苹果炭疽叶枯病菌致病相关基因CgNVF1的功能初步分析

苹果炭疽叶枯病是主要由胶孢炭疽菌(Colletotrichum gloeosporioides)引起的一种苹果重要叶部病害,严重威胁着苹果树的生长。本研究从构建的苹果炭疽叶枯病菌T-DNA突变体库中筛选获得一株致病力缺失的突变菌株A3083,采用hiTAIL-PCR方法克隆了该突变体T-DNA插入位点的右翼序列;通过与胶孢炭疽菌基因组序列比对分析,发现T-DNA插入位点位于1个预测的CGGC5_9603基因内,并将该基因命名为CgNVF1。CgNVF1基因全长2252 bp,含有2个内含子,编码709个氨基酸。CgNVF1定位于细胞质,在苹果炭疽叶枯病菌菌丝、分生孢子和附着胞中均有表达。通过构建CgNVF1敲除菌株和CgNVF1互补菌株,并结合表型分析,证实CgNVF1基因在苹果炭疽叶枯病菌附着胞形成及致病中具有重要的作用。

Functional analysis of the pathogenicity-related gene CgNVF1 in Colletotrichum gloeosporioides

Glomerella leaf spot of apple (GLSA) is mainly caused by Colletotrichum gloeosporioides, which has become a major apple leaf disease and threatens apple growth. A mutant strain A3083 that lost pathogenicity was obtained by screening the T-DNA insertional mutant library. Southern blot analysis indicated that A3083 contains a single copy of T-DNA. The T-DNA right flanking sequence of A3083 obtained by hiTAIL-PCR was aligned with the whole genome of C. gloeosporioides, which showed that the T-DNA is located in the coding region of a predicted gene CGGC5_9603. This gene was designated as CgNVF1, which is 2 252 bp in length, contains 2 introns and encodes 709 amino acids. Fluorescent signal showed that the fused protein CgNVF1-eGFP is distributed to the cytoplasm and expressed in all of the mycelium, conidium and appressorium. Phenotypic analysis of the CgNVF1 knockout mutant and the complementation strain indicated CgNVF1 is required for appressorium formation and pathogenicity in C. gloeosporioides.