Physical, Functional and Genetic Interactions between the BEACH Domain Protein SPIRRIG and LIP5 and SKD1 and Its Role in Endosomal Trafficking to the Vacuole in Arabidopsis

Beige and Chediak Higashi (BEACH) domain-containing proteins (BDCPs) are facilitators of membrane-dependent cellular processes in eukaryotes. Mutations in BDCPs cause malfunctions of endosomal compartments in various cell types. Recently, the molecular analysis of the BDCP homolog gene SPIRRIG (SPI) has revealed a molecular function in P-bodies and the regulation of RNA stability. We therefore aimed to analyze, whether SPI has also a role in membrane-dependent processes. In this study, we show that SPI physically interacts with endosomal sorting complex required for transport associated ATPase Suppressor of K+-transport growth defect1 (SKD1) and its positive regulator, LYST Interacting Protein 5 (LIP5) and report genetic interactions between SPI and SKD1 and LIP5. We further show that the endosomal transport route of soluble proteins to the lytic vacuole is disturbed in spi lip5 double mutants but not in the single mutants. These vacuolar transport defects were suppressed by additional expression of SKD1. Our results indicate that the BEACH domain protein SPI has in addition to a role in P-bodies a function in endosomal transport routes.

The LYST protein contains three conserved motifs at the C-terminus: a Pleckstrin-Homology Domain (PH-Domain), the BEACH domain and a WD40 repeat motif. WD40 repeats and PH-Domains are known to mediate heterotypic protein interactions and protein recruitments to membranes via phospholipid binding, respectively (Neer et al., 1994;Lemmon, 2007). The molecular function of BEACH domains is not known. BDCPs, exhibiting a protein domain organization like LYST, are present in all eukaryotic species and mutants have been isolated in various organisms. Some of the most prominent examples are Neurobeachin (Nbea) deficient mice, characterized by impaired retrieval and post-Golgi trafficking of neuronal neurotransmitter receptors (Wang et al., 2000;del Pino et al., 2011); Drosophila Blue Cheese (bchs) mutants in which Ras related in brain protein 11 (RAB11)-dependent vesicle trafficking events at the Trans-Golgi Network (TGN), Golgi-derived vesicles and recycling endosomes are disturbed (Khodosh et al., 2006); C. elegans Suppressor/Enhancer of Lin-12 Protein 2 (SEL2) mutants that exhibit protein sorting defects between the cell surface and lysosomes (de Souza et al., 2007); Dictyostelium deficient for LvsA or LvsB (Large Volume Sphere A and B), showing hyper-accumulations of the vacuolar H + -ATPase in early endo-and phagocytotic compartments (Gerald et al., 2002;Wu et al., 2004) and enhanced lysosomal enzyme secretion under starvation (Harris et al., 2002); and Arabidopsis thaliana bchD (BEACH domain protein D, also termed green fluorescent seed 12) mutants, in which protein sorting to the plant specific protein storage vacuoles (PSVs) in seeds, but not in vacuolated vegetative tissues, is compromised (Teh et al., 2015). Because of the commonalities between these phenotypes, BDCPs are considered to be involved in the regulation of endosomal sorting processes (Cullinane et al., 2013).
