Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole

Zhao et al. reveal amphisome compartments in plant cells. By characterizing the autophagy adaptor CFS1 that can interact with both ATG8, and the ESCRT-I complex subunit VPS23A; they show that autophagosomes are sorted at amphisomes before arriving their final destination, the central vacuole.


Introduction
we compared the localization patterns of mCherry-CFS1 and mCherry-CFS1 AIM relative to GFP-ATG8A. 140 mCherry-CFS1 formed a significantly higher number of colocalizing puncta compared to mCherry-CFS1 AIM 141 under both control and autophagy inducing conditions ( Fig. 2E-F). Collectively, these results suggest that CFS1 142 interacts with ATG8 in an AIM-dependent manner and that the CFS1-ATG8 interaction is essential for CFS1 143 function and autophagosome localization.

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To further study the residual puncta formed by mCherry-CFS1 AIM , we crossed mCherry-ATG8E with the atg5 146 mutant, which does not form autophagosomes (Thompson et al., 2005). mCherry-CFS1 was still able to form 147 a limited number of puncta in both basal and autophagy inducing conditions, indicating that CFS1 forms puncta 148 independently of autophagosomes. (Fig. 2G-H). To understand how CFS1 could still form puncta in the absence of autophagy, we looked at the other functional domains on CFS1. CFS1 has well-defined FYVE and 150 SYLF domains that bind phosphatidylinositol-3-phosphate (Pi3P) and actin, respectively (Sutipatanasomboon 151 et al., 2017) (Fig. S9A). Mutating these domains, either individually or in combination, did not alter protein 152 stability but did lead to diffuse localization patterns and disrupted ATG8 co-localization ( Fig. S9B-C). These  Bassham, 2015), which enabled us to quantify puncta within the vacuolar lumen. Upon induction of 169 autophagy with salt stress, we found significantly less CFS1 puncta compared to ATG8 or NBR1 puncta (Fig.3 170 B-C). Consistently, although both CFS1 and NBR1 colocalize with ATG8E at cytosolic autophagosomes, upon 171 conA treatment there were fewer colocalizing CFS1-ATG8 puncta inside the vacuole compared to NBR1-172 ATG8 puncta (Fig.3D). These results suggest CFS1 functions as an autophagy adaptor. Since autophagy 173 adaptors should localize on the outer autophagosome membrane, we performed immunogold-labeling TEM 174 experiments on Arabidopsis seedlings expressing mCherry-CFS1. We could readily detect gold particles on the   (Stolz et al., 2014).We next examined the role of CFS1 in this process. First, we expressed mCherry-ATG8E 183 in wild type Col-0, cfs1, or atg5, and quantified the number of mCherry-ATG8E-labelled autophagosomes in 184 root epidermal cells of these lines. Upon autophagy induction with salt stress, significantly more 185 autophagosomes accumulated in the cytosol of cfs1 cells compared to Col-0 ( Fig. 4A-B). However, after 186 treatment with conA, which stabilizes autophagic bodies in the vacuole, cfs1 mutants had significantly less 187 mCherry-ATG8E puncta ( Fig. 4A-B). atg5 mutants had no autophagosomes in either condition ( Fig. 4A-B).

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These results suggest that cfs1 mutants have defects in the delivery of autophagosomes to the vacuole, which 189 leads to the accumulation of autophagosomes in the cytosol. To support these data, we performed GFP-release 190 assays under the same conditions. When GFP-ATG8 is delivered to the vacuole, a stable GFP fragment is 191 released due to vacuolar protease activity. The ratio of free GFP to GFP-ATG8 can thus be used to quantify 192 autophagic flux (Bassham, 2015;Yoshii and Mizushima, 2017). Quantification of five independent experiments 193 showed that cfs1 had higher levels of full length GFP-ATG8A and lower levels of free GFP under nitrogen 194 starvation or salt stress conditions compared to wild type ( Fig. 4C-D). As mentioned above, the rate of NBR1 195 degradation can be also used to measure autophagic flux (Bassham, 2015;Yoshii and Mizushima, 2017).

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Following autophagy induction with salt stress NBR1 levels remained high in cfs1 compared to wild type (Fig. 197 4C,. Collectively, these results show that CFS1 is crucial for autophagic flux in Arabidopsis 198 thaliana.

