Arabidopsis SINAT Proteins Control Autophagy by MediatingUbiquitylation and Degradation of ATG13[OPEN]

Hua Qi,a,1 Juan Li,a,b,1 Fan-Nv Xia,a Jin-Yu Chen,a Xue Lei,a Mu-Qian Han,b Li-Juan Xie,a Qing-Ming Zhou,b andShi Xiaoa,2

a State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-senUniversity, Guangzhou 510275, ChinabCollege of Agronomy, Hunan Agricultural University, Changsha, 410128 China

ORCID IDs: 0000-0002-7847-5079 (H.Q.): 0000-0002-1246-9610 (J.L.); 0000-0002-2789-3609 (F.N.X.); 0000-0001-9790-998X(J.Y.C.); 0000-0003-2343-0057 (X.L.); 0000-0003-2721-6517 (M.Q.H.); 0000-0001-5721-5908 (L.J.X.); 0000-0002-9369-8641(Q.M.Z.); 0000-0002-6632-8952 (S.X.).

In eukaryotes, autophagy maintains cellular homeostasis by recycling cytoplasmic components. The autophagy-relatedproteins (ATGs) ATG1 and ATG13 form a protein kinase complex that regulates autophagosome formation; however,mechanisms regulating ATG1 and ATG13 remain poorly understood. Here, we show that, under different nutrient conditions,the RING-type E3 ligases SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA1 (SINAT1), SINAT2, and SINAT6 control ATG1and ATG13 stability and autophagy dynamics by modulating ATG13 ubiquitylation in Arabidopsis (Arabidopsis thaliana).During prolonged starvation and recovery, ATG1 and ATG13 were degraded through the 26S proteasome pathway. TUMORNECROSIS FACTOR RECEPTOR ASSOCIATED FACTOR1a (TRAF1a) and TRAF1b interacted in planta with ATG13a andATG13b and required SINAT1 and SINAT2 to ubiquitylate and degrade ATG13s in vivo. Moreover, lysines K607 and K609 ofATG13a protein contributed to K48-linked ubiquitylation and destabilization, and suppression of autophagy. Under starvationconditions, SINAT6 competitively interacted with ATG13 and induced autophagosome biogenesis. Furthermore, understarvation conditions, ATG1 promoted TRAF1a protein stability in vivo, suggesting feedback regulation of autophagy.Consistent with ATGs functioning in autophagy, the atg1a atg1b atg1c triple knockout mutants exhibited premature leafsenescence, hypersensitivity to nutrient starvation, and reduction in TRAF1a stability. Therefore, these findings demonstratethat SINAT family proteins facilitate ATG13 ubiquitylation and stability and thus regulate autophagy.

INTRODUCTION

Autophagy, a highly conserved cellular process in all eukaryotes,degrades intracellular constituents to break down toxic materialsor damaged organelles and recycle essential nutrients (Basshamet al., 2006; Xie and Klionsky, 2007; Michaeli et al., 2016; Marshalland Vierstra, 2018). To date, three distinct types of autophagy,microautophagy, macroautophagy (referred henceforth as “au-tophagy”), and mega-autophagy, have been identified in plantcells (van Doorn and Papini, 2013; Marshall and Vierstra, 2018). InArabidopsis (Arabidopsis thaliana), autophagy functions inmaintaining Glc-mediated root meristem activity in the root cells(Huang et al., 2019a). In aerial tissues, autophagy is primarilyinducibleandactivatedbyavarietyofenvironmental cues, suchasnutrient deprivation, high salt, drought, hypoxia, oxidative stress,andpathogen infection (Doelling et al., 2002;Hanaokaet al., 2002;Yoshimoto et al., 2004; Liu et al., 2005, 2009; Xiong et al., 2007;Phillips et al., 2008;Hayward et al., 2009;Chung et al., 2010;Chenet al., 2015).

Autophagy begins with the formation of phagophore, whichexpands to form a double-membraned vesicle structure, termedan “autophagosome.” In particular, the outer membrane of theautophagosome fuses with the tonoplast and releases innermembrane vesicles (named “autophagic bodies”) containingcellular contents into the vacuole, where the sequestered cargo isdegraded by the resident acid hydrolases (He andKlionsky, 2009;Liu andBassham, 2012; Li andVierstra, 2012; Zhuang et al., 2015;Michaeli et al., 2016).Over the past few decades, a large number of autophagy-

related proteins (ATGs) were discovered in plants; these ATGsplay essential roles in regulating the core autophagic machinery(Liu et al., 2018; Soto-Burgos et al., 2018; Yoshimoto andOhsumi,2018; Zhuang et al., 2018). Deletions of Arabidopsis ATG genesleads to phenotypic changes, such as premature leaf senescenceand a shortened life cycle under normal growth conditions, hy-persensitivity to fixed carbon or nitrogen starvation, decreasedtolerance tobiotic andabiotic stresses, activated innate immunity,and an altered cellular metabolome (Doelling et al., 2002; Xionget al., 2007; Hayward et al., 2009; Liu et al., 2009; Chung et al.,2010; Guiboileau et al., 2012; Avin-Wittenberg et al., 2015; Chenet al., 2015; McLoughlin et al., 2018).In plants, ATG proteins predominately assemble into four

functional protein complexes: (1) the ATG1–ATG13 protein kinasecomplex; (2) theATG6–phosphatidylinositol 3-kinase complex; (3)a complex containing the transmembrane protein ATG9; and (4)two ubiquitin-like conjugation complexes, ATG5–ATG12 and

1 These authors contributed equally to this work.2 Address correspondence to: xiaoshi3@mail.sysu.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Shi Xiao (xiaoshi3@mail.sysu.edu.cn).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00413

The Plant Cell, Vol. 32: 263–284, January 2020, www.plantcell.org ã 2020 ASPB.

ATG8–phosphatidylethanolamine, which regulate autophago-some formation (Li andVierstra, 2012; Liu andBassham, 2012; Liuet al., 2018; Soto-Burgos et al., 2018; Yoshimoto and Ohsumi,2018). Developmental and nutritional signals promote the as-sembly of theATG1–ATG13kinase complex to initiate autophagy.

In Arabidopsis, the ATG1–ATG13 kinase complex includes theSer/Thr kinase ATG1 and its accessory proteins ATG13, ATG11,and ATG101, which are key positive regulators in the induction ofautophagic vesiculation (Suttangkakul et al., 2011; Liu and Bas-sham, 2012; Li et al., 2014). Through posttranslational phos-phorylation, the Arabidopsis ATG1–ATG13 complex is regulatedby the energy signaling pathway and a variety of upstreamkinases that affect their kinase activities (Liu and Bassham, 2010;Chen et al., 2017; Pu et al., 2017; Soto-Burgos and Bassham,2017). In particular, the TARGET OF RAPAMYCIN (TOR) kinaseand SUCROSE NONFERMENTING1-RELATED KINASE1 are im-portant negative and positive regulators, respectively, of theATG1–ATG13 complex. For example, overexpression of TOR inArabidopsis inhibits autophagy (Pu et al., 2017). Furthermore,downregulation or overexpression of the KIN10 catalytic subunitof Arabidopsis SUCROSE NONFERMENTING1-RELATED KINASE1suppresses or enhances autophagy induction, respectively, in re-sponse to nutrient starvation (Chen et al., 2017; Soto-Burgos andBassham, 2017).

Increasing evidence has demonstrated that the ubiquitinmodification system regulates ATG protein stability duringautophagosome formation inyeast,mammals, andplants (Shi andKehrl, 2010; Xia et al., 2013; Popelka andKlionsky, 2015; Xie et al.,2015; Qi et al., 2017). In mammal cells, during the inductionof autophagy, the E3 ligase TUMOR NECROSIS FACTORRECEPTOR ASSOCIATED FACTOR6 (TRAF6) mediates K63-linkedubiquitylation ofUNC-51-LIKEKINASE1, a homologofATG1. Theubiquitylation stabilizes ULK1, activating its self-association andkinase activity, thereby activating autophagy (Nazio et al., 2013).Under prolonged nutrient starvation, ULK1 autophosphorylationpromotes its interaction with Cullin/KELCH-LIKE PROTEIN20,a substrate adaptor of Cul3 ubiquitin ligase and binds Cul3 andsubstrate via its BTB domain and Kelch-repeat domain, forK48-linked ubiquitylation and proteasome-mediated degradation(Leeetal., 2010). ThedegradationofULK1 leads to the terminationof autophagy and thus prevents unrestrained cellular degrada-tion (Liu et al., 2016). Moreover, during the first few hours ofstarvation, the HOMOLOGOUS TO E6-ASSOCIATED PROTEINCARBOXYL TERMINUS domain-containing E3 ubiquitin ligaseNEURALPRECURSORCELL-EXPRESSEDDEVELOPMENTALLYDOWN-REGULATED GENE 4-LIKE interacts with ULK1 andtriggers ULK1 degradation by the proteasome pathway (Nazioet al., 2016). In particular, under selenite treatment in mammaliancells, ULK1partially translocates to themitochondria, and interactswith the mitochondria-localized E3 ligase MITOCHONDRIALUBIQUITIN LIGASE ACTIVATOR OF NFKB1, which mediates theK48-linked ubiquitylation of ULK1 for degradation in selenite-inducedmitophagy (Li et al., 2015). These findings suggest thatthe protein stabilities of the ATG1–ATG13 kinase complex aretightly controlled by the ubiquitin modification system to regulateautophagy in mammalian cells.

In Arabidopsis, the protein stabilities of ATG1–ATG13 complexare also affected by the ubiquitylation system (Suttangkakul et al.,

2011); however, the underlying regulatory mechanism remainsunknown. Our recent findings reveal that under normal nutrientconditions, Arabidopsis TRAF1a and TRAF1b act as adaptors tomediate the ubiquitylation anddegradation of ATG6by interactingwith theRING-typeE3 ligasesSINAT1andSINAT2 (Qi et al., 2017).Under starvation conditions, however, TRAF1a and TRAF1b re-cruit a starvation-inducible SINAT6 protein with a truncated RINGfinger domain to stabilize ATG6 and subsequently activate au-tophagy. Here, we show that SINAT proteins regulate autophagyby interacting with ATG13 proteins andmodulating the stability ofthe ATG1–ATG13 kinase complex under different nutrient con-ditions. Moreover, we observed that the ATG1s stabilize TRAF1proteins upon nutrient starvation through a feedback mechanismin the regulation of autophagy.

