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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 .

A High-Throughput Gene Disruption Methodology for the Entomopathogenic Fungus Metarhizium robertsii