Ggne-knockout System Construction Of Peptaibols Synthetase Gene And Protease Gene Of Trichoderma Longibrachiatum SMF2and Functional Study Of Peptaibols In Plant Growth

Trichoderma species are ubiquitous fungi in the soil, root and foliar environments. They are important biological control agents for controlling plant diseases. The mechanisms employed by Trichoderma for controlling plant diseases include antibiosis, hyperparasitism, competition and plant resistance induction. The secondary metabolites, such as peptaibols, and cell wall degrading enzymes from Trichoderma play key roles in the antibiosis process. T. longibrachiatum SMF2(SMF2) is a good candidate for controlling plant diseases, which can inhibit a range of plant phytopathgens. The active secondary metabolites from SMF2were thermostable and identified as Trichokonins, a member of peptaibols with20amino acid residues. Trichokonins were synthesized by Non-ribosomal peptide synthetase gene containing20modules. And a simple protocol has been developed for preparative preparation of Trichokonins. Trichokonins exhibite antimicrobial, and antiviral activities, induce tumor cell apoptosis and elicit plant protection system elicited induced resistance in plants. In addition, TKs could promote root development in low concentration, while TK Ⅵ of high concentration could inhibit the development of plant root. However, the effect of peptaibols on plant in vivo is still unknown.The genome of SMF2has been sequenced, and two long NRPS-encoding genes encoded20modules and12modules peptaibols synthetase were found, designated as tpxl and tpx2respectively. In this study we constructed the gene knockout vectors based on homologous recombination principle and transformed the protoplasts of SMF2mediated by PEG. Hygromycinphospho-transferase (hph) and acetamidase gene (amdS) were used as transfromants selection marker. tpxl-and tpx2-deficient mutants were obtained and confirmed by PCR and Southern blotting analysis. Trichokonins could not be detected in the mutants by HPLC analysis. Moreover, it was comfirmed that tpxl-and tpx2genes were responsible for the biosynthesis of long chain and short chain peptaibols, respectively. However the deletion of tpxl-and tpx2genes has no effect on the growth of mycelium, colonial morphology and sporogenesis of SMF2.In our preliminary study, it was found that Trichokonins could promote plant growth and development at low concentration, while could inhibit plant growth and development at high concentration. In this study, we studied the effect of SMF2and tpxl-and tpx2-deficient mutants on the growth of tomato seedlings. The results showed that the wild type SMF2strain could promote the growth of tomato seedlings and improve the biomass of the root. Peptaibols synthetase deletion mutants decreased these enhancing effects, especially the deletion of tpxl in SMF2. Our study provides valuable insight into the mechanism of Trichoderma on plant growth.SprT was a kind of serine protease isolated from SMF2, exhibited antibacterial, antifungal and nematicidal activities. In this study, we constructed sprT gene knockout vector with fusion PCR technology by homologous recombination. Hph was used as the transformants selection marker. The sprT deletion mutant of SMF2was obtained and analyzed via Southern blotting. The deletion of sprT had no influence on growth, sporogenesis and colonial morphology of SMF2. SprT could not be detected in the crude extract of deletion mutant and the total protease activity decreased. However, the antifungal activities did not lost completely due to the existence of other components.