全基因组CRISPR扫描揭示少孢节丛菌3D捕虫器发育的关键分子机制

Arthrobotrys oligospora is one of typical nematophagous fungi, already demonstrating the potential applications in agricultural biological control and biomedical fields. There are two typical stages, saprophytic stage and pathogenic stage, in its asexal reproduction cycle. A plenty of three dimensional (3D) traps were developed and matured as the typical characteristics of A. oligospora when the saprophytic stage was transformed into the pathogenic stage. With the unique nematode-trapping properties and complex life cycles, A. oligospora has been one of the typical fungal strains for studying the interactions between fungi and nematodes; however, the key molecular mechanisms of the development of the 3D traps still remain elusive. In this project, we will use the whole-genome CRISPR screening as a powerful reverse genetic tool to conduct the studies of the loss of function of genes in the A. oligospora genome, with an aim to elucidate 1) the key signal pathways involved in the early development of the 3D traps, 2) the key genes and gene clusters responsible for cell division, growth, maturation, and the formation of complex topological morphology of the 3D traps, 3) the key genes and gene clusters for the regulation of the key effective molecules in the intracellular level and on the surface of the 3D traps. Investigation on the key signal pathways, genes and gene clusters will help us to understand the key molecular mechanisms of the 3D trap development of A. oligospora, and further to potentiate the use of this fungal species in the field of agricultural biological control and biomedical applications.

少孢节丛菌是一种典型捕食线虫真菌, 在生物防治和生物医药领域具有潜在应用价值。在其无性生殖阶段，具有两种典型形态：腐生营养状态和寄生捕食状态。当其从营养状态转变为捕食状态时，大量3D粘性捕食器的发育与成熟为其形态转变的典型标志。因其独特的捕食线虫能力和复杂的形态多样性，已成为发育微生物学研究的典型菌株之一，但目前其3D捕食器发育的关键分子机制仍不清晰。本项目利用全基因组CRISPR扫描策略，在基因组水平大规模采用基因功能失活的反向遗传学技术，研究：1）参与诱导3D捕食器早期发育的关键信号通路，2）负责其细胞分裂、生长、成熟及其复杂空间拓扑结构形成的关键基因，以及3）调控其表面与胞内效应分子生物合成的关键基因。研究上述关键基因功能及其作用机制，从分子水平理解该真菌从营养菌丝转变为捕食状态并发育出专门性捕食器官的关键分子机制，为利用该类真菌防治动植物线虫病或其他生物医药领域的潜在应用奠定基础。

本项目优化和完善了丝状真菌CRSPR-cas9基因组编辑的技术体系。基于Gateway cloning技术，构建了含有U6启动子调控的sgRNA元件、真菌复制子元件以及cas9表达盒等元件组成的适合于丝状真菌的基因组编辑技术体系，建立了少孢节丛菌CRISPR扫描研究的sgRNA子文库，建立了单基因扫描和多基因迭代扫描的方法，建立了sgRNA子文库扫描的方法。应有这些技术方法，探讨了121个关键候选基因在少孢节从孢菌3D捕食器发育及其捕食线虫的生物功能。. 发现8种凝集素蛋白，包括2种岩藻糖特异性凝集素、甘露糖特异性凝集素、Jacalin类凝集素、g401、g494、531、g28等凝集素对少孢节丛孢菌3D菌环的发育及其捕食线虫活性的作用。2种岩藻糖特异性凝集素基因的缺失会极大抑制3D菌环的发育，并显著性降低少孢节丛菌捕食线虫的活性。然而，其他凝集素基因的缺失并不会引起显著性的表型变化，包括3D捕食环及其捕食线虫的效率均没有发生改变。探索了少孢节丛孢菌中粘附相关蛋白Aog185、Aog55、Aog5、Aog12、Aog207、Aog231等在其3D菌环的发育及其捕食线虫活性的作用。发现上述六个粘附相关基因的缺失均抑制A. oligospora菌丝生长；对其3D捕食环形成与捕食线虫能力而言，Aog185、Aog55与Aog207的缺失对3D捕食环形成与捕食线虫能力具有显著抑制作用，Aog5与Aog12的缺失具有显著促进作用，而Aog231的缺失则无显著性影响。研究证实了Aog185基因对细胞跨膜转运系统具有重要影响，同时参与调节氨基酸代谢与生合成通路，进而调节了取决于跨膜转运而发生的生物学过程与通路。推测Aog55的缺失会导致A. oligospora中泛素-蛋白酶体系对待降解的蛋白质底物产生无效的去泛素化和泛素化循环。. 综上所述，我们部分阐明少孢节丛菌3D 捕虫器发育的分子机制，这为我们在分子水平上理解捕食性真菌从腐生营养菌丝转变为寄生捕食状态及其专门性捕食器官发育的关键过程提供了科学依据。