基于致病丧失突变体的芦笋茎枯病菌致病相关基因的鉴定与作用机制解析

Asparagus officinalis L. is a high value vegetable rich in bioactive factors such as saponins, rutin, and organic selenium. It is also considered as one of important herbal medicine plants which can prevent and treat several diseases for human such as tumour, hypertension, and hyperglycemia. At present, China is the largest asparagus planted country around the world. Asparagus stem blight is a high destructive disease of asparagus worldwide. Filamentous fungus Phmopsis asparagi is identified as the causing pathogen. However, its molecular pathogenic mechanism is poorly known. Identification of pathogenic related genes is of great importance for understanding of molecular pathogenic mechanism on asparagus stem blight. It could be considered as potential molecular targets for asparagus stem blight control and the results will also provide a solid gene fundation and new reference strategy for disease control and disease resistance breeding. Recently, our lab has completed the genome sequence and annotation project of this fungus strain Pa1100. Further more, we constructed its molecular genetic transformation system and screened 6 different pathogenicity lost T-DNA insertion mutants which identified by TAIL-PCR. In this study, further characterization will be conducted on these important mutants to identify the pathogenic related genes (PaPRG1-PaPRG6) and analyze their biological functions. These pathogenic related genes will be identified based on both these flanking sequences and whole genome sequence data. Gene knockout, complement, and over-expression technologies will be applied for genes functional analysis on biological characteristics of morphology, stress response, activity of cell wall-degrading enzymes, toxin production, and pathogenicity. Moreover, the difference of infection process and development between Paprg1-Paprg6 mutants and wild type strain Pa1100 will be analyzed by scanning electron microscope (SEM) from the point of histocytological. At the same time, these pathogenic related genes' expression level and subcellular localization of its product will be evaluated in infection progress using methods of qRT-PCR and GFP-fusion to analyze their roles in infection process from the point of genes' expression patterns. Further more, RNA-Seq will be conducted to reveal the pathogenic signaling pathway and network which these genes involved in to deeply reveal their pathogenic mechanism.

芦笋富含皂苷、芦丁，营养和保健功能突出，我国种植面积已跃居世界首位。茎枯病是芦笋生产上的毁灭性病害，致病机制解析对病害防控及抗病育种具有重要科学意义。目前国内外尚无相关报道。本项目前期对芦笋茎枯病菌Pa1100完成了全基因组测序，并筛选出6个致病丧失突变体，TAIL-PCR初步验证其T-DNA插入位点各异。为进一步鉴定这些基于突变体的致病相关基因（PaPRG1-PaPRG6，暂命名），拟利用T-DNA侧翼序列和基因组数据库将其进行高效克隆；并通过基因敲除、互补和过表达技术探讨它们对病菌形态建成、逆境响应、细胞壁降解酶活性、毒素产量和致病力调控的生物学功能。同时，借助扫描电镜观测结合qRT-PCR和GFP定位技术，从组织细胞学和分子生物学角度分析病菌入侵、定殖和扩展进程与候选基因时空表达的关系；并通过RNA-Seq技术探讨候选基因参与致病调控的信号途径和基因互作网络，深入阐明其致病作用机制。

我国芦笋种植面积已跃居世界首位，茎枯病是芦笋生产上的毁灭性病害。本项目在前期对芦笋茎枯病菌株Pa1100完成了全基因组测序的基础上，筛选获得6个致病丧失（缺陷）突变体，TAIL-PCR初步验证其T-DNA插入位点各异，为进一步研究这些基于突变体的致病相关基因（PaPRG1-PaPRG6），我们利用T-DNA侧翼序列测序和全基因组生物信息分析成功鉴定了候选基因的分子序列并完成了分子克隆。进一步，通过构建分子遗传转化体系，并利用基因敲除、互补和过表达技术探讨了它们在目标病原真菌的形态建成、细胞壁降解酶活性、毒素产量和致病力调控等方面的生物学功能。结果表明，与野生型菌株Pa1100相比，PaPRG1、PaPRG3、PaPRG5和PaPRG6基因对病菌致病力具有显著影响，突变体丧失对芦笋的致病能力。此外，PaPRG2和PaPRG3对病原菌菌落形态具有重要正向调控作用，同时对促进毒素产量具有一定作用；而PaPRG5和PaPRG6则对细胞壁降解酶活性有重要影响。PaPRG2和PaPRG4基因敲除突变体均表型出致病力显著下降（致病缺陷），且其对渗透和盐胁迫更为敏感。同时，利用GFP定位和显微镜观测技术分析了病菌入侵、定殖和扩展进程，结合不同组织样品和致病侵染进程，利用qRT-PCR分析了候选基因的时空表达特征。结果表明，在致病接种初期（24 hrs post inoculation, hpi），PaPRG1和PaPRG3表达量显著上升，直至72 hpi后出现缓慢下降；PaPRG4、PaPRG5、和PaPRG6则在致病接种72 hpi后表达显著上升；PaPRG2则持续表现出较高量表达。最后，为了阐明了其可能参与的致病相关信号调控方式，我们通过RNA-Seq技术探讨候选基因敲除突变体对其他基因的上调或下调作用。这些探索性研究对深入揭示芦笋茎枯病菌分子致病作用机制具有重要意义。