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doi:10.1111/j.1600-0854.2011.01222.x

Mechanisms of Functional Specificity AmongPlasma-Membrane Syntaxins in Arabidopsis

Ilka Reichardt1,2, Daniel Slane1, Farid El Kasmi1,

Christian Knoll1, Rene Fuchs3,4, Ulrike Mayer1,

Volker Lipka3,4 and Gerd Jurgens1,∗

1ZMBP, Entwicklungsgenetik, Universitat Tubingen, Aufder Morgenstelle 3, 72076 Tubingen, Germany2Current address: Institute of Molecular Biotechnology ofthe Austrian Academy of Sciences (IMBA), Dr. BohrGasse 3, 1030 Vienna, Austria3Sainsbury Laboratory, John Innes Centre, NorwichResearch Park, Norwich NR4 7UH, UK4Current address: Albrecht-von-Haller-Institute for PlantSciences, Georg-August-Universitat Gottingen, UntereKarspule 2, 37073 Gottingen, Germany*Corresponding author: Gerd Jurgens, gerd.juergens@zmbp.uni-tuebingen.de

Syntaxins and interacting SNARE proteins enable mem-

brane fusion in diverse trafficking pathways. The Ara-

bidopsis SYP1 family of plasma membrane-localized

syntaxins comprises nine members, of which KNOLLE

and PEN1 play specific roles in cytokinesis and innate

immunity, respectively. To identify mechanisms confer-

ring specificity of action, we examined one member

of each subfamily – KNOLLE/SYP111, PEN1/SYP121 and

SYP132 – in regard to subcellular localization, dynamic

behavior and complementation of knolle and pen1

mutants when expressed from the same promoters.

Our results suggest that cytokinesis-specific syntaxin

requires high-level accumulation during cell-plate forma-

tion, which necessitates de novo synthesis rather than

endocytosis of pre-made protein from the plasma mem-

brane. In contrast, syntaxin in innate immunity does not

need upregulation of expression but instead requires

pathogen-induced and endocytosis-dependent retarget-

ing to the infection site. This feature of PEN1 is not

afforded by SYP132. Additionally, PEN1 could not substi-

tute for KNOLLE because of SNARE domain differences,

as revealed by protein chimeras. In contrast, SYP132 was

able to rescue knolle as did KNOLLE-SYP132 chimeras.

Unlike KNOLLE and PEN1, which appear to have evolved

to perform specialized functions, SYP132 stably localized

at the plasma membrane and thus might play a role in

constitutive membrane fusion.

Key words: Arabidopsis, cell cycle, cytokinesis, innate

immunity, plasma membrane, protein dynamics, recy-

cling, syntaxin

Received 20 April 2011, revised and accepted for

publication 1 June 2011, published online 28 June 2011

SNARE proteins constitute a family of membrane-anchored proteins that play key roles in membrane fusionevents of intracellular trafficking pathways by forming

SNARE complexes that dock membranes to be fused.Their main characteristic feature is an evolutionarilyconserved domain of 60–70 amino acids arrangedin heptad repeats, which has been designated theSNARE domain (1). Based on the conserved amino-acidresidue at the center of the SNARE domain, SNAREproteins have been classified into R- (arginine) and Q-(glutamine) SNAREs. The Q-SNARE family is furtherdivided into four subfamilies (Qa-, Qb-, Qc- and Qb,c-SNAREs) based on differences in the structure of theSNARE domain (2). Each SNARE complex is formed byassociation of four interacting SNARE domains, one eachfrom VAMP/R-SNARE on the donor membrane and threefrom Q-SNAREs on the acceptor membrane: one fromsyntaxin/Qa-SNARE and two from either SNAP25/Qb,c-SNARE or one each from two t-SNARE light chains/Qb-and Qc-SNAREs (1).

Syntaxins/Qa-SNAREs are conserved among all eukaryoticorganisms, a fact that emphasizes a universal rolefor syntaxins in membrane trafficking (3). Comparedto other organisms like yeast and mammals, whichhave two and four genes encoding plasma membrane-localized syntaxins, respectively, plant genomes harbor anincreased number of genes for syntaxins involved in thelate secretory pathway (4–6). The Arabidopsis genomeencodes 18 putative syntaxins representing 5 differentSyntaxin of Plant (SYP) families, of which the 9 membersof the SYP1 family have been localized to the plasmamembrane (5–7). Although, in principle, redundancywould explain the occurrence of the numerous SYP1syntaxins in Arabidopsis, there is also evidence forfunctional diversification.

SYP1 syntaxins show different spatio-temporal expres-sion profiles (8). For instance, SYP132 is expressedubiquitously in all tissues throughout plant develop-ment, whereas SYP124, SYP125 and SYP131 are onlyexpressed in pollen, and SYP123 appears to be exclu-sively expressed in root hair cells during root develop-ment (8). The SYP1 family also includes the functionallywell-characterized syntaxins KNOLLE/SYP111 (9,10) andPEN1/SYP121/SYR1 (11–14). KNOLLE is a specializedSYP1 syntaxin of flowering plants that seems to be exclu-sively required for cytokinesis and has no ortholog in lowerplants or non-plant organisms, suggesting that other SYP1syntaxins played a comparable role in plant cytokinesisbefore KNOLLE evolved (6). KNOLLE gene expressionis confined to late G2 and M phases of the cell cycle,which is mediated by mitosis-specific activator (MSA) pro-moter elements that bind R1R2R3-Myb transcription fac-tors (9,15). In addition, KNOLLE protein only accumulatesduring mitosis, localizing to the forming cell plate that

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eventually separates the daughter cells, and is degradedimmediately after completion of cytokinesis (10,16). LikeKNOLLE, the plasma membrane-localized syntaxin PEN1also arose late in plant evolution (6). PEN1 is involvedin non-host penetration resistance against the powderymildew Blumeria graminis f. sp. hordei (B. g. hordei)and mediates vesicle fusion at B. g. hordei – Arabidopsisinteraction sites (12,13). PEN1/SYP121 also known asSYR1 was originally identified in tobacco for its involve-ment in potassium and chloride channel response to theplant hormone ABA in guard cells (11). SYR1/SYP121 hasbeen shown to affect KAT1 potassium channel activity,KAT1 trafficking to the plasma membrane, and directlyinteracts with the channel subunit KC1 via an FxRFmotif (14,17,18). PEN1/SYP121 has also been subjectedto structure–function analysis. However, that study didnot address the problem of syntaxin specificity (19). Athird member of the SYP1 family, SYP132, is ubiquitouslyexpressed in Arabidopsis (5) and has been related to theevolutionarily most ancient branch of SYP1 proteins (6). Sofar no function for SYP132 has been reported in Arabidop-sis. The tobacco ortholog NbSYP132 contributes to resis-tance against bacterial pathogens, mediating secretion ofpathogenesis-related protein 1 (20), whereas MedicagoMtSYP132 has been localized to the plasma membranesurrounding Rhizobium infection threads and to the sym-biosome membrane (21).

