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1.
Metab Eng ; 80: 216-231, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37863177

RESUMO

Transcriptomic studies have revealed that fungal pathogens of plants activate the expression of numerous biosynthetic gene clusters (BGC) exclusively when in presence of a living host plant. The identification and structural elucidation of the corresponding secondary metabolites remain challenging. The aim was to develop a polycistronic system for heterologous expression of fungal BGCs in Saccharomyces cerevisiae. Here we adapted a polycistronic vector for efficient, seamless and cost-effective cloning of biosynthetic genes using in vivo assembly (also called transformation-assisted recombination) directly in Escherichia coli followed by heterologous expression in S. cerevisiae. Two vectors were generated with different auto-inducible yeast promoters and selection markers. The effectiveness of these vectors was validated with fluorescent proteins. As a proof-of-principle, we applied our approach to the Colletochlorin family of molecules. These polyketide secondary metabolites were known from the phytopathogenic fungus Colletotrichum higginsianum but had never been linked to their biosynthetic genes. Considering the requirement for a halogenase, and by applying comparative genomics, we identified a BGC putatively involved in the biosynthesis of Colletochlorins in C. higginsianum. Following the expression of those genes in S. cerevisiae, we could identify the presence of the precursor Orsellinic acid, Colletochlorins and their non-chlorinated counterparts, the Colletorins. In conclusion, the polycistronic vectors described herein were adapted for the host S. cerevisiae and allowed to link the Colletochlorin compound family to their corresponding biosynthetic genes. This system will now enable the production and purification of infection-specific secondary metabolites of fungal phytopathogens. More widely, this system could be applied to any fungal BGC of interest.


Assuntos
Família Multigênica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiões Promotoras Genéticas , Família Multigênica/genética
2.
Bioorg Chem ; 127: 105979, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35753117

RESUMO

Chemical epigenetic manipulation of Botrytis cinerea strain B05.10 with the histone deacetylase inhibitor SAHA led to the isolation of a new cryptic metabolite, botrycinereic acid (22a). This compound was also overproduced by inactivating the stc2 gene, which encodes an unknown sesquiterpene cyclase. Its structure and absolute configuration were determined by extensive spectroscopic NMR and HRESIMS studies, and electronic circular dichroism calculations. Its biosynthesis was studied by feeding 2H and 13C isotopically labeled precursors to B. cinerea Δstc2 mutant. A detailed analysis of the labeling and coupling patterns into botrycinereic acid (22a) revealed that this compound derives from l-phenylalanine and l-leucine.


Assuntos
Botrytis
3.
Phytopathology ; 111(12): 2355-2366, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33829853

RESUMO

Many fungal plant pathogens encompass multiple populations specialized on different plant species. Understanding the factors underlying pathogen adaptation to their hosts is a major challenge of evolutionary microbiology, and it should help to prevent the emergence of new specialized pathogens on novel hosts. Previous studies have shown that French populations of the gray mold pathogen Botrytis cinerea parasitizing tomato and grapevine are differentiated from each other, and have higher aggressiveness on their host of origin than on other hosts, indicating some degree of host specialization in this polyphagous pathogen. Here, we aimed at identifying the genomic features underlying the specialization of B. cinerea populations to tomato and grapevine. Based on whole genome sequences of 32 isolates, we confirmed the subdivision of B. cinerea pathogens into two genetic clusters on grapevine and another, single cluster on tomato. Levels of genetic variation in the different clusters were similar, suggesting that the tomato-specific cluster has not recently emerged following a bottleneck. Using genome scans for selective sweeps and divergent selection, tests of positive selection based on polymorphism and divergence at synonymous and nonsynonymous sites, and analyses of presence and absence variation, we identified several candidate genes that represent possible determinants of host specialization in the tomato-associated population. This work deepens our understanding of the genomic changes underlying the specialization of fungal pathogen populations.


