RABA Members Act in Distinct Steps of Subcellular ...RABA Members Act in Distinct Steps of Subcellular Trafﬁcking of the FLAGELLIN SENSING2 ReceptorW Seung-wonChoi,a TakayukiTamaki,a

RABA Members Act in Distinct Steps of SubcellularTrafficking of the FLAGELLIN SENSING2 ReceptorW

Seung-wonChoi,a Takayuki Tamaki,a KazuoEbine,a,1 TomohiroUemura,a Takashi Ueda,a,b,2 andAkihikoNakanoa,c

a Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japanb Japan Science and Technology Agency, PRESTO, Saitama 332-0012, JapancMolecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan

Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuingsignal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integrationof 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 themechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescentprotein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PMwas transported to a compartment with properties intermediate between the trans-Golgi network (TGN) andthe multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the traffickingpathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, wedemonstrated that transport of de novo–synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our resultsdemonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABAmembers in endo-and exocytosis.

INTRODUCTION

Eukaryotic cells recognize their environment mainly through pro-teins on the plasma membrane (PM), including receptors andsensors, which evoke intracellular signal transduction to respondto environmental cues. In animal cells, endocytosis plays criticalroles in regulation of the amount of PM proteins responding toextracellular stimuli, transduction of signals from endosomes, anddownregulation 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 isknown to be regulated by endocytosis upon extracellular stimuli.For example, REQUIRES HIGH BORON1 (BOR1), a boron effluxcarrier on the PM, is endocytosed in response to changes inenvironmental boron concentration (Takano et al., 2002, 2005).The Leucine-rich repeat receptor Ser/Thr kinase FLAGELLINSENSING2 (FLS2) is another example of localization regulatedby endocytosis. FLS2 is the receptor for bacterial flagellin, andthe fls2 mutant is highly susceptible to infection by pathogenicbacteria (Zipfel et al., 2004). FLS2 recognizes 22 amino acids ofa conserved domain in the N terminus of flagellin (flg22 peptide;Felix et al., 1999), and FLS2 bound with flg22 is rapidly internalizedfrom the PM into the cytoplasm and degraded probably in vacuoles

(Robatzek et al., 2006). Intriguingly, plants expressing FLS2T867V,which has a mutation in a putative phosphorylation site, exhibitsusceptibility to pathogenic Pseudomonas syringae, and flg22-triggered internalization of this mutant protein rarely occurs, im-plying a possible link between endocytosis of FLS2 and flagellinsignaling (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 envi-ronmental cues and hormone signaling. However, our knowledgeof the molecular mechanisms of endocytosis in plant cells is stilllimited. It is apparently insufficient to simply extend the knowledgeobtained from yeast and animal systems to the plant system be-cause the plant endocytic pathway seems to operate differently inplants. The role of the trans-Golgi network (TGN) in endocytosisis a notable example. The TGN acts as the sorting platform forsecreted and vacuolar/lysosomal proteins in eukaryotic cells. Inaddition to this function, the TGN in plant cells also acts as theearly endosome; a lipophilic tracer of endocytosis, FM4-64, stainsthe TGN bearing the TGN markers Vacuolar H1-ATPase subunita1 (VHA-a1), SECRETORY CARRIER MEMBRANE PROTEIN1(SCAMP1), RABA2a, or RABA3 before this dye reaches the multi-vesicular endosomes (MVEs) marked by the conventional RAB5ortholog ARA7/RABF2b (Dettmer et al., 2006; Lam et al., 2007;Chow et al., 2008). Immunoelectron microscopy has also demon-strated that endocytosed BRI1 and BOR1 pass through the TGNand 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

1Current address: Department of Parasitology, National Institute of InfectiousDiseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.2 Address correspondence to [email protected] author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Takashi Ueda ([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.112.108803

The Plant Cell, Vol. 25: 1174–1187, March 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

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organization of RAB GTPases between animals and plants. RABGTPase is an evolutionarily conserved key player in membranetrafficking, which generally regulates the docking step of transportcarriers to target organelles through the conformational changebetween GTP-bound active and GDP-bound inactive states. RABGTPases are widely conserved in all eukaryotic lineages but seemto have diversified uniquely in each lineage. Molecular phylogeneticanalyses have suggested that most land plant RAB GTPases areclassified into eight families, each of which exhibits high similarity toanimal RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11, and RAB18(Rutherford and Moore, 2002; Vernoud et al., 2003). Plants lackclear homologs of the well-characterized animal endocytic RABs,RAB4 and RAB9, which also suggests that the regulatory mecha-nism of the plant endocytic pathway differs from the animal system.By contrast, the RAB5 and RAB11 families, which act in theendocytic pathway in animal cells, have diversified in a uniqueway in plants. The plant RAB5 family consists of two distinctsubgroups: the plant-unique ARA6 group and the conventionalRAB5 group. How the functions of these subgroups differ hasbeen unclear, but recent studies indicate that ARA6/RABF1 isinvolved in trafficking from endosomes to the PM in Arabidopsisthaliana, whereas conventional RAB5/RABF2 acts in the vacuolartrafficking pathway (Sohn et al., 2003; Bolte et al., 2004; Kotzeret al., 2004; Ebine et al., 2011). Another notable characteristic ofthe plant RAB GTPase is the variety of RAB11 homologs (referredto as RABA in Arabidopsis). A total of 26 members of the 57 RABGTPases in Arabidopsis are classified into the RABA group, whichis further divided into six subgroups (RABA1 to RABA6). RAB11regulates the endocytic pathway in animal cells; animal RAB11resides on recycling endosomes and regulates recycling of endo-cytosed proteins to the PM (Ullrich et al., 1996; Ren et al., 1998). Bycontrast, yeast homologs of RAB11, Ypt3 members, are proposedto be required for multiple steps of the exocytic pathway, includingintra-Golgi transport and transport vesicle formation at the TGN(Benli et al., 1996; Jedd et al., 1997; Cheng et al., 2002). Somemembers of the plant RABA/RAB11 group also take part in severaldifferent exocytic events unique to plants. Substantial roles in tipgrowth of pollen tubes and root hairs have been demonstrated intobacco (Nicotiana tabacum) and Arabidopsis (Preuss et al., 2004;de Graaf et al., 2005; Szumlanski and Nielsen, 2009), and expres-sion of dominant-negative RABA2a inhibits cytokinesis of root tipcells in Arabidopsis (Chow et al., 2008). RABA1b also has beenreported to regulate trafficking between the TGN and PM (Asaokaet al., 2012; Feraru et al., 2012). However, it remains unclear whethereach RABA group regulates a distinctive trafficking pathway and ifany of the subgroups are involved in endocytic trafficking.