RESULTS

ATG1 and ATG13 Are Degraded by the 26S ProteasomePathway upon Starvation and During Recovery

Recent studies have identified the importance of ubiquitin mod-ification in regulatingATGprotein stability during autophagosomeformation (Popelka and Klionsky, 2015; Xie et al., 2015; Qi et al.,2017). In Arabidopsis, TRAF1a and TRAF1b act as adaptors tocontrol the stability of ATG6 by competitively interacting with theRING finger E3 protein ligases SINAT1/SINAT2 and SINAT5/SINAT6 under different nutrient conditions (Qi et al., 2017). To furtherinvestigate the potential degradation of ATG1 and ATG13 by theUb/26S proteasome pathway, we first examined their proteinlevels in wild-type plants upon carbon or nitrogen starvation withor without treatment with the proteasome inhibitor MG132 usingATG1a- and ATG13a-specific antibodies. As shown in Figure 1,ATG1a and ATG13a levels accumulated at 12 and 24 h, butstrongly decreased under prolonged (48 and 72 h) carbon or ni-trogen starvation treatments (Figures 1A and 1B; SupplementalFigures 1A and 1B). By contrast, the degradation of ATG1a andATG13a was repressed by the application of MG132 (Figures 1Aand 1B). Interestingly, MG132 treatment also promoted the ac-cumulation of ATG8a under starvation conditions (Figures 1A and1B). As a control, ATG7 showed few substantial changes in re-sponse to the carbon or nitrogen starvation and MG132 appli-cation did not affect the level of ATG7 at any time point (Figure 1;Supplemental Figure1).Thesefindingssuggest that thestabilityofthe ATG1–ATG13 protein complex is regulated by the 26S pro-teasome pathway during autophagosome formation.To confirm the role of the 26S proteasome in modulating the

stability of ATG13 proteins, we generated the ATG13a-HA andATG13b-HA transgenic lines overexpressing HA-tagged ATG13aand ATG13b, respectively (Supplemental Figure 1). Geneticanalyses showed that the ATG13a-HA #2 and ATG13b-HA #4lines harbored single T-DNA insertions and these lines wereused for further investigation. One-week–oldATG13a-HA andATG13b-HA seedlings grown under long-day conditions inMurashige and Skoog (MS) medium were transferred to mediumwithout Suc (–C) or nitrogen (–N) for carbon or nitrogen starvationtreatments. In response to carbon (Figure 1C) or nitrogen(Figure 1D) starvation, ATG13a-HAandATG13b-HAaccumulated

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Figure 1. The ATG1 and ATG13 Proteins are Degraded through the 26S Proteasome Pathway upon Starvation and During Recovery.

(A)and (B)ATG1a,ATG13a,ATG8a, andATG7protein levels in thewild-typeplantsuponcarbon (–C;A) or nitrogen (–N;B) starvationwithorwithoutMG132.One-week–oldwild-type seedlingswere subjected to carbonor nitrogenstarvationwithorwithout 50-mMMG132 for 0, 12, 24, and48h.Relative intensity ofeach protein normalized to the loading control is shown below.(C)and (D)ATG13a-HAandATG13b-HA levels in response to carbonstarvation (–C;C) or nitrogen starvation (–N;D) with orwithoutMG132.One-week–oldtransgenic linesexpressingATG13a-HAandATG13b-HAwere treatedwithconstantdarknessornitrogenstarvationwithorwithout50-mMMG132 for0,12,24, and 48 h.

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at 12 h, but consistently decreased at 24 and 48 h after starvationtreatments. Consistent with the protein blot analyses usingATG13-specificantibodies (Figures1Aand1B), thedegradationofATG13a-HA andATG13b-HA under carbon or nitrogen starvationconditions was suppressed by the application of 50 mM of MG132(Figures 1C and 1D).

Previous studies have suggested that proteaphagy is inducedby long-term MG132 treatment (Marshall et al., 2015). To furtherexplore the involvement of the ATG13 protein degradation by the26S proteasome pathway, we examined ATG13a protein levelupon MG132 treatment for 0, 1, 3, 6, and 12 h under carbon andnitrogen deprivation conditions (–C/N). ATG13a accumulated at 1h, but decreased at 3, 6 and 12 h after starvation treatment. Bycontrast, the degradation of ATG13a was repressed by the ap-plication of MG132 (Supplemental Figure 1E). The degradation ofATG13a-HA under –C/N conditions was also suppressed byMG132 treatment (Supplemental Figure 1F).

The degradation of ATG13 proteins may also occur during therecovery stages after nutrient starvation to terminate autophagy,which is a key process that increases survival of mammalian cells(Liu et al., 2016; Antonioli et al., 2017). To test this, we monitoredthe protein levels of ATG13a-HA andATG13b-HA at various times(6, 12, 24, 48, and 72 h) after recovery from carbon or nitrogenstarvation. As expected, both ATG13a-HA and ATG13b-HA ac-cumulated rapidly within 6 h during recovery and declined dra-matically at 72 h after recovery (Supplemental Figure 1). Further,we observed that MG132 application inhibited the degradation ofATG13a-HA andATG13b-HA at recovery stages after carbon andnitrogen starvation (Figures 1E and 1F). These findings indicatedthat the 26Sproteasomepathway controls the stability of ATG13aandATG13bunderbothstarvationconditionsandduring recoveryafter starvation treatments.

TRAF1a and TRAF1b Interact with ATG13a and ATG13b

TRAF1aandTRAF1bmediate thedegradationofATG6by forminga complex, termed the “TRAFasome,”with SINAT1, SINAT2, andATG6 under nutrient-rich conditions (Qi et al., 2017). We thereforehypothesized that TRAF1 proteins may contribute to the regu-lation of ATG1 and ATG13 protein stabilities. To test this, we firstexamined the interactions between ATG13a/ATG13b and TRAF1a/TRAF1b by the yeast two-hybrid (Y2H) assay. Only ATG13a,but not ATG1a, ATG1b, ATG1c, or ATG13b interacted withTRAF1a and TRAF1b (Supplemental Figure 2A). Failure to detectan interaction between TRAF1s and ATG1s is consistent with ourprevious findings (Qi et al., 2017). Moreover, introduction ofATG13bmayhavedetrimental effects on thegrowthof yeast cells,an effect that is likely distinct from that of ATG13a.

The potential associations of ATG1s and ATG13s with TRAF1awere further confirmed by bimolecular fluorescence comple-mentation (BiFC) analyses in thewild-type Arabidopsis protoplastcells. To this end, protein fusions with yellow fluorescent protein(YFP),ATG1a-cYFP,ATG1b-cYFP,ATG1c-cYFP,ATG13a-cYFP,or ATG13b-cYFP, were transiently coexpressed with TRAF1a-nYFP in protoplast for 16 h under continuous light or dark con-ditions, followed by confocal microscopy. The BiFC assaysshowed that for all combinations, the YFP signals were detectedin the cytoplasm under light conditions, but were observedas punctate structures under dark conditions (Figure 2A;Supplemental Figure 3). By contrast, coexpression of the negativecontrols TRAF1a-nYFP/ATG7-cYFP and nYFP/cYFP failed toreconstitute an intact YFP signal in Arabidopsis leaf protoplastsunder either light or dark conditions (Figure 2A; SupplementalFigure 3). Further, we coexpressed mCherry-ATG8a (Qi et al.,2017) as an autophagasome marker with the split TRAF1a-nYFPand ATG1a-, ATG1b-, ATG1c-, ATG13a-, ATG13b-, and ATG7-cYFP fusions in protoplasts under light or dark conditions. Asshown in Supplemental Figure 3B, starvation-inducible punctatedots primarily colocalized with mCherry-ATG8a.Next, we used stable transgenic lines expressing TRAF1a-

FLAG (Qi et al., 2017) for coimmunoprecipitation (CoIP) assays.When ATG13a-HA or ATG13b-HA was transiently expressed inthe protoplasts isolated from rosettes of TRAF1a-FLAG line,TRAF1a-FLAG could be immunoprecipitated by ATG13a-HA andATG13b-HA (Figure 2B), but not by ATG7-HA, ATG1a-HA,ATG1b-HA, and ATG1c-HA (Figure 2C; Supplemental Figure 2B).We also incubated the total proteins from the TRAF1a-FLAG linewith FLAG magnetic beads and used anti-ATG13a-specific an-tibodies for immunoblot analysis. The results showed that ATG13acould be immunoprecipitated by TRAF1a-FLAG (SupplementalFigure 2C). These findings indicate that ATG13 proteins and TRAF1ainteracted in autophagosome-related structures in response tostarvation.To investigate the functional significance of protein interaction

betweenTRAF1a/TRAF1bandATG13a/ATG13b,weanalyzed thelevels of ubiquitylated ATG13a in the presence or absence ofTRAF1a and TRAF1b. After transient expression of the ATG13a-HAplasmid in the protoplasts isolated fromwild-type and traf1a-1traf1b-2 double mutant (traf1a/b) leaves (Qi et al., 2017) for 16 h,total protein was extracted and coprecipitated by HA affinityagarose beads followed by immunoblot analysis. As shown inFigure 2D, the total ubiquitylation in the traf1a/b double mutant isalmost equal to that of wild-type plants. However, the ubiq-uitylation of ATG13a-HA was reduced in the traf1a/b mutantcompared with the wild-type plants (Figure 2D). To examine therole of TRAF1a/b inmodulating ATG13a stability, we detected the

Figure 1. (continued).

(E) and (F) ATG13a-HA and ATG13b-HA levels upon carbon (–C; E) or nitrogen (–N; F) starvation or during recovery (“R”) or in the presence of MG132(R1MG132) for the indicated times. One-week–old transgenic lines expressing ATG13a-HA and ATG13b-HA were treated with constant darkness ornitrogen starvation, andmoved back toMSmedium under normal light/dark conditions in the absence or presence of 50-mMMG132 to recover for 24, 48,and 72 h.Anti-ACTINantibodies andPonceauS-stainedRubisco bands are shownbelow the blots to indicate the amount of protein loadedper lane. Numbers on theleft indicate molecular mass (kD) of each band. hpt, hours posttreatment.

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Figure 2. TRAF1s Interact with ATG13a and ATG13b In Vivo.

(A) BiFC assay of ATG1/ATG13 proteins (ATG1a and ATG13a) and TRAF1a in Arabidopsis. The split cYFP fusions ATG13a-cYFP, ATG1a-cYFP or ATG7-cYFP and TRAF1a-nYFP were coexpressed in leaf protoplasts and incubated for 16 h under light or dark conditions. The TRAF1a-nYFP/ATG7-cYFP andnYFP/cYFP vectors were coexpressed as negative controls. Confocal micrographs obtained from YFP, autofluorescent chlorophyll, bright-field, andmerged imagesareshown.Thenumbers in thecellsofATG13aorATG1a-cYFP1TRAF1a-nYFP/dark indicatetheaveragenumberofautophagosomes6SD (n53)

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protein stability of ATG13a in wild-type, traf1a/b, and TRAF1a-FLAG plants after a 48-h nutrient starvation treatment or recoveryunder nutrient-rich conditions for 48and72h. The results revealedthat the degradation of ATG13a under both nutrient-deficient andnutrient-rich recovery conditions was significantly inhibited in thetraf1a/bmutant and theTRAF1a-FLAG transgenic line (Figure 2E),suggesting that TRAF1a is involved in regulating ATG13a stabilityin planta.

