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, Δimp1 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 Δimp1 deletants (following infection) remediated interface function and cell-to-cell growth. Autophagy inhibition in wild type (following infection) recapitulated Δimp1. In addition to vacuoles, Imp1GFP 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.
Highlights
An intriguing feature of both beneficial and pathogenic plant-fungal interactions is the formation of biotrophic interfaces that facilitate nutrient acquisition and microbial growth in living host cells
In other words, was the loss of virulence in Δimp1 the cause rather than the effect of the loss of biotrophic interfacial complex (BIC) and extra-invasive hyphal membranes (EIHM) membrane integrity? To address this question, we sought more understanding on the nature of the biotrophic interface by determining its persistence in wild type (WT), we assessed whether TOR-autophagy signaling in planta controlled biotrophic interface longevity, and we explored whether the loss of membrane integrity could be reversed
Where does Imp1 act in the autophagy pathway, and how might this role relate to biotrophic interface integrity maintenance? We propose that Imp1 facilitates phagophore expansion and autophagosome formation during autophagy induction by sourcing membranes from endosomes (Fig 11B)
Summary
An intriguing feature of both beneficial and pathogenic plant-fungal interactions is the formation of biotrophic interfaces that facilitate nutrient acquisition and microbial growth in living host cells. Such interfaces comprise of a hyphal plasma membrane and cell wall, an interfacial matrix, and a plant-derived membrane [1, 2]. M. oryzae forms a focal plant-membrane rich structure outside IH called the biotrophic interfacial complex (BIC) which forms in each newly infected rice cell until the lifestyle switch to necrotrophy. Biotrophic interfaces facilitate effector deployment for the avoidance or suppression of plant innate immunity, and the intimate association between fungal hyphae and host plant cell-derived membranes is critical to the success of the infection process
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