In this study, we first visualized and characterized the endo-cytic route of FLS2, whose internalization was induced by flg22treatment, in leaf epidermal cells of Nicotiana benthamiana. Bycomparing localization of internalized FLS2 with TGN and MVEmarkers, we identified a transient compartment with proper-ties that were a hybrid of the TGN and MVE. This compartmentappears to be an intermediate mediating endocytosis of FLS2.We then explored the functions of RABA subgroups in the in-tracellular transport of FLS2 to find distinct functions of differentsubgroups of RABA. Our results indicate discrete functional dif-ferentiation among RABA subgroups in endocytic and exocyticpathways.

RESULTS

flg22-Dependent Internalization of FLS2 in N. benthamianaLeaf Epidermal Cells

In this study, we used the transient expression system in N.benthamiana leaf epidermal cells by infiltration of Agrobacteriumtumefaciens, which has been successfully used to observe intra-cellular trafficking of fluorescent protein–tagged proteins and si-multaneous 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 greenfluorescent protein (GFP)–tagged FLS2 on the PM in untreatedN. benthamiana cells (Figure 1A), as reported in Arabidopsis leafepidermal cells (Robatzek et al., 2006). Then we examined whetherflg22-dependent internalization of FLS2-GFP is also observed inthis system. Upon treatment with flg22, dot-like structures withFLS2-GFP appeared in the cytoplasm 90 and 120 min after flg22application (Figures 1B and 1C). A lipophilic dye, FM4-64, wasalso internalized in a similar time frame in N. benthamiana leafepidermal cells (see Supplemental Figure 1 online). Treatmentwith flg22A.tum, the inactive peptide derived from Agrobacteriumflagellin (Felix et al., 1999), did not result in accumulation ofFLS2-GFP at the cytoplasmic organelles (Figure 1D), indicatingthat 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 internalreflection fluorescencemicroscopy–related technique called variable-angle epifluorescence microscopy or variable incidence anglefluorescence microscopy, which enables selective visualizationof sample surface regions (Fujimoto et al., 2007; Konopka andBednarek, 2008). FLS2 signals were concentrated on the PM 40minafter applying flg22 (Figures 1E to 1G), and clear punctate signals,probably reflecting concentrated or internalized FLS2, were ob-served on or near the PM at 60 min (Figure 1H).

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

To investigate which organelles are involved in endocytosis ofFLS2, we coexpressed FLS2-GFP with monomeric red fluorescentprotein (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 com-partments in plant cells (Ueda et al., 2001; Uemura et al., 2004;Takano et al., 2005; Dettmer et al., 2006; Lam et al., 2007; Viottiet al., 2010). From 90 to 120 min after flg22 treatment, FLS2-GFPwas observed overlapping or associated with mRFP-SYP61 (Fig-ures 2A and 2B). By contrast, FLS2-GFP was not observed nearthe SYP61 compartments after 210 min (Figure 2C). For quan-titative analysis of this observation, we extracted the dot-likesignals of FLS2-GFP and mRFP-SYP61 and measured the dis-tance from the center of a FLS2-GFP–positive dot to that of thenearest mRFP-SYP61 signal using a framework run on Metamorphsoftware, which we previously developed (Ito et al., 2012). Theresults were categorized into three groups by distances: (1) colo-calized (#0.24 mm; below the resolution limit of the objective lensused in this study); (2) associated (#1 mm); and (3) independent

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(>1 mm). We compared results between two groups of samples;one was observed from 80 to 140 min after flg22 treatment (anearly endocytic stage), and the other was observed from 140 to 200min after flg22 application (a late endocytic stage). In the early stage,;27.6% 6 6.4% and 39.6% 6 6.5% of FLS2-positive compart-ments were colocalized and associated with SYP61-positive dots,respectively (Figure 2R). In the late endocytic stage, ratios ofcolocalized and associated groups were significantly decreased to5.6%6 3.9% and 19.3%6 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 localizesat SYP61-positive compartments in the early stage. Intriguingly, anexperiment using another TGN marker, VHA-a1, yielded a differentresult. When VHA-a1 and FLS2 were coexpressed and treated withflg22, we did not observe colocalization of these proteins at anytime points after the flg22 treatment (Figures 2D to 2F). Thequantitative analysis also supported this observation; only 1.0%60.9% and 1.0% 6 1.7% of FLS2-positive compartments werecategorized as colocalized in the early and late stages, respectively(Figure 2S). For further clarification of this result, we directlycompared subcellular localization of cyan fluorescent protein(CFP)-SYP61 and VHA-a1-mRFP in cells expressing FLS2-GFPand in which flg22 treatment induced endocytosis. SYP61- andVHA-a1–positive dots were mostly overlapped; however, we foundthat the SYP61-positive compartments carrying FLS2-GFP did notharbor VHA-a1-mRFP (Figures 2M to 2P). We also confirmed thatCFP-SYP61 and mRFP-SYP61 exhibited the same localizationpattern (Figure 2Q), and mRFP-SYP61 and VHA-a1-mRFP exhibitedsimilar distribution patterns in relation to the Golgi apparatusvisualized with ST-Venus (Boevink et al., 1998) (see SupplementalFigure 2 online). These results clearly indicated that the compartmentbearing SYP61 and FLS2 observed in this study has a characteristicdistinct from the TGN in cells where endocytosis of FLS2 is notinduced.We next examined whether TagRFP-tagged ARA7 and VAMP727,

an MVE-localizing RAB GTPase and R-SNARE, respectively, werecolocalized with FLS2. These endosomal proteins were localizedon 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-ARA7in the same cell and compared their subcellular localization aftertreatment with flg22. At the earlier stage (100 min after the treat-ment), 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 butoccurred 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 as-sociation of SYP61 with FLS2, while FLS2 still exhibited goodcolocalization with ARA7 (Figure 3C). These results indicated theexistence of an endosomal compartment involved in flg22-triggeredendocytosis of FLS2 that seemed to harbor an intermediate propertybetween the TGN and MVE. Of interest, the colocalization of SYP61and ARA7 was observed only at compartments carrying endo-cytosed FLS2-GFP. In Arabidopsis plants grown under normallaboratory conditions, we noted no significant colocalization be-tween the MVE and TGN proteins thus far (Ebine et al., 2008,2011). Thus, such intermediate compartments might exist only whenendocytosis is highly activated.To verify this possibility, we compared localization of SYP61

and ARA7 after flg22 treatment between two samples: leaf epi-dermal cells expressing FLS2-GFP, mRFP-SYP61, and TagRFP-ARA7, and cells expressing mRFP-SYP61 and TagRFP-ARA7 butnot FLS2-GFP. As observed above, 4.6%6 2.0% of mRFP-SYP61–positive compartments bore TagRFP-ARA7 in FLS2-expressing cellsat 100 min after application of flg22 (Figures 3D and 3E); this is

Figure 1. Endocytosis of FLS2-GFP in Leaf Epidermal Cells of N. ben-thamiana.

