Abstract
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.
Highlights
The actin cytoskeleton is a dynamic network critical for various cellular processes in eukaryotic cells, including motility, division, cytokinesis, vesicle trafficking, endocytosis and exocytosis, and cell signaling in response to biotic and abiotic stimuli [1, 2]
The actin-regulating kinase MoArk1 plays a conserved function in endocytosis and actin organization and is 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
Summary
The actin cytoskeleton is a dynamic network critical for various cellular processes in eukaryotic cells, including motility, division, cytokinesis, vesicle trafficking, endocytosis and exocytosis, and cell signaling in response to biotic and abiotic stimuli [1, 2]. By binding to the fast barbed ends of actin filaments, the CAP proteins prevent disassembly and addition of new monomers [5, 9]. CAP proteins are highly conserved in eukaryotic cells, including humans [11], plants [12], and the budding yeast Saccharomyces cerevisiae [6]. Studies have shown that null mutants or loss-of-function of CAP result in defects in cellular and developmental processes in mammals, plants, flies, and microbes [13,14,15,16,17,18]. In Arabidopsis thaliana, CAP mutants showed abnormal cell morphology but increased actin filament formation [18]. The null CAP mutants have fewer actin cables but an increased number of actin patches, and the mutants had growth defect [13, 23]
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