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Plant Physiol. Jul 2010; 153(3): 1098–1111.
Published online May 20, 2010. doi:  10.1104/pp.110.156109
PMCID: PMC2899920

The Arabidopsis Mitogen-Activated Protein Kinase Phosphatase PP2C5 Affects Seed Germination, Stomatal Aperture, and Abscisic Acid-Inducible Gene Expression1,[C][W]

Abstract

Abscisic acid (ABA) is an important phytohormone regulating various cellular processes in plants, including stomatal opening and seed germination. Although protein phosphorylation via mitogen-activated protein kinases (MAPKs) has been suggested to be important in ABA signaling, the corresponding phosphatases are largely unknown. Here, we show that a member of the Protein Phosphatase 2C (PP2C) family in Arabidopsis (Arabidopsis thaliana), PP2C5, is acting as a MAPK phosphatase. The PP2C5 protein colocalizes and directly interacts with stress-induced MPK3, MPK4, and MPK6, predominantly in the nucleus. Importantly, altered PP2C5 levels affect MAPK activation. Whereas Arabidopsis plants depleted of PP2C5 show an enhanced ABA-induced activation of MPK3 and MPK6, ectopic expression of PP2C5 in tobacco (Nicotiana benthamiana) resulted in the opposite effect, with the two MAPKs salicylic acid-induced protein kinase and wound-induced protein kinase not being activated any longer after ABA treatment. Moreover, depletion of PP2C5, whose gene expression itself is affected by ABA treatment, resulted in altered ABA responses. Loss-of-function mutation in PP2C5 or AP2C1, a close PP2C5 homolog, resulted in an increased stomatal aperture under normal growth conditions and a partial ABA-insensitive phenotype in seed germination that was most prominent in the pp2c5 ap2c1 double mutant line. In addition, the response of ABA-inducible genes such as ABI1, ABI2, RD29A, and Erd10 was reduced in the mutant plants. Thus, we suggest that PP2C5 acts as a MAPK phosphatase that positively regulates seed germination, stomatal closure, and ABA-inducible gene expression.

To cope with the limitations of a sessile lifestyle, plants have evolved a sophisticated network of responses to biotic and abiotic stress. Of the many hormones that mediate such responses, abscisic acid (ABA) has historically been one of the most intensively studied stress hormones (Koornneef et al., 1998; Christmann et al., 2006; Verslues and Zhu, 2007). In particular, ABA promotes stomatal closure and prevents stomatal opening during drought, thus reducing transpirational water loss. During late embryogenesis, ABA promotes the acquisition of desiccation tolerance and seed dormancy and inhibits seed germination. Evidence is also accumulating that ABA plays a crucial role in the plant defense response (Mauch-Mani and Mauch, 2005; Adie et al., 2007; Fan et al., 2009).

ABA signal transduction engages a complex network of both positively and negatively regulating protein kinases and Ser/Thr protein phosphatases (Leung and Giraudat, 1998; Himmelbach et al., 2003; Hirayama and Shinozaki, 2007; Umezawa et al., 2009). Protein phosphatases that dephosphorylate Ser and Thr residues are classified into two groups, the PPP family and the type 2C phosphatases (PP2Cs; Cohen, 1989). The PPP family consists of type 1 (PP1), type 2A (PP2A), and type 2B (PP2B) phosphatases (Farkas et al., 2007), which share sequence homology in their catalytic domains and are sensitive to specific inhibitors. In contrast, PP2Cs share no sequence similarity with PPPs despite striking architectural similarities of their crystal structures (Das et al., 1996). PP2Cs are monomeric enzymes that contain all 11 characteristic subdomains in the catalytic domain (Bork et al., 1996) and constitute the largest protein phosphatase family in plants, with 76 members in Arabidopsis (Arabidopsis thaliana; Kerk et al., 2002; Schweighofer et al., 2004; Kerk, 2007).

The best studied PP2Cs belong to clade A and have been demonstrated to act as negative regulators of ABA responses, most importantly ABI1, ABI2, and the cold response-linked PP2Cs AtPP2CA and HAB1 (Leung et al., 1997; Merlot et al., 2001; Saez et al., 2004; Kuhn et al., 2006; Rubio et al., 2009). Recently, progress has been made in the elucidation of the regulation of clade A phosphatases. The Regulatory Component of ABA Receptor1/Pyrabactin Resistance1 (RCAR1/PYR1) and RCAR3 were identified as interactors of ABI1 and ABI2 (Ma et al., 2009; Park et al., 2009; Szostkiewicz et al., 2010). RCAR1/PYR1 directly binds to ABA and mediates ABA-dependent inactivation of ABI1 or ABI2 in vitro, thus antagonizing PP2C action in planta. Likewise, the Bet v1-like superfamily member PYL5 antagonizes ABI1, ABI2, and HAB1 function by inhibiting their phosphatase activity in an ABA-dependent manner (Santiago et al., 2009). ABI1 and the highly homologous ABI2 have attracted most attention as partially redundant key regulators of ABA-invoked seed dormancy, stomatal closure, and growth inhibition (Merlot et al., 2001). Both phosphatases, particularly ABI2, physically interact with the Protein Kinase Salt-sensitive3 (PKS3), and ABA was shown to transiently down-regulate PKS3 kinase activity, which is required to suppress ABA action (Guo et al., 2002). ABI2 also interacts with the protein kinase Salt Overly Sensitive2 (SOS2), which is required for salt tolerance in Arabidopsis (Ohta et al., 2003). In contrast, in addition to PKS3, ABI1 can interact with the Open Stomata1 kinase, which was shown to be a positive regulator in ABA-induced stomatal closure (Yoshida et al., 2006; Geiger et al., 2009).

