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.

The ADP ribosylation factor (Arf) and the coat protein complex I (COPI) are both involved in vesicle transport. Together with GTPase-activating proteins (ArfGAPs) and guanine exchange factors (ArfGEFs) that regulate the activity of Arf, they govern vesicle formation, COPI trafficking, and the maintenance of the Golgi complex. In an ongoing effort to study the role of membrane trafficking in pathogenesis of the rice blast fungus Magnaporthe oryzae, we identified MoGlo3 as an ArfGAP protein that is homologous to Glo3p of the budding yeast Saccharomyces cerevisiae. As suspected, MoGlo3 partially complements the function of yeast Glo3p. Consistent with findings in S. cerevisiae, MoGlo3 is localized to the Golgi and that the localization is dependent on the conserved BoCCS domain. We found that MoGlo3 is highly expressed during conidiation and early infection stages, and is required for vegetative growth, conidial production, and sexual development. We further found that the ΔMoglo3 mutant is defective in endocytosis, scavenging of the reactive oxygen species (ROS), and in the response to endoplasmic reticulum (ER) stress. The combined effects result in failed appressorium function and decreased pathogenicity. Moreover, we provided evidence showing that the domains including the GAP, BoCCS and GRM are all important for normal MoGlo3 functions. Our studies further illustrate the importance of normal membrane trafficking in the physiology and pathogenicity of the rice blast fungus.

The tea-oil tree (Camellia oleifera Abel.) is a commercial edible-oil tree in China, and anthracnose commonly occurs in its plantations, causing great losses annually. We have previously revealed that CfSnf1 is essential for pathogenicity in Colletotrichum fructicola, the major pathogen of anthracnose on the tea-oil tree. Here, we identified CfGcn5 as the homolog of yeast histone acetyltransferase ScGcn5, which cooperates with ScSnf1 to modify histone H3 in Saccharomyces cerevisiae. Targeted gene deletion revealed that CfGcn5 is important in fungi growth, conidiation, and responses to environmental stresses. Pathogenicity assays indicated that CfGcn5 is essential for C. fructicola virulence both in unwounded and wounded tea-oil tree leaves. Further, we found that CfGcn5 is localized to the nucleus and this specific localization is dependent on both NLS region and HAT domain. Moreover, we provided evidence showing that the nuclear localization is essential but not sufficient for the full function of CfGcn5, and the NLS, HAT, and Bromo domains were proven to be important for normal CfGcn5 functions. Taken together, our studies not only illustrate the key functions of CfGcn5 in growth, development, and pathogenicity but also highlight the relationship between its locations with functions in C. fructicola.

BackgroundTea-oil tree (Camellia oleifera) is a unique edible-oil tree in China, and anthracnose occurs in wherever it is cultivated, causing great economic losses each year. We have previously identified the Ascomycete fungus Colletotrichum fructicola as the major pathogen of anthracnose in Ca. oleifera. The purpose of this study was to characterize the biological function of Snf1 protein, a key component of the AMPK (AMP-activated protein kinase) pathway, for the molecular pathogenic-mechanisms of C. fructicola.ResultsWe characterized CfSnf1 as the homolog of Saccharomyces cerevisiae Snf1. Targeted CfSNF1 gene deletion revealed that CfSnf1 is involved in the utilization of specific carbon sources, conidiation, and stress responses. We further found that the ΔCfSnf1 mutant was not pathogenic to Ca. oleifera, resulting from its defect in appressorium formation. In addition, we provided evidence showing crosstalk between the AMPK and the cAMP/PKA pathways for the first time in filamentous fungi.ConclusionThis study indicate that CfSnf1 is a critical factor in the development and pathogenicity of C. fructicola and, therefore, a potential fungicide target for anthracnose control.