(A) to (D) Max-intensity projection images are presented, each of whichwas reconstructed with a series of confocal Z-stack images taken at0.5-mm intervals.(A) FLS2-GFP localizes on the PM without any treatment. Bar = 10 mm.(B) FLS2-GFP is internalized into the cytoplasmic punctate compart-ments 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 flg22A.tum even after120 min.(E) to (H) Variable incidence angle fluorescence microscopy of FLS2 inclose proximity to the PM at 0 (E), 20 (F), 40 (G), and 60 min (H) afterflg22 treatment. Arrowheads indicate focal accumulation of FLS2 on thePM. Bar = 5 mm.

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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-SYP61at 90 min after flg22 treatment (A). After 120 min, FLS2-GFP is frequently observed associated with SYP61-positive compartments (B), but suchcolocalization and association are not observed after 210 min (C). Arrowheads indicate colocalized (distance#0.24 mm; below the resolution limit of theobjective lens used in this study) compartments, and arrows indicate associated (0.24 < distance #1 mm) compartments. Bars = 5 mm.(D) to (F) VHA-a1-mRFP does not overlap with FLS2-GFP. Bars = 5 mm.(G) to (I) FLS2-GFP colocalizes with TagRFP-ARA7 at all time points. Bars = 5 mm.(J) to (L) FLS2-GFP colocalizes with TagRFP-VAMP727 at all time points. Bars = 5 mm.(M) to (P) Cells expressing FLS2-GFP, VHA-a1-mRFP, and CFP-SYP61 observed after 130 min of flg22 treatments. Bars = 2 mm.(Q) CFP- and mRFP-tagged SYP61 completely overlapped when expressed in the same cell. Bar = 5 mm.(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; 189and 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 indicatethe SD values. Individual channel images are shown in Supplemental Figure 11A online. Average numbers of cells examined and numbers of punctaanalyzed in each experiment are shown in Supplemental Table 1 online.


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significantly higher than the ratio (1.1% 6 0.5%) of mRFP-SYP61compartments with TagRFP-ARA7 in cells not expressing FLS2after treatment with flg22 (n = 3 experiments, P < 0.05, Student’s ttest; Figures 3F and 3G, Table 1). These results suggested thatthe intermediate compartment identified in this study is a transientendosomal structure that is induced when endocytosis is highlyactivated. Under our experimental conditions, ;20% of SYP61-positive compartments were classified as “associated” withARA7-positive compartments, which indicated that the twocompartments were not overlapped but in close proximity(<0.24 mm distance between two compartments #1 mm), re-gardless of combinations of coexpressed proteins and timepoints (Table 1).

Effects of Mutant RABA on Endocytosis of FLS2

As described above, we found that compartments with inter-mediate properties between the TGN and MVE are involved inligand-induced endocytosis of FLS2. To identify molecular ma-chineries that control the trafficking pathway around this com-partment, we focused our interest on the RABA family becausesome RABA members have been reported to act in membranetrafficking around the TGN (Preuss et al., 2004; de Graaf et al.,2005; Chow et al., 2008), although involvement of this family inendocytosis remains unexplored. Because of the extreme ex-pansion of RABA members in Arabidopsis (see SupplementalTable 2 online), it is not practical at present to test their in-volvement 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 theeffect of a dominant-negative mutant of a representative member ofeach RABA subgroup whose expression was conditionally in-duced in cells expressing FLS2-GFP and organelle markers. Wecreated a nucleotide-free mutant of each member of the sixsubgroups of RABA (RABA1b, RABA2c, RABA3, RABA4c, RABA5c,and RABA6a) by replacing invariable Asn in the conservedGNKXD sequence with Ile (hereafter called the NI mutant) be-cause 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). Theexpression of the NI mutants was controlled by an estradiol-inducible promoter (Zuo et al., 2000) to avoid undesirable effectsof the mutant on transport of newly synthesized FLS2 to the PM.We examined the effects of these mutants on endocytic trans-port of FLS2-GFP, comparing FLS2-GFP subcellular localizationwith SYP61 and/or ARA7. For induction of mutant expression,10 mM estradiol was infiltrated into N. benthamiana leaves andincubated for 2 to 3 h, and then samples were treated with flg22.This condition is enough for accumulation of the mutant RABAmembers, as indicated by bright fluorescence from Venus fusedto the mutant RABA members (see Supplemental Figure 3 online).We examined the effects of expression of wild-type and NImutants for all six RABA subgroups, three of which (RABA2c,RABA3, and RABA5c) had no substantial effect on endocytictransport of FLS2-GFP; mutants of RABA3 and RABA5c did notexert significant effects on endocytosis of FLS2 (P > 0.05,Student’s t test), and the mutant of RABA2c had only a marginaleffect 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 sub-groups resulted in remarkable alteration in endocytic trafficking ofFLS2-GFP.

RABA6a and RABA4c Act in Distinct Stepsof FLS2 Endocytosis

In RABA6aN126I-expressing cells, we found that FLS2 colocalizedwell with SYP61 even at the late stage, at which FLS2 was observedon membrane compartments independent of SYP61-domains inwild-type RABA6a-expressing cells (Figure 4A). Quantificationfurther indicated that expression of RABA6aN126I increased co-localization of FLS2 with SYP61. We found that 41.2% 6 4.0%

Figure 3. Hybrid Nature of Endosomal Compartment Involved in EarlyFLS2 Endocytosis.

(A) to (C) Cells expressing FLS2-GFP, mRFP-SYP61, and TagRFP-ARA7observed after 100 (A), 140 (B), and 200 min (C) of flg22 treatment. Bar =2 mm.(D) to (G) The hybrid compartment is observed only when endocytosis ofFLS2-GFP is induced. Cells expressing mRFP-SYP61 and TagRFP-ARA7with FLS2-GFP ([D] and [E]) or without FLS2-GFP ([F] and [G]) were ob-served at 0 or 100 min after flg22 treatment. Arrowheads indicate com-partments bearing both mRFP-SYP61 and TagRFP-ARA7. Individualchannel images are shown in Supplemental Figure 11B online. Bars = 5 mm.