Apart from the above-described kinases, mitogen-activated protein kinases (MAPKs) have been implicated in ABA signaling (Heimovaara-Dijkstra et al., 2000; Hirayama and Shinozaki, 2007). Generally, MAPKs have been described as major components of cellular signal transduction pathways mediating various biotic and abiotic stress responses, including hormone signaling, cell division, and developmental processes (Ligterink, 2000; Asai et al., 2002; Jonak et al., 2002; Pedley and Martin, 2005; Mishra et al., 2006; Pitzschke et al., 2009). MAPK cascades are universal signal transduction modules in eukaryotes, including yeasts, animals, and plants, and are generally composed of three functionally linked kinases, a MAPK kinase kinase (MAPKKK or MEKK), a MAPK kinase (MAPKK or MKK), and a MAPK. In response to extracellular stimuli, MAPKKKs activate MAPKKs via phosphorylation of two Ser/Thr residues within the S/TXXXXXS/T motif, where X denotes any amino acid. MAPKKs, which are dual-specificity protein kinases, then activate their downstream MAPK by phosphorylating the Thr and Tyr residues within the TXY motif. Activated MAPKs phosphorylate specific effector proteins, such as transcription factors (Popescu et al., 2009), which leads to an activation of cellular responses.

In Arabidopsis, MPK3, MPK4, and MPK6 are the best characterized members of the MAPK family and have also been demonstrated to be part of the ABA signal transduction pathway. MPK3 is activated by both ABA and hydrogen peroxide in Arabidopsis seedlings, and MPK3 overexpression increases ABA sensitivity in ABA-induced postgermination arrest of growth, suggesting that the ABA signal is transmitted through MPK3 in this system (Lu et al., 2002). In addition, MPK4 and MPK6 are transiently activated after ABA application (Ichimura et al., 2000), and mpk6 mutation blocked while MPK6 overexpression enhanced ABA-dependent hydrogen peroxide production (Xing et al., 2008).

As dephosphorylation of only one residue in the highly conserved TXY motif of activated MAPKs is sufficient to abolish their activity, PP2Cs can readily act as MAPK phosphatases (MKPs). Alfalfa (Medicago sativa) MP2C was the first plant PP2C shown to negatively regulate MAPK signaling. MP2C directly interacts with the salt stress-inducible MAPK (SIMK; homologous to MPK6) and inactivates SIMK through Thr dephosphorylation of the pTEpY motif (Meskiene et al., 1998, 2003). Similarly, Arabidopsis AP2C1, as the closest MP2C homolog, was recently shown to interact with and dephosphorylate MPK4 and MPK6 (Schweighofer et al., 2007). Multiple other examples provide evidence that PP2Cs can attenuate stress-induced MAPK cascades in eukaryotes. The high-osmolarity glycerol (HOG) MAPK pathway, which controls the osmotic stress response in yeast, was shown to be negatively regulated by the PP2Cs Ptc1 and Ptc3 through direct dephosphorylation of the MAPK Hog1 (Nguyen and Shiozaki, 1999; Warmka et al., 2001). Likewise, in humans, the JNK/p38 pathway, which shares similarities with the yeast HOG pathway, is inactivated by direct binding to and dephosphorylation of p38 MAPK by PP2Cα (Takekawa et al., 1998). All these examples clearly indicate that PP2Cs are regulating diverse signaling pathways mediated by MAPK cascades.

Here, we report the identification of PP2C5 as a MAPK phosphatase. We show that PP2C5 directly interacts with and regulates the activation of stress-induced MPK3, MPK4, and MPK6. Depletion of PP2C5 and its closest homolog AP2C1 results in plants with an increased stomatal aperture, partial ABA insensitivity during seed germination, and a decreased responsiveness of ABA-inducible genes after ABA application. Thus, unlike previously described PP2Cs, PP2C5 positively regulates seed germination, stomatal closure, and ABA-inducible gene expression.

RESULTS

PP2C5 Expression Is Induced by ABA

To identify phosphatases that attenuate MAPK activities during ABA signaling, we focused on clade B of the PP2C superfamily (Supplemental Fig. S1A), of which one member, AP2C1, was recently demonstrated to act as MAPK phosphatase (Schweighofer et al., 2007). In addition, four out of the six members of clade B contain a putative MAP kinase interaction motif (KIM) similar to those found in animal MAPK kinases or MAPK phosphatases (Ho et al., 2003, 2006), suggesting that these proteins might interact with MAPKs in plants (Schweighofer et al., 2004, 2007). As the phosphatases ABI1, ABI2, AtPP2CA, and HAB1, which are all involved in ABA signaling, are transcriptionally up-regulated by ABA (Leung et al., 1997; Merlot et al., 2001; Saez et al., 2004; Kuhn et al., 2006), we analyzed ABA-inducible accumulation of transcripts encoding the four KIM-containing clade B phosphatases AP2C1 (At2g30020), AP2C2 (At1g07160), PP2C5 (At2g40180), and AP2C4 (At1g67820). Analysis of microarray data obtained from experiments conducted within the AtGenExpress initiative (Goda et al., 2008) revealed that gene induction was most prominent for PP2C5 after a 30-min treatment with ABA (Supplemental Fig. S1B). This is in agreement with an earlier report that PP2C5 belongs to an ABA-inducible gene cluster (Wang et al., 1999). Similarly, AP2C1 gene expression was weakly induced whereas gene expression of the two other PP2Cs, AP2C2 and AP2C4, was not affected.

