Mechanisms of cell-autonomous plasma membrane stabilization and repair in plants

Project Summary The long-term goal of this project is to characterize molecular mechanisms governing plasma membrane stabilization and repair in plants. Despite extensive studies in animal systems, cell-autonomous plasma membrane repair (CAPMR) in plants is an uncharted territory. This is most likely due to the fact that all plant cells (except gametes) contain a rigid cell wall that makes precise and reliable plasma membrane wounding and study very difficult. Our recent genetic studies show that knocking out three Mildew Locus O (MLO) genes MLO2, MLO6 and MLO12 in an immuno-compromised Arabidopsis mutant background results in “complete immunity” against obligate biotrophic fungal pathogens causing powdery mildew diseases. Further examination of such mutant lines revealed no signs of defense activation, indicating that these three MLO proteins serve as bona fide host susceptibility factors of powdery mildew fungi. This raises a critical question: what is the cellular function of MLO2, MLO6 and MLO12 that is co-opted by powdery mildew fungi for pathogenesis? It is known that MLO2-GFP is targeted to the fungal penetration site where the fungal feeding structure—the haustorium— is initiated. Interestingly, MLO2-GFP also exhibits focal accumulation in the sites of plasma membrane damage induced by a sharp needle. Based on these observations, it can be hypothesized that (i) MLO2, along with MLO6 and MLO12, participates in CAPMR in leaf epidermal cells and as such (ii) powdery mildew fungi co-opt this host mechanism to seal the host-fungal membranous boundary for haustorial differentiation. To test this hypothesis, higher-order mlo mutants will be created in different backgrounds and evaluated in comparison with their respective parental lines after treatments that induce membrane damages. If MLOs are indeed required for CAPMR, proximity labeling will be used to identify potential MLO2-interacting proteins. Meanwhile, multiplexed CRISPR mutagenesis-based reverse genetics will also be used to circumvent functional redundancy and identify new components that participate in MLO-dependent CAPMR. Selected genes or gene families for CRISPR mutagenesis include genes encoding glutamate receptors (GLRs), calmodulins, SNAREs and synaptotagmins, as well as additional MLO family members. The genetically identified new components will then be further evaluated for their subcellular localization and interaction with MLO2 in powdery mildew-infected cells and uninfected cells. More detailed molecular characterization will be conducted for those that play essential roles in CAPMR in plants. Information from this project will likely fill an important knowledge gap in CAPMR in plants, inspire new strategies for creating more stress-resilient plants and provide novel mechanistic insights into similar processes in animals.