The phosphatidylinositol 3-phosphate effector FYVE3 regulates FYVE2-dependent autophagy in Arabidopsis thaliana

Phosphatidylinositol 3-phosphate (PI3P) is a signaling phospholipid that play a key role in endomembrane trafficking, specifically autophagy and endosomal trafficking. However, the mechanisms underlying the contribution of PI3P downstream effectors to plant autophagy remain unknown. Known PI3P effectors for autophagy in Arabidopsis thaliana include ATG18A (Autophagy-related 18A) and FYVE2 (Fab1p, YOTB, Vac1p, and EEA1 2), which are implicated in autophagosome biogenesis. Here, we report that FYVE3, a paralog of plant-specific FYVE2, plays a role in FYVE2-dependent autophagy. Using yeast two-hybrid and bimolecular fluorescence complementation assays, we determined that the FYVE3 protein was associated with autophagic machinery containing ATG18A and FYVE2, by interacting with ATG8 isoforms. The FYVE3 protein was transported to the vacuole, and the vacuolar delivery of FYVE3 relies on PI3P biosynthesis and the canonical autophagic machinery. Whereas the fyve3 mutation alone barely affects autophagic flux, it suppresses defective autophagy in fyve2 mutants. Based on the molecular genetics and cell biological data, we propose that FYVE3 specifically regulates FYVE2-dependent autophagy.


Introduction
Endomembrane trafficking comprises dynamic membrane flow where various membrane lipids and cargo proteins are sorted and transported to their appropriate destinations. Newly synthesized proteins are targeted to various endomembrane compartments via multiple routes, including secretory and vacuolar trafficking. Proteins endocytosed from the cell surface are sorted at the endosomal compartments and either recycled to the plasma membrane or targeted to the vacuole (or lysosome in animals) for degradation. Cytoplasmic proteins may be sequestered by the autophagic membrane and delivered to the vacuole/lysosome for degradation.
The best-characterized type of autophagy is macroautophagy (hereafter referred to as autophagy), in which a membrane sac called the phagophore is expanded and sealed to form the autophagosome sequestering a small portion of the cytoplasm (Marshall and Vierstra, 2018). The autophagosome fuses with the vacuole, releasing its cargo into the vacuolar lumen as an autophagic body. Finally, the autophagic body is rapidly degraded by acid hydrolases in the vacuole. The formation of phagophores and autophagosomes is facilitated by autophagy-related (ATG) proteins, which are conserved in nearly all eukaryotes. ATG8 is a ubiquitin-fold protein conjugated to the membrane lipid phosphatidylethanolamine on the phagophore. As ATG8 resides on the autophagic membrane even after autophagosome fusion, lipidated ATG8 is a useful marker for phagophores, autophagosomes, and autophagic bodies. ATG5 and ATG7 are among the ATG proteins essential for the ATG8 conjugation and general autophagy, whereas ATG2 and ATG18A form a protein complex that transfers membrane lipids to the phagophore and contributes to phagophore growth .
Phosphatidylinositol 3-phosphate (PI3P) is a signaling phospholipid that plays regulatory roles in endomembrane trafficking (Schink et al., 2013). In eukaryotic cells, PI3P is enriched at the cytosolic leaflet of endosomal, autophagosomal, and vacuolar (or lysosomal) membranes. In budding yeast and plants, PI3P is produced from phosphatidylinositol by the phosphatidylinositol 3-kinase (PI3K) complex containing the catalytic subunit VPS34 (Vacuolar Protein Sorting 34). There are two distinct PI3K complexes which have 3 common and 1 specific subunits. The PI3K complex I, consisting of VPS34, VPS15, VPS30/ ATG6, and ATG14, is responsible for PI3P production at the autophagic membrane, while the PI3K complex II, having VPS38 instead of ATG14, generates PI3P on the endosomal membrane (Schink et al., 2013).
