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 of autophagic bodies in the vacuole (10, 13, 20, 21). formation of YFP-ATG8e–labeled abnormal structures in the Because FM4-64 is taken up into the plant cell from the plasma atg9-3 background during autophagy (Fig. 1B), suggesting that membrane via endocytosis to finally label the tonoplast and may be the formation of these tubules is indeed PI3P-dependent. delivered into the vacuole together with the tonoplast membrane To further confirm that autophagic activity is disturbed in the atg9 (22–24), to distinguish whether these abnormal structures reside mutant, we performed an YFP-ATG8e processing assay, which within the vacuole, we performed an FM4-64 uptake study in both reflects the delivery of autophagosomal membrane to the vacuole YFP-ATG8e and YFP-ATG8e/atg9-3 seedlings to label the tono- (11). Protein fractions from YFP-ATG8e and YFP-ATG8e/atg9-3 plast before BTH and Conc A treatments. As shown in Fig. 1A, plants with or without BTH treatment were subjected to immuno- Bottom, a pronounced accumulation of YFP-ATG8e–positive blotting analysis with a GFP antibody. Consistent with the confocal autophagic bodies within the vacuole was observed in the WT but data, there is much less YFP core detected in YFP-ATG8e/atg9-3 was not evident in YFP-ATG8e/atg9-3 cells. Abnormal tubules after BTH treatment, supporting that ATG9 deficiency impairs (indicated by arrow in Fig. 1A, Bottom) are clearly detected out- autophagosome formation and its subsequent delivery into the side the tonoplast in atg9-3 background, implying that the defective vacuole (Fig. 1C). Such a defect in vacuolar delivery can be reversed

Fig. 1. Dysfunction of ATG9 leads to accumulation of autophagosome-related tubular structures upon BTH induction. (A) YFP-ATG8e–labeled tubules ac- cumulate in atg9-3 after BTH treatment. Four-day- old YFP-ATG8e or YFP-ATG8e/atg9-3 seedlings were exposed to medium without BTH (Top) or with BTH (Middle) for 6 h and visualized under the confocal microscope. The YFP-ATG8e–positive dots are indicated by arrowheads whereas ring-like structures are indicated by arrows and enlarged in the Insets. (Bottom) FM4-64 was applied to label the tonoplast for 1 h, followed with additional BTH and Conc A treatment for 6 h before observation. v, vacuole. The number of autophagosome-related punctae or abnormal tubular structures per root section by Z stack projection with/without BTH treatment is quantified on the Right. The results were obtained from more than 10 individual seedlings (error bars ± SD). (Scale bars: 10 μm.) (B) Wortmannin treatment blocks the formation of YFP-ATG8e tubular structures in atg9-3 mutant. Four-day-old YFP-ATG8e/atg9-3 seedlings were transferred to medium with or without BTH for 6 h, respectively. Additional wortmannin was applied for 2 h after 4-h BTH treatment for subsequent confocal imaging. Ten slices were collected in a total thickness of 5.46 μm for generating the 3D pro- PLANT BIOLOGY jection image. (Scale bars: 10 μm.) Consistent results were obtained from at least three independent experiments. (C) Immunoblot detection of the vacuolar delivery of YFP core in YFP-ATG8e and YFP-ATG8e/ atg9-3 before/after BTH induction. Total proteins were subjected to immunoblot analysis with GFP antibodies. Immunoblotting with cFBPase antibodies was used as a loading control. h, hour. Consistent results were obtained from three independent experiments. (D) Immunoblot detection of the ATG8 lipidation level in WT, atg9-3, and atg5-1. WT, atg9-3, and atg5-1 seedlings were incubated in medium with/without BTH and Conc A treatment for 6 h, respectively. Membrane fractions were subjected to immunoblot analysis with ATG8 antibodies. Immu- noblotting with cFBPase antibodies was used as a loading control. Consistent results were obtained from three independent experiments.

