title: Systematic analysis of Zn2Cys6 transcription factors required for development and pathogenicity by high-throughput gene knockout in the rice blast fungus.
abstract: Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-Escherichia-Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn(2)Cys(6) transcription factor (TF) genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn(2)Cys(6)TF genes. A large variation in biological functions of Zn(2)Cys(6)TF genes was observed under the conditions tested. Sixty-one of 104 Zn(2)Cys(6) TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae.
authors.affiliation: School of Life Sciences Zhejiang University, Hangzhou, Zhejiang Province, China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Lu
authors.affiliation: Biotechnology Institute, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Cao
authors.affiliation: School of Life Sciences Zhejiang University, Hangzhou, Zhejiang Province, China.
authors.forename: Lilin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: School of Life Sciences Zhejiang University, Hangzhou, Zhejiang Province, China.
authors.forename: Pengyun
authors.initials: P
authors.lastname: Huang
authors.affiliation: Biotechnology Institute, Zhejiang University, Hangzhou, Zhejiang Province, China; China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou, Henan Province, China.
authors.forename: Fucheng
authors.initials: F
authors.lastname: Lin
mesh_terms: D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D055786:Gene Knockout Techniques; D020082:Magnaporthe / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014774:Virulence / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D014157:Transcription Factors