1887

Abstract

Trichoderma hypoxylon is a fungicolous species which produces rich secondary metabolites. However, no genetic transformation method is available for further studies. Here, we developed a marker-less transformation system based on the complementation of an uridine/uracil biosynthetic gene by protoplast transformation. An uridine/uracil auxotrophic mutant of Δthpyr4 was obtained by using a positive screening protocol with 5′-fluoroorotic acid as a selective reagent. To improve the homologous integration rates, the orthologues of ku70 and lig4 which play critical roles in non-homologous end-joining recombination were disrupted. The resulting thlig4 mutant showed remarkable transformation rates of 89 %, while no change was found in the thku70 deletion mutant compared with the WT strain. This suggests that thlig4 play a key role in the non-homologous recombination in this strain. Using this system, the biosynthetic gene cluster of trichothecene (tri) harzianum B was identified by deletion of the thtri5 in T. hypoxylon. Comparative genome analysis revealed that the trichothecene biosynthetic gene cluster in T. hypoxylon shared similar organizations with T. arundinaceum and T. brevicompactum, even though their encoded products are different in structures. Taken together, the highly efficient genetic system provides a convenient tool for studying the biosynthetic diversity and mining the novel natural product from the fungi.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000649
2018-03-19
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/5/769.html?itemId=/content/journal/micro/10.1099/mic.0.000649&mimeType=html&fmt=ahah

References

  1. Newman DJ, Cragg GM. Natural products as sources of new drugs from 1981 to 2014. J Nat Prod 2016; 79: 629– 661 [CrossRef] [PubMed]
    [Google Scholar]
  2. Brakhage AA. Regulation of fungal secondary metabolism. Nat Rev Microbiol 2013; 11: 21– 32 [CrossRef] [PubMed]
    [Google Scholar]
  3. Yin WB, Chooi YH, Smith AR, Cacho RA, Hu Y et al. Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans. ACS Synth Biol 2013; 2: 629– 634 [CrossRef] [PubMed]
    [Google Scholar]
  4. Wu G, Zhou H, Zhang P, Wang X, Li W et al. Polyketide production of pestaloficiols and macrodiolide ficiolides revealed by manipulations of epigenetic regulators in an endophytic fungus. Org Lett 2016; 18: 1832– 1835 [CrossRef] [PubMed]
    [Google Scholar]
  5. Zhang P, Wang X, Fan A, Zheng Y, Liu X et al. A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici. Mol Microbiol 2017; 105: 469– 483 [CrossRef] [PubMed]
    [Google Scholar]
  6. Fan A, Mi W, Liu Z, Zeng G, Zhang P et al. Deletion of a histone acetyltransferase leads to the pleiotropic activation of natural products in Metarhizium robertsii. Org Lett 2017; 19: 1686– 1689 [CrossRef] [PubMed]
    [Google Scholar]
  7. Zheng Y, Wang X, Zhang X, Li W, Liu G et al. COP9 signalosome subunit PfCsnE regulates secondary metabolism and conidial formation in Pestalotiopsis fici. Sci China Life Sci 2017; 60: 656– 664 [CrossRef] [PubMed]
    [Google Scholar]
  8. Zheng Y, Ma K, Lyu H, Huang Y, Liu H et al. Genetic manipulation of the COP9 signalosome subunit PfCsnE leads to the discovery of pestaloficins in Pestalotiopsis fici. Org Lett 2017; 19: 4700– 4703 [CrossRef] [PubMed]
    [Google Scholar]
  9. Reino JL, Guerrero RF, Hernández-Galán R, Collado IG. Secondary metabolites from species of the biocontrol agent Trichoderma. Phytochem Rev 2008; 7: 89– 123 [CrossRef]
    [Google Scholar]
  10. Hill R, Cutler H, Parker S. Trichoderma and metabolites as biological control agents. 1995; The Horticulture and Food Research Institute Of New Zealand, USA patent WO1995NZ00009
  11. Evidente A, Cabras A, Maddau L, Serra S, Andolfi A et al. Viridepyronone, a new antifungal 6-substituted 2H-pyran-2-one produced by Trichoderma viride. J Agric Food Chem 2003; 51: 6957– 6960 [CrossRef] [PubMed]
    [Google Scholar]
  12. Kishimoto N, Sugihara S, Mochida KYO, Fujita T. In vitro antifungal and antiviral activities of γ -and δ-lactone analogs utilized as food flavoring. Biocontrol Sci 2005; 10: 31– 36 [CrossRef]
