title: Development of a high-efficiency gene knockout system for Pochonia chlamydosporia.
abstract: The nematophagous fungus Pochonia chlamydosporia, which belongs to the family Clavicipitaceae (Ascomycota: Pezizomycotina: Sordariomycetes: Hypocreales), is a promising biological control agent for root-knot and cyst nematodes. Its biocontrol effect has been confirmed by pot and field trials. The genome sequence of the fungus was completed recently; therefore, genome-wide functional analyses will identify its infection-associated genes. Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150μgml(-1) G418 sulfate, representing a new selectable marker for P. chlamydosporia. The neomycin-resistance gene (neo), which was amplified from the plasmid pKOV21, conferred G418-resistance on the fungus; therefore, it was chosen as the marker gene. We subsequently developed a gene knockout system for P. chlamydosporia using split-marker homologous recombination cassettes with resistance selection and protoplast transformation. The split-marker cassettes were developed using fusion PCR, and involved only two rounds of PCR. The final products comprised two linear constructs. Each construct contained a flanking region of the target gene and two thirds of the neo gene. Alkaline serine protease and chitinase were confirmed to be produced by P. chlamydosporia during infection of nematode eggs and could participate in lysis of the eggshell of nematode eggs. Here, we knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). We obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088) and 15% (VFPPC_06535). This efficient and convenient technique will accelerate functional genomic studies in P. chlamydosporia.
authors.affiliation: College of Plant Protection, Hunan Agricultural University, Changsha 410128, China; Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China.
authors.forename: Baoming
authors.initials: B
authors.lastname: Shen
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China.
authors.forename: Jiling
authors.initials: J
authors.lastname: Xiao
authors.affiliation: College of Plant Protection, Hunan Agricultural University, Changsha 410128, China.
authors.forename: Liangying
authors.initials: L
authors.lastname: Dai
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China; Guangdong Academy of Agricultural Science, Guangzhou 510640, China.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China.
authors.forename: Zhenchuan
authors.initials: Z
authors.lastname: Mao
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China.
authors.forename: Runmao
authors.initials: R
authors.lastname: Lin
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China.
authors.forename: Yurong
authors.initials: Y
authors.lastname: Yao
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, China. Electronic address: xiebingyan@caas.cn.
authors.forename: Bingyan
authors.initials: B
authors.lastname: Xie
mesh_terms: D005659:Fungicides, Industrial / Q000494:pharmacology; D055786:Gene Knockout Techniques; D006999:Hypocreales / Q000187:drug effects / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D011523:Protoplasts; D014162:Transfection / Q000379:methods; D014170:Transformation, Genetic
chemical_list: D005659:Fungicides, Industrial
keywords: Homologous gene knockout; Pochonia chlamydosporia; Recombination; Split-marker