Despite its low abundance in biological membranes, PI3P on endosomal and vacuolar membranes is essential for plant development. Null mutations of vps34 and atg6 in Arabidopsis thaliana cause lethality (Fujiki et al., 2007;Lee et al., 2008), and vps38 mutants show pleiotropic phenotypes resulting from defective endosomal sorting and vacuolar trafficking (Lee et al., 2018;Liu et al., 2018;Yuan et al., 2018). Less obvious developmental phenotypes are observed in Arabidopsis atg14a atg14b double mutants, although they are defective in autophagy (Liu et al., 2020). Generally, plants with compromised functions of vacuoles and endosomes display lethality or severe developmental abnormality (Gonzaĺez Solıś et al., 2022), whereas autophagydeficient plants do not. Instead, the autophagy mutants are hypersensitive to different types of abiotic and biotic stress (Qi et al., 2021).
Here, we aimed to assess genetic and cell biology data to ascertain the role of Arabidopsis FYVE3/CFS2 in autophagy. We showed that FYVE3 was incorporated into the autophagic machinery via interaction with ATG8. An autophagy pathway consisting of core ATG proteins and PI3P effectors was proposed, based on our genetic data indicating a specific involvement of FYVE3 in FYVE2-dependent autophagy.

Preparation of transgenic plants
To obtain binary vectors for transgenic plants expressing ProUBQ10:GFP-FYVE3 or ProUBQ10:mCherry-FYVE3, recombination of the entry clone containing FYVE3 cDNA with pMDC99-AtUBQ10p-GFP and pMDC99-AtUBQ10p-mCherry (Suttangkakul et al., 2011) was performed via the LR Clonase II reaction (Thermo Fisher Scientific). The binary vectors were introduced into Agrobacterium tumefaciens strain GV3101. To obtain stable transformants, Agrobacterium transformants were used to infect Arabidopsis using the floral dip method (Clough and Bent, 1998).
Phenotypic analysis of the fixed carbon starvation response was performed as described (Chung et al., 2010). Briefly, after incubating seedlings on 1× MS-suc solid medium (1× MS salt including vitamins, 29 mM mannitol, and 0.25% phytagel) for 14 d, the seedlings were then deprived of light for 10 d. Seedlings resuming growth were counted after 18 d of recovery.
To prepare membrane proteins, seedlings expressing GFP-FYVE3 were homogenized with ice-chilled pestle and mortar in TNPI buffer [50 mM Tris-Cl, 150 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, 10 mM iodoacetamide, pH 8.0]. After centrifugation at 500 × g at 4°C for 5 min, 5% of the supernatant was set aside as total extract, and 95% of the supernatant was further separated into soluble and pellet fractions by centrifugation at 20,000 × g at 4°C for 15 min.

Quantitative real-time PCR analysis
To evaluate RNA abundance, seedlings were grown on 1 x MS medium for 10 d, frozen with liquid nitrogen, and homogenized in TRIsure agent (Bioline, London, UK). The RNA extract was treated with DNase I (New England Biolabs), and cDNA was synthesized using RevertAid H Minus reverse transcriptase (Thermo Fisher Scientific) with oligo(dT) 20 primers. Quantitative real-time PCR (qRT-PCR) was performed using Step One Plus Real-Time PCR System (Applied Biosystems) and EvaGreen (SolGent, Daejeon, Korea). The relative transcript abundance of target genes was calculated by the DDCT method using the UBC9 cDNA as an internal control. All primer sequences are provided in Table S1.

Yeast two-hybrid analysis
The entry clone containing FYVE3 cDNA was recombined with the destination vector pDEST22 (Thermo Fisher Scientific) via the LR Clonase II reaction. The resulting pDEST22-FYVE3 and pDEST32 expression vectors for bait proteins fused to SAR1B, SAR1C, ATG8A, ATG8E, ATG8F, ATG8I, ATG18A and FYVE2 (Kim et al., 2022) were used for transformation of the AH109 strain and Y187 strain through the Yeastmaker Yeast Transformation System 2 (Clontech, Mountain View, CA, USA). To determine protein interaction, yeast colonies isolated from Trp-and Leudeficient medium were transferred to Trp-, Leu-, and His-deficient medium containing 0 and 1 mM 3-amino-1,2,4-triazole.