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We then tested whether CFS1 is involved in autophagic flux during selective autophagy. Using our recently 200 established uncoupler-induced mitophagy assays, we compared mitophagic flux in wild type and cfs1 cells (Ma 201 et al., 2021). Ultrastructural analysis of uncoupler treated cfs1 cells showed fully formed mitophagosomes, 202 8 further confirming that CFS1 does not play a role in autophagosome biogenesis (Fig. S10). We then measured 203 mitophagic flux using western blots. Uncoupler treatment led to a decrease in levels of the mitochondrial matrix 204 protein isocitrate dehydrogenase (IDH). This decrease was restored upon conA treatment, confirming the 205 induction of mitophagy. cfs1 mutants had higher IDH levels upon uncoupler treatment, suggesting a defect in 206 mitophagic flux (Fig. 4F-G). When we examined mitochondrial ultrastructure in cfs1 by electron microscopy, 207 we saw accumulation of damaged mitochondria with distinctive electron dense precipitates, which were rare in 208 wild type but common in atg5 (Fig. 4H). Altogether, these results suggest CFS1 is also crucial for selective

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We observed multiple small vacuoles in the epidermal cells of the root meristematic zone (Fig. S12E) and intact 225 central vacuoles at the transition zone in all lines (Fig. S12F). Altogether, these results indicate that CFS1 226 specifically regulates autophagic flux without affecting other vacuolar pathways or vacuolar morphology.

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How then does CFS1 regulate autophagic flux? We hypothesized that it may interact with tethering factors, 229 such as the CORVET or the HOPS complex, and thereby bridge the autophagosomes with the tonoplast 230 (Takemoto et al., 2018). To test this hypothesis, we generated Arabidopsis lines that co-expressed mCherry-231 CFS1 with the CORVET complex component VPS3, the HOPS complex component VPS39, and the tonoplast 232 localized SNARE protein VAMP711 (Takemoto et al., 2018;Geldner et al., 2009). Under both control and 9 autophagy inducing conditions, CFS1 did not colocalize with any of those proteins, negating out our hypothesis 234 ( Fig. S13A-F).

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Consistently, neither vps23a nor vps23b mutants showed autophagic flux defects in NBR1 degradation assays 262 ( Fig. S15C-D). Finally, vps23a and vps23b were indistinguishable from wild type in nitrogen starvation plate 263 assays (Fig. S15E). Altogether, these data indicate that VPS23A and VPS23B may act redundantly or do not 264 play a role in autophagy.

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Similar to the expression of mCherry-CFS1 AIM , expression of mCherry-CFS1 PSAPP failed to rescue the nitrogen 280 starvation-sensitivity phenotype of cfs1 (Fig. 5L). Altogether, these findings demonstrate that the CFS1-VPS23A 281 interaction is critical for autophagic flux and loss of interaction leads to early senescence.

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Autophagosome maturation involves the trafficking and fusion of double-membraned autophagosomes with 284 lytic compartments. Studies in yeast and metazoans have shown that this maturation step has several similarities 285 with endocytic vesicle fusion and trafficking. Tethering complexes, RAB GTPases, SNARE proteins, and 286 adaptors facilitate the trafficking and fusion of autophagosomes with the endolysosomal compartments (Zhao 287 and Zhang, 2019;Zhao et al., 2021). While well studied in yeast and metazoans, a systematic analysis of 288 autophagosome maturation is still missing in plants. In addition, plant genomes lack homologs of key 289 maturation proteins, such as the autophagic SNARE, Syntaxin 17, suggesting plants have evolved different 290 components for autophagosome maturation. Here, we show that CFS1 is an autophagy adaptor that bridges 291 autophagosomes with multivesicular bodies and mediates the formation of amphisomes. We propose that CFS1 292 functions as a licensing factor that tethers autophagosomes to multivesicular bodies via the ESCRT-1 complex.

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This is reminiscent of COPII tethering factor p115 that targets a subpopulation of COPII vesicles to cis-Golgi 294 (Allan et al., 2000).