Zhuang et al. PNAS | Published online January 4, 2017 | E427 Downloaded by guest on September 23, 2021 by the transformation of either ATG9-GFP or YFP-ATG9 back mCherry-HDEL signals (Fig. 2B). A typical ER network pattern was into the atg9-3 mutant, which is shown by the observation of accu- observed both before and after BTH treatment in either WT or mulated autophagic bodies within the vacuole in the comple- atg9-3, indicating that the ER architecture is not altered by the BTH mentation lines (Fig. S2). treatment or loss of ATG9 (Fig. 2 A and B). These observations During phagophore development, ATG8 is conjugated to phos- suggest that the emergence of the ER-associated YFP-ATG8e– phatidylethanolamine (PE) by the conjugation system ATG5- labeled tubular structures is due to a defect during autophago- ATG7-ATG12 on the autophagosomal membrane to facilitate some formation in the atg9 mutant, rather than being the result of autophagosome formation (11, 25). Regarding the accumulation of the effect of drug treatment on the ER membrane. the abnormal YFP-ATG8e–labeled tubules in atg9-3 upon auto- To dissect the development of these autophagosome-related phagic induction, we speculate that these YFP-ATG8e–labeled tu- structures in more detail, we carriedoutinvivo3Dimagingby bules might represent increased levels of membrane-associated spinning disk confocal microscopy on YFP-ATG8e/mCherry-HDEL/ ATG8 in atg9-3. Therefore, we conducted a lipidation assay to ex- atg9-3 plants after BTH treatment (Movie S1). Representative stages amine the ATG8-PE levels in the membrane fractions from WT captured are displayed in Fig. 2C.Initially,theYFP-ATG8edot and atg9-3 plants. Immunoblotting analysis with the ATG8 antibody emerges and grows along an ER subdomain (Fig. 2 C, a and b,in- showed that ATG8-PE adducts are increased in atg9-3 after BTH dicated by arrows, and Movie S1). In the subsequent expansion and induction, suggesting that the YFP-ATG8e tubular structures in elongation steps (Fig. 2 C, c–f), the YFP-ATG8e signal turned into atg9-3 accounts for the ATG8-coated autophagosomal membrane, tubules and became more dynamic and tended to seal by forming a whereas no ATG8-PE was detected in the autophagy deficiency ring-like structure. However, it seemed that this step was interrupted, mutant atg5-1 (25) (Fig. 1D). All these results suggest that mal- as it kept moving back and forth to form protruding tubules, which function of ATG9 interrupts autophagic activity, leading to an ac- were finally sandwiched into a multilayer cage-like structure (Fig. 2 C, cumulation of abnormal autophagosome-related tubular structures g and Movies S1 and S2). Noticeably, these YFP-ATG8e-positive upon autophagic induction in Arabidopsis. structures were always accompanied by an ER signal. To get an insight into the nature of these abnormal structures, Autophagosome-Related Tubules in ATG9-Deficient Mutant Are Related we performed immuno-electron microscopy (EM) to examine to the ER Membrane. A close association between ER and auto- their ultrastructure. Four-day-old YFP-ATG8e/atg9-3 root cells phagosomal membranes has been previously reported in plant after autophagic induction were subjected to high-pressure freez- cells (10, 27); we thus hypothesized that these YFP-ATG8e–labeled ing, followed by immuno-EM analysis. Remarkably, large ex- structures might be related to the ER. To address this idea directly, panded long-tubular structures with a size of more than 1,000 nm we transformed the ER marker mCherry-HDEL into YFP-ATG8e/ were detected by labeling with the GFP antibody (Fig. 3 A and C). atg9-3 plants to analyze the correlation signals between YFP-ATG8e In addition, typical ER membranes with membrane-bound and mCherry-HDEL. Consistent with the previous observations for ribosomes were also observed (indicated by open arrowhead in SH3P2-GFP and ATG5-GFP (10, 27), the YFP-ATG8e–labeled Fig. 3A). Because previous studies used the ATG8e antibodies to ring-like signals were detected in close proximity to the mCherry- label the double-membrane autophagosome structures (10, 20), HDEL signal after BTH treatment (Fig. 2A). Remarkably, the YFP- we therefore carried out double-immunogold labeling using anti- ATG8e–labeled tubule in atg9-3 was sandwiched within the ATG8e and anti-calreticulin (ER marker) (29) to examine the

Fig. 2. Autophagosome-related tubular structures are related with ER membranes in atg9-3 mutant. (A) YFP-ATG8e–positive structures are formed in a close proximity to the ER membrane in WT. Four- day-old YFP-ATG8e/mCherry-HDEL seedlings were exposed to medium without/with BTH for 6 h and visualized under the confocal microscopy. (Scale bar: 10 μm.) (B) YFP-ATG8e-positive tubules are accompanied with the ER membrane in atg9-3. Four-day- old YFP-ATG8e/mCherry-HDEL/atg9-3 seedlings were exposed to medium without/with BTH for 6 h and visualized under the confocal microscope. (Scale bar: 10 μm.) (C) A 3D time-lapse acquisition shows the correlated growing YFP-ATG8e–positive tubular structures and the mCherry-HDEL signals. Four-day- old YFP-ATG8e/mCherry-HDEL/atg9-3 transgenic plant with BTH treatment for 5 h was observed by spinning disk confocal microscopy. Arrows indicate the development of an autophagosome-related tubular structure coherent with the ER (see also Movie S1). (Scale bar: 4 μm.)

