Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
권호 표지

A Comparison of the Phenotypic and Genetic Stability of Recombinant Trichoderma spp. Generated by Protoplast- and Agrobacterium-Mediated Transformation

  • Cardoza Rosa Elena (Spanish-Portuguese Center of Agricultural Research (CIALE), Department of Microbiology and Genetics, University of Salamanca, University of Leon, Campus of Ponferrada, Superior and Technical University College of Agricultural Engineers, Area of Microbiology) ;
  • Vizcaino Juan Antonio (Spanish-Portuguese Center of Agricultural Research (CIALE), Department of Microbiology and Genetics, University of Salamanca) ;
  • Hermosa Maria Rosa (Spanish-Portuguese Center of Agricultural Research (CIALE), Department of Microbiology and Genetics, University of Salamanca) ;
  • Monte Enrique (Spanish-Portuguese Center of Agricultural Research (CIALE), Department of Microbiology and Genetics, University of Salamanca) ;
  • Gutierrez Santiago (University of Leon, Campus of Ponferrada, Superior and Technical University College of Agricultural Engineers, Area of Microbiology)
  • Published : 2006.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 $transformants/{\mu}g$ DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 $transformants/l0^7$ spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transform ants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

Keywords

References

  1. Balance, D.J., F.P. Buxton, and G. Turner. 1983. Transformation of Aspergillus nidulans by the orotidine-5-phosphate decarboxylase gene of Neurospora crassa. Biochem. Biophys. Res. Commun. 112, 284-289 https://doi.org/10.1016/0006-291X(63)90297-3
  2. Benítez, T., M.C. Limón, J. Delgado-Jarana, and M. Rey. 1998. Glucanolytic and other enzymes and their genes, p.101-127. In C.P. Kubicek and G.E. Harman (eds.), Trichoderma and Gliocladium, Vol 2. Taylor and Francis Ltd., London, UK
  3. Beri, R.K. and G. Turner. 1987. Transformation of Penicillium chrysogenum using the Aspergillus nidulans amdS gene as a dominant selective marker. Curr. Genet. 11, 639-641 https://doi.org/10.1007/BF00393928
  4. Cantoral, J.M., B. Díez, J.L. Barredo, E. Alvarez, and J.F. Martín. 1987. High frequency transformation of Penicillium chrysogenum. Bio. Technol. 5, 494-497 https://doi.org/10.1038/nbt0587-494
  5. De Groot, M.J., P. Bundock, P.J. Hooykaas, and A.G. Beijersbergen. 1998. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat. Biotechnol. 16, 839-842 https://doi.org/10.1038/nbt0998-839
  6. Delgado-Jarana, J., A.M. Rincon, and T. Benitez. 2002. Aspartyl protease from Trichoderma harzianum CECT 2431: cloning and characterization. Microbiology 148, 1305-1315 https://doi.org/10.1099/00221287-148-5-1305
  7. Gutierrez, S., J. Velasco, A.T. Marcos, F.J. Fernández, F. Fierro, J.L. Barredo, B. Diez, and J.F. Martin. 1997. Expression of the cefG is limiting for cephalosporin biosynthesis in Acremonium chrysogenum. Appl. Microbiol. Biotechnol. 48, 606-614 https://doi.org/10.1007/s002530051103
  8. Harman, G.E. 2000. Myths and dogmas of biocontrol. Changes in percpetions derived from research on Trichoderma harzianum T-22. Plant Dis. 84, 3777-393
  9. Harman, G.E., C.R. Howell, A. Viterbo, I. Chet, and M. Lorito. 2004. Trichoderma species opportunistic, avirulent plant symbionts. Nature Rev. 2, 43-56 https://doi.org/10.1038/nrmicro797
  10. Hazell, B.W., V.S. Teo, J.R. Bradner, P.L. Bergquist, and K.M. Nevalainen. 2000. Rapid transformation of high cellulaseproducing mutant strains of Trichoderma reesei by microprojectile bombarment. Lett. Appl. Microbiol. 30, 282-286 https://doi.org/10.1046/j.1472-765x.2000.00715.x
  11. Hermosa, M.R., I. Grondona, E.A. Iturriaga, J.M. Diaz- Mínguez, C. Castro, E. Monte, and I. Garcia-Acha. 2000. Molecular characterization and identification of biocontrol isolates of Trichoderma spp. Appl. Environ. Microbiol. 66, 1890-1898 https://doi.org/10.1128/AEM.66.5.1890-1898.2000
  12. Herrera-Estrella, A., G.H. Goldman, and M. Van Montagu. 1990. High-efficiency transformation system for the biocontrol agents, Trichoderma spp. Mol. Microbiol. 4, 839-843 https://doi.org/10.1111/j.1365-2958.1990.tb00654.x
  13. Hinnen, A., J.B. Hicks, and G.R. Fink. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. USA 75, 1929-1933
  14. Hooykaas, P.J.J., C. Roobol, and R.A. Schilperoort. 1979. Regulation of the transfer of Ti-plasmids of Agrobacterium tumefaciens. J. Gen. Microbiol. 110, 99-109 https://doi.org/10.1099/00221287-110-1-99
