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ATG9 Regulates Autophagosome Progression from the Endoplasmic Reticulum in Arabidopsis

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ATG9 Regulates Autophagosome Progression from the Endoplasmic Reticulum in Arabidopsis ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis Xiaohong Zhuanga,b,1, Kin Pan Chunga,b,1, Yong Cuia,b,1, Weili Lina,b, Caiji Gaoa,b,2, Byung-Ho Kanga,b, and Liwen Jianga,b,c,3 aCentre for Cell & Developmental Biology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; bState Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and cThe Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, China Edited by Diane C. Bassham, Iowa State University, Ames, IA, and accepted by Editorial Board Member Maarten J. Chrispeels December 8, 2016 (received for review October 6, 2016) Autophagy is a conserved pathway for bulk degradationofcytoplasmic autophagy pathway because ATG9 was required for the biogenesis material by a double-membrane structure named the autophagosome. of ER-derived compartments during the unfolded protein response The initiation of autophagosome formation requires the recruitment of (9). However, whether ATG9 plays a direct role in the early stages autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal of autophagosome formation or in a specific autophagy process structure. However, the functional relationship between ATG9 vesicles remainstobeinvestigatedinplants.Onemajorchallengeisthelack and the phagophore is controversial in different systems, and the mo- of morphologically informative visualization that might correlate the lecular function of ATG9 remains unknown in plants. Here, we demon- early autophagosomal structures and ATG9 vesicles in real-time and strate that ATG9 is essential for endoplasmic reticulum (ER)-derived in three dimensions. autophagosome formation in plants. Through a combination of ge- In this study, by characterizing an Arabidopsis ATG9 deficient netic, in vivo imaging and electron tomography approaches, we show mutant, we have shown that ATG9 is essential for ER-derived that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome formation in plant cells. Through a combination of autophagosome-related tubular structures in direct membrane continu- genetic, in vivo imaging by spinning-disk confocal microscopy and ity with the ER upon autophagic induction. Dynamic analyses demon- 3D electron tomography reconstruction, we have demonstrated that strate a transient membrane association between ATG9 vesicles and the autophagosomal membrane is a clear outgrowth from an ER the autophagosomal membrane during autophagy. Furthermore, traf- subdomain, unveiling a unique role of ATG9 in autophagosome ficking of ATG18a is compromised in atg9 mutants during autophagy – progression from the ER and ATG18a trafficking during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphate in plant cells. dependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from Results the ER membrane in Arabidopsis. ATG9 Malfunction Results in Accumulation of Abnormal Autophagosome- Related Tubules upon Autophagic Induction in Arabidopsis. The auto- autophagy | ATG9 | autophagosome | endoplasmic reticulum | ATG18 phagosome formation process is conserved, and ATG8 has been used as an autophagosomal marker in Arabidopsis (10–14). In ne long-lasting question regarding autophagosome bio- Ogenesis is its membrane origin (1). The initiation site for Significance autophagosomes is termed the preautophagosomal structure or phagophore assembly site (PAS). However, the source of the phagophore membrane remains controversial in different systems, One fundamental question in the autophagy field is the mem- and exactly how the phagophore is initiated from its membrane brane origin of the autophagosome. As the sole transmembrane origin is still unclear. The core autophagy-related (ATG) ma- autophagy-related (ATG) protein, ATG9 is conserved among eu- chinery regulates phagophore assembly in a spatiotemporally co- karyotes and known to be important for autophagy, but its pre- ordinated manner whereas some of the ATG components will cise molecular function is still unknown. Through a combination of disassociate from the completed autophagosome and some are in vivo real-time imaging, 3D tomographic reconstruction, and turned over together with the autophagosome (1–3). genetic approaches, this study demonstrates that, in contrast to As the sole transmembrane protein, autophagy-related protein the atg9 mutants characterized in yeast and animal, loss of ATG9 9 (ATG9) has long been suggested to provide a lipid/membrane in Arabidopsis led to expanding autophagosome-related tubules connected to the endoplasmic reticulum during autophagy. This source for