ATG13a Is a Target of the SINAT Proteins

Previous studies revealed that Arabidopsis TRAF1a and TRAF1bare required for SINAT1- and SINAT2-associated ubiquitylationand degradation of ATG6. However, under nutrient-starvationconditions, SINAT6 is involved in inhibiting the degradation ofATG6bycompetitively interactingwithATG6 to induceautophagy(Qi et al., 2017). To further understand the molecular basis ofATG13a degradation, we used Y2H assays to assess the asso-ciations of ATG13a and all six SINAT proteins, SINAT1, SINAT2,SIANT3, SINAT4, SINAT5-S1 (a spliced form of the truncatedSINAT5 lacking the RING finger domain in theCol-0 ecotype), andSINAT6. ATG13a and ATG13b interacted only with SINAT5-S1and SINAT6 in yeast cells (Figure 3A). However, our CoIP assaysuggested thatATG13a interactedwithSINAT1,SINAT2,SINAT5-S1, andSINAT6 in plant cells (Figure 3B).WhenATG7 andSINATswere coexpressed in the wild-type protoplasts as negativecontrols, ATG7 was not coprecipitated by SINAT proteins(Supplemental Figure 4A). To investigate the domains mediatingthe interaction between SINATs and ATG13a, we used two al-ternatively spliced forms, SINAT5-S1 and SINAT5-S2 in Col-0,and a truncated form of SINAT5 for the Y2H analysis. ATG13ainteracted with SINAT5-S1 and SINAT5-S2, instead of truncatedSINAT5without theTRAFdomain, indicating that theC-terminal ofthe TRAF domain in SINAT5 is essential for the interaction be-tween ATG13a and SINAT5 (Figure 3C).

The interaction between ATG13a and SINATs was furtherconfirmed by BiFC assays in Arabidopsis cells. When SINAT1-nYFP or SINAT2-nYFP were transiently coexpressed withATG13a-cYFP or ATG13b-cYFP, respectively, in wild-type

protoplasts for 16 h, punctate YFP signals were observed(Supplemental Figure 4B). By contrast, uniform fluorescent BiFCsignalswere detected in the cytoplasmwhenSINAT5-S1-nYFPorSINAT6-nYFP were transiently coexpressed with ATG13a-cYFPor ATG13b-cYFP, respectively (Supplemental Figure 4B). Ascontrols, coexpression of SINATs-nYFP/ATG7-cYFP and nYFP/cYFP failed to reconstitute intact YFP in Arabidopsis leaf proto-plasts (Supplemental Figure 4B).Given that SINAT1, SINAT2, SINAT5-S1, and SINAT6 interact

with ATG13a (Figures 3A to 3C; Supplemental Figure 4), wefurther asked whether these SINAT proteins are involved inubiquitylationofATG13aandsubsequently affect its protein stability.To test this possibility, we coexpressed ATG13a-FLAG with GFP-tagged SINAT fusions, GFP-SINAT1-HA, GFP-SINAT2-HA, GFP-SINAT5-S1-HA, and SINAT6-GFP-HA, in wild-type Arabidopsisprotoplasts. The protein gel blots using the anti-HA or anti-FLAGantibodies showed that the ubiquitylation of ATG13a-FLAG wasinducedby theexpressionofGFP-SINAT1-HAandGFP-SINAT2-HAfusions, but very weak signals were detected with the empty vectorcontrolaswell aswithexpressionofGFP-SINAT5-S1-HAorSINAT6-GFP-HA fusions (Figure 3D).To investigate whether SINAT proteins play a role in regulating

the stability of ATG13a, we identified sinat3, sinat4, and sinat5T-DNA insertional single mutants (Supplemental Figure 5), andcrossed them to sinat1 sinat2 (Qi et al., 2017) or sinat6-2 (Qi et al.,2017)mutants, respectively, togeneratesinat1sinat2sinat3sinat4(sinat1/2/3/4) quadruple mutant and sinat5 sinat6 double mutantplants. Then we subjected the wild-type, sinat1/2/3/4 quadruplemutant, and SINAT1-OE seedlings to constant dark treatment forfixed-carbon starvation for 0, 24, 48, and 72 h, and detected theATG13 protein levels using ATG13a-specific antibodies. Asshown inFigures3Eand3F, theATG13a levelclearlydeclinedafter72 h of carbon starvation treatment in the wild-type seedlings.However, thedegradationofATG13awas inhibited in the sinat1/2/3/4 mutant and increased in the SINAT1-OE transgenic line(Figure 3E). By contrast,weobserveddecreased levels of ATG13ain the sinat5 sinat6 double mutant and an increased level in theSINAT6-OE line (compared with the wild-type control), in re-sponse to carbon starvation (Figure 3F). These findings suggest

Figure 2. (continued).

calculated from three independent experiments. For each experiment, 15 images were used for the calculation for each coexpression combination. Scalebars 5 10 mm.(B) In vivoCoIP assay of the association betweenATG13 (ATG13a andATG13b) and TRAF1a. HA-taggedATG13aor ATG13b (ATG13a-HAorATG13b-HA)was transiently expressed in protoplasts from transgenic plants expressing TRAF1a-FLAGunder light conditions for 16 h, and immunoprecipitatedwithHAaffinity agarose beads.(C) In vivoCoIPassayof the interaction betweenTRAF1a andATG1 (ATG1a, ATG1b, andATG1c) proteins.HA-taggedATG1a, ATG1b, andATG1c (ATG1a-HA,ATG1b-HA, andATG1c-HA)were transiently expressed inprotoplasts from transgenicplants expressingTRAF1a-FLAG for 16hunder continuousdarkconditions and immunoprecipitated with HA affinity agarose beads.(D)TheubiquitylationofATG13a in the traf1a/bmutant.ATG13a-HAwas transiently expressed inArabidopsisprotoplasts isolated from four-week–oldwild-type and traf1a/bmutant plants, and the ubiquitylation of ATG13awasdetected by immunoprecipitation and immunoblot analysis. Proteinswere extractedat 16 h after expression under constant light conditions and then incubatedwith HA affinity agarose beads. The blotswere probedwith anti-HA and anti-Ubantibodies.(E)ThedegradationofATG13a in traf1a/b-1andTRAF1a-FLAGplants.One-week–oldwild-type (WT), traf1a/b, andTRAF1a-FLAGplantswere subjected tocarbon (–C; top images) or nitrogen starvation (–N; bottom images) treatment for 48 h, after recovery (“R”) for 48 and 72 h. The blots were probedwith anti-ATG13a-specific antibodies. hpt, hours posttreatment.Numbers on the left indicatemolecularmass (kD) of eachband. Anti-ACTINantibodies andPonceauS-stainedRubisco bands are shownbelow theblots toindicate the amount of protein loaded per lane. The expression of GFP-HA shows the expression efficiency of each sample.

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Figure 3. Interaction of ATG13s with SINAT Proteins.

(A) Y2H analysis showing the interaction between ATG13a/b and SINAT proteins. ATG13a/b-BD and SINAT-AD (SINAT1-AD, SINAT2-AD, SINAT3-AD,SINAT4-AD, SINAT5-S1-AD, and SINAT6-AD) were coexpressed in the YH109 yeast strain and selected on SD/–Trp-Leu-His-Ade medium (–LWH). AD,empty AD plasmid.

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that ATG13a is a target of SINAT1, SINAT2, SINAT5-S1, and/orSINAT6 in Arabidopsis, and that SINAT1/SINAT2 and SINAT5/SINAT6 may play different roles in the regulation of ATG13aprotein stability.

To further investigate the degradation of ATG13a by SINAT1and SINAT2 through the 26S proteasome pathway, we coex-pressed ATG13a-HA and SINATs-FLAG in protoplasts from 4-week–old wild-type and rpn10 PAG1-GFP (Marshall et al., 2015)plants. Compared with the wild-type plants, the degradation ofATG13a bySINAT1 andSINAT2was impaired in the rpn10 PAG1-GFP plants (Supplemental Figure 6), suggesting that SINAT1 andSINAT2 mediate degradation of ATG13a by the 26S proteasomepathway.

The K607 and K609 Lys Sites Contribute to ATG13aUbiquitylation

Posttranslational polyubiquitylation, such as Lys-48-linkedubiquitylation, often targets substrates for proteasome degra-dation (Kuang et al., 2013). To investigate whether ATG13a andATG13b undergo Lys-48–linked ubiquitylation in response tostarvation treatment, we analyzed the ubiquitylation of ATG13a-HA and ATG13b-HA using K48-linked ubiquitylation antibodies.The immunoblot analyses showed that the K48-linked ubiq-uitylation levels of ATG13a and ATG13b increased after constantdark treatment for 24 and 48 h, and were further enhanced by theapplication ofMG132 (Figures 4A and 4B), indicating that ATG13aand ATG13b are modified by the K48-linked ubiquitylation inresponse to nutrient starvation.

To identify the potential ubiquitylation site of ATG13a, weused the tool UbPred (http://www.ubpred.org/; Cai et al., 2012),which predicted K607 and K609 as two potential ubiquitylationsites of ATG13a (Figure 4C). To test this possibility, we mutatedK607 and K609 to Arg (R, the abbreviation of arginine acid) togenerate ATG13aK607R-HA (ATG13a-K1-HA), ATG13aK609R-HA (ATG13a-K2-HA), and ATG13aK607/609R-HA (ATG13a-K1/2-HA) constructs. As controls, we mutated K54 and K146, whichwere not ubiquitination sites predicted by UbPred, to R to generate

ATG13aK54R-HA (ATG13a-K3-HA), and ATG13aK146R-HA(ATG13a-K4-HA) constructs. We evaluated the effects oftheK607R,K609R,K54R,andK146Rmutationsontheubiquitylationand stability of ATG13a by transiently expressing ATG13a-HA,ATG13a-K1-HA, ATG13a-K1/2-HA, ATG13a-K3-HA, and ATG13a-K4-HA in wild-type protoplasts. The ubiquitylation of ATG13a-HAdecreased in both of the K607R and K609R single mutants, andwas further reduced in the ATG13a-K1/2-HA double mutantcompared with the wild-type ATG13a-HA control (Figure 4D;Supplemental Figure 7A), suggesting that K607 and K609 arenecessary for the ubiquitylation of ATG13a. Consistent with thecorresponding ubiquitylation levels, ATG13a-HA accumulated inthe ATG13a-K1/2-HA mutant (Figure 4D). By contrast, theubiquitylation and stability of ATG13a-HA showed little differencein the K54R and K146R single mutants compared with the wild-type ATG13a-HA control (Supplemental Figure 7A). Together,these findings suggest that K607 and K609 are required forubiquitylation and degradation of ATG13a in planta.Mutations in theubiquitylationsitesof animalATG1sautophagy

proteins lead to a longer half-life comparedwith that of the control,and therefore the mutant proteins are more stable in response tothe protein translation inhibitor cycloheximide (CHX; Nazio et al.,2016; 2017). We accordingly used CHX to monitor the effects ofATG13a-K1/2 mutations on ATG13a stability. We introduced theATG13a-K1/2-HA construct into wild-type Arabidopsis to gen-erate two independent transgenic lines ATG13a-K1/2 #1 andATG13a-K1/2 #2 (Supplemental Figures 7B to 7E). We comparedtheATG13aprotein stabilities in theATG13a-HAandATG13a-K1/2-HA mutant lines in the presence or absence of CHX undercarbon starvation conditions. Under constant darkness with CHXfor 6 and 12 h, the ATG13a-K1/2-HA was more stable with longerhalf-life comparedwith that of ATG13a-HA protein (SupplementalFigure 7F). This result supports the conclusion that the K607 andK609 residues play a primary role in mediating the stability ofATG13a protein.To test the significance of ATG13a ubiquitylation in autophagy-

mediated nutrient starvation stress tolerance in Arabidopsis, wefurther analyzed the phenotypes of the ATG13a-K1/2-HA mutant

Figure 3. (continued).

