AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of thePlant Trans-Golgi Network/Early Endosome Organizationand Function[OPEN]

Michel Ruiz Rosquete,a Natasha Worden,a Guangxi Ren,a Rosalie M. Sinclair,a Sina Pfleger,a Michelle Salemi,b

Brett S. Phinney,b David Domozych,c Thomas Wilkop,a,d and Georgia Drakakakia,1

a Department of Plant Sciences University of California, Davis, California 95616bGenome Center, University of California, Davis, California 95616cDepartment of Biology and Skidmore Microscopy Imaging Center, Skidmore College, Saratoga Springs, New York 12866d Light Microscopy Core, University of Kentucky, Lexington, Kentucky 40536

ORCID IDs: 0000-0002-0373-9738 (M.R.R.); 0000-0002-0804-2884 (N.W.); 0000-0001-8068-6775 (G.R.); 0000-0003-1616-3720(R.M.S.); 0000-0003-4181-0440 (S.P.); 0000-0003-3990-7964 (M.S.); 0000-0003-3870-3302 (B.S.P.); 0000-0001-8800-0061 (D.D.);0000-0001-9066-5513 (T.W.); 0000-0002-3949-8657 (G.D.)

The dynamic trans-Golgi network/early endosome (TGN/EE) facilitates cargo sorting and trafficking and plays a vital role inplant development and environmental response. Transport protein particles (TRAPPs) are multi-protein complexes acting asguanine nucleotide exchange factors and possibly as tethers, regulating intracellular trafficking. TRAPPs are essential in alleukaryotic cells and are implicated in a number of human diseases. It has been proposed that they also play crucial roles inplants; however, our current knowledge about the structure and function of plant TRAPPs is very limited. Here, we identifiedand characterized AtTRAPPC11/RESPONSE TO OLIGOGALACTURONIDE2 (AtTRAPPC11/ROG2), a TGN/EE-associated,evolutionarily conserved TRAPP protein in Arabidopsis (Arabidopsis thaliana). AtTRAPPC11/ROG2 regulates TGN integrity, asevidenced by altered TGN/EE association of several residents, including SYNTAXIN OF PLANTS61, and altered vesiclemorphology in attrappc11/rog2 mutants. Furthermore, endocytic traffic and brefeldin A body formation are perturbed inattrappc11/rog2, suggesting a role for AtTRAPPC11/ROG2 in regulation of endosomal function. Proteomic analysis showedthat AtTRAPPC11/ROG2 defines a hitherto uncharacterized TRAPPIII complex in plants. In addition, attrappc11/rog2 mutantsare hypersensitive to salinity, indicating an undescribed role of TRAPPs in stress responses. Overall, our study illustrates theplasticity of the endomembrane system through TRAPP protein functions and opens new avenues to explore this dynamicnetwork.

INTRODUCTION

The highly dynamic plant endomembrane system mediatesintracellular cargo transport and regulates plant growth, de-velopment, and stress responses (Surpin and Raikhel, 2004;Worden et al., 2012). The trans-Golgi network/early endosome(TGN/EE) provides a main trafficking hub for the endomembranesystem and comprises a heterogeneous population of vesiclesshuttling cargo between Golgi, vacuole, plasma membrane (PM),and late endosomal compartments (Dettmer et al., 2006; Viottiet al., 2010;Wattelet-Boyer et al., 2016; Brillada andRojas-Pierce,2017; Rosquete et al., 2018). The plant SYNTAXIN OF PLANTS61(SYP61) defines a TGN/EE compartment involved in endosomalrecycling and transport of extracellular cargo (Robert et al., 2008;Drakakaki et al., 2012;Wilkopet al., 2019) andhasbeen implicatedin abiotic stress responses (Zhu et al., 2002).

Protein trafficking within the endomembrane system requiresthe orchestrated actions ofmany factors includingRabGTPases,soluble N-ethylmaleimide-sensitive factor attachment proteinreceptors (SNAREs) that mediate vesicle fusion, and tetheringfactors. Rab GTPases control the specificity and directionalityof membrane trafficking by recruiting tethers that, in turn, bringvesicles and target membranes into proximity to allowfor SNARE-mediated membrane fusion (Jahn and Scheller,2006). Rabs can switch between an active GTP-bound state(Rab-GTP) and an inactive GDP-bound form (Rab-GDP). Thisswitch requires regulatory proteins such as guanine nucleotideexchange factors (GEFs) and GTPase-activating proteins. Tofunction on membranes, the Rab GTPases require activation bya GEF, which triggers GDP release and enables GTP loading(Pfeffer, 2017).Although TGN/EE-associated Rab GTPases and tethering

factors are critical for the organelle’s function (Vuka�sinović andŽárský, 2016; Ravikumar et al., 2017), our understanding of theirroles in TGN/EE organization is in its infancy (Kim and Bassham,2011; Wang et al., 2014). Transport protein particles (TRAPPs)represent a class of multisubunit tethering complexes facilitatingvesicle trafficking (Kim et al., 2016; Lipatova et al., 2016;Vuka�sinović and Žárský, 2016; Ravikumar et al., 2017). Amongmultisubunit tethering complexes, TRAPPs are unique by virtue of

1 Address correspondence to gdrakakaki@ucdavis.edu.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: Georgia Drakakaki(gdrakakaki@ucdavis.edu).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00110

The Plant Cell, Vol. 31: 1879–1898, August 2019, www.plantcell.org ã 2019 ASPB.

http://orcid.org/0000-0002-0373-9738http://orcid.org/0000-0002-0804-2884http://orcid.org/0000-0001-8068-6775http://orcid.org/0000-0003-1616-3720http://orcid.org/0000-0003-4181-0440http://orcid.org/0000-0003-3990-7964http://orcid.org/0000-0003-3870-3302http://orcid.org/0000-0001-8800-0061http://orcid.org/0000-0001-9066-5513http://orcid.org/0000-0002-3949-8657http://orcid.org/0000-0002-0373-9738http://orcid.org/0000-0002-0804-2884http://orcid.org/0000-0001-8068-6775http://orcid.org/0000-0003-1616-3720http://orcid.org/0000-0003-4181-0440http://orcid.org/0000-0003-3990-7964http://orcid.org/0000-0003-3870-3302http://orcid.org/0000-0001-8800-0061http://orcid.org/0000-0001-9066-5513http://orcid.org/0000-0002-3949-8657http://crossmark.crossref.org/dialog/?doi=10.1105/tpc.19.00110&domain=pdf&date_stamp=2019-08-06mailto:gdrakakaki@ucdavis.eduhttp://www.plantcell.orgmailto:gdrakakaki@ucdavis.eduhttp://www.plantcell.org/cgi/doi/10.1105/tpc.19.00110http://www.plantcell.org

theirGEFactivity (Sacher et al., 1998, 2008;Wanget al., 2000;Kimet al., 2016).

In yeast, most models propose four forms (TRAPPI toTRAPPIV) of the complex. While TRAPPI constitutes the basiccore complex, the addition of distinct subunits to TRAPPIgenerates the TRAPPII to TRAPPIV complexes (SupplementalTable 1; Barrowman et al., 2000; Bröcker et al., 2010; Lipatovaet al., 2016; Ravikumar et al., 2017). In yeast, TRAPPI functionsas a tethering factor for endoplasmic reticulum (ER)–derivedCOPII vesicles en route to the Golgi apparatus (Sacher et al.,2001). TRAPPII and TRAPIII are involved in Golgi-mediatedtrafficking, while TRAPPIII and TRAPPIV are implicated in au-tophagy (Sacher et al., 2008; Lynch-Day et al., 2010; Lipatovaet al., 2016; Thomas et al., 2018). Despite their distinct functions,TRAPPI, TRAPPIII, and TRAPPIV all act as GEFs for the RabGTPase Ypt1p, while TRAPPII, in turn, is a GEF for Ypt31/32(Jones et al., 2000; Wang et al., 2000; Morozova et al., 2006;Lipatova et al., 2016). Of note, the yeast four-complexmodel hasbeen recently challenged by a study suggesting the existence ofonlyTRAPPII andTRAPPIII in thisorganism (Thomasetal., 2018).

Known mammalian TRAPP complexes exist only as TRAPPIIand TRAPPIII, both acting as GEFs on RAB1 (a Ypt1p-homologous protein), with TRAPPII additionally acting as a GEFfor RAB18 (Yamasaki et al., 2009; Scrivens et al., 2011; Bassiket al., 2013). Functionally, TRAPPII is implicated in early Golgitransport (Yamasaki et al., 2009; Sacher et al., 2019), whileTRAPPIII regulates ER-to-Golgi transport (Scrivens et al., 2011).

Notably, the mammalian TRAPPs contain two subunits notfound in yeast, TRAPPC11 and TRAPPC12, which are critical forhuman health (Supplemental Table 1; Brunet and Sacher, 2014;Sacher et al., 2019). TRAPPC11 mutations are responsible for anumber of diseases including muscular dystrophy, intellectual

disabilities, fatty liver disease, and congenital disorders of gly-cosylation (Bögershausen et al., 2013; Brunet and Sacher, 2014;DeRossi et al., 2016; Matalonga et al., 2017; Larson et al., 2018;Sacheretal., 2019). TheTRAPPC11subunit is implicated inER-to-Golgi transport and Golgi-mediated secretion in mammals(Wendler et al., 2010; Scrivens et al., 2011).Plant TRAPP complex members characterized to date are

TRS33, BET5, and the TRAPPII-specific AtTRS120/VAN4 andAtTRS130/CLUB, involved in exine pattern formation, plantcytokinesis, development, and polar protein transport (Jaberet al., 2010; Thellmann et al., 2010; Qi et al., 2011; Naramotoet al., 2014a; Zhang et al., 2018). Other putative plant TRAPPshave not been characterized (Vuka�sinović and Žárský, 2016;Ravikumar et al., 2017, 2018).Basedongeneannotation, a TRAPPIII complex likely operates

in plants; however, its presence awaits discovery and charac-terization (Ravikumar et al., 2017). Given the central role ofTRAPPs in many cellular processes in other species, theircharacterization in plants can provide insights into how plantTRAPPs regulate different trafficking pathways, plant growth,and environmental responses. Furthermore, identification andcharacterization of different plant TRAPP subunits can revealconserved and/or divergent vesicle trafficking mechanismsbetween plants and mammals.We identified At5g65950, an Arabidopsis (Arabidopsis thaliana)

ortholog of the mammalian TRAPPC11 subunit as a highlyabundant protein in the SYP61 vesicle proteome (Drakakaki et al.,2012). This protein was also recently identified in a screen forfactors involved in the response to pectin oligogalacturonides(ROG2; Kohorn et al., 2016) and is henceforth referred to asAtTRAPPC11/ROG2. Here, we found that AtTRAPPC11/ROG2regulates TGN integrity, trafficking of the SYP61 TGN/EE

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compartment, endocytic traffic, and salinity stress responses,likely as part of a TRAPPIII tethering complex in plants.

RESULTS

AtTRAPPC11/ROG2 Localizes to SYP61 Vesicles

The syntaxinSYP61 resides in aTGN/EEcompartment involved inpost-Golgi trafficking (Robert etal., 2008).At5g65950/ROG2 is thesecond most abundant protein in the SYP61 vesicle proteome(Drakakaki et al., 2012). It has a 40.3% amino acid similarity to themammalian TRAPPC11 (Supplemental Figure 1), suggesting thatAtTRAPPC11/ROG2 is a putative TRAPP protein. Based on the

role of the mammalian TRAPPC11 in Golgi-associated trafficking(Wendler et al., 2010; Scrivens et al., 2011), we reasoned that itsplant ortholog was likely also involved in plant endomembranetrafficking.We investigated the subcellular localization of AtTRAPPC11/

ROG2 in Arabidopsis using a yellow fluorescent protein (YFP)fusion expressed under the UBIQUITIN10 promoter (UBQ10pro;Grefen et al., 2010). YFP-AtTRAPPC11/ROG2 localized in thecytosol (Figure 1A), as usually observed for TRAPP subunits(Sacher et al., 1998; Loh et al., 2005; Rybak et al., 2014), andcolocalized with SYP61 at the TGN/EE (percentage of colocali-zation 5 62%, Pearson’s correlation coefficient [PCC] 5 0.61;Figures 1A to 1C). Brefeldin A (BFA) is an inhibitor of post-Golgi

Figure 1. Subcellular Localization of AtTRAPPC11/ROG2 and SYP61.