PP2C5 Is a Nuclear Protein Phosphatase

Using the PSORT analysis tool, PP2C5 was predicted to be localized to the cell nucleus, the same compartment into which MAPKs are translocated upon activation. This has been shown in Arabidopsis for MPK3 and MPK6 after ozone treatment (Ahlfors et al., 2004) and in parsley (Petroselinum crispum) for PcMPK3 and PcMPK6 after elicitation (Lee et al., 2004). Translocation of MAPKs is required to activate transcription factors, which constitute important MAPK substrates (Feilner et al., 2005; Popescu et al., 2009). In addition, the nucleus seems to be a critical site for termination of MAPK signaling by (1) nuclear sequestration of activated MAPKs away from the MAPKK as their cytoplasmic activator and (2) dephosphorylation-mediated inactivation by specific nuclear phosphatases.

To determine PP2C5 protein localization in vivo, GFP-tagged PP2C5 protein was transiently expressed either under the control of the constitutive cauliflower mosaic virus 35S promoter or its native promoter in Arabidopsis protoplasts. Irrespective of the promoter strength, PP2C5-GFP was predominantly located to the nucleus, whereas GFP alone displayed fluorescence throughout the cytoplasm and nucleus (Fig. 1A). Likewise, PP2C5-GFP transiently expressed in Nicotiana benthamiana leaves, as a heterologous plant system, also located to the nucleus (Fig. 1B).

Figure 1.
Phosphatase-active PP2C5 is located to the nucleus. A, The coding region of PP2C5 was C-terminally fused to GFP and transiently expressed in Arabidopsis protoplasts either under control of the 35S promoter (35S:PP2C5-GFP) or its native promoter (PP2C5:PP2C5-GFP). ...

To revalidate PP2C5 phosphatase activity previously described for recombinant PP2C5 (Wang et al., 1999), we first generated polyclonal antibodies against a PP2C5-specific N-terminal peptide in rabbit. Antibodies were tested with protein extracts from Arabidopsis leaves and protein extracts from N. benthamiana plants transiently expressing PP2C5-GFP (Supplemental Fig. S2). Affinity-purified anti-PP2C5 could detect PP2C5-GFP but not endogenous Arabidopsis PP2C5, which is probably present at low levels and needs prior enrichment for detection (Supplemental Fig. S2). Therefore, affinity-purified rabbit anti-PP2C5 antibody was used to immunoprecipitate transiently expressed PP2C5-GFP from N. benthamiana protein extracts. Immunoprecipitated PP2C5-GFP was subsequently subjected to an in vitro protein phosphatase assay using [32P]phospho-casein as artificial substrate (Stone et al., 1994; Wang et al., 1999). In comparison with control samples, PP2C5-GFP-containing immunoprecipitates showed a strong release of 32P into the supernatant, which is indicative of phosphatase activity (Fig. 1C). In summary, our data indicate that PP2C5 is an active PP2C that is mainly located to the nucleus.

PP2C5 Colocalizes and Interacts with Stress-Induced MAPKs

PP2C5 contains an N-terminally located KIM (Schweighofer et al., 2004), and for the PP2C5 homolog AP2C1, an interaction with the stress-induced MPK4 and MPK6 has been demonstrated (Schweighofer et al., 2007). One prerequisite for in vivo protein interaction is their colocalization; hence, we next examined the localization of the three major stress-induced MAPKs, MPK3, MPK4, and MPK6, in the Arabidopsis protoplast system. Yellow fluorescent protein (YFP) fusions of all three MAPKs localized to the same cellular compartment as PP2C5 fused to cyan fluorescent protein (CFP), which was predominantly found in the cell nucleus (Fig. 2A). This colocalization was not observed when other members of the clade B PP2Cs were investigated: AP2C1-CFP and AP2C2-CFP were both predominantly localized to plastids (confirming the PSORT prediction), whereas AP2C4-CFP was found in equal quantities both in the cytosol and the nucleus (Fig. 2B; Supplemental Fig. S3). Hence, PP2C5 is the only member of this PP2C subgroup for which gene expression is induced by ABA treatment and that colocalizes with stress-induced MAPKs in the nucleus. To analyze a direct physical interaction of PP2C5 with MPK3, MPK4, and MPK6, proteins were combined in the yeast two-hybrid system. An interaction was found between PP2C5 and MPK3, MPK4, and MPK6 in comparison with negative controls (Fig. 3A). These results were confirmed using a bimolecular fluorescence complementation (BiFC) assay based on split YFP (Walter et al., 2004). The N- and C-terminal domains of YFP were fused to PP2C5 and MPK3, MPK4, or MPK6, respectively, and transiently coexpressed in Arabidopsis protoplasts. Again, fluorescence from reconstituted YFP indicated an interaction between PP2C5 and all three MAPKs (Fig. 3B). The strongest signal was observed with the PP2C5/MPK6 complexes, which were detectable in the nucleus and in the cytoplasm, whereas PP2C5/MPK4 complexes localized predominantly in the nucleus (Fig. 3B). The weakest signal was obtained with the PP2C5/MPK3 combination. No fluorescence was detectable with the empty vector combinations as control (data not shown).

Figure 2.
PP2C5 colocalizes with stress-induced MAPKs predominantly in the nucleus. PP2C5 (A) or other group B members (B) were transiently coexpressed as CFP fusions in Arabidopsis protoplasts together with MPK3-, MPK4-, or MPK6-YPF fusions. CFP/YFP signals were ...
Figure 3.
PP2C5 interacts with MPK3, MPK4, and MPK6 through its KIM. A, For Y2H analysis, PP2C5 or point-mutated PP2C5-K90A/R91Q (PP2C5KR) was cloned into the vector pGBKT7 and used against MPK3, MPK4, or MPK6 in the vector pGADT7. The positive control was the ...