In plants, the SPIRRIG gene in Arabidopsis thaliana is the beststudied BDCP representative [SPI, also termed BEACH domain protein A1 (BchA1) (Teh et al., 2015)]. SPI deficient plants share all morphological phenotypes observed in Arabidopsis mutants with defects in the ARP2/3-(actin related proteins 2 and 3) and SCAR/WAVE-(suppressor of cAMP receptor from Dictyostelium/Wiskott Aldrich syndrome protein family verprolin-homologous protein) complex mediated actin filament polymerization and branching (Hulskamp et al., 1994;Szymanski et al., 1999;Saedler et al., 2009). These include twisted and wavy trichomes, reduced length of root hairs, cell attachment defects and less complex epidermal pavement cells (Saedler et al., 2009). In contrast to all other mutants of this class, spi mutant cells do not show any defect in actin cytoskeleton organization (Schwab et al., 2003). In addition, SPI is involved in salt stress response (Steffens et al., 2015). In this context the analysis of its molecular function revealed a role in posttranscriptional stabilization of mRNAs (Steffens et al., 2015). This raises the question, whether the BDCP SPI gene has a different molecular function as reported for other BDCPs or whether SPI has a dual role in post-transcriptional regulation of mRNA and endomembrane dynamics. A possible role of the Arabidopsis SPI protein in endomembrane dynamics was initially suggested by the finding that root hairs show fragmented vacuoles (Saedler et al., 2009). Plant vacuoles are thought to originate from the endoplasmic reticulum (ER) as part of the secretory pathway (Matile, 1968;Mesquita, 1969;Viotti et al., 2013;Zhang et al., 2014). As a consequence, vacuoles receive cargo molecules from both, the anterograde transport route from the ER and the retrograde trafficking pathway from the plasma membrane (Saint-Jore-Dupas et al., 2004;Scheuring et al., 2011). The latter pathway involves the evolutionarily highly conserved ESCRT (endosomal sorting complex required for transport) machinery that is essential for the recognition of ubiquitinated cargo molecules destined for the vacuolar/lysosomal decay at membranes of maturating endosomes (Hurley and Emr, 2006;Spitzer et al., 2009;Richardson et al., 2011;Scheuring et al., 2012;Gao et al., 2015). Following the deubiquitination of cargo molecules (Katsiarimpa et al., 2014), the AAA + -type ATPase SKD1 (Suppressor of K + -transport growth defect 1/also termed VPS4; Vacuolar Protein Sorting-associated protein 4) triggers the final steps of endosome maturation to multivesicular bodies (MVBs), the fission of intraluminal vesicles (ILVs) and dissociation of ESCRT components from the endosomal membrane (Babst et al., 1998;Sachse et al., 2004;Babst, 2005;Lata et al., 2008;Landsberg et al., 2009). The expression of dominant negative SKD1 causes the formation of abnormally large MVBs containing a reduced number of ILVs concomitant with a reduced vacuolar transport and secretion of intraluminal cargo (Raymond et al., 1992;Fujita et al., 2003;Haas et al., 2007;Shahriari et al., 2010b;Scheuring et al., 2012). A possible function of BDCPs in the ESCRT pathway is suggested by the finding that human LYST interacting protein 5 (LIP5) interacts with the LYST in yeast two-hybrid assays (Tchernev et al., 2002). LIP5 is a positive regulator of SKD1 in mammals, yeast, and plants (Fujita et al., 2004;Azmi et al., 2006;Haas et al., 2007). The expression of dominant negative SKD1 enhances the membrane association of LYST in cultured human cells (Fujita et al., 2004) suggesting that the interaction of LIP5 and LYST is functionally relevant.
In this study, we assessed the role of the BDCP SPI protein in the ESCRT regulatory pathway. We demonstrate that SPI physically interacts with LIP5 and SKD1 from Arabidopsis thaliana. By combining molecular and biochemical approaches, we demonstrate that SPI is involved in the regulation of SKD1 and LIP5 and the route of endomembrane trafficking.