(A) Confocal images showing localization of YFP-AtTRAPPC11/ROG2 in the cytosol and at the TGN/EE (arrowheads) in root cells of 3-d-old Arabidopsisseedlings.(B) CFP-SYP61 localizes at the TGN/EE (arrowheads).(C) YFP-AtTRAPPC11/ROG2 and CFP-SYP61 colocalize at the TGN/EE (arrowheads). Percentage of colocalization5 62%, PCC5 0.61, n > 15 cells perseedling, n > 10 seedlings.Bar in (A) to (C) 5 5 mm.(D) After 2 h of treatment with 12.5 mM BFA, YFP-AtTRAPPC11/ROG2 localizes to BFA bodies (arrowheads).(E) CFP-SYP61 localization to BFA bodies 2 h after 12.5 mM BFA treatment.(F)YFP-AtTRAPPC11/ROG2andCFP-SYP61colocalize inBFAbodies. Percentage of colocalization5100%,PCC50.75,n>15cells per seedling, n>10seedlings.Bar in (D) to (F) 5 10 mm.

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trafficking and endosomal recycling that causes aggregation ofpost-Golgi compartments (Nebenführ et al., 2002; Geldner et al.,2003; Naramoto et al., 2010, 2014b; Singh and Jurgens, 2017;Reynolds et al., 2018). SYP61andAtTRAPPC11/ROG2colocalizein BFA-induced bodies (percentage of colocalization 5 100%,PCC 5 0.75), implicating AtTRAPPC11/ROG2 in TGN/EE-asso-ciated trafficking (Figures 1D to 1F).

Agreenfluorescentprotein (GFP)–taggedversionofAtTRAPPC11/ROG2 under its native promoter showed the same localizationpattern as UBQ10pro:YFP-AtTRAPPC11/ROG2 (SupplementalFigures 2A and 2B). When coexpressed with mCherry-SYP32,most of the YFP-AtTRAPPC11/ROG2 signal is found in proximityto theGolgimarker (Geldner et al., 2009); however, the twoproteinsignals seldom overlap (Supplemental Figures 2C to 2E; per-centage of colocalization 5 9.1%, PCC 5 0.34). Similarly,AtTRAPPC11/ROG2-GFP colocalizes only marginally with thelate endosome/prevacuolar compartment marker mCerulean-RABF2B (Supplemental Figures 2F to 2H, percentage of coloc-alization5 4.7%, PCC5 0.27; Geldner et al., 2009). Together, ourlocalization and BFA studies indicate that TGN/EE is the mainendomembrane compartment to which AtTRAPPC11/ROG2associates, corroborating our SYP61 proteomic data (Drakakakiet al., 2012) and implicating it in TGN/EE trafficking.

Loss of AtTRAPPC11/ROG2 Alters SYP61Vesicle Trafficking

Given that AtTRAPPC11/ROG2 was identified in the SYP61compartment and the known role of TRAPPs in endomembranetrafficking (Ravikumar et al., 2017),wehypothesized that its lossoffunctionmay affect SYP61-mediated trafficking. Toward this end,we took a reverse genetics approach and identified attrappc11/rog2-2, a T-DNA insertional mutant lacking expression of the full-length transcript (Supplemental Figures 3A and 3B). We then in-vestigated the subcellular fate of SYP61 in the attrappc11/rog2-2background. Consistent with previous findings (Robert et al.,2008; Drakakaki et al., 2012; Li et al., 2017), cyan fluorescentprotein (CFP)-SYP61was localizedat theTGN/EE in theColumbiaecotype (Col-0) wild-type background (Figures 2A and 2C).However, in the attrappc11/rog2-2 mutant, CFP-SYP61 showedan aberrant pattern reminiscent of vacuolar localization, in addi-tion to the expected TGN/EE localization (Figures 2B and 2D).Staining with the vacuolar dye Seminaphtorhodafluor-1 (SNARF-1;Viotti et al., 2013) confirmed that a population of SYP61 is ec-topically localized at the tonoplast in attrappc11/rog2-2 (Figures2H to 2J). The mislocalization of SYP61 to the tonoplast likelyreflects an aberrant sorting and not altered vacuolar morphology,given the similar patterns of vacuolar staining in the wild type andattrappc11/rog2-2 (Supplemental Figures 4A to 4D). Importantly,the introduction of the UBQ10pro:YFP-AtTRAPPC11/ROG2 con-struct in the attrappc11/rog2-2 mutant background restored thepercentageof rootcellsdisplayingSYP61 localization to tonoplastto thevery low levels typicallyobserved in thewild-typebackground(Figures 2E and 2G). A similar rescue effect was observed for theAtTRAPPC11/ROG2pro:AtTRAPPC11/ROG2-GFP construct (Fig-ures 2F and 2G), also referred to as NATpro:AtTRAPPC11/ROG2-GFP. Taken together, these results demonstrate that loss ofAtTRAPPC11/ROG2 affects trafficking of SYP61 at the TGN/EE

andprove that both fluorescently tagged versions of AtTRAPPC11/ROG2 are functional. Thus, UBQ10pro:YFP-AtTRAPPC11/ROG2was used for further studies.Next, we investigated whether the localization of other TGN/EE

residents is affected in the attrappc11/rog2-2 mutant. The syn-taxin SYP41 and the small GTPase RABD2A both localize at theTGN/EE and were identified in the SYP61 vesicle proteome(Sanderfoot et al., 2001; Geldner et al., 2009; Drakakaki et al.,2012). We performed SYP41 immuno-localization experimentsand assessed the subcellular distribution of YFP-RABD2A in thewild-type and attrappc11/rog2-2 backgrounds. For both TGN/EEmarkers, a lower number of fluorescent punctae was observed inattrappc11/rog2-2 root cells (Figures 3B, 3D, and 3E), comparedwith the wild type (Figures 3A, 3C, and 3E).To further investigate the role of AtTRAPPC11/ROG2 in post-

Golgi trafficking, we used Endosidin 16 (ES16), an inhibitor forrecycling of apical, lateral, and nonpolar PM proteins. ES16 re-directs post-Golgi trafficking to vacuole transport by affecting theactivity of the small GTPaseRABA2A (Li et al., 2017). Interestingly,ES16 treatments enhanced the number of cells displaying aber-rant SYP61 tonoplast localization in attrappc11/rog2-2 roots(Figures 3F to 3H; Supplemental Movies 1 to 3). The observedsubcellular phenotype of themutant was reflected in the inhibitionof root growth of attrappc11/rog2-2 by ES16 (SupplementalFigure5), inagreementwith thepreviously reportedES16-inducedphenotype (Li et al., 2017). The synergismbetween the attrappc11/rog2-2 mutation and the ES16 effect further supports a role ofAtTRAPPC11/ROG2 in TGN/EE-mediated trafficking and sug-gests a compartmentalized function for the TRAPP protein in theTGN/EE.

Loss of AtTRAPPC11/ROG2 Alters TGN Morphologyand Dynamics

Interference with the human TRAPPC11 results in Golgi frag-mentation, indicating a role for the protein inmaintenance ofGolgiintegrity (Scrivens et al., 2011). Thus, we investigated theTGN morphology in the attrappc11/rog2-2 mutant. Interestingly,transmission electron micrographs revealed an increased size ofTGN/EE vesicles in the mutant (Figures 4A to 4D). The overall ERmorphology remained unaffected in the attrappc11/rog2-2 mu-tant (Supplemental Figures4Eand4F),pointing toaspecific roleofAtTRAPPC11/ROG2 at the TGN/EE. In addition to the changesin vesicle morphology, the number (Figure 4E) and velocity(Figure 4F) of SYP61-labeled TGN/EE vesicles were significantlyreduced in the attrappc11/rog2-2 mutant.Togetherwith themislocalization of SYP61 to the tonoplast and

the reduceddensity ofSYP41andRABD2Avesicles in themutant,these results suggest that AtTRAPPC11/ROG2 plays a role inmaintaining the morphological and functional integrity of theTGN/EE.

Loss of AtTRAPPC11/ROG2 Causes BFA Resistance andAffects Endocytic Traffic

We hypothesized that AtTRAPPC11/ROG2, as a putativetethering factor, may be involved in events of vesicle fusion.Therefore, we examined the localization of CFP-SYP61 and SYP61

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vesicle-associated YFP-RABD2A (Geldner et al., 2009; Drakakakiet al., 2012) under BFA treatment in attrappc11/rog2-2. BFA in-hibits post-Golgi trafficking and endosomal recycling, therebycausingaggregationofGolgi, TGN,andendosomalmaterial inBFAbodies (Nebenführetal.,2002;Geldneretal.,2003;Naramotoetal.,

2014b; Singh and Jurgens, 2017; Reynolds et al., 2018). While inthe wild-type background CFP-SYP61 aggregated in large BFAbodies (>20 mm3) at a ratio of 30 per 1000 mm3 (Figures 5A and 5C),formationof typicalBFAbodieswas impaired inattrappc11/rog2-2,where only punctae smaller than 20 mm3 were detectable

Figure 2. SYP61 localization in the attrappc11/rog2-2 Mutant.

(A) to (D) TGN and PM localization of CFP-SYP61 at the tip (A) and expansion zone (C) of the root, in Col-0 wild-type background. (B) and (D) AberrantCFP-SYP61 subcellular localization (arrows) at the tip (B) andexpansion zone (D)of the root in the attrappc11/rog2-2mutant background. Bar in (A) to (D)510 mm.(E) to (G)Mislocalization of CFP-SYP61 to tonoplast in attrappc11/rog2-2 is reverted by complementation of themutant with fluorescently tagged versionsof AtTRAPPC11/ROG2 expressed under either the UBIQUITIN10 (UBQ10) (E) or the native (NAT) AtTRAPPC11/ROG2 promoter (F), as shown by thequantification of thepercentageof cells displaying tonoplast SYP61 in thewild-type (WT), attrappc11/rog2-2, and complementedmutant backgrounds (G).P < 0.001, one-way ANOVA, n 5 15 confocal sections of root cells in the meristematic and transition regions from at least 10 individual seedlings pergenotype. Distinct letters indicate statistically significant differences. Error bars represent SE. Bar in (E) and (F) 5 10 mm.(H) to (J) Staining with the vacuolar (arrows) marker SNARF-1 demonstrates that the aberrant CFP-SYP61 localization corresponds to the tonoplast(arrowheads). Bar in (H) to (J) 5 10 mm.

AtTRAPPC11/ROG2 Regulates TGN/EE Trafficking 1883

(Figures 5B and 5D). A similar defect was observed for YFP-RABD2A BFA body formation in the mutant (Figures 5E and 5F).To ensure that the reduced BFA body formation was due to theATTRAPPC11/ROG2 mutation, BFA bodies labeled by thelipophilic dye FM4-64 were imaged in the complementedYFP-AtTRAPPC11/ROG2attrappc11/rog2-2 plants. Similarly to theaforementioned TGN/EE proteins, formation of FM4-64–stainedBFA bodies was affected in attrappc11/rog2-2 (Figures 5G and5H), while their presence was restored with the introduction ofYFP-AtTRAPPC11/ROG2 (Figure 5I). Treatment of seedlingswith BFA causes inhibition of root growth over a range of BFAconcentrations (Okumura et al., 2013; Cole et al., 2014). In thisstudy, attrappc11/rog2mutants displayed partial resistance at 1and 2.5 mMBFA while at a higher concentration (5 mM) the levelsof inhibition were similar to those of wild-type seedlings(Supplemental Figure 6).

