RABA Members Act in Distinct Steps of Subcellular Trafficking of the FLAGELLIN SENSING2 Receptor

This article shows endocytosed FLS2, a flagellin receptor, is initially transported to a hybrid compartment between the trans-Golgi network and multivesicular endosome. It also demonstrates that subgroups of RABA, which is a uniquely expanded RAB group in plants, have distinct roles in the endocytic and exocytic transport of FLS2, indicating functional differentiation among RABA subgroups.

Abstract

Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuing signal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. Subcellular localization of Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. However, our understanding about the mechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescent protein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PM was transported to a compartment with properties intermediate between the trans-Golgi network (TGN) and the multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the trafficking pathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, we demonstrated that transport of de novo–synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our results demonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABA members in endo- and exocytosis.

INTRODUCTION

Eukaryotic cells recognize their environment mainly through proteins on the plasma membrane (PM), including receptors and sensors, which evoke intracellular signal transduction to respond to environmental cues. In animal cells, endocytosis plays critical roles in regulation of the amount of PM proteins responding to extracellular stimuli, transduction of signals from endosomes, and downregulation of signal transduction (Sorkin and Von Zastrow, 2002; von Zastrow and Sorkin, 2007; Platta and Stenmark, 2011). Also in plants, there are several PM proteins whose localization is known to be regulated by endocytosis upon extracellular stimuli. For example, REQUIRES HIGH BORON1 (BOR1), a boron efflux carrier on the PM, is endocytosed in response to changes in environmental boron concentration (Takano et al., 2002, 2005). The Leucine-rich repeat receptor Ser/Thr kinase FLAGELLIN SENSING2 (FLS2) is another example of localization regulated by endocytosis. FLS2 is the receptor for bacterial flagellin, and the fls2 mutant is highly susceptible to infection by pathogenic bacteria (Zipfel et al., 2004). FLS2 recognizes 22 amino acids of a conserved domain in the N terminus of flagellin (flg22 peptide; Felix et al., 1999), and FLS2 bound with flg22 is rapidly internalized from the PM into the cytoplasm and degraded probably in vacuoles (Robatzek et al., 2006). Intriguingly, plants expressing FLS2^T867V, which has a mutation in a putative phosphorylation site, exhibit susceptibility to pathogenic Pseudomonas syringae, and flg22-triggered internalization of this mutant protein rarely occurs, implying a possible link between endocytosis of FLS2 and flagellin signaling (Robatzek et al., 2006). In the case of another Leucine-rich repeat receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1), its endocytosis is not induced by the ligand brassinosteroid, but brassinosteroid signaling is regulated by endocytosis of BRI1 (Geldner et al., 2007; Irani et al., 2012).

Thus, endocytosis and endocytic organelles play fundamental roles in a variety of plant functions, including responses to environmental cues and hormone signaling. However, our knowledge of the molecular mechanisms of endocytosis in plant cells is still limited. It is apparently insufficient to simply extend the knowledge obtained from yeast and animal systems to the plant system because the plant endocytic pathway seems to operate differently in plants. The role of the trans-Golgi network (TGN) in endocytosis is a notable example. The TGN acts as the sorting platform for secreted and vacuolar/lysosomal proteins in eukaryotic cells. In addition to this function, the TGN in plant cells also acts as the early endosome; a lipophilic tracer of endocytosis, FM4-64, stains the TGN bearing the TGN markers Vacuolar H1-ATPase subunit a1 (VHA-a1), SECRETORY CARRIER MEMBRANE PROTEIN1 (SCAMP1), RABA2a, or RABA3 before this dye reaches the multivesicular endosomes (MVEs) marked by the conventional RAB5 ortholog ARA7/RABF2b (Dettmer et al., 2006; Lam et al., 2007; Chow et al., 2008). Immunoelectron microscopy has also demonstrated that endocytosed BRI1 and BOR1 pass through the TGN and MVE (Viotti et al., 2010).

Moreover, the molecular machineries of endocytosis also seem to differ between plants and animals, as is evident when we compare organization of RAB GTPases between animals and plants. RAB GTPase is an evolutionarily conserved key player in membrane trafficking, which generally regulates the docking step of transport carriers to target organelles through the conformational change between GTP-bound active and GDP-bound inactive states. RAB GTPases are widely conserved in all eukaryotic lineages but seem to have diversified uniquely in each lineage. Molecular phylogenetic analyses have suggested that most land plant RAB GTPases are classified into eight families, each of which exhibits high similarity to animal RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11, and RAB18 (Rutherford and Moore, 2002; Vernoud et al., 2003). Plants lack clear homologs of the well-characterized animal endocytic RABs, RAB4 and RAB9, which also suggests that the regulatory mechanism of the plant endocytic pathway differs from the animal system. By contrast, the RAB5 and RAB11 families, which act in the endocytic pathway in animal cells, have diversified in a unique way in plants. The plant RAB5 family consists of two distinct subgroups: the plant-unique ARA6 group and the conventional RAB5 group. How the functions of these subgroups differ has been unclear, but recent studies indicate that ARA6/RABF1 is involved in trafficking from endosomes to the PM in Arabidopsis thaliana, whereas conventional RAB5/RABF2 acts in the vacuolar trafficking pathway (Sohn et al., 2003; Bolte et al., 2004; Kotzer et al., 2004; Ebine et al., 2011). Another notable characteristic of the plant RAB GTPase is the variety of RAB11 homologs (referred to as RABA in Arabidopsis). A total of 26 members of the 57 RAB GTPases in Arabidopsis are classified into the RABA group, which is further divided into six subgroups (RABA1 to RABA6). RAB11 regulates the endocytic pathway in animal cells; animal RAB11 resides on recycling endosomes and regulates recycling of endocytosed proteins to the PM (Ullrich et al., 1996; Ren et al., 1998). By contrast, yeast homologs of RAB11, Ypt3 members, are proposed to be required for multiple steps of the exocytic pathway, including intra-Golgi transport and transport vesicle formation at the TGN (Benli et al., 1996; Jedd et al., 1997; Cheng et al., 2002). Some members of the plant RABA/RAB11 group also take part in several different exocytic events unique to plants. Substantial roles in tip growth of pollen tubes and root hairs have been demonstrated in tobacco (Nicotiana tabacum) and Arabidopsis (Preuss et al., 2004; de Graaf et al., 2005; Szumlanski and Nielsen, 2009), and expression of dominant-negative RABA2a inhibits cytokinesis of root tip cells in Arabidopsis (Chow et al., 2008). RABA1b also has been reported to regulate trafficking between the TGN and PM (Asaoka et al., 2012; Feraru et al., 2012). However, it remains unclear whether each RABA group regulates a distinctive trafficking pathway and if any of the subgroups are involved in endocytic trafficking.