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and 15.4 6 3.5% of FLS2-positive compartments in early (80 to140 min) and late (140 to 200 min) stages, respectively, also boreSYP61 in RABA6aN126I-expressing cells, which was significantlyhigher than that observed in wild-type RABA6a-expressingsamples (21.6% 6 5.9% in the early stage and 5.2% 6 2.6% inthe late stage, n = 3 experiments, P < 0.05, Student’s t test;Figure 4B). This result appeared to indicate that RABA6a acts intrafficking of FLS2 from SYP61-positive compartments. We thenexamined whether ARA7 also occurs on the FLS2-positivecompartments in RABA6aN126I-expressing cells. We coexpressedTagRFP-ARA7 with FLS2-GFP and mutant or wild-type RABA6ato find that ARA7 also localized on FLS2-positive compartmentsin both cases (see Supplemental Figure 7 online). These resultsstrongly suggested that RABA6aN126I did not impair formation ofthe hybrid compartment bearing SYP61 and ARA7 but did causedelay in transport or transition from the intermediate endosometo the SYP61-free late endosomal compartment, which resultedin increased colocalization of FLS2 and SYP61 in NI mutant–expressing cells.

Intriguingly, RABA4cN128I conferred an apparently oppositeeffect from RABA6aN126I. In cells expressing the RABA4cN128I

mutant, the extent of colocalization between FLS2 and SYP61 inthe early stage was significantly reduced (5.8% 6 3.9%) com-pared with the result from coexpression of wild-type RABA4c(26.7% 6 9.6%, n = 3 experiments, P < 0.05, Student’s t test;Figures 5A and 5B). This result appeared to indicate that in-ternalized FLS2 was not transported to the SYP61 compart-ments in mutant-expressing cells. However, we found that ARA7did colocalize with FLS2 in RABA4cN128I-expressing cells (seeSupplemental Figure 8 online). Thus, there might be an alternativedirect trafficking pathway from the PM to ARA7-positive MVEswithout transit through SYP61-positive compartments.

These results clearly indicated that two distinct RABA sub-group members, RABA6a and RABA4c, regulate different stepsin the endocytic pathway of FLS2. Furthermore, the specificeffects of these RABA members also demonstrated that theinhibitory effects we observed in these experiments resultedfrom inhibition of the specific function of each RABA member inN. benthamiana, rather than reflecting the effect of titration ofgeneral RAB regulators such as RAB guanine nucleotide dis-sociation inhibitor. This notion was also supported by the resultsof coexpression of wild-type RABA members with NI mutants.We coexpressed mRFP-tagged wild-type RABA members withVenus-RABA4cN128I or Venus-RABA6aN126I for suppression ac-tivities for transport defects of FLS2 in tobacco cells expressing

FLS2-GFP and CFP-SYP61. The inhibitory effects of NI mutantswere specifically rescued by coexpression of their wild-typeversions but not by the other RABA members, which again in-dicated specific inhibition of the distinct trafficking event by eachmutant RABA, as is the case for other plant RAB GTPases (Fig-ures 4C and 5C; Batoko et al., 2000; Kotzer et al., 2004; Pinheiroet al., 2009). We then examined the order of functions of twoRABA members, RABA4c and RABA6a, in endocytic traffickingof FLS2 by double infiltration of their NI mutants. Simultaneousexpression of RABA4cN128I and RABA6aN126I caused the sametrafficking defect as RABA4cN128I (Figure 6). This result stronglysuggested that RABA4c acts in an earlier step than RABA6a inendocytic trafficking of FLS2.

RABA1b Functions in the Secretory Pathway

The NI mutant of RABA1b (RABA1bN126I) exerted another in-teresting effect. When expressed with this mutant, the distributionof mRFP-SYP61 and internalized FLS2-GFP was dramaticallychanged. mRFP-SYP61, which presented clear dot-shaped signalsin wild-type RABA1b-expressing cells, was scattered to tiny dotsthroughout the cytoplasm (Figure 7). Regarding FLS2-GFP, forwhich flg22 treatment induced endocytosis, we observed no dot-like signals in the mutant-expressing cells, but only smear signalsthat largely overlapped with mRFP-SYP61 (Figure 7). These re-sults indicated that RABA1bN126I did not block the internalizationof FLS2 from the PM but that the morphology of compartmentswith SYP61 to which FLS2 was transported was severely affected.These findings additionally suggested that expression of mu-

tant RABA1b resulted in alteration of the morphology of the TGN.Because the TGN also plays fundamental roles in the secretorypathway, we then examined the effects of NI mutants of RABA1band other RABA subgroups on the steady state localization ofFLS2-GFP at the PM. For this purpose, chimeric genes com-posed 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 memberswe examined, only RABA1bN126I caused substantial alteration inFLS2 distribution. In the RABA1bN126I-expressing cells, FLS2 wasmainly observed as scattered tiny dots throughout the cytoplasm,and weak localization to the PM was also visible (Figure 8A).Moreover, constitutive expression of RABA1bN126I affected dis-tribution of mRFP-SYP61; mRFP-SYP61 was also dispersedthroughout the cytoplasm as observed in the induced-expressionexperiment (Figure 8B). These results indicated that transport of

Table 1. Quantification of Colocalization between SYP61 and ARA7

Expression of FLS2 flg22 Treatment Colocalized Associated Independent

+FLS2 No treat. (n = 335) 0.7% (60.6%) 21.0% (61.3%) 78.3% (60.7%)Early stage (n = 539) 4.6% (62.0%) 22.5% (62.2%) 72.9% (60.9%)Late stage (n = 470) 1.0% (60.9%) 19.8% (60.9%) 79.2% (61.1%)

2FLS2 No treat. (n = 536) 0.0% (60.0%) 22.0% (62.9%) 78.0% (62.9%)Early stage (n = 624) 1.1% (60.5%) 21.6% (61.8%) 77.3% (61.3%)Late stage (n = 532) 1.2% (61.0%) 20.0% (65.3%) 78.9% (65.5%)

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 (2FLS2). Data were collected from three independent experiments.


