title: A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii.
abstract: Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the "model" fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of M. robertsii.
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Xu
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Qian
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Xiaoxuan
authors.initials: X
authors.lastname: Chen
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Liu
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Guohong
authors.initials: G
authors.lastname: Zeng
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Fang
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics; D059766:DNA End-Joining Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D016681:Genome, Fungal; D005838:Genotype; D000072200:Ku Autoantigen; D052981:Metarhizium / Q000235:genetics* / Q000472:pathogenicity; D009492:Neurospora crassa / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013172:Spores, Fungal / Q000235:genetics
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D000072200:Ku Autoantigen