Given that the TGN is also an EE in plants, an involvement ofAtTRAPPC11/ROG2 in endosomal trafficking is plausible. Re-duced incorporation of the endocytic tracer FM4-64 into BFAbodies in attrappc11/rog2-2 points to this hypothesis. To in-vestigate this, we monitored the internalization of FM4-64 in theroots of the wild type, attrappc11/rog2-2, and three additional

ATTRAPPC11/ROG2 T-DNA insertional mutants: attrappc11/rog2-4,attrappc11/rog2-5, and attrappc11/rog2-7 (SupplementalFigures 3A and 3C). Representative images 18 min post-stainingshowed a decrease in the accumulation of FM4-64–labeledpunctae within root cells of the attrappc11/rog2-2 mutant,compared with the wild-type cells (Figures 6A and 6B). A similardefect was observed in attrappc11/rog2-4, attrappc11/rog2-5,and attrappc11/rog2-7 mutants (Supplemental Figure 7). Quan-tification of FM4-64 internalization, expressed as the ratio of themean intracellular fluorescent signal versus that of PM (Rigal et al.,2015), at 12, 15, and 18 min post-staining, revealed a steadyincrease of endocytosis in the wild type; however, no such in-crease was observed in attrappc11/rog2-2 (Figure 6C). We theninvestigated the dynamic association of FM4-64–labeled endo-somes with the SYP61 compartment. A reduced colocalization ofSYP61 with FM4-64–labeled endosomes was observed in themutant atdifferent timepoints (Figures6D to6H). The reduced rateof FM4-64 accumulation in the SYP61-positive TGN/EE (Figures6C and 6F) could reflect impaired fusion of FM4-64 endosomeswith the SYP61 compartment resulting from a compromisedtethering activity of AtTRAPPC11/ROG2 along the endocyticpathway.

Figure 3. Localization of TGN Resident Proteins in the attrappc11/rog2-2 Mutant.

(A)and (B)SYP41 immuno-stainingon4-d-oldseedlings revealsa reducednumberofSYP41vesicles (redarrowheads) in theattrappc11/rog2-2mutant (B),compared with Col-0 wild type (WT) (A). Bar in (A) and (B) 5 5 mm.(C) and (D) Number of YFP-RABD2A vesicles (white arrowheads) is reduced in root cells of 4-d-old seedlings, in the attrappc11/rog2-2 background (D),compared with the Col-0 wild-type (WT) background (C). Green, YFP-RABD2A; magenta, FM4-64. Bar in (C) and (D) 5 10 mm.(E)QuantificationofSYP41- andYFP-RABD2A-labeledTGNparticle densities in root cells ofattrappc11/rog2-2 andCol-0wild-type (WT)backgrounds.P<0.001 (***), Student’s t test, n 5 15 confocal root sections from at least 10 individual seedlings per genotype. Error bars represent SE.(F) to (H)Treatmentwith 15mMES16 for 3 henhances themislocalization ofCFP-SYP61 to the tonoplast (yellowarrowheads in [F] and [G]) in root cells of 4-d-oldattrappc11/rog2-2mutants, shownbyahigherpercentageof cells displayingCFP-SYP61signal at the tonoplast afterES16 treatment (H). P

AtTRAPPC11/ROG2 Defines a TRAPPIII Complexin Arabidopsis

The sequence similarity of At5g65950 to the mammalianTRAPPC11 suggests that AtTRAPPC11/ROG2 is a putativeTRAPP protein. Yeast and mammalian TRAPP proteins exist inmultisubunit complexes (Supplemental Table 1; Kim et al., 2016).

To determine whether AtTRAPPC11/ROG2, similarly to itsmammalian ortholog, participates in a TRAPP complex, we im-munoprecipitatedYFP-AtTRAPPC11/ROG2–associatedproteinsfrom a postnuclear fraction and analyzed them by mass spec-trometry (MS). Spectral counting and MS1 label-free quantitation(LFQ) analysis revealed the presence of several TRAPPproteins inthe immunoprecipitate, including the subunits that form the coreTRAPP complex: Bet3/TRAPPC3, Trs23/TRAPPC4, and Trs31/TRAPPC5 (Figure 7; Supplemental Data Sets 1 and 2; Kim et al.,2016). Significantly, we also found the Trs85/AtTRAPPC8, At-TRAPPC12, and AtTRAPPC13 subunits that define the special-ized mammalian TRAPPIII complex (Sacher et al., 2019), but notthe Arabidopsis TRAPPII–specific isoforms AtTRS120 andAtTRS130 (Rybak et al., 2014), suggesting that the latter two arenotconstituentsof thespecificAtTRAPPC11-residingcomplex. Inan effort to dissect AtTRAPPC11/ROG2 interactions with othersubunits within the plant TRAPPIII complex, we performed bi-molecular fluorescence complementation assays (BiFCs; GrefenandBlatt, 2012) using split YFP-tagged versions of AtTRAPPC11/ROG2 and either AtTRAPPC6 or AtTRAPPC8, both TRAPPsubunits that were found in the AtTRAPPC11/ROG2 immuno-precipitate (Figure 7). Two days after infiltration of the BiFCconstructs, the analysis of YFP signal suggested that transientlyexpressed AtTRAPPC11/ROG2 interacts with AtTRAPPC6(Supplemental Figure 8A) and AtTRAPPC8 (Supplemental Figure8D) in Nicotiana benthamiana leaf epidermal cells. The detectedYFP signal appeared specific to the interaction of AtTRAPPC11/ROG2 with AtTRAPPC6 and AtTRAPPC8 since such fluorescencesignal was absent for the pair of AtTRAPPC11/ROG2 and theendosomal marker RABA1G (Geldner et al., 2009) when coex-pressed in N. benthamiana (Supplemental Figure 8G).Together, our data suggest that AtTRAPPC11 functions in

ahithertouncharacterizedTGN/EE-localizedTRAPPIII complex inArabidopsis and can be identified as a plant TRAPPIII member.

Loss of AtTRAPPC11/ROG2 Causes Hypersensitivity toSalt Stress

The endomembrane system is critical for plant development andresponse to environmental stress (Surpin andRaikhel, 2004; Baralet al., 2015). We observed a small but significant reduction in theroot growthof attrappc11/rog2-2, attrappc11/rog2-4, attrappc11/rog2-5, and attrappc11/rog2-7 mutants compared with the wildtype under normal growth conditions (Figures 8A and 8B).Furthermore, all four mutants proved hypersensitive to 100mM

salt treatment (Figures 8C and 8D). The salt hypersensitivity ofattrappc11/rog2-2 roots was corroborated in time-lapse rootgrowth experiments (Figure 8E; Supplemental Movie 4) and ata lower NaCl concentration of 50 mM (Figure 8F). Both rootelongationunder normal growthconditions andsensitivity toNaClwere restored to the wild-type levels in the complemented lineYFP-AtTRAPPC11/ROG2attrappc11/rog2-2 (Figures 8A to 8D),demonstrating the role of AtTRAPPC11/ROG2 in root growth andsalt stress response. Finally, attrappc11/rog2 mutants showeddecreased survival rates under salt treatment, compared with thewild type (Supplemental Figure 9). Consistent with the behavior inthe root growth, the complemented line YFP-AtTRAPPC11/ROG2attrappc11/rog2-2 restored survival rates to thewild-type levels.

Figure 4. TGN Morphology and Dynamics in the attrappc11/rog2-2Mutant.

(A) and (B) Electron microscopy images showing enlarged TGN vesicles(arrowheads) in root cells of attrappc11/rog2-2 seedlings (B), comparedwith Col-0 wild type (WT) (A). Bar in (A) and (B) 5 200 nm. G, Golgiapparatus.(C) and (D) Measurements of TGN vesicle diameter (C) and area (D) inelectron microscopy micrographs. P < 0.001 (***), Student’s t test, n > 5sectionspergenotype (>50particlespergenotype). Error bars represent SE.d, TGN vesicle diametermeasured along the shortest axis of the vesicle; D,TGN vesicle diameter measured along the longest axis of the vesicle.(E) The density of CFP-SYP61–labeled TGN particles is reduced in at-trappc11/rog2-2 seedlings, comparedwith Col-0wild type (WT). P < 0.001(***), Student’s t test, n5 10 seedlings per genotype, 20 cells per seedling.Error bars represent SE.(F) SYP61 vesicle velocity is reduced in attrappc11/rog2-2 root cells,compared with Col-0WT. P < 0.001 (***), Student’s t test, n5 10 seedlingsper genotype, 20 cells per seedling. Error bars represent SE. The colorscheme of the graph bars in (C) to (F) indicates the same genotypes.

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To determine whether AtTRAPPC11/ROG2 is specifically in-volved in responses to salt, a mannitol treatment was used toevaluate osmotic stress. The inhibitory effect of mannitol on rootgrowth was similar in attrappc11/rog2 mutants and the wild type(Supplemental Figure 10), indicating that AtTRAPPC11/ROG2 isspecifically involved in an ionic stress response pathway.

DISCUSSION

AtTRAPPC11 Functions in TGN/EE Trafficking

In an effort to characterize regulators of post-Golgi trafficking inplant cells, we identified AtTRAPPC11/ROG2 as a major con-stituent in the proteome of SYP61 TGN/EE vesicles. The abun-dance of AtTRAPPC11/ROG2 in TGN/EE vesicles (Drakakakiet al., 2012;Groenet al., 2014;Heardetal., 2015) and its homologywith the human tethering factor TRAPPC11 suggested a role fortheprotein inTGN/EE traffickingandvesicle fusion, likelyaspart ofa plant TRAPPIII tethering complex.

Our results place AtTRAPPC11/ROG2 in a group of plant TGN/EE residents regulating TGN/EE organization and vesicle fusion,whose mutants share mislocalization of the SYP61 SNARE pro-tein. This group includes the recently characterized TNO1 and

ECHIDNA (Gendre et al., 2011; Kim and Bassham, 2011). How-ever, attrappc11/rog2’s distinct set of subcellular phenotypespoint toward different mechanisms through which AtTRAPPC11/ROG2 regulates TGN/EE trafficking. The TGN morphology is af-fected inbothechandattrappc11/rog2-2mutants; however,whilein ech the TGNappearsmore tubular (Boutté et al., 2013), TGN/EEvesicles are enlarged in attrappc11/rog2-2. Furthermore, whileattrappc11/rog2 mutants display reduced BFA body formation,ech mutants are not impaired (Gendre et al., 2011). The fungalinhibitorBFA induces the formationofBFAcompartments likelybyfusion of TGN/EE vesicles and aggregation between the TGN/EEand other endosomes (Geldner et al., 2003; Robinson et al., 2008;Norambuena and Tejos, 2017; Singh and Jurgens, 2017). Re-duced BFA body formation in attrappc11/rog2, shown by twodifferent TGN/EE markers and the endocytic tracer FM4-64, in-dicates a possible role for AtTRAPPC11/ROG2as a tether in TGN/EE. A similar phenotype is displayed by tno1 mutants (Kim andBassham, 2011). However, AtTRAPPC11/ROG2 localizes to BFAbodies, while TNO1 protein does not, pointing to mechanisticdifferences between the two putative tethers (Kim and Bassham,2011).BFA targets a subset of the Sec7 domain-containing ADP-

ribosylation factor GEFs, including GBF/Gea and BIG/Sec7-related

Figure 5. BFA Body Formation in the attrappc11/rog2-2 Mutant.

