In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro, suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2. We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.
Plant receptor-like kinases (RLKs) are divided into three large related groups of proteins, namely receptor kinases, receptor-like cytosolic kinases (RLCKs), which lack the extracellular receptor domains, and receptor-like proteins (RLPs), which lack a protein kinase domain (Shiu and Bleecker, 2003). The RLK gene families in Arabidopsis and rice have been classified according to protein kinase domain and extracellular receptor domain homologies, with 45 subfamilies in Arabidopsis (Shiu and Bleecker, 2001, 2003; Shiu et al., 2004). Most receptor kinases and RLPs have no known ligands, and regulation of receptor kinase activity and downstream signalling is known in detail for only a few RLKs. Receptor kinases are implicated in many aspects of plant biology including growth and development, self-incompatibility in pollination, plant–microbe interactions, and defence responses (Johnson and Ingram, 2005). Receptor-like cytosolic kinases and RLPs are thought to associate with partner receptor kinases to build functional complexes, and function in self-incompatibility (Murase et al., 2004), plant development and pathogen defence (Muto et al., 2004; Rathjen et al., 1999; Rowland et al., 2005; Veronese et al., 2006).
To determine whether Rop signalling might rely on protein kinase coupling, we used yeast two-hybrid screening to obtain Rop protein kinase interactors and proceeded with their functional characterization. We identified a novel cysteine-rich receptor kinase (NCRK) and two group VI receptor-like cytosolic kinases RBK1 and RBK2 (Rop Binding protein Kinases) as direct molecular interactors of Rop GTPases. Rops interacted directly with the protein kinase domains, and binding did not require a full size catalytically active protein kinase. The NCRK protein was hyperphosphorylated and localized to endosomes in plant protoplasts, indicating efficient activation and internalization from the plasma membrane. The occurrence of a transient interaction with Rop GTPase at the plasma membrane, could be demonstrated for NCRK and RBK1 using BiFC assays. Gene expression data suggested roles for NCRK during tracheary element differentiation and for RBK1 during pathogen defence.
Molecular interaction of Rop GTPases with RLK kinase domains
To identify molecular interactors of Rop GTPases, yeast two-hybrid screening was performed on an Arabidopsis cell suspension culture cDNA library according to Soellick and Uhrig (2001), using constitutively active and prenylation-deficient AtRop4 G15V C193S as a bait. Several receptor-like protein kinase cDNAs were obtained belonging to the RLCK-VI family according to the classification by Shiu and Bleecker (2001). One partial clone, named RBK1-322 corresponded to At5g10520 and two partial clones, named RBK2-311a and b to At3g05140, respectively. Another partial clone corresponded to an unclassified receptor kinase gene NCRK (At2g28250), named NCRK-313. Prey kinase clones did not interact with a nucleotide-free and prenylation-deficient bait AtRop4 T20N C193S, used as a negative control (Figure S1). Sequencing showed that RBK1-322 and RBK2-311 contained complete kinase domains and C-termini, whereas NCRK-313 contained a partial kinase domain from subdomain V to XI and ended within the variable C-terminus outside of the catalytic domain (Figure 1a). Proper expression of bait AtRop4 and prey kinase clones was confirmed by western analysis of yeast whole cell extracts using Rop4 monoclonal antibodies and anti-HA antibodies, respectively (Figure 1b). C-terminal deletion of kinase subdomains IX–XI from NCRK-313 or subdomain XI from RBK1-322 abolished interaction with AtRop4 in the yeast two-hybrid system, while the C-termini outside the catalytic domains of RBK1-322 and RBK2-311 were not sufficient for interaction (data not shown). Using the NCRK-313 clone as a bait in a second screening, three full-length independent AtRop11 clones (At5g62880) were among several prey clones pulled out from the cell suspension culture cDNA library. The AtRop11 isoform was found also to interact with RBK1-322 and RBK2-311 in yeast two-hybrid assays (data not shown). Prey AtRop11 in yeast does not contain the G15V mutation and is therefore probably mainly in the GDP-bound form, indicating that the observed difference in interaction of the prey kinases to G15V and T20N mutants of AtRop4 baits might primarily be caused by the predicted lack of guanine nucleotide binding caused by the T20N mutation (Gibson and Wilson-Delfosse, 2001). Using an in vitro binding assay, the interaction of Rops and RLKs was examined for the RBK1 clone 322 protein. His(6×)-tagged RBK1-322 protein bound equally well to either GDP or GTPγS-loaded GST-AtRop4 (Figure 1c). In contrast, a maltose-binding protein (MBP) fusion of a GBD derived from AtGAP2 gene At4g03100 bound more efficiently to GTPγS loaded GST-Rop4. Binding of RBK1-322 protein to either GDP or GTPγS-bound AtRop4 could also be demonstrated using a protein autophosphorylation assay (Figure 1c). This indicated that binding of the RBK1-322 protein to AtRop4 was independent of the activation state of the GTPase.
