© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838

RESEARCH ARTICLE SPECIAL ISSUE: PLANT CELL BIOLOGY Signal motif-dependent ER export of the Qc-SNARE BET12 interacts with MEMB12 and affects PR1 trafficking in Arabidopsis Kin Pan Chung1, Yonglun Zeng1, Yimin Li2, Changyang Ji1, Yiji Xia2 and Liwen Jiang1,3,*

ABSTRACT A large number of SNARE proteins are encoded in the plant Soluble N-ethylmaleimide-sensitive fusion protein attachment protein genome (Sanderfoot, 2007; Sanderfoot et al., 2000). Numerous receptors (SNAREs) are well-known for their role in controlling studies have unraveled the important role of SNARE proteins membrane fusion, the final, but crucial step, in vesicular transport in plants, involving various biological processes including in eukaryotes. SNARE proteins contribute to various biological pathogen defense, cytokinesis, abiotic stress, cell expansion, processes including pathogen defense and channel activity regulation, symbiosis, gravitropism, gametophyte and seed development as well as plant growth and development. Precise targeting of SNARE (Ebine et al., 2008; El-Kasmi et al., 2011; Grefen et al., 2010a; proteins to destined compartments is a prerequisite for their proper Hachez et al., 2014; Honsbein et al., 2009; Huisman et al., 2016; functioning. However, the underlying mechanism(s) for SNARE Pan et al., 2016; Reichardt et al., 2007; Uemura et al., 2012b; Yano targeting in plants remains obscure. Here, we investigate the targeting et al., 2003). The precise targeting of SNARE proteins to a distinct – mechanism of the Arabidopsis thaliana Qc-SNARE BET12, which is compartment is essential for mediating the vesicle target- involved in protein trafficking in the early secretory pathway. Two distinct membrane fusions which secures an efficient and accurate protein signal motifs that are required for efficient BET12 ER export were trafficking. Mis-targeting of SNARE proteins results in numerous identified. Pulldown assays and in vivo imaging implicated that both the cellular defects. For instance, the cell plate formation is disrupted in COPI and COPII pathways were required for BET12 targeting. Further a mutant with an impaired trafficking of the syntaxin KNOLLE studies using an ER-export-defective form of BET12 revealed that (Park et al., 2013; Teh et al., 2013). In addition, a recent study has the Golgi-localized Qb-SNARE MEMB12, a negative regulator of revealed the novel role of the endoplasmic reticulum (ER)- pathogenesis-related protein 1 (PR1; At2g14610) secretion, was its associated SNARE SYP73 in maintaining the ER integrity and interacting partner. Ectopic expression of BET12 caused no inhibition in consequently streaming, since these features were altered in a syp73 the general ER-Golgi anterograde transport but caused intracellular mutant (Cao et al., 2016). Therefore, correct localization of SNARE accumulation of PR1, suggesting that BET12 has a regulatory role in proteins seems to be a prerequisite for their proper functioning in PR1 trafficking in A. thaliana. different cellular processes. Most SNARE proteins are tail-anchored (TA) proteins, their KEY WORDS: BET12, ER export, PR1, Protein trafficking, SNARE membrane association being conferred by the C-terminal transmembrane domain (TMD). TA proteins are post-translationally INTRODUCTION inserted into the membrane through the guided entry of TA proteins Compartmentalization of cells in eukaryotes presupposes the (GET) pathway (Schuldiner et al., 2008; Stefanovic and Hegde, development of mechanisms for protein trafficking between 2007). Recent studies have demonstrated the functional GET different membrane-enclosed organelles. Vesicular transport is components for TA protein targeting in Arabidopsis (Xing et al., the predominant pathway for protein trafficking in eukaryotic 2017) for the process whereby the SNARE SYP72 is inserted into the cells. Multiple molecular machineries are required for the ER membrane through the GET pathway (Srivistava et al., 2016). formation, transport, tethering and fusion of the vesicles to Once translocated into the ER, further targeting of membrane proteins the target compartment (Bonifacino and Glick, 2004). Soluble is determined by either specific signal motifs, the length of TMD or a N-ethylmaleimide-sensitive fusion protein attachment protein combination of both (Brandizzi et al., 2002; Hanton et al., 2006, receptors (SNAREs) have been identified as critical components 2005b; Matheson et al., 2006; Rojo and Denecke, 2008; Saint-Jore- involved in the final fusion step for the vesicular transport pathways Dupas et al., 2006). In the early secretory pathway of plants, proteins (Söllner et al., 1993). These facilitate vesicle–target-membrane that are exported from the ER and traffic to the Golgi are mediated by fusion by forming hetero-tetrameric trans-SNARE complexes coat protein complex II (COPII) machinery (Brandizzi and Barlowe, derived from a specific set of SNARE proteins (Jahn and 2013; DaSilva et al., 2004; Hawes et al., 2008; Moreau et al., 2007; Scheller, 2006; McNew et al., 2000). Robinson et al., 2015; Stefano et al., 2014). According to the model in yeast and mammals, the formation of COPII vesicles is initiated by the recruitment of the small GTPase SAR1 to the ER membrane. 1School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, Activated SAR1 then recruits the inner-coat dimeric complex New Territories, Hong Kong, China. 2Department of Biology, Hong Kong Baptist SEC23–SEC24 which captures protein cargos. Subsequent University, Hong Kong, China. 3The Chinese University of Hong Kong Shenzhen recruitment of the outer-coat complex SEC13–SEC31 stimulates Research Institute, Shenzhen 518057, China. the GTP hydrolysis of activated SAR1, and eventually leads to *Author for correspondence ([email protected]) the formation of COPII carriers containing protein cargos to be exported (Bassham et al., 2008; Chung et al., 2016; Hwang and K.P.C., 0000-0003-2786-3095; Y.Z., 0000-0002-9512-6487; C.J., 0000-0003- 4399-8272; L.J., 0000-0002-7829-1472 Robinson, 2009; Marti et al., 2010). Among the Golgi-localized SNARE proteins (Uemura et al., 2004), SYP31 and MEMB11 have

