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First published online February 20, 2009; 10.1104/pp.108.132092 Plant Physiology 149:1824-1837 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Subcellular Localization and Functional Analysis of the Arabidopsis GTPase RabE1,[W],[OA]Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824
Membrane trafficking plays a fundamental role in eukaryotic cell biology. Of the numerous known or predicted protein components of the plant cell trafficking system, only a relatively small subset have been characterized with respect to their biological roles in plant growth, development, and response to stresses. In this study, we investigated the subcellular localization and function of an Arabidopsis (Arabidopsis thaliana) small GTPase belonging to the RabE family. RabE proteins are phylogenetically related to well-characterized regulators of polarized vesicle transport from the Golgi apparatus to the plasma membrane in animal and yeast cells. The RabE family of GTPases has also been proposed to be a putative host target of AvrPto, an effector protein produced by the plant pathogen Pseudomonas syringae, based on yeast two-hybrid analysis. We generated transgenic Arabidopsis plants that constitutively expressed one of the five RabE proteins (RabE1d) fused to green fluorescent protein (GFP). GFP-RabE1d and endogenous RabE proteins were found to be associated with the Golgi apparatus and the plasma membrane in Arabidopsis leaf cells. RabE down-regulation, due to cosuppression in transgenic plants, resulted in drastically altered leaf morphology and reduced plant size, providing experimental evidence for an important role of RabE GTPases in regulating plant growth. RabE down-regulation did not affect plant susceptibility to pathogenic P. syringae bacteria; conversely, expression of the constitutively active RabE1d-Q74L enhanced plant defenses, conferring resistance to P. syringae infection.
Eukaryotic cells are compartmentalized by membranes that surround organelles having specific functions. Communication and transport between these membrane-bound compartments are vital to the cell and are accomplished through complex and tightly regulated pathways. Trafficking pathways and their players have been extensively described in yeast and mammalian cells but are still poorly characterized in plants. The main effectors and regulators of these pathways appear to be shared between all eukaryotes. Among these, Rabs are a group of small monomeric GTPases that act as molecular switches to mediate vesicle transport between membrane-bound cellular compartments (Segev, 2001  ). Rab GTPases participate in vesicle budding from a donor compartment, transport along the cytoskeleton toward a target compartment, and, eventually, tethering and fusion of the vesicles with the target membrane. Like other small GTPases, Rabs alternate between a GTP-bound active form and a GDP-bound inactive form. Their functional specificity is determined, in part, by their unique subcellular distribution (Stenmark and Olkkonen, 2001; Zerial and McBride, 2001). The Arabidopsis (Arabidopsis thaliana) genome encodes 57 Rab proteins, divided into eight subfamilies (RabA to RabH) based on sequence similarities (Rutherford and Moore, 2002; Vernoud et al., 2003). Recent studies have indicated that members of various plant Rab subfamilies participate in secretion and recycling of cell wall components (Nielsen et al., 2008), apical growth of pollen tubes (de Graaf et al., 2005; Cheung and de Vries, 2008; Cheung and Wu, 2008) and root hair (Preuss et al., 2004, 2006), formation of the phragmoplast in dividing cells (Chow et al., 2008), endocytosis (Ueda et al., 2004), and potentially osmotic stress tolerance (Mazel et al., 2004).