    [Google Scholar]
  13. Macías FA, Varela RM, Simonet AM, Cutler HG, Cutler SJ et al. Bioactive carotanes from Trichoderma virens. J Nat Prod 2000; 63: 1197– 1200 [PubMed] [Crossref]
    [Google Scholar]
  14. Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Barbetti MJ et al. A novel role for Trichoderma secondary metabolites in the interactions with plants. Physiol Mol Plant Pathol 2008; 72: 80– 86 [CrossRef]
    [Google Scholar]
  15. Ilmén M, Saloheimo A, Onnela ML, Penttilä ME. Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 1997; 63: 1298– 1306 [PubMed]
    [Google Scholar]
  16. Sun J, Pei Y, Li E, Li W, Hyde KD et al. A new species of Trichoderma hypoxylon harbours abundant secondary metabolites. Sci Rep 2016; 6: 37369– 37379 [CrossRef] [PubMed]
    [Google Scholar]
  17. Li J, Ren J, Bao L, Jin T, Wang W et al. Trichodermates A–F, new cytotoxic trichothecenes from the plant pathogenic fungus Trichoderma sp. Helv Chim Acta 2016; 99: 63– 69 [CrossRef]
    [Google Scholar]
  18. McCormick SP, Stanley AM, Stover NA, Alexander NJ. Trichothecenes: from simple to complex mycotoxins. Toxins 2011; 3: 802– 814 [CrossRef] [PubMed]
    [Google Scholar]
  19. Derntl C, Kiesenhofer DP, Mach RL, Mach-Aigner AR. Novel strategies for genomic manipulation of Trichoderma reesei with the purpose of strain engineering. Appl Environ Microbiol 2015; 81: 6314– 6323 [CrossRef] [PubMed]
    [Google Scholar]
  20. Ying SH, Feng MG, Keyhani NO. Use of uridine auxotrophy (ura3) for markerless transformation of the mycoinsecticide Beauveria bassiana. Appl Microbiol Biotechnol 2013; 97: 3017– 3025 [CrossRef] [PubMed]
    [Google Scholar]
  21. Ballance DJ, Turner G. Development of a high-frequency transforming vector for Aspergillus nidulans. Gene 1985; 36: 321– 331 [CrossRef] [PubMed]
    [Google Scholar]
  22. van Hartingsveldt W, Mattern IE, van Zeijl CM, Pouwels PH, van den Hondel CA. Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 1987; 206: 71– 75 [CrossRef] [PubMed]
    [Google Scholar]
  23. Ninomiya Y, Suzuki K, Ishii C, Inoue H. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci USA 2004; 101: 12248– 12253 [CrossRef] [PubMed]
    [Google Scholar]
  24. Ishibashi K, Suzuki K, Ando Y, Takakura C, Inoue H. Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci USA 2006; 103: 14871– 14876 [CrossRef] [PubMed]
    [Google Scholar]
  25. Qin X, Li R, Luo X, Lin Y, Feng JX. Deletion of ligD significantly improves gene targeting frequency in the lignocellulolytic filamentous fungus Penicillium oxalicum. Fungal Biol 2017; 121: 615– 623 [CrossRef] [PubMed]
    [Google Scholar]
  26. Bok JW, Keller NP. Fast and easy method for construction of plasmid vectors using modified quick-change mutagenesis. Methods Mol Biol 2012; 944: 163– 174 [CrossRef] [PubMed]
    [Google Scholar]
  27. Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y et al. Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc 2006; 1: 3111– 3120 [CrossRef] [PubMed]
    [Google Scholar]
  28. Wang X, Wu F, Liu L, Liu X, Che Y et al. The bZIP transcription factor PfZipA regulates secondary metabolism and oxidative stress response in the plant endophytic fungus Pestalotiopsis fici. Fungal Genet Biol 2015; 81: 221– 228 [CrossRef] [PubMed]
    [Google Scholar]
  29. Yelton MM, Hamer JE, Timberlake WE. Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 1984; 81: 1470– 1474 [CrossRef] [PubMed]
    [Google Scholar]
  30. Wilson TE, Grawunder U, Lieber MR. Yeast DNA ligase IV mediates non-homologous DNA end joining. Nature 1997; 388: 495– 498 [CrossRef] [PubMed]
    [Google Scholar]
  31. Mizutani O, Kudo Y, Saito A, Matsuura T, Inoue H et al. A defect of LigD (human Lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae. Fungal Genet Biol 2008; 45: 878– 889 [CrossRef] [PubMed]
    [Google Scholar]
  32. Kito H, Fujikawa T, Moriwaki A, Tomono A, Izawa M et al. MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea. Fungal Genet Biol 2008; 45: 1543– 1551 [CrossRef] [PubMed]
    [Google Scholar]
  33. Bugeja HE, Boyce KJ, Weerasinghe H, Beard S, Jeziorowski A et al. Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei. Fungal Genet Biol 2012; 49: 772– 778 [CrossRef] [PubMed]