Assuntos
Botrytis , Solanum lycopersicum , Botrytis/genética , França , Genética Populacional , Solanum lycopersicum/microbiologia , Metagenômica , Doenças das Plantas/microbiologia
4.
Environ Microbiol ; 21(12): 4808-4821, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31608584

RESUMO

The host plant is often the main variable explaining population structure in fungal plant pathogens, because specialization contributes to reduce gene flow between populations associated with different hosts. Previous population genetic analysis revealed that French populations of the grey mould pathogen Botrytis cinerea were structured by hosts tomato and grapevine, suggesting host specialization in this highly polyphagous pathogen. However, these findings raised questions about the magnitude of this specialization and the possibility of specialization to other hosts. Here we report specialization of B. cinerea populations to tomato and grapevine hosts but not to other tested plants. Population genetic analysis revealed two pathogen clusters associated with tomato and grapevine, while the other clusters co-occurred on hydrangea, strawberry and bramble. Measurements of quantitative pathogenicity were consistent with host specialization of populations found on tomato, and to a lesser extent, populations found on grapevine. Pathogen populations from hydrangea and strawberry appeared to be generalist, while populations from bramble may be weakly specialized. Our results suggest that the polyphagous B. cinerea is more accurately described as a collection of generalist and specialist individuals in populations. This work opens new perspectives for grey mould management, while suggesting spatial optimization of crop organization within agricultural landscapes.


Assuntos
Botrytis/fisiologia , Doenças das Plantas/microbiologia , Botrytis/genética , Fragaria/microbiologia , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Solanum lycopersicum/microbiologia , Vitis/microbiologia
5.
Curr Genet ; 65(4): 965-980, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30848345

RESUMO

Botcinic acid is a phytotoxic polyketide involved in the virulence of the gray mold fungus Botrytis cinerea. Here, we aimed to investigate the specific regulation of the cluster of Bcboa genes that is responsible for its biosynthesis. Our analysis showed that this cluster is located in a subtelomeric genomic region containing alternating G + C/A + T-balanced regions, and A + T-rich regions made from transposable elements that underwent RIP (Repeat-Induced Point mutation). Genetic analyses demonstrated that BcBoa13, a putative Zn2Cys6 transcription factor, is a nuclear protein with a major positive regulatory role on the expression of other Bcboa1-to-Bcboa12 genes, and botcinic acid production. In conclusion, the structure and the regulation of the botcinic acid gene cluster show similar features with the cluster responsible for the biosynthesis of the other known phytotoxin produced by B. cinerea, i.e., the sesquiterpene botrydial. Both clusters contain a gene encoding a pathway-specific Zn2Cys6 positive regulator, and both are surrounded by relics of transposons which raise some questions about the role of these repeated elements in the evolution and regulation of the secondary metabolism gene clusters in Botrytis.


Assuntos
Botrytis/genética , Doenças das Plantas/genética , Policetídeos/metabolismo , Fatores de Transcrição/genética , Elementos de DNA Transponíveis/genética , Regulação Fúngica da Expressão Gênica , Família Multigênica/genética , Doenças das Plantas/microbiologia , Mutação Puntual , Zinco/química
6.
Environ Microbiol ; 20(7): 2469-2482, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29708647

RESUMO

While abscisic acid (ABA) is known as a hormone produced by plants through the carotenoid pathway, a small number of phytopathogenic fungi are also able to produce this sesquiterpene but they use a distinct pathway that starts with the cyclization of farnesyl diphosphate (FPP) into 2Z,4E-α-ionylideneethane which is then subjected to several oxidation steps. To identify the sesquiterpene cyclase (STC) responsible for the biosynthesis of ABA in fungi, we conducted a genomic approach in Botrytis cinerea. The genome of the ABA-overproducing strain ATCC58025 was fully sequenced and five STC-coding genes were identified. Among them, Bcstc5 exhibits an expression profile concomitant with ABA production. Gene inactivation, complementation and chemical analysis demonstrated that BcStc5/BcAba5 is the key enzyme responsible for the key step of ABA biosynthesis in fungi. Unlike what is observed for most of the fungal secondary metabolism genes, the key enzyme-coding gene Bcstc5/Bcaba5 is not clustered with the other biosynthetic genes, i.e., Bcaba1 to Bcaba4 that are responsible for the oxidative transformation of 2Z,4E-α-ionylideneethane. Finally, our study revealed that the presence of the Bcaba genes among Botrytis species is rare and that the majority of them do not possess the ability to produce ABA.