To investigate the importance of the putative KIM for phosphatase-MAPK interaction, mutations K90A and R91Q in the putative KIM of PP2C5 were generated on the basis that a mutation of the corresponding two amino acids in AP2C1 blocked its interaction with MPK4 and MPK6 (Schweighofer et al., 2007). In both the yeast two-hybrid as well as the BiFC assay, interaction of mutant PP2C5-K90A/R91Q with all three MAPKs was completely abolished compared with wild-type PP2C5 (Fig. 3). These results show that the interaction with MPK3, MPK4, or MPK6 depends on an intact KIM in PP2C5.

PP2C5 Protein Levels Affect MAPK Activation

As PP2C5 is directly interacting with stress-induced MAPKs, we wanted to analyze the effect of PP2C5 depletion on MAPK activation. We selected the T-DNA insertion line N609986 (pp2c5) from the SALK collection (Fig. 4A), in which the T-DNA insertion is located in the second exon. To induce robust PP2C5 gene expression, Arabidopsis seedlings were treated with Flg22, a 22-amino acid peptide derived from bacterial flagellin that elicits plant pathogen defense responses (Felix et al., 1999) and that was shown to strongly trigger PP2C5 gene expression (Gust et al., 2007). Increased PP2C5 transcript levels could be detected in wild-type plants and, interestingly, to an even higher extent in the pp2c5 mutant when using primers amplifying fragments located upstream of the T-DNA insertion (Fig. 4B). However, a much reduced gene expression or no residual transcript accumulation was observed in the pp2c5 mutant using quantitative PCR analysis with primers either downstream of or spanning the region of the T-DNA insertion, respectively, indicating that PP2C5 transcript was strongly reduced and not present as a full-length sequence in the pp2c5 mutant (Fig. 4B).

Figure 4.
Identification of a PP2C5-T-DNA insertion line. A, A T-DNA insertion line was identified from the SALK collection, with the insertion located in the second exon (the asterisk indicates the stop codon, thick lines indicate exons, and thin lines indicate ...

Mutant seedlings were treated with ABA to analyze the activation of MAPKs using the phospho-p44/p42 antibody. In ecotype Columbia (Col-0) plants, ABA application resulted in an enhanced reactivity to the antibody of a protein band running at approximately 46 kD and to a much weaker extent of a protein band of about 44 kD, most likely representing the two major stress-induced MAPKs MPK3 and MPK6 (Fig. 5). However, compared with the wild type, the pp2c5 single mutant plants responded only slightly stronger to ABA. As the AP2C1 gene, which is a close PP2C5 homolog described to negatively regulate MPK4 and MPK6 (Schweighofer et al., 2007), is also transcriptionally induced by ABA (Supplemental Fig. S1), we generated pp2c5 ap2c1 double knockout lines to explore the possible functional redundancy of the two MAPK phosphatases (Fig. 4B). In pp2c5 ap2c1 plants, ABA-induced MAPK activation was further enhanced compared with single mutants and the wild-type plants (Fig. 5), indicating that both phosphatases are functionally redundant. Probing parallel membranes with antibodies raised against MPK3 or MPK6 indicated that the MPK protein levels remained unaltered within the tested time (data not shown), suggesting that PP2C5 and AP2C1 affect the MPK activation profile posttranslationally.

Figure 5.
pp2c5 ap2c1 double mutant plants show an enhanced ABA-induced MAPK activation. Ten to 15 6-d-old seedlings of pp2c5 and ap2c1 single mutants, the pp2c5 ap2c1 double knockout line, and the pp2c5/PP2C5 complemented line were treated with 50 μm ABA, ...

Similarly, after application of the biotic stimulus Flg22 to the leaves, pp2c5 and ap2c1 single mutants responded with a stronger MAPK activation compared with the wild-type control (Supplemental Fig. S4). The enhanced MAPK activation observed in the pp2c5 mutant could be reversed by complementation with a genomic fragment of PP2C5 (Fig. 4B). However, in contrast to ABA treatment, MAPK activities were not further increased in pp2c5 ap2c1 double knockout plants after Flg22 treatment (Supplemental Fig. S4), indicating that depletion of either phosphatase is sufficient to affect the activation of MAPKs during biotic interactions.

Differences in ABA-induced MAPK activation in the pp2c5 ap2c1 double mutant became more apparent when endogenous kinase activities of MPK3, MPK4, and MPK6 were determined after ABA treatment using an immunocomplex kinase assay. Endogenous MAPKs were immunoprecipitated from protein extracts of seedlings treated for 30 min with ABA with isoform-specific antibodies. As shown in Figure 6, in all lines except the pp2c5 ap2c1 double mutant, ABA treatment resulted only in a weak induction of MPK3 and MPK6 activities in comparison with the control samples. However, seedlings of the double mutant line responded to ABA treatment with a very strong increase in MPK3 and MPK6 and additionally also in MPK4 activity, clearly suggesting that PP2C5 and AP2C1 act as redundant MAPK phosphatases. Interestingly, MPK6 activities in the control samples were already increased in the pp2c5 and ap2c1 single and the double mutants compared with the wild-type seedlings or the complemented line.

Figure 6.
ABA-induced activation of immunoprecipitated MPK3, MPK4, and MPK6 is enhanced in pp2c5 ap2c1 mutants. MPK3 (top panels), MPK4 (middle panels), and MPK6 (bottom panels) activation was measured in the wild type, pp2c5 and ap2c1 single mutants, the pp2c5 ...