The Arabidopsis BDCP SPI Protein Interacts with LIP5 and SKD1
In a first step, we examined the protein interactions of the Arabidopsis BDCP SPI with LIP5 and SKD1. As large size of the SPI cDNA renders the molecular analysis extremely difficult, we used the evolutionarily conserved C-terminal part containing the PH-BEACH-WD40 domains (called SPI-PBW hereafter, Supplementary Figure S1A) for protein-interaction assays. Yeast two-hybrid assays revealed interactions between SPI-PBW and both, LIP5 and SKD1 ( Figure 1A). The interaction between SPI-PBW and SKD1 was confirmed by co-precipitations (co-Ps) of bacterially expressed Glutathione-S-Transferase (GST)/His 6fusions of SPI-PBW and His 6 -fusions of SKD1 ( Figure 1B). His 6 -SKD1 did not bind to GST labeled resins, confirming the specificity of the SPI-PBW/SKD1 interaction. As the bacterial expression of His 6 -LIP5 failed, we confirmed the SPI/LIP5 interaction in co-immunoprecipitation (co-IP) experiments with proteins expressed in Nicotiana benthamiana (N. benthamiana) leaf cells. Here, the expression of SPI-PBW was very weak and just above the detection level. We therefore used a slightly smaller SPI fragment lacking the WD40-repeats (termed SPI-PB; Supplementary Figure S1A). SPI-PB was C-terminally fused to a HA-tag (SPI-PB-HA). LIP5 was used as N-terminal fusion with three FLAG tags (3xFLAG-LIP5). These experiments revealed a clear co-IP suggesting an interaction between SPI-PB and LIP5. 3xFLAG-LIP5 did not bind to unlabeled beads, indicating that the SPI-PB/LIP5 interaction is specific ( Figure 1C). The specificity of the immunoprecipitation was confirmed, by showing that SPI-PB-HA and 3xFLAG-LIP5 did not bind to α-ProteinA beads (Supplementary Figure S1B).
The intracellular localization of the interaction between SPI-PBW, LIP5, and SKD1 was assessed by Bimolecular fluorescence complementation (BiFC) assays in transfected N. benthamiana leaf epidermis cells. We found BiFC interactions of SPI-PBW with LIP5 and SKD1 in cytoplasmic dots. The Manders co-efficient of the SPI-PBW/SKD1 BiFC signal was 54.96% with ARA6-mCHERRY and 45.76% with mCHERRY-ARA7 (Figures 3A,B). 49.3% of the SPI-PBW/LIP5 BiFC signal co-localized with ARA6-mCHERRY and 44.6% with mCHERRY-ARA7 (Figures 3C,D). As SPI protein fusions are ubiquitously distributed in the cytoplasm we used also a cytoplasmically localized negative control. We found no interactions of SPI-PBW and LIP5 with AtMYC1 (MYC related protein1) (Pesch et al., 2013) (Supplementary Figure S2F). The functional integrity of AtMYC1 N-terminally fused to YFP-fragments was confirmed by demonstrating its BiFC interaction with TTG1 (TRANSPARENT TESTA GLABRA1) (Zimmermann et al., 2004;  Bottom: Input of purified His 6 -SKD1 (∼50 kDa) and co-precipitations of His 6 -SKD1/GST-SPI-PBW-His 6 detected by α-His 6 antibody staining. No co-precipitation was observed between GST and His 6 -SKD1. Expected protein sizes are indicated by arrowheads. R, raw extract; TP, throughput; WF, last wash fraction; B, beads fraction. (C) Co-immunoprecipitation of SPI-PB-HA and 3xFLAG-LIP5 from lysates of transfected N. benthamiana leaves. Top: Immunoprecipitation of SPI-PB-HA (∼75 kDa) was detected by α-HA antibody staining on a Western blot. Bottom: 3xFLAG-tagged LIP5 (∼50 kDa) was detected in R, TP and the B of SPI-PB-HA co-transfected leaves by α-FLAG antibody staining on Western blots. 3xFLAG-tagged LIP5 alone was not precipitated with α-HA-beads. Expected protein sizes are indicated by arrowheads. ProteinA beads were included as a negative control for all proteins tested (Supplementary Figure S1B). Figure S2G). TTG1-YFP C was included as negative control for YFP N -SKD1 (Supplementary Figure S2F). In summary, our data suggest that the interaction between SPI-PBW and LIP5 and SKD1 can occur at endosomes. Because SPI contains a PH domain known to mediate binding to phospholipids (Ferguson et al., 1995;Lemmon, 2007) we assessed the binding-ability of bacterially expressed GST-SPI-PBW-His 6 to a selection of membrane-anchored lipids in protein-lipid overlay assays (Supplementary Figures S3A-C). GST was included as a negative control and the PH domain from PLC-d1 as a positive control (Supplementary Figure  S3B). We found no binding of GST-SPI-PBW-His 6 to any of the phospholipids tested, including the endosomal enriched Phosphatidylinositol-3-phosphate (PI3P) (Voigt et al., 2005;Vermeer et al., 2006) Figure S3C). These data suggest that the association of SPI to endosomes is not mediated by phosphoinositides.

