VAMP724 and VAMP726 are involved in autophagosome formation in Arabidopsis thaliana

ABSTRACTMacroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.Abbreviations: ATG, autophagy related; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; Conc A, concanamycin A; EM, electron microscopy; ER, endoplasmic reticulum; FRET, Förster/fluorescence resonance energy transfer; MS, Murashige and Skoog; MVB, multivesicular body; PAS, phagophore assembly site; PM, plasma membrane; PVC, prevacuolar compartment; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TEM, transmission electron microscopy; TGN, trans-Golgi network; WT, wild-type.KEYWORDS: ArabidopsisATG9autophagySNAREVAMP724VAMP726 Disclosure statementNo potential conflict of interest was reported by the author(s).Supplementary MaterialSupplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2022.2127240.Additional informationFundingThis work was supported by grants from the National Natural Science Foundation of China (91854201), the Research Grants Council of Hong Kong (AoE/M-05/12, CUHK 14100818, 14101219, C4033-19E, C4002-17G, C4002-20W, C4002-21EF, C2009-19G, and R4005-18), and The Chinese University of Hong Kong (CUHK) Research Committee and CAS-Croucher Funding Scheme for Joint Laboratories to L.J; the National Natural Science Foundation of China (32061160467 and 31870171) and Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (171014) to C.G.