(A) and (B) Confocal imaging of CFP-SYP61–labeled BFA body formation 2 h after treatment of 3-d-old attrappc11/rog2-2 (B) and wild-type (WT; see [A])seedlings with 12.5 mM BFA. Arrows indicate BFA bodies.(C) and (D) Quantitative 3D analysis of CFP-SYP61–labeled BFA bodies in the wild-type (WT; see [C]) and attrappc11/rog2-2 (D) backgrounds. Objectslarger than 20 mm3 (arrows) are only observable in the WT background. n 5 24 and 77 cells in WT and attrappc11/rog2-2 backgrounds, respectively.(E)and (F)Confocal imagingofYFP-RABD2A–labeledBFAbody formation2hafter treatmentof the3-d-oldattrappc11/rog2-2andwild-type (WT)seedlingswith12.5mMBFA.Arrows indicateBFAbodies.The imagesare representativeofconfocal root sectionsobtained frommore than10seedlingspergenotype.(G) to (I)Confocal imaging of FM4-64–labeledBFAbodies 15min after FM4-64 stainingof 4-d-old seedlings treated for 2hwith 12.5mMBFA. The formationof BFA bodies is reduced in root cells of attrappc11/rog2-2 (H), in contrast to the wild type (WT; see [G]). Complementation of the mutant with YFP-AtTRAPPC11/ROG2 restores BFA body formation to wild-type levels (I). Arrows indicate objects larger than 2.8 mm2. The images are representative ofconfocal root sections obtained from more than 10 seedlings per genotype. Bars in (G) to (I) 5 10 mm.

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proteins (Sata et al., 1998; Peyroche et al., 1999; Robineau et al.,2000; Bui et al., 2009). The absenceof aSEC7domain in TRAPPC11eliminates the possibility that AtTRAPPC11/ROG2 is a direct BFAtarget.However, lossofAtTRAPPC11/ROG2mayindirectly influenceat multiple levels the availability of BFA targeted ADP-ribosylationfactor/GEFcomplexes (Geldneretal., 2003;Mossessovaetal., 2003;

Tanaka et al., 2009; McDonold and Fromme, 2014; Richter et al.,2014). In mammalian cells, BFA resistance is consistent acrossseveral TRAPPmutants, including the loss of function of TRAPPC13andTRAPPC11 (Scrivens et al., 2011; Ramírez-Peinado et al., 2017),suggesting a conserved mechanism between different species. ATRAPPC11 functional evolutionary conservation seems to reside

Figure 6. Endocytic Traffic in the attrappc11/rog2-2 Mutant.

(A)and (B)Representative3D renderingsof4-d-oldCFP-SYPP61seedlingsshowing reducedaccumulationofFM4-64 in intracellular vesicles (arrowheads)18minafter stainingwith2mMFM4-64, in theattrappc11/rog2-2mutantbackground (B), comparedwithCol-0wild-type (WT)background (A).Bar in (A)and(B) 5 10 mm.(C)QuantificationofFM4-64accumulation,expressedas the ratiobetweenmean intracellularfluorescentsignalandfluorescent signalat thePM,12,15,and18min after staining with 2mMFM4-64. P < 0.05, two-way ANOVA followed bymultiple comparisons Tukey test, n > 20 cells per genotype. Distinct lettersindicate statistically significant differences. Error bars represent SE. Intracell., intracellular.(D)and (E)TGN/EEcolocalizationofCFP-SYP61andFM4-64 (arrowheads), 12minafter treatmentof 4-d-old seedlingswith2mMFM4-64, is less frequentlyobserved in the attrappc11/rog2-2mutant background (E), compared with the Col-0 wild-type (WT) background (D). CFP is indicated in green; FM4-64 isindicated in magenta. Bar in (D) and (E) 5 10 mm.(F)PercentagesofCFP-SYP61–labeledTGN/EEvesiclesdisplayingcolocalizationwithFM4-64, in relation to the total numberofCFP-SYP61–labeledTGN/EE vesicles, 12, 15, and 18 min after staining with 2 mM FM4-64. P < 0.05, two-way ANOVA followed by multiple comparisons Tukey test, n > 20 cells pergenotype. Distinct letters indicate statistically significant differences. Error bars represent SE.(G) and (H) 3D renderings of SYP61/FM4-64 colocalization in root cells of theCol-0wild-type (WT; see [G]) and attrappc11/rog2-2 (H) seedlings expressingCFP-SYP61, 12minafter stainingwith 2mMFM4-64.CFP-SYP61–labeledvesiclesdisplayingcolocalizationwithFM4-64 (gray) are relatively lessabundantin themutant, comparedwith thewild-typebackground.CFP-SYP61–labeledvesiclesnot colocalizedwithFM4-64are shown ingreen. FM4-64at thePM isindicated in magenta. Bar in (G) and (H) 5 5 mm.

AtTRAPPC11/ROG2 Regulates TGN/EE Trafficking 1887

Figure 7. AtTRAPPC11/ROG2 Defines an Arabidopsis TRAPP Complex.

(A)Spectral countsarepresented for thenineArabidopsisTRAPPorthologssignificantlyenriched inYFP-ROG2 immunoprecipitates (ROG2r1 to r3), and forAtTRAPPC11/ROG2 itself (bait). r, biological replicate (r1 to r3, independentlygrownseedlingsets).Note thatArabidopsisorthologsofTRS120andTRS130,defining the TRAPPII complex, were not detected (nd) in our analysis. FET, Fisher’s exact test; NC, negative control.(B)Volcanoplot illustrating log2 fold change (FC, xaxis) andstatistical significancedistribution (yaxis) of theproteomicdata set (LFQ intensities,MaxQuant).Red squares indicate proteins significantly enriched in YFP-ROG2 immunoprecipitates. Analyses were performed in triplicates.

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Figure 8. Response of attrappc11/rog2 Mutants to Salt Stress.

(A)and (B)Representative8-d-oldseedlingsof themutantsattrappc11/rog2-2,attrappc11/rog2-4,attrappc11/rog2-5, andattrappc11/rog2-7aswell as theCol-0 wild-type (WT) and the complemented attrappc11/rog2-2 mutant (YFP-AtTRAPPC11/ROG2attrappc11/rog2-2) are shown (A). Eight days after ger-mination (DAG), all four AtTRAPPC11/ROG2mutants display reduced root growth, compared with WT and the complemented attrappc11/rog2-2mutant(B). P < 0.001 (***), one-sample Student’s t test, n 5 minimum 24 seedlings per genotype. Error bars represent SE.(C) and (D) Root elongation shows hypersensitivity to salt treatment in attrappc11/rog2-2, attrappc11/rog2-4, attrappc11/rog2-5, and attrappc11/rog2-7compared with the Col-0 wild type (WT), 3 and 5 days after transfer (DAT) of 3-d-old seedlings to plates containing 100 mM NaCl. The hypersensitivity ofattrappc11/rog2-2mutantswas alleviated by complementationwith YFP-AtTRAPPC11/ROG2. The arrowhead in (C)denotes the position of root tips upontransfer. P < 0.001 (***), one-sample Student’s t test, n 5 minimum 24 seedlings per genotype. Error bars represent SE.(E) Time lapse of attrappc11/rog2-2 root growth under 75mM NaCl treatment. Root elongation measurements over the course of 110 h (only diurnal timepoints) showhypersensitivity of attrappc11/rog2-2mutants to inhibitionof root elongationby75mMNaCl treatment, comparedwith theCol-0wild type (WT;see Supplemental Movie 4). n 5 25 seedlings per genotype. Error bars represent SD.(F) The salt hypersensitivity of attrappc11/rog2-2mutants is dose dependent with 100 mMNaCl treatments resulting in an inhibitory effect on root growthstronger than the inhibition caused by 50 mM NaCl, measured 5 d after treatment. P < 0.0001 (***), one-sample Student’s t test, n 5 25 seedlings pergenotype. Error bars represent SE.

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also at the level of Golgi/TGN structural maintenance. PerturbingTRAPPC11 in human cells causes Golgi fragmentation (Scrivenset al., 2011) while interference with AtTRAPPC11/ROG2 affectsTGN/EE morphology.

AtTRAPPC11 Functions in a RABA2A-IndependentPost-Golgi Pathway

The small molecule ES16 affects recycling of apical, lateral, andnonpolar PM proteins, by interfering with the RABA2A GTPase (Liet al., 2017). The exacerbated mislocalization of SYP61 to thetonoplast in ES16-treated attrappc11/rog2 seedlings adds evi-dence to the role of AtTRAPPC11/ROG2 in post-Golgi trafficking.RAB GTPases are regulators of vesicle targeting and fusion andtheir recruitment to specific membranes is mediated by GEFs(Allanet al., 2000;Barr, 2013;Blümer et al., 2013). Several TRAPPsdisplay GEF activity (Jones et al., 2000; Morozova et al., 2006;Lipatova et al., 2016). Given the synergistic effect between ES16and attrappc11/rog2, it is likely that the complex whereAtTRAPPC11/ROG2 resides acts as a GEF on a GTPase otherthan RABA2A, likely RABD. Intriguingly, the involvement of bothRABA and RABD on recycling endosomes has been proposed inplants (Bar et al., 2013), which may account for the enhancedSYP61 mislocalization observed in the ES16-treated attrappc11/rog2-2mutants. Several RABsare present in theSYP61proteome(Drakakaki et al., 2012) and can be examined in future studies astargets of AtTRAPPC11/ROG2-containing complexes. Compli-cations in testing this GEF activity include themultisubunit natureof TRAPP complexes and the fact that only individual subunitsmay convey activity, thereby necessitating reconstitution of theintact complex together with the target protein.

Importantly, our ES16 studies suggest a compartmentalizedfunction for AtTRAPPC11/ROG2 and likely the TRAPPIII complex.Several studies usingdiversemarkers andelectronmicroscopyhavedemonstrated the heterogeneous nature of the TGN/EE population(Chow et al., 2008; Kang et al., 2011; Scheuring et al., 2011; Uemuraet al., 2014). RABA2AandSYP61 are thought to define two different,specialized TGN/EE subcompartments (Gendre et al., 2011; Doyleet al., 2015; Wattelet-Boyer et al., 2016). Colocalization of RABA2Aand SYP61 at the TGN/EE is only partial (Doyle et al., 2015), and thevesicles differ in membrane lipid composition (Wattelet-Boyer et al.,2016). The synergistic effect of attrappc11/rog2 andES16 onSYP61localization points toward a function of AtTRAPPC11/ROG2 ina RABA2A-independent post-Golgi trafficking pathway.

AtTRAPPC11 Defines a TRAPPIII Complex in Arabidopsis

Our proteomic analysis of AtTRAPPC11/ROG2 immunoprecipi-tates showed that it is a member of a TRAPPIII complex in Ara-bidopsis. This conclusion is based on (1) the presence of thecore TRAPPI subunits and the TRAPPIII-defining subunitsAtTRAPPC8/TRS85, AtTRAPPC12, and AtTRAPPC13 and (2) theabsence of the two well-characterized AtTRS120 and AtTRS130TRAPPII members in Arabidopsis (Jaber et al., 2010; Thellmannet al., 2010;Qi et al., 2011;Rybaket al., 2014), routinely detectablein proteomic studies (Drakakaki et al., 2012; Groen et al., 2014;Rybak et al., 2014). In addition, BiFC experiments corroboratedthe presence of AtTRAPPC6 andAtTRAPPC8 in theAtTRAPPC11/

ROG2 complex. Answering questions about the stoichiometry andstructure of the TRAPPIII plant complex will benefit from the latestadvances in cryo-electron microscopy (Waltz et al., 2019).Arabidopsis TRAPPII localizes at the TGN/EE (Qi et al., 2011;

Naramoto et al., 2014a; Ravikumar et al., 2017, 2018). However,neither the AtTRS120 nor the AtTRS130 TRAPPII-specific sub-units immunoprecipitatedwithAtTRAPPC11/ROG2. It is plausiblethat several TRAPP complexes operate at the plant TGN/EE andthat they have both partially overlapping and distinct roles.Functional compartmentalization is expected based on theirputative GTPase targets, with RABA and RABD being the likelytargets of TRAPPII and TRAPPIII, respectively (Vuka�sinović andŽárský, 2016). A constitutively activated version of the RABA1cGTPase (a RAB11 or Ypt31/32 homolog) partially complementstheTRAPPIImutant trs130, whileRABD (aRAB1orYpt1 homolog)does not (Qi et al., 2011; Qi and Zheng, 2011). The lack of SYP61mislocalization to the tonoplast in the TRAPPII mutant attrs120(Ravikumar et al., 2018) supports the distinct roles of the tworespective complexes at the TGN/EE.Multisubunit tetheringcomplexes includingTRAPPscanexist in

a variety of modular forms as result of subunit exchanges,a property referred to asmodularity (Desfougères et al., 2015; Kimet al., 2016; Ravikumar et al., 2017). Given the very limited in-formation we have on plant TRAPPs, we cannot exclude thepossibility that AtTRAPC11/ROG2 takes part in more than onemultisubunit complex. Another layer of complexity arises fromplausible functions of AtTRAPPC11/ROG2 outside the TRAPIIIcomplex as suggested for its mammalian ortholog (Sacher et al.,2019). This could account for the diverse subcellular effects ob-served in its absence. Future studies can assess the likelihood ofsuch an intriguing scenario.

