Dicot-specific ATG8-interacting ATI3 proteins interact with conserved UBAC2 proteins and play critical roles in plant stress responses

Abstract

Selective macroautophagy/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 3 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/UBAC2B (Ubiquitin-associated [UBA] protein 2a/b), 2 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 lumenal 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.

A Family of NAI2-Interacting Proteins in the Biogenesis of the ER Body and Related Structures

“…We have recently reported identification of three related AUTOPHAGY-RELATED PROTEIN8 (ATG8)interacting proteins, ATI3a, ATI3b, and ATI3c, from Arabidopsis and provided evidence that these ATI3 proteins function as selective autophagy receptors through interacting with two ER-localized UBIQUITIN ASSOCIATED DOMAIN CONTAINING2 (UBAC2) proteins and target degradation of specific unknown ER components during plant stress responses (Zhou et al, 2018). To further study the ATI3/UBAC2-mediated selective autophagy pathway, we attempted to identify UBAC2-interacting proteins using yeast twohybrid screens.…”

“…Arabidopsis (Arabidopsis thaliana) plants were grown in growth chambers or growth rooms at 23°C with 120 mE$m 22 $s 21 light intensity and a photoperiod of 12 h of light and 12 h of dark. The ubac2a-1 and ubac2b-1 mutants have been previously described (Zhou et al, 2018). Homozygous nai2-2 (Salk_005896), naip1-1 (FLAG_512D04), naip2-1 (GABI_529B11), naip2-2 (GABI_922B07), and naip3-1 (Salk_200721) were identified by PCR using gene-specific primers flanking the T-DNA/transposon insertions ( Supplemental Table S1).…”

“…In order to find UBAC2a-interacting proteins, a Gal4-based yeast two-hybrid system was utilized as previously described (Zhou J et al, 2018). In brief, UBAC2a was PCR amplified using gene-specific primers (59-agcgaattcatgaac ggcggtccctcc-39 and 59-agcgtcgacttagtttctgtcgaatcccatt-39) and cloned into pBD-GAL4 vector to generate the bait vector.…”

“…The samples were incubated at 100°C for 15 min in the Laemmli sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The myc-tagged NAI2 and PYK10 proteins were detected by western blotting using an anti-myc monoclonal antibody as previously described (Zhou et al, 2018).…”

Arabidopsis Endoplasmic Reticulum-Localized UBAC2 Proteins Interact with PAMP-INDUCED COILED-COIL to Regulate Pathogen-Induced Callose Deposition and Plant Immunity

“…We have recently reported that the mutants for the two close Arabidopsis homologs of UBAC2 are compromised in heat tolerance and resistance to the necrotrophic pathogen Botrytis cinerea (Zhou et al, 2018). To further analyze the roles of the evolutionarily conserved proteins, we analyzed the ubac2 mutants for response to a virulent strain of the bacterial pathogen P. syringae DC3000 (PstDC3000).…”