摘要：

希金斯炭疽菌是一种半活体营养型真菌,能够引起十字花科炭疽病,对华南地区的菜心产业造成了严重危害。病原菌分泌的效应子能够帮助病原菌侵入寄主植物,在病原菌与植物互作过程中起到重要作用。为探究希金斯炭疽菌效应子在十字花科炭疽病致病过程中的作用。基于华南农业大学热带亚热带真菌研究室前期的研究结果,筛选得到一个候选效应子ChEP085,该效应子在N端含有一段21个氨基酸的信号肽。为明确该效应子的功能,以希金斯炭疽菌侵染拟南芥Col-0的cDNA为模板,利用qRT-PCR技术测定了ChEP085基因的表达情况,发现ChEP085基因在侵染24 h后有最大表达量。利用农杆菌介导马铃薯X病毒烟草瞬时表达体系对ChEP085基因进行了验证,结果表明:该基因能够稳定诱导烟草细胞坏死。将ChEP085基因构建到增强型绿色荧光蛋白载体pBinceGFP上,在烟草上瞬时表达ChEP085基因进行亚细胞定位,发现该效应子同时定位于细胞质和细胞膜上。删除位于N端的信号肽后,瞬时表达的效应子ChEP085在细胞核、细胞膜和细胞质中均有分布,说明信号肽影响ChEP085的定位。最后利用同源重组技术敲除ChEP085基因,发现该基因的缺失对希金斯炭疽菌的菌落形态及菌丝生长速度均无明显影响,但会导致希金斯炭疽菌的孢子产量降低,致病力明显减弱,说明ChEP085在希金斯炭疽菌产孢及致病过程中均发挥重要作用。

关键词: 希金斯炭疽菌, 效应子, ChEP085基因, 致病力

Abstract:

Colletotrichum higginsianum is a hemibiotrophic fungus that causes cruciferous anthracnose, a serious damage to the flowering Chinese cabbage industry in South China. Effectors secreted by pathogens can help pathogens invade host plants and play an important role in the interaction between pathogens and plants. To investigate the role of C. higginsianum effectors in the pathogenesis of cruciferous anthracnose. Based on previous research results from the Laboratory of Tropical and Subtropical Fungi, South China Agricultural University, a potential effector ChEP085 with a 21 amino acid signal peptide at the N-terminus was screened out. To clarify the function of this effector, the cDNA of Arabidopsis thaliana Col-0 infected by C. higginsianum was used as the template, and the expression of ChEP085 gene was determined by qRT-PCR. It was found that the gene ChEP085 had the maximum expression after 24 h of infection. The gene ChEP085 was verified by Agrobacterium tumefaciens mediated Potato virus X(PVX) tobacco transient expression system. The results showed that the gene could stably induce tobacco cell necrosis. The gene ChEP085 was constructed into the enhanced green fluorescent protein vector pBinceGFP and transiently expressed on tobacco leaves for subcellular localization. It was found that the gene was localized in cytoplasm and cell membrane. After deletion of the signal peptide located at the N terminus, the transiently expressed effector ChEP085 was distributed in the nucleus, cell membrane and cytoplasm, indicating that the signal peptide affects the localization of ChEP085. Finally, the gene ChEP085 was knockout by homologous recombination technology, and it was found that the deletion of this gene had no significant effects on the colony morphology and mycelial growth rate of C. higginsianum, but it would lead to the reduction of conidiation and significantly weakened the pathogenicity of C. higginsianum, indicating that ChEP085 play an important role in the conidiation and pathogenesis of C. higginsianum.

Key words: Colletotrichum higginsianum, Effector, ChEP085 gene, Pathogenicity