Confocal microscopy, image processing, and quantification
To observe fluorescence signals from Arabidopsis plants or leaf protoplasts, either an LSM 510 or AxioObserver LSM 800 confocal microscope (Carl Zeiss) was used. For LSM 510, a 488 nm laser and BP500-530IR emission filter were used to detect GFP signals. For LSM 800, a 488 nm laser was used for excitation and fluorescence was detected at the 410-546 nm range to acquire GFP, citrine, and YFP signals. To detect mRFP and mCherry, a 587 nm laser was used for excitation, and fluorescence was detected at the 595-700 nm range.

FYVE3 interacts with specific ATG8 isoforms
To gain insight into the FYVE3 protein, we studied its physical interaction with other proteins. An Arabidopsis interactome study identified yeast two-hybrid (Y2H) interactions of FYVE3 with ATG8D (Arabidopsis Interactome Mapping Consortium, 2011). To identify additional interactors, we performed Y2H assay using FYVE3 as a prey and select ATG proteins, FYVE2, and SAR1 isoforms as baits. Our Y2H assay demonstrated interaction of FYVE3 with select ATG8 isoforms such as ATG8A and ATG8F but not with ATG8E and ATG8I ( Figure 1A).
The positive Y2H interactions were further tested using in planta interaction assays using bimolecular fluorescence complementation (BiFC) in Arabidopsis leaf protoplasts. The BiFC interaction of NYFP-FYVE3 with CYFP-ATG8A was verified ( Figure 1B), whereas no considerable fluorescence was reconstituted from negative controls of NYFP-FYVE3 paired with either CYFP or CYFP-TRAF1A (Qi et al., 2017) (Figure 1B). In addition, interactions of FYVE3 with SAR1B, ATG18A, ATG6, and FYVE2 were detected in BiFC experiments ( Figure 1B), although these interactions were not apparent in Y2H ( Figure 1A). We previously showed that FYVE2 interacts with SAR1B and ATG18A (Kim et al., 2022). Thus, FYVE3 may be brought into proximity of SAR1B, FYVE2, and ATG18A, for example, by their simultaneous binding to a scaffold protein. These data suggested that FYVE3 associates with ATG8A, FYVE2, SAR1B, ATG18A, and ATG6 in plant cells.

FYVE3 is recruited from the cytosol to autophagic and endosomal membranes
To investigate the subcellular distribution and trafficking of FYVE3 proteins, we prepared Arabidopsis transgenic plants expressing FYVE3 fused to either GFP or mCherry. The membrane fractionation experiment indicated that GFP-FYVE3 was primarily observed in the soluble fraction ( Figure S2). We then determined the subcellular distribution of mCherry-FYVE3 by co-localizing it with various organelle markers ( Figure 2). Root cells expressing mCherry-FYVE3 had diffuse fluorescence with relatively scarce punctate signals. The mCherry-FYVE3 puncta often overlapped with the puncta of the PI3P biosensor citrine-2xFYVE (Figures 2A, G), the late endosome marker YFP-ARA7 ( Figures 2B,  G), and the autophagic marker GFP-ATG8A ( Figures 2C, G).
We also located mCherry-FYVE3 puncta relative to GFP-FYVE2 ( Figure 2F). GFP-FYVE2 were mostly localized at cytoplasmic puncta, some of which were also decorated with mCherry-FYVE3. Although mCherry-FYVE3 puncta were fewer than GFP-FYVE2 puncta, the former mostly overlapped with the latter (Figures 2F, G). As FYVE2 was shown to localize on the autophagic membrane and, to a lesser extent, on the endosomal membrane (Kim et al., 2022), these data indicated that mCherry-FYVE3 puncta mainly represent either autophagic or endosomal compartments that are abundant in PI3P, whereas the diffuse signal corresponds to a cytosolic pool of mCherry-FYVE3.