In this study, we first visualized and characterized the endocytic route of FLS2, whose internalization was induced by flg22 treatment, in leaf epidermal cells of Nicotiana benthamiana. By comparing localization of internalized FLS2 with TGN and MVE markers, we identified a transient compartment with properties that were a hybrid of the TGN and MVE. This compartment appears to be an intermediate mediating endocytosis of FLS2. We then explored the functions of RABA subgroups in the intracellular transport of FLS2 to find distinct functions of different subgroups of RABA. Our results indicate discrete functional differentiation among RABA subgroups in endocytic and exocytic pathways.

RESULTS

flg22-Dependent Internalization of FLS2 in N. benthamiana Leaf Epidermal Cells

In this study, we used the transient expression system in N. benthamiana leaf epidermal cells by infiltration of Agrobacterium tumefaciens, which has been successfully used to observe intracellular trafficking of fluorescent protein–tagged proteins and simultaneous expression of multiple proteins (Goodin et al., 2007a, 2007b; Tardif et al., 2007; Martin et al., 2009; Wang et al., 2011; Denecke et al., 2012). First, we confirmed localization of green fluorescent protein (GFP)–tagged FLS2 on the PM in untreated N. benthamiana cells (Figure 1A), as reported in Arabidopsis leaf epidermal cells (Robatzek et al., 2006). Then we examined whether flg22-dependent internalization of FLS2-GFP is also observed in this system. Upon treatment with flg22, dot-like structures with FLS2-GFP appeared in the cytoplasm 90 and 120 min after flg22 application (Figures 1B and 1C). A lipophilic dye, FM4-64, was also internalized in a similar time frame in N. benthamiana leaf epidermal cells (see Supplemental Figure 1 online). Treatment with flg22^A.tum, the inactive peptide derived from Agrobacterium flagellin (Felix et al., 1999), did not result in accumulation of FLS2-GFP at the cytoplasmic organelles (Figure 1D), indicating that flg22 specifically triggers endocytosis of FLS2 in this system. To examine earlier events of endocytosis of FLS2 in this system, we then observed the behavior of FLS2-GFP using a total internal reflection fluorescence microscopy–related technique called variable-angle epifluorescence microscopy or variable incidence angle fluorescence microscopy, which enables selective visualization of sample surface regions (Fujimoto et al., 2007; Konopka and Bednarek, 2008). FLS2 signals were concentrated on the PM 40 min after applying flg22 (Figures 1E to 1G), and clear punctate signals, probably reflecting concentrated or internalized FLS2, were observed on or near the PM at 60 min (Figure 1H).

Figure 1.

Endocytosis of FLS2-GFP in Leaf Epidermal Cells of N. benthamiana.

(A) to (D) Max-intensity projection images are presented, each of which was reconstructed with a series of confocal Z-stack images taken at 0.5-μm intervals.

(A) FLS2-GFP localizes on the PM without any treatment. Bar = 10 μm.

(B) FLS2-GFP is internalized into the cytoplasmic punctate compartments within 90 min of treatment with flg22.

(C) Punctate compartments observed at 120 min after flg22 treatment.

(D) Endocytosis of FLS2-GFP is not induced by flg22^A.tum even after 120 min.

(E) to (H) Variable incidence angle fluorescence microscopy of FLS2 in close proximity to the PM at 0 (E), 20 (F), 40 (G), and 60 min (H) after flg22 treatment. Arrowheads indicate focal accumulation of FLS2 on the PM. Bar = 5 μm.

FLS2 Is Transported to Unknown Compartments That Show Hybrid Characteristics between the TGN and MVE

To investigate which organelles are involved in endocytosis of FLS2, we coexpressed FLS2-GFP with monomeric red fluorescent protein (mRFP)-SYP61 or VHA-a1-mRFP, markers of the TGN, or with TagRFP-ARA7 or TagRFP-VAMP727, markers for the MVE, because these organelles are known to act as endocytic compartments in plant cells (Ueda et al., 2001; Uemura et al., 2004; Takano et al., 2005; Dettmer et al., 2006; Lam et al., 2007; Viotti et al., 2010). From 90 to 120 min after flg22 treatment, FLS2-GFP was observed overlapping or associated with mRFP-SYP61 (Figures 2A and 2B). By contrast, FLS2-GFP was not observed near the SYP61 compartments after 210 min (Figure 2C). For quantitative analysis of this observation, we extracted the dot-like signals of FLS2-GFP and mRFP-SYP61 and measured the distance from the center of a FLS2-GFP–positive dot to that of the nearest mRFP-SYP61 signal using a framework run on Metamorph software, which we previously developed (Ito et al., 2012). The results were categorized into three groups by distances: (1) colocalized (≤0.24 μm; below the resolution limit of the objective lens used in this study); (2) associated (≤1 μm); and (3) independent (>1 μm). We compared results between two groups of samples; one was observed from 80 to 140 min after flg22 treatment (an early endocytic stage), and the other was observed from 140 to 200 min after flg22 application (a late endocytic stage). In the early stage, ∼27.6% ± 6.4% and 39.6% ± 6.5% of FLS2-positive compartments were colocalized and associated with SYP61-positive dots, respectively (Figure 2R). In the late endocytic stage, ratios of colocalized and associated groups were significantly decreased to 5.6% ± 3.9% and 19.3% ± 6.6%, respectively (n = 3 experiments, P < 0.01, Student’s t test) (Figure 2R). These results suggested that FLS2 that is internalized upon flg22 application transiently localizes at SYP61-positive compartments in the early stage. Intriguingly, an experiment using another TGN marker, VHA-a1, yielded a different result. When VHA-a1 and FLS2 were coexpressed and treated with flg22, we did not observe colocalization of these proteins at any time points after the flg22 treatment (Figures 2D to 2F). The quantitative analysis also supported this observation; only 1.0% ± 0.9% and 1.0% ± 1.7% of FLS2-positive compartments were categorized as colocalized in the early and late stages, respectively (Figure 2S). For further clarification of this result, we directly compared subcellular localization of cyan fluorescent protein (CFP)-SYP61 and VHA-a1-mRFP in cells expressing FLS2-GFP and in which flg22 treatment induced endocytosis. SYP61- and VHA-a1–positive dots were mostly overlapped; however, we found that the SYP61-positive compartments carrying FLS2-GFP did not harbor VHA-a1-mRFP (Figures 2M to 2P). We also confirmed that CFP-SYP61 and mRFP-SYP61 exhibited the same localization pattern (Figure 2Q), and mRFP-SYP61 and VHA-a1-mRFP exhibited similar distribution patterns in relation to the Golgi apparatus visualized with ST-Venus (Boevink et al., 1998) (see Supplemental Figure 2 online). These results clearly indicated that the compartment bearing SYP61 and FLS2 observed in this study has a characteristic distinct from the TGN in cells where endocytosis of FLS2 is not induced.

Figure 2.

FLS2 Is Transported via SYP61- and ARA7-Positive Endosomal Compartments.