Received 24 February 2017; Accepted 23 May 2017 been reported to have a critical role in mediating ER-to-Golgi Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838 anterograde transport (Bubeck et al., 2008; Chatre et al., 2005). Similarly, the fluorescence pattern of YFP–BET12 changed from Although the roles of SNARE proteins in the early secretory pathway punctate to aggregates upon BFA treatment (Fig. 1D). However, have been investigated, the mechanism for SNARE targeting to the unlike ST–YFP and VHAa1–GFP, the YFP–BET12 aggregate was Golgi membrane remains elusive. Until now, only a single study has not only found in the dense-core FM4-64 aggregate but also at its demonstrated that ER export and Golgi targeting of SYP31 depends periphery (Fig. 1D). Fluorescence intensity line plots along the on the di-acidic motif in its N-terminus (Chatre et al., 2009). YFP–BET12 aggregate showed a different distribution pattern from In the present study, we aimed to elucidate the targeting ST–YFP and VHAa1–GFP in that the YFP–BET12 signal intensity mechanism and the functional role of the Qc-SNARE BET12 remained high in both the periphery and the core region of the (also termed Bs14b) in the early secretory pathway. Previous studies FM4-64 peak (Fig. 1E), indicating that YFP–BET12 is being suggested that BET12 is involved in plant fertility and displayed a trapped in both the Golgi-derived and TGN-derived aggregates. Golgi localization in Arabidopsis protoplasts (Bolanos-Villegas To further confirm the localization of YFP–BET12 from the et al., 2015). BET11 (also termed Bs14a), shares a 78% amino acid CLSM analysis, we performed immunogold electron microscopy similarity to BET12, and was also suggested to localize on Golgi (EM) with anti-GFP antibodies (recognizing YFP) on ultrathin membranes (Uemura et al., 2004). Unlike SYP31 and MEMB11, sections prepared from high-pressure frozen and freeze-substituted BET11 overexpression did not severely affect protein ER-to-Golgi root cells of transgenic Arabidopsis seedlings expressing YFP– anterograde transport (Bubeck et al., 2008; Chatre et al., 2005). BET12. Consistent with our confocal findings, EM observations Strikingly, an in vitro study in yeast has suggested that BET11 and showed that gold particles (on secondary antibodies recognizing BET12 tend to form a distinct quaternary SNARE complex with anti-GFP) were present on both the Golgi stacks as well as the different yeast Golgi SNAREs, as BET11 had a SNARE-binding associated TGN (Fig. 1F). Quantification of immunogold labeling profile that resembled that of Sft1p, whereas the binding profile of indicated that ∼37% and ∼52% of the gold particles were associated BET12 resembled that of Bet1p (Tai and Banfield, 2001). In order with the Golgi (including on both the cis- and trans-side) and TGN, to characterize the role of BET12 in ER-to-Golgi protein trafficking, respectively (Fig. 1G). Taken together, CLSM and EM studies we first determined the subcellular localization of BET12 in demonstrated that YFP–BET12 localized to both the Golgi and transgenic plants and uncovered a signal motif-dependent targeting TGN in transgenic Arabidopsis plants. mechanism that was necessary for efficient ER export. We further identified the Qb-SNARE MEMB12 as the interacting partner of BET12 is an integral membrane protein with an N-terminus BET12 and performed experiments that suggested they have a facing the cytosol function in regulating the secretion of pathogenesis-related protein 1 In order to elucidate the BET12-targeting mechanism, we first needed (PR1; At2g14610) in Arabidopsis. to know its protein topology. Most SNARE proteins are type II membrane protein, with the N-terminus that contains the SNARE motif RESULTS exposed to the cytosol and with a C-terminal TMD. To determine Golgi and trans-Golgi network localization of BET12 whether BET12 is a membrane protein, total proteins were extracted in transgenic Arabidopsis plants from Arabidopsis cells expressing YFP–BET12 and were then To determine the subcellular localization of BET12, we generated separated into soluble and membrane fractions by ultracentrifugation. transgenic plants expressing N-terminally yellow fluorescent protein Immunoblot analysis using anti-GFP antibodies showed that YFP– (YFP)-tagged BET12 (YFP–BET12) driven by the UBQ10 promoter. BET12 was found in the membrane fraction, but not the soluble fraction Confocal laser-scanning microscopy (CLSM) analysis revealed a (Fig. S2A, lane 1 and 2). To distinguish integral from peripheral punctate distribution pattern of YFP–BET12 in Arabidopsis root cells. membrane protein, microsomes isolated were then subjected to high Immunofluorescence labeling using antibodies against organelle- salt, high pH and detergent washes, followed by immunoblotting with specific markers was performed, and revealed that YFP–BET12 was anti-GFP antibodies. The reliability of the assay was verified by using partially colocalized with the Golgi marker EMP12 (also known as anti-VSR antibody (this antibody recognizes several VSR proteins), as TMN1) and the trans-Golgi network (TGN) marker SYP61 but was VSR is known to be an integral membrane protein (Paris et al., 1997). distinct from the ER marker calreticulin (Fig. 1A–C), suggesting that Immunoblot analysis showed that YFP–BET12 remained associated YFP–BET12 localizes to both the Golgi and TGN. with microsomal membrane under high salt and high pH condition but Previous studies have reported that the fungal toxin Brefeldin A released to the soluble fraction upon detergent washes (Fig. S2A, lanes (BFA) causes aggregation of both Golgi and TGN, with the Golgi- 3–10). The response of YFP–BET12 towards different conditions was derived and TGN-derived aggregates being distinct from each other similar to that of VSR, indicating that BET12 is most likely to be an (Lam et al., 2009; Zhang et al., 2011a). To further prove the integral membrane protein. localization of YFP–BET12, we carried out BFA treatment As determined by sequence analysis (TMHMM server 2.0), BET12 followed by a styryl dye FM4-64 uptake experiment, since the is predicted to have a typical SNARE topology (Fig. S2B). To dye can be used as an endocytic tracer to label endosomal determine whether the YFP–BET12 fusion retains the same topology, compartments, including the TGN (Bolte et al., 2004), using we carried out a protease protection assay using microsomes isolated transgenic plants expressing ST–YFP (a trans-Golgi marker; ST is from Arabidopsis cells expressing YFP–BET12, followed by the rat sialyl transferase ST6GAL1), VHAa1–GFP (TGN marker) immunoblotting with anti-GFP antibodies. GFP–VSR2, a membrane and YFP–BET12. After BFA treatment, VHAa1–GFP was found in protein with a known topology that the N-terminus is facing into the dense-core aggregates, termed BFA bodies, together with FM4-64, lumen (Cai et al., 2011), was used as a control in this assay. No band while ST–YFP was distinctly found at the periphery of the FM4-64- was detected in the immunoblot analysis of YFP–BET12 in the labeled BFA body (Fig. S1A,B). Line plots were constructed to presence of the protease trypsin, indicating that the N-terminus show the corresponding fluorescence intensity along the BFA- together with the YFP face the cytosol and can therefore be digested by induced aggregates. From these, the VHAa1–GFP peak was seen to trypsin (Fig. S2C, lane 2). By contrast, a band was detected in the completely overlap with the FM4-64 peak, whereas the ST–YFP immunoblot of GFP–VSR2, showing that its lumen-facing N-terminus peak was largely separate from the FM4-64 peak (Fig. S1A,B). was protected from trypsin digestion (Fig. S2C, lane 5). As expected, Journal of Cell Science

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Fig. 1. YFP–BET12 localizes to the Golgi and trans-Golgi network in transgenic Arabidopsis plants. (A–C) Confocal immunofluorescence labeling showing that YFP–BET12 partially colocalized with the Golgi marker (A) EMP12 and the TGN marker (B) SYP61 but was distinct from the ER marker (C) calreticulin in transgenic Arabidopsis root cells. The insert is a 2× enlargement of the box outlined with a dashed line. The linear Pearson correlation coefficient (rp) and scatterplot (right-hand panels) was obtained using ImageJ with the PSC colocalization plug-in by analyzing 20 individual confocal images for each study. An rp value of +1.0 represents a complete colocalization. Scale bars: 10 μm. (D) Confocal images showing the colocalization of the BFA-induced aggregation of YFP–BET12 and the endocytic tracer FM4-64. 5-day-old transgenic seedlings were treated with 10 μg/ml BFA for 30 min, followed by FM4-64 uptake for another 30 min before imaging. The insert is a 2× enlargement of the box outlined with a dashed line. Enlargement showing that YFP–BET12 aggregates formed in the periphery as well as the core BFA compartment labeled by FM4-64. Confocal images were collected from 10 individual seedlings. Scale bar: 10 μm. (E) Line plot generated by ImageJ showing the fluorescence intensity of YFP–BET12 (green) and FM4-64 (red) along the BFA-induced aggregates (line marked by a and b) shown in D. (F) Immunogold electron microscopy labeling showing the presence of YFP–BET12 in the Golgi and TGN. Gold particles were present on both the Golgi (solid arrows) and TGN (open arrows). Scale bars: 100 nm. (G) The relative percentage (mean±s.d.) of the distribution of gold particles found in the Golgi, TGN and organelles other than the Golgi and TGN. 20 electron micrographs showing the anti-GFP labeling were used for quantification. no band could be detected for YFP–BET12 and GFP–VSR2 in the by transient expression in Arabidopsis protoplasts to identify the presence of both trypsin and Triton X-100 (Fig. S2C, lane 3 and 6). regions that are responsible for BET12 trafficking. Upon transient The results from these biochemical assays suggest that YFP–BET12 is expression of YFP–BET12 and CLSM analysis with organelle- an integral membrane protein, which maintains a typical SNARE specific markers, it could be seen that YFP–BET12 partially topology with its N-terminus facing the cytosol. colocalized with the Golgi marker Man1–RFP and TGN marker mRFP–SYP61 (Fig. 2A,B). As YFP–BET12 displayed a partial The N-terminal region and the region in-between the SNARE Golgi and TGN localization in both the transiently expressing motif and the TMD are important for BET12 trafficking protoplasts and the transgenic plants, we decided to use the transient With the determined BET12 protein topology, we next generated setup to screen for targeting defects caused by truncation and various truncation and deletion versions of YFP–BET12, followed deletion of YFP–BET12. We first deleted the whole cytosolic Journal of Cell Science

