Shen Baoming, Xiao Jiling, Dai Liangying, Huang Yonghong, Mao Zhenchuan, Lin Runmao, Yao Yurong, Xie Bingyan
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.
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.
Microbiol Res. 2015 Jan;170:18-26. doi: 10.1016/j.micres.2014.10.001. Epub 2014 Oct 18.
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.
食线虫真菌厚垣孢普可尼亚菌属于麦角菌科（子囊菌门：粪壳菌纲：肉座菌目：虫草菌科），是一种很有前景的防治根结线虫和孢囊线虫的生防菌。其生防效果已通过盆栽试验和田间试验得到证实。该真菌的基因组序列最近已完成；因此，全基因组功能分析将鉴定其与感染相关的基因。基因敲除技术是研究基因功能的有用分子工具。然而，厚垣孢普可尼亚菌的培养物对多种高水平的真菌抑制剂具有抗性，这使得在这种真菌中应用基因敲除技术变得困难。幸运的是，我们发现野生厚垣孢普可尼亚菌菌株PC - 170不能在含有150μg/ml硫酸庆大霉素的培养基上生长，这代表了厚垣孢普可尼亚菌的一种新的选择标记。从质粒pKOV21中扩增得到的新霉素抗性基因（neo）赋予了该真菌对庆大霉素的抗性；因此，它被选为标记基因。随后，我们利用带有抗性选择和原生质体转化的分裂标记同源重组盒，为厚垣孢普可尼亚菌开发了一种基因敲除系统。分裂标记盒通过融合PCR构建，仅涉及两轮PCR。最终产物由两个线性构建体组成。每个构建体包含目标基因的侧翼区域和三分之二的neo基因。已证实厚垣孢普可尼亚菌在感染线虫卵期间会产生碱性丝氨酸蛋白酶和几丁质酶，并且它们可能参与线虫卵壳的裂解。在此，我们敲除了一个几丁质酶基因VFPPC_01099和两个蛋白酶基因（VFPPC_10088、VFPPC_06535）。每次转化后我们获得了大约100个疑似突变体。通过PCR筛选后，基因敲除的平均效率为13%：VFPPC_01099为11%，VFPPC_10088为13%，VFPPC_06535为15%。这种高效且便捷的技术将加速厚垣孢普可尼亚菌的功能基因组学研究。
