Endosomal sorting machineries regulate the transport of their cargoes among intracellular compartments. However, the molecular nature of such intracellular trafficking processes in pathogenic fungal development and pathogenicity remains unclear. Here, we dissect the roles and molecular mechanisms of two sorting nexin proteins and their cargoes in endosomal recycling in Fusarium graminearum using high-resolution microscopy and high-throughput co-immunoprecipitation strategies. We show that the sorting nexins, FgSnx41 and FgSnx4, interact with each other and assemble into a functionally interdependent heterodimer through their respective BAR domains. Further analyses demonstrate that the dimer localizes to the early endosomal membrane and coordinates endosomal sorting. The small GTPase FgRab5 regulates the correct localization of FgSnx41-FgSnx4 and is consequently required for its trafficking function. The protein FgSnc1 is a cargo of FgSnx41-FgSnx4 and regulates the fusion of secreted vesicles with the fungal growing apex and plasma membrane. In the absence of FgSnx41 or FgSnx4, FgSnc1 is mis-sorted and degraded in the vacuole, and null deletion of either component causes defects in the fungal polarized growth and virulence. Overall, for the first time, our results reveal the mechanism of FgSnc1 endosomal recycling by FgSnx41-FgSnx4 heterodimer which is essential for polarized growth and pathogenicity in F. graminearum.

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SR protein-specific kinases (SRPKs) uniquely with a spacer region are important splicing factors from yeast to human. However, little is known about their biological functions in filamentous fungi. Therefore, we characterized a SRPK called SRK1 in wheat scab fungus Fusarium graminearum. Our data showed that Srk1 is required for vegetative growth, sexual reproduction and plant infection, and plays critical roles in pre-mRNA alternative splicing and gene expression. Remarkably, we found that Srk1 displayed dynamic shuttling between cytoplasm and the nucleus, which is regulated by the divergent spacer domain rather than its kinase activity, suggesting a regulatory mechanism for Srk1. Interestingly, Srk1-GFP also localized to the septal pores, indicating a possible role of Srk1 unrelated to mRNA processing. Although both K1 and K2 lobes of the kinase domain are essential for Srk1 functions, the K2 but not K1 lobe is responsible for the septal pore localization. Lastly, we established that Srk1 physically interacts with the two SR proteins, FgNpl3 and FgSrp1. Overall, our results indicated that SRK1 regulates fungal development, plant infection and mRNA processing by phosphorylation of other splicing factors including SR proteins, and the spacer domain regulates the functions of Srk1 by modulating its nucleocytoplasmic shuttling.

Fusarium head blight (FHB) caused by the ascomycete fungus Fusarium graminearum can result in significant crop losses and render the crops harmful to human health due to contamination with mycotoxin. Although the pathogenesis of F. graminearum is widely investigated by molecular genetics approaches, detailed studies about its cellular and developmental processes at the initial stages of infection are very limited. We applied live-cell imaging approach to characterize the spatial and temporal development of growing hyphae and plant responses during F. graminearum and wheat coleoptile interactions. The present investigation demonstrates that F. graminearum uses two strategies to penetrate the host epidermal cells. The pathogen breaks through the host cell wall with appressoria-like structures derived from surficial hyphae, and also with the narrow pegs from thick and bulbous intracellular hyphae. Live cell imaging in the presence of the endocytic tracker FM4-64 showed that the plasma and intermembranes of the invaded wheat coleoptile cells were intact. Invasive hyphae exhibit branching, budding, pseudohyphae-like growth, cell-to-cell spreading ability, and were sealed within a plant membrane, indicating a biotrophic lifestyle of this fungus inside the invaded cells. Time-lapse imaging suggested that there were callose depositions at the plant cell walls in the form of continuous lines and also on the outer linings of the fungal invasive hyphae at colonization stage. In addition, our studies demonstrate that the activation of the toxisomerelated gene (TRI4) requires external stimuli and is spatio-temporally modulated. Generally, this study provides new insights into the colonization strategies and host response features during F. graminearum-plant interactions. Further tracing of cellular details will significantly contribute to our understanding of molecular mechanisms of F. graminearum-host interactions.

Summary Secretion is a fundamental process that plant pathogens utilize to deliver effectors into the host to downregulate immunity and promote infection. Here, we uncover a fascinating membrane trafficking and delivery route that originates from vacuolar membranes in Magnaporthe oryzae and conduits to the host interface and plasma membrane. To perform such secretory/trafficking function, MoRab7 first recruits the retromer complex to the vacuolar membrane, enabling recognition of a family of SNARE proteins, including MoSnc1. Live‐cell imaging confirmed a highly dynamic vesicular trafficking of the retromer complex component(s) and MoSnc1 toward and across the host interface or plasma membrane, and subsequent fusion with target membranes. Interestingly, disruption of the MoRab7/Retromer/MoSnc1‐based endolysosomal cascade affects effector secretion and fungal pathogenicity. Taken together, we discovered an unconventional protein and membrane trafficking route starting from the fungal endolysosomes to the M. oryzae–rice interaction interface and dissect the role of MoRab7/Retromer/MoSnc1 sorting machinery in effector secretion during biotrophy and invasive growth in rice blast fungus.