NCRK contains a novel cysteine-rich receptor domain
The extracellular receptor domain encoded by NCRK genes contains two repeats of about 40 amino acids sharing a novel conserved cysteine-rich motif, WXCXCX13–18CX3CXC (Figure S2a). The C-terminus encoded by NCRK-like sequences from different plant species such as the magnoliids Persea americana and Liriodendron tulipifera and the conifers Cryptomeria japonica and Picea glauca contains an absolutely conserved DLTEPR core motif (Figure S2b), within an acidic PEST-like sequence (Rechsteiner and Rogers, 1996). The RLCK-VI gene subfamily in Arabidopsis consists of two separate clades of seven genes each (Shiu and Bleecker, 2001), with RBK1 and RBK2 belonging to the same clade, here named RLCK-VIb. RLCK-VIb homologous genes were represented by expressed sequence tags (ESTs) from the gymnosperms Cycas rumphii and different conifers (not shown) indicating that this group of RLCKs is likely to be conserved in all seed plants.
Preferential binding of GTP-bound AtRop4 to NCRK in insect cells
We decided to study the molecular interaction between Rop GTPase and NCRK by flow-cytometric fluorescence resonance energy transfer (FRET) assay in insect cells. This method has the advantage over in vitro binding assays of determining the interaction of full-length trans-membrane NCRK with prenylated AtRop4 in a more natural membrane environment. The combination of a YFPv-tagged GBD derived from AtGAP2 gene At4g03100 and enhanced cyan fluorescent protein (ECFP)-tagged AtRop4 G15V was used as a positive control FRET pair (Figure 2a). Comparable high FRET efficiency was also measured with a control CFP–yellow fluorescent protein (YFP) fusion protein. Low but significant FRET was observed between NCRK-YFPv and either wildtype or G15V ECFP-AtRop4. The interaction with the G15V mutant version was consistently higher than with wildtype AtRop4, indicating a preferential binding of NCRK to GTP-bound AtRop4. Fluorescence resonance energy transfer between AtRop4 T20N and NCRK did not exceed background levels, in agreement with a lack of interaction observed in yeast two-hybrid assays. Histogram analysis of the cells in the FRET channel showed a shift of the distribution maximum to higher signal strength when using ECFP-AtRop4 G165V (Figure S5). C-terminal deletion of NCRK protein kinase subdomains IX–XI, also abolished FRET with AtRop4 as expected from yeast two-hybrid results (Figure 2a). Proper expression of YFPv- and ECFP-tagged proteins was confirmed by the levels of fluorescence observed in FRET analysis and by western analysis of total transfected insect cell lysates using GFP monoclonal antibodies, indicating comparable levels of expression for ECFP-Rops and NCRK-YFPv, and higher levels of expression for cytosolic YFPv-GBD (Figure 2b). Yellow fluorescent protein serves as an acceptor fluorophor and thus lower expression levels of the transmembrane protein NCRK-YFPv correlated well with lower FRET efficiency. When transformed into plant protoplasts, NCRK-YFPv displayed a large range of mobility on low-percentage SDS-PAGE due to hyperphosphorylation (see below), but not so in insect cells, possibly due to the absence of a plant-specific ligand required for kinase activation. NCRK remained unphosphorylated in the presence of interacting constitutively active AtRop4 G15V, indicating that GTP-bound Rop was not sufficient to activate NCRK in insect cells (Figure 2c).
To determine the possibility of the Rop GTPases being RLK substrates, we used immunoprecipitated NCRK-YFPv and RBK1-YFPv expressed in insect cells in protein kinase assays with added recombinant Rop4 protein. Autophosphorylation activity was detected for RBK1, but not for NCRK (not shown). Cross-phosphorylation of either GDP- or GTPγS-loaded Rop4 by RBK1 did not exceed background levels (Figure S3), suggesting that Rop4 is not a RBK1 substrate.
