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

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.

MoPpe1 partners with MoSap1 to mediate TOR and cell wall integrity signalling in growth and pathogenicity of the rice blast fungus Magnaporthe oryzae

“…Previously, studies in M. oryzae showed that the nuclear division is critical in appressorium function (Marroquin‐Guzman et al ., ). To further understand whether the deletion of MoPPE1 caused a nuclear division defect that may account for restricted invasion, a histone H1 protein fused to a red fluorescent protein (H1‐RFP) construct was introduced into the wild‐type strain and Δ Moppe1 mutant (Li et al ., ,b; Marroquin‐Guzman et al ., ). However, the nuclear number were of no significant difference between the Δ Moppe1 mutant and the wild‐type strains at 0, 4, 12, 24 h and infection phase when observed, suggesting that MoPpe1 may be dispensable for nuclear division which was in coordination with cell growth (Supporting Information Fig.…”

“…The GFP‐MoNut1 fusion construct was introduced into Δ Moppe1 and Δ Monut1 mutants respectively. The proteins extracted from mycelium were resolved on 8% SDS‐polyacrylamide gels prepared with 50 μM acrylamide‐dependent Phos‐tagligand and 100 μM MnCl 2 as described (Li et al ., ,b). Gel electrophoresis was run at 60 V for 6 ± 8 h. Prior to transfer, gels were equilibrated in transfer buffer containing 5 mM EDTA for 30 min two times and then in transfer buffer without EDTA for 15 min.…”

VPS9 domain‐containing proteins are essential for autophagy and endocytosis in Pyricularia oryzae

“…graminearum have been verified to be involved in endocytosis and pathogenicity (Dou et al ., ; Zhang et al ., ). Through functional analysis of endocytic proteins such as the ArfGAP protein MoGlo3 (Zhang et al ., ), the class V myosin MoMyo5 (Tang et al ., ), the capping protein MoCap (Li et al ., ), the verprolin protein MoVrp1 (Huang et al ., ), the ADP‐ribosylation factor Arf6 (Zhu et al ., ), the actin‐regulating kinase homologue MoArk1 (Wang et al ., ) and an early endocytic protein‐MoEnd3 (Li et al ., ), it was revealed that endocytosis is essential for conidiogenesis, appressorium formation and pathogenicity in P . oryzae .…”

Arginine methylation is required for remodelling pre‐mRNA splicing and induction of autophagy in rice blast fungus

“…The recombination plasmid was isolated using the Yeast Plasmid Mini Preparation Kit (Sangon Biotech, SK1246, Shanghai, China), and plasmids containing the correct inserts were transformed into protoplasts of M. oryzae , as previously described (Yang et al ., ). The CDS of MoHMT1 was cloned and ligated into the pYF11 vector (L. Li et al ., ) to produce the MoHMT1‐GFP fusion construct (primers shown in Table S1), driven by its 1.5 kb native promoter, and was subsequently transformed into the MoHMT1 ko mutant strain. The transformed strain was selected by bleomycin resistance on OA plates.…”