TOR-autophagy branch signaling via Imp1 dictates plant-microbe biotrophic interface longevity

Abstract

Like other intracellular eukaryotic phytopathogens, the devastating rice blast fungus Magnaporthe ( Pyricularia) oryzae first infects living host cells by elaborating invasive hyphae (IH) surrounded by a plant-derived membrane. This forms an extended biotrophic interface enclosing an apoplastic compartment into which fungal effectors can be deployed to evade host detection. M. oryzae also forms a focal, plant membrane-rich structure, the biotrophic interfacial complex (BIC), that accumulates cytoplasmic effectors for translocation into host cells. Molecular decision-making processes integrating fungal growth and metabolism in host cells with interface function and dynamics are unknown. Here, we report unanticipated roles for the M. oryzae Target-of-Rapamycin (TOR) nutrient-signaling pathway in mediating plant-fungal biotrophic interface membrane integrity. Through a forward genetics screen for M. oryzae mutant strains resistant to the specific TOR kinase inhibitor rapamycin, we discovered IMP1 encoding a novel vacuolar protein required for membrane trafficking, V-ATPase assembly, organelle acidification and autophagy induction. During infection, Δ imp1 deletants developed intracellular IH in the first infected rice cell following cuticle penetration. However, fluorescently labeled effector probes revealed that interface membrane integrity became compromised as biotrophy progressed, abolishing the BIC and releasing apoplastic effectors into host cytoplasm. Growth between rice cells was restricted. TOR-independent autophagy activation in Δ imp1 deletants (following infection) remediated interface function and cell-to-cell growth. Autophagy inhibition in wild type (following infection) recapitulated Δ imp1. In addition to vacuoles, Imp1 ^GFP localized to IH membranes in an autophagy-dependent manner. Collectively, our results suggest TOR-Imp1-autophagy branch signaling mediates membrane homeostasis to prevent catastrophic erosion of the biotrophic interface, thus facilitating fungal growth in living rice cells. The significance of this work lays in elaborating a novel molecular mechanism of infection stressing the dominance of fungal metabolism and metabolic control in sustaining long-term plant-microbe interactions. This work also has implications for understanding the enigmatic biotrophy to necrotrophy transition.

Author summary

Plant-associated fungi can form intimate connections with living host cells. Clarifying the molecular drivers of these interactions, and which partner is dominant, might be important in understanding how beneficial plant-fungal relationships can be enhanced to improve crop yields while pathogenic interactions that threaten crop health are disrupted. In common with other symbionts and phytopathogens, the devastating rice blast fungus Magnaporthe oryzae elaborates invasive hyphae in living host cells surrounded by plant-derived membranes. Nothing is known at the molecular signaling level about how such plant-microbe biotrophic interfacial zones are maintained as the fungus grows in and between host cells. Here, we report that fungal membrane trafficking processes controlled by nutrient signaling pathways are critical for maintaining biotrophic interface integrity during M. oryzae growth in rice cells. Impairing these processes resulted in erosion of the plant-microbe interface and failure of the fungus to thrive. To our knowledge, this work presents the first evidence indicating that the fungal partner is dominant in propagating the plant-microbe boundary. This suggests that the biotrophic interface is a fungal construct and provides clues on how such interfaces might be modulated to benefit the host plant.