GENETIC ANALYSIS OF ARABIDOPSIS AUTOPHAGY-RELATED 8 FAMILY PROTEINS REVEALS THEIR REDUNDANT AND REGULATORY ROLES IN PLANT AUTOPHAGY

Autophagy, a critical process for the vacuolar degradation of proteins and organelles, is governed by multiple conserved autophagy-related (ATG) proteins. The central component of the ATG machinery is the ubiquitin-like protein ATG8, which is essential for multiple steps of the autophagy process, including phagophore expansion, autophagosome closure, trafficking and fusion with the lysosome/vacuole, and selective cargo recruitment. Currently, our understanding of the roles of ATG8 in plant autophagy and the functional specialization of ATG8 family members is limited due to genetic redundancy. To assess the roles of ATG8 genes in plant autophagy, here we used CRISPR/Cas9 technology to systematically knockout the Arabidopsis ATG8 genes. By analyzing the atg8 mutants, we found that in contrast to mammalian ATG8s, in which the LC3s and GABARAP subfamilies play distinct roles in the autophagic process, Arabidopsis ATG8s perform an overlapping function in controlling autophagic flux. Combinatorial mutations of Clade I and Clade II ATG8s resulted in severely impaired autophagy under nutrient-starved conditions. Furthermore, we found that RABG3 proteins, members of the RAB7/RABG GTPase family, interact with ATG8s through AIM-LDS interfaces, and that such interaction is essential for the association of RABG3 proteins with the autophagosomal membrane and probably for the fusion of autophagosome with the vacuole, but is not required for endosomal trafficking. With the collection of multiple high-order atg8 mutants generated in this study, we now provide a venue to study the roles of ATG8 genes in canonical autophagy and non-canonical autophagy in Arabidopsis.