E428 | www.pnas.org/cgi/doi/10.1073/pnas.1616299114 Zhuang et al. Downloaded by guest on September 23, 2021 PNAS PLUS

Fig. 3. Immuno-EM analysis reveals the autophagosome-related tubular structures are accompanied with the ER membrane in atg9-3 mutant. (A) Immunolabeling with GFP antibodies shows an autophagosome-related tubular structure in YFP- ATG8e/atg9-3 transgenic plants upon DTT induction. Root cells of YFP-ATG8e/atg9-3 after 4 h DTT treatment were high-pressure freezing fixed and sections were subjected to immunolabeling. An overview is shown on the Left. Open arrows indicate gold particles for anti-GFP (10 nm). Open arrowheads indicate the ER membrane. (Scale bars: 500 nm.) (B) Double immunolabeling with ATG8e and calreticulin antibodies are detected on autophagosome-related tubular structures in YFP-ATG8e/atg9-3 after 5 h BTH treatment. Open arrowheads indicate the ER membrane. (Bottom) The enlarged cropped regions (indicated by dashed square) with labeling of gold particles. Open arrows and arrows indicate gold particles for ATG8e (10 nm) and calreticulin antibodies (6 nm), respectively. M, MVB. (Scale bar: 500 nm.) (C) Quantitative analysis of transmission electron microscope immunolocalization signal density in YFP- ATG8e/atg9-3.

possible correlation between the autophagosome-related struc- We therefore used electron tomography to reconstitute the 3D tures and ER membranes. Consistent with the confocal analysis, organization of these tubular structures (Movie S3). A repre- large gold particles for anti-ATG8e labeled autophagosome-like sentative example of tubule-like autophagosome structures in structures with high-curvature stacked tubules whereas small gold the atg9-3 mutant after autophagic induction is depicted in Fig. particles for anti-calreticulin were observed on the same structure 4A, showing a connection with the ER membrane. It would seem with rough ER (indicated by open arrowhead in Fig. 3B) nearby that these multilayer structures also contain small vesicles and (Fig. 3 B and C). ER fragments inside. However, when examined in detail, these vesicle-like structures in fact are narrow tubules that have a di- PLANT BIOLOGY Three-Dimensional Tomographic Reconstruction Reveals a Direct rect membrane continuity with the ribosome-free ER membrane. Connection Between Developing Autophagosomal Membranes and A previous study using an ATG4 overexpression mutant indicated the ER via Multiple Narrow Membranes in atg9-3. The above re- that the isolation membrane and ER are limited to one connection sults, together with previous studies (10, 27), suggest that the (30). In contrast, multiple membrane connection sites (indicated by autophagosomal structures are related to the ER in Arabidopsis. red and blue arrows in Fig. 4 B and D) between the autophagosome- However, it remains to be determined whether these YFP- related structures and theERwereobservedintheatg9-3 mutant, ATG8e–positive tubules and the ER membrane are directly with narrow interfaces ranging from 50 to 100 nm (Fig. 4 B and D). connected to one another or whether they are transiently con- The corresponding 3D tomogram models were drawn (Fig. 4 C and fluent, as in the model proposed for ATG5-GFP and ER (27). E and Movie S4), showing how the large multilayer structure was

Zhuang et al. PNAS | Published online January 4, 2017 | E429 Downloaded by guest on September 23, 2021 interconnected. Taken together, all these data demonstrate a direct cause there is little information on ATG9 in plants, we tried, as a connection between the abnormal autophagosomal tubular struc- first step, to explore the nature of ATG9-containing vesicles in tures and the ER in the atg9-3 mutant, implying that ATG9 is re- plants by accessing the membrane topology of ATG9. We con- quired for efficient budding of autophagosomal intermediates from ducted a protease protection assay with microsomes isolated the ER membrane. from Arabidopsis cells expressing either ATG9-GFP or YFP- ATG9, respectively, followed by immunoblot analysis using a ATG9 Vesicles Are Transiently Associated with the Autophagosome GFP antibody (Fig. S3A). Both the C terminus of ATG9-GFP Membrane upon Autophagic Induction in Arabidopsis. The data and N terminus of YFP-ATG9 were found to be sensitive to obtained so far indicate that ATG9 is essential for the auto- protease digestion because no band was detected in the presence phagosome to develop from the ER membrane. However, the of protease (Fig. S3A, lanes 4 and 5 and 7 and 8) whereas, in the nature of ATG9-positive vesicles in yeast and mammalian cells control using a GFP fusion of the type I integral membrane remains elusive (1), and in plant cells it is totally unknown. Se- protein GFP-VSR2, the luminal GFP remained intact after quence alignment analysis shows that the N- and C-terminal protease digestion (Fig. S3A, lanes 1 and 2) (31). This result regions on ATG9 are highly disordered in different species, but indicates that both the N and C terminus of ATG9 are exposed there is a highly conserved region in the core region compared to the cytosol (Fig. S3A), exhibiting a similar topology to that in with other species (6). Both Arabidopsis and human ATG9 lack a yeast and mammals (32, 33). Interestingly, when ATG9-GFP and long N terminus although it has been suggested that the major mCherry-ATG9 were coexpressed in Arabidopsis cells, they function of the long tail is for protein–protein interactions. Be- colocalized with each other, suggesting that the N or C terminus