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newly synthesized FLS2 to the PM was disturbed by defectivetrafficking around the SYP61-positive TGN in the mutant-expressingcells.We also tested for an inhibitory effect of RABA1bN126I on transport

of another PM protein, BOR1, a boron efflux carrier whose lo-calization is regulated by external boron concentration (Takanoet al., 2002, 2005). Steady state localization of BOR1 was slightlydifferent from that of FLS2; BOR1-GFP was observed on punctateorganelles in addition to the PM, probably reflecting its constitutiveinternalization (Takano et al., 2005; Figure 9A). Coexpression ofRABA1bN126I also resulted in alteration of this localization pattern;BOR1 predominantly localized in scattered small dots in thecytoplasm, and only weak PM localization was detected in cellsexpressing RABA1bN126I (Figure 9B). This result again suggestedthat RABA1b acts in the secretory pathway. By contrast, the NImutants of RABA4c and RABA6a had no visible effect on pre-sentation 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 localizationof FLS2 (see Supplemental Figure 9 online). The inhibitory effectof RABA1bN126I in secretory trafficking of FLS2 was attributable tospecific inhibition of the RABA1 function because estradiol-inducedcoexpression of wild-type RABA1b, but not RABA4c or RABA6a,restored the PM localization of FLS2-GFP (Figure 8C). Theseresults again support the specific and distinct functions of RABAsubgroups in plant cells.

Distinct Subcellular Localizations of RABA1b, RABA4c,and RABA6a

We next asked whether three RABA members with differentfunctions, RABA1b, RABA4c, and RABA6a, reside on differentsubcellular compartments or on the same organelle. We thus di-rectly compared the localization of these proteins using combi-nations of different fluorescent proteins. Expression of fluorescentprotein–tagged RABA members was induced by incubation with10 mM estradiol for 1 h. RABA1b, RABA4c, and RABA6a localizedat various sizes of dot-like structures (Figure 10A). These RABAmembers exhibited good colocalization on comparatively largecompartments; however, they did not overlap at small vesicularstructures (Figure 10A). This different localization is not due tothe difference in fused fluorescent proteins because RABAmembers tagged with different fluorescent proteins localized tothe same compartments (Figure 10B).In Arabidopsis root epidermal cells, comparatively large com-

partments bearing RABA1b partly overlap with the TGN marked

Figure 4. The RABA6a NI Mutant Increases the Ratio of Compartmentswith Both FLS2-GFP and mRFP-SYP61.

(A) Cells expressing FLS2-GFP and mRFP-SYP61 with wild-type RA-BA6a (top panels) or RABA6aN126I (bottom panels) were observed after120 or 200 min after flg22 treatment. Expression of RABA6a and RA-BA6aN126I was induced by estradiol treatment. Arrowheads and arrowsindicate FLS2-GFP signals colocalized and associated with TagRFP-SYP61, respectively. Individual channel images are shown in SupplementalFigure 11C online. Bar = 5 mm.(B) Stacked bar graph indicating results of quantitative analyses of co-localization between FLS2-GFP and mRFP-SYP61. WT, wild-type RA-BA6a-expressing cell; NI, RABA6aN126I-expressing cells. Data werecollected from three independent experiments, and 178 and 162 (wildtype) or 204 and 209 (NI) FLS2-positive dots in total were observed inearly (80 to 140) and late (140 to 200) stages, respectively. Error barsindicate SD values. Average numbers of cells examined and numbers of

puncta analyzed in each experiment are shown in Supplemental Table 1online.(C) Stacked bar graph indicating results of quantitative analyses of co-localization between FLS2-GFP and CFP-SYP61 in RABA6aN126I-expressingcells. RABA6a, RABA6a-coexpressing cells; RABA4c, RABA4c-coexpressingcells. Data were collected from three independent experiments; 64 and51 (RABA6a) and 118 and 87 (RABA4c) FLS2-positive dots in total wereobserved in early (80 to 140) and late (140 to 200) stages, respectively.Error bars indicate SD values. Average numbers of cells examined andnumbers of puncta analyzed in each experiment are shown in SupplementalTable 1 online.

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by SYP43 or VHA-a1 (Asaoka et al., 2012). Thus, we examinedwhether RABA members are also localized on the TGN in our ex-perimental system. When coexpressed in FLS2-GFP–expressingN. benthamiana leaf epidermal cells, Venus-tagged RABA mem-bers and mRFP-SYP61 frequently colocalized on larger particles,while mRFP-SYP61 did not target to RABA-positive smallervesicles (Figure 10C). The colocalization of SYP61 and RABA1bwas not affected by RABA4cN128I or RABA6aN126I, which exertedinhibitory effects on endocytic transport of FLS2 (see SupplementalFigure 10 online). These results indicated that three RABAmemberslocalize on different compartments with overlap on the TGN, whichis consistent with their different functions in transport of FLS2.

DISCUSSION

Internalized FLS2 Is Transported to IntermediateCompartments between the TGN and MVE

Endocytic transport of the flagellin receptor FLS2 is tightly coupledwith the plant defense response and thus is of great interest andimportance for understanding defense regulatory mechanisms(Robatzek et al., 2006). Recent work showed that FLS2 internalizesinto cells in a ligand-inducedmanner, passing through an endocyticpathway distinct from that of constitutively recycled FLS2. Thisreport also indicated that RAB5 is responsible for endocytosis ofFLS2 (Beck et al., 2012). However, the identity of endosomalcompartments mediating endocytic traffic of this receptor andthe molecular details of its mechanisms remain largely unknown.In this study, we showed that internalized FLS2 passed througha compartment with a hybrid nature between the TGN and MVEand that localized both mRFP-SYP61 and TagRFP-ARA7. Theplant TGN is also recognized as an early endosome because aninternalized lipophilic dye, FM4-64, stains the TGN earlier thanthe 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 theendocytosed cargo proteins BOR1 and BRI1 reach the TGN.Direct maturation from the TGN to MVE has also been proposedrecently (Bottanelli et al., 2011; Scheuring et al., 2011), sug-gesting the sequential action of the TGN and MVE along theendocytic pathway. However, recent studies have indicated thatplants also harbor a clathrin-independent endocytosis mech-anism (Li et al., 2012). Thus, the endocytic route could varydepending on each cargo, which would not be distinguishableby monitoring bulk membrane flow using FM dyes. What isnow needed is to monitor protein cargos to obtain robust andspecific information.

Figure 5. The RABA4c NI Mutant Decreases the Ratio of Compartmentswith Both FLS2-GFP and mRFP-SYP61.