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Fig. 2. Efficient export of BET12 from the ER depends on both its N-terminal region and the region between the SNARE motif and the TMD. (A,B) Confocal images showing the partial colocalization of YFP–BET12 with the Golgi marker (A) Man1–RFP and the TGN marker (B) mRFP–SYP61 in Arabidopsis protoplasts. The right panel is a 3× enlargement of the box outlined with a dashed line. The degree of colocalization was quantified and is represented by the Pearson correlation coefficient (rp), with the rp value of +1.0 for complete colocalization. Scale bar: 10 μm. (C–H) Confocal images showing distinct subcellular localizations of different YFP–BET12 truncation and deletion fusions (schematically shown at top of figure) upon co-expression with the ER marker CNX–RFP and the Golgi marker Man1–RFP. The (C) ER network, (D) Golgi punctate and (E–H) the combination of ER and Golgi punctate pattern of the corresponding truncation and deletion fusions was observed in Arabidopsis protoplasts. The right panel is a 3× enlargement of the box outlined with a dashed line.

The degree of colocalization was quantified and represented by the Pearson correlation coefficient (rp), where an rp value of +1.0 represents complete colocalization. Scale bars: 10 μm.

N-terminus of BET12 to determine its effect on trafficking. CLSM (Joglekar et al., 2003), we also deleted the BET12 SNARE motif analysis showed that YFP–BET12(107-130) colocalized with the to test for any targeting defect. Upon transient expression, ER marker CNX–RFP and displayed an ER pattern (Fig. 2C), YFP–BET12(1-32)(98-130) showed a punctate pattern which indicating that the presence of the TMD and C-terminus is not colocalized with Man1–RFP (Fig. 2D), suggesting that SNARE sufficient for its ER export, and the cytosolic N-terminal probably motif is not essential for BET12 ER export. To identify the region contains ER export signals. Since, in previous studies, the SNARE containing the ER export signals, we further deleted the nine amino motif has been suggested to play a role in SNARE targeting acids (a.a.) between the SNARE motif and the TMD. Interestingly, Journal of Cell Science

4 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838 instead of only showing a punctate pattern, YFP-BET12(1-32)(107- (a.a. 98–106) in order to identify more precisely amino acid residues 130) partially colocalized with CNX–RFP and displayed an ER responsible for COPII-mediated ER export. By sequence alignment, pattern as well (Fig. 2E). This result suggests that the very we identified motifs that bind and interact with Sar1 and Sec24, as N-terminus (a.a. 1–32) contains ER export signal and aids in reported previously. In yeast, it has been shown that the COPII inner targeting to the Golgi which lead to the punctate pattern (Fig. 2F). coat complex Sec23–Sec24 binds to LxxLE and mediates SNARE The deletion of the nine amino acids (a.a. 98–106) inhibits ER Bet1 ER export (Mossessova et al., 2003). Interestingly, the COPII- export to some extent, thus leading to the appearance of the ER binding motif LxxLE was present within the N-terminus region (a.a. pattern. Similarly, the presence of both the punctate and ER pattern 1–32) of BET12 (Fig. 4A, in blue). In addition, the dibasic motif of was also observed when expressing YFP–BET12(98-130) (Fig. 2G, glycosyltransferase was found to interact with Sar1 in mammalian H), supporting the notion that the nine amino acids (a.a. 98–106) cells (Giraudo and Maccioni, 2003). A similar dibasic motif was also contain an ER export signal and aid in BET12 targeting, while identified in the region between the BET12 SNARE motif and the the absence of the very N-terminus (a.a. 1–32) hampers its ER TMD (a.a. 98–106) (Fig. 4A, in blue). To prove the conserved role export efficiency. Taken together, in vivo expression of truncated of these motifs in ER export, we generated two mutated BET12 and deleted versions of YFP–BET12 revealed that both the constructs with point mutations in the LxxLE and RK dibasic motif N-terminus (a.a. 1–32) and the region between the SNARE motif respectively, which are termed YFP–BET12(L18A,L21A,E22A) and TMD (a.a. 98–106) of BET12 contain ER export signals and are and YFP–BET12(R102A,K103A). We then performed a transient responsible for its efficient trafficking. expression experiment using Arabidopsis protoplasts, and the subcellular localization of these mutated BET12 proteins was BET12 trafficking depends on functional COPI and COPII determined by CLSM. Unlike the sole punctate pattern displayed by machineries YFP–BET12 (Fig. 4B), both the YFP-BET12(L18A,L21A,E22A) To elucidate the mechanism of BET12 trafficking, we made use of the and YFP-BET12(R102A,K103A) showed a punctate and ER region identified as containing the ER export signals to find out the pattern which partially colocalized with CNX–RFP (Fig. S3A,B), potential interaction partners and the related trafficking machinery. A suggesting that the separate mutations in one of the potential COPII- synthetic peptide of the nine amino acids (a.a. 98–106 containing the binding motifs causes a partial defect in BET12 ER export. To ER export motif) was conjugated onto Sepharose beads as bait for further assess the role of these motifs, we generated a construct with pulldown experiments. Sepharose beads conjugated with or without five point mutations so that all five residues became alanine this peptide were incubated with total proteins extracted from residues, termed YFP–BET12-m (Fig. 4A, in red). Strikingly, YFP– Arabidopsis suspension cells. After washing, proteins were eluted BET12-m displayed a dominant ER pattern and colocalized with from the beads and subjected to SDS-PAGE followed by silver CNX–RFP (Fig. 4C), suggesting that the mutations severely inhibit staining (Fig. 3A). In duplicate experiments, intense bands that were its ER export. Interestingly, a few puncta with faint fluorescence repeatedly observed in the lane with the conjugated peptide but signals were localized close to the ER, suggesting that a limited not with the empty Sepharose were isolated for tandem mass amount of YFP–BET12-m may still exit the ER and reach the Golgi spectrometry (MS/MS) analysis. MS/MS analysis showed that certain (Fig. 4C; Fig. S4). The failure in ER export of YFP–BET12-m was proteins pulled out by the nine-amino-acid peptide were identified not only observed in protoplasts but also in intact Arabidopsis as components of the COPI (ε-COP and ζ-COP) and COPII seedlings; YFP–BET12 and YFP–BET12-m was expressed in (Sar1) machineries (Table S1). It has been reported that COPI and 7-day-old seedlings by particle bombardment and the COPII machinery are important for regulating ER-to-Golgi protein corresponding transformed cells were imaged using CLSM. In trafficking (Gao et al., 2014; Hanton et al., 2005a; Paul and Frigerio, leaf pavement and trichome cells, YFP–BET12 displayed a punctate 2007). As a Golgi-localized SNARE, BET12 likely interacts with the pattern resembling the Golgi localization (Fig. 4D). By contrast, COPI and COPII machinery components to maintain its proper YFP–BET12-m exhibited an ER network pattern in both the leaf localization. To determine whether this was indeed the case, we pavement and trichome cells, which was obviously observed transiently expressed YFP–BET12 together with a GDP-fixed mutant throughout the z-stack projection of multiple confocal layers form of ADP-ribosylation factor 1 (Arf1-GDP), which interferes with (Fig. 4E). Point mutagenesis of the five residues significantly the COPI machinery (Pimpl et al., 2003; Takeuchi et al., 2002), and changed the localization of YFP–BET12 from a punctate to an ER with the GTP-locked version of SAR1 (SAR1-GTP) mutant, which pattern. The fact that there are two putative COPII-binding motifs in interferes with the COPII machinery and inhibits protein export from different regions of the protein may explain our above observation the ER (Osterrieder et al., 2010; Takeuchi et al., 2000; Zeng et al., that the absence of any one ER export signal leads to both the 2015). CLSM analysis showed that YFP–BET12 relocated to the ER punctate and ER localization pattern. and colocalized with the ER marker CNX–RFP upon co-expression with Arf1-GDP (Fig. 3B). Similarly, YFP–BET12 displayed an ER ER-export-defective YFP–BET12-m retained MEMB12 but not pattern when co-expressed with the SAR1 GTP-locked mutant form other Golgi-localized SNAREs in the ER Sar1C-DN–RFP, indicating its failure to undergo ER export when the The role of the SNARE BET12 in ER–Golgi trafficking is unclear. COPII machinery is disrupted (Fig. 3C). Pulldown MS/MS analysis, We speculated that the overexpression of an ER-export-defective together with the evidence obtained from the in vivo cell biological form of BET12 might interfere with protein trafficking in the early study, suggested that BET12 trafficking depends on functional COPI secretory pathway. To test this hypothesis, we co-expressed and COPII machineries. YFP–BET12-m with protein cargos known to be transported through the conventional secretory pathway. Aleurain–mRFP and The LxxLE motif in the N-terminus and the dibasic motif prior RFP–SCAMP1 were used as soluble and membrane cargo markers, to the TMD are responsible for ER export of BET12 respectively (Lam et al., 2007; Miao et al., 2008). However, CLSM The intimate relationship of BET12 with COPII machinery analysis showed that the trafficking of both the Aleurain–mRFP and prompted us to take a detailed look into the N-terminus RFP–SCAMP1 was not affected by YFP–BET12-m and they were

