禾谷镰刀菌CDC2A/2B介导的侵染生长与营养生长细胞周期调控不同的分子机理

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most important diseases of wheat and barley worldwide. Understanding the molecular mechanisms of F. graminearum pathogenesis is critical for developing more effective disease control strategies. Since F. graminearum and other hemi-biotrophic pathogens generally produce infectious hyphae with distinct morphology from saprophytic (or vegetative) hyphae, cell cycle regulation may be different during saprophytic and infection growth but the underlying mechanisms are not well understood. In a previous study, we found that F. graminearum uniquely has two co-orthologs (CDC2A and CDC2B) of the CDC2 gene, which encodes a cyclin-dependent kinase that is the key regulator of mitosis in eukaryotes. Both Cdc2A and Cdc2B are functionally in saprophytic growth. They are functionally redundant. Deletion both of them is lethal. However, the cdc2B mutant has no defects in pathogenesis. Only CDC2A is functionally and plays a critical role in invasive growth. These results suggest that cell cycle regulation is different during saprophytic and infection growth in F. graminearum. In this project, we will characterize the functional difference between Cdc2A and Cdc2B by identifying sequence features and specific cyclins or targets responsible for their functional specificity. Our goal is to determine molecular mechanism of different cell cycle regulation between saprophytic and infectious growth in F. graminearum. Overall, results from proposed project will be helpful not only to better understand molecular regulation mechanisms of pathogenic growth in hemi-biotrophic pathogens, but also to develop new drug targets for control of FHB and other fungal diseases.

禾谷镰刀菌引起的赤霉病是影响小麦产量和品质最严重的病害之一，阐明病原菌致病机理对制定和发展有效病害防控策略至关重要。禾谷镰刀菌等一些半活体营养真菌侵染生长与营养生长的菌丝形态不同，具有不同的细胞周期调控，但其分子机理尚不清楚。细胞周期蛋白依赖性激酶Cdc2是细胞周期调控中最重要的因子，项目前期研究发现大多数真菌只编码一个CDC2基因，而禾谷镰刀菌编码两个：CDC2A和CDC2B。二者均调控营养生长的细胞周期且功能冗余，只CDC2A作用于侵染生长，为研究侵染生长与营养生长细胞周期调控差异的机理提供了契机。本项目拟进一步鉴定决定二者功能差异的序列位点，筛选其特异结合的细胞周期蛋白、磷酸化底物及其它互作蛋白，明确二者作用机制不同的分子基础，最终揭示侵染生长与营养生长细胞周期调控差异的机理。研究结果对明确半活体营养病原真菌致病机制具有重要价值，抑制侵染菌丝形成有望成为小麦赤霉病防治的有效策略之一。

禾谷镰刀菌引起的小麦赤霉病是影响小麦产量和品质最严重的病害之一，病菌有性生殖产生的子囊孢子是小麦赤霉病发生的初侵染源，阐明禾谷镰刀菌有性生殖和侵染致病的调控机理，对制定和发展有效病害防控策略至关重要。细胞周期蛋白依赖性激酶CDC2是细胞周期调控中最重要的因子，大多数真菌只编码一个CDC2基因，本项目前期研究发现禾谷镰刀菌编码两个CDC2基因（CDC2A和CDC2B），并证明二者在营养生长阶段的功能相同，但在有性生殖和侵染致病阶段的功能不同。本项目进一步鉴定了导致CDC2A和CDC2B功能差异的蛋白序列位点，验证了两者启动子的功能差异，鉴定并功能验证了与其互作的细胞周期蛋白的功能，筛选和验证了与CDC2A特异互作的蛋白和磷酸化底物的磷酸化位点。研究结果证明CDC2A和CDC2B第126位氨基酸差异（R126E）以及启动子表达强度差异共同决定了其生物学功能的差异。同时，本项目鉴定到3个CDC2A/CDC2B依赖的细胞周期蛋白CLN1、CLN2、CLN3，并证明其具有不同的生物学功能和亚细胞定位，CLN3很可能与CDC2A有性生殖阶段的特异功能有关，而CLN1和CLN2很可能与CDC2A侵染致病阶段的特异功能有关。此外，本项目筛选获得134个与CDC2A潜在特异互作的蛋白和39个磷酸化底物，功能验证了5个CDC2A候选特异性磷酸化底物和6个磷酸化位点的功能，证明这些位点的磷酸化并非是CDC2A特异功能的主要决定因素。综上所述，本项目阐明了CDC2A和CDC2B功能差异的分子基础，揭示了禾谷镰刀菌有性生殖和侵染致病特异的细胞周期调控机制。