We have previously analyzed mechanisms of KNOLLEspecificity in cytokinesis and identified the mitosis-specificexpression of KNOLLE as a major determinant (22). Theclosest paralog of KNOLLE named SYP112 was func-tionally equivalent to KNOLLE when expressed from theKNOLLE promoter. In contrast, PEN1 was not able tosubstitute for KNOLLE in cytokinesis when expressedlike KNOLLE. The results suggested functional diver-gence of the two SYP1 proteins but did not offer anymechanistic explanation. To analyze mechanisms thatensure the specific biological activity of KNOLLE andPEN1, we expressed these specialized SYP1 syntaxinsand SYP132 from the same set of promoters in sta-bly transformed Arabidopsis plants. The transgenicallymade proteins were analyzed for their ability to substi-tute for KNOLLE or PEN1 in the respective mutant aswell as their subcellular localization and dynamics. Wealso tested chimeric proteins that were generated byexchanging the SNARE domain between KNOLLE andPEN1 or SYP132. We show that KNOLLE is completelyfunctional during cytokinesis when carrying the SNAREmotif of SYP132 but fails to fulfill proper cell-plate for-mation when carrying the SNARE motif of PEN1. Ourresults suggest that functional specificity of the spe-cialized SYP1 syntaxins KNOLLE and PEN1 divergedfrom the presumably ancient SYP132. KNOLLE functionappears to require high-level expression during the mitosisimmediately preceding cytokinesis, whereas PEN1 func-tion displays striking subcellular protein dynamics thatseems to enable rapid retargeting to infection sites viaendocytosis.

Results

Unlike KNOLLE, PEN1 and SYP132 syntaxins

are stable proteins

In dividing cells of the Arabidopsis seedling root, KNOLLEaccumulates at the trans-Golgi network (TGN) in earlymitosis, localizes to the cell plate during cytokinesis andtakes the degradation route via multivesicular bodies(MVBs) to the lytic vacuole shortly after completion ofthe newly made plasma membrane (10,16,23). To analyzethe subcellular localization and protein behavior of PEN1and SYP132 in comparison to KNOLLE, we generatedtransgenic lines stably expressing Myc-tagged PEN1(Myc-PEN1) or SYP132 (Myc-SYP132) under control ofthe KNOLLE cis-regulatory sequences (22). Colocalizationanalyses indicated that both Myc-PEN1 and Myc-SYP132accumulated at KNOLLE-positive compartments: at theTGN in early mitosis and at the cell plate duringcytokinesis, although the SYP132 signal was weakerthan the PEN1 signal at the cell plate (Figure 1A–F;22). After formation of the cell plate, neither Myc-PEN1nor Myc-SYP132 colocalized at KNOLLE-labeled MVBs(Figure 1G–L). Both Myc-PEN1 and Myc-SYP132 proteinswere more stable than endogenous KNOLLE as well astransgenically made Myc-KNOLLE and still detectable ininterphase cells, when the KNOLLE promoter is not active(Figure 1N,O; compare with Figure 1M). Intriguingly, PEN1accumulated much more strongly at the cell plate thanat the plasma membrane during cytokinesis, whereasSYP132 accumulated evenly at both the cell plate andthe plasma membrane (Figure S1). Thus, both PEN1 andSYP132 localize at the division plane in dividing cells andat the plasma membrane in interphase, indicating thatthey do not take the KNOLLE degradation pathway aftercell-plate formation.

SYP132 but not PEN1 can substitute for KNOLLE

function

Previously we have shown that PEN1 cannot substitutefor KNOLLE function; however, the reason for thishas not been clarified so far (22). To characterize thisinability in more detail we performed a comparativeanalysis of the knolle complementation competenceof PEN1, SYP132 and KNOLLE when expressed fromthe KNOLLE promoter. Progeny from five KN:Myc-KNOLLE, nine KN:Myc-PEN1 and five KN:Myc-SYP132transgenic lines that were also knolle heterozygouswere phenotypically analyzed. All five KN:Myc-KNOLLEtransgenic lines completely rescued the knolle mutantphenotype. Two out of five KN:Myc-SYP132 transgeniclines rescued the knolle mutant phenotype completely,which was not the case for any of the nine KN:Myc-PEN1 transgenic lines. Examples of high-level and low-level expression lines for each transgene are shown inFigure 2 and Table S1. We observed phenotypic variationbetween different transgenic lines ranging from severeknolle seedlings (no rescue) to normal seedlings (completerescue) (Figure 2A/1). Partially rescued seedlings initiallydeveloped like wild type but were later arrested in

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Figure 1: Subcellular localization of SYP1 syntaxins in

seedling root-tip cells. A–F) MYC-PEN1 (red; A–C) and MYC-SYP132 (red; D–F) colocalize with KNOLLE (green) at the cell plate(asterisks in B, E) in dividing cells. Blue, DAPI. G–L) MYC-PEN1(G–I) and MYC-SYP132 (J–L) colocalize with KNOLLE (green) atthe TGN in early mitotic cells but not at MVBs (arrowheads in I,L). M–O) KNOLLE (M) only labels mitotic cells, whereas MYC-PEN1 (N) and MYC-SYP132 (O) are more stable and also label theplasma membrane in interphase cells. Scale bars, 5 μm.

growth and eventually died (Figure 2A/2,3). To addresswhy different Myc-PEN1 and Myc-SYP132 transgeniclines rescued knolle mutants to different degrees, weanalyzed their expression levels (Figure 2B,C). WhereasPEN1 transgenic lines only rescued knolle partially at bothlow and high levels of expression, SYP132 transgenic linesrescued knolle partially or completely, depending on thelevel of expression (Figure 2A,B). Surprisingly, the proteinlevels of the low expression lines were still much higherthan the Myc-KNOLLE protein level required for rescuingthe knolle mutant completely (Figure 2B). As PEN1 andSYP132 are more stable than KNOLLE, the proteinlevel we detected by immuno-blotting analysis does notrepresent their expression levels in mitotic cells. KNOLLEmRNA and protein are only present in dividing cells (9,10)and thus, the transcript level of each SYP1 transgeneshould represent the respective de novo synthesizedprotein amount in dividing cells. Therefore, we comparedlevels of transcript accumulation between the different

SYP1 transgenes. Intriguingly, although all Myc-KNOLLEtransgenic lines rescued the knolle mutant, some linesdisplayed a lower level of transcript accumulation than didMyc-PEN1 or Myc-SYP132 transgenic lines (Figure 2C).Thus, the rescue ability appeared not to depend on mereexpression quantity but rather might reflect a qualitativedifference between the SYP1 syntaxins in regard to theirability to promote cytokinesis.

