Li Yuan-Bao,Shen Ningning,Deng Xianya,Liu Zixuan,Zhu Shuai,Liu Chengyu,Tang Dingzhong,Han Li-Bo
The dynamic assembly of the actin cytoskeleton is vital for Magnaporthe oryzae development and host infection. The actin-related protein MoFim1 is a key factor for organizing the M. oryza e actin cytoskeleton. Currently, how MoFim1 is regulated in M. oryzae to precisely rearrange the actin cytoskeleton is unclear. In this study, we found that MoFim1 associates with the M. oryzae mitogen-activated protein (MAP) kinase Pmk1 to regulate actin assembly. MoFim1 directly interacted with Pmk1, and the phosphorylation level of MoFim1 was decreased in Δ pmk1 , which led to a change in the subcellular distribution of MoFim1 in the hyphae of Δ pmk1. Moreover, the actin cytoskeleton was aberrantly organized at the hyphal tip in the Δ pmk1 , which was similar to what was observed in the Δ mofim1 during hyphal growth . Furthermore, phosphorylation analysis revealed that Pmk1 could phosphorylate MoFim1 at serine 94. Loss of phosphorylation of MoFim1 at serine 94 decreased actin bundling activity. Additionally, the expression of the site mutant of MoFim1 S94D (in which serine 94 was replaced with aspartate to mimic phosphorylation) in Δ pmk1 could reverse the defects in actin organization and hyphal growth in Δ pmk1 . It also partially rescues the formation of appressorium failure in Δ pmk1. Taken together, these findings suggest a regulatory mechanism in which Pmk1 phosphorylates MoFim1 to regulate the assembly of the actin cytoskeleton during hyphal development and pathogenesis.
Eukaryotic cells respond to environmental stimuli when cell surface receptors are bound by environmental ligands. The binding initiates a signal transduction cascade that results in the appropriate intracellular responses. Studies have shown that endocytosis is critical for receptor internalization and signaling activation. In the rice blast fungus Magnaporthe oryzae , a non-canonical G-protein coupled receptor, Pth11, and membrane sensors MoMsb2 and MoSho1 are thought to function upstream of G-protein/cAMP signaling and the Pmk1 MAPK pathway to regulate appressorium formation and pathogenesis. However, little is known about how these receptors or sensors are internalized and transported into intracellular compartments. We found that the MoEnd3 protein is important for endocytic transport and that the Δ Moend3 mutant exhibited defects in efficient internalization of Pth11 and MoSho1. The Δ Moend3 mutant was also defective in Pmk1 phosphorylation, autophagy, appressorium formation and function. Intriguingly, restoring Pmk1 phosphorylation levels in Δ Moend3 suppressed most of these defects. Moreover, we demonstrated that MoEnd3 is subject to regulation by MoArk1 through protein phosphorylation. We also found that MoEnd3 has additional functions in facilitating the secretion of effectors, including Avr-Pia and AvrPiz-t that suppress rice immunity. Taken together, our findings suggest that MoEnd3 plays a critical role in mediating receptor endocytosis that is critical for the signal transduction-regulated development and virulence of M . oryzae .
During the interaction between the rice blast fungus Magnaporthe oryzae and the host, the pathogen employs a series of receptors and sensors at the plasma membrane to recognize host surface cues and to activate signal transduction pathways required for appressorium formation and pathogenicity. We found that MoEnd3-mediated endocytosis is responsible for internalization of non-canonical GPCR Pth11 and the sensor MoSho1 to endosomal compartments. This is important for activating the downstream Pmk1 MAPK pathway to control appressorium formation and penetration. MoEnd3 is regulated through phosphorylation by the actin-regulating kinase MoArk1. In addition, MoEnd3 has a role in establishing effector secretion required for suppressing rice immunity. Our studies provide evidence that endocytosis is required for normal signaling and virulence in M . oryzae .
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