Genetic Analysis of SPI, LIP5, and SKD1 in Arabidopsis
To test the functional relevance of the SPI/LIP5/SKD1 interactions, we performed a genetic analysis. We focused on the seed coat mucilage phenotype because plants expressing dominant negative SKD1 produce seeds lacking seed mucilage completely (Shahriari et al., 2010a). Seed coat mucilage is enriched in polymeric sugars that are secreted from the outer seed coat cells. Its presence can be monitored by Ruthenium Red staining of whole seeds (Western et al., 2000). We found no difference between spi and wild-type seeds (Figures 4A,B). The mucilage layer of lip5 mutant seeds was still formed, but its area was significantly smaller than in wild-type seeds. This observation is consistent with the finding that LIP5 acts as an enhancer of SKD1 ATPase activity (Haas et al., 2007). The spi lip5 double mutant seeds exhibited a strong reduction of the mucilage layer that was significantly different to wild type as well as lip5 single mutant seeds (Figures 4A,B). Apart from this mucilage phenotype we did not note any differences to spi mutants in spi lip5 double mutant plants with respect to growth and morphological features. These data indicate that the secretion of seed coat mucilage requires the SKD1/SPI/LIP5 pathway and that SPI and LIP5 act synergistically.

Synergistic Function of SPI and LIP5 in Vacuolar Transport
To test whether SPI is functionally involved in the endosomalvacuolar transport route, we examined the intracellular localization of two soluble vacuolar enzymes CPY and AALP in spi mutants (Rojo et al., 2003;Shahriari et al., 2010b). Both, CPY-CFP and AALP-mCHERRY were found in the vacuole in more than 90% of the cells. Their distribution was unaffected in both spi and lip5 single mutants. However, in spi lip5 double mutants we found a severe reduction of cells displaying vacuolar CPY and AALP (Figures 5A-D). The vacuolar sorting defect was even more obvious after controlled induction of CPY-mCHERRY expression using an EtOH-inducible vector system (Roslan et al., 2001) (Figures 5E,F and Supplementary Figure S4).
We reasoned that the vacuolar sorting defect might result in an increased secretion similar as observed in plants expressing dominant negative SKD1 (Shahriari et al., 2010b). We therefore performed a secretion assay using Col-0 and spi lip5 leaf mesophyll protoplasts. We compared the amounts of CPY present in the fraction of protoplasts with those secreted to the surrounding medium after 1 h of incubation ( Figure 6). As compared to wild type, we found a stronger signal of CPY in the medium of spi lip5 protoplasts in two independent experiments (one shown in Figure 6). We excluded that the CPY signals from the medium samples were caused by impurities from damaged protoplast by using α-cytoplasmic Fructose-1.6-Bisphosphatase (cF1.6BPase) as a control (Figure 6).

The Absence of SPI and LIP5 Can Be Compensated by Overexpression of SKD1
The finding that the spi lip5 double mutants share many phenotypes with the dominant negative SKD1 mutants (mucilage phenotype, vacuolar transport defects, aberrant protein secretion) suggests that they may act together in promoting SKD1 activity. In this case, one would expect that the overexpression of SKD1 can compensate the reduced activation of SKD1 in spi lip5 double mutants. We therefore tested whether overexpression of SKD1 can rescue the spi lip5 vacuolar transport phenotype. Toward this end, we assessed the localization of AALP-mCHERRY and CPY-CFP in spi lip5 double mutants co-expressing SKD1. The vacuolar transport of AALP was restored in 100% (Figures 7A,B) and of CPY-CFP in 80% of cells (Figures 7C,D). Co-expression of the late endosomal marker ARA7 did not restore the vacuolar transport of CPY-CFP (Figures 7E,F), highlighting the SKD1-specificity of the rescue. These data suggest that LIP5 and SPI regulate endosomal-vacuolar transport by acting as positive regulators of SKD1.

DISCUSSION
Initially spi has been described as a member of the class of Arabidopsis distorted mutants. In contrast to all other mutants in this class, spi displays no actin phenotype suggesting that it may regulate cell morphogenesis in a cytoskeleton-independent manner. As SPI encodes a BDCP homolog it was initially postulated that SPI may function in endomembrane dynamics. The finding, however, that SPI has a molecular function in the post-transcriptional regulation of mRNA stability raised the question, whether SPI has an additional role in membrane dynamics. Here, we provide genetic and molecular data that functionally link SPI and SKD1 and LIP5. A connection between the distorted phenotype and endosomal transport has not been revealed so far. Neither the lip5 mutant nor expression of dominant negative SKD1 has been reported to trigger a distorted phenotype. Further studies might aim to investigate if there is any direct connection between these fields of research.

BDCPs -How Do They Control Membrane Dynamics?
The common phenotypes of BDCP mutants points to their function in the control of membrane fusion and fission events at different steps along the endomembrane system (Cullinane et al.,

2013). Recently, a working model was developed that proposes
BDCPs to act as scaffold proteins that regulate membrane dynamics in a binding partner-dependent manner (Cullinane et al., 2013). However, only few binding partners of BDCPs have been identified (Adam-Klages et al., 1996;Segui et al., 2001;Tchernev et al., 2002;Filimonenko et al., 2010;Hocking et al., 2010) and their contribution to the pathophysiology observed in BDCP mutants was not characterized in detail so far. A possible link between BDCPs and the LIP5/SKD1 complex had been suggested by the findings that LIP5 interacts with LYST in yeast two-hybrid assays (Tchernev et al., 2002) and that over-expression of a dominant negative SKD1 protein leads to an association of LYST to membranes (Fujita et al., 2004). Our finding that SPI directly interacts not only with LIP5 but also with SKD1 strongly suggest that a functional link between BDCPs and LIP5/SKD1 is evolutionarily conserved.