Assuntos
Ácido Abscísico/biossíntese , Botrytis/metabolismo , Carbono-Carbono Liases/metabolismo , Ácido Abscísico/análogos & derivados , Sequência de Bases , Botrytis/enzimologia , Botrytis/genética , Carotenoides/metabolismo , Genes Fúngicos , Oxirredução , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo
7.
Mol Microbiol ; 100(2): 247-62, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26691528

RESUMO

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 Zn2 Cys6 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.


Assuntos
Botrytis/metabolismo , Proteínas Fúngicas/metabolismo , Ácidos Hexurônicos/metabolismo , Fatores de Transcrição/metabolismo , Botrytis/genética , Sequência Conservada , Cisteína , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Genoma Fúngico , Solanum lycopersicum , Micélio/metabolismo , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas , Nicotiana , Fatores de Transcrição/genética , Zinco
8.
Org Biomol Chem ; 15(25): 5357-5363, 2017 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-28617493

RESUMO

Botrytis cinerea is a polyphagous fungal parasite which causes serious damage to more than 200 plant species and consequent economic losses for commercial crops. This pathogen produces two families of phytotoxins, the botryanes and botcinins, which are involved in the infection mechanism. The B. cinerea genome has provided a complete picture of the genes involved in the biosynthesis of its secondary metabolites. The botrydial biosynthetic gene cluster has been identified. This cluster consists of seven genes, where the genes BcBOT1, BcBOT3 and BcBOT4 encode three mono-oxygenases. A study of the Bcbot4Δ null mutant revealed that this mono-oxygenase was involved in the hydroxylation at C-4 of the probotryane skeleton (C-11 of the presilphiperfolane skeleton). A detailed study of the Bcbot4Δ null mutant has been undertaken in order to study the metabolic fate of the presilphiperfolan-8-ol intermediate biosynthesized by this organism and in particular by this strain. As a result three new presilphiperfolanes and three new cameroonanes have been identified. The results suggest that the absence of the oxygen function at C-11 of the presilphiperfolane skeleton permits rearrangement to a cameroonane whilst hydroxylation at C-11 precludes this rearrangement. It is possible that the interactions of the C-11 hydroxylated derivatives perturb the stereo-electronic requirements for the migration of the C-11:C-7 sigma bond to C-8.


Assuntos
Botrytis/metabolismo , Sesquiterpenos/metabolismo , Botrytis/genética , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Mutação , Sesquiterpenos/química
9.
PLoS Genet ; 10(1): e1004040, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24415947

RESUMO

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, but 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 over-expression of bcltf1, we confirmed the predicted role of the transcription factor 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 light-responsive genes, and that the expression levels of the majority of these genes (66%) are modulated by BcLTF1. In addition, the deletion of bcltf1 affects the expression of 1,539 genes irrespective of the light conditions, including the overexpression of known and so far uncharacterized secondary metabolism-related genes. Increased expression of genes encoding alternative respiration enzymes, such as the alternative oxidase (AOX), suggest a mitochondrial dysfunction in the absence of bcltf1. The hypersensitivity of Δbctlf1 mutants to exogenously applied oxidative stress--even in the absence of light--and the restoration of virulence and growth rates in continuous light by antioxidants, indicate that BcLTF1 is required to cope with oxidative stress that is caused either by exposure to light or arising during host infection.


Assuntos
Fatores de Transcrição GATA/genética , Doenças das Plantas/genética , Plantas/genética , Virulência/genética , Aspergillus nidulans/genética , Botrytis/genética , Botrytis/patogenicidade , Fatores de Transcrição GATA/isolamento & purificação , Regulação Fúngica da Expressão Gênica , Luz , Proteínas Mitocondriais/genética , Estresse Oxidativo/genética , Oxirredutases/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo
10.
Environ Microbiol ; 18(11): 4068-4086, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27347834

RESUMO

Botrytis cinerea is a plant pathogenic fungus known for its utilization of light as environmental cue to regulate asexual differentiation: conidia are formed in the light, while sclerotia are formed in the dark. As no orthologues 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 simultaneously to suppress sclerotial development. Light-induced expression of bcltf2 is mediated via a so far unknown pathway, and is attenuated in a (blue) light-dependent 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.