Fig. 4. A 3D tomographic analysis reveals multiple- direct connections between the autophagosome- related tubules and the ER membranes in atg9-3. (A) Tomographic slice showing a representative example of tubule-like autophagosome structures that connected with the ER in atg9-3 mutant Arabidopsis roots upon autophagic induction with DTT treatment. (Right) A gallery of serial tomographic slice images display close-up views of membrane connection sites between autophagosomes and the smooth ER (indicated by red arrows). (B–E) To- mographic slices and 3D models show membrane connection sites (indicated by arrows) between autophagosome structures and the smooth ER. The 3D tomographic models of the autophagosome and the ER element in A are shown in C and E.Theout- ermost lamella (green) was continuous with the ER tubule (yellow). Inner lamellae and enclosed compartments were differently color-coded. (Insets) Higher- magnification view of the contact site (dashed square) disclosing the membrane continuity (see also Movies S3 and S4). (Scale bars: A,500nm;B and D,100nm.)

E430 | www.pnas.org/cgi/doi/10.1073/pnas.1616299114 Zhuang et al. Downloaded by guest on September 23, 2021 tag does not affect the targeting of ATG9 (Fig. S3B). ATG9 may efficient development of the phagophore from its initiation site, PNAS PLUS transport through the ER–Golgi pathway, as ATG9-GFP were which leads to the extending ATG8-labeled tubules associated retained on the ER when coexpressed with an Arf mutant (31), with the ER membrane. In this scenario, it is very likely that which interrupts the ER–Golgi traffic (Fig. S3C). ATG9 is essential for mediating the trafficking of certain key To further examine the subcellular localization of ATG9 in the regulator(s) during this process. plant cell, we generated ATG9-GFP transgenic plants. When crossed In both yeast and mammalian cells, ATG9 has been shown to form with different endosomal markers, it seems that the ATG9-GFP a complex with ATG18, a PI3P effector; thus, disturbing ATG9 signals were in close proximity to the trans-Golgi network (TGN) transit from PAS will interfere with the recruitment of both ATG18 marker (VHA-a1-RFP) and late endosome marker (mCherry- and ATG8 onto the phagophore (35). ATG18a and ATG2 have Rha1), but were separate from the trans-Golgi marker (ST-RFP) been reported to function during autophagy in Arabidopsis (36–38), (Fig. 5 A–C, Top). The TGN/early endosome is sensitive to Brefeldin implying that a conserved ATG9–ATG18 complex may participate in A (BFA) treatment by producing “BFA bodies” whereas late autophagosome formation during autophagy. Our coimmunopreci- endosomes will form ring-like structures with the additional PI3K pitation analysis showed that ATG9 indeed forms a complex with inhibitor wortmannin treatment (34). Interestingly, a portion of the ATG18a (Fig. 7C). Transient expression in Arabidopsis protoplasts ATG9-GFP–labeled compartments are sensitive to both BFA and was then used to analyze the subcellular association between auto- wortmannin treatments (Fig. 5 A–C, Bottom). However, the ATG9 phagosomal markers as before (10, 13). We observed that most of the aggregates induced by BFA did not fully colocalize with the VHA- ATG18a signals overlapped with ATG9 whereas a few are associated a1-RFP–labeled signals, implying that they are not identical com- with the autophagosome marker mCherry-ATG8e (Fig. 7D). partments. Upon wortmannin treatment, although the ATG9-GFP To check whether ATG9 mediates the trafficking of ATG18a signals were observed on ring-like structures, they were restricted to a during autophagosome formation, we transformed YFP-ATG18a certain subdomain, distinct from the pattern in mCherry-Rha1. into the WT, atg9-3,andatg5-1 plants, respectively. In the WT Taken together, these results indicate that the ATG9 vesicles may be background, YFP-ATG18a mainly displayed a cytosolic pattern derived from the Golgi compartments. with tiny dots whereas larger YFP-ATG18a foci appeared in the To figure out how the ATG9 vesicles contribute to the auto- cytoplasm after BTH treatment (Fig. 7E, Left), presumably rep- phagosome membrane during autophagy in the plant cell, we resenting a translocation of YFP-ATG18a onto the autophago- crossed the ATG9-GFP transgenic plant with autophagosome re- somal membrane. Noticeably, no obvious YFP-ATG18a–labeled porters, including mCherry-ATG8e and SH3P2-RFP, respectively, autophagic bodies were accumulated in the vacuole after BTH followed by a dynamic analysis of their subcellular distributions after and Conc A treatments (Fig. S5A, Center), and no elevated free autophagic induction. It seems that the