Functional Analysis of ER Localized Ubac2 and Ubac2- Interacting Proteins From Arabidopsis

Selective autophagy targets specific cargo by autophagy receptors through interaction with ATG8 (autophagy-related protein 8)/MAP1LC3 (microtubule associated protein 1 light chain 3) for degradation in the vacuole. Here, we report the identification and characterization of three related ATG8-interacting proteins (AT1G17780/ATI3A, AT2G16575/ATI3B and AT1G73130/ATI3C) from Arabidopsis. ATI3 proteins contain a WxxL LC3-interacting region (LIR) motif at the C terminus required for interaction with ATG8. ATI3 homologs are found only in dicots but not in other organisms including monocots. Disruption of ATI3A does not alter plant growth or development but compromises both plant heat tolerance and resistance to the necrotrophic fungal pathogen Botrytis cinerea. The critical role of ATI3A in plant stress tolerance and disease resistance is dependent on its interaction with ATG8. Disruption of ATI3B and ATI3C also significantly compromises plant heat tolerance. ATI3A interacts with AT3G56740/ubac2A and AT2G41160/ubac2 B (Ubiquitin-associated [UBA] protein 2a/b), two conserved proteins implicated in endoplasmic reticulum (ER)-associated degradation. Disruption of ubac2A and ubac2B also compromised heat tolerance and resistance to B. cinerea. Overexpression of ubac2 induces formation of ATG8- and ATI3-labeled punctate structures under normal conditions, likely reflecting increased formation of phagophores or autophagosomes. The ati3 and ubac2 mutants are significantly compromised in sensitivity to tunicamycin, an ER stress-inducing agent, but are fully competent in autophagy-dependent ER degradation under conditions of ER stress when using an ER luminal marker for detection. We propose that ATI3 and ubac2 play an important role in plant stress responses by mediating selective autophagy of specific unknown ER components. Further analysis shows that ubac2 also plays a role in Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). PTI is initiated upon recognition of PAMPs by pattern recognition receptors (PRRs), which is then transmitted through a complex signaling cascade including changes of the phosphorylation status of regulators and enzymes, ultimately leading to the production of a variety of signaling and antimicrobial molecules such as reactive oxygen species (ROS) and callose deposition. Here, we report that mutants for two Arabidopsis genes encoding close homologs of UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2 (ubac2), a conserved component of endoplasmic reticulum (ER) protein quality control (ERQC), were compromised in PTI based on their hypersusceptibility to a type III secretion system-deficient strain of the bacterial pathogen Pseudomonas syringae. The ubac2 mutants were normal in the biogenesis of PRRs, activation of MAPKs, production of ROS and PTI-associated gene expression. Pathogen- and PAMP-induced callose deposition, however, was compromised in the ubac2 mutants. ubac2 proteins interact with plant-specific long coiled-coil protein PAMP-induced Coiled Coil (PICC), whose mutants were also compromised in PTI and callose deposition. Compromised callose deposition in both the ubac2 and picc mutants was associated with reduced biogenesis of PMR4 calllose synthase responsible for pathogen-induced callose synthesis. Constitutive overexpression of PMR4 restored callose synthesis and PTI in both the ubac2 and picc mutants. These results indicate that ubac2 and PICC are components of a specific ERQC pathway with a critical role in PMR4 accumulation and callose deposition in plant innate immunity. Using yeast two-hybrid screening, we found that ubac2 interacts with three unknown proteins weakly which were demonstrated to interact with ER body component NAI2 strongly later, thus these three unknown proteins were named NAIP. ER body is one type of vesicles produced by ER in plants. These vesicles accumulate and transport proteins, lipids and metabolites. In the Brassicales including Arabidopsis, ER body is found throughout the epidermis of cotyledons, hypocotyls and roots. NAI2 is a key factor for the ER body formation in Arabidopsis. Homologs of NAI2 are found only in the Brassicales and, therefore, may have evolved specifically to enable the ER body formation. Here, we report that three related NAI2-interacting proteins (NAIP1, 2 and 3) from Arabidopsis play a critical role in the biogenesis of the ER bodies and related structures. Confocal microscopy using GFP fusions revealed that all three NAIPs are components of the ER bodies found in the cotyledons, hypocotyls and roots. Genetic analysis with the mutants for the NAIPs supports that they have a critical and redundant role in the ER body formation. NAIP2 and NAIP3 are also components of other vesicular structures likely derived from the ER that are formed independent of NAI2 and are present not only in the cotyledons, hypocotyls and roots but also in the rosette leaves. These results indicate that while NAIP1 is a specialized ER body component, NAIP2 and NAIP3 are less specialized and can be components of different types of ER-derived structures. Analysis with chimeric NAIP proteins revealed that their N-terminal domains play a major role in the functional specialization in the biogenesis of distinct types of ER-derived compartments. Unlike NAI2, NAIPs have homologs in all plants and, therefore, NAIP-containing ER structures are likely to be present widely in plants. The ER bodies in the Brassicales with a specialized function may have evolved from some of these NAIP-containing ER structures likely present widely in plants