SYP132 cannot replace PEN1 in pathogen

penetration resistance

PEN1 was shown to play a role in non-host resis-tance to fungal pathogens by contributing to localizedcell wall deposition (formation of papillae), which pre-vents barley powdery mildew Blumeria graminis f. sp.hordei (B. g. hordei) spores from invading Arabidopsisleaves (12). Although pen1 mutants display no obviousphenotype in the absence of pathogen attack, papillaformation upon attempted fungal ingress is delayedand penetration resistance significantly reduced (12,24).To analyze whether SYP1 syntaxins are able to sub-stitute for PEN1 in penetration resistance, transgenicplant lines constitutively expressing SYP1 syntaxins fromthe UBIQUITIN 10 (UBQ10) promoter (25) were gener-ated and these plants were crossed with pen1-1 mutantplants (12). pen1-1 mutant plants expressing UBQ10:RFP-PEN1 or UBQ10:RFP-SYP132 were inoculated withpowdery mildew B. g. hordei. B. g. hordei–Arabidopsisinteraction sites were visualized by established coomassieblue and callose staining protocols that allow quantificationof fungal invasion rates (Figure 3C; 12). Only about 20% ofthe fungal penetration attempts were successful in wild-type control plants, whereas the success rate increasedto about 90% in the pen1-1 mutant (Figure 3C). This wasalso reflected in significantly different levels of fluores-cent epidermal cells showing hypersensitive-like cell deathresponse (Figure 3D,E) because of activation of post-invasion defense mechanisms (26). UBQ10:RFP-PEN1fully restored wild-type invasion resistance, whereasUBQ10:RFP-SYP132 complemented pen1-1 only partially,allowing an intermediate 60% of fungal sporelings toinvade epidermal leaf cells (Figure 3C). We did notrecover any UBQ10:RFP-KNOLLE transgenic plants thatexpressed KNOLLE at detectable levels. To analyze howthis functional difference between the two SYP1 syntaxinsPEN1 and SYP132 in pathogen resistance comes about,we investigated their behavior at the subcellular level.

Quantitative live-cell imaging revealed that RFP-PEN1expressed from the UBQ10 promoter strongly accu-mulated at the Arabidopsis–B. g. hordei interactionsites 16 h after inoculation (Figure 4A–C). At sites ofattempted fungal penetration, RFP-PEN1 signal inten-sity was about 4.5 times higher than at other plasmamembrane regions of the same cell (Figures 4C andS2), indicative of the recently described active translo-cation to and accumulation in a pathogen-induced plasma-membrane microdomain (24,27). In contrast, RFP-SYP132expressed from the UBQ10 promoter showed only about

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Figure 2: knolle rescue ability of transgenic PEN1 and SYP132. A) Improved morphology (asterisks) of partially rescued transgenicseedlings (1) which, however, die on soil as growth-retarded plants (2,3). Partially rescued transgenic seedlings from each line analyzedwere genotyped by PCR after transfer to soil. Lines analyzed: (1a) KN::MYC-PEN1 #6, (1b) KN::MYC-PEN1 #9, (1c) KN::MYC-SYP132#8 and (1d) KN::MYC-SYP132 #5. Arrowheads indicate wild-type seedlings; kn, knolle. Scale bars, 2 mm. B) Transgenic proteins fromseedling extracts of the same transgenic lines detected with anti-Myc monoclonal antibody and anti-KNOLLE antiserum. Rubisco loadingcontrol stained with Ponceau S. C) Analysis of transcript levels (MYC-SYP1) from the same transgenic SYP1 lines as in (B). Actin2,loading control.

1.4 times higher signal intensity at plant–pathogen interac-tion sites than elsewhere at the same plasma membrane(Figures 4D–F and S2). In summary, the poor ability ofUBQ10:RFP-SYP132 to prevent pathogen entry correlatedwell with its comparatively inefficient recruitment to fun-gal invasion sites rather than with any conceivable activitydifferences to PEN1 at the penetration site.

PEN1 but not SYP132 constitutively cycles between

the plasma membrane and endosomes

To address why PEN1 and SYP132 differed in their abilityto substitute for knocked-out SYP1 family members,we analyzed their subcellular localization and dynamicsin seedling root cells. Both Myc-PEN1 and Myc-SYP132 expressed from the KNOLLE promoter localizeat the plasma membrane and at the cell plate.Additionally, Myc-PEN1 stained some endomembranecompartments in interphase cells, unlike SYP132 (FigureS3). To identify these compartments we did double-labeling experiments with specific subcellular markers(Figure 5A–H). Myc-PEN1-positive puncta were distinctfrom the Golgi stacks that were labeled by the γCOPsubunit of the coat protein I (COPI) complex mediatingretrograde transport from the cis-Golgi to the endoplasmicreticulum (ER) (Figure 5A; 28). Additionally, there wasalso no colocalization with the trans-Golgi labeled by

the yellow fluorescent protein (YFP)-tagged rat sialyltransferase, N-ST-YFP (Figure 5E; 29). In contrast, thevesicle formation-initiating GTPase ARF1 that localizes tothe Golgi/TGN/early endosome (16,23) labeled some Myc-PEN1-positive punctate structures (Figure 5B–D). ARF1was localized mainly to the TGN and also to the Golgistacks by immunogold labeling (23). Thus, we wouldexpect some ARF1-positive compartments not to belabeled by endocytosed RFP-PEN1. In addition, live-cellimaging revealed complete colocalization of GFP-PEN1-positive puncta with the endocytic tracer FM4-64 within10 min of incubation (Figure 5F–H; 12). Thus, in interphasecells, PEN1 localizes to the plasma membrane and to theTGN, which functionally corresponds to early endosomesin plants (30,31).