majority of the ATG9-GFP YFP core in YFP-ATG18a was detected after autophagic in- and mCherry-ATG8e signals remained separate after autophagic duction (Fig. S5B). These results indicate that at least a portion of induction (Fig. S4A) although a close association occasionally oc- YFP-ATG18a are recycled from the autophagosome membrane curred among more than one ATG9-GFP punctae and mCherry- during autophagy. By contrast, YFP-ATG18a–labeled tubules af- ATG8e foci (Fig. S4B). We further used a spinning disk confocal ter autophagic induction were observed in atg9-3 but not in atg5-1 microscope to capture time-lapse acquisitions, as shown in Fig. 6A. (Fig. 7E, Center and Fig. S5A, Bottom). However, the PI3K in- The ATG9 vesicles seemed to transiently interact with the auto- hibitor wortmannin treatment abrogated the formation of large phagosomal membrane labeled by mCherry-ATG8e, and move YFP-ATG18a foci and YFP-ATG18a–labeled tubules in the WT away after a short period of interaction (Fig. 6A and Movie S5). A and atg9 mutant, respectively (Fig. 7E, Bottom), implying that similar dynamic coherence between ATG9-GFP and SH3P2-RFP translocation of ATG18a onto the autophagosomal membrane is wasalsoobserved(Fig.6B and Movie S6). PI3P-dependent. Taken together, our results reveal that ATG18a It was previously shown that SH3P2 is localized on phagophore- requires a functional ATG9 and PI3K activity for its proper traf- like structures associated with the ER membrane and is delivered ficking during autophagy. into the vacuole together with the autophagosome during autophagy (10); thus, the transient interaction between ATG9-GFP and the Discussion SH3P2-RFP–labeled tubule suggests a possible contribution of Despite current efforts to describe the function of the autophagy ATG9 vesicles to the growing autophagosome. Interestingly, there machinery in plants, the molecular basis underlying the hierar- was no obvious delivery of ATG9-GFP together with mCherry- chical interaction of different ATG proteins is still poorly char- ATG8e in the vacuole even after additional vacuolar inhibitor acterized, thus clouding our understanding of autophagosome treatment (Fig. S4C). A GFP processing assay also confirmed that no formation in plants (3, 39, 40). In particular, dynamic studies and GFP core was detected in ATG9-GFP after BTH treatment, com- ultrastructural information on the initial stages of phagophore paredwiththatinYFP-ATG8e(Fig. S4D). It would therefore seem formation are lacking. However, a previous study using ATG5- that, after the transient association with the autophagosome mem- GFP by live-cell imaging nicely showed the ATG5-decorated brane, ATG9 vesicles are subsequently recycled back into the cyto- phagophore localized to the external surface of the ER (27). In plasmic pool but do not follow the autophagosome into the vacuole. another report, an ER-associated omegasome-like structure and phagophore labeled by SH3P2-GFP have also been observed (10, ATG9 Regulates the Trafficking of ATG18a on the Autophagosomal 41). These observations suggest that the ER might serve as a Membrane in a PI3P-Dependent Manner. To gain an insight into the platform for autophagosome formation, but whether direct possible hierarchical relationship between ATG9 and other ATG membrane continuity exists between the growing phagophore proteins, we first generated a double mutant atg9-3/atg5-1 and the ER is unclear. In this study, by in vivo imaging and expressing YFP-ATG8e to test whether the formation of ab-

electron tomographic reconstructions (Figs. 2 and 4 and Movies PLANT BIOLOGY normal tubular structures is dependent on the ATG5-ATG12 S1 and S2), we provide genetic evidence in Arabidopsis to dem- conjugation system (11) (Fig. 7 A and B). Interestingly, no YFP- onstrate a role of ATG9 for the efficient outgrowth of an auto- ATG8e–labeled abnormal tubules were observed in atg9-3/atg5-1 phagosome from the ER with morphological details. The abnormal after BTH treatment (Fig. 7A), suggesting that this ATG5-de- tubular structures in atg9-3 after BTH autophagic induction exhibit pendent accumulation of YFP-ATG8e–labeled tubular struc- drastic dynamics for tethering with the ER membrane (Fig. 2 and tures is probably impeded after the initial recruitment of ATG8 Movies S1 and S2) via a direct interconnection between the auto- onto the preautophagosomal structures because neither atg5 nor phagosomal membrane and the ER (Fig. 4 and Movies S3 and S4), atg7 loss-of-function mutants formed such tubules (Fig. S1B). placing the ER as one membrane source for plant autophagosome We hypothesized that ATG9 deficiency may interfere with the formation.