AtTRAPPC11 Is Involved in TGN/EE Endocytic Trafficking

In line with a TRAPIII function in recycling from early endosome toGolgi in yeast (Thomas et al., 2018), TGN/EE endocytic traffickingis affected in attrappc11/rog2 Arabidopsis mutants. FM4-64 in-ternalization in TGN/EE compartments is reduced in attrappc11/rog2, suggesting a function of ATTRAPPC11/ROG2 in endocyticpathways and reflecting the function of the plant TGN as an earlyendosome (Dettmer et al., 2006; Viotti et al., 2010). A plausiblehypothesis is that AtTRAPPC11/ROG2 participates in tethering ofendosomal vesicleswithTGNcompartments,whichcouldexplainthe reduced colocalization of FM4-64 with SYP61 in the mutantbackground. The function of scaffolding or tethering proteins thatmediate reassembly and fusion of small endocytic vesicles isproposed in tobacco BY-2 cells (Ito et al., 2017). AtTRAPPC11/ROG2 could serve a similar tethering function, accounting for thereduced colocalization of FM4-64 with SYP61 in the mutant;however, such mechanism awaits confirmation in Arabidopsis.Interestingly, impaired endocytosis has also been described inmutants of the Arabidopsis TRAPPII complex (Ravikumar et al.,2018), suggesting that both complexes share a function in en-docytic trafficking. Similar to attrappc11/rog2, single mutants ofthe STOMATAL CYTOKINESIS DEFECTIVE complex, an inter-actor of Arabidopsis EXOCYST and RabE1 family of GTPases,display deficiencies in endocytic traffic (McMichael et al., 2013;Mayers et al., 2017).

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Loss of AtTRAPPC11 Affects Root Growth and PlantStress Response

Root growth is affected in attrappc11/rog2 mutants, which isparticularly evidentwhen seedlingsenter their secondweekof life.The seedling lethality of the TRAPPII complex mutations attrs120and attrs130, however, reflects a specific role of these twoTRAPPII members in cytokinesis (Thellmann et al., 2010; Qi et al.,2011). This further points to possible distinct functions of theTRAPPII and TRAPPIII complexes in plants.

The effect of attrappc11/rog2 on root growth is subtle, assimilarly observed in several TGN single mutants (Kim and Bas-sham, 2011; Gendre et al., 2013; Renna et al., 2018). Severalscenarios are plausible to account for this. Functional redundancybetween subunits of the TRAPP complex where AtTRAPPC11/ROG2 resides might exist with core complex subunits compen-sating, as suggested for the yeast TRAPIII subunit Trs85 (Thomaset al., 2018). Alternatively, partial redundancy may lie in otherTGN/EE-resident regulators associated with AtTRAPPC11/ROG2,asobservedwithGTPases (Pinheiroetal.,2009). Inaddition,based on our transcript analysis, we cannot exclude the possibilitythat partially functional truncated versions of the AtTRAPPC11/ROG2 protein are produced by the different AtTRAPPC11/ROG2T-DNA insertional mutants included in this study.

Our salinity studies revealed a role for AtTRAPPC11/ROG2 inabiotic stress. Salinity induces osmotic stress across the PM andinhibits ion transporters andcytosolic enzymes (Baral et al., 2015).Mutants of TGN/EE vesicle constituents, includingSYP61 (osm1),TNO1, SYP42, and SYP43, all exhibit salt stress–hypersensitivephenotypes (Zhu et al., 2002; Kim and Bassham, 2011; Uemuraet al., 2012). Conversely, knockouts of the SNAREs SYP22 andVAMP7C, and overexpression of the Golgi SNARE SFT12, exhibitincreased salt tolerance (Leshem et al., 2006; Hamaji et al., 2009;Tarte et al., 2015). Furthermore, mutants of RAB proteins ARA6/RABF1, RABA1, and the RAB5-GEF VPS9 show salt hypersen-sitivity, while overexpression ofRABG3enhances salt tolerance inArabidopsis (Mazel et al., 2004; Ebine et al., 2011; Asaoka et al.,2013; Baral et al., 2015). Collectively, these observations un-derscore an involvement of post-Golgi trafficking in stress re-sponses. It is likely, given the similar phenotype of osm1, that thesalt hypersensitivity of attrappc11/rog2 mutants is the result ofaltered TGN/EE-mediated trafficking. Notably, RABA1 quadruplemutants, which are orthologs of TRAPP-GEF targets in otherspecies, are sensitive to salt stress, but not to mannitol (Asaokaet al., 2013; Vuka�sinović and Žárský, 2016), pointing to a role ofTRAPPs in salinity stress.

In a recent study, AtTRAPPC11/ROG2 was identified in a re-sponse to oligogalacturonide, a pectin oligosaccharide normallyelicitedaspartof thedefense responseagainstcertainpathogens.The rog2 mutant suppressed the phenotype of a dominant wall-associated kinase allele, implicating AtTRAPPC11/ROG2 in bioticstress (Kohorn et al., 2016). It is possible that the defects in TGN/EE-mediated trafficking seen in attrappc11/rog2 also interferewith the responses to biotic stress or with cell wall–mediatedsignaling, thus suppressing the kinase mutant phenotype. Giventhe role of AtTRAPPC11/ROG2 in both biotic and abiotic stressresponses, further efforts into its detailed characterization mightbring about a better understanding of plant defensemechanisms.

The wide range of TRAPPopathies associated with the humanTRAPC11mutations include, among others, muscular dystrophy,myopathy, and liver disease (Sacher et al., 2019) and point towardyet-undiscovered functions of the AtTRAPPC11/ROG2 in plantphysiology. The role of human TRAPPC11 upstream of auto-phagosome formation was recently demonstrated (Stanga et al.,2019); a similar function warrants exploration in plant cells, es-pecially under biotic and abiotic stress.How the plant TGN/EE is organized and which factors maintain

its integrity remain crucial questions for a comprehensive un-derstanding of this organelle’s complexity. We have identifiedAtTRAPPC11/ROG2 as a regulator of TGN/EE integrity and post-Golgi/endosomal trafficking. Furthermore, our studies have re-vealed the existence of a plant-specific TRAPPIII complex and anunprecedented role for a TRAPP subunit in plant abiotic stressresponse. This opens up new avenues toward dissecting TGN/EEpopulations through the study of evolutionarily conserved traf-ficking regulators in eukaryotes.

METHODS

Plant Expression Constructs

The plant expression vector UBQ10pro:YFP-AtTRAPPC11/ROG2 wascreated using Gateway cloning to insert the PCR-amplifiedAtTRAPPC11/ROG2 coding sequence (CDS) into pUBN-YFP (Grefen et al., 2010). TheAtTRAPPC11/ROG2 CDS was amplified from Arabidopsis (Arabidopsisthaliana) using the primers ROG2Cf and ROG2Cr and Phusion PCRpolymerase (New England Biolabs). The PCR product was cloned inpENTR/D-TOPO (Invitrogen/Thermo Fisher Scientific) and shuttled intopUBN-YFP (Grefen et al., 2010) using Gateway cloning. The plant ex-pression vector NATpro:AtTRAPPC11/ROG2-GFP, where NAT indicatesnative, was created by amplifying the genomic fragments of both theAtTRAPPC11/ROG2promoter regionand thegenic regionand introducingthem into the pGWB4-GFP (Nakagawa et al., 2007) vector using a two-fragment multisite Gateway system. The promoter region was amplifiedusing the Phusion PCR polymerase (New England Biolabs) and primersROG2pro-f and ROG2pro-r. The genic region was amplified using primersROG2gnm-f and ROG2gnm-r. The PCR products were introduced intovectors pDONR221 P1-P5r and pDONR221 P5-P2 for the promoter andgenic regions, respectively, using the BP reaction (Life Technologies). Theshuttling of the gene coding and promoter fragments into the pGWB4 finaldestination vector was achieved using the LR reaction (Life Technologies).

Theplasmidswere introduced intoGV3101Agrobacterium tumefaciensand used to transform Arabidopsis Col-0 wild type, SYP61pro:CFP-SYP61(Robert et al., 2008), and attrappc11/rog2-2 via floral dip (Clough andBent,1998). Transformants were selected using Basta (glufosinate-ammonium,Sigma-Aldrich) or hygromycin (Thermo Fisher Scientific), respectively.

For BiFC experiments, the AtTRAPPC11/ROG2, AtTRAPPC6/TRS33,RABA1G, andAtTRAPPC8/TRS85CDSswere amplified fromArabidopsisusing the primers ROG2GWf/ROG2GWr, TRS33GWf/TRS33GWr,RABA1GGWf/RABA1GGWr, and TRS85GWf/TRS85GWr, respectively,and Phusion PCR polymerase. The ROG2GWf/ROG2GWr PCR productwas inserted into pDONR221-P1P4, while the TRS33GWf/TRS33GWr,RABA1Gf/RABA1Gr, and TRS85GWf/TRS85GWr PCR products wereinserted intopDONR221-P3P2viaBP (Invitrogen/ThermoFisherScientific)reactions. The resulting Gateway entry vectors were then used in 3-vectorLR reactions (Invitrogen/Thermo Fisher Scientific) for the shuttling of theCDSs into the pBiFCt-2in1-CCdestination vector (Grefen andBlatt, 2012).

The final BiFC constructs carrying AtTRAPPC11/ROG2-CtYFP andeither AtTRAPPC6-NtYFP, AtTRAPPC8-NtYFP, or RABA1G-NtYFP, all

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four expressed under the control of the Cauliflower mosaic virus 35Spromoter (Benfey and Chua, 1990), were introduced into GV3101 (Agro-bacterium tumefaciens) and used to infiltrate Nicotiana benthamianaleaves. The sequences of the primers used for the generation of the dif-ferent constructs are presented in Supplemental Table 2.

Plant Material and Growth

Arabidopsis seedlings of Col-0 were used in this study. T-DNA insertionalmutant linesofAtTRAPPC11/ROG2, CS827843 (SAIL) (attrappc11/rog2-1),SALK119008 (attrappc11/rog2-2), SALK079993 (attrappc11/rog2-3),GABI620C04 (attrappc11/rog2-4), SALK071556 (attrappc11/rog2-5),CS805803 (SAIL) (attrappc11/rog2-6), and CS901090 (WiscDsLox)(attrappc11/rog2-7), were obtained from the Arabidopsis Biological Re-sources Center (http://www.arabidopsis.org; Alonso et al., 2003). Themutant genotypes and the AtTRAPPC11/ROG2 transcripts produced inthe different mutants were characterized using primers described inSupplemental Table 2. Because the genetic characterization of CS827843(attrappc11/rog2-1), SALK079993 (attrappc11/rog2-3), and CS805803(attrappc11/rog2-6) proved complex, only the mutant lines SALK119008(attrappc11/rog2-2), GABI620C04 (attrappc11/rog2-4), SALK071556 (at-trappc11/rog2-5), andCS901090 (attrappc11/rog2-7), were further used inthis study.

