related gene encoding a GATA transcription factor (BcLTF1 for light-responsive TF1) with characterized homologues in Aspergillus nidulans (NsdD) and Neurospora crassa (SUB- 1). By deletion and overexpression of bcltf1, we confirmed the predicted role of the TF in virulence, and discovered furthermore its functions in regulation of light-dependent differentiation, the equilibrium between production and scavenging of reactive oxygen species (ROS), and secondary metabolism. Microarray analyses revealed 293 lightresponsivegenes in B. cinerea B05.10 including five further TF-encoding genes (BcLTF2-6), and that the expression levels of the majority of these genes (66%) are modulated by BcLTF1. Bcltf2 encodes a C2H2-TF which is the homologue of N. crassa SAH-1 (short aerial-hyphae-1). Expression levels are increased in mutants exhibiting a hyper-conidiation phenotype such as deletion mutants of BcLTF1, the VELVET protein BcVEL1 and the WHITE COLLAR-like TF BcWCL1, suggesting a role of BcLTF2 in regulation of conidiation. Indeed, Δbcltf2 mutants do not produce conidia in axenic culture and in planta. Notably, mutants form sclerotia under all illumination conditions indicating that the suppression of sclerotial development by light is likely due to the induction of conidiation rather than due to a direct suppression of the sclerotium differentiation program.

absent

absent

absent

absent

absent

absent

absent

POSTER P11 - p.133; absent

absent

absent

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

International audience

International audience

International audience

International audience

International audience

International audience

International audience

International audience

International audience

International audience

International audience

National audience

Fusarium graminearum est un champignon phytopathogène responsable de la fusariose de l'épi, maladie affectant les céréales telles que le blé ou le maïs. Le champignon peut également produire des métabolites secondaires toxiques ou mycotoxines, tels que les trichothécènes de type B qui sont stables d'un point de vue chimique et thermique. Ces mycotoxines peuvent contaminer les grains avant récolte. Aucun procédé à l'heure actuelle ne permet de les éliminer ou de limiter leur toxicité. Un moyen de lutte efficace est donc de limiter la biosynthèse de ces toxines. Cette voie implique les gènes Tri qui sont regroupés dans un cluster dont la régulation est complexe. Lors de l'infection de la plante hôte, le champignon se retrouve potentiellement en contact avec des molécules pro ou antioxydantes intervenant dans les mécanismes de défense de la plante. In vitro, ces molécules pro-oxydantes activent la production de trichothécènes via la surexpression des gènes Tri alors que les composés antioxydants la répriment, via la répression des gènes Tri.Le facteur de transcription Fgap1, homologue au facteur Yap1 de levure impliqué dans la réponse au stress oxydant, a été identifié chez F. graminearum et son rôle potentiel dans la régulation de la biosynthèse des trichothécènes a été étudié. Des souches recombinantes ont été construites et ont permis de montrer l'implication de ce facteur non seulement dans l'activation de l'expression des gènes de détoxification, mais aussi dans la modulation de la production de trichothécènes en réponse au stress oxydant. Ce facteur n'intervient cependant pas dans la réponse aux composés antioxydants.Une analyse RNA-seq a été entreprise pour identifier plus globalement les réseaux de régulation impliqués en réponse aux variations redox., Fusarium graminearum is a pathogenic fungus responsible for "Fusarium Head Blight", a diseaseaffecting cereals, including wheat or maize. The fungus can produce toxic secondary metabolitesbelonging to the type B trichothecene family. These metabolites are heat and chemically stablemolecules. These toxins can be found in grains before harvest and no available decontaminationprocess can eliminate or detoxify trichothecenes. The best way to restrict their occurrence is to limittheir biosynthesis. The Tri genes involved in the biosynthetic pathway are clustered and theirregulation is complex. During plant/pathogen interaction, the fungus must cope with pro orantioxidants, involved in plant defense mechanisms. In vitro, prooxidant molecules stimulatetrichothecenes biosynthesis via Tri genes overexpression, whereas antioxidants compounds repress Trigenes expression and toxin accumulation.The transcription factor Fgap1, homologous to Yap1 in yeast and involved in response to oxidativestress, was identified in F. graminearum and its potential role in the regulation of trichothecenesbiosynthesis was investigated. Using recombinant strains, we demonstrated that, in response tooxidative stress, Fgap1 is not only involved in expression of detoxifying activities, but also in themodulation of trichothecene accumulation. Nonetheless, Fgap1 is not involved in response toantioxidant compounds. RNA-seq analysis has been initiated to identify regulatory network involvedin response to redox variations.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to, Author Summary Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with very wide host ranges. They cause vast economic damage during crop cultivation as well as in harvested produce. These fungi are typical examples of necrotrophs: they first kill host plant cells and then colonize the dead tissue. The genome sequences of the two fungi were determined in order to examine commonalities in structure and content and in order to find unique features that may distinguish them from other pathogenic fungi and from saprotrophic fungi. The genomes show high sequence identity and a similar arrangement of genes. S. sclerotiorum and B. cinerea differ in their regulation of sexual reproduction, and the genetic basis and its evolution could be explained from the genome sequence. The genome sequence revealed a striking difference in the number and diversity of secondary metabolism gene clusters, which may be involved in the adaptation to different ecological niches. Altogether, there were no unique features in the genomes of S. sclerotiorum and B. cinerea that could be identified as “silver bullets,” which distinguish these aggressive pathogens from other pathogenic and non-pathogenic fungi. These findings reinforce the quantitative, multigenic nature of necrotrophic pathogenesis.