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Our findings are consistent with a role in autophagosome maturation as opposed to autophagosome biogenesis 297 as was recently suggested (Kim et al., 2022): (i) our differential centrifugation-based autophagosome enrichment 11 biogenesis components but did identify trafficking related proteins such as Myosin 14 (Fig 1B, Table S3), (ii) 300 we observe fully formed autophagosomes in micrographs obtained from cfs1 mutants indistinguishable from 301 and in similar numbers to wild type (Fig. 3A, Fig. S10), and (iii) we observe a clear colocalization with the 302 ESCRT-1 protein VPS23A in both confocal and electron micrographs (Fig. 5). Although we see significant 303 defects in bulk and selective autophagic flux in cfs1, CFS1 AIM , and CFS1 PSAPP mutants, autophagic flux is not 304 fully blocked in any of them. These findings suggest two, not mutually exclusive, explanations: 1) There could 305 be other autophagy adaptors that mediate the maturation of a sub-population of autophagosomes. Consistently, 306 in metazoans, there are several autophagy adaptors that are either involved in SNARE recruitment, Rab GTPase 307 activation or autophagosome trafficking (Zhao et al., 2021). Performing autophagosome enrichment 308 experiments in a GFP-ATG8A/cfs1 line could reveal these adaptors. 2) Some autophagosomes may be 309 trafficked directly to the vacuole without an intermediary amphisome step. A systematic characterization of the 310 autophagosome fusion machinery would shed light on both of these possibilities.

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There are multiple trafficking routes to the vacuole: endocytic trafficking, post-Golgi trafficking, ER to vacuole 313 transport, and autophagy (Aniento et al., 2022). Although we are starting to understand how these routes 314 operate individually, how distinct vacuolar trafficking pathways are coordinated in response to changes in 315 metabolic demands and external stimuli remains elusive Here, based on our findings, we propose that vacuolar 316 trafficking is organized as a hub and spoke type distribution system, where amphisomes serve as sorting hubs 317 for multivesicular bodies and autophagosomes ( Figure 6). The hub and spoke model was developed by Delta

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Airlines in the 1950s, and has since then been successfully employed by various types of supply chain logistics 319 and aviation industry. The model implies that, rather than transportation taking place from A to B as in the 320 point-to-point system, all materials transit through centralized hubs. It thereby reduces logistical costs as fewer 321 routes are necessary. It also permits economies of scale, since complicated operations can be performed in the 322 hubs rather than separately organized in each node (Oti, 2013). Organising vacuolar trafficking via hubs (i) 323 could allow the cell to intricately balance the anabolic and catabolic needs and seamlessly integrate various 324 intrinsic and extrinsic signals, (ii) could facilitate the crosstalk between post-Golgi trafficking, endocytosis, and 325 autophagy, and (iii) could provide a route for loading and secretion of extracellular vesicles.

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One well-known drawback of hub and spoke distribution models is that the hub represents a single point of 328 failure. Any congestion in the hub can severely impact the whole system. Therefore, building a robust hub is 329 critical for a successful supply chain (Oti, 2013). This could be informative for translational approaches that   (Cui et al., 2019(Cui et al., , 2020Krüger and Schumacher, 2018).      Table 1). The primers used for genotyping are listed in Table 4. Coding sequences from gene of interest were 381 amplified from Col-0 cDNA with primers listed in Table 4. Plasmids were assembled via the GreenGate cloning 382 method (Lampropoulos et al., 2013). In short, CFS1 (At3g43230) and CFS2 (At1g29800) were cloned in two 383 parts to remove internal BsaI sites (see Table 4 for primers). For introducing point mutations in mCherry-384 CFS1 AIM (WLNL-267-ALNA), mCherry-CFS1 FYVE (RHHCR-195-AHACA) and mCherry-CFS1 PSAPP (PSAPP-385 145-AAAAA) site-directed mutagenesis was applied using primers listed in Table 4. For mCherry-CFS1 SYLF 386 (K282A, R288A, K320A), the SYLF domain was mutagenized by ordering a synthetic DNA sequence carrying 387 point mutations (Table 4) and replaced by restriction enzyme digestion with NcoI (NEB) and XbaI (NEB). All 388 constructs used in this study are listed in Table 3. Transgenic plant lines were generated via the Agrobacterium-389 mediated floral-dip method (Clough and Bent, 1998) and are listed in Table 1.