The following Arabidopsis lines have been described previously:SYP61pro:CFP-SYP61 (Robert et al., 2008; Drakakaki et al., 2012),UBQ10pro:YFP-RABD2A (WAVE 29Y; Geldner et al., 2009), UBQ10pro:mCherry-SYP32 (WAVE 22R), UBQ10pro:mCerulean-RABF2B (WAVE 2C;Geldner et al., 2009), and 35Spro:HDEL-GFP (Nelson et al., 2007). Geneticcrosses of SYP61pro:CFP-SYP61, UBQ10pro:YFP-RABD2A, NATpro:AtTRAPPC11/ROG2-GFP, and 35Spro:HDEL-GFPwith attrappc11/rog2-2mutants were established in this study. The same applies for the crossesUBQ10pro:mCherry-SYP32 3 UBQ10pro:YFP-AtTRAPPC11/ROG2 andUBQ10pro:mCerulean-RABF2B 3 NATpro:AtTRAPPC11/ROG2-GFP.Seedswere sterilized using 30% (v/v) sodiumchlorate in ethanol (absolute)with0.06%(v/v) of TritonX-100 (Sigma-Aldrich). Seedswereplatedon0.25Murashige and Skoog medium (1.15 g L21 Murashige and Skoog minimalorganicsmedium, 10gL21Suc, 5gL21Phytagel (Sigma-Aldrich), andcoldvernalized for 48 h at 4°C in the dark, after which plates were transferred toa plant growth chamber for seedling growth. Plants were grown in tem-perature-andphotoperiod-controlledenvironments, set to long-day (16-h-light/8-h-dark cycle) conditions, using fluorescent light (at 100 to150mmolquanta photosynthetically active radiation [PAR] m–2 s–1) at 22 to 24°C.

Chemical Treatments

Treatments with BFA (Sigma-Aldrich) were performed on 3- to 4-d-oldArabidopsis seedlings, grown as described in the "Plant Material andGrowth" section. Seedlings were treated for 2 h in 1 mL of liquid 0.25Murashige and Skoog (2.3 g L21 Murashige and Skoog minimal organicsmedium and 10 g L21 Suc) supplemented with 12.5 mM BFA in lightconditions at room temperature. Treated seedlings were mounted in BFA-supplemented media for imaging. FM4-64 (Synaptored C2, Biotium) la-beling of BFA bodies involved a pre-treatment of the seedlings with12.5 mM BFA for 2 h followed by a 5-min incubationin 1 mL of liquid 0.25Murashige andSkoogmediumcontaining12.5mMBFAand2mMFM4-64,on ice. Seedlings were then washed twice in 1 mL of liquid Murashige andSkoog medium containing 12.5 mM BFA, on ice, mounted in BFA-supplemented medium and imaged 15 min post washing (Rigal et al.,2015). For root growth studies, seeds were germinated in solid 0.25Murashige and Skoog medium supplemented with either 1, 2.5, or 5 mMBFA or DMSO (control) and grown for 4 d. Root elongation was measuredusing the segmented line tool of the ImageJ software (Schneider et al.,2012). For FM4-64 internalization experiments, 4-d-old seedlings were

incubated for 5 min in the dark with cold 0.25 Murashige and Skoogmedium supplemented with 2 mM FM4-64, followed by a quick washingstep in FM4-64–free Murashige and Skoog medium (Rigal et al., 2015)before mounting on washing medium for imaging over a period of 18 min.For tonoplast visualization, seedlings were incubated for 3 h under lightgrowth conditions in 1 mL of liquid Murashige and Skoog medium con-taining 2 mM FM4-64 and subsequently mounted in the same treatmentsolution for imaging. Three-hour treatments of 4-d-old seedlings with10mMSNARF-1 (ThermoFisherScientific)wereperformed in 1mLof liquid0.25 Murashige and Skoog medium in the dark at room temperature, asdescribed previously (Viotti et al., 2013).

Three-hour treatments of 4-d-old seedlings with 15 mM ES16 (5615,470,964; ChemBridge) were performed in 1 mL of liquid 0.25 MurashigeandSkoogmedium, in six-well plates, afterwhich seedlingsweremountedusing the treatment medium. For root growth studies, seeds were ger-minated in solid 0.25 MS Murashige and Skoog medium supplementedwith 20 mM ES16 and grown for 5 d. Root elongation was measured usingthe segmented line tool of the ImageJ software (Schneider et al., 2012).

Light Microscopy and Image Analysis

A Leica SP8 confocal microscope was used for localization studies ofYFP-AtTRAPPC11/ROG2, AtTRAPPC11/ROG2-GFP, CFP-SYP61, YFP-RABD2A, mCherry-SYP32, and mCerulean-RABF2B, for the detection ofa fluorescein isothiocyanate (FITC)-conjugated secondary antibody andthe endocytic tracer FM4-64. Fluorescence signals of YFP (excitation 513nm, emission 518 to 582 nm), CFP (excitation 405 nm, emission 465 to 500nm), FITC (excitation488nm, emission495 to550nm),mCherry (excitation587 nm, emission 598 to 684 nm), mCerulean (excitation 405 nm, emission410 to 509 nm), and FM4-64 (excitation 488 nm, emission 652 to 759 nm)were collected with 403 (water), 633 (oil), and 1003 (oil) objectives.

Localization of YFP-RABD2A (excitation 514 nm, emission 519 to 558nm) and HDEL-GFP (excitation 488 nm, emission 493 to 549 nm) fusions,the vacuolar stain with SNARF-1 (excitation 561 nm, emission 619 to 675nm), and the YFP (excitation 514 nm, emission 519 to 579 nm) and redfluorescent protein (excitation 594 nm, emission 599 to 685) signals fromBiFC experiments were acquired using a Zeiss 710 confocal microscopeusing 403 (water) and 633 (oil) objectives. Image analysis was performedusing LAS AF lite (Leica) and Zen Blue (Zeiss) software. Data representimages from more than five independent seedlings.

BFA body image analysis was performed using a combination ofsoftware tools. CFP-SYP61 Z stacks were deconvolved with AutoQuantX3, and volumes were determined with Imaris 7 (Bitplane) following ap-propriate segmentation. Data for the object volumes and seedling volumeswere exported, and histograms were generated in Origin (OriginLab). Avolume of 20 mm3 was set as cut-off for the definition of BFA bodies(>20 mm3). In the case of FM4-64–labeled BFA bodies, two-dimensionalconfocal data were used to measure the area of aggregates using thepolygon tool of the ImageJ software. BFA bodies were defined as objectswithanarea>2.8mm2thatupon three-dimensional (3D)projectiongeneratea sphere of 20 mm3, coinciding with the cut-off established for the in-terpretation of Imaris-based 3D BFA body quantifications. Data for BFAtreatments and controls are an aggregate from five seedlings of eachgenotype. Data are presented normalized to the imaged volume of theseedling or cell area, depending on the experiment.

CFP-SYP61, SYP41, and YFP-RABD2A particles were counted man-ually within 100- to 200-mm2 regions of interest, drawn using the polygontool of ImageJ. For quantification of FM4-64 uptake, ImageJ was used tomeasure the mean fluorescence intensity of the PM and inside the cell,using the segmented line and polygon tools, respectively, as describedpreviously (Rigal et al., 2015).

For the determination of SYP61 particle velocities, 200 single frameswere obtained over a period of 1.5 min. The track tool of Imaris 8.2.0

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(Bitplane) was used to determine the velocities of individual particles. ForCFP-SYP61/FM4-64 colocalization studies, Z stacks were generatedevery 3 min over a period of 18 min and subsequently deconvolved withHuygens (SVI). Thenumbersof totalSYP61punctaeand thesubpopulationof SYP61 punctae colocalized with FM4-64 signal were obtained withImaris 9.1.2 for individual cells, using the cells and spots detectionmodules. The colocalization of SYP61 and FM4-64 was expressed as theratio between the number of SYP61 vesicles associated with FM4-64fluorescent punctae and the number of total SYP61 fluorescent punctaedetected in the cell.

Colocalization analysis between YFP-AtTRAPPC11/ROG2 and CFP-SYP61wasperformedonHuygens deconvolved Z stacks. The FIJI Coloc2plugin, which uses theCostes test for statistical significance (Costes et al.,2004), was used to determine the PCC (automated thresholding, PointSpread Function 3, 10 iterations). Percentages of colocalizationbetween YFP-AtTRAPPC11/ROG2 and mCherry-SYP32 and betweenAtTRAPPC11/ROG2-GFP and mCerulean-RABF2B were estimated usingImageJ.

Immunoprecipitation and MS Analysis

Sample Preparation and Digestion

Extracts from 15-d-old YFP-AtTRAPPC11/ROG2 seedlings growing inliquid cultures were used for immunoprecipitation as described previously(Drakakaki et al., 2012), with the modification that the postnuclear (S1000)plant fraction was used, and the GFP-Trap (Chromatek) system was usedwithGFP-free beads as controls. For proteomeanalysis, proteins bound tomagnetic beads were precipitated using the manufacturer’s recom-mended protocol and the ProteoExtract Protein Precipitation kit (http://www.emdmillipore.com/life-science-research/proteoextract-protein-precipitationkit/EMD_BIO-539180/p_xdCb.s1ORVMAAAEjpxp9.zLX; Cal-Biochem). The resulting protein pellet was solubilized in 100mL of 6Mureain 50mMammonium bicarbonate. Next, 200mMDTTwas added to a finalconcentration of 5 mM, and samples were incubated for 30 min at 37°C.Next, 20 mM iodoacetamide was added to a final concentration of 15 mMand incubated for 30 min at room temperature, followed by the addition of20 mL of DTT to quench the iodoacetamide reaction. Next, Lys-C/trypsin(Promega) was added in a 1:25 (enzyme:protein) ratio and incubated at37°C for 4 h. Samples were then diluted to a concentration of less than 1Murea by the addition of 50 mM ammonium bicarbonate and digestedovernight at 37°C. The following day, samples were desalted usingC18 Macro Spin Columns (Nest Group) and dried down by vacuumcentrifugation.

LC MS-MS Analysis

Liquid chromatography (LC) separation was performed on a ProxeonEasy-nLC II HPLC (Thermo Fisher Scientific) with a Proxeon nanospraysource. Ten microliters of digested peptides, reconstituted in 2% (v/v)acetonitrile:0.1% trifluoroacetic acid, was loaded onto a 100 mm3 25-mmMagic C18 100Å 5U reverse phase trap, where they were desalted onlinebefore being separated on a 75 mm3 150-mmMagicC18200Å3U reversephase column. Peptides were eluted using a gradient of 0.1% (v/v) formicacid and 100% (v/v) acetonitrile with a flow rate of 300 nL/min. A 35-mingradientwas runwith 5%to35% (v/v) acetonitrile over 50min, 35%to80%(v/v) over 3min, 80% (v/v) acetonitrile with 0.1% (v/v) formic acid for 1min,80% to 5% (v/v) acetonitrile with 0.1% (v/v) formic acid over 1 min, andfinally held at 5% (v/v) acetonitrile for 5 min.

Mass spectra were collected on an Orbitrap Q-Exactive mass spec-trometer (ThermoFisher Scientific) in a data-dependentmodewith oneMSprecursor scan followedby15 tandemmassspectrometry (MS-MS) scans.A dynamic exclusion of 5 s was used. MS spectra were acquired witha resolution of 70,000 and a target of 13 106 ions or a maximum injection

time of 20 ms. MS-MS spectra were acquired with a resolution of 17,500and a target of 53 104 ions or amaximum injection timeof 250ms. Peptidefragmentation was performed using higher energy collision dissociationwithanormalizedcollisionenergyvalueof27.Unassignedchargestatesaswell as 11 and ions greater than 15 were excluded from MS-MSfragmentation.