Fungal and bacterial infections in plants are often associated with the activation (or suppression) of extracellular defense responses, including secretion of defense-related proteins and antimicrobial phytoalexins into the apoplast, and the formation of callose-rich cell wall appositions, known as papillae (Snyder and Nicholson, 1990
Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) is a Gram-negative bacterium that causes bacterial speck of tomato (Solanum lycopersicum) and Arabidopsis. Like other Gram-negative bacterial pathogens of plants and animals, Pst DC3000 delivers virulence effector proteins directly into the host cell via the type III secretion system (TTSS; Buttner and Bonas, 2003
A previous yeast two-hybrid (Y2H) screening of a tomato cDNA library for AvrPto-interacting proteins yielded two small GTPases, named Api2 and Api3 (Bogdanove and Martin, 2000
The identification of Rab GTPases as AvrPto interactors in both tomato and Arabidopsis suggested that, as part of its virulence mechanism, this effector may perturb intracellular vesicle trafficking in the plant (Bogdanove and Martin, 2000
Identification of Arabidopsis RabE Proteins as Y2H Interactors of AvrPto
We conducted a Y2H screening of two separate Arabidopsis cDNA libraries, using AvrPto as bait. Several AvrPto-interacting Arabidopsis proteins were identified, including a member of the RabE family of small GTPases (At5g59840), a putative cytoplasmic kinase (At4g11890), an auxin signaling repressor, IAA7 (At3g23050), two hypothetical proteins (At3g26600 and At5g16840), and several putatively chloroplast- or mitochondria-targeted proteins. The interaction with the small GTPase RabE was particularly interesting because RabE was predicted to be membrane localized, as is AvrPto (Shan et al., 2000 To characterize the specificity of the AvrPto-RabE interaction, we investigated whether AvrPto interacts with other members of the RabE family and with other Rab proteins. Of the other Arabidopsis Rab protein families (i.e. RabA to -D, -F, and -G), we cloned and expressed in yeast RabA1a, -B1b, -C1, -D2a, -F2a, and -G3a; none of these representatives interacted with AvrPto in the Y2H system (Fig. 1A ). Of the five RabE genes, all but RabE1c were successfully cloned and expressed in yeast. All four RabE proteins tested (RabE1a, -b, -d, and -e) interacted with AvrPto (Fig. 1B). Thus, it appears that AvrPto interacts specifically with the Arabidopsis RabE family of GTPases.
Small GTPases normally cycle between the active GTP-bound and the inactive GDP-bound states. Mutation of highly conserved residues can be used to alter the nucleotide binding and hydrolysis activities of Rab proteins (Nielsen et al., 2008 ; Supplemental Fig. S1). The mutant forms RabE1d-S29N (predicted to be GDP bound) and RabE1d-Q74L (predicted to be GTP bound) were engineered. In the Y2H system, AvrPto interacted only with wild-type RabE1d or with GTP-restricted RabE1d-Q74L but not with GDP-restricted RabE1d-S29N (Fig. 1C), suggesting that AvrPto preferably binds to the active form.
Analysis of the Arabidopsis genome showed that 44 of the 57 Rab genes reside in duplicated regions. Of the five RabE genes, RabE1d and -E1e appear to be derived from a major duplication event between chromosomes III and V, and the same holds true for RabE1b and -E1c (Rutherford and Moore, 2002
To investigate potential up- or down-regulation of the RabE family members in response to pathogens or other stresses, we analyzed the expression patterns of the RabE genes with the AtGenExpress Visualization Tool (http://jsp.weigelworld.org/expviz/expviz.jsp; Schmid et al., 2005 ) across several publicly available microarray data sets. None of the five genes appeared to be significantly (more than 2.5-fold) up- or down-regulated in response to pathogens or elicitors. In the absence of any expression-based indication of whether the five RabE proteins have redundant or distinct functions, we chose the highly expressed RabE1d (At5g03520) for further study.
All Rab proteins are normally present in cells in two pools, one cytoplasmic and the other membrane associated (Novick and Brennwald, 1993
Leaf epidermal cells typically contain a very large vacuole that accounts for most of the cell volume. Fluorescence detected at the cell periphery may represent the PM, the vacuolar membrane (tonoplast), or the thin layer of cytoplasm that is between the PM and the tonoplast. To more precisely determine whether GFP-RabE1d was also localized at the PM, we stained live leaf tissue with the lipophylic dye FM4-64 (Fischer-Parton et al., 2000 ; Bolte et al., 2004). FM4-64 produces a bright red fluorescence in membranes but not in aqueous solutions and is rapidly incorporated into the PM. It is often used in microscopy as an endocytic tracker, because it is retained in the portions of PM that are internalized by endocytosis (Ueda et al., 2001). Within the first 15 to 30 min after incorporation (depending on the system used), FM4-64 selectively stains the PM. CLSM analysis revealed a precise overlap of GFP-RabE1d fluorescence with FM4-64 fluorescence immediately after staining, indicating that RabE1d is associated with the plasma membrane (Fig. 3D).