The fungal toxin brefeldin A (BFA) reversibly inhibitsvesicle trafficking by blocking the activity of sensitive ARFguanine-nucleotide exchange factors (ARF-GEFs) (32,33).BFA treatment of Arabidopsis seedling roots trapscycling plasma-membrane proteins in endosomal BFAcompartments by inhibiting ARF-GEFs required forrecycling (31,32). In contrast, secretory traffic from theER to the plasma membrane is not inhibited by BFAin Arabidopsis so that newly synthesized proteins arenot trapped in BFA compartments (16,31,34). When

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SYP132. A and B) SYP1 syntaxins expressed from the KNOLLEpromoter in flowers (A) and leaves (B). Note the absence ofMyc-tagged KNOLLE in leaf extracts. Vertical bars indicatejunction sites in the same gel edited with imaging software.In brief, bands of no interest for the experiment were cutout and the band corresponding to myc-SYP132 transgenicprotein moved to the right of the junction site. C) Frequencyof cell death reflecting successful penetration events at B.g. hordei–Arabidopsis interaction sites in leaves of transgenicplants. n = 6; error bars indicate standard deviation (∗∗p < 0.01,t-test). D–H) Aniline-blue staining of B. g. hordei–Arabidopsisinteraction sites in infected leaves. Leaf cells respond with papillaformation in wild type (D), in KN:MYC-PEN1 transgenic pen1lines (F) and in KN:MYC-SYP132 transgenic lines (H). B. g. hordeisuccessfully penetrated leaf cells, leading to cell wall deposition,in pen1 (E) and in KN:MYC-SYP132 transgenic pen1 lines (G).Col-0, wild-type control. Scale bars, 100 μm.

KN:Myc-SYP1 transgenic root tips were treated with BFA,all three transgenically made SYP1 proteins accumulatedin large BFA compartments in mitotic cells (Figure S4A–C).

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cells. A and B) UBQ10:RFP-PEN1 and (D, E) UBQ10:RFP-SYP132 label the plasma membrane (PM) and the B. g.hordei–Arabidopsis interaction sites (PS); (A and D) fluorescence,(B and E) fluorescence superimposed on bright-field images.Fungal appressoria (FA) and scan lines (between arrowheads)are outlined in (A, D). C and F) Quantitative scans of RFP-SYP1protein accumulation from the plasma membrane (PM) acrossthe cell to the penetration site (PS; arrowheads in A, D). Scalebars, 5 μm.

In non-dividing cells, however, Myc-PEN1 localized to BFAcompartments, whereas Myc-SYP132 still labeled theplasma membrane (Figure S4B,C) and KNOLLE proteinwas not detected because of its specific degradationshortly after completion of cytokinesis (Figure S4A).Because the transgenically made SYP1 proteins wereexpressed from the KNOLLE promoter, and thereforenot synthesized during interphase, only labeled SYP1

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seedling root cells. A–E) MYC-PEN1 (red) does not colocalizewith the Golgi-markers (green) γCOP (A) or N-ST-YFP (E) butcolocalizes with the TGN/endosomal marker ARF1 (green, B–D).F–H) Live imaging: GFP-PEN1 completely colocalizes with FM4-64 after 10 min of treatment. I–P) Immunostaining of gnl1 mutantwithout (I, M) or with 25 μM BFA treatment for 1 h (J–L, N–P).Blue, DAPI. I, M) MYC-PEN1 (I) and MYC-SYP132 (M) localizeat the cell plate in gnl1 mutant cells (asterisks). J–L) AfterBFA treatment, MYC-PEN1 (red) accumulates at the ER (openarrowhead, K), as does N-ST-YFP, and also in endosomal BFAcompartments (closed arrowhead, K). N–P) After BFA treatment,MYC-SYP132 (red) only accumulates at the ER (open arrowhead,O) as does BFA-sensitive GNL1-YFP. Q–V) Immuno-localizationof RFP-PEN1 (Q–S) and RFP-SYP132 (T–V) expressed from theHISTONE 4 (H4) promoter. Q–S) RFP-PEN1 (red) colocalizes withKNOLLE (green) at the cell plate, although it is not expressed individing cells. T–V) RFP-SYP132 (red) does not colocalize withKNOLLE (green) at the cell plate in dividing cells but labels theplasma membrane. Blue, DAPI. Scale bars, 5 μm.

protein that was endocytosed from the plasma membraneaccumulated in BFA compartments. These results indicatethat PEN1, but not SYP132, cycles constitutively betweenthe plasma membrane and endosomal compartment(s) ininterphase cells.

In mitotic cells, both PEN1 and SYP132 could be detectedin BFA compartments. To determine whether PEN1 andSYP132 are newly synthesized or endocytosed beforeaccumulating in BFA compartments during mitosis, weblocked the secretory pathway by BFA treatment of gnl1mutant seedlings expressing KN:Myc-PEN1 and KN:Myc-SYP132 transgenes. GNL1 is a BFA-resistant ARF-GEFthat mediates retrograde transport from the Golgi stacksto the ER (34,35). Treating gnl1 mutant seedlings with BFAleads to the inhibition of ER–Golgi traffic, as shown bythe ER accumulation of KNOLLE and the Golgi markerN-ST-YFP as well as the fusion of Golgi stacks withthe ER (16,35). Without BFA treatment, both Myc-PEN1and Myc-SYP132 accumulated at the cell plate in mitoticcells of gnl1 mutant seedlings (Figure 5I,M). After BFAtreatment, however, the newly synthesized Myc-SYP1syntaxins were trapped in the ER and, thus, did notreach the plane of cell division (Figure 5J–L,N–P, openarrowheads). Intriguingly, Myc-PEN1 also accumulated inBFA compartments, whereas Myc-SYP132 did not butrather labeled the plasma membrane (Figure 5K; closedarrowhead, compare with Figure 5O). This indicates thatPEN1 is both secreted and endocytosed during mitosis,whereas SYP132 is not endocytosed and only the de novosynthesized protein is transported through the secretorypathway to the cell plate. It should be noted that genomicGFP fusions of SYP132 also label the cell plate in dividingcells, in addition to labeling the plasma membrane inall cells (8). Considering our results, this observationsuggests that the endogenous promoter of SYP132 isalso active during M phase such that newly synthesizedSYP132 is targeted to the cell plate.