We next wanted to investigate the effect of PP2C5 overexpression on MAPK activation. As we could not obtain any stably transformed PP2C5-overexpressing Arabidopsis plants, we investigated MAPK activation in N. benthamiana leaves transiently expressing PP2C5 as a GFP fusion (Fig. 1B). Leaves were treated with ABA or the flagellin-derived peptide Flg22 to trigger MAPK activation, and extracted proteins were subjected to an in gel kinase assay. Complementary to the enhanced MAPK activation observed in PP2C5- and AP2C1-depleted Arabidopsis leaves, N. benthamiana leaves expressing the PP2C5-GFP fusion protein were strongly impaired in both ABA- and Flg22-induced activation of two major MAPKs with approximate sizes of 48 and 46 kD, respectively, presumably representing salicylic acid-induced protein kinase (SIPK; an MPK6 ortholog) and wound-induced protein kinase (WIPK; an MPK3 ortholog; Fig. 7; Supplemental Fig. S5). These results strongly suggest that PP2C5 acts as a negative regulator of MAPK signaling, particularly of MPK3 and MPK6.

Figure 7.
PP2C5-overexpressing leaves are impaired in ABA-stimulated MAPK activation. Constructs for 35S:GFP or 35S:PP2C5-GFP were transiently expressed in N. benthamiana leaves as described in Figure 1B. After 2 d, leaves were treated without (control) or with ...

PP2C5 Is Involved in a Subset of ABA-Mediated Responses

As PP2C5 gene expression is affected by ABA treatment (Supplemental Fig. S1) and because MPK3, MPK4, and MPK6 have been implicated in ABA signaling (Ichimura et al., 2000; Lu et al., 2002; Gudesblat et al., 2007; Xing et al., 2008), the effect of PP2C5 depletion on typical ABA responses was investigated. As ABA induces stomatal closure and as MAPK activities are associated with stomatal movements (Lu et al., 2002; Gudesblat et al., 2007), we measured stomatal aperture in pp2c5 mutant plants. Compared with the wild type, leaves of both pp2c5 and ap2c1 T-DNA insertion lines showed a slightly, but significantly, increased stomatal aperture under normal growth conditions (Fig. 8), whereas stomatal aperture in the pp2c5 line complemented with a genomic PP2C5 fragment was indistinguishable from that measured in the wild type. This effect was even more pronounced in the pp2c5 ap2c1 double knockout line, indicating that the two highly similar proteins are functionally partially redundant. However, despite the increased stomatal opening in the pp2c5 and ap2c1 single and double mutant lines, they did not show an increased water loss during drought stress (Supplemental Fig. S6).

Figure 8.
PP2C5 knockout lines show an increased stomatal aperture. Wild-type Arabidopsis Col-0, knockout lines pp2c5 and ap2c1, the double knockout line pp2c5 ap2c1, and the complemented pp2C5 line pp2c5/PP2C5 were grown on soil, and stomatal apertures on abaxial ...

ABA is also an important hormone during pathogen defense (Mauch-Mani and Mauch, 2005; Adie et al., 2007; Fan et al., 2009), and pathogen-induced stomatal closure is part of the plant innate immune response (Melotto et al., 2006). Therefore, we next examined the effect of pp2c5 mutation on resistance to bacterial pathogens. When infected with the virulent bacterium Pseudomonas syringae pv tomato DC3000 (Pto DC3000), pp2c5, ap2c1 as well as pp2c5 ap2c1 plants did not show a significantly altered susceptibility (Supplemental Fig. S7A). We also tested the coronatine-deficient strain Pto DC3661, which is normally less pathogenic on Arabidopsis, as these bacteria cannot induce coronatine-dependent reopening of stomata after their pathogen-associated molecular pattern (PAMP)-triggered closure during infection (Melotto et al., 2006). Although pp2c5 and ap2c1 single mutants and the pp2c5 ap2c1 double knockout line exhibited an increased stomatal aperture, all mutants were still fully resistant to Pto DC3661 infection (Supplemental Fig. S7B).

Another well-established function of ABA is to promote seed dormancy and to inhibit seed germination (Christmann et al., 2006). When seeds of pp2c5 mutants were sown on ABA-containing Murashige and Skoog (MS) medium, the germination rate was significantly increased compared with wild-type seeds, indicating that the pp2c5 mutant displays partial insensitivity toward ABA (Fig. 9A). The germination rate of ap2c1 seeds was intermediate to that of the pp2c5 mutant and the wild type; however, seeds of the pp2c5 ap2c1 double knockout line showed an even further increased ABA insensitivity.

Figure 9.
PP2C mutants display altered ABA responses. A, Seeds of wild-type Arabidopsis Col-0 and knockout lines pp2c5, ap2c1, pp2c5 ap2c1, and pp2c5/PP2C5 were germinated on medium supplemented with the indicated concentrations of ABA. Seedlings were scored for ...

Moreover, gene expression of ABA-inducible genes was investigated. As exemplified by the genes ABI1, ABI2, RD29A, and Erd10 (Yamaguchi-Shinozaki and Shinozaki, 1993; Leung et al., 1997; Merlot et al., 2001), the application of ABA caused a transcriptional up-regulation in Col-0 wild-type plants and the pp2c5/PP2C5 complemented line. However, except for ABI1 gene expression, which was only affected in the pp2c5 ap2c1 double mutant, ABA-induced gene expression for the other genes was significantly diminished in both the single and double mutant lines (Fig. 9B). Thus, the partial ABA-insensitive phenotype in the pp2c mutants observed in the germination assay is reflected in a partial ABA insensitivity with respect to ABA-triggered gene expression.

In summary, PP2C5 and AP2C1 appear to be involved in stomatal opening, seed germination, and ABA-regulated gene expression as functionally partially redundant MAPK phosphatases.