To investigate whether the punctate structures labeled by GFP-RabE1d correspond to the Golgi apparatus, we examined colocalization with rat sialyl transferase, a Golgi marker protein (Wee et al., 1998
GFP-RabE1d expression in the transgenic plants was driven by the cauliflower mosaic virus 35S constitutive promoter. To exclude the possibility that the observed RabE localization reflects patterns of only the transgenically expressed protein, we conducted additional analyses to determine the localization of endogenous RabE in transgenic as well as wild-type Arabidopsis plants. We performed subcellular membrane fractionation by centrifugation of clarified plant extracts on Suc step gradients (Zeng and Keegstra, 2008
We also produced transgenic plants expressing RabE1d-Q74L, which is predicted to be constitutively active, as a GFP fusion. Unlike wild-type GFP-RabE1d, the GFP-RabE1d-Q74L fusion was not detected in intracellular punctate structures (i.e. Golgi) but was primarily found at the cell periphery (Fig. 4A
). PM staining with FM4-64 revealed that the bulk of GFP-RabE1d-Q74L fluorescence did not overlap with the PM but was most likely localized in the tonoplast (Fig. 4B). We obtained from the Arabidopsis Biological Resource Center Arabidopsis transgenic lines expressing various endomembrane markers fused to GFP (Cutler et al., 2000
Challenge with P. syringae Bacteria Promotes Focal Accumulation of GFP-RabE1d To investigate whether intracellular distribution of the RabE1d protein was perturbed in the presence of bacteria, we performed microscopic observation of transgenic GFP-RabE1d plants challenged with various strains of Pst DC3000. Leaves were syringe inoculated with bacteria at 1 x 108 colony-forming units (cfu) mL–1 and analyzed by CLSM at 5 to 6 h after inoculation. We observed that bacterial inoculation caused polarized GFP-RabE1d accumulation in mesophyll cells (Fig. 6A ). Interestingly, whereas such a phenomenon was restricted to a few sparse cells in leaves challenged with wild-type virulent Pst DC3000 (Fig. 6D) or with the nonpathogenic TTSS-defective mutant hrpA– (Fig. 6C), focal accumulation of GFP-RabE1d was widespread in leaves inoculated with the avirulent strain Pst DC3000 (avrRpt2; Fig. 6E). These results suggest that the GFP-RabE1d subcellular localization is dynamic and can respond to bacterial infection, especially in a gene-for-gene interaction.
RabE1d Cosuppression in Transgenic Plants Resulted in Altered Plant Growth and Morphology A large percentage of GFP-RabE1d transgenic Arabidopsis plants generated in this study showed cosuppression of the transgene and of endogenous RabE, as demonstrated by western-blot analysis (Fig. 7A ). Severe reduction of the endogenous RabE protein level in transgenic plants invariably correlated with a distinct morphological phenotype (Fig. 7, B and C). Rosette leaves developed normally for the first 3 weeks, when Arabidopsis development is usually slower, and plants were indistinguishable from Col-0 plants. In the following 2 weeks, when Arabidopsis size increases more rapidly, the leaves of RabE-cosuppressed plants did not fully elongate (Fig. 7D); midribs remained short, while the leaf lamina continued to expand, producing a characteristic wavy phenotype. Mature (6- to 7-week-old) RabE-cosuppressed plants were significantly smaller than wild-type plants and had short midribs and petioles. RabE-cosuppressed plants flowered at the same time as wild-type Arabidopsis, although their stems were much shorter than wild-type stems and the plants produced fertile seeds. The progeny of selected silenced lines (B8, B11, and B13) maintained silencing and had the same phenotype as the parental plants.