Association of FYVE3 with autophagic membranes requires ATG8 lipidation and PI3P
The subcellular distribution of FYVE3 is consistent with their protein interaction with select ATG8 isoforms, suggesting that FYVE3 binds to ATG8 on phagophores during autophagosome biogenesis. As fluorescent fusions of FYVE3 are largely co-localized with autophagic puncta, we then examined whether these proteins are delivered together to the vacuole for degradation and detected as autophagic bodies. Observation of the autophagic bodies requires At the subcellular level, mCherry-FYVE3 is mainly cytosolic but can be found at the PI3P-enriched membrane of autophagic and endosomal organelles. (A-F), Representative images of root tip cells co-expressing mCherry-FYVE3 with a variety of organelle markers, such as PI3P biosensor (A), late endosome marker (B), phagophore and autophagosome marker (C), trans-Golgi network marker (D), Golgi stack marker (E), and GFP-FYVE2 (F), which associates with autophagic and endosomal membranes. Scale bars = 5 µm. Arrows indicate mCherry-FYVE3 puncta that overlap with coexpressed markers, whose quantification is shown in (G). Columns marked with different letters represent significantly different means according to the t-test (mean ± S.E.; n = 11-12 images; p < 0.01). the treatment of GFP-ATG8 transgenic plants with concanamycin A (ConA), an inhibitor of vacuolar proton pumps. Transgenic plants expressing both GFP-ATG8A and mCherry-FYVE3 were treated with ConA, and their root cell images were compared with DMSO-treated controls ( Figure 3A). In ConA-treated roots, 81% of the intravacuolar mCherry-FYVE3 bodies emitted a GFP-ATG8A signal ( Figure 3A). ConA-treated roots expressing GFP-FYVE2 and mCherry-FYVE3 also showed a moderate level of co-localization in the vacuole ( Figure 3B). Similarly, ConA-treated root cells expressing mCherry-FYVE3 alone showed intravacuolar puncta resembling autophagic bodies ( Figure S3A). These data indicated that fluorescent fusions of FYVE3 are degraded in the vacuolar lumen, together with ATG8A and FYVE2.
To define the molecular mechanism underlying the vacuolar trafficking of FYVE3 fluorescent fusions, we tested the effect of ConA and the PI3K inhibitor wortmannin (Wm) treatment on the Fluorescent fusions of FYVE3 are delivered to the vacuole in a manner dependent on canonical autophagy machinery and PI 3-kinase activity. (A) and (B), Confocal images of mature root cells co-expressing mCherry-FYVE3 with either GFP-ATG8A (A) or GFP-FYVE2 (B). Seedlings grown in a nitrogen-rich medium were treated with DMSO or 0.5 µM ConA for 16 h prior to confocal microscopy. In the images of DMSO-treated cells, arrows indicate cytoplasmic puncta showing both GFP and mCherry signal. Percentages of vacuolar bodies showing both GFP and mCherry signal were calculated from ConA-treated cells. (C), Confocal images of mature root cells expressing GFP-FYVE3 in the genetic background of WT, atg5-1, and atg2-1. Transgenic seedlings were incubated in nitrogen-rich liquid medium and treated with DMSO, 0.5 µM ConA, or 0.5 µM ConA plus 30 µM Wm for 16 h. The graph on the right shows the number of vacuolar puncta (mean ± S.E.; n = 12-13 images) per 1,000 µm 2 area of the central vacuole.