SPI Localization to MVBs by Binding to SKD1 and/or LIP5
As shown for human LYST, the SPI protein is evenly distributed in the cytoplasm (Perou et al., 1997;Fujita et al., 2004). The previous finding that LYST relocates to membranes only upon co-expression of dominant negative SKD1 (E235Q) suggests that it is either recruited under these conditions, or that it is present at endosomal membranes at low levels and becomes detectable because of the formation of large endosomal class E compartments. Consistent with both scenarios, we observe SPI interactions with LIP5 and SKD1 at endosomes in BiFC experiments. As BiFC tends to stabilize protein-protein interactions (Kerppola, 2008) a recruitment of SPI to endosomes by its binding partners is conceivable.
Membrane association of other BDCPs has been shown to depend on their binding to phospholipids. The human BDCP factor associated with neutral sphingomyelinase activation (FAN) binds directly to plasma membrane associated phosphoinositides (Haubert et al., 2007). Other BDCPs such as the mammalian homologs NBEA and lipopolysaccharide-responsive and beigelike anchor protein (LRBA) do not bind to phospholipids. Crystal structure analysis of LRBA revealed an uncommon PH domain, lacking a cluster of basic residues essential to form the classical phospholipid binding fold (Gebauer et al., 2004;Haubert et al., 2007;Lemmon, 2007). SPI protein does not appear to bind to phosphoinositides suggesting that the recruitment of SPI to membranes may be mediated by binding to other proteins.

SPI and LIP5 Are Positive Regulators of the Protein Transport between MVBs and Plant Lytic Vacuoles
The ATPase activity of SKD1 is essential for the disassembly of the ESCRT machinery from membranes, and thereby the formation of ILVs in late endosomal compartments (Babst et al., 1998;Azmi et al., 2006;Cai et al., 2014). The depletion of SKD1 and expression of its dominant negative SKD1 versions cause the formation of aberrantly enlarged pre-vacuolar structures (class E compartments) in various species (Fujita et al., 2003;Shahriari et al., 2010b). In plants, the expression of dominant negative SKD1 causes vacuole fragmentation suggesting that the membrane transport between MVBs and the vacuole is impaired (Shahriari et al., 2010b). Although spi mutants exhibit fragmented vacuoles (Saedler et al., 2009), we found no obvious defects in endosomal trafficking. Neither the internalization of the styryl dye FM4-64 (Saedler et al., 2009) nor the vacuolar transport of soluble cargo proteins (this study) were affected in spi mutants. Thus, the cellular spi mutant phenotype differs to other class E (Fujita et al., 2003;Haas et al., 2007) or BDCP mutants (Faigle et al., 1998;Cornillon et al., 2002). Similar results were reported for lip5 mutant plants (Cai et al., 2014). Although LIP5 promotes the enzymatic activity of SKD1, lip5 mutants exhibit no global mutant phenotypes (Haas et al., 2007). However, a role of LIP5 in MVB biogenesis was reported in the context of stress responses (Wang et al., , 2015. In addition, trafficking defects of PIN proteins in roots were correlated with impaired gravitropism (Buono et al., 2016). Together these data suggest that LIP5 is a regulator of SKD1 but not an absolute requirement for SKD1 activity. Our genetic studies, showing a redundant function of LIP5 and SPI suggest that the two proteins act together in the regulation of SKD1. As the additional expression of SKD1 can overcome the requirement of LIP5 and SPI it is likely that SKD1 acts genetically downstream of LIP5 and SPI. It is conceivable that SPI acts together with LIP5 as a positive regulator of SKD1. This conclusion appears to be in conflict with data  Figure S4). Data denote the average of three biological replicates (n = 15 cells each). Error bars in A,C,E represent SDs. Asterisks represent statistically significant changes in comparison to Col-0 (two-tailed Student's t-test; * * p < 0.01, * * * p < 0.001). (F) Representative maximum projections of alcR pro :CPY-mCHERRY (column I) before (Upper) and after induction of gene expression (Lower) in transfected leaf epidermis cells of Col-0 co-expressing free YFP (column II) as transformation control. Column III presents the overlay picture of columns I and II. Scale bars: 30 µm.
Frontiers in Plant Science | www.frontiersin.org FIGURE 6 | CPY is aberrantly secreted in protoplasts from spi lip5 double mutants. Detection of CPY in total protein samples from (A) Col-0 and (B) spi lip5-derived leaf mesophyll protoplasts before their incubation in secretion medium (raw), the secretion medium (sec) after 60 min of incubation and the corresponding intact protoplasts fraction (pro), on α-CPY (Rojo et al., 2003) stained Western blots. The positions of CPY precursor and intermediate forms are indicated by arrowheads. cF1.6BPase was detected by α-cF1.6BPase antibody (Agrisera antibodies) staining and used as control. Please note that the contrasts of α-CPY and α-cF1.6BPase stained Western blots have been adjusted to easily visualize any protein present in the secretory fraction. Non-manipulated α-cF1.6BPase stained Western blots are presented in the lowest row (indicated by * ).The same pattern of CPY and cF1.6BPase was observed in a second independent experiment. obtained in human cell culture systems. Here, overexpression of SKD1 was not sufficient to restore the giant lysosome phenotype in LYST deficient fibroblasts suggesting that LYST acts downstream of SKD1-mediated membrane transport (Fujita et al., 2004). This suggests that the molecular function we describe for the BDCP SPI is mechanistically different to that of BDCPs in humans. An alternative explanation would be that a rescue of protein/membrane trafficking by additional expression of SKD1 is independent of a rescue of endosomal sizes.

