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Functional analysis of polyketide synthase genes in the biocontrol fungus Clonostachys rosea.

ABSTRACT: Clonostachys rosea is a mycoparasitic fungus used for biological control of plant diseases. Its genome contains 31 genes putatively encoding for polyketide synthases (PKSs), 75% of which are arranged in biosynthetic gene clusters. Gene expression analysis during C. rosea interactions with the fungal plant pathogens Botrytis cinerea and Fusarium graminearum showed common and species-specific induction of PKS genes. Our data showed a culture media dependent correlation between PKS gene expression and degree of antagonism in C. rosea. The pks22 and pks29 genes were highly induced during fungal-fungal interactions but not during pigmentation, and gene deletion studies revealed that PKS29 was required for full antagonism against B. cinerea, and for biocontrol of fusarium foot rot on barley. Metabolite analysis revealed that ?pks29 strains has a 50% reduced production (P?=?0.001) of an unknown polyketide with molecular formula C15H28O3, while ?pks22 strains lost the ability to produce four previously unknown polyketides named Clonorosein A-D. Clonorosein A and B were purified, their structures determined, and showed strong antifungal activity against B. cinerea and F. graminearum. These results show that PKS22 is required for production of antifungal polyketide Clonorosein A-D, and demonstrate the role of PKS29 in antagonism and biocontrol of fungal plant diseases.

SUBMITTER: Fatema U

PROVIDER: S-EPMC6177402 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Functional analysis of polyketide synthase genes in the biocontrol fungus Clonostachys rosea.

Fatema Umma U Broberg Anders A Jensen Dan Funck DF Karlsson Magnus M Dubey Mukesh M

Scientific reports 20181009 1

Clonostachys rosea is a mycoparasitic fungus used for biological control of plant diseases. Its genome contains 31 genes putatively encoding for polyketide synthases (PKSs), 75% of which are arranged in biosynthetic gene clusters. Gene expression analysis during C. rosea interactions with the fungal plant pathogens Botrytis cinerea and Fusarium graminearum showed common and species-specific induction of PKS genes. Our data showed a culture media dependent correlation between PKS gene expression ...[more]

PMID: 30301915

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Project description:Necrotrophic mycoparasitism is an intricate process involving recognition, physical mycelial contact, and killing of host fungi (mycohosts). During such interactions, mycoparasites undergo a complex developmental process involving massive regulatory changes of gene expression to produce a range of chemical compounds and proteins that contribute to the parasitism of the mycohosts. Small RNAs (sRNAs) are vital components of posttranscriptional gene regulation, although their role in gene expression regulation during mycoparasitisms remain understudied. Here, we investigated the role of sRNA-mediated gene regulation in mycoparasitism by performing sRNA and degradome tag sequencing of the mycoparasitic fungus Clonostachys rosea interacting with the plant-pathogenic mycohosts Botrytis cinerea and Fusarium graminearum at two time points. The majority of differentially expressed sRNAs were downregulated during the interactions with the mycohosts compared to a C. rosea self-interaction control, thus allowing desuppression (upregulation) of mycohost-responsive genes. Degradome analysis showed a positive correlation between high degradome counts and antisense sRNA mapping and led to the identification of 201 sRNA-mediated potential gene targets for 282 differentially expressed sRNAs. Analysis of sRNA potential gene targets revealed that the regulation of genes coding for membrane proteins was a common response against both mycohosts. The regulation of genes involved in oxidative stress tolerance and cellular metabolic and biosynthetic processes was exclusive against F. graminearum, highlighting common and mycohost-specific gene regulation of C. rosea. By combining these results with transcriptome data collected during a previous study, we expand the understanding of the role of sRNA in regulating interspecific fungal interactions and mycoparasitism. IMPORTANCE Small RNAs (sRNAs) are emerging as key players in pathogenic and mutualistic fungus-plant interactions; however, their role in fungus-fungus interactions remains elusive. In this study, we employed the necrotrophic mycoparasite Clonostachys rosea and the plant-pathogenic mycohosts Botrytis cinerea and Fusarium graminearum and investigated the sRNA-mediated gene regulation in mycoparasitic interactions. The combined approach of sRNA and degradome tag sequencing identified 201 sRNA-mediated putative gene targets for 282 differentially expressed sRNAs, highlighting the role of sRNA-mediated regulation of mycoparasitism in C. rosea. We also identified 36 known and 13 novel microRNAs (miRNAs) and their potential gene targets at the endogenous level and at a cross-species level in B. cinerea and F. graminearum, indicating a role of cross-species RNA interference (RNAi) in mycoparasitism, representing a novel mechanism in biocontrol interactions. Furthermore, we showed that C. rosea adapts its transcriptional response, and thereby its interaction mechanisms, based on the interaction stages and identity of the mycohost.

