大丽轮枝菌效应蛋白VdSRP2的功能分析

为明确效应蛋白VdSRP2在大丽轮枝菌Verticillium dahliae中的生物学功能，从大丽轮枝菌落叶型菌株V592中克隆VdSRP2基因并进行生物信息学分析，利用酵母转化酶分泌系统对其信号肽活性进行测定，采用实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)技术分析VdSRP2基因在大丽轮枝菌中的表达模式，并以V592菌株为材料获得VdSRP2基因的敲除突变体和过表达体菌株，通过表型分析和致病性测定确定VdSRP2基因的生物学功能。结果显示，VdSRP2基因编码232个氨基酸，含有5个半胱氨酸残基，N-端信号肽具有分泌活性，为真菌的典型效应蛋白；VdSRP2基因主要在大丽轮枝菌菌丝和微菌核中表达，其中经棉花根系诱导培养24 h时表达量最高；与野生型菌株V592相比，VdSRP2基因敲除导致大丽轮枝菌的产孢量和孢子萌发率显著下降，不能形成微菌核，对棉花的致病力明显减弱；但VdSRP2基因敲除不影响大丽轮枝菌的穿透能力；VdSRP2基因在本氏烟和棉花叶片瞬时表达不会诱导细胞死亡。表明VdSRP2是大丽轮枝菌微菌核形成必需...

Functional analysis of the effector protein VdSRP2 in fungal plant pathogen Verticillium dahliae

To determine the function of the secreted protein VdSRP2 in fungal plant pathogen Verticillium dahliae, the VdSRP2 gene was cloned from the virulent defoliating V. dahliae isolate V592 from cotton and bioinformatic analysis of its amino acid sequence was performed using online software. The secretory function of the putative N-terminal signal peptide of the VdSRP2 protein was tested using the yeast invertase secretion assay. The expression pattern of the VdSRP2 gene were analyzed by real-time fluorescence quantitative PCR (qPCR). The biological functions of the VdSRP2 gene was investigated using the knockdown mutants and overexpression strains of the VdSRP2 gene generated from the V592 strain. The results showed that the VdSRP2 gene encodes 232 amino acids containing five cysteine residues, and N-terminal signal peptide had secretory function, indicating VdSRP2 was a conventional fungal effector protein. The VdSRP2 gene was mainly expressed in mycelia and microsclerotia, and the highest expression was reached at 24 h post-induction by cotton roots. Compared with the wild-type strain V592, VdSRP2 gene knockout resulted in significantly decreased conidial production and germination rate, complete loss of microscletotial formation and reduced virulence to cotton. However, VdSRP2 gene knockout did not affect the penetration ability of V. dahliae, and transient expression of the VdSRP2 gene in the leaves of tobacco and cotton did not induce cell death. In conclusion, the ^VdSRP2 gene is required for the microsclerotial formation, and plays an important role in conidial production and germination, and virulence in V. dahliae, but VdSRP2 protein could not act as an elicitor to activate host resistance.