Assuntos
Botrytis/crescimento & desenvolvimento , Botrytis/efeitos da radiação , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Fatores de Transcrição/metabolismo , Botrytis/genética , Botrytis/metabolismo , Proteínas Fúngicas/genética , Luz , Fenótipo , Plantas/microbiologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/efeitos da radiação , Fatores de Transcrição/genética
11.
Fungal Genet Biol ; 96: 33-46, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27721016

RESUMO

Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G+C/A+T-equilibrated regions that contain the Bcbot genes alternate with A+T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)2Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the ΔBcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle.


Assuntos
Aldeídos/metabolismo , Botrytis/genética , Compostos Bicíclicos com Pontes/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Família Multigênica , Fatores de Transcrição/metabolismo , Sequência Rica em At , Botrytis/metabolismo , Botrytis/patogenicidade , Elementos de DNA Transponíveis , DNA Fúngico , Proteínas Fúngicas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Virulência
12.
Eukaryot Cell ; 14(7): 636-51, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25934690

RESUMO

Important for the lifestyle and survival of every organism is the ability to respond to changing environmental conditions. The necrotrophic plant pathogen Botrytis cinerea triggers an oxidative burst in the course of plant infection and therefore needs efficient signal transduction to cope with this stress. The factors involved in this process and their precise roles are still not well known. Here, we show that the transcription factor Bap1 and the response regulator (RR) B. cinerea Skn7 (BcSkn7) are two key players in the oxidative stress response (OSR) of B. cinerea; both have a major influence on the regulation of classical OSR genes. A yeast-one-hybrid (Y1H) approach proved direct binding to the promoters of gsh1 and grx1 by Bap1 and of glr1 by BcSkn7. While the function of Bap1 is restricted to the regulation of oxidative stress, analyses of Δbcskn7 mutants revealed functions beyond the OSR. Involvement of BcSkn7 in development and virulence could be demonstrated, indicated by reduced vegetative growth, impaired formation of reproductive structures, and reduced infection cushion-mediated penetration of the host by the mutants. Furthermore, Δbcskn7 mutants were highly sensitive to oxidative, osmotic, and cell wall stress. Analyses of Δbap1 bcskn7 double mutants indicated that loss of BcSkn7 uncovers an underlying phenotype of Bap1. In contrast to Saccharomyces cerevisiae, the ortholog of the glutathione peroxidase Gpx3p is not required for nuclear translocation of Bap1. The presented results contribute to the understanding of the OSR in B. cinerea and prove that it differs substantially from that of yeast, demonstrating the complexity and versatility of components involved in signaling pathways.


Assuntos
Botrytis/patogenicidade , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Estresse Oxidativo , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Virulência , Adaptação Fisiológica , Proteínas Fúngicas/genética , Germinação , Oxirredução , Phaseolus/genética , Phaseolus/metabolismo , Doenças das Plantas/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
13.
Mol Plant Microbe Interact ; 28(6): 659-74, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25625818

RESUMO

Botrytis cinerea, the gray mold fungus, is an important plant pathogen. Field populations are characterized by variability with regard to morphology, the mode of reproduction (conidiation or sclerotia formation), the spectrum of secondary metabolites (SM), and virulence. Natural variation in bcvel1 encoding the ortholog of Aspergillus nidulans VeA, a member of the VELVET complex, was previously shown to affect light-dependent differentiation, the formation of oxalic acid (OA), and virulence. To gain broader insight into the B. cinerea VELVET complex, an ortholog of A. nidulans LaeA, BcLAE1, a putative interaction partner of BcVEL1, was studied. BcVEL1 but not its truncated versions interacts with BcLAE1 and BcVEL2 (VelB ortholog). In accordance with the expected common as well as specific functions of BcVEL1 and BcLAE1, the deletions of both genes result in similar though not identical phenotypes. Both mutants lost the ability to produce OA, to colonize the host tissue, and to form sclerotia. However, mutants differ with regard to aerial hyphae and conidia formation. Genome-wide expression analyses revealed that BcVEL1 and BcLAE1 have common and distinct target genes. Some of the genes that are underexpressed in both mutants, e.g., those encoding SM-related enzymes, proteases, and carbohydrate-active enzymes, may account for their reduced virulence.