Zhuang et al. PNAS | Published online January 4, 2017 | E431 Downloaded by guest on September 23, 2021 Fig. 5. ATG9-GFP vesicles are adjacent to TGN and PVC/MVB. Dual observation of ATG9-GFP and trans-Golgi marker (ST-RFP) (A) and TGN marker (VHA1-a1- RFP) (B), as well as PVC/MVB marker (mCherry-Rha1) (C), in root tip cells reveals that ATG9-GFP–labeled punctae are adjacent to the TGN and PVC/MVB (Top), and sensitive to both BFA and wortmannin treatment (A–C, Bottom). For drug treatments, 4-d-old plants were incubated in medium with either BFA for 1 h or wortmannin for 2 h before observation. Colocalization relationship was calculated by Pearson–Spearman correlation. (Scale bars: 10 μm.)

Compared with other autophagy deficiency mutants (e.g., atg5-1 phagic defect in atg9, ATG9 vesicles are therefore probably and atg7-2) that fail to form autophagosomal structures, it is recruited onto the autophagosomal membrane for efficient auto- intriguing that malfunction of ATG9 leads to the formation of ab- phagosome progression from the ER. normal autophagosome-related tubules (Fig. 1A and Fig. S1B). We Autophagosome formation requires a retrieval disassociation found that ATG9 dysfunction does not affect ATG8 conjugation of core ATG proteins from the autophagosomal membrane, onto the autophagosomal membrane in Arabidopsis (Fig. 1D). On which is mainly regulated by retrograde transport of ATG9 from the contrary, the formation of extending YFP-ATG8e tubules along the phagophore (42). A previous study indicates that recycling of theERmembraneinatg9-3 requires PI3K activity and the ATG5 ATG18a during autophagosome formation is mediated by ATG9 conjugation system (Figs. 1B and 7A), thus indicating an in- (35). In this context, we found that ATG18a forms a complex volvement of ATG9 in autophagosome progression downstream of with ATG9 in Arabidopsis (Fig. 7 C and D) and that both the PI3P and the initial recruitment of ATG8 onto the autophagosome ATG9 and ATG18a are recycled and dissociated from the membrane. Our observation shows that, in the normal condition, completed autophagosome during autophagy, suggesting that a ATG9 reside on cytoplasmic compartments adjacent to the TGN conserved ATG9–ATG18 complex functions in autophagy across and prevacuolar compartment (PVC)/multivesicular body (MVB) eukaryotes. In Arabidopsis, it is reported that knockdown of in Arabidopsis. However, after autophagic induction, transient as- ATG18a impairs autophagosome formation (36). Our observa- sociation between ATG9-GFP–positive punctae and the autopha- tion shows that, upon autophagic induction, ATG9 dysfunction gosomal membrane, labeled by mCherry-ATG8e or SH3P2-RFP, leads to the formation of abnormal YFP-ATG18a–labeled tu- was observed by live-cell imaging (Fig. 6). Together with the auto- bules in a PI3P-dependent manner after autophagic induction

E432 | www.pnas.org/cgi/doi/10.1073/pnas.1616299114 Zhuang et al. Downloaded by guest on September 23, 2021 PNAS PLUS

Fig. 6. ATG9 vesicles are transiently associated with the autophagosome membrane during autophagosome formation. (A) Time-lapse dynamics analysis shows transient association between the ATG9-GFP punctae and the mCherry-ATG8e–labeled foci (see also Movie S5). Four-day-old ATG9-GFP/mCherry- ATG8e transgenic plants were treated with DTT for 4 h and observed under the spinning disk confocal microscope. The dashed square indicates the cropped region for the time-lapse analysis. s, second. (Scale bar: 10 μm.) (B) Time-lapse dynamics analysis shows the transient association between the ATG9-GFP punctae and the SH3P2-RFP–labeled structure (see also Movie S6). Four-day-old ATG9-GFP/SH3P2-RFP transgenic plants were exposed to medium with BTH for 5 h and observed under the spinning disk confocal microscope. The dashed square indicates the cropped region for the time-lapse analysis. s, second. (Scale bar: 10 μm.)