Database Searching and Analysis of LFQ Intensity Data

LC-MS-MS data were searched against The Arabidopsis InformationResource 10 database (https://www.arabidopsis.org/35,686sequences)using MaxQuant version 1.5.7.4 with the default options and the followingexceptions: the variable modifications deamidation (NQ) and phosphor-ylation (STY) were enabled, the iBAQoption andmatch between runswereenabled, and quantitation was enabled for modified peptides. Peptide andprotein identificationswerefilteredusingadecoy falsediscovery rateof1%(Cox and Mann, 2008). Perseus 1.6.02 (Tyanova et al., 2016) was used togenerate the volcano plot using the above MaxQuant LFQ values.

Database Searching and Analysis for Spectral Counting

Tandemmass spectrawere extractedbyProteomeDiscoverer version2.1.Charge state deconvolution and deisotoping were not performed. All MS-MS samples were analyzed using X! Tandem (The GPM, thegpm.org;version CYCLONE [2013.02.01.1]). X! Tandem was set up to search theUniprot Arabidopsis reference proteome (updated May 2017; 63,066entries including decoy and common laboratory contaminant sequences),assuming the digestion enzyme trypsin. X! Tandem was searched witha fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 20ppm. Carbamidomethylation of cysteine was specified as a fixed modi-fication. Glu->pyro-Glu of the N terminus, ammonia loss of the N terminus,Gln->pyro-Glu of the N terminus, deamidation of Asn andGln, oxidation ofMet and Trp, dioxidation of Met and Trp and acetylation of the N terminuswere specified in X! Tandem as variable modifications. Data analysis wasperformed using Scaffold 4.8.2 (Proteome Software).

Criteria for Protein Identification

Scaffold 4.8.2 was used to validate MS-MS–based peptide and proteinidentifications. Peptide identifications were accepted if they exceededspecificdatabase search engine thresholds. X! Tandem identifications andat least two identified peptideswereminimal requirements for acceptance.Proteins that contained similar peptides and could not be differentiatedbased on MS-MS analysis alone were grouped to satisfy the principles ofparsimony. This resulted in a spectrum decoy false discovery rate of 0%and aprotein decoy false discovery rate of 0%. Proteins sharing significantpeptideevidenceweregrouped intoclusters.ProteinswereannotatedwithGO terms from gene association.goa_uniprot (downloaded May 1, 2013;Ashburner and The Gene Ontology Consortium et al., 2000).

Electron Microscopy and TGN Vesicle Size Determination

Four-day-old Arabidopsis seedlings were fixed in 2.5% (v/v) para-formaldehyde and 0.25% (v/v) glutaraldehyde in phosphate buffer on ice.Roots were then excised and embedded in thin sheets of agarose andwashed three times with cold phosphate buffer over a 30-min period. Theroots were then dehydrated stepwise over 4 h in ethanol (10 to 100%) andinfiltrated overnight in a 1:1 mixture of ethanol:London resin. All stepsoccurred at 4°C. The rootswere then placed in London resin at 4°C for 24 hwith three changes of fresh resin therein. The roots were then placed in flatembeddingmolds, and the resin was polymerized in UV light for 8 h at 4°C.Sectionswere cut on a LeicaUC6 ultramicrotome and collected on coppergrids. Cells from the outermost two or three layers of the cortex wereanalyzed. The grids were conventionally stained with uranyl acetate:lead

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citrateandviewedwithaZeissLibra120transmissionelectronmicroscopeat120kV. Electron micrographs were analyzed using ImageJ. The diameter of TGNvesicleswas estimated using the software’s line tool. Data represent analysis ofmore than five sections per genotypewithmore than 50 particles per genotype.

Immunostaining of SYP41

Four-day-old seedlings were fixed in 4% (v/v) paraformaldehyde. Sampleswere washed once with microtubule-stabilizing buffer and transferred intomedium baskets of an InsituPro VSi instrument (Intavis) for automatedin situ hybridization and immunohistochemistry. A standard protocol forbasic whole-mounted Arabidopsis seedlings was used (Sauer and Friml,2010; Park et al., 2014). Briefly, samples were successively washed threetimes with PBS containing 0.1% (v/v) Triton X-100, incubated in 2% (w/v)Driselase (Sigma-Aldrich) for 45 min at 37°C for cell wall hydrolysis, andtreated with 10% (v/v) DMSO and 3% (v/v) octylphenoxypolyethoxy-ethanol (IGEPAL CA-630, Sigma-Aldrich) in PBS containing 0.1% (v/v)Triton X-100 for membrane permeabilization. The rabbit primary antibodyagainst SYP41 (Sanderfoot et al., 2001) and a goat anti-rabbit IgG sec-ondary antibody conjugated to FITC (656111, Invitrogen) were used ata dilution of 1:500. For imaging, samples were mounted in CitiFluor AF1antifade mounting medium (Electron Microscopy Sciences).

BiFC Experiments

Agrobacterium-mediated transformation of leaves of 4-week-old N.benthamiana plants with the BiFC constructs described herein (see “PlantExpression Constructs”) was performed as described previously(Mehlhorn et al., 2018, with the following changes: 4 mL of overnightbacterial cultures was used to inoculate 16 mL of fresh media. The sub-cultures were grown to an OD600 5 0.8 and were centrifuged at 8000g for20 min. The pellets were resuspended in 10 mL of infiltration medium,composed of 50 mM MES, pH 5.6 (Sigma-Aldrich), 2 mM Na3PO4(Spectrum Chemical), 0.5% (w/v) Glc (Sigma-Aldrich), and 100 mM ace-tosyringone (AcrosOrganics). Approximately 560.5-mm2N.benthamianaleaf sections were excised 48 h after transient expression andmounted forsubsequent imaging.

Salt and Mannitol Stress Treatments

Seedlings were grown as described in the "Plant Material and Growth"section. After 3 d of growth, seedlings were transferred to new 0.25Murashige and Skoog plates containing various concentrations of NaCl ormannitol. Seedlings were grown for fivemore days and imaged daily usinga flatbed scanner or recorded every 30 min using a digital camera asdescribed previously (French et al., 2009). Root elongation was measuredusing the segmented line tool of ImageJ (Schneider et al., 2012) orRootTrace (French et al., 2009) for time-lapse image analysis. One-sampleStudent’s t test and two-way analysis of variance (ANOVA) statisticalanalyses were performed using R (R Development Core Team, 2006) orSAS version 9.1.

Statistical Analysis

P-values were calculated with a two-tailed Student’s t test or a two-wayANOVA followed by multiple comparisons Tukey test R (R DevelopmentCore Team, 2006) or SAS version 9.1. See Supplemental Table 3 for de-tailed statistical results.

Accession Numbers

Sequence data from this article can be found at the Arabidopsis GenomeInitiative and/or GenBank/EMBL databases under the following accession

numbers: AtTRAPPC11/ROG2 (At5g65950), Mus musculus TRAPPC11(ID: 320714), AtTRAPPC12 (At4G39820), AtTRAPPC13 (At2G47960),AtTRAPPC2 (At1G80500), AtTRAPPC2(L) (At2G20930), AtTRAPPC4(At5g02280), AtTRAPPC5 (At5g58030), AtTRAPPC6 (At3g05000),AtTRAPPC3 (At5g54750), AtTRAPPC8/TRS85 (At5g16280), AtTRAPPC9/TRS120 (At5g11040), AtTRAPPC10/TRS130 (At5g54440), RABD2A(AT1G02130), SYP61 (AT1G28490), SYP41 (AT5G26980), RABA1G(AT3G15060), RABF2B (AT4G19640), SYP32 (AT3G24350).

Supplemental Data

Supplemental Figure 1. Protein sequence alignment ofMus musculusTRAPPC11 and AtTRAPPC11/ROG2.

Supplemental Figure 2. Colocalization of AtTRAPPC11/ROG2 withthe Golgi marker SYP32 and the late endosomal marker RABF2B.

Supplemental Figure 3. Analysis of AtTRAPPC11 transcripts inattrappc11/rog2 mutants.

Supplemental Figure 4. Vacuolar and endoplasmic reticulum mor-phologies in attrappc11/rog2-2.

Supplemental Figure 5. Effect of ES16 on attrappc11/rog2-2 mutantroot growth.

Supplemental Figure 6. Root growth response of attrappc11/rog2mutants to BFA.

Supplemental Figure 7. Intracellular accumulation of the endocytictracer FM4-64 in mutants of AtTRAPPC11/ROG2.

Supplemental Figure 8. Bimolecular fluorescence complementationassays.

Supplemental Figure 9. Survival of attrappc11/rog2 mutants undersalinity stress.

Supplemental Figure 10. Root growth response of attrappc11/rog2mutants to mannitol.

Supplemental Table 1. TRAPP complex subunits in yeast, mammals,and Arabidopsis.

Supplemental Table 2. Primers used in this study.

Supplemental Table 3. Summary of statistical tests.

Supplemental Data Set 1. Spectral count analysis of Arabidopsis TRAPPmembers enriched in YFP-AtTRAPPC11/ROG2 immunoprecipitates.

Supplemental Data Set 2. Label free quantitation (LFQ) analysis ofArabidopsis TRAPP members enriched in YFP-AtTRAPPC11/ROG2immunoprecipitates.

Supplemental Movie 1. 3D rendering of CFP-SYP61 localization inCol-0 wild-type seedlings.

Supplemental Movie 2. 3D rendering of CFP-SYP61 localization inattrappc11/rog2-2 seedlings.

Supplemental Movie 3. 3D rendering of CFP-SYP61 localization inattrappc11/rog2-2 seedlings under ES16 treatment.

Supplemental Movie 4. Root growth time-lapse imaging of attrappc11/rog2-2 under salt treatment.

ACKNOWLEDGMENTS

We thank the Arabidopsis Biological Resources Center for ArabidopsisT-DNA insertion mutants and Nico Geldner (University of Lausanne),Christopher Grefen (University of Tübingen), Andreas Nebenführ

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(University of Tennessee), Natasha Raikhel (University of California, Riv-erside), and Chris Somerville (University of California, Berkeley) for fluo-rescent marker lines and the SYP41 antibody. We thank ShahabMadahhosseini (University ofCalifornia, Davis) andPaul Bilinski (Universityof California, Davis) for assistance with the statistical analyses. We thankMarkus Pauly (Heinrich Heine University, Düsseldorf), Bo Liu (University ofCalifornia, Davis), Mary Tierney (University of Vermont), and Destiny Davis(University of California, Davis) for critical discussions and reading of thearticle. We thank anonymous reviewers for constructive comments. Thiswork was supported by National Science Foundation (MCB 1818219 andIOS-1258135 awards to G.D) and United States Department of Agriculture(Hatch CA-D-PLS-2132-H to G.D.). Partial support provided by the ChinaScholarship Council to G. R.

AUTHOR CONTRIBUTIONS

G.D., M.R.R., N.W., and T.W. designed research. G.D., D.S.D., S.P., M.S.,R.M.S., G.R., M.R.R., N.W., and T.W. performed research. B.S.P., G.D.,D.D.,M.S., R.S., G.R.,M.R.R., N.W., and T.W. analyzed data. G.D.,M.R.R.,N.W., andT.W.wrote thearticle.All authors read, revised, andapproved thearticle.

Received February 22, 2019; revisedMay 6, 2019; accepted June 2, 2019;published June 7, 2019.

REFERENCES

Allan, B.B., Moyer, B.D., and Balch, W.E. (2000). Rab1 recruitment ofp115 into a cis-SNARE complex: Programming budding COPIIvesicles for fusion. Science 289: 444–448.

Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis ofArabidopsis thaliana. Science 301: 653–657.

Asaoka, R., Uemura, T., Nishida, S., Fujiwara, T., Ueda, T., andNakano, A. (2013). New insights into the role of Arabidopsis RABA1GTPases in salinity stress tolerance. Plant Signal. Behav. 8: 8.

Ashburner, M., et al.; The Gene Ontology Consortium (2000).Gene ontology: Tool for the unification of biology. Nat. Genet. 25:25–29.

Bar, M., Leibman, M., Schuster, S., Pitzhadza, H., and Avni, A.(2013). EHD1 functions in endosomal recycling and confers salttolerance. PLoS One 8: e54533.