(A) Cells expressing FLS2-GFP and mRFP-SYP61 with wild-type RA-BA4c (top panels) or RABA4cN128I (bottom panels) were observed after80 or 170 min after flg22 treatment. Expression of RABA4c and RA-BA4cN128I was induced by estradiol treatment. Arrowheads indicateFLS2-GFP signals colocalized with TagRFP-SYP61. Note that colocali-zation was not observed in RABA4cN128I-expressing cells. Individualchannel images are shown in Supplemental Figure 11D online. Bar = 5 mm.(B) Stacked bar graph indicating results of quantitative analyses of co-localization between FLS2-GFP and mRFP-SYP61. WT, wild-type RA-BA4c-expressing cell; NI, RABA4cN128I-expressing cells. Data werecollected from three independent experiments, and 174 and 153 (wildtype) 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 indicateSD values. Average numbers of cells examined and numbers of punctaanalyzed in each experiment are shown in Supplemental Table 1 online.

(C) Stacked bar graph indicating results of quantitative analyses ofcolocalization between FLS2-GFP and CFP-SYP61 in RABA4cN128I-expressing cells. RABA4c, RABA4c-coexpressing cells; RABA6a,RABA6a-coexpressing cells. Data were collected from three independentexperiments; 32 (RABA4c) and 30 (RABA6a) FLS2-positive dots intotal were observed in the early (80 to 140) stage. Error bars indicateSD values. Average numbers of cells examined and numbers of punctaanalyzed in each experiment are shown in Supplemental Table 1online.


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To investigate the function of the TGN and MVE in ligand-triggered endocytosis of FLS2 and to examine which RabGTPases are involved, we used an N. benthamiana transient ex-pression system, which allowed us time-sequential and multicolorobservation of endocytosed FLS2 and other trafficking compo-nents in living cells. In this system, we found that induction ofendocytosis of FLS2 led to formation of a novel compartment thatharbored both SYP61, a TGN-resident SNARE protein, and ARA7,the MVE-resident Rab GTPase, suggesting that the nature of thiscompartment is intermediate between the TGN and MVE. Of in-terest, VHA-a1, another well-characterized TGN protein, was notrecruited to this hybrid compartment. Both SYP61 and VHA-a1colocalize with SYP43, a SNARE protein residing on the TGN inArabidopsis root epidermal cells (Uemura et al., 2012). Thus, ourresults could further indicate the unique character of the in-termediate compartment, as it harbors a limited set of TGN proteinsin addition to endosomal RAB5 GTPase. Because quantitativeanalyses in previous studies have shown that the known TGNmarkers are not colocalized completely (Dettmer et al., 2006; Chowet al., 2008; Boutté et al., 2010; Gendre et al., 2011), it is also

possible that a specific population of the TGN (or a specificmembrane domain around the TGN) serves in flg22-triggeredendocytic transport of FLS2. An interesting future project wouldbe studying how endocytic trafficking and remodeling of theTGN are integrated in plant cells.Another important finding is that SYP61 appeared to segre-

gate to distinct membrane domains from ARA7 and FLS2 andwas gradually removed from the hybrid compartment, whileFLS2 exhibited good colocalization with ARA7 even after a longincubation. This result suggests that the TGN components aregradually eliminated from the hybrid compartment, leading tomaturation to an MVE with a fully late endosomal nature. In agree-ment with this, endosomal sorting complex required for transport(ESCRT) components are gradually recruited to the TGN to leadmaturation of the TGN to the MVE, and colocalization of SYP61and ARA7 is also observed when the function of the TGN is ham-pered by concanamycin A or the function of the ESCRT complex isinhibited (Scheuring et al., 2011). Thus, our results, together withthe findings by Scheuring et al., might suggest that maturationof 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 observedbecause of too-rapid maturation kinetics. Another possibility isthat internalized FLS2 is transported directly to the hybrid en-dosomes bearing both SYP61 and ARA7. Although we have not

Figure 6. Coexpression of NI Mutants of RABA4c and RABA6a.

(A) Cells expressing FLS2-GFP and CFP-SYP61 with both RABA6aN126I

and RABA4cN128I were observed after 100 or 170 min following flg22treatment. Expression of RABA6aN126I and RABA4cN128I was induced byestradiol treatment. Note that colocalization was not observed in cellsexpressing RABA6aN126I and RABA4cN128I. Individual channel imagesare shown in Supplemental Figure 11E online. Bar = 5 mm.(B) Stacked bar graphs indicating results of quantitative analyses ofcolocalization between FLS2-GFP and CFP-SYP61. Data were collectedfrom three independent experiments; 69 and 63 FLS2-positive dots intotal were observed in early (80 to 140) and late (140 to 200) stages,respectively. Error bars indicate SD values. Average numbers of cellsexamined and numbers of puncta analyzed in each experiment areshown in Supplemental Table 1 online.

Figure 7. The RABA1b NI Mutant Affects the Localization of mRFP-SYP61.

Localization of FLS2-GFP and mRFP-SYP61 was observed at the in-dicated times after flg22 treatment in RABA1b- (top panels) or RABA1bN126I-expressing cells (bottom panels). Expression of RABA1b and RABA1bN126I

was induced by estradiol treatment. Individual channel images are shownin Supplemental Figure 11F online.

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detected colocalization of ARA7 and TGN markers at the steadystate in our experimental system, another group reported thatARA7 is also detected at the TGN (Stierhof and El Kasmi, 2010).Thus, a small number of the hybrid compartments could exist inplant cells, with a population that might increase under specificconditions that require enhanced endocytosis. Consistent withthis concept, a recent study on FLS2 trafficking in Arabidopsis alsosuggested 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 revealits nature and function. Whether other endocytic cargos also passthrough this intermediate compartment and whether this hybridcompartment is observed when endocytosis is activated in otherplants including Arabidopsis would be interesting questions toaddress in future research.

RABA/RAB11 Groups Have Distinct Functions in MembraneTrafficking around the TGN

We succeeded in finding RABA members whose respectivedominant-negative mutants affect different steps of endocytosisof FLS2. RABA6aN126I affected the maturation step from thehybrid endosomes to the late endosome/MVE; FLS2 retainedlocalization on the hybrid endosomes with both SYP61 andARA7 in RABA6aN126I-expressing cells even after a long in-cubation, suggesting a delay in the maturation process. In theanimal system, early-to-late maturation of endosomes is asso-ciated with replacement of Rab GTPases, which is referred to as“Rab conversion.” Rab5 on the early endosomes in animal cellsis gradually replaced by Rab7, which is responsible for lateendosomal trafficking mediated by effector complexes of theseRab GTPases, CORVET and HOPS (Rink et al., 2005). Consideringthe subcellular localization of RABA members around the TGN(Preuss et al., 2004; de Graaf et al., 2005; Chow et al., 2008), ourresults may indicate that plants also employ a similar molecularmechanism in endosomal maturation, which is associated withRab conversion from RABA6a to ARA7.However, coexpression of RABA4cN128I resulted in a signifi-

cant decrease in the endosome population with both SYP61 andFLS2, while internalization of FLS2 was not markedly affected.