  15. Howell, C.R. 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts. Plant Dis. 87, 4-10 https://doi.org/10.1094/PDIS.2003.87.1.4
  16. Lazo, G.R., P.A. Stein, and R.A. Ludwig. 1991. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9, 963-967 https://doi.org/10.1038/nbt1091-963
  17. Lorito, M., C.K. Hayes, A. Di Pietro, and G.E. Harman. 1993. Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA. Curr. Genet. 24, 349-356 https://doi.org/10.1007/BF00336788
  18. Lorito, M. 1998. Chitinolytic enzymes and their genes, p.73- 79. In C.P. Kubicek and G.E. Harman (eds.), Trichoderma and Gliocladium, vol 2. Taylor and Francis Ltd., London, UK
  19. Margollez-Clark, E., C.K. Hayes, G.E. Harman, and M. Penttila. 1996. Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei. Appl. Environ. Microbiol. 62, 2145-2151
  20. Mishra, N.C. 1985. Gene transfer in fungi. Adv. Genet. 23, 73-178 https://doi.org/10.1016/S0065-2660(08)60512-X
  21. Mohr, G. and K. Esser. 1990. Improved transformation frequency and heterologous promoter recognition in Aspergillus niger. Appl. Microbiol. Biotechnol. 34, 63-70
  22. Monte, E. 2001. Editorial paper: Understanding Trichoderma: Between agricultural biotechnology and microbial ecology. Int. Microbiol. 4, 1-4
  23. Mozo, T. and P.J. Hooykaas. 1991. Electroporation of megaplasmids into Agrobacterium. Plant Mol. Biol. 16, 917-918 https://doi.org/10.1007/BF00015085
  24. Penttila, M., H. Nevalainen, M. Ratto, E. Salminen, and J. Knowles. 1987. A versatile transformation system for the celluylolytic filamentous fungus Trichoderma reesei. Gene 61, 155-164 https://doi.org/10.1016/0378-1119(87)90110-7
  25. Punt, P.J., R.P. Oliver, M.A. Dingemanse, P.H. Pouwels, and C.A.M.J.J. van den Hondel. 1987. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56, 117-124 https://doi.org/10.1016/0378-1119(87)90164-8
  26. Queener, S.W., T.D. Ingolia, P.L. Skatrud, J.L. Chapman, and K.R. Kaster. 1985. A system for genetic transformation of Cephalosporium acremonium. Microbiology 468-472
  27. Ramboseck, J.A. and J. Leach. 1987. Recombinant DNA in filamentous fungi: progress and prospects. Crit. Rev. Biotechnol. 6, 357-93 https://doi.org/10.3109/07388558709089387
  28. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA
  29. Sanchez-Torres, P., R. Gonzalez, J.A. Perez-Gonzalez, L. Gonzalez-Candelas, and D. Ramon. 1994. Development of a transformation system for Trichoderma longibrachiatum and its use for constructing multicopy transformants for the egl1 gene. Appl. Microbiol. Biotechnol. 41, 440-446
  30. Sivan, A., T.E. Stasz, M. Hemmat, C.K. Hayes, and G.E. Harman, 1992. Transformation of Trichoderma spp. with plasmids coferring hygromycin B resistance. Mycologia 84, 687-694 https://doi.org/10.2307/3760378
  31. Sivasithamparam, K.Y. and E.L. Ghisalberti. 1998. Secondary metabolism in Trichoderma and Gliocladium, p.139-191. In C.P. Kubicek and G.E. Harman (eds.), Trichoderma and Gliocladium, Vol 2. Taylor and Francis Ltd., London, UK
  32. Specht, C.A., C.C. DiRusso, C.P. Novotny, and R.C. Ullrich 1982. A method for extracting high-molecular weight deoxyribonucleic acid from fungi. Anal. Biochem. 119, 158-163 https://doi.org/10.1016/0003-2697(82)90680-7
  33. Teo, V.S., P.L. Bergquist, and K.M. Nevalainen. 2002. Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta adaptor system. J. Microbiol. Methods 51, 393-399 https://doi.org/10.1016/S0167-7012(02)00126-4
  34. Vicente, M.F., A. Cabello, A. Platas, M.T. Basilio, S. Diez, and R.A. Dreikorn. 2001. Antimicrobial activity of ergokonin A from Trichoderma longibrachiatum. J. Appl. Microbiol. 91, 806-813 https://doi.org/10.1046/j.1365-2672.2001.01447.x
  35. Vizcaino, J.A., L. Sanz, A. Basilio, F. Vicente, S. Gutierrez, M.R. Hermosa, and E. y Monte. 2005. Screening of antiantimicrobial activities in Trichoderma isolates representing three Trichoderma sections. Mycol. Res. 109, 1397-1406 https://doi.org/10.1017/S0953756205003898
  36. Vizcaino, J.A., R.E. Cardoza, M. Hauser, M.R. Hermosa, M. Rey, A. Llobell, J.M. Becker, S. Gutierrez, and E. Monte. 2006. ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum. Fungal Genet. Biol. 43, 234-246 https://doi.org/10.1016/j.fgb.2005.12.003
  37. Yelton, M.M., J.E. Hamer, and W.E. Timberlake. 1987. Transformation of Aspergillus nidulans by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81, 1470-1474
  38. Zeilinger, S. 2004. Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation. Curr. Genet. 45, 54-60 https://doi.org/10.1007/s00294-003-0454-8