The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Generation of an Arginine Auxotrophic Mutant of Colletotrichum acutatum as a Recipient Host for Insertional Mutagenesis

  • Kim, Hee-Kyoung (Department of Medical Biotechnology, Soonchunhyang University) ;
  • Lee, Sun-Hee (Department of Medical Biotechnology, Soonchunhyang University) ;
  • Kim, Heung-Tae (Department of Plant Medicine, Chungbuk National University) ;
  • Yun, Sung-Hwan (Department of Medical Biotechnology, Soonchunhyang University)
  • Published : 2009.09.30
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Abstract

Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding N-acetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.

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References

  1. Antal, Z., Manczinger, L. and Ferenczy, L. 1997. Transformation of a mycoparasitic Trichoderma harzianum strain with the argB gene of Aspergillus nidulans. Biotechnol. Tech. 11:205-208 https://doi.org/10.1023/A:1018417917255
  2. Brooker, N. L., Mischke, C. F., Patterson, C. D., Mischke, S., Bruckart, W. L. and Lydon, J. 1996. Pathogenicity of bar-transformed Colletotrichum gloeosporioides f. sp. aeschynomene. BioI. Control 7:159-166 https://doi.org/10.1006/bcon.1996.0079
  3. Chen, H. Q., Dekkeres, K. L., Rollins, J. A., Ko, N. J., Timmer, L. W. and Chung, K. R. 2005. A gene with domains related to transcription regulation is required for pathogenicity in Colletotrichum acutatum causing Key lime anthracnose. Mol. Plant Pathol. 6:513-525 https://doi.org/10.1111/j.1364-3703.2005.00300.x
  4. Chung, K. R., Shilts, T., Li, W. and Timmer, L. W. 2002. Engineering a genetic transformation system for Colletotrichum acutatum, the causal fungus of lime anthracnose and postbloom fruit drop of citrus. FEMS Microbiol. Lett. 213:33-39 https://doi.org/10.1111/j.1574-6968.2002.tb11282.x
  5. Chung, W., Ishii, H., Nishimura, K., Fukaya, M. and Yano, K. 2006. Fungicide sensitivity and phylogenetic relationship of anthracnose fungi isolated from various fruit crops in Japan. Plant Dis. 90:506-512 https://doi.org/10.1094/PD-90-0506
  6. Dickman, M. B. 1988. Whole cell transformation of the alfalfa fungal pathogen Colletotrichum trifolii. Curr. Genet. 14:241-246 https://doi.org/10.1007/BF00376744
  7. Epstein, L., Lusnak, K. and Kaur, S. 1998. Transformation-mediated developmental mutants of Glomerella graminicola (Colletotrichum graminicola). Fungal Genet. Biol. 23:189-203 https://doi.org/10.1006/fgbi.1997.1029
  8. Fincham, J. R. S. 1989. Transformation in fungi. Microbiol. Rev. 53:148-170
  9. Fu, J., Hettler, E. and Wickes, B. L. 2006. Split marker transformation increases homologous integration frequency in Cryptococcus neoformans. Fungal Genet. Biol. 43:200-212 https://doi.org/10.1016/j.fgb.2005.09.007
  10. Hahm, Y. T. and Batt, C. A. 1988. Genetic transformation of an argB mutant of Aspergillus oryzae. Appl. Environ. Microbiol. 54:1610-1611
  11. Han, Y.-K., Lee, T., Han, K.-H., Yun, S.-H. and Lee, Y.-W 2004. Functional analysis of the homo serine O-acetyltransferase gene and its identification as a selectable marker in Gibberella zeae. Curr. Genet. 46:205-212 https://doi.org/10.1007/s00294-004-0528-2
  12. Horowitz, S., Freeman, S. and Sharon, A. 2002. Use of green fluorescent protein-transgenic strains to study pathogenic and nonpathogenic lifestyles in Colletotrichum acutatum. Phytopathology 92:743-749 https://doi.org/10.1094/PHYTO.2002.92.7.743
  13. John, M. A. and Peberdy, J. F. 1984. Transformation of Aspergillus nidulans using the argB gene. Enz. Microb. Technol. 6:386-389 https://doi.org/10.1016/0141-0229(84)90011-5
  14. Johnstone, I. L., Hughes, S. G. and Clutterbuck, A. J. 1985. Cloning an Aspergillus nidulans developmental gene by transformation. EMBO J. 4:1307-1311
  15. Karunakaran, M., Nair, V., Rho, H.-S., Choi, J., Kim, S. and Lee, Y.-H. 2008. Agrobacterium tumefaciens-mediated transformation in Colletotrichum falcatum and C. acutatum. J. Microbiol. Biotechnol. 18:234-241