Figure 8. Constitutive Expression of RABA1bN126I Alters Steady StateLocalization of FLS2-GFP and mRFP-SYP61.

(A) Localization of FLS2-GFP in cells expressing RABA1bN126I (leftpanel), RABA4cN128I (middle panel), or RABA6aN126I (right panel) underregulation of the 35S promoter. Max intensity projection images arepresented, each of which is reconstructed with a series of confocal Z-stack images taken at 0.5-mm intervals. Bar = 5 mm.(B) Localization of FLS2-GFP and mRFP-SYP61 in RABA1b- or RA-BA1bN126I-expressing cells under regulation of the 35S promoter. Leftpanels show the middle plane of cells, and right panels show the con-focal plane near the PM in the same cells shown in left panels. Individualchannel images are shown in Supplemental Figure 11G online. Bars =5 mm.(C) Localization of FLS2-GFP in cells expressing 35S promoter-drivenRABA1bN126I and estradiol-inducible promoter-driven RABA1b (leftpanel), RABA4c (middle panel), or RABA6a (right panel). Top and bottompanels indicate cells before and after estradiol treatment, respectively.Bar = 5 mm.

Figure 9. Constitutive Expression of RABA1bN126I Alters Steady StateLocalization of BOR1-GFP.

(A) Localization of BOR1-GFP. Bar = 5 mm.(B) to (D) RABA1bN126I (B), RABA4cN128I (C), or RABA6aN126I (D) werecoexpressed under regulation of the 35S promoter in cells expressingBOR1-GFP. Max intensity projection images are presented, each ofwhich is reconstructed with a series of confocal Z-stack images taken at0.5-mm intervals.


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This result appears to indicate that FLS2 does not reach theTGN. Curiously, however, we found that ARA7 resided on theFLS2-positive compartment also in RABA4cN128I-expressingcells. This observation might be explained by accelerated mat-uration from the hybrid compartment to the late endosome;however, that would not be likely because a dominant-negativeRab GTPase generally inhibits or delays trafficking events, which

should also be the case for RABA4cN128I. Thus, our finding mayindicate an alternative trafficking pathway that mediates trans-port from the PM to the ARA7-positive endosome directly whenthe early endocytic pathway to the TGN/early endosome iscompromised. It is also possible that the effects of RABA6aN126I

and RABA4cN128I on colocalization of FLS2 and SYP61 repre-sent altered distribution of SYP61; these mutant RABA memberscould affect transport of SYP61 to or from ARA7- and FLS2-positive endosomes. However, this situation would not be aslikely because the localization pattern of SYP61 and RABA1b wasnot affected by coexpression of RABA6aN126I or RABA4cN128I.Another interesting finding is that RABA1b, a member of the

largest RABA subgroup, is involved in delivery of newly syn-thesized FLS2 and BOR1 to the PM. Coexpression of RA-BA1bN126I resulted in dispersed localization of SYP61, whichalso indicates that this RABA member is responsible for main-tenance of TGN distribution. However, RABA1bN126I did notseem to affect internalization of FLS2 to the SYP61-positivecompartment, as shown by relocalization to a dispersed patternsimilar to that of SYP61 in RABA1bN126I-expressing cells afterflg22 treatment. We did not see any effects on FLS2 and SYP61distribution for equivalent mutants of other members includingRABA4cN128I and RABA6aN126I in both estradiol-induced and35S promoter–driven constitutive expression. These resultsstrongly suggest that the RABA1 group specifically acts in thesecretory pathway but not in the endocytic pathway, which isalso required for maintenance of TGN morphology. RABA1bfunction in the secretory pathway is also suggested by our studyin Arabidopsis plants (Asaoka et al., 2012)

Functional Diversification of Plant RABA Members

Our data clearly indicate that different RABA members havedistinct functions in intracellular trafficking of FLS2. Consistentwith this, three members identified in this study as being in-volved, RABA1b, RABA4c, and RABA6a, exhibited distinctsubcellular localization, supporting that these members act atdifferent membrane domains around the TGN. Recent compara-tive genomics indicate that extreme expansion of Rab11 homo-logs is one of the most remarkable characteristics in organizationof plant Rab GTPases. How Rab functions have diverged duringthe expansion of plant Rab11 homologs has remained unclear,but our results indicate that the functions of RABA/Rab11members have diversified from each other. Another importantfinding is that plant RABA/Rab11 is involved in both secretoryand endocytic pathways. The functional diversity together withtheir important roles in plant-unique physiological events (Preusset al., 2004; de Graaf et al., 2005; Chow et al., 2008; Szumlanskiand Nielsen, 2009) indicate that expansion of this family couldplay pivotal roles in the increased complexity of membranetrafficking pathways in plant cells, which are recruited to plant-unique physiological events during plant evolution. The nextinteresting question is how the RABA/Rab11 members encodedby a paralogous set of genes acquired diverse functions in mem-brane trafficking. Further functional analyses of this group, aswell as identification and characterization of upstream regulatorsand downstream effectors, would be needed to answer thisquestion.

Figure 10. Subcellular Localization of RABA1b, RABA4c, and RABA6a.

(A) All three RABA members localized on large clear punctate com-partments and tiny scattered vesicles when expressed in N. benthamianacells separately (top panels). When two RABA members tagged with theindicated fluorescent proteins were expressed in the same cells, theycolocalized on the large compartments but not on the scattered vesicles(bottom panels). Green, magenta, and white arrowheads indicate theVenus, mRFP, and overlapped signals, respectively. Bar = 5 mm.(B) When the same RABA members were tagged with different fluores-cent proteins and expressed in the same cell, localization patternsoverlapped almost completely. Bar = 5 mm.(C) mRFP-SYP61 was coexpressed with RABA1b (left panel), RABA4c(middle panel), or RABA6a (right panel) in FLS2-GFP–expressing cells.Bar = 5 mm.Individual channel images are shown in Supplemental Figure 11H online.

