Comprehensive genetic analyses of Arabidopsis autophagy-related 8 family reveal redundant regulatory roles during autophagy.

The ubiquitin-like protein ATG8 is a central component of the autophagy process and is required at multiple steps during both bulk and selective autophagy. Currently, our understanding of the roles of ATG8 in plants and the possible functional specialization of its family members is limited by genetic redundancy. Here, we employed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 targeting technology to systematically inactivate all nine Arabidopsis thaliana ATG8 loci. Subsequent analyses of the resulting mutants revealed that, unlike mammalian ATG8 family members, which have distinct roles, Arabidopsis isoforms largely overlap in their functions controlling autophagic flux. Notably, combinatorial mutations have similarly impaired autophagy and misregulated proteomes much like other autophagy mutants. We further examined the functional redundancy of Arabidopsis ATG8s in late autophagy stages by investigating their interactions with Rab GTPase (RABG)3/RAB7 proteins. We found that all ATG8 representatives could interact with RABG3 proteins via ATG8-interacting motif-LC3-interacting region-docking site interfaces. Such interactions are crucial for RABG3 binding to the autophagosome membrane and probably for the fusion of autophagosomes with the vacuole. However, they are not necessary for endosomal trafficking. With this collection of multiple high-order atg8 mutants, we provide a venue to selectively study the roles of individual ATG8 isoforms during both canonical and noncanonical autophagy in Arabidopsis.