Plant Material, Protoplast Isolation, Growth Conditions, and Mucilage Staining
Arabidopsis thaliana spi-2 (GK_205G08), spi-3 (SALK_065311), spi-4 (GK_420D09), lip5-1 (SAIL_854F08) and lip5-2 (GK_351B10) have been previously described (Haas et al., 2007;Wang et al., 2014); and were obtained from the National Arabidopsis Stock Center. Positions of T-DNA insertions were confirmed by sequencing the flanking genomic regions. Seeds were grown on soil under long day conditions (22 • C; 110 µmol m −2 s −1 ). Ruthenium Red Staining of seed coat mucilage was performed in H 2 O (+0.1% Tween20) as described (Western et al., 2000). Isolation of protoplasts from Arabidopsis leaves was performed according to the Tape-Arabidopsis Sandwich method (Wu et al., 2009). Fifty microliters protoplasts were harvested and washed with 150 µl of W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KCl, 5 mM glucose, and 2 mM MES, pH 5.7) in a 1.5 ml Eppendorf tube at 100g for 5 min. For determination of protein secretion 50 µl of washed protoplasts were incubated in 50 µl of W5 solution for 60 min at room temperature in the dark. After centrifugation (5 min, 100g), supernatant (secretion fraction) and intact protoplasts were divided and boiled in protein loading buffer for further analysis.
were analyzed by immunoblotting (Steffens et al., 2014). HA-and 3xFLAG fusions were (co)-immunoprecipitated using antibody conjugated µ-columns according to the manufacturer's instructions (Miltenyi; µMACS TM ) from total protein lysates derived from infiltrated N. benthamiana leaves. Bound proteins were eluted and analyzed by immunoblotting, using either a monoclonal horseradish peroxidase (HRP) coupled with anti-HA antibody from rat (Roche; 1:1000 dilution) or a primary monoclonal anti-FLAG antibody from mouse (Sigma-Aldrich; 1:2000 dilution) and a secondary anti-mouse antibody from goat coupled to HRP (Sigma-Aldrich; 1:10,000).

Transient Expression in Plants, Induction Conditions and Confocal Laser Scanning Microscopy (CLSM)
Nicotiana benthamiana leaves were transformed as described (Yang et al., 2000). Biolistic transformation of Arabidopsis leaves was performed as described  and analyzed after 12 to 16 h by Confocal Laser Scanning Microscopy (CLSM). CLSM was performed as described (Steffens et al., 2014). Expression of alcR pro -fusion proteins (pCAMPARI-vector) was induced by fumigation of transfected leaves with 2% ethanol (v/v) for 4 h (Roslan et al., 2001). Before induction of protein expression, transfected cells were identified by the presence of constantly co-expressed free YFP. To ensure comparisons between equally strong transfected cells, laser intensities and exposure times were fixed.

AUTHOR CONTRIBUTIONS
AS and MH designed this study. AS performed the research and analyzed the data. AS and MJ generated plasmids and established mutant lines. AS and MH wrote the paper. MH directed the project.