(D), GFP-FYVE3 cleavage assay. WT, atg5-1, or atg2-1 seedlings expressing GFP-FYVE3 were incubated as described in (C), and protein extract was prepared for immunoblot analysis using anti-GFP (upper) and anti-histone H3 (lower; for loading control) antibodies. Open diamonds indicate the position of protein bands that are not stabilized by ConA and migrates slightly slower than free GFP bands (indicated by a solid arrowhead). Representative images selected from 3 repeat experiments are shown. (E), Confocal images of mature root cells expressing GFP-ATG8A and mCherry-FYVE3 in WT (left images) and atg2-1 (right images) background. Seedlings were incubated in a nitrogen-lacking liquid medium for 48 h and treated with DMSO or 30 µM Wm for 1 h prior to microscopic observation. Arrows indicate GFP-ATG8A puncta overlapping with mCherry-FYVE3 signal. The Pearson correlation coefficients (PCCs) were calculated from 10 (WT) and 13 (atg2-1) images (mean ± S.E.). Scale bars = 5 µm. subcellular localization of GFP-FYVE3 ( Figure 3C). As expected, autophagic body-like puncta were observed in the vacuole of ConAtreated wild-type (WT) seedlings expressing GFP-FYVE3 ( Figure 3C, WT, + ConA). Accumulation of the GFP-FYVE3 bodies was suppressed by Wm treatment (Figure 3C, WT, + ConA, Wm), suggesting that GFP-FYVE3 is also targeted to the vacuole via PI3K-dependent trafficking. To test whether core ATG genes are involved in this trafficking, we introgressed the GFP-FYVE3 transgene into atg2-1 and atg5-1 background by a genetic cross. atg5-1 is defective in ATG8 lipidation (Chung et al., 2010), whereas atg2-1 accumulates a high level of lipidated ATG8 (Kang et al., 2018). When we observed their progenies by confocal microscopy, no autophagic body-like GFP-FYVE3 puncta were observed in ConA-treated atg5-1 and atg2-1 vacuoles ( Figure 3C), demonstrating that canonical autophagy mediates the vacuolar targeting of GFP-FYVE3.
The vacuolar targeting of GFP-FYVE3 was validated by the immunoblot analysis using anti-GFP antibodies, similar to the GFP-ATG8 and GFP-FYVE2 cleavage assays (Kim et al., 2022). In WT, a very faint band corresponding to GFP-FYVE3 was detected, whereas free GFP released from GFP-FYVE3 was readily observed and its intensity increased by ConA ( Figure 3D) and dark ( Figure S3B) treatments, indicating that the free GFP band is a partial degradation product in the vacuole. In atg5-1 and atg2-1, the GFP-FYVE3 band intensity was increased, whereas ConA and dark treatments did not affect the intensity of protein bands (indicated by open diamonds in Figures 3D and S3B) that migrated slightly slower than free GFP bands (solid arrowheads in Figures 3D and S3B). Combined with confocal microscopy data ( Figure 3C), these immunoblot results indicated that GFP-FYVE3 is delivered to the vacuole for degradation via canonical, ATG2-and ATG5-dependent autophagy pathway. Consistently, nitrogen starvation increased the ratio of free GFP to GFP-FYVE3 in WT but not in an atg7-2 background ( Figure S3C). The intensity of the slower-migrating protein band appeared to increase by nitrogen starvation ( Figure S3C). Although we do not know the nature of this protein band, the same phenomenon was observed in atg7-2 mutants expressing the autophagy marker GFP-ATG8 (Spitzer et al., 2015). These data indicated that the vacuolar trafficking of GFP-FYVE3 showed a typical pattern of the autophagy marker.
To define the molecular requirement for the formation of GFP-FYVE3 puncta, we examined the effect of atg5 and atg2 mutations and Wm treatment. The abundance of GFP-FYVE3 puncta decreases and increases in atg5 and atg2, respectively ( Figure  S3D). Again, Wm effectively diminished the accumulation of GFP-FYVE3 puncta in atg2 ( Figure S3D). The PI3P-dependent accumulation of GFP-FYVE3 puncta in atg2 cytoplasm is very similar to that of GFP-ATG8A (Kang et al., 2018), and may result from arrested elongation/maturation of phagophores in atg2.
