Dataset Information

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

ABSTRACT: Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669bp) and pksT-2 (7901bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase-acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

SUBMITTER: Yao L

PROVIDER: S-EPMC4874624 | biostudies-literature | 2016 Apr-Jun

REPOSITORIES: biostudies-literature

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Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

Yao Lin L Tan Chong C Song Jinzhu J Yang Qian Q Yu Lijie L Li Xinling X

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20160302 2

Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analy ...[more]

PMID: 26991299

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Project description:BackgroundThe species of T. harzianum are well known for their biocontrol activity against plant pathogens. However, few studies have been conducted to further our understanding of its role as a biological control agent against S. sclerotiorum, a pathogen involved in several crop diseases around the world. In this study, we have used RNA-seq and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum gene expression during growth on cell wall of S. sclerotiorum (SSCW) or glucose. RT-qPCR was also used to examine genes potentially involved in biocontrol, during confrontation between T. harzianum and S. sclerotiorum.ResultsData obtained from six RNA-seq libraries were aligned onto the T. harzianum CBS 226.95 reference genome and compared after annotation using the Blast2GO suite. A total of 297 differentially expressed genes were found in mycelia grown for 12, 24 and 36 h under the two different conditions: supplemented with glucose or SSCW. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on SSCW or glucose. We identified various genes of biotechnological value encoding proteins with functions such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. To validate the expression profile, RT-qPCR was performed using 20 randomly chosen genes. RT-qPCR expression profiles were in complete agreement with the RNA-Seq data for 17 of the genes evaluated. The other three showed differences at one or two growth times. During the confrontation assay, some genes were up-regulated during and after contact, as shown in the presence of SSCW which is commonly used as a model to mimic this interaction.ConclusionsThe present study is the first initiative to use RNA-seq for identification of differentially expressed genes in T. harzianum strain TR274, in response to the phytopathogenic fungus S. sclerotiorum. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against S.sclerotiorum. The RNA-seq data presented will facilitate improvement of the annotation of gene models in the draft T. harzianum genome and provide important information regarding the transcriptome during this interaction.

Project description:The fungus Trichoderma arundinaceum exhibits biological control activity against crop diseases caused by other fungi. Two mechanisms that likely contribute to this activity are upregulation of plant defenses and production of two types of antifungal secondary metabolites: the sesquiterpenoid harzianum A (HA) and the polyketide-derived aspinolides. The goal of the current study was to identify aspinolide biosynthetic genes as part of an effort to understand how these metabolites contribute to the biological control activity of T. arundinaceum. Comparative genomics identified two polyketide synthase genes (asp1 and asp2) that occur in T. arundinaceum and Aspergillus ochraceus, which also produces aspinolides. Gene deletion and biochemical analyses in T. arundinaceum indicated that both genes are required for aspinolide production: asp2 for formation of a 10-member lactone ring and asp1 for formation of a butenoyl subsituent at position 8 of the lactone ring. Gene expression and comparative genomics analyses indicated that asp1 and asp2 are located within a gene cluster that occurs in both T. arundinaceum and A. ochraceus. A survey of genome sequences representing 35 phylogenetically diverse Trichoderma species revealed that intact homologs of the cluster occurred in only two other species, which also produced aspinolides. An asp2 mutant inhibited fungal growth more than the wild type, but an asp1 mutant did not, and the greater inhibition by the asp2 mutant coincided with increased HA production. These findings indicate that asp1 and asp2 are aspinolide biosynthetic genes and that loss of either aspinolide or HA production in T. arundinaceum can be accompanied by increased production of the other metabolite(s). KEY POINTS: • Two polyketide synthase genes are required for aspinolide biosynthesis. • Blocking aspinolide production increases production of the terpenoid harzianum A. • Aspinolides and harzianum A act redundantly in antibiosis of T. arundinaceum.

Dataset Information

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

Publications

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

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