Project description:Biological control is a promising approach to reduce plant diseases caused by nematodes to ensure high productivity in agricultural production. Large-scale analyses of genetic variation in fungal species used for biocontrol can generate knowledge regarding interaction mechanisms that can improve efficacy of biocontrol applications. In this study, we performed a genome-wide association study (GWAS) for in vitro antagonism against the root lesion nematode Pratylenchus penetrans in 53 previously genome re-sequenced strains of the biocontrol fungus Clonostachys rosea. Nematode mortality in C. rosea potato dextrose broth (PDB) culture filtrates was highly variable and showed continuous variation (p < .001) between strains, indicating a polygenic inheritance. Twenty-one strains produced culture filtrates with higher (p ≤ .05) nematode mortality compared with the PDB control treatment, while ten strains lowered (p ≤ .05) the mortality. The difference in in vitro antagonism against P. penetrans correlated with antagonism against the soybean cyst nematode Heterodera glycines, indicating lack of host specificity in C. rosea. An empirical Bayesian multiple hypothesis testing approach identified 279 single nucleotide polymorphism markers significantly (local false sign rate < 10-10) associated with the trait. Genes present in the genomic regions associated with nematicidal activity included several membrane transporters, a chitinase and genes encoding proteins predicted to biosynthesize secondary metabolites. Gene deletion strains of the predicted nonribosomal peptide synthetase genes nps4 and nps5 were generated and showed increased (p ≤ .001) fungal growth and conidiation rates compared to the wild type. Deletion strains also exhibited reduced (p < .001) nematicidal activity and reduced (p ≤ .05) biocontrol efficacy against nematode root disease and against fusarium foot rot on wheat. In summary, we show that the GWAS approach can be used to identify biocontrol factors in C. rosea, specifically the putative nonribosomal peptide synthetases NPS4 and NPS5.

Project description:BackgroundClonostachys rosea strain IK726 is a mycoparasitic fungus capable of controlling mycotoxin-producing Fusarium species, including F. graminearum and F. culmorum, known to produce Zearalenone (ZEA) and Deoxynivalenol (DON). DON is a type B trichothecene known to interfere with protein synthesis in eukaryotes. ZEA is a estrogenic-mimicing mycotoxin that exhibits antifungal growth. C. rosea produces the enzyme zearalenone hydrolase (ZHD101), which degrades ZEA. However, the molecular basis of resistance to DON in C. rosea is not understood. We have exploited a genome-wide transcriptomic approach to identify genes induced by DON and ZEA in order to investigate the molecular basis of mycotoxin resistance C. rosea.ResultsWe generated DON- and ZEA-induced cDNA libraries based on suppression subtractive hybridization. A total of 443 and 446 sequenced clones (corresponding to 58 and 65 genes) from the DON- and ZEA-induced library, respectively, were analysed. DON-induced transcripts represented genes encoding metabolic enzymes such as cytochrome P450, cytochrome c oxidase and stress response proteins. In contrast, transcripts encoding the ZEA-detoxifying enzyme ZHD101 and those encoding a number of ATP-Binding Cassette (ABC) transporter transcripts were highly frequent in the ZEA-induced library. Subsequent bioinformatics analysis predicted that all transcripts with similarity to ABC transporters could be ascribed to only 2 ABC transporters genes, and phylogenetic analysis of the predicted ABC transporters suggested that they belong to group G (pleiotropic drug transporters) of the fungal ABC transporter gene family. This is the first report suggesting involvement of ABC transporters in ZEA tolerance. Expression patterns of a selected set of DON- and ZEA-induced genes were validated by the use of quantitative RT-PCR after exposure to the toxins. The qRT-PCR results obtained confirm the expression patterns suggested from the EST redundancy data.ConclusionThe present study identifies a number of transcripts encoding proteins that are potentially involved in conferring resistance to DON and ZEA in the mycoparasitic fungus C. rosea. Whilst metabolic readjustment is potentially the key to withstanding DON, the fungus produces ZHD101 to detoxify ZEA and ABC transporters to transport ZEA or its degradation products out from the fungal cell.

Project description:BackgroundFilamentous fungi produce small cysteine rich surface active amphiphilic hydrophobins on the outer surface of cell walls that mediate interactions between the fungus and the environment. The role of hydrophobins in surface hydrophobicity, sporulation, fruit body formation, recognition and adhesion to host surface and virulence have been reported. The aim of the present study was to characterize the biological function of hydrophobins in the fungal biocontrol agent Clonostachys rosea in order to understand their potential roles in biocontrol mechanisms.ResultsBased on the presence of hydrophobin domains, cysteine spacing patterns and hydropathy plots, we identified three class II hydrophobin genes in C. rosea. Gene expression analysis showed basal expression of Hyd1, Hyd2 and Hyd3 in all conditions tested with the exception of induced Hyd1 expression in conidiating mycelium. Interestingly, up-regulation of Hyd1, Hyd2 and Hyd3 was found during C. rosea self interaction compared to interactions with the fungal plant pathogens Botrytis cinerea or Fusarium graminearum in dual culture assays. Phenotypic analysis of C. rosea deletion and complementation strains showed that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity, although no difference in mycelia hydrophobicity was found between wild type (WT) and mutant strains. Interestingly, mutant strains showed increased growth rates, conidiation and enhanced tolerances of conidia to abiotic stresses. Antagonism tests using in vitro dual culture and detached leaf assays showed that the mutant strains were more aggressive towards B. cinerea, F. graminearum or Rhizoctonia solani, and that aggression was partly related to earlier conidial germination and enhanced tolerance of mutant strains to secreted fungal metabolites. Furthermore, in vitro Arabidopsis thaliana root colonization assays revealed reduced root colonization ability of the ΔHyd3 strain, but not for the ΔHyd1 strain. Furthermore, enhanced root colonization ability for the ΔHyd1ΔHyd3 strain was found in comparison to WT.ConclusionsThese results show a role for hydrophobins in conidial hydrophobicity, control of conidial germination under stress conditions, and in root colonization in C. rosea. However, functional studies of Hyd2 remains to be performed in order to fully assess the role of hydrophobins in C. rosea.

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Functional analysis of polyketide synthase genes in the biocontrol fungus Clonostachys rosea.

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Functional analysis of polyketide synthase genes in the biocontrol fungus Clonostachys rosea.

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