ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis

Abstract

Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphate-dependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.

Keywords: ATG18; ATG9; autophagosome; autophagy; endoplasmic reticulum.

Fig. 1.

Dysfunction of ATG9 leads to accumulation of autophagosome-related tubular structures upon BTH induction. (A) YFP-ATG8e–labeled tubules accumulate 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 projection 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. Immunoblotting with cFBPase antibodies was used as a loading control. Consistent results were obtained from three independent experiments.

Fig. S1.

YFP-ATG8e forms abnormal tubules in atg9 mutants upon autophagic induction. (A) Z-stack projection for the 4-d-old YFP-ATG8e and YFP-ATG8e/atg9-3 root sections with/without BTH treatment. (Scale bar: 10 μm.) (B) No YFP-ATG8e labeled autophagic bodies or abnormal tubular structures are accumulated in atg5-1 and atg7-2 mutant plants upon BTH and Conc A treatment. (Scale bar: 10 μm.) (C) Four individual YFP-ATG8e/atg9-3 transgenic lines display a similar defect in forming the YFP-ATG8e–labeled tubular structures upon BTH treatment. (Scale bar: 10 μm.) (D) Abnormal YFP-ATG8e–labeled tubular structures are observed in two additional independent atg9 T-DNA insertion mutants atg9-2 and atg9-4 upon BTH treatment. (Scale bar: 10 μm.) (E) YFP-ATG8e–labeled autophagic bodies are observed upon DTT and Conc A treatment in WT background whereas abnormal YFP-ATG8e–labeled tubular structures are detected in atg9-3 mutants. (Scale bar: 10 μm.)

Fig. S2.

ATG9-GFP or YFP-ATG9 restored the defective vacuolar delivery of autophagosome in atg9-3. Differential interference contrast micrographs show the root epidermal cells exposed to DTT and Conc A treatment for 6 h. Similar to the WT cell, accumulation of autophagic bodies in the vacuole is restored in atg9-3 root cells expressing ATG9-GFP or YFP-ATG9, which is not evident in atg9-3. (Scale bar: 10 μm.)

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.)

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.

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) Tomographic 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. The outermost 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, 500 nm; B and D, 100 nm.)

Fig. S3.

Topology analysis of ATG9. (A) Topology analysis of ATG9 by trypsin digestion assay, showing that ATG9 has its both N and C terminus facing to the cytosol. Microsomes isolated from Arabidopsis cells expressing either ATG9-GFP or YFP-ATG9, respectively, were subjected to protease protection assay and immunoblotted with GFP antibody. The type I integral membrane protein GFP-VSR2 is used as a control. A diagram depicting the topology of ATG9 is shown on the Right. (B) ATG9-GFP and YFP-ATG9 are colocalized in Arabidopsis protoplasts. (Scale bar: 50 μm.) (C) ARF1(Q71L) blocks ER export of ATG9-GFP and the Golgi marker Man1-RFP upon their coexpression together with ARF1(Q71L) in Arabidopsis protoplast. (Scale bar: 50 μm.)

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.)

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. S4.

ATG9-GFP has transiently interactions with autophagosomal membrane and is recycled during autophagy. (A) ATG9-GFP vesicles are rarely overlapped with mCherry-ATG8e–labeled ring-like structure. Four-day-old ATG9-GFP/mCherry-ATG8e transgenic plants were exposed to medium without/without BTH for 6–8 h, followed by confocal imaging. (Scale bars: 10 μm.) (B) Example shows an association between the ATG9-GFP vesicles and an mCherry-ATG8e–labeled structure. (Scale bars: 10 μm.) (C) Confocal analysis shows that ATG9-GFP are not delivered together with autophagosome into the vacuole. Four-day-old ATG9-GFP/mCherry-ATG8e transgenic plants were exposed to medium with BTH and Conc A for 6 h. (Bottom) The 3D projection of 10 slices in a total thickness of 5.46 μm. (Scale bar: 10 μm.) (D) GFP processing assay shows that ATG9-GFP are not degraded upon autophagic induction. Four-day-old ATG9-GFP and YFP-ATG8e seedlings were exposed to medium without/with BTH for 6 h, and protein extracts were subjected to Immunoblot by GFP antibody. Immunoblotting with cFBPase antibodies was used as a loading control.

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 antibodies as indicated. (D) Subcellular localization analysis among ATG18a, ATG9, and ATG8e. (Top) Coexpression of ATG9-GFP and mCherry-ATG18a. (Middle) Coexpression 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.)

Fig. S5.

YFP-ATG18a recycles from the autophagosomal membrane during autophagy. (A) YFP-ATG18a is translocated onto the autophagosomal membrane in a process that requires ATG5 during autophagy. Four-day-old YFP-ATG8e, YFP-ATG18a, and YFP-ATG18a/atg5-1 were transferred to medium with/without BTH and Conc A and observed under the confocal microscopy. (Scale bar: 10 μm.) (B) GFP processing assay shows that YFP core is not increased in YFP-ATG18a after autophagic induction. Four-day-old YFP-ATG18a and YFP-ATG8e were exposed to medium without/with BTH for 6 h, and protein extracts were subjected to immunoblot by GFP antibody. Immunoblotting with cFBPase antibodies was used as a loading control.