Although BFA is commonly believed to be a highly specificinhibitor of membrane trafficking with known moleculartargets, there is always a remote possibility of non-specificside effects (36). To examine in a different way whetherSYP132 is indeed not endocytosed during cytokinesis,we expressed SYP132 exclusively during interphase andinvestigated its ability to reach the cell plate. PEN1 wasused as a control and both SYP1 proteins were expressedfrom the cis-regulatory elements of the HISTONE 4 (H4)gene because H4 mRNA appears in S-phase and iscompletely degraded before the onset of mitosis (37,38).Both RFP-PEN1 and RFP-SYP132 were present at theplasma membrane in interphase cells as they werewhen expressed from the KNOLLE promoter (FigureS4D–F,H–J, compare with Fig. 1H,I). Additionally, RFP-PEN1 but not RFP-SYP132 colocalized with ARF1-labeledendosomes and accumulated in BFA compartments uponBFA treatment (Figure S4G,K). In mitotic cells, weobserved RFP-PEN1 fluorescence at the division plane,colocalizing with KNOLLE at the cell plate (Figure 5Q–S).In contrast, RFP-SYP132 did not label the developing cellplate at all, indicating that the protein is not internalizedduring mitosis (Figure 5T–V). Taken together, our resultsdemonstrate that SYP1 syntaxins behave differentlyat the subcellular level: PEN1 cycles constitutivelybetween the plasma membrane and endosomes, whereas

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SYP132, once delivered, remains statically at the plasmamembrane. This difference in localization dynamics mightalso explain two observations reported above. First, PEN1but not SYP132 accumulates more strongly at the cellplate than at the plasma membrane in dividing cells(Figure S1). In the case of cycling PEN1, both de novosynthesized and endocytosed proteins are targeted to thecell plate, whereas in the case of SYP132, only de novosynthesized protein accumulates at the cell plate. Second,SYP132 when expressed from the UBQ10 promoterwas not able to completely rescue the pen1 mutantduring pathogen attack because only newly synthesizedprotein was targeted to the infection site. To performa more rigorous test of this idea, we crossed KN:Myc-SYP1 transgenic plants with pen1-1 mutant plants andanalyzed their homozygous pen1-1 mutant progeny thatalso expressed the transgene. In accordance with theexclusive activity of the KNOLLE promoter in proliferatingtissues (10), all transgenically made SYP1 proteins weredetected in flowers (Figure 3A). However, Myc-PEN1 andMyc-SYP132 also accumulated in leaves, again indicatingthat these proteins were more stable than KNOLLE(Figure 3B). We tested their ability to restrict pathogenentry in pen1-1 mutants (Figure 3C–H; 12). Myc-PEN1completely rescued pen1-1, reducing fungal entry rates tothe wild-type level (Figure 3C,F). In contrast, Myc-SYP132entirely failed to rescue pen1-1 (Figure 3C,G). In addition,Myc-SYP132 expressed in the wild-type backgrounddid not interfere with endogenous PEN1 function(Figure 3C,H). Thus, when expressed from the KNOLLEpromoter, SYP132 cannot take over PEN1 function inpathogen resistance. Thus, the ability of syntaxin to rescuepen1 requires dynamic localization behavior. However,this cannot explain the ability of SYP132 to rescue knollein cytokinesis. As SYP132 but not PEN1 can substitutefor KNOLLE function (Figure 2), differences in amino-acidsequence rather than protein abundance at the cell plateaccount for their different rescuing ability.

Significance of the SNARE domain for KNOLLE

function

Although PEN1 localized at the cell plate, it did notsubstitute for KNOLLE during cell-plate formation. Apossible reason for its failure might be an insufficientinteraction with other SNARE partners of the cytokinesisSNARE complex. The SNARE domain was identified asthe main determinant of specificity between interactingSNARE proteins (39–43). Although the SNARE domainof SYP1 syntaxins is very conserved by amino-acidsequence, we cannot rule out that subtle differencesaccount for the efficiency of interaction. To test if theSNARE domain contributes to functional specificity ofSYP1 syntaxins, we generated chimeric proteins byswapping the SNARE domains between KNOLLE andPEN1 or SYP132, and expressed these chimeric proteinsfrom the KNOLLE promoter (Figure 6A). Both KNOLLEcarrying the SNARE domain of PEN1 (KNOLLE-PEN1SND)

and PEN1 carrying the SNARE domain of KNOLLE (PEN1-KNOLLESND) localized at the cell plate and the plasma

membrane in dividing cells (Figure 6B,C). In interphasecells both chimeric proteins were detected at the plasmamembrane, indicating that they were stable and notdegraded after cytokinesis. The same was observedfor the KNOLLE-SYP132 chimeras as both ‘KNOLLE-SYP132SND’ and ‘SYP132-KNOLLESND’ localized at thecell plate as well as the plasma membrane in dividing cellsand at the plasma membrane in non-dividing cells, furtherdemonstrating high protein stability (Figure 6D,E). Thus,replacing the SNARE domain of KNOLLE with that of PEN1or SYP132 leads to increased protein stability, suggestingthat the SNARE domain of KNOLLE in conjunction withthe remainder of the KNOLLE protein promotes its rapidturnover. All chimeric constructs were tested for theirability to rescue the knolle mutant. Remarkably, all proteinsharboring the SNARE domain of KNOLLE were able tocompletely rescue the knolle mutant phenotype (TableS1). In addition, chimeric KNOLLE protein harboring theSNARE domain of SYP132 was also able to rescue theknolle mutant phenotype, consistent with the rescueability of SYP132 (Table S1). On the contrary, chimericKNOLLE protein carrying the SNARE domain of PEN1did not rescue the knolle phenotype, and thus behavedlike the wild-type PEN1 protein (Table S1). These resultssuggest that the SNARE domain contributes to syntaxinfunction in cytokinesis.

knolle mutant rescue by PEN1-KNSND is limited

to expression during mitosis

Replacing the SNARE domain of PEN1 with that ofKNOLLE rendered PEN1 competent to rescue the knollemutant when expressed during mitosis. Considering thatPEN1 is endocytosed and reaches the plane of celldivision during cytokinesis, we addressed the possibilitythat chimeric PEN1-KNOLLESND protein can rescue theknolle mutant if expressed before mitosis. To this endwe generated plants expressing RFP-PEN1-KNOLLESNDorthe reciprocal construct RFP-KNOLLE-PEN1SND under thecontrol of the S-phase-specific HISTONE 4 promoter. Thechimeric proteins accumulated at the plasma membranein non-dividing cells and at the cell plate during mitosis(Figure 6F,G). H4:RFP-KNOLLE-PEN1SND was not able torescue the knolle mutant phenotype as might have beenexpected because KN:RFP-KNOLLE-PEN1SND also didnot rescue knolle. Surprisingly, H4:RFP-PEN1-KNOLLESNDalso did not rescue the knolle mutant phenotypein contrast to KN:RFP-PEN1-KNOLLESND (Table S1),although expression levels of the RFP-PEN1-KNOLLESNDprotein were comparable for either promoter (Figure S5).These results clearly demonstrate that KNOLLE functiondepends on (i) high-level de novo protein synthesis duringmitosis and (ii) its specific SNARE domain.