Baral, A., Shruthi, K.S., and Mathew, M.K. (2015). Vesicular traf-ficking and salinity responses in plants. IUBMB Life 67: 677–686.

Barr, F.A. (2013). Review series: Rab GTPases and membrane iden-tity: causal or inconsequential? J. Cell Biol. 202: 191–199.

Barrowman, J., Sacher, M., and Ferro-Novick, S. (2000). TRAPPstably associates with the Golgi and is required for vesicle docking.EMBO J. 19: 862–869.

Bassik, M.C., et al. (2013). A systematic mammalian genetic in-teraction map reveals pathways underlying ricin susceptibility. Cell152: 909–922.

Benfey, P.N., and Chua, N.H. (1990). The cauliflower mosaic virus35S promoter: Combinatorial regulation of transcription in plants.Science 250: 959–966.

Blümer, J., Rey, J., Dehmelt, L., Mazel, T., Wu, Y.W., Bastiaens, P.,Goody, R.S., and Itzen, A. (2013). RabGEFs are a major de-terminant for specific Rab membrane targeting. J. Cell Biol. 200:287–300.

Bögershausen, N., et al. (2013). Recessive TRAPPC11 mutationscause a disease spectrum of limb girdle muscular dystrophy and

myopathy with movement disorder and intellectual disability. Am.J. Hum. Genet. 93: 181–190.

Boutté, Y., Jonsson, K., McFarlane, H.E., Johnson, E., Gendre, D.,Swarup, R., Friml, J., Samuels, L., Robert, S., and Bhalerao, R.P.(2013). ECHIDNA-mediated post-Golgi trafficking of auxin carriersfor differential cell elongation. Proc. Natl. Acad. Sci. USA 110:16259–16264.

Brillada, C., and Rojas-Pierce, M. (2017). Vacuolar trafficking andbiogenesis: A maturation in the field. Curr. Opin. Plant Biol. 40:77–81.

Bröcker, C., Engelbrecht-Vandré, S., and Ungermann, C. (2010).Multisubunit tethering complexes and their role in membrane fusion.Curr. Biol. 20: R943–R952.

Brunet, S., and Sacher, M. (2014). In sickness and in health: The roleof TRAPP and associated proteins in disease. Traffic 15: 803–818.

Bui, Q.T., Golinelli-Cohen, M.P., and Jackson, C.L. (2009). LargeArf1 guanine nucleotide exchange factors: Evolution, domainstructure, and roles in membrane trafficking and human disease.Mol. Genet. Genomics 282: 329–350.

Chow, C.M., Neto, H., Foucart, C., and Moore, I. (2008). Rab-A2 andRab-A3 GTPases define a trans-Golgi endosomal membrane do-main in Arabidopsis that contributes substantially to the cell plate.Plant Cell 20: 101–123.

Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana.Plant J. 16: 735–743.

Cole, R.A., McInally, S.A., and Fowler, J.E. (2014). Developmentallydistinct activities of the exocyst enable rapid cell elongation anddetermine meristem size during primary root growth in Arabidopsis.BMC Plant Biol. 14: 386.

Costes, S.V., Daelemans, D., Cho, E.H., Dobbin, Z., Pavlakis, G.,and Lockett, S. (2004). Automatic and quantitative measurement ofprotein-protein colocalization in live cells. Biophys. J. 86: 3993–4003.

Cox, J., and Mann, M. (2008). MaxQuant enables high peptideidentification rates, individualized p.p.b.-range mass accuraciesand proteome-wide protein quantification. Nat. Biotechnol. 26:1367–1372.

DeRossi, C., et al. (2016). trappc11 is required for protein glycosyl-ation in zebrafish and humans. Mol. Biol. Cell 27: 1220–1234.

Desfougères, Y., D’Agostino, M., and Mayer, A. (2015). A modulartethering complex for endosomal recycling. Nat. Cell Biol. 17:540–541.

Dettmer, J., Hong-Hermesdorf, A., Stierhof, Y.D., and Schumacher, K.(2006). Vacuolar H1-ATPase activity is required for endocytic and se-cretory trafficking in Arabidopsis. Plant Cell 18: 715–730.

Doyle, S.M., Haeger, A., Vain, T., Rigal, A., Viotti, C., Łangowska,M., Ma, Q., Friml, J., Raikhel, N.V., Hicks, G.R., and Robert, S.(2015). An early secretory pathway mediated by GNOM-LIKE 1 andGNOM is essential for basal polarity establishment in Arabidopsisthaliana. Proc. Natl. Acad. Sci. USA 112: E806–E815.

Drakakaki, G., van de Ven, W., Pan, S., Miao, Y., Wang, J., Keinath,N.F., Weatherly, B., Jiang, L., Schumacher, K., Hicks, G., andRaikhel, N. (2012). Isolation and proteomic analysis of the SYP61compartment reveal its role in exocytic trafficking in Arabidopsis.Cell Res. 22: 413–424.

Ebine, K., et al. (2011). A membrane trafficking pathway regulated bythe plant-specific RAB GTPase ARA6. Nat. Cell Biol. 13: 853–859.

French, A., Ubeda-Tomás, S., Holman, T.J., Bennett, M.J., andPridmore, T. (2009). High-throughput quantification of root growthusing a novel image-analysis tool. Plant Physiol. 150: 1784–1795.

Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W.,Muller, P., Delbarre, A., Ueda, T., Nakano, A., and Jürgens, G.(2003). The Arabidopsis GNOM ARF-GEF mediates endosomal

AtTRAPPC11/ROG2 Regulates TGN/EE Trafficking 1895

http://dx.doi.org/10.13039/100000001

recycling, auxin transport, and auxin-dependent plant growth. Cell112: 219–230.

Geldner, N., Dénervaud-Tendon, V., Hyman, D.L., Mayer, U.,Stierhof, Y.D., and Chory, J. (2009). Rapid, combinatorial analy-sis of membrane compartments in intact plants with a multicolormarker set. Plant J. 59: 169–178.

Gendre, D., Oh, J., Boutté, Y., Best, J.G., Samuels, L., Nilsson, R.,Uemura, T., Marchant, A., Bennett, M.J., Grebe, M., andBhalerao, R.P. (2011). Conserved Arabidopsis ECHIDNA proteinmediates trans-Golgi-network trafficking and cell elongation. Proc.Natl. Acad. Sci. USA 108: 8048–8053.

Gendre, D., McFarlane, H.E., Johnson, E., Mouille, G., Sjödin, A.,Oh, J., Levesque-Tremblay, G., Watanabe, Y., Samuels, L., andBhalerao, R.P. (2013). Trans-Golgi network localized ECHIDNA/Yptinteracting protein complex is required for the secretion of cell wallpolysaccharides in Arabidopsis. Plant Cell 25: 2633–2646.

Grefen, C., and Blatt, M.R. (2012). A 2in1 cloning system enablesratiometric bimolecular fluorescence complementation (rBiFC). Bi-otechniques 53: 311–314.

Grefen, C., Donald, N., Hashimoto, K., Kudla, J., Schumacher, K.,and Blatt, M.R. (2010). A ubiquitin-10 promoter-based vector setfor fluorescent protein tagging facilitates temporal stability andnative protein distribution in transient and stable expression stud-ies. Plant J. 64: 355–365.

Groen, A.J., Sancho-Andrés, G., Breckels, L.M., Gatto, L., Aniento,F., and Lilley, K.S. (2014). Identification of trans-Golgi networkproteins in Arabidopsis thaliana root tissue. J. Proteome Res. 13:763–776.

Hamaji, K., et al. (2009). Dynamic aspects of ion accumulation byvesicle traffic under salt stress in Arabidopsis. Plant Cell Physiol.50: 2023–2033.

Heard, W., Sklená�r, J., Tomé, D.F., Robatzek, S., and Jones, A.M.(2015). Identification of regulatory and cargo proteins of endosomaland secretory pathways in Arabidopsis thaliana by proteomic dis-section. Mol. Cell. Proteomics 14: 1796–1813.

Ito, Y., Toyooka, K., Fujimoto, M., Ueda, T., Uemura, T., andNakano, A. (2017). The trans-Golgi network and the Golgi stacksbehave independently during regeneration after brefeldin A treat-ment in tobacco BY-2 cells. Plant Cell Physiol. 58: 811–821.

Jaber, E., Thiele, K., Kindzierski, V., Loderer, C., Rybak, K.,Jürgens, G., Mayer, U., Söllner, R., Wanner, G., and Assaad,F.F. (2010). A putative TRAPPII tethering factor is required for cellplate assembly during cytokinesis in Arabidopsis. New Phytol. 187:751–763.

Jahn, R., and Scheller, R.H. (2006). SNAREs--engines for membranefusion. Nat. Rev. Mol. Cell Biol. 7: 631–643.

Jones, S., Newman, C., Liu, F., and Segev, N. (2000). The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt31/32. Mol. Biol.Cell 11: 4403–4411.

Kang, B.H., Nielsen, E., Preuss, M.L., Mastronarde, D., andStaehelin, L.A. (2011). Electron tomography of RabA4b- and PI-4Kb1-labeled trans Golgi network compartments in Arabidopsis.Traffic 12: 313–329.

Kim, S.J., and Bassham, D.C. (2011). TNO1 is involved in salt tol-erance and vacuolar trafficking in Arabidopsis. Plant Physiol. 156:514–526.

Kim, J.J., Lipatova, Z., and Segev, N. (2016). TRAPP complexes insecretion and autophagy. Front. Cell Dev. Biol. 4: 20.

Kohorn, B.D., Hoon, D., Minkoff, B.B., Sussman, M.R., andKohorn, S.L. (2016). Rapid oligo-galacturonide induced changesin protein phosphorylation in Arabidopsis. Mol. Cell. Proteomics 15:1351–1359.

Larson, A.A., et al. (2018). TRAPPC11 and GOSR2 mutations asso-ciate with hypoglycosylation of a-dystroglycan and muscular dys-trophy. Skelet. Muscle 8: 17.

Leshem, Y., Melamed-Book, N., Cagnac, O., Ronen, G., Nishri, Y.,Solomon, M., Cohen, G., and Levine, A. (2006). Suppression ofArabidopsis vesicle-SNARE expression inhibited fusion of H2O2-containing vesicles with tonoplast and increased salt tolerance.Proc. Natl. Acad. Sci. USA 103: 18008–18013.

Li, R., Rodriguez-Furlan, C., Wang, J., van de Ven, W., Gao, T.,Raikhel, N.V., and Hicks, G.R. (2017). Different endomembranetrafficking pathways establish apical and basal polarities. Plant Cell29: 90–108.

Lipatova, Z., Majumdar, U., and Segev, N. (2016). Trs33-containingTRAPP IV: A novel autophagy-specific Ypt1 GEF. Genetics 204:1117–1128.

Loh, E., Peter, F., Subramaniam, V.N., and Hong, W. (2005). Mam-malian Bet3 functions as a cytosolic factor participating in transportfrom the ER to the Golgi apparatus. J. Cell Sci. 118: 1209–1222.

Lynch-Day, M.A., Bhandari, D., Menon, S., Huang, J., Cai, H.,Bartholomew, C.R., Brumell, J.H., Ferro-Novick, S., and Klionsky,D.J. (2010). Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore topromote autophagy. Proc. Natl. Acad. Sci. USA 107: 7811–7816.

Matalonga, L., et al. (2017). Mutations in TRAPPC11 are associatedwith a congenital disorder of glycosylation. Hum. Mutat. 38:148–151.

Mayers, J.R., Hu, T., Wang, C., Cárdenas, J.J., Tan, Y., Pan, J., andBednarek, S.Y. (2017). SCD1 and SCD2 form a complex thatfunctions with the exocyst and RabE1 in exocytosis and cytokine-sis. Plant Cell 29: 2610–2625.

Mazel, A., Leshem, Y., Tiwari, B.S., and Levine, A. (2004). Inductionof salt and osmotic stress tolerance by overexpression