Identification of soluble secreted proteins from appressoria of Colletotrichum higginsianum by analysis of expressed sequence tags
Identification of soluble secreted proteins from appressoria of Colletotrichum higginsianum by analysis of expressed sequence tags
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DOI：
10.1099/mic.0.2007/014944-0
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发表时间：
2008-04-01
期刊：
影响因子：
2.8
通讯作者：
O'Connell, Richard
中科院分区：
文献类型：
--
作者：
Kleemann, Jochen;Takahara, Hiroyuki;O'Connell, Richard
半活体营养型子囊菌Colletotrichum higginsianum在芸苔属作物和模式植物拟南芥上引起炭疽病。黑变附着胞穿透寄主表皮和细胞壁，在活表皮细胞内形成特化的活体营养菌丝。为了鉴定附着胞分泌的可能作为毒力效应子的蛋白质，从体外形成的成熟附着胞制备cDNA文库。对980个克隆进行双向测序，产生了1442个高质量的表达序列标签（EST），包括518个独特的序列。BLASTx分析显示，其中353个（68%）与NCBI非冗余蛋白质数据库中的条目具有显著相似性，其中49个也与实验验证的真菌致病性基因同源。从独特序列从头开始预测ORF，并使用SignalP筛选潜在的信号肽。预计有53个独特序列（10%）编码进入分泌途径的蛋白质，其中26个可能是可溶性分泌蛋白。对于这些选定的子集，RT-PCR显示，7个基因，编码分泌蛋白的未知功能，包括两个Colletotrichum特异性基因，上调附着胞和表达早期在植物感染，因此代表候选效应。
The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis. Melanized appressoria pierce the host cuticle and cell wall to form specialized biotrophic hyphae inside living epidermal cells. To identify proteins secreted by appressoria that may function as virulence effectors, a cDNA library was prepared from mature appressoria formed in vitro. Bidirectional sequencing of 980 clones generated 1442 high-quality expressed sequence tags (ESTs), comprising 518 unique sequences. BLASTx analysis showed that 353 (68%) of these had significant similarity to entries in the NCBl non-redundant protein database, of which 49 were also homologous to experimentally verified fungal pathogenicity genes, ORFs were predicted ab initio from the unique sequences and screened for potential signal peptides using SignalP. Fifty-three unique sequences (10 %) were predicted to encode proteins entering the secretory pathway, of which 26 were likely to be soluble secreted proteins. For a selected subset of these, RT-PCR showed that seven genes that encode secreted proteins of unknown function, including two Colletotrichum-specific genes, are upregulated in appressoria and expressed early during plant infection, and therefore represent candidate effectors.