Expression of the RabE gene family was analyzed by RT-PCR in the cosuppressed lines and compared with expression in wild-type plants. RT-PCR demonstrated that not all RabE gene family members are equally affected by cosuppression. RabE1d and RabE1e were knocked down most severely, followed by RabE1b and RabE1c. RabE1a showed only slight down-regulation (Fig. 7E). Given the high degree of sequence similarity among small GTPases of the Arabidopsis Rab superfamily, we tested whether other closely related Rabs were affected by silencing. The closest relatives of the RabE clade in Arabidopsis are the four RabD proteins (RabD1, -D2a, -D2b, and -D2c). RabD was previously characterized as a regulator of the early secretory pathway, being involved in transport from the endoplasmic reticulum to the Golgi (Batoko et al., 2000 ; Zheng et al., 2005). RT-PCR revealed that transcripts of all four RabD genes were present at similar levels in RabE1d-silenced plants and in wild-type Arabidopsis (Fig. 7E). Silencing in the transgenic plants, therefore, is specific and limited to the RabE genes, primarily RabE1d and -E1e.
We performed pathogenesis assays with Pst DC3000 to establish whether the partially RabE-cosuppressed plants are impaired in their defenses against P. syringae. Pst DC3000 consistently caused disease symptoms and multiplied on RabE-cosuppressed plants, reaching similar population levels as on wild-type Arabidopsis (Supplemental Fig. S2). In several instances, however, we observed that older (6- to 7-week-old) or environmentally stressed RabE-cosuppressed plants displayed some degree of basal resistance, possibly due to stress caused by RabE down-regulation (Supplemental Fig. S2). We did not observe a consistent and reproducible difference between wild-type Col-0 plants and RabE-cosuppressed plants in the multiplication of Pst DC3000 (avrRpt2) or a TTSS-defective mutant of Pst DC3000, the hrpA– mutant (Supplemental Fig. S2).
GFP-RabE-Q74L plants did not exhibit significant alterations in growth and development, other than the appearance of minute sparse indentations in mature rosette leaves about 2 weeks prior to bolting. The origin and significance of these indentations are unclear; however, we have been unable to show, using trypan blue staining, that they develop as a consequence of cell death. Although transgenic expression of constitutively active GFP-RabE1d-Q74L did not globally affect plant growth or development (as did RabE cosuppression), it had a remarkable effect on plant responses to P. syringae infection. Upon challenge with Pst DC3000, the GFP-RabE1d-Q74L-expressing plants displayed a considerable degree of resistance, reflected by bacterial multiplication being consistently restricted (10- to 100-fold) compared with multiplication on wild-type Arabidopsis (Fig. 8A ). This observation was consistent across several experiments on different transgenic lines. Visible disease symptoms, namely chlorosis and necrosis, were also markedly reduced (Fig. 8B). This enhanced resistance phenotype apparently requires the constitutively active form of RabE1d, as transgenic expression of GFP-RabE1d did not result in enhanced resistance (Supplemental Fig. S2).
Constitutive Secretion of Extracellular Proteins in GFP-RabE1d-Q74L-Expressing Plants
Up-regulation of the secretory pathway was recently demonstrated in systemic acquired resistance (SAR; Wang et al., 2005
Plant RabE GTPases are predicted to be involved in mediating secretory vesicle traffic from the Golgi to the PM. Therefore, they could play fundamental roles in the secretion of extracellular matrix molecules, thereby influencing plant growth and development, as well as in the secretion of extracellular defense molecules in response to pathogen infections. RabE proteins from both tomato (Bogdanove and Martin, 2000 ) and Arabidopsis (this study) interact with a pathogen effector protein, AvrPto, in Y2H assays. Because a number of studies have implicated vesicle trafficking in pathogen defense (Collins et al., 2003; Assaad et al., 2004; Wang et al., 2005; Nomura et al., 2006; Kalde et al., 2007), physical interaction between AvrPto and RabE GTPases could indicate a potential role of RabE GTPases in plant defense. However, to date, only one report has documented subcellular localization of Arabidopsis GFP-RabE1d transiently expressed in heterologous tobacco epidermal cells (Zheng et al., 2005). Subcellular localization of RabE GTPases in native Arabidopsis cells and the biological role of RabE GTPases in regulating plant growth or defense at the whole-plant level remained unknown.