To investigate the effect of atg2 mutation on mCherry-FYVE3 to the autophagic membrane, we co-localized mCherry-FYVE3 with GFP-ATG8A in WT and atg2 background. atg2 root cells overaccumulated cytoplasmic mCherry-FYVE3 puncta, which extensively overlapped with GFP-ATG8A puncta ( Figure 3E). Thus, ATG2 is essential for the targeting of mCherry-FYVE3 to the vacuole, but is dispensable for mCherry-FYVE3 recruitment to the phagophore.

FYVE3 is required for the overaccumulation of autophagic structures in fyve2 background
To test the potential functions of FYVE3 in autophagy, we employed fyve3-1, a T-DNA insertional allele (Kim et al., 2022). RT-PCR analysis indicated that fyve3-1 is a knock-out allele, and there is no apparent transcriptional feedback between FYVE2 and FYVE3 genes ( Figure S1B). Because of the FYVE and SYLF domains shared by FYVE2 and FYVE3, we initially postulated a genetic redundancy of FYVE2 and FYVE3. In fyve2 mutant background, phagophores are over-accumulated and autophagic flux is partially compromised (Kim et al., 2022). However, no significant change in autophagic flux was measured in fyve3 single mutants, and fyve2 fyve3 double mutants did not show further reduction in autophagic flux, when compared with fyve2 single mutants (Kim et al., 2022; Figure S4). These data were inconsistent with a simple model that FYVE2 and FYVE3 are duplicates. To further investigate the genetic relationship between these two genes, we quantified the abundance of autophagic organelles in N-starved WT, fyve2, fyve3, and fyve2 fyve3 double mutant cells expressing the autophagic marker GFP-ATG8A. Confocal microscopy analysis confirmed previous observation that fyve2 root cells overaccumulated GFP-ATG8A puncta in the cytoplasm, compared with WT ( Figure 4A; Kim et al., 2022). As previously reported (Kim et al., 2022), Wm effectively blocked the formation of GFP-ATG8A puncta in fyve2 ( Figure 4A). In contrast, fyve3 single mutants did not accumulate GFP-ATG8A puncta ( Figure 4A). Notably, less than 10% of GFP-ATG8A puncta were detected in fyve2 fyve3 double mutants, relative to fyve2 mutants ( Figure 4A). Complementation experiments revealed that the expression of mCherry-FYVE3 transgene in fyve2 fyve3 mutants restored the accumulation of GFP-ATG8A puncta ( Figure S5). These results indicate that FYVE3 is responsible for the over-accumulation of immature autophagic structures in fyve2.
Based on autophagy reporter analysis using fyve3 single and fyve2 fyve3 double mutants, we hypothesized that FYVE3 might serve a specific function during autophagosome formation. This hypothesis was tested using a different mutant combination of atg2 and fyve3 ( Figure 4B). Compared with fyve2, atg2 mutants accumulate even more autophagic puncta, and autophagic flux is compromised almost completely (Kang et al., 2018;Kim et al., 2022). Unlike fyve2 fyve3 double mutants, atg2 fyve3 double mutant cells showed over-accumulation of GFP-ATG8A puncta as atg2 single mutants did (compare Figure 4B with Figure 4A). As fyve3 did not suppress the over-accumulation of GFP-ATG8A puncta in atg2, FYVE3 appears to play a specific role in the formation of GFP-ATG8A puncta in fyve2. Of note, treatment with Wm only partially suppressed the GFP-ATG8A puncta in atg2 fyve3 double mutants ( Figure 4B).