(a.a. 1–32) and the region between the SNARE motif and TMD transported to the vacuole and plasma membrane correspondingly Journal of Cell Science

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Fig. 3. COPI and COPII machinery components bind to the BET12 peptide and affect BET12 targeting. (A) Synthetic peptide of the defined region (a.a. 98–106) of BET12 was conjugated to Sepharose and incubated with proteins extracted from Arabidopsis suspension cells. Eluted proteins were subjected to silver staining, followed by MS/MS analysis. Proteins identified as COPI and COPII components are indicated by arrows. The full list of proteins identified from the MS/MS analysis is included in the supplementary information (Table S1). M, molecular mass marker. (B,C) Confocal images showing the effect on YFP–BET12 trafficking when it is co-expressed with Arf1–GTP and Sar1C-DN–RFP (a GTP-locked mutant SAR1). YFP–BET12 displayed an ER pattern upon co-expression with (B) Arf1-GTP and (C) Sar1C-DN-RFP in Arabidopsis protoplasts. Scale bar=10 μm.

(Fig. S4A,B). Man1–RFP still maintained a typical punctate pattern (Zhang et al., 2011b). As BET12 was shown to interact with upon co-expression with YFP–BET12-m (Fig. S4C), suggesting the MEMB12, we decided to test whether BET12 would also affect Golgi was not disrupted. We then screened a set of Golgi-localized PR1 secretion. Upon transient expression of PR1–RFP in SNARE proteins for any trafficking defect as YFP–BET12-m may Arabidopsis protoplasts, we could not detect any intracellular interact with its partner SNARE proteins and are retained together in fluorescence signal using confocal microscopy. We speculated that the ER. Interestingly, CLSM analysis revealed that only mCherry– the majority of PR1–RFP was being secreted to the culture medium. MEMB12 but not mCherry–SYP31 nor mCherry–GOS12 was To prove this hypothesis, we therefore separated the protoplasts trapped in the ER together with YFP–BET12-m in protoplasts from the culture medium by low-speed centrifugation. The collected (Fig. 5A; Fig. S4D,E). The ER-trapping effect of YFP–BET12-m on culture medium was then concentrated and total proteins were mCherry–MEMB12 was also observed in intact Arabidopsis leaf and extracted from the protoplast pellet. Both the medium and protein trichome cells (Fig. 5B,C). Consistent with the previous findings, extracts were then subjected to immunoblot analysis using anti-RFP mCherry–MEMB12 displayed a punctate pattern when co-expressed antibodies. As expected, a band was detected in the medium fraction with YFP–BET12 (Fig. S5). The ER trapping of mCherry–MEMB12 but not in the proteins extracted from pellet (Fig. 6A, lane 1, 2), is probably caused by the physical interaction with the ER- suggesting that PR1–RFP is constantly secreted out of the export-defective YFP–BET12-m. To confirm this, we performed a protoplasts. Interestingly, co-expression of PR1–RFP with YFP– fluorescence resonance energy transfer-acceptor photobleaching BET12 or YFP–MEMB12 resulted in intracellular accumulation of (FRET-AB) assay to verify the potential protein–protein interaction PR1–RFP, as evidenced by the band detection in the pellet fraction between YFP–BET12-m and Cerulean–MEMB12 (Xing et al., 2016) (Fig. 6A, lane 4, 6), although the majority of PR1–RFP was still in which YFP–linker–Cerulean, and YFP–BET12-m with secreted to the medium (Fig. 6A, lane 3, 5). The amounts of CNX–Cerulean was used as a positive and negative control, PR1–RFP retained intracellularly and secreted extracellularly were respectively. FRET-AB analysis suggested an in vivo interaction quantified (Fig. 6B), showing that the ectopic expression of between YFP–BET12-m and Cerulean–MEMB12 as their FRET YFP–BET12 and YFP–MEMB12 affects PR1 trafficking. CLSM efficiency was significantly high compared to the negative control analysis using Arabidopsis seedlings expressing PR1–RFP was (Fig. 5D). Co-immunoprecipitation (Co-IP) assay using GFP-trap performed to further confirm its secretion behavior. When was further performed to show the interaction between YFP–BET12- PR1–RFP was expressed alone, PR1–RFP showed a fluorescence m and HA–MEMB12, as HA–MEMB12 was immunoprecipitated by signal in the extracellular space surrounding the leaf pavement cell YFP–BET12-m as shown in the immunoblot using anti-GFP and (Fig. 6C). Consistent with this, co-expression of PR1–RFP with -HA antibodies (Fig. 5E). Taken together, results from the CLSM YFP–BET12 affected PR1–RFP trafficking as red fluorescence foci analysis and protein–protein interaction assays strongly suggest that were found inside the cell while some PR1–RFP was still secreted to YFP–BET12-m interacts with MEMB12 and its ER-export-defective the apoplast (Fig. 6D). To further verify the relationship between nature causes the trapping of both proteins in the ER. BET12 overexpression and PR1 trafficking, we repeated the secretion assay with a gradual increment in the expression of Ectopic expression of BET12 and MEMB12 causes HA–BET12 and determined its effect in PR1 trafficking by intracellular accumulation of PR1–RFP in Arabidopsis performing immunoblot analysis. The greater the abundance of A previous study suggested that MEMB12 is involved in regulating HA–BET12 is, the more PR1–RFP was trapped in the pellet fraction the secretion of the antimicrobial protein PR1 in Arabidopsis (Fig. 6E), suggesting that the effect of HA–BET12 in PR1 intracellular Journal of Cell Science