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Plasmids were assembled via the Gateway (Ishizaki et al., 2015) or GreenGate (Lampropoulos et al., 2013) 398 system and introduced through Agrobacterium tumefaciens transformation (Kubota et al., 2013). cfs1 knockout 399 mutants were achieved through CRISPR/Cas9 gene editing via 2 gRNAs. gRNAs were inserted at the BsaI 400 (NEB) sites of the pMpGE_En03 entry vector and subsequently inserted into the pMpGE010 destination 401 vector via LR clonase II reaction (Invitrogen) (Sugano et al., 2018). Successful transformants were verified via 402 sequencing of PCR products from amplified genomic DNA. Stable plant lines and constructs used are listed in 403 Table 2 and Table 3 respectively.

446
For Arabidopsis, protein concentration was measured using the Amido black method (Popov et al., 1975). Ten 447 μl of protein sample was diluted in 190 μl water and added with 1 ml Amido black staining solution (90% 448 methanol, 10% acetic acid, 0.005% (w/v) Amido black 10B (SIGMA)). Samples were mixed thoroughly and 449 centrifuged at 15000 rpm for 10 min. After removal of supernatant, pellets were washed with 1 ml washing 450 solution (90% ethanol and 10% acetic acid) and centrifuged at 15000 rpm for 10 min. Resulting pellets were 451 dissolved in 1 ml 0.2 N NaOH and the corresponding optical density (OD) at 630 nm was measured via a plate

471
Afterwards lysates underwent several differential centrifugation steps where each time the supernatant was 472 transferred. Samples were spun for (1) 10 min at 1000 g, to remove cell debris and nuclei; (2) 10 min at 10000 473 g, to remove bigger organelles like mitochondria and chloroplasts; (3) 10 min at 15000 g, to further remove 474 organelles (S3 fraction) and finally (4) 60 min at 100000 g (S4 and P4 fraction) (LaMontagne et al., 2016). Protein 475 concentration in S3 was normalized via Bradford (SIGMA) to ensure that equal amount of protein was loaded 476 before ultracentrifugation step. The P4 fraction was dissolved gently in GTEN-based buffer (without PVPP)

In vitro pull-downs 484
Recombinant proteins were expressed using Rosetta2(DE3)pLysS E. coli strain. Bacteria were grown to an 485 OD600 of 0.6 -0.7 followed by induction with 300 mM IPTG and overnight incubation at room temperature

In vivo co-immunoprecipitation 501
For co-immunoprecipitation, forty seeds per genotype were grown in 1/2 MS media for 7 d. Proteins were 502 extracted by adding 800 μl of EDTA-free buffer with 2% PVPP. Lysates were cleared by centrifugation at 503 15000 rpm at 4 °C for 10 min twice. 500 μl supernatant was incubated with 20 μl RFP-Trap ® Magnetic Agarose 504 beads (Chromotek) for 1 h. Beads were washed 3 and 5 times with EDTA-free buffer, without TCEP, before 505 and after incubation with lysate respectively. Beads were eluted in 50 μl 2x Laemmli buffer, boiled for 10 min 506 at 95°C and subjected to western blot analysis with indicated antibodies.

509
For affinity purification, S4, P4, and P4 + proteinase K samples described in Fig. 1A were prepared as same as 510 the method described above for the protease protection sample preparation, except that samples were incubated for 1 h with 40 μl RFP-Trap ® Magnetic Agarose beads (Chromotek) after proteinase K treatment. Mass spectrometry sample preparation and measurement were performed as previously described in Stephani and 513 Picchianti et al., 2020(Stephani et al., 2020.

515
Mass spectrometry data processing

516
The total number of MS/MS fragmentation spectra was used to quantify each protein (Table S1). The data 517 matrix of spectral count values (Table S2) was submitted to a negative-binomial test using the R package 518 IPinquiry4 (https://github.com/hzuber67/IPinquiry4) that calculates fold change and p-values using the quasi-  Table   530 S4.