(A) to (C) Time-course observation of FLS2-GFP endocytosis in mRFP-SYP61–expressing cells. Internalized FLS2-GFP colocalizes with mRFP-SYP61 at 90 min after flg22 treatment (A). After 120 min, FLS2-GFP is frequently observed associated with SYP61-positive compartments (B), but such colocalization and association are not observed after 210 min (C). Arrowheads indicate colocalized (distance ≤0.24 μm; below the resolution limit of the objective lens used in this study) compartments, and arrows indicate associated (0.24 < distance ≤1 μm) compartments. Bars = 5 μm.

(D) to (F) VHA-a1-mRFP does not overlap with FLS2-GFP. Bars = 5 μm.

(G) to (I) FLS2-GFP colocalizes with TagRFP-ARA7 at all time points. Bars = 5 μm.

(J) to (L) FLS2-GFP colocalizes with TagRFP-VAMP727 at all time points. Bars = 5 μm.

(M) to (P) Cells expressing FLS2-GFP, VHA-a1-mRFP, and CFP-SYP61 observed after 130 min of flg22 treatments. Bars = 2 μm.

(Q) CFP- and mRFP-tagged SYP61 completely overlapped when expressed in the same cell. Bar = 5 μm.

(R) to (U) Stacked bar graphs representing results of quantification of colocalization between FLS2-GFP and mRFP-SYP61 (R), FLS2-GFP and VHA-a1-mRFP (S), FLS2-GFP and TagRFP-ARA7 (T), or FLS2-GFP and TagRFP-VAMP727 (U). Data were collected from three independent experiments; 189 and 195 (FLS2-GFP and mRFP-SYP61), 209 and 186 (FLS2-GFP and VHA-a1-mRFP), 259 and 278 (FLS2-GFP and TagRFP-ARA7), and 178 and 158 (FLS2-GFP and TagRFP-VAMP727) FLS2-positive dots were observed in early (80 to 140) and late (140 to 200) stages, respectively. Error bars indicate the sd values. Individual channel images are shown in Supplemental Figure 11A online. Average numbers of cells examined and numbers of puncta analyzed in each experiment are shown in Supplemental Table 1 online.

We next examined whether TagRFP-tagged ARA7 and VAMP727, an MVE-localizing RAB GTPase and R-SNARE, respectively, were colocalized with FLS2. These endosomal proteins were localized on FLS2-positive puncta observed in both early and late stages (Figures 2G to 2L, 2T, and 2U).

To investigate the relationship between SYP61- and ARA7-positive compartments in flg22-induced endocytosis of FLS2, we then expressed FLS2-GFP, mRFP-SYP61, and TagRFP-ARA7 in the same cell and compared their subcellular localization after treatment with flg22. At the earlier stage (100 min after the treatment), both SYP61 and ARA7 were colocalized on the same FLS2-positive compartments (Figure 3A). However, after 140 min, most SYP61 localization did not overlap with FLS2 or ARA7 but occurred instead at the membrane domains in close proximity to (or associated with) the FLS2- and ARA7-positive domains (Figure 3B). After 200 min, we did not see colocalization or association of SYP61 with FLS2, while FLS2 still exhibited good colocalization with ARA7 (Figure 3C). These results indicated the existence of an endosomal compartment involved in flg22-triggered endocytosis of FLS2 that seemed to harbor an intermediate property between the TGN and MVE. Of interest, the colocalization of SYP61 and ARA7 was observed only at compartments carrying endocytosed FLS2-GFP. In Arabidopsis plants grown under normal laboratory conditions, we noted no significant colocalization between the MVE and TGN proteins thus far (Ebine et al., 2008, 2011). Thus, such intermediate compartments might exist only when endocytosis is highly activated.

Figure 3.

Hybrid Nature of Endosomal Compartment Involved in Early FLS2 Endocytosis.

(A) to (C) Cells expressing FLS2-GFP, mRFP-SYP61, and TagRFP-ARA7 observed after 100 (A), 140 (B), and 200 min (C) of flg22 treatment. Bar = 2 μm.

(D) to (G) The hybrid compartment is observed only when endocytosis of FLS2-GFP is induced. Cells expressing mRFP-SYP61 and TagRFP-ARA7 with FLS2-GFP ([D] and [E]) or without FLS2-GFP ([F] and [G]) were observed at 0 or 100 min after flg22 treatment. Arrowheads indicate compartments bearing both mRFP-SYP61 and TagRFP-ARA7. Individual channel images are shown in Supplemental Figure 11B online. Bars = 5 μm.

To verify this possibility, we compared localization of SYP61 and ARA7 after flg22 treatment between two samples: leaf epidermal cells expressing FLS2-GFP, mRFP-SYP61, and TagRFP-ARA7, and cells expressing mRFP-SYP61 and TagRFP-ARA7 but not FLS2-GFP. As observed above, 4.6% ± 2.0% of mRFP-SYP61–positive compartments bore TagRFP-ARA7 in FLS2-expressing cells at 100 min after application of flg22 (Figures 3D and 3E); this is significantly higher than the ratio (1.1% ± 0.5%) of mRFP-SYP61 compartments with TagRFP-ARA7 in cells not expressing FLS2 after treatment with flg22 (n = 3 experiments, P < 0.05, Student’s t test; Figures 3F and 3G, Table 1). These results suggested that the intermediate compartment identified in this study is a transient endosomal structure that is induced when endocytosis is highly activated. Under our experimental conditions, ∼20% of SYP61-positive compartments were classified as “associated” with ARA7-positive compartments, which indicated that the two compartments were not overlapped but in close proximity (<0.24 μm distance between two compartments ≤1 μm), regardless of combinations of coexpressed proteins and time points (Table 1).

Table 1. Quantification of Colocalization between SYP61 and ARA7.

Expression of FLS2	flg22 Treatment	Colocalized	Associated	Independent
+FLS2	No treat. (n = 335)	0.7% (±0.6%)	21.0% (±1.3%)	78.3% (±0.7%)
	Early stage (n = 539)	4.6% (±2.0%)	22.5% (±2.2%)	72.9% (±0.9%)
	Late stage (n = 470)	1.0% (±0.9%)	19.8% (±0.9%)	79.2% (±1.1%)
−FLS2	No treat. (n = 536)	0.0% (±0.0%)	22.0% (±2.9%)	78.0% (±2.9%)
	Early stage (n = 624)	1.1% (±0.5%)	21.6% (±1.8%)	77.3% (±1.3%)
	Late stage (n = 532)	1.2% (±1.0%)	20.0% (±5.3%)	78.9% (±5.5%)

SYP61-positive compartments were classified into three classes according to the criteria mentioned in the text in cells expressing GFP-FLS2 (+FLS2) and cells that were not transformed with GFP-FLS2 (−FLS2). Data were collected from three independent experiments.