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Fig. 4. Signal motifs identified in distinct regions are important for YFP–BET12 targeting in both Arabidopsis protoplast and plants. (A) Amino acid sequence showing the putative COPII-binding motif (in blue) of BET12 and the targeted mutagenesis into alanine residues (in red) in BET12-m,withtheir corresponding subcellular localization listed on the right. (B,C) Confocal images showing the (B) punctate pattern of YFP–BET12 and (C) the colocalization of YFP–BET12-m with CNX–RFP in Arabidopsis protoplasts. The right panel is a 4× enlargement of the box outlined with a dashed line. The degree of colocalization was quantified and represented by the Pearson correlation coefficient (rp), where an rp value of +1.0 represents complete colocalization. Scale bars: 10 μm. (D,E) Confocal images showing the subcellular distribution pattern of (D) YFP–BET12 and (E) YFP–BET12-m in Arabidopsis seedlings. Mutation of the putative COPII-binding motif of BET12 shifted its localization from the (D) punctate pattern (YFP–BET12) to the (E) ER network pattern (YFP–BET12-m)inboththeleaf pavement (left three columns) and trichome cells (right-hand column). The third column shows a 4× enlargement of the box outlined with a dashed line. Scale bars: 10 μm. accumulation is dosage dependent. Interestingly, overexpression of whether the antibacterial defense was affected, we performed HA–BET12-m also interfered with PR1 secretion and caused its bacterial growth assays using wild-type and transgenic plants intracellular retention, although to a lesser extent than HA–BET12 did overexpressing YFP–BET12 infected by both virulent and avirulent (Fig. S6). Taken together, both the biochemical assay and in vivo (avrRpt2) strains of the bacterial pathogen Pseudomonas syringae pv. imaging data suggest that ectopic expression of BET12 affects PR1 tomato (Pst DC3000). Bacterial growth assays showed that there was trafficking and causes its intracellular accumulation. no significant difference in the growth of the virulent or avirulent PR1 is known as an antimicrobial protein that plays an important Pst strains when comparing the wild-type and YFP–BET12 role in plant immunity (Van Loon and Van Strien, 1999). As ectopic overexpression plants (Fig. 6F), suggesting that YFP–BET12 expression of YFP–BET12 interferes with PR1 trafficking, to test transgenic plants are not more susceptible to pathogen infection. Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838

Fig. 5. An ER-export-defective form of YFP–BET12-m interacted with MEMB12 and caused its ER retention. (A–C) Confocal images showing the colocalization of YFP–BET12-m and mCherry–MEMB12 in Arabidopsis (A) protoplasts, (B) leaf pavement and (C) trichome cells. Both YFP–BET12-m and mCherry–MEMB12 displayed an ER network-like pattern when co-expressed. The box outlined with a dashed line is shown as a 5× enlargement in A,B and 4× enlargement in C. The degree of colocalization was quantified and represented by the Pearson correlation coefficient (rp), where an rp value of +1.0 represents complete colocalization. Scale bars: 10 μm. (D) FRET-AB analysis showing the in vivo interaction between YFP–BET12-m and Cerulean–MEMB12. Confocal images showing an example of FRET sample before and after photobleaching (mean±s.d.). The oval highlighted with a dashed line represents the region targeted for photobleaching. FRET efficiency was quantified by measuring the FRET event from 20 protoplasts expressing YFP–linker–Cerulean; CNX–Cerulean with YFP–BET12-m, and YFP–BET12-m with Cerulean–MEMB12, respectively. (E) Co-IP assay showing the interaction between YFP–BET12-m and HA–MEMB12. Arabidopsis protoplasts expressing GFP or YFP–BET12-m with HA–MEMB12 were subjected to protein extraction and IP via GFP-trap followed by immunoblotting with anti-HA- and GFP-antibodies. The asterisk represents the full-length size of YFP–BET12-m. The experiment was repeated three times showing similar results.

DISCUSSION TMD (Brandizzi et al., 2002; Langhans et al., 2008; Robinson et al., Multiple mechanisms for membrane protein targeting have been 2007; Rojo and Denecke, 2008; Schoberer et al., 2009; Wang et al., proposed in plants, including distinct signal motif recognition by 2014). However, the targeting mechanisms for most of the SNAREs trafficking machineries, as well as the various properties of the to their destined compartments, remain obscure in plants. It has been Journal of Cell Science

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Fig. 6. See next page for legend. reported that the entire longin domains of the VAMP7 SNAREs and we demonstrated the Golgi and TGN localization of the Qc-SNARE the di-acidic motif of the SYP31 are essential for the vacuolar and BET12 in Arabidopsis and revealed its COPII-dependent ER export Golgi targeting, respectively (Chatre et al., 2009; Uemura et al., mechanism. Interestingly, subcellular localization studies of BET12 2005). However, additional factors and the nature of the trafficking using transgenic plants and protoplasts yielded results with minor machinery involved in SNARE targeting is not known. In this study, discrepancy: YFP–BET12 was more highly localized to the TGN in Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838

