MoCAP proteins regulated by MoArk1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae

Actin organization is a conserved cellular process that regulates the growth and development of eukaryotic cells. It also governs the virulence process of pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae, with mechanisms not yet fully understood. In a previous study, we found that actin-regulating kinase MoArk1 displays conserved functions important in endocytosis and actin organization, and MoArk1 is required for maintaining the growth and full virulence of M. oryzae. To understand how MoArk1 might function, we identified capping protein homologs from M. oryzae (MoCAP) that interact with MoArk1 in vivo. MoCAP is heterodimer consisting of α and β subunits MoCapA and MoCapB. Single and double deletions of MoCAP subunits resulted in abnormal mycelial growth and conidia formation. The ΔMocap mutants also exhibited reduced appressorium penetration and invasive hyphal growth within host cells. Furthermore, the ΔMocap mutants exhibited delayed endocytosis and abnormal cytoskeleton assembly. Consistent with above findings, MoCAP proteins interacted with MoAct1, co-localized with actin during mycelial development, and participated in appressorial actin ring formation. Further analysis revealed that the S85 residue of MoCapA and the S285 residue of MoCapB were subject to phosphorylation by MoArk1 that negatively regulates MoCAP functions. Finally, the addition of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) failed to modulate actin ring formation in ΔMocap mutants, in contrast to the wild-type strain, suggesting that MoCAP may also mediate phospholipid signaling in the regulation of the actin organization. These results together demonstrate that MoCAP proteins whose functions are regulated by MoArk1 and PIP2 are important for endocytosis and actin dynamics that are directly linked to growth, conidiation and pathogenicity of M. oryzae.

肌动蛋白组织（actin organization）是一类保守的细胞过程，调控真核细胞的生长与发育。其同时调控病原真菌的致病过程，以稻瘟病菌（Magnaporthe oryzae）为例，但其具体分子机制尚未完全阐明。本团队此前的研究发现，肌动蛋白调控激酶MoArk1在胞吞作用（endocytosis）与肌动蛋白组织中发挥保守功能，且该激酶对维持稻瘟病菌的正常生长与完全致病力不可或缺。为解析MoArk1的作用机制，本研究从稻瘟病菌中鉴定得到与其在体内存在互作的加帽蛋白同源物（MoCAP）。MoCAP为由α亚基MoCapA与β亚基MoCapB组成的异二聚体。对MoCAP亚基进行单基因或双基因敲除，均会导致菌丝生长与分生孢子形成异常。ΔMocap突变体还表现出附着胞（appressorium）侵染能力下降，以及在寄主细胞内的侵入菌丝生长受抑的表型。进一步研究发现，ΔMocap突变体的胞吞过程延迟，且细胞骨架组装异常。上述表型与实验结果一致：MoCAP蛋白可与肌动蛋白MoAct1互作，在菌丝发育阶段与肌动蛋白共定位，并参与附着胞的肌动蛋白环形成过程。深入分析表明，MoCapA的S85位点与MoCapB的S285位点可被MoArk1磷酸化，该修饰对MoCAP的功能具有负调控作用。最后，与野生型菌株不同，外源添加磷脂酰肌醇4,5-二磷酸（phosphatidylinositol 4,5-bisphosphate, PIP2）无法恢复ΔMocap突变体的肌动蛋白环形成缺陷，这提示MoCAP可能还通过介导磷脂信号通路参与调控肌动蛋白组织过程。综上，本研究结果证实，受MoArk1与PIP2共同调控的MoCAP蛋白，在胞吞作用与肌动蛋白动态调控过程中发挥关键作用，而该过程直接关联稻瘟病菌的生长、产孢与致病力。