autophagosome formation because ATG9-deficient mu- work thus provides functional evidence for a unique role of ATG9 tants in yeast or mammal fail to form autophagosomes (4, 5). Al- in autophagosome progression from the endoplasmic reticulum in though ATG9 is conserved in all eukaryotes (6), it seems that ATG9 plant cells, shedding new light on the membrane origins of auto- might perform its function divergently in different systems. In yeast, phagosome in plants. ATG9 participates in an early step by shuttling from a non-PAS site to the PAS site and supports an assembly model for yeast auto- Author contributions: X.Z., K.P.C., Y.C., and L.J. designed research; X.Z., K.P.C., Y.C., W.L., phagosome biogenesis (4). In contrast, mammalian ATG9 is not and C.G. performed research; X.Z., K.P.C., Y.C., and B.-H.K. analyzed data; and X.Z., Y.C., stably incorporated into the isolation membrane or autophagosomes and L.J. wrote the paper. but is instead transiently associated with the omegasome, a phos- The authors declare no conflict of interest. phatidylinositol 3-phosphate (PI3P)-enriched endoplasmic reticulum This article is a PNAS Direct Submission. D.C.B. is a Guest Editor invited by the (ER) subdomain (5). Cryomicroscopy studies have shown a close Editorial Board. 1 association between ATG9 vesicles and the omegasome structure X.Z., K.P.C., and Y.C. contributed equally to this work. 2 (7), together with the presence of ATG9 on tubulovesicular mem- Present address: Guangdong Provincial Key Laboratory of Biotechnology for Plant De- velopment, School of Life Sciences, South China Normal University, Guangzhou 510631, branes surrounding autophagosomes (5). A recent finding by live- China. cell imaging indicates that autophagosome formation occurs where 3To whom correspondence should be addressed. Email: [email protected]. ATG9 vesicles coalesce with the ER (8). In addition, it is suggested This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in yeast that ATG9 may play a distinct role in the ER-dependent 1073/pnas.1616299114/-/DCSupplemental. E426–E435 | PNAS | Published online January 4, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1616299114 Downloaded by guest on September 23, 2021 Arabidopsis, an ATG9 counterpart has also been identified and YFP-ATG8e–labeled structures are compromised before their PNAS PLUS shown to affect autophagic activity during starvation or pathogen delivery into the vacuole. Consistent with previous studies, neither infection (6, 15–17). To investigate the functional role of ATG9 in autophagic bodies nor abnormal tubules were detected after BTH autophagosome formation in plant cells, we transformed the auto- and Conc A treatments in the autophagy-deficient mutants atg5-1 phagosomal marker YFP-ATG8e into an ATG9-deficient mutant, (25) and atg7-2 (15) (Fig. S1B). Similar defects were also observed atg9-3 (9). Previous studies have shown that exogenous benzothia- in four individual YFP-ATG8e/atg9-3 transgenic lines and with diazole (BTH) or dithiothreitol (DTT) treatment can trigger another two independent ATG9 alleles, atg9-2 (26) and atg9-4(16, autophagy in plants (10, 18, 19). After BTH application, more YFP- 17), as well as under a different autophagy inducer, DTT (Fig. S1 ATG8e–labeled dots (indicated by arrowheads in Fig. 1A, Left)and C–E). These results indicate that the formation of YFP-ATG8e– ring-like structures (indicated by arrows in Fig. 1A, Left) were labeled tubular structures upon autophagic induction is specifically observed in wild-type (WT) root cells. Surprisingly, in the atg9-3 caused by lack of a functional ATG9. mutant background, the YFP-ATG8e signals accumulated on long- It has been reported previously that the phagophore is initiated extending tubular structures after BTH treatment (Fig. 1A, Center). from an omegasome-like structure that arises from a PI3P- Quantitative analysis from Z-stacking sections (Fig. S1A) showed a enriched ER subdomain, where the conjugation of ATG8-PE significant suppression of YFP-ATG8e foci formation in atg9-3 occurs (1). The phosphoinositide 3-kinase (PI3K) inhibitor wort- during autophagy, compared with that in the WT, whereas abnor- mannin has been used to block this process, in which the forma- mal tubules accumulated in atg9-3 after BTH treatment (Fig. 1A, tion of autophagosome-related structures [labeled by ATG5, Right). To further examine the effect of ATG9 deficiency on the ATG8, or SH3 domain-containing protein (SH3P2)] were sup- autophagic flux, we next treated the cells with BTH and con- pressed upon wortmannin treatment in plant (10, 27, 28). In- canamycin A (Conc A), a V-ATPase inhibitor, to prevent the terestingly, we found that the wortmannin treatment abrogates the degradation
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