Isolation of mononuclear propagules from coenocytic hyphae of the mucormycosis pathogen Rhizopus delemar

1. 摘要 德雷马根霉 (Rhizopus delemar) 是毛霉菌病的主要致病菌，毛霉菌病是一种危及生命的真菌感染。 免疫功能低下的患者。尽管其临床重要性，R. delemar 由于其 有限的遗传易处理性和缺乏可用的研究资源。具体来说，R. delemar 生产 共核菌丝和多核孢子，导致获得稳定的基因特异性难度很大 突变株。我们之前采用过连续菌丝尖端显微切割，并使用 CRISPR-Cas9 技术，分离单核突变株。然而，这种隔离方法费力且低效。 为了绕过这个障碍，我们提出菌丝破碎和原生质体生成，再加上 荧光激活细胞分选（FACS），可分离单核繁殖体以获得突变体 遗传转化后的克隆稳健。我们首先会优化菌丝碎片和原生质体 生产富集单核细胞（目标 1a）并用荧光核标记 R. delemar 菌株 通过 DNA 转化标记（目标 1b）。然后我们将分类并分离单核菌丝 通过 FACS 分析片段/原生质体（目标 2a）。此外，我们将改进 DNA 质粒 对 R. delemar 进行进一步的基因操作（目标 2b）。我们的研究计划的成功实施将 为促进 R. delemar 诱导的毛霉菌病机制的遗传学研究提供了宝贵的工具。

1. Abstract Rhizopus delemar is the leading casual-agent of mucormycosis, a life-threatening fungal infection in immunocompromised patients. Despite its clinical importance, R. delemar remains understudied due to its limited genetic tractability and lack of available research resources. Specifically, R. delemar produces coenocytic hyphae and multinucleated spores, resulting in a high difficulty obtaining stable gene-specific mutant strains. We have previously employed consecutive hyphal tip microdissection, and with CRISPR-Cas9 techniques, to isolate mononuclear mutant strains. However, this isolation method is laborious and inefficient. To circumvent this obstacle, we propose hyphal fragmentation and protoplast generation, coupled with fluorescence-activated cell sorting (FACS), which allows isolation of mononuclear propagules to obtain mutant clones following genetic transformation robustly. We will first optimize hyphal fragmentation and protoplast production to enrich single nucleated cells (Aim 1a) and label R. delemar strains with fluorescent nuclear markers through DNA transformation (Aim 1b). We will then sort and isolate mononuclear hyphal fragments/protoplasts through FACS analysis (Aim 2a). Additionally, we will improve the DNA plasmid for further genetic manipulation of R. delemar (Aim 2b). Our research plan's successful implementation will provide an invaluable tool to facilitate genetic studies of R. delemar-induced mucormycosis mechanisms.