(B) In vivo Co-IP analysis showing the interaction between ATG13a and SINATs. FLAG-tagged ATG13a (ATG13a-FLAG) was coexpressed with GFP-HA-tagged SINATs (GFP-SINAT1-HA, GFP-SINAT2-HA, GFP-SINAT5-S1-HA, and SINAT6-GFP-HA) in Arabidopsis protoplasts and immunoprecipitated byGFP agarose beads.(C) Truncation analysis of SINAT5 to identify the functional domain mediating the ATG13a/b-SINAT5 association. Full-length SINAT5 was amplified fromecotype Ler containing a RING finger (RING), a zinc finger (ZINC), and a TRAF domain (TRAF). SINAT5-S1 and SINAT5-S2 are two alternatively splicedproducts produced in ecotypeCol-0 andcontaining aTRAFdomainwith impairedRINGorZINCdomains.△183 to△309 is an artificially truncated proteinwithout the TRAF domain. Truncated SINAT5 was fused to the BD domain and coexpressed with ATG13a/b-AD in yeast. Positive clones were selected onSD medium lacking Trp, Leu, His, and Ade (–LWH). AD, empty AD plasmid.(D) In vivo ubiquitylation of ATG13a by SINAT1, SINAT2, SINAT5-S1, and SINAT6. ATG13a-FLAGwas coexpressed with GFP-SINAT1-HA, GFP-SINAT2-HA, GFP-SINAT5-S1-HA, or SINAT6-GFP-HA in Arabidopsis protoplasts for 16 h under continuous light conditions, and its ubiquitylationwas detected byimmunoblot analysis.(E)ATG13aprotein level in the sinat1/2/3/4mutant andSINAT1-OE line in response tocarbonstarvation.One-week–oldwild type (WT), sinat1/2/3/4mutant,and SINAT1-OE lines were treated with darkness for 0, 24, 48, and 72 h. The blots were probed with anti-ATG13a-specific antibodies.(F)ATG13a accumulation after treatmentwith constant darkness in the sinat5 sinat6mutant andSINAT6-OE line.One-week–oldwild type, sinat5/6mutant,and SINAT6-OE lines were treated with darkness for 0, 24, 48, and 72 h. The blots were probed with anti-ATG13a antibodies.Numbers on the left indicatemolecularmass (kD) of eachband. Theblot expressionofGFP-HAshows theexpressionefficiencyof eachsample.Anti-ACTINantibodies, or Ponceau S-stained Rubisco bands are shown below the blots to indicate the amount of protein loaded per lane. Relative intensity of eachprotein normalized to the loading control is shown below. hpt, hours posttreatment; WT, wild type.

270 The Plant Cell

Figure 4. Identification of ATG13a/b Ubiquitylation Sites.

(A) and (B) K48-linked ubiquitylation in response to carbon starvation in ATG13a-HA (A) and ATG13b-HA (B). One-week–old transgenic lines expressingATG13a-HA and ATG13b-HA were treated with darkness or 50 mM of MG132 for 0, 12, 24, and 48 h. Total protein was immunoprecipitated by HA affinityagarose beads and immunoblotted with Lys48 anti-ubiquitin-specific antibodies. Numbers on the left indicate molecular mass (kD) of each band.

Regulation of ATG13s by SINAT Proteins 271

in response to nutrient starvation treatment. The 1-week–oldwild-type, ATG13a-OE, ATG13a-K1/2 #1, and ATG13a-K1/2 #2seedlingswere grownonMSand transferred to liquidMSmedium(N1) or nitrogen-deficient liquid MS medium (N–) for 4 d. All theplants had very similar phenotypes to the wild-type plants undernitrogen-sufficient (N1) conditions (Figure 4E). However, theATG13a-OE, ATG13a-K1/2 #1, and ATG13a-K1/2 #2 lines ex-hibited increased tolerance to nitrogen starvation (Figure 4E). Inparticular, the twomutant linesATG13a-K1/2#1 andATG13a-K1/2#2weremore tolerant than theATG13a-OE line, as calculatedbythe relative chlorophyll contents of the seedlings (Figure 4F).

Similarly, when1-week–oldwild-type,ATG13a-OE,ATG13a-K1/2#1, andATG13a-K1/2 #2 seedlingswere subjected to constant darktreatment for fixed-carbon starvation (C–) for 9 d, the ATG13a-OE,ATG13a-K1/2#1, andATG13a-K1/2#2 seedlings showed improvedsurvival with green true leaves compared with the wild type(Figure 4G; Supplemental Figure 8A). Consistent with the nitrogenstarvation, theATG13a-K1/2#1andATG13a-K1/2#2showedbettertolerance than that of the ATG13a-OE line, and the three lines hadsignificantly higher relative chlorophyll contents and fresh weightsthan the wild type (Figure 4H; Supplemental Figure 8B).

To further investigate the functional relevance of ATG13aubiquitylation in autophagy-associated nutrient starvation toler-ance, we performed a complementation test by introducingATG13a-K1/2-HA into the atg13a atg13b (atg13a/b) double mu-tant (Suttangkakul et al., 2011) to generate the ATG13a-K1/2-HAatg13a/b lines (ATG13a-K1/2-HAatg13a/b #1 andATG13a-K1/2-HA atg13a/b #3). When 1-week–old seedlings of wild type, theatg13a/bdoublemutant, and theATG13a-K1/2-HAatg13a/b lineswere subjected to 4-d nitrogen starvation, the double mutantsshowed increased sensitivity, with yellowing seedlings and sig-nificantly lower chlorophyll contents (Supplemental Figures 8Cand 8D). However, the sensitivity of atg13a/b double mutant tonitrogen starvation was completely complemented by ATG13a-K1/2-HA and showed comparable relative chlorophyll contents tothe wild-type seedlings (Supplemental Figures 8C and 8D).

TRAF1s Are Required for SINAT1- and SINAT2-MediatedUbiquitylation and Degradation of ATG13 Proteins

To determine the involvement of TRAF1a and TRAF1b in theSINAT-mediated ubiquitylation and degradation of ATG13a, wecoexpressed ATG13a-FLAG and GFP-SINAT1-HA in the proto-plasts isolated from rosettes of the wild type and the traf1a/b double mutant. As shown in Figure 5A, ATG13a-FLAG ubiq-uitylation was enhanced by the presence of GFP-SINAT1-HA inthe wild-type background. However, it strongly declined in thetraf1a/b mutant either in the presence or in the absence of GFP-SINAT1-HA (Figure 5A), suggesting that TRAF1a and TRAF1b arerequired for SINAT-mediated ubiquitylation of ATG13a. Further-more, we observed that the degradation of ATG13a-FLAG in-duced by the expression of GFP-SINAT1-HA was impaired in thetraf1a/bmutant (Figure 5B). Together, these findings indicate thatTRAF1a and TRAF1b contribute to SINAT1-mediated ubiq-uitylation and degradation of ATG13a.In Arabidopsis, SINAT6 plays an opposite role to SINAT1 in

ubiquitylation and destabilization of ATG6 by competitivelyinteracting with ATG6 to form a different TRAFasome, TRAF1-SINAT6-ATG6 (Qi et al., 2017). Given the evidence that SINAT6promoted the accumulation of ATG13a (Figure 3F), we thereforehypothesized that SINAT6 may be involved in maintaining thestability of ATG13a by associating with ATG13a under certaingrowth conditions. To test this possibility, we transiently ex-pressed ATG13a-FLAG with GFP-SINAT1-HA alone or GFP-SINAT1-HA together with SINAT6-GFP-HA in wild-type protoplastsand detected the ubiquitylation and degradation of ATG13a byimmunoblot analyses. Competition analyses showed that theSINAT1-mediated ubiquitylation of ATG13awas strongly reducedby coexpression of SINAT6 (Figure 5C). Furthermore, SINAT1-mediated degradation of ATG13a was strongly inhibited by theaddition of SINAT6 in a dose-dependent manner (Figure 5D),implying that SINAT1 and SINAT6 may play opposing roles in theregulation of the stability of ATG13 proteins.

Figure 4. (continued).

(C) Alignment of ATG13a and the ATG13a ubiquitylation site mutant. The alignment was analyzed using the T-Coffee website (http://tcoffee.crg.cat/apps/tcoffee/index.html). ATG13a-K1 indicates the K607Rmutation, ATG13a-K2 indicates the K609Rmutation, and ATG13a-K1/2 indicates both the K607 andK609 point mutations to R.(D)ATG13a levels in the ubiquitylation sitemutants. HA-tagged ATG13a (ATG13a-HA), andATG13a ubiquitylation sitemutants (ATG13a-K1-HA, ATG13a-K2-HA, andATG13a-K1/2-HA)were expressed inwild-type protoplast. Total proteinwas extracted after incubation for 16hunder constant light conditions,and immunoprecipitated by HA affinity agarose beads. The blots were probedwith anti-HA and anti-Ub antibodies. Numbers on the left indicatemolecularmass (kD) of each band. Anti-ACTIN antibodies and Ponceau S-stained membranes are shown below the blots to indicate the amount of protein loadedper lane.(E) Response of the ATG13a ubiquitylation mutant to nitrogen-starvation treatment. One-week–old seedlings grown on MS medium were transferred tonitrogen-rich (N1) or nitrogen-deficient (N–) liquid medium for an additional 5 d before photographing and chlorophyll measurement.(F)Relative chlorophyll contents of seedlingswith orwithout nitrogen-deficient treatment shown in (E). The relative chlorophyll contentswere calculated bycomparing the values in N– seedlings versus N1 seedlings.(G) Phenotypes of the ATG13a ubiquitylation mutant in response to carbon starvation. One-week–old wild-type, ATG13a-OE, ATG13a-K1/2 #1, andATG13a-K1/2#2 seedlingsgrownonMSsolidmediumwere transferred toMSplateswithSuc (C1) orwithoutSuc followedby constant dark treatment (C–)for 9 d. The images were recorded after a 7-d recovery.(H) Relative chlorophyll contents of wild-type, ATG13a-OE, ATG13a-K1/2 #1, and ATG13a-K1/2 #2 seedlings described in (G) after recovery. The relativechlorophyll contents were calculated by comparing the values of C–treated versus C1 treated seedlings.Relative chlorophyll contents are average values6SD (n5 3) calculated from three independent experiments. For each experiment, five technical replicatespooledwith at least 15 seedlingswereusedper genotype.Asterisks indicate significant differences from thewild type (WT) (A)orATG13a-OE (B); *P<0.05;**P < 0.01 by one-way ANOVA.

272 The Plant Cell

ATG1s Stabilize TRAF1a In Vivo

The ATG1 Arabidopsis Ser/Thr kinases proteins interact withATG13s and other regulatory proteins, such as ATG11 andATG101, to form a protein kinase complex that stimulates auto-phagic vesiculation (Liu and Bassham, 2010; Suttangkakul et al.,

2011; Li et al., 2014). Although ATG1s did not interact with TRAF1proteins in the Y2H and CoIP assays, they reconstituted an intactYFP signal in the BiFC analysis (Figure 2; Supplemental Figures 2and 3). In the CoIP assay, particularly, when TRAF1a-FLAG andATG1a-HA, ATG1b-HA, or ATG1c-HA were transiently coex-pressed, TRAF1a-FLAG mobility was clearly shifted by ATG1

Figure 5. TRAF1a Is Required for SINAT-Mediated Ubiquitylation and Degradation of ATG13a.