DISCUSSION

PP2C5 Is a MAPK Phosphatase

MAPKs function as key signal integration points for a vast number of external stimuli that affect the proper functioning of cells. Hence, they must be subject to rigorous regulation to control appropriate intensity and timing of their activation. Our results identify PP2C5 as an Arabidopsis PP2C of the B subgroup that can act as MAPK phosphatase. PP2C5 not only colocalizes with the stress-induced MAPKs MPK3, MPK4, and MPK6 (Fig. 2) but also directly interacts with those MAPKs via its KIM (Fig. 3). The fact that PP2C5 and the MAPKs form complexes mainly in the nucleus (Fig. 3B) is consistent with the proposed shuffling of the MAPKs into the nucleus upon stress (Ahlfors et al., 2004; Lee et al., 2004) and with the involvement particularly of MPK6 in the regulation of stress-induced gene expression (Asai et al., 2002). Moreover, the activation of MAPKs is affected by PP2C5 expression, and we observed stronger stress activation of MPK3, MPK4, and MPK6 in Arabidopsis when PP2C5 was depleted (Figs. 5 and and6;6; Supplemental Fig. S4). In agreement with that, ectopic expression of PP2C5-GFP in Nicotiana tabacum abolished ABA- and elicitation-induced activation of SIPK and WIPK, the orthologs of MPK6 and MPK3, respectively (Fig. 7; Supplemental Fig. S5). These results suggest that PP2C5 is a bona fide protein phosphatase that attenuates MAPK activation by regulating the strength and duration of MAPK activation. Another indirect piece of evidence for PP2C5 being a MAPK phosphatase is the observation that cotransfection of PP2C5 with MPK6 and the MPK6 substrate ERF104 abolished MPK6-mediated ERF104 phosphorylation (Bethke et al., 2009).

Notably, PP2C5 is not the only phosphatase regulating MPK3, MPK4, and MPK6. Arabidopsis AP2C1, another clade B member and a close homolog of PP2C5, was also recently shown to interact with and inactivate MPK4 and MPK6 (Schweighofer et al., 2007). In addition to PP2Cs, dual-specificity phosphatases (DsPTPs) are thought to dephosphorylate and thereby regulate MAPKs (Martin et al., 2005). The class of DsPTPs, which can remove both phosphates in the TXY activation motif of MAPKs, were regarded for a long time as classical MAPK phosphatases in yeast and mammals (Keyse, 1998; Camps et al., 2000). The Arabidopsis genome encodes five potential DsPTPs, MKP1, MKP2, DsPTP1, PHS1, and IBR5 (Kerk et al., 2002). The first of those phosphatases identified in plants was DsPTP1, which can dephosphorylate and thereby inactivate stress-induced MPK4 (Gupta et al., 1998). MPK4, MPK3, and particularly MPK6 also interact with MKP1, which was demonstrated to be involved in signal transduction during genotoxic and salt stress in Arabidopsis (Ulm et al., 2001, 2002). Additionally, during the oxidative stress response, MPK3 and MPK6 can be inactivated by a third DsPTP, MKP2 (Lee and Ellis, 2007).

In summary, MAPK cascades are involved in multiple cellular signaling pathways; thus, it is not surprising that plants have developed a highly complex network to fine-tune cellular responses to internal and external stimuli. Our results demonstrate that PP2C5 is one of the corresponding phosphatases regulating MAPK activity during cellular adaptation to different stress responses.

PP2C5 and AP2C1 Are Required for ABA Signaling

ABA is a universal stress hormone in higher plants that also plays a major role in various aspects of plant growth and development (Koornneef et al., 1998; Mauch-Mani and Mauch, 2005; Christmann et al., 2006). We show here that PP2C5 and AP2C1 as members of clade B of PP2Cs are also involved in different ABA responses apart from the well-characterized clade A PP2Cs. Stomatal aperture of leaves of unstressed pp2c5 and ap2c1 mutants is slightly but significantly increased compared with the wild type (Fig. 8). Notably, pp2c5 ap2c1 double mutants showed an even more pronounced stomatal aperture, indicating that PP2C5 and AP2C1 have partially redundant functions in stomata regulation. ABA generally promotes stomatal closure and inhibits stomatal opening (Koornneef et al., 1998). Based on our observations (Supplemental Figs. S6 and S7), we postulate that PP2C5 and AP2C1 affect ABA-mediated stomatal opening but are not the major regulators involved in the stomatal closure control. First, ABA normally causes stomatal closure in response to drought stress, and the abi1 and abi2 mutants show an increased water loss and a wilty phenotype under water stress conditions due to failure to close their stomata (Roelfsema and Prins, 1995; Leung et al., 1997). However, although stomatal aperture was increased in pp2c5, ap2c1, and pp2c5 ap2c1 mutant plants, their response to drought stress was not significantly altered compared with wild-type plants (Supplemental Fig. S6). Second, ABA-mediated stomatal closure is part of plant innate immunity against bacterial pathogens and is triggered by surface-exposed PAMPs. During infection, stomatal closure is reversed by the jasmonic acid mimic coronatine; hence, Pto strains depleted of coronatine are normally less infectious. In accordance, plants defective in ABA biosynthesis, such as the aba3 mutant, are not able to close their stomata (Leon-Kloosterziel et al., 1996) and are more susceptible to coronatine-deficient Pto bacteria (Melotto et al., 2006). However, pp2c5, ap2c1, and pp2c5 ap2c1 plants were still fully resistant to the less virulent coronatine-deficient strain Pto DC3661 and showed a normal response to infection with virulent Pto DC3000 bacteria (Supplemental Fig. S7). Therefore, our results indicate that ABA-induced stomatal closure, triggered for instance by drought stress or PAMP recognition, is not affected by PP2C5 and AP2C1 depletion.