Accumulation of autophagic puncta can result from either induction of autophagy or retarded biogenesis of autophagosomes. Autophagic flux analysis is necessary to distinguish these two possibilities. As fyve3 alone did not significantly affect autophagic flux (Kim et al., 2022; Figure S4), we assessed the effect of fyve3 on autophagic flux in atg2 and fyve2 mutant background. To quantify autophagic flux in different mutants, we used the GFP-ATG8A cleavage assay (Chung et al., 2010;Shin et al., 2014), where anti-GFP immunoblot analysis of seedlings expressing GFP-ATG8A is performed, and autophagic flux is determined from the protein band intensity of free GFP moiety, relative to that of GFP-ATG8A. GFP-ATG8A transgenic seedlings in WT, atg2, fyve2, and fyve3, or their double/triple mutant background were exposed to either N starvation ( Figure 5C) or AZD ( Figure 5D) and subjected to GFP-ATG8A cleavage assay. In both conditions, autophagic flux was fully inhibited in any genetic background containing atg2 and also greatly interfered in fyve2 (Figures 5C, D). In contrast, fyve2 fyve3 double mutant was indistinguishable from WT and fyve3, confirming that fyve3 suppresses fyve2 (Figures 5C, D).
Finally, the physiological function of FYVE3 gene expression was tested by phenotypic analysis of fyve3 single and fyve2 fyve3 double mutants. Autophagy-deficient mutants are hypersensitive to carbon starvation (Hanaoka et al., 2002;Chung et al., 2010). Our fixed-carbon starvation experiments showed that fyve2-2 seedlings showed hypersensitivity to light deprivation, whereas fyve3-1 did not ( Figure 5E). Importantly, fyve2-2 fyve3-1 double mutants showed a WT-like sensitivity, demonstrating that the suppression effect of fyve3 on fyve2 is physiologically significant.

Discussion
FYVE3 is a putative PI3P effector involved in autophagy (Chung, 2019), but its function remains unelucidated. In this study, we investigated the role of FYVE3 in plant autophagy. We demonstrated that FYVE3 interacts with ATG8 isoforms (Figure 1), associates with ATG8-positive autophagic membranes (Figure 2), and is degraded in the vacuole in a manner dependent on core ATG genes and PI3K ( Figure 3). As autophagic flux is not significantly altered by fyve3 single mutation under N starvation (Figures 4, 5), FYVE3 could be considered an autophagic cargo that does not play a critical role in autophagosome biogenesis. However, this scenario is unlikely, because genetic analysis of fyve2 fyve3 double mutants indicated that fyve3 suppressed fyve2 phenotypes, such as overaccumulation of autophagic puncta, reduction in autophagic flux, and starvation response (Figures 4,5). This suppression by fyve3 is in contrast with the hyperaccumulation of autophagic markers in atg2 fyve3 double mutants, where no suppression was observed. Based on these genetic interactions, we propose that FYVE3 acts upstream of FYVE2 but downstream of ATG2 during autophagosome formation ( Figure 5F).
The phenotypic consequence of fyve3 mutation is not evident unless it is combined with fyve2 mutation, which prevented us and others from identifying its involvement in autophagy (Kim et al., 2022;Zhao et al., 2022). In our previous work (Kim et al., 2022), we employed 48-h nitrogen starvation for autophagic flux assay, whereas 24-h starvation was used for the assay in this study. Nevertheless, we observed a similar suppression in the autophagic flux analysis in which 21-d-old leaves of fyve2 fyve3 mutants were used (Kim et al., 2022). Zhao et al. (2022) reported that the steadystate level of the autophagic cargo receptor NBR1 was comparable between fyve2 and fyve2 fyve3, but did not directly assess the fyve3 suppresses the accumulation of autophagic puncta in fyve2 but not those in atg2. (A, B) Representative confocal microscopy images (left) and quantification (right) of GFP-ATG8A puncta (arrows at the images) acquired from mature root cells with indicated genetic background. Transgenic Arabidopsis seedlings expressing GFP-ATG8A were incubated in a nitrogen-lacking liquid media for 48 h and treated with either wortmannin (Wm) or solvent control (DMSO) for 1 h prior to image acquisition. Scale bars = 5 µm. Columns marked with asterisks represent means that significantly differ from each other, according to the t-test. Mean ± S.E.; n = 12 to 26 images; **p < 0.01.