Using a combination of confocal microscopy and cell membrane fractionation on transgenic plants expressing GFP-RabE1d and on wild-type plants, we show not only that RabE proteins are localized in the Golgi, as reported by Zheng and colleagues (2005)
Interestingly, the RabE-Q74L protein expressed in Arabidopsis as a GFP fusion displayed an unanticipated localization pattern. The bulk of fluorescent protein was localized at the vacuolar membrane, and we could not detect, by CLSM fluorescence in the Golgi or in the PM, the predicted destination of the active form of RabE. After delivery of vesicles to the target membrane, Rab proteins are usually recycled back to the cytoplasm and then incorporated again into the donor membrane for multiple rounds of vesicle transport. Such recycling is dependent on hydrolysis of GTP into GDP, because inactivated GDP-bound Rabs are extracted from the target membrane by accessory proteins that mediate their recycling (Segev, 2001 Analysis of RabE-cosuppressed plants clearly indicates that RabE GTPases play a role in plant growth, as evidenced by dwarf plant sizes and altered leaf morphology in RabE-cosuppressed plants. Rosette leaves of RabE-cosuppressed lines developed relatively normally for the first 3 weeks, when Arabidopsis development is usually slower. However, when Arabidopsis size increases more rapidly, in the 4th and 5th weeks, the leaves of RabE-cosuppressed plants were not able to fully elongate; leaf midribs and petioles remained short, yielding characteristically wavy leaves. Inflorescences emerged at the same time as in the wild type, but the overall stature of the RabE-cosuppressed plants remained much shorter. These observations lead us to suggest that RabE-mediated vesicle traffic from the Golgi to the PM is required for the rapid elongation of certain tissues (e.g. leaf midribs and stems) associated with rapid plant growth. The dwarf phenotype of RabE-cosuppressed plants was likely caused by simultaneous down-regulation of more than one RabE gene, as suggested by reduced expression of RabE1d and -E1e and, to a lesser extent, RabE1a, -E1b, and -E1c (Fig. 6E). Consistent with this speculation, knockout mutants of RabE1d, caused by T-DNA insertions, did not exhibit any defect in growth and development (data not shown). Notably, cosuppression did not affect the expression of RabD genes, the closest homologues of RabE genes; therefore, the observed phenotypes can be ascribed to partial silencing of multiple RabE gene family members.
Besides our interest in determining the subcellular localization of RabE GTPases in native Arabidopsis cells and a possible role of RabE GTPases in growth and development at the whole plant level, another major motivation for this work was to investigate a potential role of RabE GTPases in plant defense. Bogdanove and Martin (2000)
Interestingly, GFP-RabE1d plants challenged with the avirulent strain Pst DC3000 (avrRpt2) manifested dramatic focal accumulation of GFP-RabE1d in mesophyll cells (Fig. 5). GFP-RabE1d focal accumulation was limited to a few sporadic cells when plants were inoculated with wild-type virulent Pst DC3000 (i.e. in a susceptible interaction) or with the hrpA– mutant (which activates plant basal defenses). Polarized accumulation of RabE GTPase during a gene-for-gene interaction could be part of the host cell response to direct defense-associated vesicle trafficking toward invading pathogens. If so, the GFP-RabE1d fusion may be a useful cellular marker for future study of vesicle trafficking in the gene-for-gene response in Arabidopsis. Accumulation of other vesicle traffic-associated regulators (such as t-SNAREs and v-SNAREs) at the site of pathogen penetration has also been observed in fungal infections (Assaad et al., 2004 RabE cosuppression in Arabidopsis, under the conditions reported in this study, did not result in increased susceptibility to P. syringae bacterial strains. RabE proteins, therefore, may not be required for establishing defenses against this pathogen. Alternatively, partial down-regulation of RabE proteins may be insufficient to confer a discernible defense phenotype. A complete knockout of all five RabE genes would be necessary to resolve this question. However, the dwarf phenotype of partially RabE-cosuppressed plants suggests that it is unlikely that completely RabE-deficient plants would be viable and/or suitable for bacterial infection assays.