(Fig. 7E). Therefore, we presume that, in Arabidopsis,ATG9 DTT in the atg9 mutant (Fig. 4) would reflect a specific role of deficiency interferes with autophagosome formation by disturbing ATG9 in ER-phagy rather than in general autophagy. Further in- the recycling of ATG18a from the autophagosomal membrane, vestigations are obviously needed to better understand the role of which subsequently leads to impaired autophagosome formation ATG9 in regulating this ER-dependent autophagosome formation. and increased levels of ATG8-PE. This presumption does not necessarily exclude the proposal that ATG4 is required for the Materials and Methods deconjugation of ATG8-PE (43), and it is possible that the in- Additional materials and methods, including plant materials, antibodies, crement of ATG8-lipidation in atg9-3 is also due to a reduced plasmid construction, immunoprecipitation, confocal microscopy, and a list of turnover of ATG8-PE mediated by ATG4. primers are described in SI Materials and Methods and Table S1. An increasing number of studies have placed emphasis on the role of ATG9 in the ER-related trafficking machinery for autophagosome Plant Materials and Growth and Treatment Conditions. Arabidopsis thaliana biogenesis. For example, ATG9 is able to recruit the vesicle tethering transfer DNA (T-DNA) insertional mutants atg9-2 (SALK_130796) (26), atg9-3 machinery, TRAPPIII complex and Ypt1, to the PAS whereas ab- (SALK_128991) (16), and atg5-1 (SAIL_129_B07) (25) were obtained from the sence of ATG9 impairs the trafficking of Trs85 or Ypt1 onto the Arabidopsis Information Resource (TAIR) (www.arabidopsis.org/). atg7-2 (GABI_655B06) (15) T-DNA insertion mutant was obtained from the Not- autophagosome membrane (44, 45). It has also been shown that tingham Arabidopsis Stock Centre. atg9-4 (SALK_145980) mutant was as UVRAG, another component of the ER tethering complex, works previously reported (16, 17). Seeds were surface sterilized and sown on together with the PI3K complex to mobilize ATG9 translocation plates with Murashige and Skoog (MS) salts plus 0.8% agar. The seeded during autophagosome formation (46). Therefore, ATG9 deficiency plates were kept at 4 °C for 3 d before being moved to the growth chamber. may result in a failure to deliver the tethering machinery that is es- The plates were incubated at 22 °C under a long-day (LD) (16 h light and 8 h sential for autophagosome fission from the ER membrane in Ara- dark) photoperiod. Plants exposed to LD conditions were transferred to soil bidopsis. In the future, efforts are required to focus on the molecular after 2 wk. For autophagic induction, 4- or 5-d-old seedlings were trans- mechanism of ATG9 with the ER-related machineries in plant cells. ferred in liquid MS with methanol (1:100) as control or 100 μM BTH for at We noticed that, in the atg9-3 mutant, some YFP-ATG8e signals least 5 h or 2 mM DTT for 4 h before observation or as indicated. Conc A was are not associated with the ER membrane (Fig. 2 A and B)andthat used at a concentration of 0.5 μM. PI3K activity was inhibited by the addition the whole autophagic activity is not fully blocked in the atg9-3 of 8.95 μM wortmannin for 2–4 h in the medium. Then, 10 μg/mL BFA was mutant (Fig. 1 A and C), suggesting that ATG9 is not necessarily applied in the medium and incubated for 1 h before observation. required for the whole autophagic flux. Because multiple membrane sources have been implicated to contribute to autophago- ATG8 Lipidation Assay. An ATG8 lipidation assay was carried out essentially as some biogenesis (30, 47–49), other membrane sources that are described previously (11). The 4- or 5-d-old seedlings were transferred in PLANT BIOLOGY liquid MS with methanol (1:100) as control, or with 100 μM BTH and 0.5 μM ATG9- or ER-independent might also contribute to autophago- Conc A for 6 h, respectively, followed by extraction in lysis buffer containing some formation. On the other hand, autophagy in plant can be 25 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1× Complete Pro- induced by different stimuli (e.g., starvation, hydrogen peroxide, tease Inhibitor Mixture (Roche). The total cell extracts were centrifuged at DTT, and BTH) to trigger certain autophagic processes like 500 × g for 5 min at 4 °C. The supernatant was centrifuged at 100,000 × g for chlorophagy, pexophagy, ribophagy, or ER-phagy (10, 13, 18, 19, 30 min, and the membrane pellets were solubilized in an equal volume of 38). Because both BTH and DTT treatments may induce ER stress lysis buffer with additional 1% Triton X-100. Protein samples were subjected (19, 50), our observation of the tight coherence between the ER to SDS/PAGE in the presence of 6 M urea and analyzed by immunoblotting membrane and autophagosome structures induced by BTH and with anti-ATG8 (Agrisera) at a dilution of 1:1,000.