Assuntos
Botrytis , Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Vitis/microbiologia , Aspergillus nidulans/genética , Botrytis/genética , Botrytis/metabolismo , Botrytis/patogenicidade , Frutas/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Hifas , Luz , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Folhas de Planta/microbiologia , Metabolismo Secundário , Deleção de Sequência , Esporos Fúngicos , Técnicas do Sistema de Duplo-Híbrido , Virulência
14.
Mol Plant Microbe Interact ; 28(11): 1167-80, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26267356

RESUMO

Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea, while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infection process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus spreading. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes upregulated during infection of MB are enriched in functional categories related to necrotrophy, such as degradation of the plant cell wall, proteolysis, membrane transport, reactive oxygen species (ROS) generation, and detoxification. Quantitative-polymerase chain reaction on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but is stopped at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathway switch during berry ripening. In response to B. cinerea inoculation, VB activated a burst of ROS, the salicylate-dependent defense pathway, the synthesis of the resveratrol phytoalexin, and cell-wall strengthening. On the contrary, in infected MB, the jasmonate-dependent pathway was activated, which did not stop the fungal necrotrophic process.


Assuntos
Botrytis/genética , Resistência à Doença/genética , Frutas/genética , Doenças das Plantas/genética , Vitis/genética , Botrytis/patogenicidade , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/microbiologia , Ciclopentanos/metabolismo , Frutas/crescimento & desenvolvimento , Frutas/microbiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Interações Hospedeiro-Patógeno/genética , Análise de Sequência com Séries de Oligonucleotídeos , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salicilatos/metabolismo , Sesquiterpenos/metabolismo , Estilbenos/metabolismo , Virulência/genética , Vitis/crescimento & desenvolvimento , Vitis/microbiologia , Fitoalexinas
15.
PLoS Genet ; 7(8): e1002230, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21876677

RESUMO

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 <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.


Assuntos
Ascomicetos/genética , Botrytis/genética , Genoma Fúngico , Doenças das Plantas/microbiologia , Elementos de DNA Transponíveis , Genes Fúngicos , Genômica , Filogenia , Doenças das Plantas/genética , Sintenia
16.
Fungal Genet Biol ; 52: 9-19, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23396263

RESUMO

Botrytis cinerea, the grey mould fungus, secretes non-host-specific phytotoxins that kill the cells of many plant species. Phytotoxic assays performed about ten years ago, have highlighted the role in the infection mechanism of one of these secondary metabolites, the sesquiterpene botrydial. We recently showed that BcBOT1 to BcBOT5 genes, which are required for botrydial biosynthesis, are organised into a physical cluster. However, this cluster includes no gene encoding a transcription factor (TF) that might specifically coregulate the expression of BcBOT genes. To identify which TF(s) are implicated in the regulation of this cluster and thereby to decipher DNA-protein interactions in the phytopathogenic fungus B. cinerea, we developed a strategy based on the yeast one-hybrid (Y1H) method. In this study, a Y1H library was generated with the TFs predicted from complete genome sequencing. The screening of this library revealed an interaction between a promoter of the botrydial biosynthesis gene cluster and a new Cys2His2 zinc finger TF, that we called BcYOH1. Inactivation of the BcYOH1 gene and expression analyses demonstrated the involvement of this TF in regulating expression of the botrydial biosynthesis gene cluster. Furthermore, whole-transcriptome analysis suggested that BcYOH1 might act as a global transcriptional regulator of phytotoxin and other secondary metabolism gene clusters, and of genes involved in carbohydrate metabolism, transport, virulence and detoxification mechanisms.


Assuntos
Botrytis/genética , Família Multigênica , Micotoxinas/genética , Fatores de Transcrição/genética , Botrytis/metabolismo , Botrytis/patogenicidade , Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Fatores de Transcrição/isolamento & purificação
17.
Chembiochem ; 14(1): 132-6, 2013 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-23203902

RESUMO

Isotopic labelling experiments and the study of mutants with disrupted genes encoding botcinic acid have revealed a common link in the biosynthesis of the polyketide toxins excreted by Botrytis cinerea: botcinins and botrylactones. Furthermore, the results reported here shed light on the origin of the starter unit, thereby solving a long-standing mystery in the biosynthesis of botcinins.


