多主棒孢CcCas2基因纯合敲除突变体构建及致病功能  

Construction of homozygous deletion mutants and pathogenic function of the gene CcCas2 in Corynespora cassiicola parasitizing cucumber

作　　者：侯梦圆 武海燕[2] 何雨芯 郭雅双 耿月华[1] 臧睿[1] 张猛[1] 徐超 HOU Mengyuan;WU Haiyan;HE Yuxin;GUO Yashuang;GENG Yuehua;ZANG Rui;ZHANG Meng;XU Chao(College of Plant Protection,Henan Agricultural University,Zhengzhou 450046,Henan,China;Analytical Instrument Center,Henan Agricultural University,Zhengzhou 450002,Henan,China)

机构地区：[1]河南农业大学植物保护学院,河南郑州450046 [2]河南农业大学测试中心,河南郑州450002

出　　处：《菌物学报》2023年第8期1701-1717,共17页Mycosystema

基　　金：河南农业大学青年英才项目(30500603)。

摘　　要：多主棒孢引起的棒孢叶斑病为我国黄瓜生产上重要的新兴流行病害。多主棒孢寄主广泛,其cassiicolin(Cas)毒素是多主棒孢侵染橡胶早期的关键致病因子,但在黄瓜/多主棒孢病害系统中的毒力功能目前尚不明确。本研究从来自黄瓜多主棒孢HG3菌株的基因组序列中鉴定到单拷贝的Cas2亚型基因,命名为CcCas2。在敲除该基因的过程中,发现CcCas2缺失突变体均为杂合,荧光染色试验进一步证实多主棒孢是多核真菌。对杂合突变体进行潮霉素筛选并单孢纯化,获得了纯合的CcCas2缺失突变体菌株(检出率≥20%)。致病力测定显示,CcCas2纯合突变体和野生型菌株接种黄瓜叶片4 d后的发病程度相当。通过转录组数据、RT-PCR和qRT-PCR分析均发现CcCas2在转录水平上受到了强烈地抑制。本研究明确了多主棒孢CcCas2基因由于在转录水平受到抑制而对黄瓜没有毒力;所提出的多主棒孢纯合突变体高效获取方法为该菌进一步的功能基因研究奠定了基础。Corynespora leaf spot caused by Corynespora cassiicola has become an important emerging epidemic disease in the cucumber production of China.C.cassiicola has a wide host range,and its cassiicolin(Cas)toxin is proved to be a key pathogenic factor in the early infection phase of C.cassiicola on rubber tree.However,virulence function of this toxin in the cucumber/C.cassiicola pathosystem remains unclear.In this study,a single-copy Cas2 gene,named CcCas2,was identified in the genomic sequences of C.cassiicola strain HG3 isolated from cucumber.During the process of the gene knockout,it was found that the deletion mutants of CcCas2 were all heterozygous,and subsequent fluorescence staining further confirmed that C.cassiicola is a multinucleate fungus.The heterozygous mutants were used to perform single-spore purification on PDA media containing hygromycin B,thus the homozygous mutants of CcCas2 were screened out(detection rates≥20%).The pathogenicity tests showed that there was no significant difference in disease severity between the CcCas2 homozygous mutant and wild-type strain on the 4th day post-inoculation on cucumber leaves.Analyses of transcriptome data,RT-PCR and qRT-PCR showed that the CcCas2 gene was strongly suppressed at the transcriptional level.Our research certifies that due to being transcriptionally inhibited,the CcCas2 gene of C.cassiicola has no virulence to cucumber.The proposed high-efficient way to achieve C.cassiicola homozygous mutants lays the foundation for further research on functional genes of this fungus.

关 键 词：多主棒孢 效应蛋白 多核丝状真菌 杂合敲除突变体 基因表达 

分 类 号：S436.421.1[农业科学—农业昆虫与害虫防治]