Zhuang et al. PNAS | Published online January 4, 2017 | E433 Downloaded by guest on September 23, 2021 Fig. 7. ATG9 regulates the trafficking of ATG18a on the autophagosomal membrane in a PI3P-dependent manner. (A) Formation of YFP-ATG8e tubules in atg9-3 during autophagy in a process that requires ATG5. Four-day-old YFP-ATG8e/atg9-3/atg5-1 seedlings were exposed to medium without BTH (Left), with BTH (Center), or with BTH and Conc A (Right) treatments for 6 h, respectively, and visualized under the confocal microscope. (Scale bar: 10 μm.) (B) Genotyping of the atg9-3/atg5-1 double transgenic plants. Similar results were obtained from three independent experiments. (C) Immunoprecipitation assay shows that YFP-ATG18a is associated with ATG9-5Flag. Cell lysate from Arabidopsis protoplasts transiently expressing GFP or YFP-ATG18a together with ATG9-5Flag for 12 h were subjected to a GFP trap assay. The resulting immunoprecipitation and cell lysate were analyzed by immunoblotting using Flag or GFP antibodiesas indicated. (D) Subcellular localization analysis among ATG18a, ATG9, and ATG8e. (Top) Coexpression of ATG9-GFP and mCherry-ATG18a. (Middle) Coex- pression of YFP-ATG18a, ATG9-CFP, and mCherry-ATG8e. (Bottom) The merged image from the Center and the area indicated in the dash box was enlarged on the Right. Constructs were transiently expressed in Arabidopsis protoplasts for 12 h before observation. Colocalization relationship was calculated by Pearson–Spearman correlation. (Scale bars: 50 μm.) (E) Accumulation of ATG18a tubules in atg9-3 after BTH induction in a PI3P-dependent manner. Four-day- old YFP-ATG18a and YFP-ATG18a/atg9-3 seedlings were transferred to medium with or without BTH for 6 h, respectively. Additional wortmannin was applied for 2 h after 4 h BTH treatment for subsequent confocal imaging. The number of autophagosome-related punctae or abnormal tubular structures per root section by Z stack projection with indicated treatment was quantified on the Right. The results were obtained from more than 10 individual seedlings (error bars ± SD). (Scale bar: 10 μm.)

EM Analysis of Resin-Embedded Cells. The general procedures used to prepare by a poststaining procedure using aqueous uranyl acetate/lead citrate. Sections transmission electron microscopy samples and ultrathin sectioning of samples were examined using a Hitachi H-7650 transmission electron microscope with a have been described previously (10). For high-pressure freezing, 4-d-old charge-coupled device (CCD) camera operating at 80 kV (Hitachi High-Technol- transgenic root tips with indicated treatment were cut from the seedlings ogies Corporation, www.hitachi-hightech.com/jp/). and immediately frozen in a high-pressure freezing apparatus (EM PACT2, Leica, www.leica-microsystems.com/home/), with subsequent freeze substitution in dry Electron Tomography, 3D Reconstruction, and Modeling. Sample sectioning, acetone containing 0.1% uranyl acetate at −85 °C. Infiltration with HM20, em- poststaining, and electron microscopy imaging were performed as described bedding, and UV polymerization were performed stepwise at −35 °C. For previously (51, 52). In brief, 220-nm-thick sections were cut and poststained immunolabeling, standard procedures were performed as described previously with uranyl acetate and lead citrate. Electron tomography observations (10). The working concentration of rat anti-ATG8e (10) and rabbit anti-GFP an- were performed with an FEI Tecnai F20 transmission electron microscope. tibodies was 40 μg/mL Calreticulin antibodies (29) were used at a dilution of For each grid, a tilt image stack from +60° to −60° with 2° increments was 1:200. Gold particle-coupled secondary antibodies were diluted at 1:40, followed collected, and the second tilt image stack was collected by rotating the grid

E434 | www.pnas.org/cgi/doi/10.1073/pnas.1616299114 Zhuang et al. Downloaded by guest on September 23, 2021 by 90°. Dual-axis tomograms were calculated from pairs of image stacks with of Leeds) for the calreticulin antibody, and Professor Chris Hawes (Oxford PNAS PLUS the etomo program of the IMOD software package (bio3d.colorado.edu). Brookes University) for the ST-RFP seed. This work was supported by grants from The 3D models were generated using the 3dmod program of the IMOD the Research Grants Council of Hong Kong (CUHK465112, 466613, and 14130716 software package. and CUHK2/CRF/11G, C4011-14R, and AoE/M-05/12), the National Natural Science Foundation of China (31270226 and 31470294), NSFC/RGC (N_CUHK406/12), the ACKNOWLEDGMENTS. We thank Professor Diane Bassham (Iowa State Chinese Academy of Sciences-Croucher Funding Scheme for Joint Laborato- University) for sharing the atg9-4 seed, Professor Jurgen Denecke (University ries, and the Shenzhen Peacock Project (KQTD201101) (to L.J.).

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