Preparation of Arabidopsis thaliana samples for confocal microscopy 533
For all experiments except PIN2 endocytosis imaging, Arabidopsis seeds were vapor-phase sterilized by 534 chlorine (generated by a 10:1 mixture of 13% sodium hypochlorite and 36% HCl) for 15 min and were 535 subsequently stored at 4˚C for 2 d for vernalization. Vernalized seeds were spread on 1/2 MS media plates (+ 536 1% plant agar (Duchefa)) and grown at 21˚C at 60% humidity under LEDs with 50 mM/m 2 s a and a 16 h 537 light/8 h dark photoperiod for 5 d. Plates were placed vertically to let the roots elongate along the media 538 surface. Five-day-old seedlings were placed in 1/2 MS media and treated with salt or chemicals as indicated in 539 each experiment before confocal imaging. For nitrogen starvation, 1/2 MS media was replaced by nitrogen-540 deficient 1/2 MS media. For PIN2 endocytosis imaging, Arabidopsis seeds were vapor-phase sterilized by 541 chlorine (generated by a 25:1 mixture of 2.6% sodium hypochlorite and 36% HCl) for 3 to 4 hours. The 542 sterilized seeds were spread on 1/2 MS media plates (+ 1% plant agar (Duchefa)). The plated seeds were 543 subsequently stored at 4˚C for 2 d for vernalization. Vernalized seeds were then grown at 21˚C at 60% humidity The Marchantia polymorpha asexual gemmae were incubated in 1/2 Gamborg B5 media for 2 d before imaging.

557
Two-day-old M. polymorpha thalli were placed on a microscope slide with water and covered with a coverslip.

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The apical meristem region was used for image acquisition.

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For TEM samples preparation, high-pressure freezing, freeze substitution, resin embedding, and 614 ultramicrotomy were performed as described before (Ma et al., 2021;Kang, 2010;Wang et al., 2017). Briefly, 20 seven-day-old Arabidopsis seedlings were incubated in 150 mM NaCl or 50 μM DNP for 1 hour and were then rapidly frozen with an EM ICE high-pressure freezer (Leica Microsystems).
with acetone at room temperature. Root samples were separated from planchettes and embedded in Embed-

637
The significance level of differences between two experimental groups was marked as *, p<0.05; **, p<0.01; 638 ***, p<0.001; ns, not significant. For comparing the significance of differences between multiple experimental 639 groups, one-way ANOVA was performed as indicated in each experiment.

641
For PIN2 endocytosis qualitative quantification, the Arabidopsis seedling with at least 5 root epidermal cells 642 that contain visible PIN2-GFP in the vacuole is considered as the one with high PIN2 endocytic activities.

645
Coding sequences of CFS1 and CFS2 homologs were obtained by using the BLAST tool against representative 646 species of different plant lineages in Phytozome (Goodstein et al., 2012). The tree was inferred from a 2283-647 nt-long alignment by using the Maximum Likelihood method and Tamura-Nei model as implemented by 21 (Felsenstein, 1985). Best-scoring ML tree (-98686.19      . Bars indicate the mean ± standard deviation of 10 biological replicates. Brown-Forsythe and Welch one-way ANOVA test was performed to analyze the differences of the mCherry-CFS1 puncta number between each group. Unpaired t-tests with Welch's correction were used for multiple comparisons. Family-wise significance and confidence level, 0.05 (95% confidence interval) was used for analysis. Seven-day-old Arabidopsis seedlings were incubated in 150 mM NaClcontaining 1/2 MS media for 1 h for autophagy induction before cryofixation. Scale bars, 500 nm. AP, autophagosome. M, mitochondrion. (B) Confocal microscopy images of Arabidopsis root epidermal cells expressing pUBQ::mCherry-ATG8E, pNBR1::TagRFP-NBR1 or pUBQ::mCherry-CFS1. Five-day-old seedlings were first incubated in 5 μΜ BCECF-AM-containing 1/2 MS media for 30 minutes for vacuole staining and were subsequently transferred to 1/2 MS media containing 90 mM NaCl and 1 μΜ concanamycin A (conA) for 2 hours before imaging. Scale bars, 5 μm.