Rop4 GTPase interacts with NCRK and RBK1 kinases in plant protoplasts
To demonstrate in vivo interaction between Rop4 and NCRK and RBK1 kinases, we used the bimolecular fluorescence complementation (BiFC) method, which is well suited to monitoring transient molecular interactions in plants (Bhat et al., 2006). In co-transformed Arabidopsis seedling protoplasts, split YFP-tagged GTP-bound Rop4 G15V interacted with both split YFP-tagged NCRK and RBK1 kinases (Figure 3a), as evident from the presence of reconstituted YFP fluorescence at the plasma membrane. In contrast, YFP-tagged NCRK and RBK1 localized predominantly to endomembrane compartments (Figure 3b). Nucleotide-free Rop4 T20N was relatively ineffective in BiFC, whereas comparable amounts of the split YFP proteins could be detected by immunoblot analysis (not shown). The observation that the T20N mutation in Rop4 led to a decreased BiFC signal, in agreement with the yeast two-hybrid and FRET data, indicated that specific molecular interactions between Rop4 and the kinases caused the BiFC effect in protoplasts.
NCRK partially localizes to RabF2a positive endosomes in plant protoplasts
The intracellular localization of YFPv-tagged kinases was examined in transformed plant protoplasts using a plant expression plasmid that allowed relatively high expression levels (Reichel et al., 1996). In tobacco mature leaf protoplasts, NCRK localized to a few (one to ten) large globular endomembrane compartments of 2–4 μm diameter with visible lumen. In protoplasts derived from Arabidopsis cell suspension, NCRK-YFPv was mainly associated with numerous smaller vesicles, although a few larger compartments were present as well (Figure 4). A few cells with low amounts of plasma membrane-localized NCRK-YFPv could also be observed (not shown). In Arabidopsis protoplasts, the larger NCRK positive endomembrane compartments and a subpopulation of the NCRK-positive small vesicles were also positive for ECFP-RabF2a used commonly as a marker for early endosomes and prevacuolar components (PVCs) (Ueda et al., 2004). ECFP-RabF2a was present in endomembranes and also showed a diffuse fluorescence consistent with the cycling of Rab GTPases between membranes and cytosol (Figure 4h,k), whereas NCRK-YFPv was confined to membranes. The GFP-AtRop4 localized to both plasma membrane and cytosol, while ECFP-AtRop11 was predominantly associated with the plasma membrane (Figure 4), probably related to the different lipid modifications predicted for these Rop isoforms. The presence of NCRK in early endosomes and PVCs indicated that the protein was internalized from the plasma membrane, suggesting that molecular association of Rop GTPases and NCRK might occur transiently at the plasma membrane. Dominant negative AtRop4 T20N did not change the endomembrane localization of NCRK, indicating that active GTP-bound Rops are not required for internalization of NCRK (Figure 5). Co-expression of NCRK with wildtype or constitutively active Rops resulted in retention of NCRK in a perinuclear membrane compartment (Figure 5), which partially co-localized with the endoplasmic reticulum (ER) and Golgi marker ER-CFP (not shown). In transgenic Arabidopsis expressing NCRK-YFPv under the control of the 35S promoter, the receptor kinase was also exclusively present in endomembranes, including the globular compartments of diameter up to 5 μm with visible lumen, showing that NCRK internalization is not specific for protoplasts (Figure S4).
RBK1 localizes to cytosol and endomembranes
RBK1-YFPv was mainly cytosolic in tobacco leaf protoplasts, as indicated by a diffuse fluorescence (Figure 4), but was also associated with endomembrane compartments in Arabidopsis protoplasts, with occasional nuclear localization as shown (Figure 5). Since RBK1 is predicted from its sequence to be a cytosolic protein this indicated the presence of endogenous endomembrane-localized RBK1-interacting proteins in Arabidopsis protoplasts. Co-expression of RBK1 with either dominant negative or constitutively active Rop mutants had no effect on localization of RBK1 (Figure 5). RBK1-YFPv and ECFP-Rops did not co-localize at the plasma membrane, which was indicated as the site of interaction in the BiFC experiments, suggesting the molecular interaction at the plasma membrane was transient.