Effects of Mutant RABA on Endocytosis of FLS2

As described above, we found that compartments with intermediate properties between the TGN and MVE are involved in ligand-induced endocytosis of FLS2. To identify molecular machineries that control the trafficking pathway around this compartment, we focused our interest on the RABA family because some RABA members have been reported to act in membrane trafficking around the TGN (Preuss et al., 2004; de Graaf et al., 2005; Chow et al., 2008), although involvement of this family in endocytosis remains unexplored. Because of the extreme expansion of RABA members in Arabidopsis (see Supplemental Table 2 online), it is not practical at present to test their involvement in endocytosis of FLS2 using classical mutant analyses. Thus we took advantage of the transient expression system in N. benthamiana leaf epidermal cells, which allowed us to monitor the effect of a dominant-negative mutant of a representative member of each RABA subgroup whose expression was conditionally induced in cells expressing FLS2-GFP and organelle markers. We created a nucleotide-free mutant of each member of the six subgroups of RABA (RABA1b, RABA2c, RABA3, RABA4c, RABA5c, and RABA6a) by replacing invariable Asn in the conserved GNKXD sequence with Ile (hereafter called the NI mutant) because these mutants have been successfully used as dominant-negative forms for functional analyses of RABA members in plants (Cheung et al., 2002; Chow et al., 2008; Bottanelli et al., 2011). The expression of the NI mutants was controlled by an estradiol-inducible promoter (Zuo et al., 2000) to avoid undesirable effects of the mutant on transport of newly synthesized FLS2 to the PM. We examined the effects of these mutants on endocytic transport of FLS2-GFP, comparing FLS2-GFP subcellular localization with SYP61 and/or ARA7. For induction of mutant expression, 10 μM estradiol was infiltrated into N. benthamiana leaves and incubated for 2 to 3 h, and then samples were treated with flg22. This condition is enough for accumulation of the mutant RABA members, as indicated by bright fluorescence from Venus fused to the mutant RABA members (see Supplemental Figure 3 online). We examined the effects of expression of wild-type and NI mutants for all six RABA subgroups, three of which (RABA2c, RABA3, and RABA5c) had no substantial effect on endocytic transport of FLS2-GFP; mutants of RABA3 and RABA5c did not exert significant effects on endocytosis of FLS2 (P > 0.05, Student’s t test), and the mutant of RABA2c had only a marginal effect on colocalization with SYP61 at the later stage (P = 0.0493, Student’s t test; see Supplemental Figures 4 to 6 online). However, expression of NI mutants of the other three RABA subgroups resulted in remarkable alteration in endocytic trafficking of FLS2-GFP.

RABA6a and RABA4c Act in Distinct Steps of FLS2 Endocytosis

In RABA6a^N126I-expressing cells, we found that FLS2 colocalized well with SYP61 even at the late stage, at which FLS2 was observed on membrane compartments independent of SYP61-domains in wild-type RABA6a-expressing cells (Figure 4A). Quantification further indicated that expression of RABA6a^N126I increased colocalization of FLS2 with SYP61. We found that 41.2% ± 4.0% and 15.4 ± 3.5% of FLS2-positive compartments in early (80 to 140 min) and late (140 to 200 min) stages, respectively, also bore SYP61 in RABA6a^N126I-expressing cells, which was significantly higher than that observed in wild-type RABA6a-expressing samples (21.6% ± 5.9% in the early stage and 5.2% ± 2.6% in the late stage, n = 3 experiments, P < 0.05, Student’s t test; Figure 4B). This result appeared to indicate that RABA6a acts in trafficking of FLS2 from SYP61-positive compartments. We then examined whether ARA7 also occurs on the FLS2-positive compartments in RABA6a^N126I-expressing cells. We coexpressed TagRFP-ARA7 with FLS2-GFP and mutant or wild-type RABA6a to find that ARA7 also localized on FLS2-positive compartments in both cases (see Supplemental Figure 7 online). These results strongly suggested that RABA6a^N126I did not impair formation of the hybrid compartment bearing SYP61 and ARA7 but did cause delay in transport or transition from the intermediate endosome to the SYP61-free late endosomal compartment, which resulted in increased colocalization of FLS2 and SYP61 in NI mutant–expressing cells.

Figure 4.

The RABA6a NI Mutant Increases the Ratio of Compartments with Both FLS2-GFP and mRFP-SYP61.

(A) Cells expressing FLS2-GFP and mRFP-SYP61 with wild-type RABA6a (top panels) or RABA6a^N126I (bottom panels) were observed after 120 or 200 min after flg22 treatment. Expression of RABA6a and RABA6a^N126I was induced by estradiol treatment. Arrowheads and arrows indicate FLS2-GFP signals colocalized and associated with TagRFP-SYP61, respectively. Individual channel images are shown in Supplemental Figure 11C online. Bar = 5 μm.

(B) Stacked bar graph indicating results of quantitative analyses of colocalization between FLS2-GFP and mRFP-SYP61. WT, wild-type RABA6a-expressing cell; NI, RABA6a^N126I-expressing cells. Data were collected from three independent experiments, and 178 and 162 (wild type) or 204 and 209 (NI) FLS2-positive dots in total were observed in early (80 to 140) and late (140 to 200) stages, respectively. Error bars indicate sd values. Average numbers of cells examined and numbers of puncta analyzed in each experiment are shown in Supplemental Table 1 online.

(C) Stacked bar graph indicating results of quantitative analyses of colocalization between FLS2-GFP and CFP-SYP61 in RABA6a^N126I-expressing cells. RABA6a, RABA6a-coexpressing cells; RABA4c, RABA4c-coexpressing cells. Data were collected from three independent experiments; 64 and 51 (RABA6a) and 118 and 87 (RABA4c) FLS2-positive dots in total were observed in early (80 to 140) and late (140 to 200) stages, respectively. Error bars indicate sd values. Average numbers of cells examined and numbers of puncta analyzed in each experiment are shown in Supplemental Table 1 online.

Intriguingly, RABA4c^N128I conferred an apparently opposite effect from RABA6a^N126I. In cells expressing the RABA4c^N128I mutant, the extent of colocalization between FLS2 and SYP61 in the early stage was significantly reduced (5.8% ± 3.9%) compared with the result from coexpression of wild-type RABA4c (26.7% ± 9.6%, n = 3 experiments, P < 0.05, Student’s t test; Figures 5A and 5B). This result appeared to indicate that internalized FLS2 was not transported to the SYP61 compartments in mutant-expressing cells. However, we found that ARA7 did colocalize with FLS2 in RABA4c^N128I-expressing cells (see Supplemental Figure 8 online). Thus, there might be an alternative direct trafficking pathway from the PM to ARA7-positive MVEs without transit through SYP61-positive compartments.

Figure 5.

The RABA4c NI Mutant Decreases the Ratio of Compartments with Both FLS2-GFP and mRFP-SYP61.

(A) Cells expressing FLS2-GFP and mRFP-SYP61 with wild-type RABA4c (top panels) or RABA4c^N128I (bottom panels) were observed after 80 or 170 min after flg22 treatment. Expression of RABA4c and RABA4c^N128I was induced by estradiol treatment. Arrowheads indicate FLS2-GFP signals colocalized with TagRFP-SYP61. Note that colocalization was not observed in RABA4c^N128I-expressing cells. Individual channel images are shown in Supplemental Figure 11D online. Bar = 5 μm.