Additionally, the seed germination rate that is normally decreased by ABA treatment was significantly increased in pp2c5 mutants compared with the wild-type control or the pp2c5/PP2C5 complemented line (Fig. 9A). Again, PP2C5 and AP2C1 seem to have redundant functions, as the double mutant showed an even more pronounced ABA insensitivity during seed germination. This effect was also observed with respect to ABA-inducible gene expression. Transcriptional activation of the ABA-responsive genes ABI1, ABI2, RD29A, and Erd10 was strongly reduced after external ABA application in the pp2c single and double mutant plants compared with the wild type (Fig. 9B). Hence, PP2C5 and AP2C1 are involved in various ABA-mediated cellular responses.

PP2C5 Is a Positive Regulator of ABA Signaling

So far, PP2Cs have only been described as negative regulators of ABA signaling and loss-of-function mutations in ABI1, ABI2, HAB1, and AtPP2CA render the plants ABA hypersensitive (Gosti et al., 1999; Tahtiharju and Palva, 2001; Saez et al., 2004). Although the abi1-1 mutant was described as being ABA insensitive (Koornneef et al., 1984), this initially isolated mutation had a dominant effect, and follow-up reports showed that loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA (Gosti et al., 1999). Thus, the wild-type ABI1 phosphatase, and most likely also ABI2 as close ABI1 homolog, are negative regulators of ABA responses. Likewise, antisense inhibition of AtPP2CA as well as depletion of HAB1 resulted in an ABA-hypersensitive inhibition of seed germination (Tahtiharju and Palva, 2001; Saez et al., 2004), suggesting that also AtPP2CA and HAB1 function as negative regulators in ABA signaling. Beside PP2Cs, DsPTPs have also been demonstrated to regulate ABA signaling. Whereas the phs1 mutant reacts hypersensitively to ABA during seed germination (Quettier et al., 2006), ibr5 mutants showed a reduced responsiveness to auxin and ABA (Monroe-Augustus et al., 2003). Hence, PHS1 can be regarded as a negative regulator of ABA responses in contrast to IBR5, which seems to act as a positive regulator of ABA signaling. However, the only known IBR5-interacting protein is MPK12, which itself does not seem to be involved in ABA signaling (Lee et al., 2009), suggesting that although IBR5 might play a role in ABA signaling, this is most likely not mediated via a MAPK cascade.

Our results show that PP2C5 acts as regulator of the major stress-induced MAPKs MPK3, MPK4, and MPK6 and that depletion of PP2C5 function causes a partial ABA insensitivity with regard to seed germination and ABA-induced gene expression (Fig. 9). Thus, PP2C5 and also its close homolog AP2C1 act as positive regulators that are required for full ABA responsiveness.

Does PP2C5 Regulate ABA Signaling via Action on MAPKs?

Although MAPKs are involved in ABA signaling and several phosphatases have been described to regulate MPK3, MPK4, and MPK6, little is known about a direct link between phosphatase-mediated MAPK regulation and the ABA response. One prominent example is ABI1, a PP2C of the A subgroup that attenuates ABA-dependent stress signaling and that was also shown to directly bind to MPK6, thereby inhibiting its kinase activity (Leung et al., 2006). Another putative MAPK regulator during ABA signaling is PHS1, one of the five Arabidopsis DsPTPs. Loss-of-function mutants of PHS1 displayed impaired microtubule organization (Naoi and Hashimoto, 2004) and ABA hypersensitivity (Quettier et al., 2006). Recently, it has been demonstrated that PHS1 physically interacts with MPK18, and mpk18 seedlings have defects in microtubule-related functions (Walia et al., 2009). However, an involvement of MPK18 in ABA responses has so far not been shown. A second DsPTP implicated in ABA signaling is IBR5, which was initially reported to confer reduced sensitivity to auxin and ABA in Arabidopsis roots (Monroe-Augustus et al., 2003). However, IBR5 only interacts with MPK12, and reduced expression of the MPK12 gene resulted in root growth that is hypersensitive to exogenous auxins but shows normal ABA sensitivity (Lee et al., 2009). Therefore, it is still elusive if PHS1 and IBR5 regulate MAPKs during ABA signaling.

We show here that PP2C5 is a negative regulator of MPK3, MPK4, and MPK6, three MAPKs implicated in the signaling pathways mediating the ABA response (Lu et al., 2002; Gudesblat et al., 2007; Xing et al., 2008). ABA has been shown to activate MPK3, and the activated kinase is thought to phosphorylate the transcription factor ABI5 (Lu et al., 2002). Constitutive expression of MPK3 in transgenic plants resulted in no visible phenotype but hypersensitivity to exogenous ABA (Lu et al., 2002). Interestingly, antisense plants with reduced levels of MPK3 mRNA in the guard cells displayed partial insensitivity to ABA in inhibition of stomatal opening but responded normally to this hormone in stomatal closure (Gudesblat et al., 2007). Likewise, MPK6 overexpression led to enhanced ABA responses, while the mpk6 mutant was impaired in ABA signaling (Xing et al., 2008). These results indicate that a depletion of the corresponding MKP(s) as negative regulators should lead to an enhanced signaling through those MAPKs and hence should result in ABA hypersensitivity similar to that observed for MAPK overexpression. Unexpectedly, depletion of PP2C5 and AP2C1 resulted in a partial ABA insensitivity phenotype. As there are no data available for the involvement of MPK4 in the ABA response, apart from the fact that MPK4 enzyme activity is ABA inducible (Xing et al., 2008), we cannot rule out that PP2C5 and/or AP2C1 act specifically on MPK4 during ABA signaling. Moreover, PP2C5 gene expression is induced by various other phytohormones apart from ABA, such as ethylene and GA3 (data not shown). However, we could not observe alterations in the cellular response to ethylene or GA3 in pp2c5, ap2c1, or pp2c5 ap2c1 mutant plants when compared with the wild type (data not shown). As multiple other kinases also have been demonstrated to be involved in ABA signaling, such as calcium-dependent protein kinases (Mori et al., 2006; Zhu et al., 2007), SNF1-related protein kinases (Fujii et al., 2007; Jossier et al., 2009; Umezawa et al., 2009), or the LRR kinase RPK1 (Osakabe et al., 2005), our future work will now address if PP2C5 solely acts on MAPKs or if other kinases can be targeted by PP2C5 and/or AP2C1.