Assuntos
Botrytis/genética , Botrytis/metabolismo , Deleção de Genes , Lactonas/metabolismo , Pironas/metabolismo , Lactonas/química
18.
Sci Rep ; 13(1): 1417, 2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-36697464

RESUMO

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.


Assuntos
Fungos , Anotação de Sequência Molecular , Metabolismo Secundário , Software , Sequência de Aminoácidos , Anotação de Sequência Molecular/métodos , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Metabolismo Secundário/genética , Fungos/enzimologia , Fungos/genética , Triptofano Sintase/genética , Sequência Conservada/genética
19.
Phytopathology ; 101(12): 1433-45, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21830954

RESUMO

Botrytis cinerea is a major crop pathogen infesting >220 hosts worldwide. A cryptic species has been identified in some French populations but the new species, B. pseudocinerea, has not been fully delimited and established. The aim of this study was to distinguish between the two species, using phylogenetic, biological, morphological, and ecological criteria. Multiple gene genealogies confirmed that the two species belonged to different, well-supported phylogenetic clades. None of the morphological criteria tested (spore size, germination rate, or mycelial growth) was able to discriminate between these two species. Sexual crosses between individuals from the same species and different species were carried out. Only crosses between individuals from the same species were successful. Moreover, population genetics analysis revealed a high level of diversity within each species and a lack of gene flow between them. Finally, a population survey over time showed that B. cinerea was the predominant species but that B. pseudocinerea was more abundant in spring, on floral debris. This observation could not be explained by temperature adaptation in tests carried out in vitro or by aggressiveness on tomato or bean leaves. This study clearly establishes that B. cinerea and B. pseudocinerea constitute a complex of two cryptic species living in sympatry on several hosts, including grapevine and blackberry. We propose several biological or molecular tools for unambiguous differentiation between the two species. B. pseudocinerea probably makes a negligible contribution to gray mold epidemics on grapevine. This new species has been deposited in the MycoBank international database.


Assuntos
Botrytis/classificação , Proteínas Fúngicas/genética , Especiação Genética , Magnoliopsida/microbiologia , Doenças das Plantas/microbiologia , Simpatria , Sequência de Bases , Botrytis/enzimologia , Botrytis/isolamento & purificação , Botrytis/fisiologia , Chaperonina 60/genética , DNA Fúngico/química , DNA Fúngico/genética , Ecossistema , França , Frutas/microbiologia , Loci Gênicos , Variação Genética , Genótipo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Dados de Sequência Molecular , Fenótipo , Filogenia , RNA Helicases/genética , Análise de Sequência de DNA , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Temperatura , Vitis/microbiologia
20.
Phytochemistry ; 183: 112627, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33429353

RESUMO

Botrytis cinerea is a necrotrophic fungal pathogen that affects a total of 586 genera representing approximately 1400 plant species. This pathogen produces two families of phytotoxins involved in its infection process i.e. botrydial and its relatives, and botcinic and botcineric acids and their relatives, botcinins. The botrydial biosynthetic cluster consists of seven genes, where the gene BcBOT4 encodes a cytochrome P450 monooxygenase that was shown to catalyse regio- and stereospecific hydroxylation at position C-4 of the presilphiperfolan-8-ß-ol skeleton. The null mutant bcbot4Δ halted the production of botrydial and its derivatives, and instead accumulated tricyclic presilphiperfolane alcohol and overproduced a significant number of polyketides. A detailed study of the bcbot4Δ mutant led us to the isolation and characterization of five undescribed polyketides, three derived from botcinic and botcineric acids (botcinins H, I, J), one derived from the initial pentaketide (botcinin K), and one cinbotolide derivative (cinbotolide D). Botcinins are tetra-methylated tetraketides biosynthesized by the sequential assembly of a pentaketide (C10) based on an acetate primer unit which is lost through a retro-Claisen type C-C bond cleavage. The structural characterization of botcinin K showed a basic chemical structure corresponding to a botcinin (C14) derivative obtained directly from the original per-methylated pentaketide leading to the biosynthesis of botrylactone and other botcinins, confirming the previously proposed biosynthetic route.


Assuntos
Botrytis , Policetídeos , Aldeídos , Vias Biossintéticas , Compostos Bicíclicos com Pontes , Doenças das Plantas
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