Fig. 6. Ectopic expression of BET12 and MEMB12 affects PR1-RFP experiments were performed. Synthetic peptide pulldown trafficking in Arabidopsis protoplasts and plants. (A) Secretion assay experiments indicated that Sar1, one of the major constituents for – showing the intracellular accumulation of PR1 RFP when co-expressed with COPII vesicle formation, may interact with the region between the YFP–BET12 and YFP–MEMB12 in Arabidopsis protoplasts. Total proteins were extracted from the protoplasts (P) and the culture medium (M) for cells SNARE motif and the TMD (a.a. 98-106) (Fig. 3). Consistent with that (I) singly expressed PR1-RFP, (II) co-expressed PR1–RFP with YFP– this, by sequence analysis, we found that the presence of a dibasic BET12, and (III) co-expressed PR1–RFP with YFP–MEMB12, respectively, motif, which is reported to be Sar1 binding (Giraudo and Maccioni, followed by immunoblot (IB) analysis using anti-RFP antibodies. Anti-GFP 2003; Srivastava et al., 2012; Yuasa et al., 2005), in the same region. antibodies were used to detect the expression of YFP–BET12 and YFP– It has been shown that mutation of the dibasic motif residues MEMB12. Ponceau S staining was used as a loading reference for quantifying proximal to the TMD of bZIP28 to alanine residues interferes with the relative abundance of PR1 proteins among these three samples using ImageJ (shown below blot). The relative abundance of total PR1 proteins from its interaction with Sar1 and inhibits its ER export under ER stress the (I) singly expressed PR1–RFP sample was set as 1.00. (B) Quantification (Srivastava et al., 2012). In addition, a conserved motif, LxxLE, in (mean±s.d.) of the percentage of PR1–RFP secreted to the culture medium for yeast Bet1 which binds to the B site of the Sec23–Sec24 complex the I–III for experiments shown in A. Three independent experiments were (Mossessova et al., 2003), was identified in the BET12 N-terminal performed to obtain the quantification results. (C) Confocal images showing region (a.a. 1–32). Although no direct interaction between the the secretion of PR1–RFP to the extracellular space in Arabidopsis seedlings. SNAREs and Sec24 has been reported in plants, Sec24 has been The box outlined with a dashed line is shown as a 4× enlargement. The shown to interact with the potassium channel KAT1 via its di-acidic asterisk represents the intracellular region of the leaf pavement cell. Scale bar: 10 μm. (D) Confocal images showing the intracellular retention and motif and facilitate its ER export (Sieben et al., 2008). Independent extracellular secretion of PR1–RFP in Arabidopsis seedlings upon co- mutagenesis of the putative Sec24-binding motif LxxLE (a.a. 18–22) expression with YFP–BET12. PR1–RFP signals were found both inside and and the Sar1-binding dibasic motif RK (a.a. 102–103) into alanine outside of the cell. The box outlined with a dashed line is shown as a 4× residues results in a partial defect in ER export of BET12 (Fig. S3). enlargement. The asterisk represents the intracellular region of the pavement Simultaneous mutagenesis of all the five residues severely inhibited μ cell. Scale bar: 10 m. (E) Secretion assay showing the intracellular BET12 trafficking, which resulted in the ER localization of YFP– accumulation of PR1–RFP upon HA–BET12 overexpression is dosage dependent. Total proteins were extracted from the protoplasts co-expressing BET12-m (Fig. 4), as well as in an ER-trapping effect upon PR1–RFP with increasing amounts of HA–BET12 (from 1 to 3), followed by co-expression with Sar1C-DN–RFP. These data indicate that the ER immunoblot analysis using anti-RFP and HA antibodies. Anti-cFBPase was export of BET12 is signal motif-dependent and is mediated by used as loading control. The experiment was repeated three times with each functional COPII machinery. showing similar results. (F) Bacterial growth assay showing that YFP–BET12 In addition to COPII, the COPI machinery component Arf1 has transgenic plants were not susceptible nor resistant to pathogen infection. been reported to interact with another Golgi-localized SNARE, 4-week-old wild-type and YFP–BET12 transgenic plants were infiltrated with Pst (DC3000) (1×106 cfu/ml) and Pst (avrRpt2) (5×106 cfu/ml). Growth of both MEMB11 (Marais et al., 2015). It has been proposed that membrin Pst strains was measured at 0 and 3 days post inoculation (dpi). Results are the (also known as GOSR2), the mammalian MEMB11, act as a mean±s.d. obtained from eight leaf discs. Similar results were obtained in three recruiter for Arf1 recruitment to the Golgi membrane to initiate biological replicates. COPI vesicle formation (Honda et al., 2005). EMP12 was also shown to interact with the COPI machinery to maintain its Golgi transgenic plant root cells while YFP–BET12 showed a slightly localization (Gao et al., 2012). Interestingly, certain COPI higher colocalization rate with the Golgi than the TGN marker in machinery components were identified in our pulldown MS/MS protoplasts. This variation may be due to the different data (Fig. 3), suggesting that BET12 may also bind to COPI and methodologies (immunolabeling and transient expression) and become incorporated into COPI vesicles for its proper targeting. plant materials (transgenic plants and protoplasts) used for the Impaired trafficking and mis-targeting of SNARE proteins has localization studies. Previous studies indicated that the TMD length been reported to be detrimental to cells. For instance, the correct is unlikely to be the sole determinant that dictates subcellular trafficking of KNOLLE is important for plant cell cytokinesis (Park localization of SNARE proteins (Chatre et al., 2009; Uemura et al., et al., 2013; Reichardt et al., 2007), while a reduced salt tolerance 2005). This is consistent with our findings showing that the may be caused by the partial mislocalization of SYP61 in tno1 presence of the TMD of BET12 is alone insufficient for its ER mutants (Kim and Bassham, 2011). To determine whether there is export (Fig. 2). It has been reported that the mammalian ortholog any adverse cellular effect caused by the ER-export-defective form rat BET1 (rBET1) depends on its SNARE motif for targeting of BET12, we monitored the trafficking of both the soluble and (Joglekar et al., 2003). However, the absence of the BET12 SNARE membrane proteins known to employ the ER-to-Golgi secretory motif did not affect its ER export in Arabidopsis cells. Instead, pathway. However, no trafficking defect was observed as both the through truncation experiments, we identified two regions that Aleurain–mRFP and RFP–SCAMP1 were transported to their contain signal motifs that are responsible for efficient ER export of respective destined compartments and even YFP–BET12-m was BET12: the N-terminus (a.a. 1–32) and the region in-between the significantly trapped in the ER (Fig. S4). Interestingly, it is reported SNARE motif and the TMD (a.a. 98–106). BET12 ER export is that the overexpression of the ER-export-defective form of SYP31 hampered but not completely abrogated by deleting either one of the severely inhibits tobacco plant growth (Melser et al., 2009), as the signal-motif-containing regions (either a.a. 1–32 or a.a. 98–106), as accumulation of ER-trapped SYP31 is toxic to the secretory evidenced by the observation of both the ER and punctate (Golgi) pathway by potentially disturbing the homeostasis of the SNARE localization pattern of the truncated YFP–BET12 (Fig. 2). Indeed, machinery as well as the ER–Golgi interface. Although protein similar experimental approaches have been applied in elucidating trafficking was not affected by YFP–BET12-m overexpression, the the targeting mechanism of another Golgi-localized SNARE defective ER export of BET12 could trap the interacting SNARE SYP31. A novel di-acidic motif ExxD, residing in a region partner together in the ER. Among the Golgi-localized SNAREs between the SNARE helices, was found to facilitate the ER export screened, only MEMB12 was found to be trapped in the ER upon of SYP31 (Chatre et al., 2009). To precisely identify the amino acid co-expressing with YFP–BET12-m (Fig. 5). Bos1, the yeast residues constituting the signal motifs for BET12 ER export and homolog of MEMB12, does not have any Sec24-binding motif the trafficking machinery involved, mutagenesis and pulldown (Mossessova et al., 2003). Unlike other Golgi-localized SNAREs Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838 that can bind to Sec24 for their ER export, it is plausible that Bos1 overabundance of SNARE proteins may result in uncontrolled binds to its partner SNAREs to form a complex, which is then co- SNARE partner interaction and thus disrupt the SNARE machinery packaged into COPII vesicles for ER export (Mossessova et al., homeostasis. It has been suggested that SNARE proteins could 2003). Similar to its ortholog Bos1, no putative Sec24-binding become non-fusogenic when over-accumulated, and these SNAREs motif could be identified in MEMB12, indicating that its ER export are termed inhibitory SNAREs (i-SNAREs) (Di Sansebastiano, may be dependent on its partner SNARE. In this sense, the ER 2013), as evidenced by a study using an in vitro fusion assay retention of YFP–BET12-m, which interacts with MEMB12, may (Varlamov et al., 2004). Both the yeast and rat orthologs of BET12, result in the defective ER export of MEMB12 as well. Interestingly, Bet1 and rBET1, show an inhibitory effect on SNARE fusion when a recent study suggested that the preassembled ternary Golgi in excess (Varlamov et al., 2004). In plants, a previous study Q-SNARE complex was preferred for ER export in mammalian proposed that SYP51 and SYP52 behave as the i-SNAREs and cells, a finding that is supported by the sorting defect present in all affected vacuolar trafficking when they accumulate on the tonoplast other Q-SNAREs upon the mutation of the binding site for Sec24C– (De Benedictis et al., 2013). In that sense, considering our result in Syntaxin 5 (Adolf et al., 2016). In our study, the pulldown assay PR1 trafficking, it is plausible that overexpressed BET12 could act followed by the MS/MS analysis failed to identify the potential as an i-SNARE in the early secretory pathway, and therefore SNARE partners interacting with BET12, probably due to the fact prevents the fusion of PR1-containing vesicles and thus inhibit its that only the defined region (a.a. 98–106) of BET12 but not its secretion. Interestingly, overexpression of the Golgi-localized SNARE coiled-coil domain was used as a bait for the pulldown SNAREs SYP31 and MEMB11 strongly inhibited the ER-to- assay. Indeed, BET12 was shown to preferentially form a SNARE Golgi anterograde transport, as evidenced by the redistribution of complex with the yeast Bos1 and certain Golgi SNAREs in an Man1–RFP into the ER (Bubeck et al., 2008). It is noteworthy that in vitro study (Tai and Banfield, 2001). Similar in vitro binding BET12 overexpression did not affect the distribution of Man1–RFP assays using purified Arabidopsis SNARE proteins may represent (Fig. 2A), indicating that the general anterograde transport pathway a good approach to identify the SNARE partners of BET12. is not perturbed by BET12. Instead, PR1 trafficking was interfered, Characterization of the interacting domain between BET12 and suggesting a potential role of BET12 in regulating pathogenesis- MEMB12, as well as the use of in vitro budding assays to determine related protein secretion and plant immunity. Further studies, whether BET12 and MEMB12 are co-packaged into vesicles, combining in vitro