(B) Stacked bar graph indicating results of quantitative analyses of colocalization between FLS2-GFP and mRFP-SYP61. WT, wild-type RABA4c-expressing cell; NI, RABA4c^N128I-expressing cells. Data were collected from three independent experiments, and 174 and 153 (wild type) or 143 and 82 (NI) FLS2-positive dots in total were observed in early (80 to 140) and late (140 to 200) stages, respectively. Error bars indicate sd values. Average numbers of cells examined and numbers of puncta analyzed in each experiment are shown in Supplemental Table 1 online.

(C) Stacked bar graph indicating results of quantitative analyses of colocalization between FLS2-GFP and CFP-SYP61 in RABA4c^N128I-expressing cells. RABA4c, RABA4c-coexpressing cells; RABA6a, RABA6a-coexpressing cells. Data were collected from three independent experiments; 32 (RABA4c) and 30 (RABA6a) FLS2-positive dots in total were observed in the early (80 to 140) stage. Error bars indicate sd values. Average numbers of cells examined and numbers of puncta analyzed in each experiment are shown in Supplemental Table 1 online.

These results clearly indicated that two distinct RABA subgroup members, RABA6a and RABA4c, regulate different steps in the endocytic pathway of FLS2. Furthermore, the specific effects of these RABA members also demonstrated that the inhibitory effects we observed in these experiments resulted from inhibition of the specific function of each RABA member in N. benthamiana, rather than reflecting the effect of titration of general RAB regulators such as RAB guanine nucleotide dissociation inhibitor. This notion was also supported by the results of coexpression of wild-type RABA members with NI mutants. We coexpressed mRFP-tagged wild-type RABA members with Venus-RABA4c^N128I or Venus-RABA6a^N126I for suppression activities for transport defects of FLS2 in tobacco cells expressing FLS2-GFP and CFP-SYP61. The inhibitory effects of NI mutants were specifically rescued by coexpression of their wild-type versions but not by the other RABA members, which again indicated specific inhibition of the distinct trafficking event by each mutant RABA, as is the case for other plant RAB GTPases (Figures 4C and 5C; Batoko et al., 2000; Kotzer et al., 2004; Pinheiro et al., 2009). We then examined the order of functions of two RABA members, RABA4c and RABA6a, in endocytic trafficking of FLS2 by double infiltration of their NI mutants. Simultaneous expression of RABA4c^N128I and RABA6a^N126I caused the same trafficking defect as RABA4c^N128I (Figure 6). This result strongly suggested that RABA4c acts in an earlier step than RABA6a in endocytic trafficking of FLS2.

Figure 6.

Coexpression of NI Mutants of RABA4c and RABA6a.

(A) Cells expressing FLS2-GFP and CFP-SYP61 with both RABA6a^N126I and RABA4c^N128I were observed after 100 or 170 min following flg22 treatment. Expression of RABA6a^N126I and RABA4c^N128I was induced by estradiol treatment. Note that colocalization was not observed in cells expressing RABA6a^N126I and RABA4c^N128I. Individual channel images are shown in Supplemental Figure 11E online. Bar = 5 μm.

(B) Stacked bar graphs indicating results of quantitative analyses of colocalization between FLS2-GFP and CFP-SYP61. Data were collected from three independent experiments; 69 and 63 FLS2-positive dots in total were observed in early (80 to 140) and late (140 to 200) stages, respectively. Error bars indicate sd values. Average numbers of cells examined and numbers of puncta analyzed in each experiment are shown in Supplemental Table 1 online.

RABA1b Functions in the Secretory Pathway

The NI mutant of RABA1b (RABA1b^N126I) exerted another interesting effect. When expressed with this mutant, the distribution of mRFP-SYP61 and internalized FLS2-GFP was dramatically changed. mRFP-SYP61, which presented clear dot-shaped signals in wild-type RABA1b-expressing cells, was scattered to tiny dots throughout the cytoplasm (Figure 7). Regarding FLS2-GFP, for which flg22 treatment induced endocytosis, we observed no dot-like signals in the mutant-expressing cells, but only smear signals that largely overlapped with mRFP-SYP61 (Figure 7). These results indicated that RABA1b^N126I did not block the internalization of FLS2 from the PM but that the morphology of compartments with SYP61 to which FLS2 was transported was severely affected.

Figure 7.

The RABA1b NI Mutant Affects the Localization of mRFP-SYP61.

Localization of FLS2-GFP and mRFP-SYP61 was observed at the indicated times after flg22 treatment in RABA1b- (top panels) or RABA1b^N126I-expressing cells (bottom panels). Expression of RABA1b and RABA1b^N126I was induced by estradiol treatment. Individual channel images are shown in Supplemental Figure 11F online.

These findings additionally suggested that expression of mutant RABA1b resulted in alteration of the morphology of the TGN. Because the TGN also plays fundamental roles in the secretory pathway, we then examined the effects of NI mutants of RABA1b and other RABA subgroups on the steady state localization of FLS2-GFP at the PM. For this purpose, chimeric genes composed of a 35S promoter, mutant cDNA for each RABA member, and a 35S terminator were introduced with FLS2-GFP into N. benthamiana cells. Among mutants of the six RABA members we examined, only RABA1b^N126I caused substantial alteration in FLS2 distribution. In the RABA1b^N126I-expressing cells, FLS2 was mainly observed as scattered tiny dots throughout the cytoplasm, and weak localization to the PM was also visible (Figure 8A). Moreover, constitutive expression of RABA1b^N126I affected distribution of mRFP-SYP61; mRFP-SYP61 was also dispersed throughout the cytoplasm as observed in the induced-expression experiment (Figure 8B). These results indicated that transport of newly synthesized FLS2 to the PM was disturbed by defective trafficking around the SYP61-positive TGN in the mutant-expressing cells.

Figure 8.

Constitutive Expression of RABA1b^N126I Alters Steady State Localization of FLS2-GFP and mRFP-SYP61.

(A) Localization of FLS2-GFP in cells expressing RABA1b^N126I (left panel), RABA4c^N128I (middle panel), or RABA6a^N126I (right panel) under regulation of the 35S promoter. Max intensity projection images are presented, each of which is reconstructed with a series of confocal Z-stack images taken at 0.5-μm intervals. Bar = 5 μm.

(B) Localization of FLS2-GFP and mRFP-SYP61 in RABA1b- or RABA1b^N126I-expressing cells under regulation of the 35S promoter. Left panels show the middle plane of cells, and right panels show the confocal plane near the PM in the same cells shown in left panels. Individual channel images are shown in Supplemental Figure 11G online. Bars = 5 μm.

(C) Localization of FLS2-GFP in cells expressing 35S promoter-driven RABA1b^N126I and estradiol-inducible promoter-driven RABA1b (left panel), RABA4c (middle panel), or RABA6a (right panel). Top and bottom panels indicate cells before and after estradiol treatment, respectively. Bar = 5 μm.