The ER localized NCRK is unphosphorylated, while endosomal NCRK is hyperphosphorylated
Tagged proteins were correctly expressed in plant protoplasts (Figure 6a). In contrast to NCRK-YFPv expressed in insect cells, plant NCRK-YFPv showed a large range of electrophoretic mobility on low-percentage SDS-PAGE gels (Figure 6b). This mobility shift could be eliminated by lambda protein phosphatase treatment, indicating the protein was mostly hyperphosphorylated when expressed in plant protoplasts (Figure 6c, lanes 1 and 2). To determine a possible effect of Rops on regulation of kinase activity, we examined the phosphorylation status of NCRK in protoplasts co-transformed with RopGAP and different Rop constructs (Figure 6b). Co-transformation experiments with either dominant negative ECFP-Rop4 T20N or RopGAP2, which are predicted to deplete the cell of endogenous active Rops, indicated that Rop-GTP is not necessary for hyperphosphorylation of NCRK (Figure 6b, lanes 3 and 4). When NCRK-YFPv was co-expressed with either wildtype or constitutively active ECFP-Rops, the mobility of NCRK-YFPv in SDS PAGE was similar to protein phosphatase-treated NCRK-YFPv from single transformations (Figure 6c, lanes 3 and 4), and was resistant to protein phosphatase treatment (Figure 6c, lanes 5 and 6), indicating that NCRK remained inactive under these conditions, where it was retained in the ER. The mobility range for RBK1 was smaller and more difficult to resolve on 6% acrylamide gels, but lower-mobility forms were visible as a ladder, suggesting that RBK1 was also partly phosphorylated in plant protoplasts irrespective of the presence of co-expressed Rop mutants (Figure 6d).
Gene expression analysis of Rop-interacting protein kinases
Examination of public Arabidopsis micro-array databases indicated that RLCK-VIb genes are ubiquitously expressed, with several members of the gene family especially highly upregulated during differentiation of tracheary elements and in pollen. The single-copy NCRK gene was also highly expressed during differentiation of tracheary elements but not in pollen (Table S1). Transgenic Arabidopsis lines containing an NCRK promoter GUS fusion construct were used to analyse gene expression in 1–3-week-old seedlings. The NCRK gene appeared ubiquitously expressed, with the highest levels of GUS activity in leaf primordia, in the root apical meristem, in lateral root primordia, and in the stele of older roots and hypocotyls (Figure 7a,d–f). In cotyledons and leaves, expression was highest in vasculature and hydathode endothem (Figure 7a–c). Trichomes expressed relatively high levels of GUS activity (Figure 7a). In situ mRNA detection on 6-day-old Arabidopsis seedling sections showed that NCRK gene expression was upregulated in the apical shoot meristem and young leaf primordia (Figure 7g,h), and confirmed higher levels of gene expression associated with vascular strands as shown for cotyledon and hypocotyl sections (Figure 7g–i).
Arabidopsis RBK1 promoter GUS seedlings indicated relatively high expression levels in vasculature, hydathode endothem, leaf mesophyll cells and trichomes. In contrast to NCRK, the RBK1 gene was not expressed at high levels in the root apical meristem and lateral root primordia (Figure 8e,f). In situ mRNA detection experiments confirmed the relatively high levels of RBK1 gene expression associated with vasculature, as shown for cotyledon and hypocotyl sections (Figure 8g–i).
Locally induced expression of the RBK1 gene upon pathogen exposure
Public microarray data indicated strong upregulation of RBK1 gene expression in leaves exposed to oomycete and fungal pathogens (Table S1). Since several RLK genes have been functionally characterized in relation to pathogen defence, we decided to study this phenomenon in more detail. When Phytophthora infestanszoospores were applied to Arabidopsis rosette leaves, RBK1 mRNA accumulated peaking at 6 h after infection as demonstrated by northern analysis (Figure 9a). Independent RBK1 promoter GUS lines showed local induction of gene expression at the sites of P. infestans application (Figure 9b). Local application of Botrytis cinerea spores resulted in RBK1 mRNA upregulation at 24 h after inoculation as measured by real-time RT-PCR (Figure 9c), and likewise resulted in local induction of GUS activity in RBK1 promoter GUS lines (Figure 9d). A general ethylene responsiveness of the RBK1 promoter has been shown by Millenaar et al. (2005) and Zimmermann et al. (2004), and could be confirmed in RBK1 promoter GUS lines upon ethylene treatment as an induction of transcription of the gene in the vascular cylinder of primary roots and in the root cortex in the root/hypocotyl junction zone (Figure 9e). Upregulation of RBK1 transcription during B. cinerea exposure could be reduced to levels comparable to those detected in the mock-treated samples by the inhibitor of ethylene perception 1-MCP (Figure 9c), suggesting that ethylene production during B. cinerea attack was a major RBK1-inducing signal.