(A) SINAT-mediated ATG13a ubiquitylation in the traf1a/b mutant. ATG13a-FLAG and GFP-SINAT1-HA were transiently coexpressed in Arabidopsisprotoplasts prepared from wild-type or traf1a/b plants for 16 h under constant light conditions.(B)SINAT1-associateddegradationofATG13a isdependentonTRAF1a.ATG13a-FLAGandGFP-SINAT1-HAwere transiently coexpressed inArabidopsisprotoplasts prepared from wild-type and traf1a/b plants for 16 h under continuous light conditions.(C) SINAT1-mediated ATG13a ubiquitylation in response to coexpression of SINAT6. ATG13a-FLAGwas coexpressed with SINAT1 or SINAT1/SINAT6 inArabidopsis protoplasts for 16 h under constant light conditions.(D) SINAT1-associated degradation of ATG13a in response to SINAT6. ATG13a-FLAGwas coexpressed with GFP-SINAT1-HA in the presence of variousamounts (0, 10, 20, and 30 mg) of SINAT6-GFP-HA in Arabidopsis protoplasts for 16 h under continuous light conditions.The blot expression of GFP-HA shows the expression efficiency of each sample. Ponceau S-stained Rubisco bands are shown below the blots to indicatethe amount of protein loadedper lane. Numbers on the left indicatemolecularmass (kD) of each sizemarker. Relative intensity of each protein normalized tothe loading control is shown below. WT, wild type.

Regulation of ATG13s by SINAT Proteins 273

proteins (Figure2C); thispromptedus toaskwhetherATG1saffectthe patterns of TRAF1 proteins. Given that ATG1s function asSer/Thr kinases in response to autophagy induction, the shiftedmobility of TRAF1a-FLAG is likely due to the phosphorylation byATG1s. To confirm this, we expressed ATG1a-HA, ATG1b-HA, orATG1c-HA in protoplast cells from the rosettes of stable TRAF1a-FLAG transgenic plants. The immunoblot analysis detected twobands of TRAF1a-FLAG using anti-FLAG antibodies, and thehigher-molecular–weight bandappearedwith thecoexpressionofATG1 proteins (Figure 6A). TRAF1a-FLAG patterns were evalu-atedbyadding lambdaprotein phosphataseand thephosphataseinhibitor PhosSTOP to the total proteins (Suttangkakul et al.,2011). As shown in Figure 6B, the lambda phosphatase treatmentreduced the levels of the higher-molecular–weight species ofTRAF1a-FLAG, and PhosSTOP blocked this shift, implying thatATG1 proteins are involved in the phosphorylation-like modifi-cation of TRAF1a in vivo.

To investigate the function of ATG1a in modulating TRAF1astability, we crossed TRAF1a-FLAG to YFP-ATG1a stabletransgenic plants (Chen et al., 2017) to generate a TRAF1a-FLAGYFP-ATG1a double combination line and determined theTRAF1a-FLAG stability in response to nutrient starvation treat-ment at various times. TRAF1a-FLAG degraded after exposure toconstant darkness at 16 and 24 h. By contrast, in the YFP-ATG1aline, TRAF1a-FLAGaccumulated continuously from0 to 24 h aftercarbon starvation treatment (Figure 6C), suggesting that ATG1aplays a crucial role in maintaining the stability of TRAF1a inresponse to nutrient starvation. In particular, in the presenceof YFP-ATG1a, potential phosphorylated TRAF1a-FLAG was thepredominant form at 24 h after carbon starvation (Figure 6C).

ATG1 Null Mutants Are Hypersensitive to NutrientDeprivation

To further investigate the potential roles of ATG1s in modulatingthe stability of TRAF1 proteins, we identified four T-DNA in-sertional mutants, atg1a-2, atg1b-1, atg1c-1, and atg1t-1, whichcompromise the expression of ATG1a, ATG1b, ATG1c, andATG1t, respectively (Supplemental Figure 9; Suttangkakul et al.,2011).Reverse transcriptionPCRanalysesshowed that these fourT-DNA insertions blocked the transcription of ATG1a, ATG1b,ATG1c, and ATG1t, respectively (Supplemental Figure 9C), in-dicating that all of these lines are null mutants.

Previous studies demonstrate that the classic autophagy-defective mutants exhibit premature leaf senescence and hy-persensitivity to nutrient deprivation (Doelling et al., 2002;Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompson et al., 2005;Xiong et al., 2005; Phillips et al., 2008; Chung et al., 2010;Suttangkakuletal.,2011;Qietal.,2017).Allof theatg1singlemutantsshowed little or no phenotypic change compared with wild-typeplants grown in either nutrient-rich or nutrient-deprived conditions(SupplementalFigure10),confirmingpreviousfindings (Suttangkakulet al., 2011).

To test their functional redundancy, we crossed the atg1a-2,atg1b-1, atg1c-1, and atg1t-1 mutants to generate atg1a atg1cand atg1b atg1t double mutants, the atg1a atg1b atg1c (atg1abc)triple mutant, and the atg1a atg1b atg1c atg1t (atg1abct) qua-druple mutant for further phenotypic analyses. The atg1a atg1c

and atg1b atg1t double mutants also showed few morphologicaldifferences from the wild-type plants under normal growth con-ditions and after starvation treatments (Supplemental Figure 10).Similar to theother identifiedautophagy-deficientatgmutants, theatg1abc triple and atg1abct quadruple mutants did not displayobvious phenotypic differences from the wild-type plants undernutrient-rich conditions for up to 3 weeks of growth (Figure 7;Supplemental Figures 11 and 12).In contrast with their phenotypes on nutrient-rich medium, the

atg1abc and atg1abct mutants showed significant hypersensi-tivity when grown in fixed-carbon- or nitrogen-deficient medium(Figure 7; Supplemental Figures 11 and 12). After fixed-carbonstarvation induced by constant darkness for 7 d, the mutantsshowed yellowing leaves, in contrast to the green leaves andsignificantly higher chlorophyll contents in the wild-type plants(Figures 7A to 7C; Supplemental Figure 11). After a 7-d recoveryunder normal light/dark conditions, 60% of the wild-type plantssurvived, while only 20–30% of the triple or quadruple mutantssurvived (Figure 7D).Toconfirm the responseof theatg1abcandatg1abctmutants to

fixed-carbon starvation, 3-week–old soil-grown mutants weresubjected to constant dark treatment. Indeed, the atg1abc andatg1abct mutants displayed hypersensitivity to carbon depriva-tionwith lower relative chlorophyll contentsandsurvival rates thanthat of the wild-type plants (Supplemental Figures 12A to 12C).When 1-week–old wild-type, atg1abc triple mutant, and atg1abctquadruplemutant plantswere subjected to a nitrogen-deprivationtreatment on solid medium for 5 d or in liquid medium for 4 d, allcotyledons of the triple and quadruple mutants exhibited in-creased yellowing, as calculated by the relative chlorophyllcontents of the plants (Figures 7E to 7G; Supplemental Figure 11,12D and 12H). The sensitivities of the atg1abc and atg1abctmutants to nutrient starvations observed in this studywere similarto that of the atg13a atg13bdoublemutant (Supplemental Figures12F to12H;Suttangkakul et al., 2011), but slightlyweaker than thatof the atg10-1mutant (Figure 7; Supplemental Figures 11 and 12;Phillips et al., 2008).We monitored the natural senescence of these mutants, ob-

serving that the rosettes of the atg1abc and atg1abct mutantswere green like the wild-type plants during the first four weeks(Figure 7H). However, similar to the atg10-1 mutant, thecotyledons and some true leaves of the 5-week–old atg1abcand atg1abct mutants were yellowing, indicating the onset ofsenescence (Figure 7H). By contrast, all of the leaves in the wild-type plants were still green at the same stage. By measuring thechlorophyll contents, we found that the relative chlorophyll con-tents in the rosette leavesof theatg1abcandatg1abctmutantsat5and 6 weeks old were significantly lower than that of wild-typeplants (Figure 7I). Together, these results indicate that, similar toother autophagy-deficient mutants, the atg1abc triple and at-g1abct quadruple mutants showed enhanced sensitivities tonutrient starvation and premature leaf senescence.

ATG1s Are Required for the Regulation of TRAF1 Stability

To investigate the role of ATG1s in ATG protein turnover andTRAF1 maintenance, we first examined the levels of ATG1a,ATG13a, and ATG8a, in the atg1abc and atg1abctmutants using

274 The Plant Cell

Figure 6. Phosphorylation of TRAF1 by ATG1s.

(A) Migration of TRAF1a in cells expressing ATG1 proteins. HA-tagged ATG1a, ATG1b, and ATG1c (ATG1a-HA, ATG1b-HA, and ATG1c-HA) weretransiently expressed in protoplasts from TRAF1a-FLAG transgenic plants. Proteins were extracted at 16 h after expression under continuous darkconditions, and the blots were probed with anti-HA and anti-FLAG antibodies.(B) TRAF1a phosphorylation by ATG1a. HA-tagged ATG1a (ATG1a-HA) was transiently expressed in protoplasts from TRAF1a-FLAG transgenic plants.Proteins were extracted at 16 h after expression under constant dark conditions. The phosphorylation of TRAF1a was confirmed by digestion withphosphatase and phosphatase inhibitor, and the blots were probed with anti-HA and anti-FLAG antibodies.(C)Degradation of TRAF1a in theYFP-ATG1a transgenic line. One-week–oldTRAF1a-FLAG andTRAF1a-FLAG/YFP-ATG1a seedlingswere transferred toMS medium without Suc followed by dark treatment for the indicated times. Anti-FLAG and anti-YFP were used for immunoblot analysis. The arrowheadindicates the YFP-ATG1a bands. hpt, hours posttreatment.Numbers on the left indicatemolecularmass (kD) of eachband. Anti-ACTINantibodies andPonceauS-stainedRubisco bands are shownbelow theblots toindicate the amount of protein loaded per lane.

Regulation of ATG13s by SINAT Proteins 275

Figure 7. Deletion of ATG1a, ATG1b, and ATG1c Confers Hypersensitivity to Carbon and Nitrogen Starvation.

