Illuminating the journey of autophagosomes in plants

Autophagy is an essential quality control pathway that mediates the recycling of damaged cellular components that would otherwise harm the cell. It involves encapsulating the damaged macromolecules in a double membrane vesicle termed the autophagosome, which is then carried to the vacuole for recycling. Despite extensive molecular studies in yeast and metazoans, in plants, how autophagosomes are transported and fuse with the vacuole remain largely unknown. Intriguingly, although the core autophagy machinery is highly conserved, plant genomes lack some of the key players that are involved in autophagosome transport. This suggests that the green lineage may have evolved novel means to deliver autophagic cargo to the vacuole. To unravel the molecular mechanism of autophagic cargo delivery in plants, we focused on identification of the autophagy adaptors that are recruited to autophagosomes by interacting with ATG8 proteins on the outer autophagosome membrane. Through extensive fractionation-coupled mass spectrometry experiments, we identified two candidate autophagy adaptors that are critical for autophagic flux in Arabidopsis. We have obtained preliminary evidence suggesting both of these proteins directly interact with ATG8 proteins via short linear motifs termed ATG8 interacting motifs. In this proposal, we will use functional cross-complementation assays in Arabidopsis and Marchantia to elucidate the molecular functions and evolution of these adaptor proteins. We will also establish correlative light and electron microscopy (CLEM) techniques to visualize these autophagy adaptors on outer autophagosome membranes at the ultrastructural level. Finally, we will perform recovery assays to see if increasing the level of these autophagy adaptors could facilitate autophagy and thereby enhance stress tolerance in plants. Altogether, our studies will shed light on a hitherto unknown group of molecular players in plant autophagy and reveal if they can be modulated to engineer more robust plants.

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