autophagic flux of fyve2 fyve3 (i.e., cfs1 cfs2) double mutants. As NBR1 is transcriptionally regulated in an autophagy-independent manner , further work is needed to investigate how NBR1 is regulated in fyve2 and fyve3 mutants.
The biochemical function of FYVE3 on the autophagic membrane is not clear. As autophagic flux is almost completely inhibited by atg2 but only partially by fyve2 (Kang et al., 2018;Kim et al., 2022), we postulate two separate autophagic routes, FYVE2dependent and FYVE2-independent routes ( Figure 5F). FYVE3 may specifically participate in FYVE2-dependent autophagy.
Considering the suppression effect indicating the involvement of FYVE3 in FYVE2-dependent autophagy, the absence of fyve3 single mutant phenocopying fyve2 is noteworthy. This can be explained if FYVE3 is a negative regulator of FYVE2-mediated autophagy. For example, FYVE3 may compete with autophagosome biogenesis factors for ATG8 binding. Alternatively, it is possible that FYVE3 both positively mediates the FYVE2-dependent autophagy and negatively regulates the FYVE2-independent route. In the absence of FYVE3, vacuolar delivery of ATG8 through the FYVE2independent pathway may increase and compensate for a reduced FYVE3 acts upstream of FYVE2 and downstream of ATG2 in a genetic pathway defined by the autophagy marker GFP-ATG8A. (A, B) Representative confocal microscopy images (left) and quantification (right) of GFP-ATG8A puncta (arrows at the images) acquired from mature root cells with indicated genetic background. Arabidopsis transgenic seedlings expressing GFP-ATG8A were incubated in either nitrogen-lacking liquid media for 12 h (A) or nitrogen-sufficient liquid media containing AZD8055 for 1 h (B), and treated with either wortmannin (Wm) or solvent control (DMSO) for 1 h prior to image acquisition. Scale bars = 5 µm. (C, D), GFP-ATG8A cleavage immunoblot assays. Arabidopsis transgenic seedlings expressing GFP-ATG8A with various genetic backgroundsa were incubated in either nitrogen-lacking liquid media for 24 h (C) or nitrogen-sufficient liquid media containing AZD8055 for 3 h (D) prior to protein extraction. Quantification of relative protein band intensities is shown below. (E) Phenotypic analysis for fixed-carbon starvation response. (F) A model for FYVE2 and FYVE3 functions in autophagosome dynamics. Columns marked with asterisks represent means that significantly differ from each other, according to the t-test. Mean ± S.E.; n = 14 to 16 images (A, B), or 4 seedling populations (C, D, E); *0.01 < p < 0.05; **p < 0.01.
autophagic flux of ATG8 through the FYVE2-dependent autophagy. In the latter model, the interaction of FYVE3 with ATG8 may be responsible for recruiting FYVE2 on expanding phagophores (Kim et al., 2022). Further investigation is needed to reveal the nature of FYVE2-dependent autophagy and a specific role for FYVE3 in this pathway. In summary, our study uncovers a regulatory module of FYVE2 and FYVE3 and its potential role in autophagosome biogenesis ( Figure 5F). Our study suggests that FYVE3 participates in FYVE2dependent autophagy via its interaction with PI3P and ATG8 isoforms. Thus, future investigation should ask what regions of FYVE3 are responsible for these interactions, whether FYVE3 is a positive or negative regulator of FYVE2-dependent autophagy, and how core ATG proteins and PI3P collaborate with FYVE2 and FYVE3.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.