Discussion

Our study addressed functional divergence versusredundancy among members of the SYP1 syntaxinfamily, focusing on one representative from each of

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KN::KNOLLE-PEN1SND

KN::PEN1-KNOLLESND

KN::KNOLLE-SYP132SND

KN::SYP132-KNOLLESND

H4::KNOLLE-PEN1SND

H4::PEN1-KNOLLESND

ASYP111/KNOLLE

SYP121/PEN1

N-terminus Ha Hb Hc SNARE domain TM

KNOLLE-PEN1SND

PEN1-KNOLLESND

SYP132

KNOLLE-SYP132SND

SYP132-KNOLLESND

B

C

D

E

F

G

Figure 6: Subcellular localization of chimeric SYP1 proteins. A) Diagram of chimeric proteins of KNOLLE (blue), PEN1 (red) andSYP132 (green). B–I) Live imaging of RFP-tagged syntaxins with swapped SNARE-domains. B–E) When expressed from the KNOLLEpromoter, KNOLLE-PEN1SND (B), PEN1-KNOLLESND (C), KNOLLE-SYP132SND (D) and SYP132-KNOLLESND (E) localize at the cell plate(asterisks) in dividing cells and show high protein stability. F–G) When expressed from the HISTONE 4 promoter, KNOLLE-PEN1SND(F) and PEN1-KNOLLESND (G) localize at the cell plate (asterisks) in dividing cells and show high protein stability. Scale bars, 5 μm.

the three subgroups, KNOLLE/SYP111, PEN1/SYP121and SYP132. KNOLLE and PEN1 have their specificbiological roles in cytokinesis and non-host pathogendefense, respectively (9,12). To identify mechanismsdefining specificity of protein function, we expressedSYP1 proteins from a specific set of promoters, whicheliminated differences in gene expression conferred bythe endogenous promoters (8), and we swapped proteindomains.

KNOLLE plays a unique role in somatic cytokinesis, whichcannot easily be substituted for by other syntaxins (22). Itsclosest paralog SYP112 is essentially functionally equiva-lent, including rapid degradation at the end of cytokinesis,but lacks the strong expression of KNOLLE during mito-sis preceding cytokinesis (22). The same study revealedthe inability of PEN1 to substitute for KNOLLE whenexpressed from the KNOLLE cis-regulatory sequences butdid not identify a plausible molecular mechanism for thisfailure. As PEN1 when expressed like KNOLLE accumu-lated at the cell plate there seemed to be some functional

difference between the two proteins, although the lev-els of protein accumulation had not been compared. Ourpresent study revealed that PEN1 was indeed expressedduring M phase at least as strongly as KNOLLE but failedto rescue the knolle mutant. By contrast, SYP132 whenexpressed like KNOLLE rescued the knolle mutant com-pletely, indicating a clear functional difference betweenPEN1 on one hand and KNOLLE and SYP132 on the other.SNARE domain swaps between KNOLLE and PEN1 orSYP132 yielded chimeric proteins of which PEN1 proteinwith the SNARE domain of KNOLLE was able to rescuethe knolle mutant completely. Thus, the SNARE domainappears to be a critical determinant of KNOLLE proteinfunctional specificity. This is a surprising result, consider-ing the earlier observation that both KNOLLE and PEN1interact with the same Qb,c-SNARE SNAP33 (13,44).Although the same interacting Qb,c-SNARE is involvedin cytokinesis and in non-host pathogen defense, theR-SNARE might be different between the two SNAREcomplexes. Alternatively, the rate of assembly or disas-sembly of the two SNARE complexes might be different.

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KNOLLE expression outside mitosis appears to bedetrimental, although 35S promoter-driven expressionresulted in KNOLLE accumulation near the apical plasmamembrane in growing root hairs (45). However, using theS-phase-specific H4 promoter, KNOLLE accumulation wasseverely impaired, whereas both PEN1 and SYP132 wereexpressed and accumulated at the plasma membrane. AsPEN1-KNOLLESND protein was able to rescue the knollemutant completely when expressed from the KNOLLEpromoter, i.e. during M phase immediately precedingcytokinesis, and this chimeric protein was stable and notdeleterious, we expressed the same protein from the S-phase-specific H4 promoter. Surprisingly, early expressionof the PEN1-KNOLLESND followed by its transient storageat the plasma membrane and subsequent endocytosisduring cytokinesis enabled its accumulation in the planeof cell division but did not rescue the knolle mutant,although the protein level was comparable to that of thesame protein made from the KNOLLE promoter. Thisstrongly suggests that the cytokinesis-specific syntaxinneeds to be synthesized immediately before cell-plateformation, whereas the same protein when endocytosedis ineffective. This is in line with the observation thatBFA-induced inhibition of ER–Golgi traffic in gnl1 mutantseedlings impairs cytokinesis (16). There is no obviousreason for this difference in efficacy between newlymade and endocytosed syntaxin in cytokinesis, especiallybecause the TGN acts as a sorting station that directsboth secretory and endocytosed proteins to the plane ofcell division in cytokinesis (31).

The comparative analysis of PEN1 and SYP132 revealedan important difference in the dynamic behavior of thetwo proteins. Whereas PEN1 cycled continually betweenthe plasma membrane and endosomal compartments,SYP132 appeared to associate stably with the plasmamembrane. This was observed in the root cells inwhich ER–Golgi traffic was inhibited by BFA treatmentof gnl1 mutant seedlings: when expressed from theKNOLLE promoter, PEN1 was trapped in the ER but stillaccumulated in endosomal BFA compartments, whereasSYP132 was only detected in the ER and at the plasmamembrane. The same difference was observed when thetwo proteins were expressed from the S-phase-specificH4 promoter: only PEN1 accumulated at the plane ofdivision (cell plate) during cytokinesis, whereas SYP132stayed at the plasma membrane. Thus, PEN1 appears tobe a highly dynamic protein, whereas SYP132 once madeappears to be firmly anchored at the plasma membrane.