  16. Kim, J.-E., Myong, K., Shim, W.-B., Yun, S.-H. and Lee, Y. W. 2007. Functional characterization of acetylglutamate synthase and phosphoribosylamine-glycine ligase genes in Gibberella zeae. Curr. Genet. 51:99-108 https://doi.org/10.1007/s00294-006-0110-1
  17. Kim, J. T., Park, S.-Y., Choi, W., Lee, Y.-H. and Kim, H. T. 2008. Characterization of Colletotrichum isolates causing anthracnose of pepper in Korea. Plant Pathol. J. 24:17-23 https://doi.org/10.5423/PPJ.2008.24.1.017
  18. Lee, T., Oh, D. W., Kim, H. S., Lee, J., Kim, Y. H., Yun, S. H. and Lee, Y. W. 2001. Identification of deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae by using PCR. Appl. Environ. Microbiol. 67:2966-2972 https://doi.org/10.1128/AEM.67.7.2966-2972.2001
  19. Manczingerx, L., Antal, Z. and Ferenczy, L. 1995. Isolation of uracil auxotrophic mutants of Trichoderma harzianum and their transformation with heterologous vectors. FEMS Microbiol. Lett. 130:159-162 https://doi.org/10.1111/j.1574-6968.1995.tb07713.x
  20. Nakaune, R. and Nakano, M. 2007. Benomyl resistance of Colletotrichum acutatum is caused by enhanced expression of betatubulin 1 gene regulated by putative leucine zipper protein CaBEN1. Fungal Genet. Biol. 44:1324-1335 https://doi.org/10.1016/j.fgb.2007.03.007
  21. Namiki, F., Matsunaga, M., Okuda, M., Inoue, I., Nishi, K., Fujita, Y. and Tsuge, T. 2001. Mutation of an arginine biosynthetic gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis. Mol. Plant-Microbe Interact. 14:580-584 https://doi.org/10.1094/MPMI.2001.14.4.580
  22. Park, K. S. and Kim, C. H. 1992. Identification, distribution and etiological characteristics of anthracnose fungi of red pepper in Korea. Korean J Plant Pathol. 8:61-69
  23. Parsons, K. A., Chumley, F. G. and Valent, B. 1987. Genetic transformation of the fungal pathogen responsible for rice blast disease. Proc. Natl. Acad. Sci. USA 84:4161-4165 https://doi.org/10.1073/pnas.84.12.4161
  24. Sambrook, J. and Russell, D. W. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA
  25. Sreenivasaprasad, S. and Talhinhas, P. 2005. Genotypic and phenotypic diversity in Colletotrichum acutatum, a cosmopolitan pathogen causing anthracnose on a wide range of hosts. Mol. Plant Pathol. 6:361-378 https://doi.org/10.1111/j.1364-3703.2005.00291.x
  26. Stabb, J. F. and Sundstrom, P. 2003. URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes. Trends Microbiol. 11:69-73 https://doi.org/10.1016/S0966-842X(02)00029-X
  27. Sunagawa, M. and Magae, Y. 2002. Transformation of the edible mushroom Pleurotus ostreatus by particle bombardment. FEMS Microbiol. Lett. 211:143-146 https://doi.org/10.1111/j.1574-6968.2002.tb11216.x
  28. Talhinhas, P., Muthumeenakshi, S., Neves-Martins, J., Oliveira, H. and Sreenivasaprasad, S. 2008. Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles. Mol. Biotechnol. 39:57-67 https://doi.org/10.1007/s12033-007-9028-1
  29. Venard, C. and Vaillancourt, L. 2007. Colonization of fiber cells by Colletotrichum graminicola in wounded maize stalks. Phytopathology 97:438-447 https://doi.org/10.1094/PHYTO-97-4-0438
  30. Ventura, L., Ramon, D. and Perez-Gonzalez, J. A. 1992. Isolation of an Aspergillus terreus mutant impaired in arginine biosynthesis and its complementation with the argB gene from Aspergillus nidulans. FEMS Microbiol. Lett. 99:187-192 https://doi.org/10.1111/j.1574-6968.1992.tb05564.x
  31. Xue, T., Nguyen, C. K., Romans, A., Kontoyiannis, D. P. and May, G. S. 2004. lsogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293. Arch. Microbiol. 182:346-353 https://doi.org/10.1007/s00203-004-0707-z
  32. You, B. J., Choquer, M. and Chung, K. R. 2007. The Colletotrichum acutatum gene encoding a putative pH-responsive transcription regulator is a key virulence determinant during fungal pathogenesis on citrus. Mol. Plant-Microbe Interact. 20:1149-1160 https://doi.org/10.1094/MPMI-20-9-1149