(E) Quantification of confocal experiments in (D)
showing the mCherry-ATG8E colocalization ratio of NBR1-GFP and GFP-CFS1 to mCherry-ATG8E. The mCherry-ATG8E colocalization ratio is calculated as the ratio between the number of mCherry-ATG8E puncta that colocalize with NBR1-GFP or GFP-CFS1 puncta compared with the number of total mCherry-ATG8E puncta. Bars indicate the mean ± standard deviation of 10 biological replicates. Two-tailed and unpaired t tests with Welch's corrections were performed to analyze the differences of mCherry-ATG8E colocalization ratio between GFP-CFS1 and NBR1-GFP. ***, p value < 0.001. (F) TEM images showing immuno-gold labeled GFP-CFS1 at the autophagosomes in Arabidopsis root cells. Seven-day-old seedlings were incubated in 150 mM NaCl-containing 1/2 MS media for 2 h for autophagy induction before cryofixation. Sections from pUBQ::GFP-CFS1 expressing cell samples were labelled with an anti-GFP primary antibody and a secondary antibody conjugated to 10 nm gold particles. Yellow arrowheads mark the gold particles associated with autophagosomes. Scale bars, 500 nm. AP, autophagosome.
(G) Quantification of the localization of the GFP-specific gold particles imaged in the experiment shown in (F). Approximately 900 gold particles in 50 TEM images captured from 5 independent samples were grouped into autophagosomes, cytosol or other organelles according to their locations. One-way ANOVA was performed to analyze the significant difference between different gold particle locations. ****, p value < 0.0001.  The predicted complex interaction interface involving AtCFS1 PSAPP motif is highlighted as a zoom in with the side chains of relevant residues represented as sticks.
Arabidopsis seeds were first grown in 1/2 MS media under continuous light for one week and 7-day-old seedlings were subsequently transferred to 1/2 MS media ± 1 µM conA or nitrogen-deficient (-N) 1/2 MS media ±1 µM conA for 12 h. Ten μg of total protein extract was loaded and immunoblotted with anti-GFP and anti-NBR1 antibodies.
(J) Quantification of (I) showing the relative autophagic flux. Values were calculated as the protein band intensities of GFP divided by the protein band intensity of GFP-ATG8A and were normalized to untreated (Control) Col-0. Results are shown as the mean ± standard deviation of 5 independent replicates. Onetailed and paired Student t-tests were performed to analyze the significance of the relative autophagic flux differences. ns, not significant. **, p < 0.01. ***, p < 0.001.
(K) Quantification of (I) showing the relative NBR1 level in respect to untreated (Control) Col-0. Values were calculated via normalization of protein bands to Ponceau S and shown as the mean ± standard deviation of 5 independent replicates. One-tailed and paired Student t-tests were performed to analyze the significance of the relative NBR1 level difference. ns, not significant. *, p < 0.05. **, p < 0.01. (L) Phenotypic characterization of Arabidopsis cfs1 mutants complemented with pUBQ::mCherry-CFS1, pUBQ::mCherry-CFS1 AIM , pUBQ::mCherry-CFS1 PSAPP or pUBQ::mCherry-CFS1 AIM+PSAPP upon nitrogen starvation. Twenty-five seeds per genotype were grown on 1/2 MS media plates (+1% plant agar) for 1 week and 7-day-old seedlings were subsequently transferred to nitrogen-deficient 1/2 MS media plates (+0.8% plant agar) and grown for 2 weeks. Plants were grown at 21°C under LEDs with 85 µM/m²/sec and a 14 h light/10 h dark photoperiod. d0 depicts the day of transfer. Brightness of pictures was enhanced ≤19% with Adobe Photoshop (2020). Representative images of 4 independent replicates are shown. Western blot analysis of 7-day-old Col-0 seedlings expressing pUBQ::GFP-ATG8A. Arabidopsis seedlings were treated with 3 μM Torin for 90 min prior to differential centrifugation described in Figure  1A. A total of 5 μg of protein was loaded in each lane. Protein extracts were immunoblotted with anti-GFP and anti-NBR1 antibodies.