In this study we identified a cysteine-rich receptor kinase NCRK and two RLCK-VIb cytosolic kinases RBK1 and RBK2 as novel molecular interactors of Rop GTPases by means of yeast two-hybrid screening. Plant Rops are unique among the Rho GTPases in interacting directly with receptor protein kinase domains. A molecular interaction of full-length NCRK and Rop GTPase was confirmed by flow cytometric FRET analysis of YFPv and ECFP-tagged proteins expressed in insect cells. The occurrence of the Rop/NCRK and Rop/RBK1 interactions in plant cells was demonstrated by BiFC assay, which established the plasma membrane as the site of interaction. The NCRK gene is highly expressed during tracheary differentiation, while RBK1 gene expression is upregulated by pathogen exposure and ethylene. Our data indicate that different groups of Rop GTPases have a general potential to interact with specific RLKs, and mark these previously uncharacterized protein kinases as participating in Rop-regulated aspects of plant development and pathogen defence.
Consequences of the Rop/RLK molecular interactions
Rho-associated kinases are an important class of Rho effectors in yeast and animal cells, whereas target kinases for Rops in plants have not yet been found. Rho GTPases commonly bind to conserved binding domains outside the catalytic domain of their target kinases (Zhao and Manser, 2005), which is not a feature of the Rop/RLK interaction. An important question is whether these RLKs might phosphorylate Rops or whether Rops might modulate RLK signalling. The presence of conserved putative phosphorylation sites in Rop GTPases has been noted previously, and the possibility of Rops being substrates for RLK kinase activity has been suggested by Agrawal et al. (2003). However, the lack of efficient phosphorylation of Rop-GDP and Rop-GTP by RBK1 as observed in in vitro kinase assays argued against Rop being a natural substrate for phosphorylation by RLCK-VIb kinases, even though this will have to be clarified by in vivo experiments. Although yeast two-hybrid results and pull-down experiments showed that both GDP- and GTP-bound Rops are able to interact with NCRK and RLCK-VIb proteins, FRET experiments with full-size NCRK and prenylated Rop in a membranous environment indicated a preferential interaction with the GTP-bound conformation, suggesting that GTP-bound Rop might be the preferred interactor in vivo. NCRK expressed in insect cells, with or without co-expressed constitutively active Rop GTPase, was not hyperphosphorylated, indicating that GTP-Rop binding is not sufficient for NCRK activation and also suggesting the absence of the relevant activating plant ligand.
NCRK localization and activation in plant protoplast
Partial co-localization of NCRK with the endosomal and PVC marker RabF2a (Ueda et al., 2004) in co-transformed plant protoplasts indicated that the NCRK protein is internalized from the plasma membrane. NCRK was hyperphosphorylated in transformed plant protoplasts but not in insect cells, which indicated the presence of a functional activating stimulus in protoplasts, presumably a ubiquitous higher plant ligand. Since Rops and NCRK expressed in protoplasts were not co-localized, it is likely that a transient molecular interaction of Rop and NCRK preceded internalization of NCRK from the plasma membrane, as was also indicated by the occurrence of BiFC at the plasma membrane. Depletion of active endogenous Rop in protoplasts co-transformed with NCRK and either dominant negative Rop4 T20N or RopGAP2 did not inhibit NCRK hyperphosphorylation, indicating that receptor kinase activation did not require Rop GTPase. When NCRK-YFPv and wildtype or constitutively active CFP-Rops were co-expressed from plasmids that allowed high expression levels, NCRK-YFPv was retained in the ER. We think this protein trafficking blockage might be due to ectopic molecular interaction of Rops with ER-localized NCRK, and was not caused by a general inhibition of membrane cycling in the presence of high intracellular Rop protein levels as described by Bloch et al. (2005), since the plasma membrane localization of a control CFP-tagged RLK BRL3 (Cano-Delgado et al., 2004) in the presence of over-expressed Rops was unchanged (not shown). Taken together, the data are consistent with a general characteristic of receptor kinase signalling, i.e. ligand-induced activation at the plasma membrane and internalization of receptor kinase oligomers.