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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 tumefaciensstrain GV3101:pMP90. A single colony of each transformant was culturedin 5mL YEBmedium (5 g beef extract, 1 g yeast extract, 5 g Suc, and 0.49 gMgSO4$7H2O dissolved in 1 liter of water) supplemented with 10 mg/mLrifampicin at 30°C overnight. The bacteria were collected and re-suspended in infiltration buffer (10 mMMES, 10 mMMgCl2, and 0.15 mMacetosyringone, pH 5.5) at 0.02 (FLS2-GFP) or 0.1 (other constructs)OD600. The agrobacteria carrying p19 were resuspended together with allsamples (Voinnet et al., 2003). The resuspended agrobacteria were in-filtrated into leaves ofN. benthamianawith gentle pressure using syringeswithout needles. For coexpression of proteins, bacterial strains withdifferent constructs were mixed before inoculation. Samples were ob-served within 48 h after infiltration. For induction of endocytosis of FLS2,100 mM flg22 peptide was infiltrated into leaves expressing FLS2-GFP.For estradiol-dependent induction of gene expression, b-estradiol (10 mM)was applied for 1 to 3 h before treatment with flg22.

FM4-64 Staining

FM4-64 (50 mM; Invitrogen/Molecular Probes; T13320) dissolved in distilledwater was infiltrated to the leaf tissue of N. benthamiana from the abaxialsurface. Samples were incubated at room temperature for indicated timesand 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 werecloned into pGWB1 containing the 35S promoter (Nakagawa et al., 2007).For RABA members (RABA1b, RABA2c, RABA3, RABA4c, RABA5c, andRABA6a), cDNA fragments were amplified by PCR using the primers listedin Supplemental Table 3. Amplified wild-type and mutant cDNA fragmentswere conjugated with cDNA for fluorescent proteins and cloned intopMDC7 (Curtis and Grossniklaus, 2003) for estradiol-inducible expression.For constitutive expression, cDNA fragments of RABAwere introduced intopB7WGY2 and pH7WGR2 containing YFP and RFP, respectively (Karimiet al., 2002).

Confocal Laser Scanning Microscopy

For single-color imaging, GFPwas excited by a diode-pumped solid-statelaser (Cobolt Blues) at 473 nm and observed under a microscope (BX51;Olympus) equipped with a confocal scanner unit (CSU10; YokogawaElectric) and a cooled charge-coupled device camera (ORCA-AG;Hamamatsu Photonics). Images were processed with IPLab software (BDBiosciences) and Photoshop CS5 (Adobe). Multicolor observation wasperformed using an LSM710 or LSM780 confocal microscope (Carl Zeiss)with the oil immersion lens (363, numerical aperture = 1.40). Spectralunmixing (if necessary) and processing of obtained images were per-formed using ZEN 2008 or ZEN 2011 software (Carl Zeiss). For dual-colorimaging, GFP and Venus were excited at 488 nm, and the emission wascollected between 501 and 545 nm. mRFP and TagRFP were excited at561 nm, and the emission was collected between 570 and 615 nm. Forthree-color imaging, samples expressing fluorescent proteins (combi-nations of three of four XFPs: GFP, Venus, mRFP, and TagRFP) wereexcited at 488 and 561 nm, and the emission was collected between 484and 640 nm. For three- or four-color imaging using CFP, samples wereexcited at 405, 488, and 561 nm, and the emission was collected between

411 and 633 nm. The colocalization analysis was performed as describedpreviously (Ito et al., 2012) with Metamorph software (Molecular Devices).

Variable Incidence Angle Fluorescence Microscopy

The leaves from infiltratedN.benthamianawere placed ona glass slide (76326 mm; Matsunami) and covered with a cover slip 0.12- to 0.17-mm thick(24 3 60 mm; Matsunami), and epidermal cells were observed undera fluorescence microscope (Nikon Eclipse TE2000-E and a CFI ApoTIRF 3100 H/1.49–numerical aperture objective) equipped with a NikonTIRF2 system. GFP was simultaneously excited with a 488-nm laser. Allimages were acquired with an Andor iXonEM electron multiplying chargecoupled device (EMCCD) camera; each frame was exposed for 100 ms. Theacquired images were prepared and analyzed using Photoshop CS5 (AdobeSystems) and NIS-Elements software (Nikon).

Accession Numbers

The Arabidopsis Genome Initiative locus identifiers for the genesmentioned inthis 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 CompartmentsAre Frequently Associated with the Golgi Apparatus.

Supplemental Figure 3. Estradiol Treatment Efficiently Induces GeneExpression.

Supplemental Figure 4. The NI Mutant of RABA2c Does Not AffectTrafficking Kinetics of FLS2-GFP.

Supplemental Figure 5. The NI Mutant of RABA3 Does Not AffectTrafficking Kinetics of FLS2-GFP.

Supplemental Figure 6. The NI Mutant of RABA5c Does NotAffect Trafficking Kinetics of FLS2-GFP.

Supplemental Figure 7. Colocalization of FLS2-GFP and TagRFP-ARA7 in RABA6aN126I-Expressing Cells.

Supplemental Figure 8. FLS2-GFP and TagRFP-ARA7 Colocalize inRABA4c- and RABA4cN126I-Expressing Cells after flg22 Treatment.

Supplemental Figure 9. Localization of FLS2-GFP in N. benthamianaLeaf Epidermal Cells Expressing RABA2cN125I, RABA3N141I, or RA-BA5cN125I Is Constitutively under Regulation of the 35S Promoter.

Supplemental Figure 10. Quantitative Analysis of Colocalizationbetween SYP61 and RABA1b.

Supplemental Figure 11. Individual Channel Images for All MulticolorFigures.

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.

ACKNOWLEDGMENTS

We thank Nam-Hai Chua (Rockefeller University) for providing the plasmidpMDC7, Shigeyuki Betsuyaku and Minobu Shimizu (University of Tokyo) for


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their generous support in experiments using N. benthamiana, and SilkeRobatzek for providing the FLS2-GFP construct and fruitful discussions.This work was supported by Grants-in-Aid for Scientific Research and theTargeted Proteins Research Program from the Ministry of Education,Culture, Sports, Science, and Technology of Japan (A.N. and Ta.U.) andJapan Science and Technology Agency, PRESTO (Ta.U.).

AUTHOR CONTRIBUTIONS

S.-w.C. performed the main parts of the experiments and wrote thearticle. T.T. constructed the experimental system using N. benthamiana.K.E. constructed plasmids used in this study. To.U. constructed plasmidsused in this study. Ta.U. designed the research and wrote the article. A.N.supervised the study.

Received December 20, 2012; revised February 26, 2013; acceptedMarch 14, 2013; published March 26, 2013.

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