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 Δ Mocap mutants also exhibited reduced appressorium penetration and invasive hyphal growth within host cells. Furthermore, the Δ Mocap 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 (PIP 2 ) failed to modulate actin ring formation in Δ Mocap 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 PIP 2 are important for endocytosis and actin dynamics that are directly linked to growth, conidiation and pathogenicity of M . oryzae . Author summary The actin-regulating kinase MoArk1 plays a conserved function in endocytosis and actin organization and is also essential for growth and full virulence of the rice blast fungus Magnaporthe oryzae . To understand how MoArk1 functions, we identified the F-actin capping protein α (MoCapA) and β (MoCapB) subunits that interact with MoArk1. We showed that single and double deletions of MoCAPA and MoCAPB result in slowed growth, reduced conidia production, abnormal morphogenesis, and attenuated virulence. We found that Δ Mocap mutants are defective in endocytosis and actin organization and that MoCAP proteins are subject to regulation by MoArk1 through protein phosphorylation. Finally, we provided evidence demonstrating that MoCAP proteins modulate actin dynamics in response to phosphatidylinositol 4,5-biphosphate (PIP 2 ). These combined results suggest that MoCAP proteins play an important role in endocytosis, actin organization, and virulence. Further studies of MoCAP proteins could lead to a better understanding of the connections between actin organization and host infection by M . oryzae .
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