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Background and Aims: Laccase enzymes produced by Botrytis cinerea are involved in the oxidation of phenolic substances during the development of grey mould, which causes significant economic losses. The aim of this work was to study the structural and activity characteristics of the laccase enzymes secreted by three B. cinerea strains that are involved in the development of grey mould. Methods and Results: Laccase enzymes obtained from three B. cinerea strains [one reference strain (B05.10) and two strains obtained from two French vineyards (VA612 and RM344)] were characterised. Analysis by LC-QTOF-MS revealed that the three strains contained a mixture of Laccase-2-BcLCC2 and Laccase-3-BcLCC7. The structural characteristics of the laccases from the three strains, such as molecular weight and glycosylation degree, were identical. Nevertheless, their catalytic activities were significantly different. Conclusions: Differences in catalytic activities could be due either to possible differences in the relative amount of Laccase-2- BcLCC2 and Laccase-3-BcLCC7 produced by each strain or to differences in the glycosidic fraction of the enzymes. Significance of the Study: The severity of the infection caused by B. cinerea may be not only related to the infection level but also to the strain involved.

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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.

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.