    [Google Scholar]
  34. Malmierca MG, Barua J, McCormick SP, Izquierdo-Bueno I, Cardoza RE et al. Novel aspinolide production by Trichoderma arundinaceum with a potential role in Botrytis cinerea antagonistic activity and plant defence priming. Environ Microbiol 2015; 17: 1103– 1118 [CrossRef] [PubMed]
    [Google Scholar]
  35. Nielsen KF, Gräfenhan T, Zafari D, Thrane U. Trichothecene production by Trichoderma brevicompactum. J Agric Food Chem 2005; 53: 8190– 8196 [CrossRef] [PubMed]
    [Google Scholar]
  36. Mukherjee PK, Horwitz BA, Kenerley CM. Secondary metabolism in Trichoderma–a genomic perspective. Microbiology 2012; 158: 35– 45 [CrossRef] [PubMed]
    [Google Scholar]
  37. Mukherjee PK, Horwitz BA, Herrera-Estrella A, Schmoll M, Kenerley CM. Trichoderma research in the genome era. Annu Rev Phytopathol 2013; 51: 105– 129 [CrossRef] [PubMed]
    [Google Scholar]
  38. Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM et al. Trichoderma: the genomics of opportunistic success. Nat Rev Microbiol 2011; 9: 749– 759 [CrossRef] [PubMed]
    [Google Scholar]
  39. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. Trichoderma species–opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2004; 2: 43– 56 [CrossRef] [PubMed]
    [Google Scholar]
  40. Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 1987; 61: 155– 164 [CrossRef] [PubMed]
    [Google Scholar]
  41. Zhong YH, Wang XL, Wang TH, Jiang Q. Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis. Appl Microbiol Biotechnol 2007; 73: 1348– 1354 [CrossRef] [PubMed]
    [Google Scholar]
  42. Zeilinger S. Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation. Curr Genet 2004; 45: 54– 60 [CrossRef] [PubMed]
    [Google Scholar]
  43. Mitra G, Bachhawat BK. Enhanced in vivo catalytic activity of PEG-modified cellulase complex from Trichoderma reesei. Biochem Mol Biol Int 1997; 42: 93– 102 [CrossRef] [PubMed]
    [Google Scholar]
  44. Kück U, Hoff B. New tools for the genetic manipulation of filamentous fungi. Appl Microbiol Biotechnol 2010; 86: 51– 62 [CrossRef] [PubMed]
    [Google Scholar]
  45. Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L et al. A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics 2006; 172: 1557– 1566 [CrossRef] [PubMed]
    [Google Scholar]
  46. Critchlow SE, Jackson SP. DNA end-joining: from yeast to man. Trends Biochem Sci 1998; 23: 394– 398 [CrossRef] [PubMed]
    [Google Scholar]
  47. Alexander NJ, Proctor RH, McCormick SP. Genes, gene clusters, and biosynthesis of trichothecenes and fumonisins in Fusarium. Toxin Rev 2009; 28: 198– 215 [CrossRef]
    [Google Scholar]
  48. Cardoza RE, Malmierca MG, Hermosa MR, Alexander NJ, McCormick SP et al. Identification of loci and functional characterization of trichothecene biosynthesis genes in filamentous fungi of the genus Trichoderma. Appl Environ Microbiol 2011; 77: 4867– 4877 [CrossRef] [PubMed]
    [Google Scholar]
  49. Malmierca MG, Cardoza RE, Alexander NJ, McCormick SP, Collado IG et al. Relevance of trichothecenes in fungal physiology: disruption of tri5 in Trichoderma arundinaceum. Fungal Genet Biol 2013; 53: 22– 33 [CrossRef] [PubMed]
    [Google Scholar]
  50. Brown DW, Dyer RB, McCormick SP, Kendra DF, Plattner RD. Functional demarcation of the Fusarium core trichothecene gene cluster. Fungal Genet Biol 2004; 41: 454– 462 [CrossRef] [PubMed]
    [Google Scholar]
  51. Yin WB, Baccile JA, Bok JW, Chen Y, Keller NP et al. A nonribosomal peptide synthetase-derived iron(III) complex from the pathogenic fungus Aspergillus fumigatus. J Am Chem Soc 2013; 135: 2064– 2067 [CrossRef] [PubMed]
    [Google Scholar]
  52. Alexander NJ, Hohn TM, McCormick SP. The TRI11 gene of Fusarium sporotrichioides encodes a cytochrome P-450 monooxygenase required for C-15 hydroxylation in trichothecene biosynthesis. Appl Environ Microbiol 1998; 64: 221– 225 [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000649
Loading
/content/journal/micro/10.1099/mic.0.000649
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error