RLCK-VIb gene expression is suggestive of a role in plant development and pathogen defence
Specific Rop genes in Arabidopsis are highly upregulated in elongating pollen tubes and support tip growth (Gu et al., 2003, 2005; Vernoud et al., 2003). Other Rops are associated with morphogenesis of epidermal leaf cells (Fu et al., 2002, 2005) or are highly expressed in vasculature (Brembu et al., 2005). Apart from their role in different aspects of plant development, Rops are also involved in plant defence responses (see reviews by Agrawal et al., 2003; Gu et al., 2004). Therefore, gene expression patterns of Rop interactors may indicate their importance in different Rop-mediated processes. Arabidopsis RLCK-VIb genes are differentially expressed during plant development with specific genes upregulated during xylogenesis or in pollen, as in case of the RBK2 gene (Pina et al., 2005), and during pathogen attack as for the RBK1 gene. The RBK1 promoter was ethylene responsive and maximal RBK1 mRNA levels during B. cinerea exposure required ethylene perception. Higher gene expression levels of the seven-member RLCK-VIb gene clade generally correlated with higher levels of cell wall biosynthesis and/or ROS production, whether during tracheary differentiation, in pollen, trichomes or during the hypersensitive response caused by P. infestans and B. cinerea pathogens. This expression profile might indicate that RLCK-VIb protein kinases function in cell wall sensing, similar to WAK and THE1 kinases (Decreux and Messiaen, 2005; Hematy et al., 2007).The presence of a universal stress protein (USP) domain in a number of RLCK-VIa and -VIb protein kinases (Kerk et al., 2003) might also suggest a role for this family of protein kinases in oxidative stress signalling, although the activating stimulus for RLCK-VIb kinases remains unknown.
NCRK, an orphan receptor kinase gene highly upregulated during tracheary differentiation
Homozygous Arabidopsis NCRK gene knock-out lines or overexpressing lines did not show obvious abnormalities (results not shown), offering no preliminary indications for gene function. High NCRK gene expression levels in trichomes were consistent with a relatively large number of plant NCRK ESTs originating from cotton fibre cDNA libraries in public databases (not shown), and seem to argue against a role for NCRK in cell type specification or intercellular communication, but instead for a role in cell wall sensing and detection of an autosecretory ligand. Relatively high levels of NCRK gene expression in vasculature were consistent with data by Kubo et al. (2005), which show that the NCRK gene is highly upregulated in developing tracheary cells in Arabidopsis cell suspensions treated with brassinolide to undergo trans-differentiation to xylem elements. The changes in cell morphology during differentiation of tracheary elements are accompanied by extensive rearrangements in the microtubular and actin cytoskeletons and altered cell wall synthesis (Fukuda, 2004), which will require Rop action and possibly a number of cell wall sensing RLKs.
We currently hypothesize that ligand-dependent NCRK signalling and RLCK-VIb kinase signalling are modulated by Rop GTPases. It will be interesting to identify RLCK-VIb- and NCRK-interacting proteins in future and elucidate the related signalling pathways.
Yeast two-hybrid screening
AtRop4 constructs were cloned into the bait vector pAS2 (Clontech, http://www.clontech.com/), and yeast two-hybrid screening of an Arabidopsis cell suspension culture cDNA library in pACT2 (Nemeth et al., 1998) was carried out according to Soellick and Uhrig (2001). Protein extracts of yeast double transformants were prepared according to the Clontech yeast protocol handbook and assayed by western blotting. Prey kinase proteins were detected using an anti-HA monoclonal (Covance; http://www.covance.com), while AtRop4 bait proteins were detected using either affinity-purified AtRop4 polyclonals (Eurogentec, http://www.eurogentec.com/) or monoclonal AtRop4 antibodies (Nanotools, http://www.nanotools.de/).