We also tested for an inhibitory effect of RABA1b^N126I on transport of another PM protein, BOR1, a boron efflux carrier whose localization is regulated by external boron concentration (Takano et al., 2002, 2005). Steady state localization of BOR1 was slightly different from that of FLS2; BOR1-GFP was observed on punctate organelles in addition to the PM, probably reflecting its constitutive internalization (Takano et al., 2005; Figure 9A). Coexpression of RABA1b^N126I also resulted in alteration of this localization pattern; BOR1 predominantly localized in scattered small dots in the cytoplasm, and only weak PM localization was detected in cells expressing RABA1b^N126I (Figure 9B). This result again suggested that RABA1b acts in the secretory pathway. By contrast, the NI mutants of RABA4c and RABA6a had no visible effect on presentation of FLS2-GFP and BOR1-GFP on the PM (Figures 8A, 9C, and 9D). In a similar manner, NI mutants of RABA2c, RABA3, and RABA5c caused no substantial change in the PM localization of FLS2 (see Supplemental Figure 9 online). The inhibitory effect of RABA1b^N126I in secretory trafficking of FLS2 was attributable to specific inhibition of the RABA1 function because estradiol-induced coexpression of wild-type RABA1b, but not RABA4c or RABA6a, restored the PM localization of FLS2-GFP (Figure 8C). These results again support the specific and distinct functions of RABA subgroups in plant cells.

Figure 9.

Constitutive Expression of RABA1b^N126I Alters Steady State Localization of BOR1-GFP.

(A) Localization of BOR1-GFP. Bar = 5 μm.

(B) to (D) RABA1b^N126I (B), RABA4c^N128I (C), or RABA6a^N126I (D) were coexpressed under regulation of the 35S promoter in cells expressing BOR1-GFP. Max intensity projection images are presented, each of which is reconstructed with a series of confocal Z-stack images taken at 0.5-μm intervals.

Distinct Subcellular Localizations of RABA1b, RABA4c, and RABA6a

We next asked whether three RABA members with different functions, RABA1b, RABA4c, and RABA6a, reside on different subcellular compartments or on the same organelle. We thus directly compared the localization of these proteins using combinations of different fluorescent proteins. Expression of fluorescent protein–tagged RABA members was induced by incubation with 10 μM estradiol for 1 h. RABA1b, RABA4c, and RABA6a localized at various sizes of dot-like structures (Figure 10A). These RABA members exhibited good colocalization on comparatively large compartments; however, they did not overlap at small vesicular structures (Figure 10A). This different localization is not due to the difference in fused fluorescent proteins because RABA members tagged with different fluorescent proteins localized to the same compartments (Figure 10B).

Figure 10.

Subcellular Localization of RABA1b, RABA4c, and RABA6a.

(A) All three RABA members localized on large clear punctate compartments and tiny scattered vesicles when expressed in N. benthamiana cells separately (top panels). When two RABA members tagged with the indicated fluorescent proteins were expressed in the same cells, they colocalized on the large compartments but not on the scattered vesicles (bottom panels). Green, magenta, and white arrowheads indicate the Venus, mRFP, and overlapped signals, respectively. Bar = 5 μm.

(B) When the same RABA members were tagged with different fluorescent proteins and expressed in the same cell, localization patterns overlapped almost completely. Bar = 5 μm.

(C) mRFP-SYP61 was coexpressed with RABA1b (left panel), RABA4c (middle panel), or RABA6a (right panel) in FLS2-GFP–expressing cells. Bar = 5 μm.

Individual channel images are shown in Supplemental Figure 11H online.

In Arabidopsis root epidermal cells, comparatively large compartments bearing RABA1b partly overlap with the TGN marked by SYP43 or VHA-a1 (Asaoka et al., 2012). Thus, we examined whether RABA members are also localized on the TGN in our experimental system. When coexpressed in FLS2-GFP–expressing N. benthamiana leaf epidermal cells, Venus-tagged RABA members and mRFP-SYP61 frequently colocalized on larger particles, while mRFP-SYP61 did not target to RABA-positive smaller vesicles (Figure 10C). The colocalization of SYP61 and RABA1b was not affected by RABA4c^N128I or RABA6a^N126I, which exerted inhibitory effects on endocytic transport of FLS2 (see Supplemental Figure 10 online). These results indicated that three RABA members localize on different compartments with overlap on the TGN, which is consistent with their different functions in transport of FLS2.

DISCUSSION

Internalized FLS2 Is Transported to Intermediate Compartments between the TGN and MVE

Endocytic transport of the flagellin receptor FLS2 is tightly coupled with the plant defense response and thus is of great interest and importance for understanding defense regulatory mechanisms (Robatzek et al., 2006). Recent work showed that FLS2 internalizes into cells in a ligand-induced manner, passing through an endocytic pathway distinct from that of constitutively recycled FLS2. This report also indicated that RAB5 is responsible for endocytosis of FLS2 (Beck et al., 2012). However, the identity of endosomal compartments mediating endocytic traffic of this receptor and the molecular details of its mechanisms remain largely unknown. In this study, we showed that internalized FLS2 passed through a compartment with a hybrid nature between the TGN and MVE and that localized both mRFP-SYP61 and TagRFP-ARA7. The plant TGN is also recognized as an early endosome because an internalized lipophilic dye, FM4-64, stains the TGN earlier than the Rab5-positive MVEs (Dettmer et al., 2006; Lam et al., 2007; Chow et al., 2008). Consistent with these findings, Viotti et al. (2010) demonstrated by immunoelectron microscopy that the endocytosed cargo proteins BOR1 and BRI1 reach the TGN. Direct maturation from the TGN to MVE has also been proposed recently (Bottanelli et al., 2011; Scheuring et al., 2011), suggesting the sequential action of the TGN and MVE along the endocytic pathway. However, recent studies have indicated that plants also harbor a clathrin-independent endocytosis mechanism (Li et al., 2012). Thus, the endocytic route could vary depending on each cargo, which would not be distinguishable by monitoring bulk membrane flow using FM dyes. What is now needed is to monitor protein cargos to obtain robust and specific information.

To investigate the function of the TGN and MVE in ligand-triggered endocytosis of FLS2 and to examine which Rab GTPases are involved, we used an N. benthamiana transient expression system, which allowed us time-sequential and multicolor observation of endocytosed FLS2 and other trafficking components in living cells. In this system, we found that induction of endocytosis of FLS2 led to formation of a novel compartment that harbored both SYP61, a TGN-resident SNARE protein, and ARA7, the MVE-resident Rab GTPase, suggesting that the nature of this compartment is intermediate between the TGN and MVE. Of interest, VHA-a1, another well-characterized TGN protein, was not recruited to this hybrid compartment. Both SYP61 and VHA-a1 colocalize with SYP43, a SNARE protein residing on the TGN in Arabidopsis root epidermal cells (Uemura et al., 2012). Thus, our results could further indicate the unique character of the intermediate compartment, as it harbors a limited set of TGN proteins in addition to endosomal RAB5 GTPase. Because quantitative analyses in previous studies have shown that the known TGN markers are not colocalized completely (Dettmer et al., 2006; Chow et al., 2008; Boutté et al., 2010; Gendre et al., 2011), it is also possible that a specific population of the TGN (or a specific membrane domain around the TGN) serves in flg22-triggered endocytic transport of FLS2. An interesting future project would be studying how endocytic trafficking and remodeling of the TGN are integrated in plant cells.