PEN1 plays an important role in Arabidopsis non-hostresistance to fungal pathogens (12,24). Endogenous PEN1appears to be moderately up-regulated in response topathogen attack and accumulates rapidly at the site ofinfection. We used two different promoters to analyzethe relevance of syntaxin retargeting for mounting asuccessful defense against non-adapted powdery mildewfungi. The UBQ10 promoter is constitutively activeand thus provides, during pathogen attack, both newly

synthesized syntaxin and syntaxin made earlier and thenstored at the plasma membrane. In contrast, the KNOLLEpromoter is only active in proliferating cells but not inmature leaf cells. Thus, in the latter case, only syntaxinmade earlier and then stored at the plasma membrane isavailable during pathogen attack. Our data suggest thatPEN1 is highly dynamic such that endocytic retargetingof plasma membrane-localized syntaxin is sufficient andno newly made syntaxin is necessary for mounting asuccessful defense during pathogen attack. In contrast,SYP132 only partially rescued the compromised pathogendefense of pen1 leaves when expressed from the UBQ10promoter but had no effect when expressed from theKNOLLE promoter. Thus, even newly synthesized SYP132might not be efficiently targeted to the site of infection.One possible explanation for this difference in subcellulardynamics between PEN1 and SYP132 might be that thestrong accumulation of syntaxin at the infection site doesnot result from directional secretion but rather requiresendosomal retargeting. Similar observations were madein polar targeting of PIN proteins in Arabidopsis (46,47).

Compared to the two specialized SYP1 syntaxins KNOLLEand PEN1, SYP132 might represent a rather general syn-taxin function at the plasma membrane, possibly involvedin constitutive fusion of secretory vesicles. No knockoutmutants of SYP132 are known. However, SYP132 appearsto be broadly if not ubiquitously expressed during develop-ment (8). Furthermore, sequence comparisons with SYP1syntaxins from lower plants suggest that SYP132 mightplay an ancient role in secretory traffic to the plasmamembrane (6).

In an evolutionary scenario that associates SYP132 withplasma-membrane syntaxins of primitive land plants,KNOLLE and PEN1 appear to have evolved divergently toserve their respective highly specialized function. KNOLLEhas adopted an exclusive role in membrane fusion duringcytokinesis, which for yet unknown reasons requireshigh-level expression immediately before cytokinesis.In addition to, and possibly as a consequence of, thedramatic change in gene regulation leading to high-levelprotein accumulation, KNOLLE protein has become highlyunstable, being targeted to the vacuole for degradationat the end of cytokinesis. Interestingly, during KNOLLEevolution, there seems to have been no substantialfunctional change from the presumably ancient SNAREdomain of SYP132, unlike the SNARE domain of PEN1.In contrast, PEN1 displays dramatic subcellular proteindynamics, as evidenced by its continual cycling in non-infected cells and the retargeting to the plane of celldivision during cytokinesis. This dynamic behavior alsoenables PEN1 to act in plant innate immunity, facilitatingits rapid relocation from the plasma membrane tofungal infection sites via endocytosis and retargeting. Itremains to be determined how the attacked plant cellreorganizes its membrane trafficking to fend off fungalintruders.

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Methods

Plant growth, transformation and selectionArabidopsis thaliana plants were grown on half-strength Murashige andSkoog (MS) medium (+1% sucrose for microscopy) or on soil at 18–23◦C,with cycles of 16 h light and 8 h dark. Landsberg/Niederzenz (Ler/Nd) plantsheterozygous for the knolle mutation X37-2 (9) or Columbia (Col) plantshomozygous for the pen1-1 mutation (12) were transformed with Agrobac-terium tumefaciens, using the floral-dip method (48). T1 plants frombulk-harvested seeds were selected for transformants either grown on soilby spraying twice with a 1:1000 dilution of Basta® (45) (183 g/L Glufosinate-ammonium, Bayer) or grown on half-strength MS medium containing 50 μM

kanamycin. BASTA-resistant plants were genotyped for knolle X37-2 (22)and kanamycin-resistant plants for pen1-1 (12) as described.

Molecular biologyTransgenic constructs for subcellular localization and for knolle rescuewere expressed from the mitosis-specific KNOLLE promoter. KN andPEN1 coding sequences were cloned into the KNOLLE cassette asdescribed (22). SYP132 CDS was amplified from a flower and siliquecDNA library of the Landsberg ecotype (49) by polymerase chain reaction(PCR) according to standard procedures using Taq DNA Polymerase (PeqLab Biotechnologie GmbH). SYP132 CDS was directionally cloned into theKNOLLE cassette in the pBluescript vector via restriction sites SmaI andEcoRI (22). The KN:Myc-SYP132 insert was introduced into the pBAR-Bvector via the restriction sites HpaI and SpeI.

Constructs for SNARE domain swaps were generated by primer extensionPCR according to standard procedures using Taq DNA Polymerase (Peq LabBiotechnologie GmbH). Constructs were cloned into the multiple cloningsite (MCS) of the pGreenIIB containing the KNOLLE cassette carrying anN-terminal RFP-tag.

A HISTONE 4 (H4) expression cassette was generated by amplifying 543 bpupstream and 177 bp downstream of the H4 gene. The 5′ and 3′ sequenceswere directionally cloned into the binary pGreenIIB-vector using the restric-tion sites SacI/XbaI and EcoRI/KpnI, respectively, surrounding an MCScontaining XbaI, SmaI and EcoRI. An N-terminal RFP-tag was introduced byXbaI/SmaI. SYP1 coding sequences were introduced via SmaI/EcoRI sites.

The UBQ10 promoter (25) constructs for pen1-1 rescue were generated bydirectionally cloning Arabidopsis syntaxin coding sequences into the binarypGreenIIB-vector downstream of the UBQ10 promoter via the restrictionsites SmaI and SpeI.

The constructs were checked by restriction digest and sequencingusing the ABI PRISM Big Dye Terminator Cycle Sequencing Kit andthe ABI-Sequencer 310 (Applied Biosystems) or using GATC (Konstanz)service before transformation into A. tumefaciens strain GV3101. Standardprotocols were used for molecular biology(50). Restriction enzymes werepurchased from MBI Fermentas and synthetic oligonucleotides from ARK(Sigma-Aldrich).

For RT-PCR analysis, total RNA was isolated from 100 mg Arabidopsisseedlings, using the ’Trizol-method’(51) or the RNAeasy plant mini kit(Qiagen). After removal of contaminating DNA (DNase I, Fermentas),first strand cDNA was synthesized with Superscript II RNaseH-ReverseTranscriptase (Invitrogen), using the dT-anchor-random II primer. As acontrol, we used ACTIN2. All primer sequences are listed in Table S2.

Western blot analysisPreparation of protein extracts and Western blots was performed asdescribed (10). For protein extraction we used one inflorescence, fiverosette leaves or 50 mg seedlings. Rabbit anti-KNOLLE antiserum wasused at 1:5000 dilution (10), mouse anti-α-tubulin monoclonal antibodyat 1:4000 (Sigma-Aldrich), rat anti-RFP monoclonal antibody at 1:1500

(chromotek), sheep anti-rabbit IgPOD polyclonal antibody at 1:1000(Boehringer), goat anti-mouse IgPOD polyclonal antibody at 1:10 000(Boehringer), goat anti-rat IgPOD at 1:1000 (Sigma-Aldrich) and mouseanti-Myc-POD monoclonal antibody at 1:1000 (Roche). Independent T1knolle heterozygous lines for each transgene (KNOLLE, PEN1 and SYP132)were investigated for expression level. For phenotypic and transcript levelanalyses, we used the strongest and weakest expression line of eachtransgenic construct.