SNARE fusion assay with the in vivo monitoring would certainly shed light on the ER export mechanism of SNARE of protein trafficking, will help to characterize i-SNARE activity in proteins. plants. It will also open the possibility that, in addition to the well- The involvement of the Golgi-localized SNARE MEMB12 in the known fusogenic role of SNARE proteins, the regulation and plant defense against pathogens has been previously reported (Zhang targeting of non-fusogenic SNAREs to compartments could fine- et al., 2011b). Arabidopsis MEMB12 knockout mutants exhibit tune protein trafficking in response to various stress conditions. enhanced resistance to the bacterial pathogen Pst as the absence of It has been recently reported that the single homozygous bet11 or MEMB12 promotes the exocytosis of the antimicrobial protein PR1 bet12 T-DNA insertional mutants display no obvious vegetative (Zhang et al., 2011b). Consistent with its role as a negative regulator phenotype. Reduced seed set was observed in homozygous and for PR1 secretion, we found in our study that the MEMB12 heterozygous bet11/bet12 (and vice versa) double mutants, overexpression caused intracellular accumulation of PR1. probably caused by defective pollen tube growth (Bolanos- Interestingly, ectopic expression of BET12 affects PR1 trafficking Villegas et al., 2015). These findings imply that the function of just as MEMB12 does. Although PR1 trafficking was affected, BET11 and BET12 may partially overlap, as functional redundancy transgenic plants overexpressing BET12 displayed no significant has been reported between SNARE family members (Kim and difference in resistance to the Pst infection (Fig. 6), probably due to Bassham, 2013; Shirakawa et al., 2010; Uemura et al., 2010). the fact that the amount of secreted PR1 for defense was not Further studies using either bet12 or bet11/bet12 double mutants in significantly reduced. SNARE proteins are known to play an pathogen response will certainly help to elucidate the role of ER-to- important role in plant defense (Assaad et al., 2004; Collins et al., Golgi SNAREs in plant immunity. 2003; Kwon et al., 2008; Uemura et al., 2012a; Wang et al., 2016). Strikingly, a previous study has revealed the essential role of the plant secretory pathway for the plant immunity, as mutants of the secretory MATERIALS AND METHODS Plasmid construction pathway component showed reduced PR1 secretion and were The cDNA of BET12 was amplified and cloned into the pBI121 backbone susceptible to bacterial pathogen (Wang et al., 2005). Another containing an UBQ10 promoter (Grefen et al., 2010b), the YFP coding study reported that the plasma membrane-localized SNARE SYP132 sequence and the nopaline synthase terminator for the generation of YFP– underwent phosphorylation upon elicitor treatment and the efficient BET12 transgenic plants. pBI221 vectors containing an UBQ10 promoter, PR1 secretion was SYP132-dependent (Kalde et al., 2007). the YFP or the HA coding sequence, the BET12 coding sequence and the Mounting evidence suggests that the absence of certain SNARE nopaline synthase terminator were generated for the transient expression in proteins or secretory components makes plants more susceptible to protoplasts. All the truncation and deletion versions and point mutagenesis pathogens; our finding that the presence of BET12 and MEMB12 mutants of BET12 were amplified from YFP–BET12 and cloned into the play a repressive role in PR1 secretion may therefore seem pBI221 vector. All the primers used in this study are listed in Table S2. contradictory. It has been proposed that MEMB12 is involved in Plant materials and growth conditions retrograde protein trafficking from the Golgi back to the ER, thus – PR1 may be recycled back and instead of being secreted (Zhang To generate YFP BET12 transgenic plants, pBI121 constructs containing YFP–BET12 were introduced into Agrobacterium tumefaciens and et al., 2011b). As BET12 shows interaction with MEMB12, it seems transformed into wild-type Arabidopsis thaliana (Col-0) by the floral dip reasonable that the overexpression of BET12 would inhibit PR1 method (Clough and Bent, 1998). Arabidopsis seeds were surface sterilized secretion by promoting its retrograde transport. Although we cannot and plated on standard Murashige and Skoog (MS) growth medium (pH rule out this possibility, a possible alternative explanation may be 5.7) supplemented with 1% sucrose and 1% agar. Seedlings were grown on the excess SNARE proteins present due to overexpression. The vertically oriented plates in growth chambers at 22°C under a long-day (16 h Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838 light and 8 h dark) photoperiod. Plants used for the bacteria growth assay Proteins were separated by SDS-PAGE and stained by silver staining. were grown on soil in a growth room under a short-day condition with a 10 h Protein bands with significantly higher intensity in the conjugated-peptide light and 14 h dark photoperiod for an extended vegetative growth phase. lane than in the Sepharose control lane were cut out for in-gel trypsin digestion. Peptides were then extracted from the digested gel and further Transient expression by electroporation and particle subjected to liquid chromatography-tandem mass spectrometry analysis as bombardment described previously (Gao et al., 2012). Maintenance of Arabidopsis suspension cells and transient expression in protoplasts were performed by electroporation as described previously Co-IP assay and FRET-AB analysis (Miao and Jiang, 2007). For particle bombardment, 7-day-old Arabidopsis Co-IP assays were performed using proteins extracted from protoplasts seedlings were transferred and placed horizontally on a MS agar plate. Gold expressing YFP–BET12-m and HA–MEMB12. Extracted proteins were particles were coated with plasmid DNA and bombarded into seedlings as incubated with GFP-TRAP magnetic beads following the recommended described previously (Wang and Jiang, 2011). protocol (ChromoTek, http://www.chromotek.com/). After the washing steps, proteins were eluted and boiled in SDS sample buffer, followed by immunoblot Immunofluorescence labeling analysis using anti-GFP and anti-HA antibodies. FRET-AB analysis was Preparation and fixation of 5-day-old Arabidopsis roots for performed using the Leica SP8 confocal microscope as described previously immunofluorescence labeling was performed as described previously (Gao et al., 2015). Target proteins were transiently expressed in Arabidopsis (Sauer et al., 2006). Fixed roots were incubated with anti-EMP12, anti- protoplasts. Fixation wasthen performed by incubating the protoplasts with 3% SYP61 and anti-calreticulin at 4°C overnight, followed by probing with formaldehyde in PBS for 15 min at room temperature. After two rounds of Alexa Fluor 568-conjugated goat anti-rabbit-IgG (Invitrogen) secondary washing with PBS, fixed samples were then subjected to FRET-AB analysis. antibody (1:1000 dilution) for confocal observation. Defined region of interest was selected for photobleaching using a high- intensity laser (514 nm). The signal intensity of the donorand acceptor proteins BFA treatment and FM4-64 uptake study before and after photobleachingwere measured forcalculatingFRETefficiency 5-day-old Arabidopsis seedlings were treated with BFA at 10 μg/ml for through the built-in SP8 algorithm. For each testing FRET protein pair, 20 30 min, followed by FM4-64 uptake for another 30 min before imaging individual cells expressing the target proteins were used for the analysis. For the (Lam et al., 2009). All experiments were repeated at least three times with positive control, an amino acid linker peptide, SSSELSGDEVGGTSGSEF, similar results. was used to fuse the C-terminus of Cerulean and the N-terminus of YFP to create the Cerulean–linker–YFP fusion, while CNX–Cerulean and YFP– Confocal microscopy analysis BET12-m were used as a negative control. CLSM analysis was performed using the Leica SP8 confocal microscope with a sequential line scanning setting using 63× water lens. For each experiment, PR1 secretion assay more than 20 confocal images were collected for quantification and Secretion assays were performed using protoplasts expressing PR1–RFP or colocalization analysis. The Pearson correlation coefficient was calculated PR1–RFP co-expressed with YFP–MEMB12 or YFP–BET12. Protoplasts by using the PSC plugin and a line plot was made using ImageJ (Wayne and culture medium were collected separately by centrifugation at 100 g for Rasband, NIH, https://imagej.nih.gov/) as described (French et al., 2008). 5 min. Total proteins were extracted from the protoplasts, while collected medium was concentrated using Amicon® Ultra-4 Centrifugal Filter Units Electron microscopy study with a 3 kDa molecular mass cut-off by centrifugation at 3200 g for 30 min. EM sample preparation, ultrathin sectioning and immunogold labeling Proteins were extracted and the concentrated medium was subjected to SDS- using 10-nm gold particles were performed as previously described (Tse PAGE followed by immunoblot analysis using anti-RFP and -GFP et al., 2004). Anti-GFP antibodies were used for labeling of root cells in antibodies. Ponceau S staining was used as a loading reference for YFP–BET12 transgenic plants. Transmission electron microscopy was calculating the relative abundance of PR1 proteins in each sample. performed using a Hitachi H-7650 transmission electron microscope with a charge-coupled device camera (Hitachi High-Technologies) operating at Bacterial growth assay 80 kV. Bacterial growth assays were performed using 4-week-old wild-type and YFP–BET12 transgenic Arabidopsis plants as described previously (Li Topology analysis and protease protection assay et al., 2013). 1×106 cfu/ml of Pst (DC3000) and 5×106 cfu/ml of Pst Total proteins were extracted from protoplasts expressing YFP–BET12 avrRpt2 were used for infection by infiltration into Arabidopsis leaves with without the addition of detergent as described (Zhuang et al., 2017). To obtain syringes. Eight leaf-discs were collected at 0 dpi and 3 dpi for counting proteins in soluble and membrane fraction, total proteins were ultracentrifuged bacterial number. For each growth assay, the results from three biological at 100,000 g for 30 min, and the membrane pellets were washed and replicates were used for bacterial quantification. solubilized in an equal volume of extraction buffer with additional 1% Triton X-100. For integral membrane protein determinations, membrane pellets were Antibodies incubated with 1 M KCl, 0.1 M Na2CO3, 1% Triton X-100 and 1% SDS for The anti-GFPantibody(1:1000dilution)wasgenerated as described previously 30 min on ice, followed by ultracentrifugation at 100,000 g for 30 min. (Shen et al., 2014). Anti-cFBPase antibody (1:2000 dilution) was purchased Soluble and membrane fraction were subjected to SDS-PAGE and from Agrisera (cat. no. AS04043). Anti-HA antibody (1:1000 dilution) was immunoblot analysis using anti-GFP, anti-VSR and anti-cFPBase purchased from Abcam (catalog no. ab18181). Anti-RFP antibody (1:1000 antibodies . For protease protection assays, microsomes isolated from dilution) was purchased from chromotek (rat monoclonal 5F8). Anti-VSR, protoplasts expressing YFP–BET12 were subjected to trypsin digestion as -SYP61, -EMP12 and -calreticulin antibody were used as described previously previously described (Gao et al., 2012), followed by immunoblot analysis (Gao et al., 2012; Sanderfoot et al., 2001; Shen et al., 2014; Tse et al., 2004). using anti-GFP antibodies.