(A) and (B) Phenotypes of the atg1abc triple mutant and atg1abct quadruple mutant in response to carbon starvation. One-week–old wild-type (WT),atg1abc, atg1abct, and atg10-1 seedlingswere grown onMSsolidmedium for 1week. The seedlingswere transferred toMSagarwith Suc (C1) orMSagarplates without Suc followed by constant dark treatment (C–) for 7 d. The images were recorded after a 7-d recovery.(C)and (D)Here, (C)Relativechlorophyll contentsand (D)survival ratesof thewild type (WT),atg1abc,atg1abct, andatg10-1seedlingsweredescribed in (A)after recovery. The relative chlorophyll contents were calculated by comparing the values of C– treated seedlings versus C1 treated seedlings. Data are

276 The Plant Cell

the corresponding specific antibodies. Protein gel blot analysesrevealed that compared with the wild-type seedlings, ATG1adecreased significantly, but ATG8a and ATG13a accumulated tohigh levels in the atg1abc and atg1abct mutants under eithernutrient-rich or starvation conditions (Figure 8A), suggesting thatloss of ATG1s prevents starvation-induced autophagy proteinturnover. Moreover, we coexpressed TRAF1a-HA andATG1a-HAin wild type to detect the possible phosphorylation and proteinstability of TRAF1a. As shown in Figure 8B, the phosphorylation-like modification of TRAF1a-HA (pTRAF1a-HA) was predominatelyincreased by constant darkness for 16 h anddisappeared after 6 hof recoveryunder light conditions in thewild-typeplants (lanes1 to3), while thewild-type (lanes 4 to 6) and atg1abcmutant (lanes 7 to9) cells expressing only TRAF1a-HA did not show this increase.Compared with the TRAF1a-HA levels in the wild-type cells undereither light ordark conditions, thedeletionsofATG1proteins in theatg1abc mutant significantly destabilized TRAF1a-HA (lanes 7 to9, Figure 8B). Taken together, these findings suggest that ATG1proteins are redundantly essential for the regulation of TRAF1astability.

DISCUSSION

Autophagy is required for the degradation of cellular nutrients,toxic materials, and damaged organelles to promote survival andcellular homeostasis at certain developmental stages or in re-sponse to biotic and abiotic stresses and is an evolutionarilyconserved process in all eukaryotes (Xie and Klionsky, 2007;Michaeli et al., 2016).

Thus far, more than 40 ATG proteins and their associatedregulatory proteins have been identified in plants. Among these,ATG1 andATG13 form aprotein kinase complex that plays crucialroles in the initiation of autophagy and autophagic vesicle as-sembly by interacting with the regulatory proteins ATG11 andATG101 (Suttangkakul et al., 2011; Liu and Bassham, 2012; Liet al., 2014).

ATG13 is one of the core components of the ATG1–ATG13complex, which is regulated by a series of upstream effectorsdependent on nutrient availability. In yeast cells, ATG13 isstructurally divided into an N-terminal globular domain (Jao et al.,

2013), and a C-terminal region, which is predicted to be an in-trinsically disordered region (Kamada et al., 2000). The C-terminalintrinsically disordered region of ATG13 is dephosphorylated inresponse to starvation and interacts with ATG1 and ATG17,thereby leading to the formation of the ATG1–ATG13 complex(Fujioka et al., 2014; Yamamoto et al., 2016). In yeast, the TORkinase acts as a key negative regulator to phosphorylate ATG13under nutrient-rich conditions, which reduces the ability of theATG1–ATG13 complex to alleviate autophagy (Rabinowitz andWhite, 2010).Previous findings revealed that in response to nutrient starva-

tion, Arabidopsis ATG1a and ATG13a are dramatically degradedin the vacuole in an autophagy-dependent manner, demon-strating a feedback turnover mechanism during the biogenesis ofstarvation-induced autophagy (Suttangkakul et al., 2011; Li et al.,2014).We further discovered herein that ATG13a andATG13b aresubject to ubiquitylation and proteasomal degradation uponprolonged nutrient starvation and during recovery after starvation(Figure 1; Supplemental Figure 1) implying that, similar to mam-malian ULK1 and ATG13, Arabidopsis ATG13 turnover is alsocontrolled by ubiquitylation-mediated proteolysis. Moreover, thisprocess involves the RING-type E3 ligases SINAT1 and SINAT2,and RING-finger–truncated SINAT6 (Figure 3) to maintain theautophagy dynamics under different nutrient conditions. Partic-ularly, we observed that ATG1a and ATG13a levels declined at48 h after MG132 treatment compared with those of 12 or 24 h(Figure 1), confirming the regulation of these two proteins by analternativepathwaysuchasautophagy (Suttangkakul et al., 2011).Based on these findings, it is conceivable that: (1) under

nutrient-rich conditions, SINAT1 and SINAT2 accumulate toubiquitylate and destabilize ATG13 proteins to maintain a rela-tively low autophagy level; (2) in response to prolonged nutrientstarvation, SINAT1 and SINAT2 likely contribute by targetingATG13proteins forubiquitylationanddegradation tomodulate thehighly activated autophagy to proper cellular levels; and (3) duringrecovery after starvation, the proteolysis of ATG13 proteins by theaction of SINAT1 and SINAT2 is necessary for the termination ofactivate autophagy. By contrast, SINAT6 is likely to play an op-posing role in suppressing the ubiquitylation and degradation ofthe ATG1/ATG13 complex by competitively interacting with

Figure 7. (continued).

average values6SD (n5 3) calculated from three independent experiments. For each experiment, five technical replicates pooled with 15 seedlings wereused per genotype. Asterisks indicate significant differences from the wild type; *P < 0.05; **P < 0.01 by one-way ANOVA.(E) and (F) Phenotype of the atg1abc triple mutant and atg1abct quadruple mutant in response to nitrogen starvation. One-week–old wild-type (WT),atg1abc, atg1abct, and atg10-1 seedlingswere grownonMSagar for 1week. The seedlingswere transferred toN-rich (N1) or N-deficient (N–)mediumandphotographed at 7 d after treatment.(G)Relativechlorophyll contentsof thewild type (WT),atg1abc,atg1abct, andatg10-1seedlingswithorwithoutnitrogenstarvationshown in (E). The relativechlorophyll contents were calculated by comparing the values of N– treated seedlings versus N1 treated seedlings. Data are average values6SD (n5 4)calculated from four independent experiments. For each experiment, five technical replicates pooled with 15 seedlings were used per genotype. Asterisksindicate significant differences from the wild type; **P < 0.01 by one-way ANOVA.(H) Images showing the onset of leaf senescence in thewild type (WT), atg1abc triplemutant, atg1abctquadruplemutant, and atg10-1mutant plants grownunder normal light/dark growth conditions. Photographs were taken at 4, 5, and 6 weeks after germination. Arrows indicate senescent leaves.(I) Relative chlorophyll content of plants grown under normal light/dark conditions for the indicated times in (H). The values of 4-week–old wild-type (WT),atg1abc triplemutant,atg1abctquadruplemutant, andatg10-1mutantplantswereset at100%,and the relativechlorophyll contentsofWTandatg1abcandatg1abct mutant leaves at 5- and 6-weeks–old were calculated accordingly. Data are average values 6SD (n 5 3) calculated from three independentexperiments. For each experiment, fivewhole plants (technical replicates) were used per genotype. Asterisks indicate significant differences from theWT;**P < 0.01 by one-way ANOVA.

Regulation of ATG13s by SINAT Proteins 277

ATG13 proteins to promote autophagy in response to nutrientdeprivation.

Consistent with the autophagy-associated phenotypes andautophagosome formation in the root cells of their knockoutmutants (Qi et al., 2017), we further showed that SINAT1/SINAT2and SINAT6 act as negative and positive regulators, respectively,in the regulation of autophagy by modulating ATG1 and ATG13stabilities (Figure 5). Our previous findings have suggested that inresponse tocarbonstarvation, theGFP-SINAT1andGFP-SINAT2fusion proteins are rapidly degraded, while that of SINAT6-GFP isaccumulated from 6 to 24 h upon treatment (Qi et al., 2017).

Genetically, weobserved that the sinat1 sinat2doublemutant andSINAT1/SINAT2-Cas9 deletion lines showed increased toleranceand enhanced autophagosome formation under carbon and ni-trogen starvation conditions, while the sinat6 knockout mutantsdisplayed opposite phenotypes (Qi et al., 2017), suggesting SI-NAT1/SINAT2 and SINAT6 play opposing roles in autophagy.In this study, we provided further evidence to support the idea

that SINAT1/SINAT2 and SINAT6 proteins differentially modulateATG13 stabilities under various nutrient conditions. Particularly,SINAT1 and SINAT2 were involved in ubiquitylation and degra-dation of ATG13, which were strongly decreased in the presence

Figure 8. ATG1s Are Required for ATG Protein Turnover and TRAF1 Phosphorylation.

(A)ATG1a, ATG13a, andATG8 levels in thewild type (WT) and the atg1abc and atg1abctmutants after carbon starvation treatments for the indicated times.An immunoblot with anti-ACTIN antibodies is shown below the blots to indicate the amount of protein loaded per lane. hpt, hours posttreatment.(B) Phosphorylation and stability of TRAF1a in the wildtype (WT) and atg1abcmutant. TRAF1a-HA and ATG1a-HA were expressed in theWT and atg1abcmutant protoplasts for 16 h under light (“L”) or dark conditions (“D”) or followed by recovery under light conditions for 6 h (“R”). Ponceau S-stainedmembranes are shown below the blots to indicate the amount of protein loaded per lane. The expression of GFP-HA indicates the expression efficiency ofeach sample. Relative intensity of TRAF1a-HA and ATG1a-HA normalized to the loading control is shown below. Numbers on the left indicate molecularmass (kD) of each band.

278 The Plant Cell

of SINAT6 (Figures 3 and 5). We thus propose that under nutrient-rich conditions, TRAF1a/TRAF1b proteins could interact withSINAT1/SINAT2 for ubiquitylation and degradation of ATG13s.Instead, under nutrient-starvation conditions, TRAF1a/TRAF1bproteins promote the stabilization of ATG13s by interacting withSINAT6, which acts as a positive regulator in maintaining ATG13stability and autophagy induction.

Besides the TRAF-domain-containing SINAT proteins, we andother groups have identified two Arabidopsis TRAF proteins,TRAF1a and TRAF1b (also termed “MUSE14” and “MUSE13,”respectively), which contain only an N-terminal TRAF domain andserve as molecular adaptors rather than E3 ligases to regulateplant immunity, development, andabiotic stress tolerance (Huanget al., 2016; Qi et al., 2017). In Arabidopsis, TRAF1a and TRAF1bregulate plant autoimmunity and pathogen resistance by inter-acting with the E3 ubiquitin ligase SCFCPR1 complex to forma plant-type TRAFasome that modulates the ubiquitylation anddegradation of the NLR immune sensors SNC1 (suppressor ofnpr1-1, constitutive 1) and RPS2 (resistant to Pseudomonas sy-ringae 2; Huang et al., 2016). Moreover, Arabidopsis TRAF1proteins regulate autophagy by interacting with the E3 ligasesSINAT1, SINAT2, and SINAT6 to modulate the stability of ATG6under differential nutrient conditions (Qi et al., 2017).

In this study, we further showed that Arabidopsis TRAF1a andTRAF1b interact with SINAT1, SINAT2, and SINAT6 to modulateautophagy dynamics and form two different plant TRAFasomes,TRAF1–SINAT1/SINAT2–ATG13 and TRAF1–SINAT6–ATG13,providing evidence to demonstrate the central roles of TRAF1-mediated ubiquitylation of the ATG1–ATG13 complex in au-tophagy initiation in plant cells. Consistent with this notion, wesuggested thatATG13adegradationwasstrongly inhibited inboththe traf1a/bdoublemutantand theTRAF1a-FLAGoverexpressingtransgenic lines (Figure 2E), indicating that TRAF1a and TRAF1bact as both positive and negative regulators in modulating theprotein stabilities of the ATG1–ATG13 complex.