Transgenic expression of the RabE1d-Q74L variant, on the other hand, conferred in Arabidopsis a significant degree of resistance to Pst DC3000. It remains to be determined whether this resistance is caused by a direct effect of RabE1d-Q74L, due to enhancement of defense-related vesicle traffic, or rather is triggered by an indirect effect, due to overall perturbation of cellular vesicle traffic. We found that the IWF collected from water-treated GFP-RabE1d-Q74L plants contained PR1 and several unknown proteins that were absent from the IWF of water-treated wild-type Arabidopsis, indicating constitutive activation of secretory and defense pathways in these plants. BTH application resulted in similar levels of secreted PR1 and other proteins in the apoplast in both wild-type and RabE1d-Q74L transgenic plants (Fig. 8C). However, some of these extracellular proteins are detected only in the IWF of water- and BTH-treated RabE1d-Q74L-expressing plants but not in the IWF of BTH-treated wild-type plants. These unique extracellular proteins associated with the expression of RabE1d-Q74L suggest the activation of other secretory pathways in these plants in addition to the SAR pathway. Interestingly, activation of SAR and resistance to Pst DC3000 in the GFP-RabE1d-Q74L transgenic plants did not correlate with a dwarf phenotype, a common phenotype of Arabidopsis mutants that are constitutively resistant to pathogens (Lorrain et al., 2003
Y2H Assay We identified Arabidopsis (Arabidopsis thaliana) proteins that interacted with AvrPto of Pseudomonas syringae pv tomato strain DC3000 using the Matchmaker LexA-based Y2H system (Clontech Laboratories). Two Arabidopsis cDNA libraries, constructed from infected and uninfected Landsberg erecta plants (kindly provided by J. Jones), were screened. The AvrPto coding sequence was amplified from Pst DC3000 genomic DNA by PCR (sense primer, 5'-GCGAATTCCGAACCATGGGAAATATATGTGTC-3'; antisense primer, 5'-GCCTCGAGATTGCCAGTTACGGTA-3') and cloned into pNLexA to serve as bait in the Y2H screen.
We amplified the RabE1d (At5g03520) coding sequence from Arabidopsis Col-0 cDNA using the rabE-5' and rabE-3' primers (Supplemental Table S1), containing the EcoRI and BamHI restriction sites, respectively. The PCR product was ligated into a TOPO vector (pCR2.1; Invitrogen) and sequenced. Single nucleotide changes were introduced in the RabE1d sequence by two-step overlapping PCR, to generate the RabE1d-S29N and RabE1d-Q74L mutant derivatives. RabE1d-S29N was obtained through a G
RabE1d and the mutant variant RabE1d-Q74L were subcloned in the EcoRI and BamHI sites of the binary expression vector pEGAD (Cutler et al., 2000
Total proteins were extracted as follows: approximately 20 mg (fresh weight) of fresh or frozen leaf tissue was ground with a pestle in a microfuge tube in the presence of 100 µL of 1x SDS-PAGE loading buffer (90 mM Tris-HCl, pH 8.0, 100 mM dithiothreitol, 3% SDS, 22.5% Suc, 10 µL mL–1 Protease Inhibitor Cocktail for plant cell extracts [Sigma], and bromphenol blue [to saturation]). Extracts were immediately heated at 80°C for 10 min and then frozen at –20°C. Before loading, extracts were thawed at room temperature and centrifuged at 20,000g for 2 min to pellet debris. An equal volume of each sample was used for SDS-PAGE. Total proteins were separated on precast gradient gels (4%–20%; ISC BioExpress), then transferred onto Immobilon-P membranes (Millipore) using a semidry transfer apparatus (SEMI PHOR; Hoefer Scientific Instruments). Protein detection was carried out with the following primary antibodies: anti-RabE (raised in chicken against recombinant RabE protein expressed in Escherichia coli; Cocalico Biological), anti-XT1 (Faik et al., 2002
Leaves were harvested and weighed immediately prior to extraction. Leaf tissue (2.5 g) was ground with a cold mortar and pestle in the presence of 5 mL of ice-cold extraction buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 10 mM EDTA, 1 mM dithiothreitol, and 10 µL mL–1 Protease Inhibitor Cocktail for plant cell extracts [Sigma]) containing 34% Suc (w/v). The extract was homogenized with a Polytron immersion blender (three pulses of 10 s each), filtered through a single layer of Miracloth, and centrifuged for 10 min at 10,000g to remove most unbroken chloroplasts and nuclei. The supernatant was adjusted to 40% Suc in about a 10-mL final volume (concentration was determined with a refractometer) and layered on a 5-mL cushion of 50% Suc in clear