Accession numbers In vitro peptide binding assay and MS/MS analysis Sequence data for the proteins analyzed in this article can be found in the A synthetic peptide of the nine amino acids 98–106 of BET12 was EMBL/GenBank data libraries under the following accession numbers: conjugated to CnBr-activated Sepharose as described previously (Contreras AT4G14455 (BET12); AT1G10950 (EMP12); AT1G28490 (SYP61); et al., 2004). Total proteins were extracted from Arabidopsis suspension AT2G30290 (VSR2); AF126550 (MAN1); AT5G20990 (CNX); AT4G02080 cells and were incubated with the conjugated peptide for 4 h at 4°C in a (SAR1C); AT5G50440 (MEMB12); AT2G14610 (PR1); AT2G28520 (VHA- rotator. After incubation, the Sepharose were washed five times with a1); AT5G05760 (SYP31); AT2G45200 (GOS12); AT2G01770 (VIT1)and incubation buffer and proteins were eluted by boiling in SDS sample buffer. OS07G0564600 (SCAMP1). Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838

Acknowledgements Contreras, I., Yang, Y. D., Robinson, D. G. and Aniento, F. (2004). Sorting signals We gratefully acknowledge Mr Kwok Wai Kwan for technical assistance. We thank in the cytosolic tail of plant p24 proteins involved in the interaction with the COPII Professor Jurgen Denecke (University of Leeds, UK) for providing anti-calreticulin coat. Plant Cell Physiol. 45, 1779-1786. antibodies. DaSilva, L. L. P., Snapp, E. L., Denecke, J., Lippincott-Schwartz, J., Hawes, C. and Brandizzi, F. (2004). Endoplasmic reticulum export sites and golgi bodies behave as single mobile secretory units in plant cells. Plant Cell 16, 1753-1771. Competing interests De Benedictis, M., Bleve, G., Faraco, M., Stigliano, E., Grieco, F., Piro, G., The authors declare no competing or financial interests. Dalessandro, G. and Di Sansebastiano, G. P. (2013). AtSYP51/52 functions diverge in the post-Golgi traffic and differently affect vacuolar sorting. Mol. Plant 6, Author contributions 916-930. Conceptualization: K.C., L.J.; Methodology: K.C., Y.Z., Y.L., Y.X., L.J.; Software: Di Sansebastiano, G. P. (2013). Defining new SNARE functions: the i-SNARE. K.C.; Validation: K.C.; Formal analysis: K.C.; Investigation: K.C., Y.Z., Y.L., C.J.; Front Plant Sci. 4, 99. Resources: K.C., Y.Z., Y.L., C.J., Y.X.; Data curation: K.C., Y.Z., Y.L.; Writing - Ebine, K., Okatani, Y., Uemura, T., Goh, T., Shoda, K., Niihama, M., Morita, M. T., original draft: K.C.; Writing - review & editing: K.C., Y.Z., L.J.; Visualization: K.C.; Spitzer, C., Otegui, M. S., Nakano, A. et al. (2008). A SNARE complex unique to Supervision: Y.X., L.J.; Project administration: L.J.; Funding acquisition: L.J. seed plants is required for protein storage vacuole biogenesis and seed development of Arabidopsis thaliana. Plant Cell 20, 3006-3021. Funding El-Kasmi, F., Pacher, T., Strompen, G., Stierhof, Y. D., Muller, L. M., Koncz, C., Mayer, U. and Jurgens, G. (2011). 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