Prevalence and Functional Significance of Regulated Alternative Splicing in Plant Pathogenic Fungi

了解微生物致病性的分子基础对于破译疾病的生物学过程和设计新的有效的疾病控制方法至关重要。尽管在微生物疾病的治疗方面取得了进展，但真菌疾病继续对人类、动物和植物构成越来越大的威胁，往往危及粮食安全和人类福祉。特别是在过去的几十年里，新出现的传染性真菌疾病出现了前所未有的增加，而微生物对现有的疾病控制化学物质的耐药性不断丧失，使问题更加严重，需要开发新的治疗策略。这一过程的关键是阐明微生物感染宿主的分子机制。在这方面，选择性剪接（AS）是真核基因表达中的一个关键因素，通过该因素，同一基因可以产生不同的蛋白质变体，这是微生物致病性的一个未被探索的特征，它可以通过调节感染期间毒力因子的最佳产生来促进微生物的毒力。因此，该项目将侧重于系统分析植物病原真菌中的AS，重点是破译其对寄生虫感染的功能意义。这一发现将极大地促进我们对真菌发病机制的理解，并可用于通过在宿主感染过程中对真菌AS进行药物操作来开发新的植物疾病治疗方法。该项目还将被用作提高公众对传染性真菌疾病的认识和培训分子植物科学方面多样化学生群体的基础。关于真菌对植物的致病机制的研究大多集中在发现致病致病的毒力因子及其生化特性上，而对控制毒力相关基因转录组多样性的表观遗传机制关注较少。AS剪接可能是基因表达的最重要的转录后调控机制，但是，尽管它在真核细胞中产生蛋白质组多样性和功能复杂性方面很重要，但它的流行程度、生物学作用和真菌致病性的功能后果几乎没有研究。该项目的目标是系统地识别和表征植物病原真菌在其宿主的完整感染周期中的AS事件，以便为其普遍性，物种保护和微生物毒力贡献等基本问题提供关键答案。这一研究领域很重要，因为毒力相关基因的AS调控是微生物致病性的一个相对未被探索的特征，它可能使微生物能够快速调节和微调与宿主的相互作用。为了实现其目标，该项目将整合全基因组转录组分析，通过靶向蛋白质组学方法选择性检测新的剪接异构体，并通过rnai介导的异构体特异性基因靶向对候选AS基因进行功能分析。为了评估结果的普遍性，分析将在两种系统发育相关的植物病原真菌感染其共同宿主时进行。该项目的成功完成可能会引发宿主-微生物相互作用领域的范式转变，强调as介导的转录后调控在寄生虫适应性和毒力中的重要性。

Understanding the molecular basis of microbial pathogenicity is vital for deciphering the biological process of disease, and for designing new and effective disease control methods. In spite of advances in treatment of microbial diseases, fungal diseases continue to pose an increasing threat to humans, animals, and plants that often endanger food security and human well-being. The last few decades, in particular, have seen an unprecedented increase in emerging infectious fungal diseases, while the continuous loss to microbial drug resistance of available chemistries for disease control, intensifies the problem and necessitates the development of novel therapeutic strategies. Key to this process is the elucidation of the molecular mechanisms employed by microbes to infect their hosts. In this respect, alternative splicing (AS), a key element in eukaryotic gene expression by which different protein variants are generated from the same gene, is an unexplored feature of microbial pathogenicity which could promote microbial virulence by regulating the optimal production of virulence factors during infection. Thus, the project will focus on a systematic analysis of AS in plant pathogenic fungi, with an emphasis on deciphering its functional significance for parasitic infections. The findings will significantly advance our understanding of fungal pathogenesis and could be used for the development of novel therapeutic treatments for plant diseases through the pharmaceutical manipulation of fungal AS during infection of the host. The project will also be used as a basis for raising public awareness on infectious fungal diseases and training of a diverse student body in molecular plant sciences. Most studies on fungal pathogenesis on plants have focused on the discovery and biochemical characterization of virulence factors that contribute to infections and disease, but much less attention has been placed on the epigenetic mechanisms that control transcriptome diversity of virulence-associated genes. AS splicing is perhaps the most crucial posttranscriptional regulatory mechanism of gene expression but, despite its importance in generating proteome diversity and functional complexity in eukaryotic cells, its prevalence, biological role, and functional consequence for fungal pathogenicity have been barely studied. The goal in this project is to systematically identify and characterize AS events in plant pathogenic fungi during a complete infection cycle of their host, in order to provide critical answers to fundamental questions regarding its pervasiveness, species conservation, and contribution to microbial virulence. This area of study is important because regulated AS of virulence-associated genes is a relatively unexplored feature of microbial pathogenicity, which potentially enables microbes to quickly modulate and fine-tune interactions with their host. To achieve its goals, the project will integrate genome-wide transcriptome profiling, with selective detection of novel splice isoforms through targeted proteomics approaches, and functional analyses of candidate AS genes by RNAi-mediated isoform-specific gene targeting. To assess the generality of the findings, the analysis will be done in two phylogenetically related plant pathogenic fungi when infecting their common host. The successful completion of this project could trigger a paradigm-shift in the field of host-microbe interactions by highlighting the importance of AS-mediated post-transcriptional regulation in parasite fitness and virulence.