Inhibitor treatment and FM 4-64 stainingThree- to five-day-old seedlings were incubated in 1 mL of liquid medium(half-strength MS medium) containing 50 μM BFA. FM 4-64 dissolved inwater was used at 4 μM final concentration. Seedlings were incubatedwith inhibitors and dye at room temperature for the indicated timesfollowed by fixation with 4% paraformaldehyde in microtuble stabilizingbuffer (MTSB) (50 mM PIPES, 5 mM EGTA, 5 mM MgSO4, adjust pH withKOH). The following stock solutions were used: 50 mM BFA (Sigma-Aldrich)in dimethyl sulphoxide (DMSO):ethanol (1:1), and 2 mM FM 4-64 (MolecularProbes) in water. Control treatments were performed with equal amountsof the respective solvents.

Antibody staining and confocal laser-scanning

microscopyWhole-mount immunofluorescence was performed as described (10).Antibodies and dilutions were as follows: rabbit anti-KNOLLE anti-serum (1:2000) (10), mouse anti-Myc monoclonal antibody 9E10 (1:600;Santa Cruz Biotechnology), rabbit anti-SEC21/γCOP polyclonal antibody(1:1000) (16), rabbit anti-ARF1 polyclonal antibody (1:5000) (52), fluores-cein isothiocyanate (FITC)-conjugated secondary goat anti-rabbit antibody(1:600, Dianova), Cy3-conjugated secondary goat anti-mouse antibody(1:600, Dianova). DAPI (4’,6-diamidino-2-phenylindole) staining was per-formed as described (45). Immunofluorescence and live-cell microscopywere done with a Leica TCS-SP2/SP5 confocal laser-scanning microscope.All confocal laser-scanning microscopy (CLSM) images were obtainedusing the LEICA CONFOCAL software and a 63× water-immersion objective.Images were processed using ADOBE PHOTOSHOP CS3.

B. g. hordei inoculation and quantification of pen1

mutant phenotypeFour-week-old Arabidopsis plants were inoculated with powdery mildewBlumeria graminis hordei from barley. Live-cell imaging of single leaveswas performed at 12–16 h after inoculation. Penetration rescue analyseswere performed at 72 h after inoculation.

Individual B. g. hordei–Arabidopsis interaction sites were characterizedmicroscopically for failed and successful invasion (efficient papilla forma-tion versus haustorium formation and hypersensitive-response-like celldeath) using aniline blue and coomassie blue as recently described (12).The experiment was repeated three times, and 100 interaction sites pergenotype were scored each time.

Acknowledgments

We thank Stefan Driessen, Ulrike Hiller and Alexandra Matei fortechnical assistance. This work was supported by the DeutscheForschungsgemeinschaft through an AFGN grant to G. J.

Supporting Information

Additional Supporting Information may be found in the online version ofthis article:

Figure S1: Subcellular localization of PEN1 and SYP132 during

cytokinesis. A–F) Like KNOLLE (A, D), MYC-PEN1 (B) and MYC-SYP132

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(E) accumulate at the cell plate (arrows). MYC-PEN1 accumulates lessat the plasma membrane, whereas MYC-SYP132 shows no difference(arrowheads). C and F) Overlay counterstained with DAPI (DNA, blue). Gand H) Quantitative scans of MYC-PEN1 (G) and MYC-SYP132 (H) proteinaccumulation from the plasma membrane (PM) across the cell includingthe cell plate (CP). Scale bars, 5 μm.

Figure S2: Quantification of RFP-SYP1 protein accumulation at

infected leaf cells. A–C, upper panels) UBQ:RFP-PEN1 and (D–F, upperpanels) UBQ:RFP-SYP132 label the plasma membrane and the B. g.hordei–Arabidopsis interaction sites. Scale bars, 5 mm. A–F, lower panels)Quantitative scans of RFP-SYP1 protein accumulation from the plasmamembrane across the cell to the penetration site. G) Quantification ofsignal intensity of the RFP-SYP1 proteins at the penetration site comparedto the plasma membrane. n = 5; error bars indicate standard deviation.

Figure S3: Co-labeling of GFP-PEN1 and RFP-SYP132. A–C) Subcellularlocalization of GFP-PEN1 (A) and RFP-SYP132 (B) in Arabidopsis root-tipcells. Both SYP1 proteins label the plasma membrane (arrows), whereasonly PEN1 labels some endosomes (arrowheads); (C) merged image. D–F)After BFA treatment PEN1 (D) but not SYP132 (E) accumulates in BFAcompartments; (F) merged image. Scale bars, 5 μm.

Figure S4: Subcellular behavior of SYP1 syntaxins. A–C) Like KNOLLE(A), MYC-PEN1 (B, red) and MYC-SYP132 (C, red; arrowhead) accumulatein BFA compartments in mitotic cells. Only PEN1 accumulates in BFAcompartments in interphase cells (B). D–K) Immuno-localization of RFP-PEN1 (D–G) and RFP-SYP132 (H–K) expressed from the HISTONE 4 (H4)promoter. D–F) RFP-PEN1 (red) localizes at the plasma membrane andcolocalizes with the TGN/endosomal marker ARF1 (green). G) RFP-PEN1accumulates in BFA compartments. H–J) RFP-SYP132 (red) localizes at theplasma membrane but does not colocalize with ARF1 (green). K) KNOLLE(green) but not RFP-SYP132 (red) accumulates in BFA compartments(arrowhead). Scale bars, 5 μm.

Figure S5: Protein levels of PEN1-KNOLLESND. Protein expression oftransgenic PEN1-KNOLLESND from several independent transgenic lineswas analyzed with anti-RFP antibody. Note that protein levels are nearlyequal when expressed from the KNOLLE (KN) promoter or from theHISTONE 4 (H4) promoter. KN:RFP-PEN1-KNOLLESND transgenic lines#2 and #4 were shown to rescue knolle mutants like wild type (Table S1).

Table S1: Rescue analysis of SYP1 syntaxins. Progeny of transgenicplant lines were grown on agar plates to seedling stage and phenotypicallyanalyzed. knolle and partial rescue phenotypes were counted.

Table S2: Oligonucleotide sequences.

Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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