核盘菌双分病毒SsPV1抑制灰葡萄孢分生孢子萌发及其机制解析

Botrytis cinerea is an important plant pathogen fungus and its conidia play important roles in disease cycle. Inhibition of conidia germination (this stage includes spores inflation, germination, germ tube elongation, and conversion into mycelium) is one of effective control strategies for control gray mold disease. Previous research the partitivirus SsPV1 isolated from Sclerotinia sclerotiorum was successfully induced into B. cinerea strain KY-1. It is interesting that conidia germination of the SsPV1-infected strain KY-1V is inhibited and fails to conversion from germ tube to mycelium. In the present project, serial experiments were designed to decipher the mechanisms of conidia germination via SsPV1-Botrytis cinerea interaction system. Firstly, the biological features of conidia and germinated germ tube, ROS accumulation and nuclear division during conidia germination will be compared between strain KY-1 and KY-1V. Secondly, subcellular localization of SsPV1 in conidia and germ tube will be confirmed via FISH or immunogold techniques, and frequency of conidia carrying SsPV1 will be calculated. Thirdly, differential expression genes will be obtained based on levels of proteome, transcriptome and metabolome. Finally, combining the S. sclerotiorum proteins directly interacted with SsPV1, the specifically expressed new genes in conidia germination will be mined based on the bioinformatics methods, and an expected model of genes interaction net for conidia germination will be constructed. Our research results expect to elucidate the molecular mechanisms on inhibition of conidia germination by partitivirus SsPV1 in B. cinerea and will supply theory evidence for SsPV1 application to control gray mold disease.

灰葡萄孢是重要的植物病原真菌，分生孢子在其病害循环中发挥关键作用，抑制分生孢子萌发（包括分生孢子膨大、萌发、芽管延伸及芽管转化为菌丝体的过程）是防治灰霉病的有效途径之一。申请者前期将核盘菌双分病毒SsPV1转染至灰葡萄孢KY-1后，发现携带SsPV1的菌株KY-1V分生孢子萌发异常。本项目拟在此基础上，通过KY-1和KY-1V生物学性状比较，解析SsPV1灰葡萄孢的分生孢子结构、芽管形态、ROS积累和核分裂等孢子萌发相关性状的影响；明确SsPV1在分生孢子、芽管中的亚细胞定位及孢子携带SsPV1频率；对KY-1和KY-1V分生孢子萌发前后转录组、蛋白质组和代谢组数据关联分析，筛选差异表达基因；明确与SsPV1互作的寄主蛋白，鉴定新的孢子萌发相关基因，并构建分生孢子萌发相关信号途径的调控网络模型。研究期望阐明SsPV1抑制灰葡萄孢分生孢子萌发机制，为灰霉病绿色高效防治提供科学依据和理论指导。

灰葡萄孢（Botrytis cinerea）是一类世界性分布的死体营养型植物病原真菌，其寄主范围广泛，可以侵染1400多种植物，每年造成的经济损失高达100-1000亿美元。利用低毒相关真菌病毒防控灰霉病是一种潜在生物防治策略，亦是解析灰葡萄孢致病机理的材料。前期研究中，发现源自核盘菌的双分病毒SsPV1可以导致灰葡萄孢明显的弱毒特性。本研究项目中，采用病毒粒子转染的方法，成功将双分病毒SsPV1转染至菌株B05.10菌株中，证实SsPV1可以导致菌株B05.10生长速度减慢、菌落形态及致病力异常。采用转录组比较分析的策略，筛选出了受病毒SsPV1调控的灰葡萄孢基因，发现在SsPV1侵染的B05.10V菌株中，共有483个上调表达的基因，239个下调表达的基因；通过gene ontology和KEGG对差异基因进行富集分析，上调的基因中transporter 相关基因较多，而下调的基因主要包括核糖体的合成和次级代谢（如毒素等）。经GO组成分分析，发现多数差异的基因都定位在细胞膜上。利用PEG介导的原生质体转化的方法，获得了差异表达基因Bcin01g0493、Bcin03g01250、Bcin16g04990的敲除转化子，部分敲除转化子致病力明显降低。基于灰葡萄孢的转录组分析，采用遗传学策略，在灰葡萄孢的基因组中鉴定了两个编码枯草杆菌蛋白酶的基因Bcser1和Bcser2，并对他们进行了功能分析。它们都包含一个inhibitor I9结构域和一个peptidase S8结构域。Bcser1和Bcser2在菌核形成阶段以及菌丝侵染寄主叶片后期的表达量均升高，但Bcser1的上调倍数明显高于Bcser2。基因敲除和互补结果表明Bcser1缺失对灰葡萄孢生长和致病均无明显影响。但Bcser2敲除严重影响了灰葡萄孢的菌核形成和孢子形成，菌丝生长速度也有所降低。∆Bcser2和∆Bcser1/2在载玻片和洋葱表皮上均不能形成完整的侵染垫，丧失了侵染拟南芥和番茄叶片的能力，但是可以侵染受伤的叶片。表明Bcser2在灰葡萄孢的菌核发育和致病过程中发挥重要作用，为防治作物灰霉病提供了理论依据。在本项目资助下，发表SCI源研究论文1篇，会议摘要2篇，培养博士研究生3人，其中毕业1人；主持人在项目执行期间获得国家自然科学基金优秀青年基金资助。