Insect cell transfection and FRET measurements
Full-length NCRK cDNA was obtained from the RIKEN Bioresource Center (RBC) and tagged at the C-terminus with YFPv (Venus version of EYFP; Nagai et al., 2002). GTPase-binding domain cDNA was tagged at the N-terminus with YFPv, while Rop4 wildtype and mutant Rop4 cDNAs G15V and T20N (Molendijk et al., 2001) were tagged at the N-terminus with CFP (ECFP, Clontech). Tagged plant cDNAs were subcloned into pRmHa-3 for transfection of Drosophila melanogasterS2 cells as described (Rolli et al., 2002). Flow cytometric FRET measurements were performed on transfected cells expressing fluorescent proteins essentially according to Chan et al. (2001). For western analysis, frozen insect cell lysates were dissolved in SDS-PAGE loading buffer and samples were run on 15% acrylamide gels. Expressed ECFP/YFPv-tagged proteins were detected using GFP monoclonal antibodies (Roche, http://www.roche.com/) and an ECL detection kit (Amersham, http://www5.amershambiosciences.com/).
Immunoprecipitation and protein kinase assays
Full-length RBK1 cDNA was obtained from RBC, tagged at the C-terminus with YFPv and subcloned into pRMHa-3. NCRK-YFPv and RBK1-YFPv were expressed in Drosophila S2 cells. Immunoprecipitation was performed on cell lysates using monoclonal GFP antibodies (Roche). AtRop4 cDNA lacking the polybasic tail (amino acids 1–179) was cloned in pET45b (Novagen; http://www.merckbiosciences.com). Bacterially expressed AtRop4 was affinity purified using nickel nitrilotriacetic acid (NiNTA), followed by HPLC gel filtration. Kinase assays were performed according to standard procedures using 32P γATP.
Plant protoplast transformation and confocal microscopy
The ECFP-tagged wildtype AtRop4, ECFP-tagged G15V and T20N mutant versions of AtRop4, YFPv-tagged NCRK and YFPv-tagged RBK1 inserts were transferred from pRMHa-3 constructs to plant expression vector pCKGFP (Reichel et al., 1996) from which the GFP encoding DNA had been removed (pCK). AtRabF2a (Rha1; At5g45130) and BRL3 (At3g13380) cDNAs were obtained from the BRC. AtRabF2a and BRL3 were tagged with ECFP at the N-terminus and C-terminus, respectively, and subcloned into pCK. Full-length RopGAP2 (At4g03100) cDNA was also subcloned into pCK. The ER-CFP marker was used as in Boevink et al. (1998) but substituting GFP for ECFP and cloning the marker cDNA into pCK. Protoplasts were transformed according to the polyethylene glycol-mediated transformation protocol (Koop et al., 1996) using 0.5 × 106 cells and 25–50 μg of each plasmid DNA per transformation. As visualized by fluorescence microscopy, transformation efficiencies of approximately 50–80% were routinely obtained, with co-transformation efficiencies at almost 100% (our observations). For microscopy, a Zeiss LSM 510 confocal microscope equipped with a 32-channel spectral detector (Zeiss LSM 510 META) was used with LSM software (Zeiss, http://www.zeiss.com/).
NCRK and RBK1 cDNAs were cloned in BiFC vectors pUC-SPYCE and pUC-SPYNE for C-terminal split YFP tagging (Walter et al., 2004). Rop4 T20N and Rop4 G15V cDNAs were cloned in two home-made vectors for N-terminal split YFP tagging. Twenty-five micrograms of plasmid DNA for each BIFC construct was used to co-transform Arabidopsis seedling protoplasts. The percentage of BiFC-positive protoplasts was determined by cell counting using an Axiovert 200M microscope (Zeiss) with Sensi Cam camera (PCO Computer Optics, http://www.pco.de/) and Metamorph software (MDS; http://www.moleculardevices.com). A minimum of 600 cells were counted for each transformation sample.
Lambda protein phosphatase treatments and SDS-PAGE mobility shift assay
Transformed protoplast pellets were resuspended in phosphatase buffer containing protease inhibitor cocktail, 2 mm MnCl2 and 1% Triton X-100. Protein dephosphorylation reactions using lambda protein phosphatase (λ PPase, 400 U, 30 min, 30°C) were performed according to the manufacturer’s instructions (New England Biolabs, http://www.neb.com/) with phosphatase inhibitor cocktail (Sigma, http://www.sigmaaldrich.com/) added to untreated controls. Samples corresponding to 0.5 × 106 cells were analysed using 20 cm-long 6% acrylamide SDS-PAGE gels, followed by western blotting according to standard protocols.