Although we did not detect an interaction between ATG1sand TRAF1 proteins by Y2H and CoIP assays, we observed bythe BiFC assay that ATG1s and TRAF1 proteins were associatedin the autophagosome-related punctate structures (Figure 2;Supplemental Figures 2 and 3). Interestingly, our data revealedthat ATG1 proteins prevent the degradation of TRAF1a, possiblyby phosphorylation, in response to carbon starvation (Figures 6Ato 6C), suggestive of a feedback regulatory mechanism betweenthe ATG1–ATG13 kinase complex and TRAF1 proteins.

Given that the potential role of Arabidopsis ATG1s in auto-phagosome formation has not been well understood, we furthercharacterized the atg1abc triple mutant and the atg1abct qua-druple mutant. Similar to other autophagy-defective atgmutants,a previous finding reported that the Arabidopsis atg13a atg13bdouble mutants exhibit premature leaf senescence and hyper-sensitivity to fixed carbon and nitrogen limitations (Suttangkakulet al., 2011). In this study, we showed that the atg1abc triplemutant and the atg1abct quadruple mutant were similar to theatg13a atg13b double mutants in their phenotypes of age-dependent and starvation-induced leaf senescence (Figure 7;Supplemental Figures 11 and 12). Moreover, the turnover ofATG13a and ATG8a was repressed in the atg1abc and atg1abctmutants compared with the wild-type seedlings (Figure 8A),

suggesting that ATG1a, ATG1b, and ATG1c function redundantlyin the regulation of autophagy in Arabidopsis. Because the at-g1abct quadruple mutant did not show an enhanced senescencephenotype comparedwith the atg1abc triplemutant (Figure 7), theroles of ATG1t in this process are still obscure.More interestingly, a recent work validated the role of ATG1s in

autophagosome formation using GFP-ATG8e transgenic lines.Specifically,Huanget al. (2019b) found that in response tonutrientstarvation, the formation of GFP-ATG8e-labeled punctuatestructures (autophagosomesor their intermediates)wasmarkedlyinduced in the wild-type root cells. However, such accumulationwas not evident in the atg1abc and atg1abct backgrounds undereither nutrient-rich or starvation conditions (Huang et al., 2019b),further confirming that deletion of ATG1s leads to deficiency ofautophagosome formation.Asexpected, thephosphorylation-likemodificationandstability

of TRAF1a were significantly reduced in the atg1abc triple mutant(Figure 8B), confirming the involvement of TRAF1a regulation byATG1s for modulating its stability. Although we have confirmedthatATG1swere involved in thephosphorylation-likemodificationof TRAF1a in vitro, the shift in molecular weight of TRAF1a mayalso be caused by other larger posttranslational modifications.Consistent with this, a recent study revealed that the degradationof both TRAF1a and TRAF1b is mediated by the SCFSNIPER4

complex to control the turnover of TRAF1 proteins in plant cells(Huang et al., 2018). Thus, further investigations of the interactionof posttranslational modifications, such as ubiquitylation andphosphorylation, indetermining thestabilityofTRAF1proteinswillbe needed to better understand the molecular mechanism ofTRAF1 proteins in the regulation of autophagy initiation.In conclusion, our observations present strong evidence

that, under normal nutrient conditions, the RING-type E3 ligasesSINAT1andSINAT2 regulate theubiquitylationanddegradationofATG13a, leading to disassociation of the ATG1–ATG13 complex,and therefore suppress autophagy (Figure 9). Under starvationconditions, however, the ATG1s stabilize TRAF1 proteins bya feedback regulatory mechanism (Figure 9). Given that in re-sponse to nutrient starvation, SINAT1 and SINAT2 are degraded,but SINAT6 is accumulated (Qi et al., 2017), the TRAF1-SINAT6-ATG13 TRAFasome is therefore predominant under starvationconditions to promote autophagy induction in Arabidopsis (Fig-ure 9). The SINATs competitively interact with ATG13 and ATG6under different nutrient conditions reminiscent of the role ofHordeum vulgare Armadillo1 and H. vulgare Plant U-Box15 inregulating powdery mildew infection in barley (Rajaraman et al.,2018). This suggests that this regulatory strategy may be widelyused by plants (Rajaraman et al., 2018).

METHODS

Plant Materials, Growth Conditions, and Treatments

All wild-type, mutants, and transgenic Arabidopsis (Arabidopsis thaliana)plants used in this study are in the Columbia (Col-0) background. TheT-DNA insertional mutants described in this study were obtained fromThe Arabidopsis Information Resource (http://www.arabidopsis.org), withthe locus names atg1a-2 (SALK-054351), atg1b-1 (CS446939), atg1c-1(CS920806), atg1t-1 (SALK-062634), sinat3 (SALK-125517), sinat4

Regulation of ATG13s by SINAT Proteins 279

(CS415212), and sinat5 (SALK-069496). The mutants were identified byPCR using a gene-specific primer paired with a T-DNA border-specificprimer (Supplemental Data Set 1). The atg1a-2, atg1b-1, atg1c-1, andatg1t-1 mutants were crossed to each other to generate the atg1ac andatg1bt double mutants, the atg1abc triple mutant, and the atg1abctquadruple mutant. The sinat1 sinat2 double mutant (Qi et al., 2017) wascrossed to sinat3 and sinat4 single mutant to generate the sinat1/2/3/4quadruple mutant. The sinat5 single mutant was crossed to sinat6-2 (Qiet al., 2017) to generate the sinat5 sinat6doublemutant. The atg10-1 singlemutant, atg13ab double mutant, rpn10 PAG1-GFP, and traf1a traf1bdouble mutant were described by Phillips et al. (2008), Suttangkakul et al.(2011),Marshall et al. (2015), andQi et al. (2017), respectively. Themutantsand transgenic lines generated in this study are listed in SupplementalTables1and2.AllArabidopsis seedsweresurface-sterilizedwith20%(v/v)bleach containing 0.1% (v/v) Tween 20 for 20 min and washed withsterilized water five times. The seeds were sown on MS medium (Sigma-Aldrich) containing 2%Suc (w/v) and 0.8%agar (w/v). After cold treatmentunderdarkconditions for3d, theplateswere incubatedat22°Cunder long-day (16-h-light/8-h-dark) or short-day (8-h-light/16-h-dark) photoperiodswith a light intensity of 170 mmol/m2/s using fluorescent bulbs (cat. no.F17T8/ TL841 17W; Philips). After germination for 7 d, the seedlings weretransferred to soil for further growth.

For the carbon-deprivation treatment, 1-week–old seedlings grown onMS medium or 3-week–old soil-grown plants were transferred to contin-uous darkness for the indicated duration, followed by recovery undernormal growth conditions for 7 d. Samples were photographed at theindicated timepoints.The ratioof survivingplants, asdefinedby thegrowthof new leaves, to dead plants was calculated from 10 plants per genotype.

The effects of nitrogen starvation on plant growth were determinedaccording to Qi et al. (2017). Briefly, 1-week–old seedlings grown on MSmedium were transferred to MS or nitrogen-deficient MSmedium (solid orliquid) and grown under normal growth conditions for the indicated times.

Arabidopsis seedlings (1-week–old) grown on solid MS with 2% (w/v)Suc for biochemical analysis were transferred to the sterile 12-well plateswith liquid MS medium (–C, –N, or –C/N) and 50 mM of MG132 or 0.5mMCHX for treatment. After the specified treatments, seedlings were dried onpaper and flash-frozen in liquid nitrogen for protein extraction beforeprotein blot analysis.

Plasmid Construction

All plasmids used in this study were generated using an In-Fusionmethod.The gene-specific primers with 15-bp extensions homologous to thecorresponding vectors are listed in Supplemental Data Set 1. Plasmids fortransient expression analyses were derived from the pUC119 vector (Liet al., 2013). For the ATG13a-HA, ATG13a-FLAG, ATG13b-HA, ATG13a-K1-HA,ATG13a-K2-HA,ATG13a-K1/2-HA,ATG13a-K3-HA,ATG13a-K4-HA, ATG7-HA, and ATG7-FLAG constructs, the full-length coding regionsof ATG13a, ATG13b, ATG13a-K1, ATG13a-K2, ATG13a-K1/2, ATG13a-K3, ATG13a-K4, and ATG7 were inserted into BamHI- and StuI-digestedpUC119plasmids.SINAT1,SINAT2,SINAT5-S1, andSINAT6were clonedinto StuI (SINAT1, SINAT2, and SINAT5-S1) or BamHI (SINAT6) digestedpUC119 to generate GFP and HA-tagged SINAT constructs. TRAF1a-HA,SINAT1-FLAG, SINAT2-FLAG, SINAT5-S1-FLAG, and SINAT6-FLAGwere constructed as described previously by Qi et al. (2017). To generatestable transgenic plants expressing ATG13a-HA, ATG13b-HA, ATG13a-K1/2-HA, GFP-SINAT1-HA, and SINAT6-GFP-HA, the UBQ10pro:ATG13a-HA, UBQ10pro:ATG13b-HA, UBQ10pro:ATG13a-K1/2-HA,UBQ10pro:GFP-SINAT1-HA, andUBQ10pro:SINAT6-GFP-HA fragmentsderived frompUC119constructsweredigestedbyAscI andcloned into thebinary vector pFGC-RCS (Li et al., 2013). The expression cassettes weresubsequently introduced into wild-type Arabidopsis (Col-0) by Agro-bacterium tumefaciens-medium transformation via the floral dip method(Clough and Bent, 1998). The TRAF1a-FLAG transgenic plants were de-scribed by Qi et al. (2017). To generate plasmids for Y2H analysis, full-length coding sequence fragments of ATG13a, ATG13b, ATG1a, ATG1b,ATG1c, and SINAT5-S1 were amplified and inserted into pGADT7 andpGBKT7 vectors digested by EcoRI. The fragments of SINAT1/SINAT2/SINAT3/SINAT4/SINAT6were inserted into pGADT7vectors digestedwithEcoRI and BamHI to generate SINAT1/SINAT2/SINAT3/SINAT4/SINAT6-AD plasmids for Y2H. The SINAT5-BD plasmids for Y2Hwere constructedas described previously by Qi et al. (2017). To generate plasmids for theBiFC assay, the full-length coding sequence fragments of ATG13a,ATG13b, ATG1a, ATG1b, ATG1c, ATG7, SINAT1, SINAT2, SINAT5-S1,SINAT6, and TRAF1a were inserted into BamHI-digested pHBT-YC (Qiet al., 2017) or pHBT-YN (Qi et al., 2017) respectively, to generate fusionswith nYFP or cYFP.

Measurement of Chlorophyll Contents

Measurement of chlorophyll contents was performed as described prvi-ously by Porra et al.. (1989) and Xiao et al. (2010). Arabidopsis leaves wereharvested after nitrogenor carbonstarvation or at different growth periods.Arabidopsis total chlorophyll was extracted by immersing the samples in2mLof N, N-dimethylformamide for 48h in thedarkat 4°C.Absorbancewas

Figure9. WorkingModel forTwoDistinct TRAFasomes,TRAF1s-SINAT1/SINAT2-ATG13s, and TRAF1s-SINAT6-ATG13s in the Regulation of Au-tophagy Dynamics in Arabidopsis.