ultracentrifugation tubes. The homogenate was subsequently layered with 10 mL of 34% Suc, 8 mL of 25% Suc, and 8 mL of 18% Suc (w/v). All Suc solutions were prepared in the same buffer used for extraction. Gradients were centrifuged at 100,000g for 3 h, at 4°C, in an SW28 rotor (Beckman). After centrifugation, the membrane-containing interphases were collected and diluted with Suc-free extraction buffer, and membranes were collected by ultracentrifugation (1 h at 100,000g). Membrane pellets were resuspended in equal volumes of SDS-PAGE loading buffer and heated at 80°C for 10 min. Equal volumes were loaded on SDS-PAGE gels. Protein electrophoresis and western blotting were performed as described above.
Pieces of leaves were sampled randomly and mounted in water. Imaging was performed using an LSM510 META inverted confocal laser scanning microscope (Zeiss) and either a 20x or a 40x oil-immersion objective. For GFP-RabE fluorescence analysis, the 488-nm excitation line of an argon ion laser was used, with a 505- to 530-nm band-pass filter, in the single-track facility of the microscope. Images were processed with the LSM Image Browser version 3.1 (Zeiss) and with the Adobe Photoshop Elements version 5.0 software (Adobe Systems). For FM4-64 staining, detached Arabidopsis leaves were submerged in 8.2 µM FM4-64 (Molecular Probes) in water for 15 min. Leaves were rinsed in distilled water and observed immediately. For imaging GFP-RabE1d and FM4-64 fluorescence, the 488-nm excitation line was used; GFP fluorescence was collected with a 505- to 530-nm band-pass filter, and FM4-64 fluorescence was collected with a 615-nm long-pass filter.
ST-RFP transient expression in Arabidopsis leaves was achieved by biolistic transformation. The binary vector was a gift of Dr. F. Brandizzi (Saint-Jore et al., 2002
Arabidopsis plants were grown in soil, in growth chambers, under a 12-h-dark/12-h-light cycle. The light intensity averaged 100 µE m–2 s–1, and the temperature was kept constant at 20°C. Pst DC3000 bacteria were cultured in low-salt Luria-Bertani medium (10 g L–1 tryptone, 5 g L–1 yeast extract, and 5 g L–1 NaCl) supplemented with 100 µg mL–1 rifampicin. For multiplication assays in plants, bacterial liquid cultures were incubated at 30°C to the mid to late logarithmic phase. Bacteria were collected by centrifugation and resuspended in sterile water with the addition of 0.004% Silwet L-77 (OSI Specialties). Titer of the bacterial inoculum was 1 x 105 cfu mL–1, unless otherwise indicated. Arabidopsis leaves were inoculated by syringe infiltration, and bacteria enumeration in leaves was conducted as described previously (Katagiri et al., 2002
Total RNA was extracted from 100 mg of Arabidopsis leaf tissue with the RNeasy Plant Mini Kit (Qiagen), according to the manufacturer's specifications. RNA concentration in samples was determined with a NanoDrop ND-1000 Spectrophotometer (NanoDrop). RNA RT and target gene amplification (RT-PCR) were performed using the RNA LA PCR Kit (AMV), version 1.1 (TaKaRa). RT reaction mixture was prepared according to the manufacturer's protocol (5 mM MgCl2, 1x RNA PCR buffer, 1 mM deoxynucleoside triphosphate mixture, 1 unit µL–1 RNase inhibitor, 0.25 units µL–1 AMV reverse transcriptase, 0.125 µM oligo[dT] Adaptor Primer, RNase-free water, and 1 µg of total RNA). For amplification of the RabE and RabD transcripts, a single RT reaction was carried out in a total volume of 50 µL and incubated in a thermal cycler for 30 min at 45°C, followed by 5 min at 99°C and 5 min at 5°C. Five microliters of reverse-transcribed cDNA was used as template in each of 10 PCRs with gene-specific primer pairs designed to amplify the five RabE gene family members, the four RabD genes, and the Actin8 gene as a control. Primer sequences are listed in Supplemental Table S2. Each PCR sample contained 2.5 mM MgCl2, 1x LA PCR buffer II, 0.2 µM forward primer and 0.2 µM reverse primer, sterilized distilled water, and 5 µL of the RT reaction described above in a final volume of 25 µL. The reactions were placed in a thermal cycler, and amplification was performed under the following conditions: 94°C for 2 min (one cycle); 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min (22 or 25 cycles); 72°C for 1 min (one cycle); and then 4°C. Ten microliters of the PCR samples was loaded on a 1% agarose gel. Gels were photographed with a Bio-Rad Gel Documentation System, and band intensity was analyzed with the Quantity One software (Bio-Rad).