Another important finding is that SYP61 appeared to segregate to distinct membrane domains from ARA7 and FLS2 and was gradually removed from the hybrid compartment, while FLS2 exhibited good colocalization with ARA7 even after a long incubation. This result suggests that the TGN components are gradually eliminated from the hybrid compartment, leading to maturation to an MVE with a fully late endosomal nature. In agreement with this, endosomal sorting complex required for transport (ESCRT) components are gradually recruited to the TGN to lead maturation of the TGN to the MVE, and colocalization of SYP61 and ARA7 is also observed when the function of the TGN is hampered by concanamycin A or the function of the ESCRT complex is inhibited (Scheuring et al., 2011). Thus, our results, together with the findings by Scheuring et al., might suggest that maturation of the TGN to the MVE is at least partly responsible for ligand-induced endocytic trafficking of FLS2 to the vacuole.

Curiously, we did not observe a compartment harboring FLS2 and SYP61 without ARA7. This result raises two possibilities. The first is that internalized FLS2 is transported to the SYP61-positive but ARA7-negative TGN first, which cannot be observed because of too-rapid maturation kinetics. Another possibility is that internalized FLS2 is transported directly to the hybrid endosomes bearing both SYP61 and ARA7. Although we have not detected colocalization of ARA7 and TGN markers at the steady state in our experimental system, another group reported that ARA7 is also detected at the TGN (Stierhof and El Kasmi, 2010). Thus, a small number of the hybrid compartments could exist in plant cells, with a population that might increase under specific conditions that require enhanced endocytosis. Consistent with this concept, a recent study on FLS2 trafficking in Arabidopsis also suggested that some population of ARA7-positive compartments could act as early endosomes (Beck et al., 2012). Regardless, further studies of this hybrid compartment are needed to reveal its nature and function. Whether other endocytic cargos also pass through this intermediate compartment and whether this hybrid compartment is observed when endocytosis is activated in other plants including Arabidopsis would be interesting questions to address in future research.

RABA/RAB11 Groups Have Distinct Functions in Membrane Trafficking around the TGN

We succeeded in finding RABA members whose respective dominant-negative mutants affect different steps of endocytosis of FLS2. RABA6a^N126I affected the maturation step from the hybrid endosomes to the late endosome/MVE; FLS2 retained localization on the hybrid endosomes with both SYP61 and ARA7 in RABA6a^N126I-expressing cells even after a long incubation, suggesting a delay in the maturation process. In the animal system, early-to-late maturation of endosomes is associated with replacement of Rab GTPases, which is referred to as “Rab conversion.” Rab5 on the early endosomes in animal cells is gradually replaced by Rab7, which is responsible for late endosomal trafficking mediated by effector complexes of these Rab GTPases, CORVET and HOPS (Rink et al., 2005). Considering the subcellular localization of RABA members around the TGN (Preuss et al., 2004; de Graaf et al., 2005; Chow et al., 2008), our results may indicate that plants also employ a similar molecular mechanism in endosomal maturation, which is associated with Rab conversion from RABA6a to ARA7.

However, coexpression of RABA4c^N128I resulted in a significant decrease in the endosome population with both SYP61 and FLS2, while internalization of FLS2 was not markedly affected. This result appears to indicate that FLS2 does not reach the TGN. Curiously, however, we found that ARA7 resided on the FLS2-positive compartment also in RABA4c^N128I-expressing cells. This observation might be explained by accelerated maturation from the hybrid compartment to the late endosome; however, that would not be likely because a dominant-negative Rab GTPase generally inhibits or delays trafficking events, which should also be the case for RABA4c^N128I. Thus, our finding may indicate an alternative trafficking pathway that mediates transport from the PM to the ARA7-positive endosome directly when the early endocytic pathway to the TGN/early endosome is compromised. It is also possible that the effects of RABA6a^N126I and RABA4c^N128I on colocalization of FLS2 and SYP61 represent altered distribution of SYP61; these mutant RABA members could affect transport of SYP61 to or from ARA7- and FLS2-positive endosomes. However, this situation would not be as likely because the localization pattern of SYP61 and RABA1b was not affected by coexpression of RABA6a^N126I or RABA4c^N128I.

Another interesting finding is that RABA1b, a member of the largest RABA subgroup, is involved in delivery of newly synthesized FLS2 and BOR1 to the PM. Coexpression of RABA1b^N126I resulted in dispersed localization of SYP61, which also indicates that this RABA member is responsible for maintenance of TGN distribution. However, RABA1b^N126I did not seem to affect internalization of FLS2 to the SYP61-positive compartment, as shown by relocalization to a dispersed pattern similar to that of SYP61 in RABA1b^N126I-expressing cells after flg22 treatment. We did not see any effects on FLS2 and SYP61 distribution for equivalent mutants of other members including RABA4c^N128I and RABA6a^N126I in both estradiol-induced and 35S promoter–driven constitutive expression. These results strongly suggest that the RABA1 group specifically acts in the secretory pathway but not in the endocytic pathway, which is also required for maintenance of TGN morphology. RABA1b function in the secretory pathway is also suggested by our study in Arabidopsis plants (Asaoka et al., 2012)

Functional Diversification of Plant RABA Members

Our data clearly indicate that different RABA members have distinct functions in intracellular trafficking of FLS2. Consistent with this, three members identified in this study as being involved, RABA1b, RABA4c, and RABA6a, exhibited distinct subcellular localization, supporting that these members act at different membrane domains around the TGN. Recent comparative genomics indicate that extreme expansion of Rab11 homologs is one of the most remarkable characteristics in organization of plant Rab GTPases. How Rab functions have diverged during the expansion of plant Rab11 homologs has remained unclear, but our results indicate that the functions of RABA/Rab11 members have diversified from each other. Another important finding is that plant RABA/Rab11 is involved in both secretory and endocytic pathways. The functional diversity together with their important roles in plant-unique physiological events (Preuss et al., 2004; de Graaf et al., 2005; Chow et al., 2008; Szumlanski and Nielsen, 2009) indicate that expansion of this family could play pivotal roles in the increased complexity of membrane trafficking pathways in plant cells, which are recruited to plant-unique physiological events during plant evolution. The next interesting question is how the RABA/Rab11 members encoded by a paralogous set of genes acquired diverse functions in membrane trafficking. Further functional analyses of this group, as well as identification and characterization of upstream regulators and downstream effectors, would be needed to answer this question.