In response to different nutrient signals, the RING-type E3 ligases SINAT1,SINAT2, and SINAT6 control the stability of ATG13 proteins and the dy-namics of autophagy by modulating the ubiquitylation of ATG13s. Undernormal conditions,TRAF1aandTRAF1b interact inplantawithATG13aandATG13b and require the presence of SINAT1 and SINAT2 to ubiquitylateand degrade ATG13s in vivo. Under nutrient starvation conditions, SINAT6competitively interact with ATG13 and induce autophagy. Furthermore,under starvation conditions, theATG1kinasephosphorylated TRAF1a andpromoted its protein stability in vivo, suggesting a feedback mechanismregulating autophagy.

280 The Plant Cell

determined at 664 and 647 nm, and the total chlorophyll content wasmeasured and normalized to fresh weight per sample.

Protein Isolation and Immunoblot Analysis

For total protein extraction, 1-week–old Arabidopsis seedlings grown onMS medium or after nutrient starvation were ground in liquid nitrogen andhomogenized in ice-cold protein extraction buffer (50 mM of sodiumphosphate at pH 7.0, 200 mM of NaCl, 10 mM of MgCl2, 0.2%b-mercaptoethanol, and 10% glycerol) supplemented with protease in-hibitor cocktail (Roche 04693132001). The samples were placed on ice for30 min and centrifuged at 4°C at 12,000g for 30 min. The supernatant wastransferred to a new microfuge tube before electrophoresis.

For immunoblot analysis, clarified extracts were subjected to SDS-PAGE and transferred to a Hybond-C membrane (Amersham). Specificanti-ATG1a (Suttangkakul et al., 2011; 1:8,000), anti-ATG13a (Suttangkakulet al., 2011; 1:5,000), anti-ATG7 (cat. No. ab99001, 1:2,000; Abcam), anti-ATG8a (cat. No. ab77003, 1:1,500; Abcam), anti-HA (cat. No. H6533,1:5,000; Sigma-Aldrich), anti-FLAG (cat. No. A8592, 1:5,000; Sigma-Aldrich), anti-Ub (cat. No. 10201-2-AP, 1:2,000; Proteintech), anti-K48Ub(cat. No.12930, 1:1,000; Cell Signaling Technology), anti-GFP (cat.No.2955, 1:1,000; Cell Signaling Technology), anti-YFP (cat. No. ab290;1:2,500; Abcam), and anti-ACTIN (cat. No. 58169; 1:1,000; Cell SignalingTechnology) antibodies were used in the protein blotting analysis.Quantification of the protein immunoblot signal was determined with thesoftware ImageJ (Gallo-Oller et al., 2018).

Lambda Protein Phosphatase Treatment

ATG1-HA proteins were expressed in the protoplasts isolated from theTRAF1-FLAG transgenic line for 16h, followedbyextractionof total proteinby immunoprecipitation (IP) buffer (10 mM of HEPES at pH 7.4, 150mM ofNaCl, 2 mM of EDTA, and 10% [v/v] glycerol) with 0.5% (v/v) Triton X-100supplemented with protease inhibitor cocktail (catalog no. 04693132001;Roche).After thesampleswerecentrifugedat4°Cat12,000g for30min, thesupernatants were incubated with Lambda protein phosphatase (NewEngland Biolabs) according to the instructions, with or without applicationof a 13 concentration of the phosphatase inhibitor PhosSTOP (Roche) for30 min at 30°C. The reactions were heated to 95°C for 5 min before im-munoblot analysis.

Y2H, CoIP, and BiFC Assays

Determination of protein–protein interactions by the Y2H assay wasconducted as described previously by Chen et al. (1992) with minormodifications. Both plasmids with AD and BD were transformed intoyeast strain YH109. The protein interactions were identified by growthafter 2 d onmedium lackingHis, Leu, andTrp. To avoid self-activation ofthe transformants, 5 mM of 3-amino-1,2,4-triazole was added to themedium.

Preparation and transfection of Arabidopsis mesophyll protoplastswere performed according to Yoo et al. (2007). Protoplasts isolatedfrom 4-week–old rosetteswere transfectedwith the indicated plasmidsand cultured for 16 h before protein extraction. For the CoIP assays, thecells were collected and lysed in IP buffer (10 mM of HEPES at pH 7.4,150 mM of NaCl, 2 mM of EDTA, and 10% [v/v] glycerol) with 0.5% (v/v)Triton X-100. A 10% volume of total lysis (10%) was used for input, andthe remainder was incubated with GFP, HA, or FLAG affinity beads(Sigma-Aldrich) for 4 h at 4°C. The beads were then collected andwashed five times with IP buffer containing 0.1% (v/v) Triton X-100,followedby adding 53SDS-PAGEsample buffer andheated at 95°C for5 min before protein blot analysis.

For the BiFC assay, the split nYFP and cYFP plasmids or withmCherry-ATG8a were coexpressed in leaf protoplasts prepared fromthe wild-type plants for 16 h under light or dark conditions, and theYFP and mCherry signals were detected by confocal microscopyirradiated with 488-nm (YFP) or 516-nm (mCherry) light, and visual-ized with the band-pass 500- to 530-nm (YFP) or 560- to 610-nm(mCherry) IR filters.

In Vivo Ubiquitylation Assay

For the in vivo ubiquitylation assay, the ATG13a-HA plasmid was eitherexpressed in Arabidopsismesophyll protoplasts isolated fromwild-type ortraf1a/b-1 doublemutant protoplasts, or ATG13a-FLAG coexpressedwithGFP-SINAT1/SINAT2/SINAT5-S1-HAor SINAT6-GFP-HAplasmids inwild-type protoplasts. After a 16-h incubation, the cells were collectedand lysed in the IP buffer containing 0.5% (v/v) Triton X-100 withvigorous vortexing. The supernatants were incubated with HA affinityagarose beads or FLAG magnetic beads before immunoblot analysis.The empty vector pUC119-UBQ10-GFP-HA was coexpressed withother constructs for determining the expression efficiency. Theubiquitylation patterns of ATG13a-HA or ATG13a-FLAGwere detected byanti-ubiquitin antibodies (cat. No. 10201-2-AP, 1:2,000; Proteintech).

RNA Extraction and RT-qPCR

Total RNA was extracted from 3-week–old Arabidopsis leaves usinga HiPure Plant RNA Mini kit (E.Z.N.A.; Omega Bio-Tek) according to themanufacturer’s instructions.

Onemgof total RNAwasused to convert into cDNAwith theHiScript IIQRT Super Mix kit with gDNA Wiper (Vazyme). cDNA samples were diluted1:10 in water before use.

RT-qPCRwas performed in 10-mL reaction volumeswith gene-specificprimers (Supplemental Data Set 1), and conducted with a StepOne PlusReal-time PCR System (Applied Biosystems) using ChamQ SYBR ColorqPCR Master Mix (Vazyme). The RT-qPCR was performed with the fol-lowing protocol: 95°C for 5min followedby 40 cycles of 95°C for 15 s, 55°Cfor 150 s, and 72°C for 30 s, and a subsequent standard dissociationprotocol to validate the presence of a unique PCR product.

For calculation of relative transcription levels, the delta of thresholdcycle (△Ct) values were calculated by subtracting the arithmetic mean Ctvalues of the target ATG13a from the normalizing ACTIN2. The relativetranscription level (22DDCt) was calculated from three independentexperiments.

Statistical Analysis

Data reported in this study are means 6 SD of three independent experi-ments unless otherwise indicated. The significance of the differencesbetween groups was determined by one-way ANOVA. The P values < 0.05or<0.01wereused todetermine significance. Theone-wayANOVA resultsare listed in Supplemental Data Set 2.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/European Molecular Biology Laboratory databasesunder the following accession numbers: TRAF1a (At5g43560), TRAF1b(At1g04300),SINAT1 (At2g41980),SINAT2 (At3g58040),SINAT3 (At3g61790),SINAT4 (At4g27880), SINAT5 (At5g53360), SINAT6 (At3g13672), ATG13a(At3g49590), ATG13b (At3g18770), ATG1a (At3g61960), ATG1b (At3g53930),ATG1c (At2g37840), ATG1t (At1g49780), ATG8a (At4g21980), ATG7(At5g45900), ATG10 (At3g07525).

Regulation of ATG13s by SINAT Proteins 281

Supplemental Data

Supplemental Figure 1. Degradation of ATG13 proteins duringrecovery after starvation (Supports Figure 1)

Supplemental Figure 2. Interaction of ATG1/ATG13 and TRAF1ain vivo and in vitro (Supports Figure 2)

Supplemental Figure 3. Interaction of ATG1/ ATG13 and TRAF1a byBiFC assay (supports Figure 2)

Supplemental Figure 4. Interaction of ATG13a/b and SINATs in vivo(supports Figure 3)

Supplemental Figure 5. Identification of sinat single mutants (sup-ports Figure 3)

Supplemental Figure 6. ATG13a is degraded via the 26S proteasomepathway (supports Figure 3)

Supplemental Figure 7.. Identification of ATG13a ubiquitylation sitemutants (supports Figure 4)

Supplemental Figure 8. Phenotypic analyses of ATG13a ubiquityla-tion mutants in the atg13ab double mutant background (supportsFigure 4)

Supplemental Figure 9. Isolation of atg1 single mutants (supportsFigure 7)

Supplemental Figure 10. Phenotypic analyses of atg1 single anddouble mutants (supports Figure 7)

Supplemental Figure 11. Phenotypic analyses of atg1abc andatg1abct mutants (supports Figure 7)

Supplemental Figure 12. Phenotypic analyses of atg1abc and at-g1abct mutants (supports Figure 7)

Supplemental Table 1. Mutants generated in this study

Supplemental Table 2. Transgenic Arabidopsis plants generated inthis study

Supplemental Data Set 1. Sequence of primers used in this study

Supplemental Data Set 2. ANOVA analysis in this study

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation ofChina (grants 31725004, 31670276, and 31461143001 to S.X. and grant31800217 to H.Q.), Natural Science Foundation of Guangdong Province(grant 2018A030313210 to H.Q.), and by the Major Project of China onNew Varieties of GMO Cultivation (2018zx08011-01B to J.L.).

We thank the Arabidopsis Biological Resource Center (www.arabidopsis.org) for providing atg1a-2, atg1b-1, atg1c-1, and atg1t-1mutant seeds.

AUTHOR CONTRIBUTIONS

S.X. designed the research; H.Q., J.L., F.N.X., J.Y.C., X.L., M.Q.H., andL.J.X. carried out the experiments; S.X., H.Q., and Q.M.Z. analyzed thedata; S.X., H.Q., and J.L. wrote the article.

ReceivedMay28, 2019; revisedSeptember 30, 2019; acceptedNovember11, 2019; published November 15, 2019.

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DOI 10.1105/tpc.19.00413; originally published online November 15, 2019; 2020;32;263-284Plant Cell

Shi XiaoHua Qi, Juan Li, Fan-Nv Xia, Jin-Yu Chen, Xue Lei, Mu-Qian Han, Li-Juan Xie, Qing-Ming Zhou and

ATG13Arabidopsis SINAT Proteins Control Autophagy by Mediating Ubiquitylation and Degradation of

 This information is current as of March 16, 2020

 

Supplemental Data /content/suppl/2019/11/14/tpc.19.00413.DC1.html

References /content/32/1/263.full.html#ref-list-1

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