基因CfATG6和CfATG14参与调控果生刺盘孢细胞自噬和致病力

CfATG6 and CfATG14 regulate the autophagy and pathogenicity of Colletotrichum fructicola

摘要:

【目的】炭疽病是油茶的主要病害，由刺盘孢属的多种真菌引起，其中果生刺盘孢分布范围最广、分离率最高，是油茶炭疽病的主要致病菌。研究自噬相关蛋白CfAtg6和CfAtg14的生物学功能，为进一步揭示果生刺盘孢通过细胞自噬调控致病的分子机制，并为油茶炭疽病的防治提供理论基础。【方法】根据同源重组原理，通过聚乙二醇(polyethylene glycol, PEG)介导的方法，在果生刺盘孢中敲除基因CfATG6和CfATG14，并进一步获得回补菌株ΔCfatg6-C和ΔCfatg14-C。【结果】酵母双杂交试验结果显示，果生刺盘孢蛋白CfAtg6和CfAtg14可能存在互作关系。生物学表型测定结果表明，相较于野生型和回补菌株，突变体ΔCfatg6和ΔCfatg14均表现出营养生长速率显著减慢，附着胞形成率分别只有野生型的5%和18%；突变体ΔCfatg6和ΔCfatg14致病力均极显著减弱，造成的油茶叶片病斑面积少于野生型和回补菌株的1/3；CfATG6和CfATG14基因缺失突变体均丧失转运和降解CfAtg8蛋白的能力，并对细胞壁胁迫更敏感。突变体ΔCfatg6的分生孢子产量显著降低，仅为野生型的20%左右；氧化胁迫试验结果表明，相较于野生型和回补菌株，过氧化氢对突变体的生长抑制率升高10%左右。内质网压力胁迫试验表明，ΔCfatg14对二硫苏糖醇抑制率升高5%以上。【结论】自噬相关基因CfATG6和CfATG14参与调控了果生刺盘孢生长发育、细胞自噬和致病力。

Abstract:

[Objective] Anthracnose is a major disease attacking Camellia oleifera plants. Colletotrichum fructicola with a wide distribution scope and a high isolation rate is the major pathogen causing anthracnose in C. oleifera. This study explored the roles of autophagy-related proteins CfAtg6 and CfAtg14 and the molecular mechanism for the pathogenicity of C. fructicola, aiming to provide a theoretical basis for the prevention and control of anthracnose in C. oleifera. [Methods] The homologous recombination principle and polyethylene glycol (PEG)-mediated transformation method were employed to construct the gene-deleted strains ΔCfATG6 and ΔCfATG14 and the complemented strains ΔCfatg6-C and ΔCfatg14-C. [Results] The yeast two-hybrid assay results showed that ΔCfatg6 and ΔCfatg14 might interact with each other. Compared with the wild type and complemented strains, ΔCfatg6 and ΔCfatg14 demonstrated significantly slow vegetative growth, and their appressorium formation rates were only 5% and 18% that of the wide type. In addition, ΔCfatg6 and ΔCfatg14 showed significantly weakened pathogenicity, causing the lesion areas only 1/3 of the wild type and complemented strains on C. oleifera leaves. In addition, ΔCfatg6 and ΔCfatg14 lost the ability of transporting and degrading CfAtg8 protein and became more sensitive to the cell wall stress. The conidium production of ΔCfatg6 decreased significantly, being only 20% that of wild type. The inhibition rate of hydrogen peroxide on the growth of the deleted strains was 10% higher than those on the wild type and complemented strains. ΔCfatg14 showed increased sensitivity to dithiothreitol stress. [Conclusion] The autophagy-related genes CfATG6 and CfATG14 are involved in the regulation of the growth, autophagy, and pathogenicity of C. fructicola.