In situ RNA detection
The DNAs corresponding to the transcriptional trailer regions of NCRK (207 nt) and RBK1 (157 nucleotides) genes were obtained using PCR (Platinum Taq; Invitrogen, http://www.invitrogen.com/) on whole seedling cDNA, cloned in transcription vector pGEM-T easy (Promega, http://www.promega.com/) and sequenced. Biotin-labelled probe RNA was transcribed using the Ribomax kit as recommended by the manufacturer (Promega). Six-day-old Arabidopsis seedlings were embedded in paraffin and processed for microtome sectioning. Tissue sections on slides were hybridized with probe RNAs, using a slide processing robot (Cnops et al., 2006). Immunological detection of hybridized probe was performed using alkaline phosphatase conjugated anti-biotin monoclonal antibody (Sigma).
Arabidopsis transformation and binary promoter GUS constructs
The complete intergenic region and transcriptional leader sequences including ATG start codons of the NCRK (1.94 kb) and RBK1 (2.07 kb) genes were obtained using PCR (AccuPrime pfx DNA polymerase; Invitrogen) on whole Arabidopsis genomic DNA. Promoter DNAs were cloned into Gateway® donor plasmid pDONR207 (Invitrogen), and from there into promoter GUS reporter destination vector pMDC163 (Curtis and Grossniklaus, 2003). Binary constructs were sequenced, and electroporated into Agrobacterium tumefaciensGV3101 pMP90. Agrobacterium-mediated Arabidopsis transformation was carried out using the floral dip method (Clough, 2005).
Pathogen experiments, plant treatments and expression analyses
Phytophthora infestans isolate 208 M2 (Si-Ammour et al., 2003) was grown on oat-bean medium at 18°C in the dark. Ten to twelve-day-old mycelium was incubated with 10 ml of sterile distilled water at 4°C for 3 h. After filtration, zoospores were counted and the appropriate concentration of the zoospore suspension was adjusted. Arabidopsis plants were inoculated with 10 μl drops of the zoospore suspension. After inoculation, plants were incubated in a phytochamber with 16 h of light (200 μE) at 20°C, 8 h dark at 18°C and 100% humidity. Botrytis cinerea (strain SAS 56) inoculation was carried out on mature rosette leaves by spotting four drops of 5μl each (100 spores μl−1) per leaf and collecting infected and mock-treated leaf material after 24 h of incubation at the above described conditions. Botrytis cinerea spores were resuspended in potato dextrose broth (PDB) medium and with mock treatment consisting of 5 μl drops of PDB medium only. 1-Methylcyclopropene (1-MCP) treatments were carried out by incubating plants in closed containers with a saturating concentration (10 p.p.m.) of the chemical. Extraction of RNA was performed by using a commercial kit (NucleoSpin® RNA Plant; Macherey Nagel, http://www.macherey-nagel.com/) following the manufacturer’s instructions. Ethylene treatment was carried out by flushing 7-day-old seedlings, grown on Arabidopsis medium agar plates, in gas-tight jars with 10 p.p.m. ethylene or air (control) for 24 h. Quantitative real-time PCR was carried out on a DNA Engine Opticon2 (Biorad; http://www.bio-rad.com) using SYBR Green RealMasterMix (Eppendorf, http://www.eppendorf.com/) as described by Nonis et al. (2007).
The work was supported by grants from the Deutsche Forschungsgemeinschaft to KP (SFB, 746), the Alexander von Humbold Foundation, the FCI, a Marie Curie Fellowship of the European Community programme ‘Improving Human Research Potential’ under contract number HPMF-CT-2002-01880 to BR, and a DFG grant under contract number Hu794/3-2 to MH. Additional support was provided by the Freiburg Initiative in Systems Biology (# 03139219), a program of the German Federal Ministry in Research and Education. We are grateful to Dr Bingshan Wang for providing plant BiFC vectors, to Drs Olaf Tietz and Taras P. Pasternak for advice on histochemical analysis, to Dr C. S. V. Rajendrakumar for providing RopGAP2 cDNA, to Claudia Gilles for plant transformation and propagation, to Dr Roland Nitschke at the Freiburg University Life Imaging Centre for advice on confocal microscopy, and to Dr William Teale for critical reading of the manuscript. We are also grateful to Professor Giuseppe Firrao and Dr Patrick De Marta, University of Udine, Dipartimento di Biologia Applicata alla Difesa delle Piante, for their kind help with the Botrytis experiments and to dr. Alberto Nonis, Dipartimento di Scienze Agrarie e Ambientali, for his help with real-time RT-PCR.