BTH (Actigard) was prepared at a final concentration of 300 µM in water and sprayed onto potted GFP-RabE1d-Q74L and Col-0 gl1 Arabidopsis plants. A separate set of plants was sprayed with water as a control. Plants were covered with a tight-fitting clear plastic dome and assayed for responses at 3 d after BTH application.
BTH-treated and control plants were harvested 3 d after treatment. Whole plants were vacuum infiltrated for 2 min with distilled water containing 0.002% Silwet L-77 (OSI Specialties). The plants were placed in conical centrifuge tubes (Nalgene) containing a mesh septum placed about 2 cm above the bottom. IWF was collected by centrifuging the infiltrated plants at 400g for 20 min at 4°C. The IWF volume was measured with a micropipette, and the appropriate volume of 5x SDS-PAGE loading buffer was immediately added. Samples were heated at 85°C for 5 min, then frozen or loaded onto an acrylamide gel.
The following materials are available in the online version of this article.
We thank colleagues who kindly shared the antibodies used in this study: Dr. Marc Boutry (anti-PM ATPase), Dr. Xinnian Dong (anti-PR1), Dr. Ken Keegstra (anti-XT1), Dr. Kinya Nomura (anti-RabE), and Dr. Natasha Raikhel (anti-  -TIP). We are thankful to Dr. Federica Brandizzi and Dr. Melinda Frame for assistance with confocal microscopy, Dr. Shuo Cheng Zhang for help with particle bombardment, and Beth Rzendzian and Zach Ferguson for plant care. The ST-RFP construct was a kind gift of Dr. Federica Brandizzi. Dr. Jonathan Jones provided the Arabidopsis cDNA libraries that we used in our Y2H screen. Thanks to members of the He laboratory for helpful comments and to Karen Bird for editorial review of the manuscript. Received November 13, 2008; accepted February 15, 2009; published February 20, 2009.
1 This work was supported by the U.S. Department of Energy (grant no. DEFG02–91ER20021) and the National Institutes of Health (grant no. R01AI060761). 
2 Present address: Center for Research in College Science Teaching and Learning, Michigan State University, East Lansing, MI 48824.
3 Present address: Department of Medicine, Virginia Commonwealth University and McGuire Veterans Affairs Medical Center, Richmond, VA 23284. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Sheng Yang He (hes{at}msu.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.132092 * Corresponding author; e-mail hes{at}msu.edu.
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ABSTRACT
RESULTS
A substitution; in the first PCR step, two overlapping RabE1d fragments were amplified using the primer combinations rabE-5'/S29N-rev and rabE-3'/S29N-for (Supplemental Table S1). The products were purified from agarose gel, mixed, and used as template for a second PCR amplification step, with the rabE-5' and rabE-3' primers. The presence of an overlapping region allowed annealing of the two gene fragments and amplification of the full-length coding sequence. A similar procedure was used to introduce the Q74L mutation through an A