METHODS

Transient Expression in Nicotiana benthamiana and flg22 Treatment

We used 3- to 5-week-old N. benthamiana plants for agroinfiltration. Expression vectors were introduced into Agrobacterium tumefaciens strain GV3101:pMP90. A single colony of each transformant was cultured in 5 mL YEB medium (5 g beef extract, 1 g yeast extract, 5 g Suc, and 0.49 g MgSO₄·7H₂O dissolved in 1 liter of water) supplemented with 10 μg/mL rifampicin at 30°C overnight. The bacteria were collected and resuspended in infiltration buffer (10 mM MES, 10 mM MgCl₂, and 0.15 mM acetosyringone, pH 5.5) at 0.02 (FLS2-GFP) or 0.1 (other constructs) OD₆₀₀. The agrobacteria carrying p19 were resuspended together with all samples (Voinnet et al., 2003). The resuspended agrobacteria were infiltrated into leaves of N. benthamiana with gentle pressure using syringes without needles. For coexpression of proteins, bacterial strains with different constructs were mixed before inoculation. Samples were observed within 48 h after infiltration. For induction of endocytosis of FLS2, 100 μM flg22 peptide was infiltrated into leaves expressing FLS2-GFP. For estradiol-dependent induction of gene expression, β-estradiol (10 μM) was applied for 1 to 3 h before treatment with flg22.

FM4-64 Staining

FM4-64 (50 μM; Invitrogen/Molecular Probes; T13320) dissolved in distilled water was infiltrated to the leaf tissue of N. benthamiana from the abaxial surface. Samples were incubated at room temperature for indicated times and observed under the confocal laser scanning microscope.

Plasmid Construction

The plasmid containing pFLS2:FLS2-GFP was described previously (Robatzek et al., 2006). For mRFP-SYP61, VHA-a1-mRFP, TagRFP-ARA7, and TagRFP-VAMP727 constructs, PCR-amplified cDNA fragments were cloned into pGWB1 containing the 35S promoter (Nakagawa et al., 2007). For RABA members (RABA1b, RABA2c, RABA3, RABA4c, RABA5c, and RABA6a), cDNA fragments were amplified by PCR using the primers listed in Supplemental Table 3. Amplified wild-type and mutant cDNA fragments were conjugated with cDNA for fluorescent proteins and cloned into pMDC7 (Curtis and Grossniklaus, 2003) for estradiol-inducible expression. For constitutive expression, cDNA fragments of RABA were introduced into pB7WGY2 and pH7WGR2 containing YFP and RFP, respectively (Karimi et al., 2002).

Confocal Laser Scanning Microscopy

For single-color imaging, GFP was excited by a diode-pumped solid-state laser (Cobolt Blues) at 473 nm and observed under a microscope (BX51; Olympus) equipped with a confocal scanner unit (CSU10; Yokogawa Electric) and a cooled charge-coupled device camera (ORCA-AG; Hamamatsu Photonics). Images were processed with IPLab software (BD Biosciences) and Photoshop CS5 (Adobe). Multicolor observation was performed using an LSM710 or LSM780 confocal microscope (Carl Zeiss) with the oil immersion lens (×63, numerical aperture = 1.40). Spectral unmixing (if necessary) and processing of obtained images were performed using ZEN 2008 or ZEN 2011 software (Carl Zeiss). For dual-color imaging, GFP and Venus were excited at 488 nm, and the emission was collected between 501 and 545 nm. mRFP and TagRFP were excited at 561 nm, and the emission was collected between 570 and 615 nm. For three-color imaging, samples expressing fluorescent proteins (combinations of three of four XFPs: GFP, Venus, mRFP, and TagRFP) were excited at 488 and 561 nm, and the emission was collected between 484 and 640 nm. For three- or four-color imaging using CFP, samples were excited at 405, 488, and 561 nm, and the emission was collected between 411 and 633 nm. The colocalization analysis was performed as described previously (Ito et al., 2012) with Metamorph software (Molecular Devices).

Variable Incidence Angle Fluorescence Microscopy

The leaves from infiltrated N. benthamiana were placed on a glass slide (76 × 26 mm; Matsunami) and covered with a cover slip 0.12- to 0.17-mm thick (24 × 60 mm; Matsunami), and epidermal cells were observed under a fluorescence microscope (Nikon Eclipse TE2000-E and a CFI Apo TIRF ×100 H/1.49–numerical aperture objective) equipped with a Nikon TIRF2 system. GFP was simultaneously excited with a 488-nm laser. All images were acquired with an Andor iXonEM electron multiplying charge coupled device (EMCCD) camera; each frame was exposed for 100 ms. The acquired images were prepared and analyzed using Photoshop CS5 (Adobe Systems) and NIS-Elements software (Nikon).

Accession Numbers

The Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are At5g46330 (FLS2), At1g28490 (SYP61), At2g28520 (VHA-a1), At4g19640 (ARA7), At3G54300 (VAMP727), At1g16920 (RABA1b), At3g46830 (RABA2c), At1g01200 (RABA3), At5g47960 (RABA4c), At2g43130 (RABA5c), and At1g73640 (RABA6a).

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure 1. Internalization of FM4-64 in Leaves of N. benthamiana.

• Supplemental Figure 2. SYP61- and VHA-a1–Positive Compartments Are Frequently Associated with the Golgi Apparatus.

• Supplemental Figure 3. Estradiol Treatment Efficiently Induces Gene Expression.

• Supplemental Figure 4. The NI Mutant of RABA2c Does Not Affect Trafficking Kinetics of FLS2-GFP.

• Supplemental Figure 5. The NI Mutant of RABA3 Does Not Affect Trafficking Kinetics of FLS2-GFP.

• Supplemental Figure 6. The NI Mutant of RABA5c Does Not Affect Trafficking Kinetics of FLS2-GFP.

• Supplemental Figure 7. Colocalization of FLS2-GFP and TagRFP-ARA7 in RABA6a^N126I-Expressing Cells.

• Supplemental Figure 8. FLS2-GFP and TagRFP-ARA7 Colocalize in RABA4c- and RABA4c^N126I-Expressing Cells after flg22 Treatment.

• Supplemental Figure 9. Localization of FLS2-GFP in N. benthamiana Leaf Epidermal Cells Expressing RABA2c^N125I, RABA3^N141I, or RABA5c^N125I Is Constitutively under Regulation of the 35S Promoter.

• Supplemental Figure 10. Quantitative Analysis of Colocalization between SYP61 and RABA1b.

• Supplemental Figure 11. Individual Channel Images for All Multicolor Figures.

• Supplemental Table 1. Sample Information for Quantitative Analyses.

• Supplemental Table 2. A List of RABA Members in Arabidopsis.

• Supplemental Table 3. A List of Primers Used in This Study.

Glossary

PM

plasma membrane

TGN

trans-Golgi network

MVE

multivesicular endosome