Plasma
(B) Protease protection assay of enriched autophagosomes in (A). Autophagosomes were treated with 30 ng/μl proteinase K in absence or presence of 1% Triton X-100. A total of 5 μg of protein was loaded in each lane. Protein extracts were immunoblotted with anti-GFP and anti-NBR1 antibodies. (B) Confocal microscopy images of Arabidopsis root epidermal cells co-expressing pUBQ::GFP-CFS1 and pUBQ::mScarlet-CFS2. Five-day-old Arabidopsis seedlings were incubated in either control, nitrogendeficient (-N) or 150 mM NaCl-containing 1/2 MS media before imaging. Note that no CFS2 puncta signals could be detected. Scale bars, 5 μm. (C) Western blot analysis of Arabidopsis seedlings expressing pUBQ::mScarlet-CFS2 used in (B). Total lysates were immunoblotted with anti-RFP antibodies. Images of two biological replicates are shown. (D) Phenotypic characterization of Arabidopsis cfs1 and cfs2 mutants upon nitrogen starvation. Twenty-five Arabidopsis seeds per genotype were first grown on 1/2 MS media plates (+1% plant agar) for 1 week and 7-day-old seedlings were subsequently transferred to nitrogen-deficient (-N) 1/2 MS media plates (+0.8% plant agar) and grown for 2 weeks. Plants were grown at 21°C under LEDs with 85 µM/m²/sec and a 14 h light/10 h dark photoperiod. d0 depicts the day of transfer. Brightness of pictures was enhanced ≤19% with Adobe Photoshop (2020). Representative images of 4 biological replicates are shown. (E) Western blots showing the endogenous NBR1 level in Col-0, cfs1, cfs2, cfs1cfs2 or atg5 under control or nitrogen starved (-N) conditions. Arabidopsis seeds were first grown in 1/2 MS media under continuous light for one week and 7-day-old seedlings were subsequently transferred to control or nitrogen-deficient 1/2 MS media for 12 h. Ten μl of total seedling extract was loaded and immunoblotted with anti-NBR1 antibodies.
(F) Quantification of (E) showing the relative NBR1 level of Col-0, cfs1, cfs2, cfs1cfs2 or atg5 under control or nitrogen starved conditions. Values were calculated via normalization of protein bands to Ponceau S and to untreated (Control) Col-0 and shown as the mean ± standard deviation of 3 independent replicates. Onetailed and paired Student t-tests were performed to analyze the significance of the relative NBR1 level differences between Col-0 and each mutant. ns, not significant. *, p value < 0.05. **, p value < 0.01. (G) Western blot showing the endogenous NBR1 level in Col-0, cfs1, cfs2, cfs1cfs2, or atg5 under control or salt stressed (NaCl) conditions. Arabidopsis seeds were first grown in 1/2 MS media under continuous light for one week and 7-day-old seedlings were subsequently transferred to control or 150 mM NaCl-containing 1/2 MS media for 16 h. Ten μl of total seedling extract was loaded and immunoblotted with anti-NBR1 antibodies.
(H) Quantification of (G) showing the relative NBR1 level of Col-0, cfs1, cfs2, cfs1cfs2, or atg5 under control or salt stress (NaCl) conditions. Values were calculated via normalization of protein bands to Ponceau S and to untreated (Control) Col-0 and shown as the mean ± standard deviation of 3 independent replicates. One-tailed and paired Student t-tests were performed to analyze the significance of the relative NBR1 level difference between Col-0 and each mutant. ns, not significant. *, p value < 0.05. **, p value < 0.01.   Coding sequences of CFS1 and CFS2 homologs were obtained by using the BLAST tool against representative species of different plant lineages in Phytozome (Goodstein et al., 2012). The tree was inferred from a 2283-nt-long alignment by using the Maximum Likelihood method and Tamura-Nei model as implemented by MEGA X (Tamura and Nei, 1993;Kumar et al., 2018). 100 bootstrap method and a discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.8072)) (Felsenstein, 1985). The tree is represented using Interactive Tree Of Life (iTOL) v4 (Letunic and Bork, 2019). Best-scoring ML tree (-98686.19) is shown with purple circles indicating bootstrap values above 80 on their respective clades. The scale bar indicates the evolutionary distance based on the nucleotide substitution rate. All CFS1 homologs are grouped in the blue region while all CFS2 homologs are grouped in the orange region. Genes that encode Arabidopsis thaliana CFS1 (AtCFS1), A. thaliana CFS2 (AtCFS2) and Marchantia polymorpha CFS1 (MpCFS1) are highlighted with purple, green and orange colors, respectively.  (E) GFP cleavage assay of pEF1::GFP-MpATG8A in M. polymorpha wild-type (Tak-1) or cfs1 mutants. Tenday-old propagules were treated with 12 μM Torin for 5 h before protein extraction. Fifteen μg of total protein extract was loaded and immunoblotted with anti-GFP antibodies. Representative images of 2 biological replicates are shown.
(F) GFP cleavage assay of pEF1::GFP-MpATG8B in M. polymorpha wild-type (Tak-1) or cfs1 mutants. Tenday-old propagules were treated with 12 μM Torin for 5 h before protein extraction. Fifteen μg of total protein extract was loaded and was immunoblotted with anti-GFP antibodies. Representative images of 2 biological replicates are shown.