Transcriptional changes caused by cpst12 gene disruption in Cryphonectria parasitica

A putative homologue of the Saccharomyces cerevisiae Ste12 transcription factor was identified in a series of expressed sequence tag (EST)-based microarray analyses as being down regulated in strains of the chestnut blight fungus, Cryphonectria parasitica, infected by virulence-attenuating hypoviruses. Cloning of the corresponding gene, cpst12, confirmed a high level of similarity to Ste12 homologues of other filamentous fungi. Disruption of cpst12 resulted in no alterations in in vitro growth characteristics or colony morphology and an increase in the production of asexual spores, indicating that CpST12 is dispensable for vegetative growth and conidiation on artificial medium. However, the disruption mutants showed a very substantial reduction in virulence on chestnut tissue and a complete loss of female fertility, two symptoms normally conferred by hypovirus infection. Both virulence and female fertility were restored by complementation with the wild-type cpst12 gene. Analysis of transcriptional changes caused by cpst12 gene disruption with a custom C. parastica cDNA microaray chip identified 152 responsive genes. A significant number of these putative CpST12-regulated genes were also responsive to hypovirus infection. Thus, cpst12 encodes a cellular transcription factor, CpST12, that is down-regulated by hypovirus infection and required for female fertility, virulence and regulated expression of a subset of hypovirus responsive host genes. Keywords: Genetic modification Transcriptional profiles between cpst12 deletion mutant strain cpst-E1 and isogenic wild-type strain EP155 were compared in this experiment. Two sets of RNA samples with dye-swab (total of four hybridizations) were analyzed.

本研究在一系列基于表达序列标签（expressed sequence tag, EST）的基因芯片分析中，于被致病力衰减型低毒病毒感染的板栗疫病菌（*Cryphonectria parasitica*）菌株中，鉴定出酿酒酵母（*Saccharomyces cerevisiae*）Ste12转录因子（Ste12 transcription factor）的推定同源蛋白，且该蛋白在感染菌株中呈下调表达。对对应基因cpst12的克隆证实，其与其他丝状真菌的Ste12同源物具有高度序列相似性。敲除cpst12后，菌株的体外生长特性与菌落形态未发生明显改变，但其无性孢子产量有所提升，这表明CpST12对于人工培养基上的营养生长与产孢并非必需。然而，该cpst12敲除突变体在板栗组织上的致病力出现显著下降，且完全丧失雌性育性，而这两种表型正是低毒病毒感染通常所诱导的典型症状。通过转入野生型cpst12基因进行遗传互补实验，可完全恢复突变体的致病力与雌性育性。利用定制化的板栗疫病菌cDNA基因芯片（cDNA microarray chip）分析cpst12基因敲除所引发的转录组变化，共鉴定出152个差异应答基因。其中相当数量的推定CpST12调控基因，同时也对低毒病毒感染产生转录应答。综上，cpst12编码一种细胞转录因子CpST12，该因子可被低毒病毒感染下调表达，且对于雌性育性、致病力以及低毒病毒应答宿主基因子集的调控表达均为必需。关键词：遗传修饰；本实验比较了cpst12缺失突变株cpst-E1与同基因背景野生型菌株EP155的转录谱差异。本次分析共使用两组经染料互换（dye-swap）标记的RNA样本，总计完成4次杂交实验。