In summary, we identified PP2C5 and its close homolog AP2C1 as functionally partially redundant protein phosphatases that positively regulate ABA-induced gene expression, ABA-mediated seed dormancy, and stomatal closure. It remains to be demonstrated if PP2C5 action on MAPKs can be functionally linked to its role in ABA signaling.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana Col-0) plants were routinely grown on a 1:3 vermiculite:soil mixture for 5 to 6 weeks in a phytochamber (8-h photoperiod, 22°C, 40%–60% humidity). For in vitro culture, seeds were surface sterilized by overnight treatment with chlorine gas (released from a mixture of 100 mL of 12% commercial bleach and 3 mL of 37% hydrochloric acid) in a desiccator. Then, seeds were sown on half-strength MS medium (Duchefa) containing 0.8% (w/v) Select agar (Sigma) and cultivated in long-day conditions (16-h photoperiod). Stratification of the seeds was conducted in the dark at 4°C for 2 d. Arabidopsis cells were propagated as described (Dettmer et al., 2006). Nicotiana benthamiana plants were cultivated in the greenhouse with a photoperiod of 14 h, 60% humidity, and a temperature of 25°C during the day and 19°C at night. Seeds for the T-DNA insertion lines for PP2C5 (SALK_109986) and AP2C1 (SALK_065126) were obtained from the SALK Institute collection (Alonso et al., 2003), and individual plants with a single T-DNA insertion were selected after Southern-blot analysis (Supplemental Fig. S8). To generate the pp2c5 ap2c1 double mutant, ap2c1 homozygous plants were crossed with the homozygous pp2c5 mutant. Plants were further self-fertilized, and pp2c5 ap2c1 double homozygous seedlings were then used for experiments in the F3 generation.

Plant Transformation and Protein Localization Studies

For GFP fusions driven by the cauliflower mosaic virus 35S promoter, the 1,493-bp genomic PP2C5 fragment was cloned into vector pK7FWG2.0 (Karimi et al., 2005) via the Gateway recombination system (Invitrogen). For plasmid PP2C5:PP2C5-GFP, the 35S promoter was replaced by a 1,502-bp PP2C5 promoter fragment. As GFP control, the vector pK7WGF2.0 (Karimi et al., 2005) was used. For colocalization experiments, fusion constructs were generated in vectors pEXSG-YFP (for MPK6), pENSG-YFP (for MPK3 and MPK4), and pEXSG-CFP (for PP2Cs) as described (Bethke et al., 2009).

Transient protein expression in Arabidopsis protoplasts of ecotype Col-0 and N. benthamiana leaves was performed as described (Ludwig et al., 2005; Dettmer et al., 2006). Localization studies of the PP2C5-GFP fusion protein and GFP as a control were carried out 15 to 18 h after transformation using a confocal laser-scanning microscope as described elsewhere (Diepold et al., 2007). For complementation experiments, the PP2C5:PP2C5-GFP construct in pK7FWG2.0 was stably transformed into pp2c5 plants by the floral dip procedure (Clough and Bent, 1998). Restored gene transcription in transformed seedlings was verified by reverse transcription (RT)-PCR, and plants were used for experiments in the F3 generation.

Transcript Analysis

Total RNA from seedlings was isolated using the Tri Reagent method according to the manufacturer's recommendations (Sigma). First-strand cDNA was synthesized from 1 μg of total RNA using RevertAid Moloney murine leukemia virus reverse transcriptase (Fermentas). RT-quantitative PCR amplifications and measurements were performed with the iQ5 Multicolour Real Time PCR detection system (Bio-Rad). RT-quantitative PCR amplifications were monitored using the ABsolute SYBR Green Fluorescein Mix (Thermo Scientific). Relative quantification of gene expression data was performed using the 2–ΔΔCT method (Livak and Schmittgen, 2001). Expression levels were normalized using the threshold cycle values obtained for the EF-1α gene. The presence of a single PCR product was further verified by dissociation analysis in all amplifications. All quantifications were made in duplicate on RNA samples obtained from three independent experiments. The sequences of the primers used for PCR amplifications are indicated in Supplemental Table S1.

Microarray data were obtained from the AtGenExpress initiative (http://www.arabidopsis.org/info/expression/ATGenExpress.jsp) and were analyzed using the digital northern tool of the Genevestigator program (http://www.genevestigator.ethz.ch).

Enzymatic Activity Assays

For phosphatase assays, PP2C5-GFP was transiently expressed in N. benthamiana leaves and immunoprecipitated with rabbit anti-PP2C5 antibody from crude protein extracts as described (Romeis et al., 2001). Phosphatase activity was measured using 32P-labeled casein (11.2 μg per reaction; Wang et al., 1999) as artificial substrate and incubated with immunoprecipitated PP2C5-GFP at 25°C for 30 min according to the protocol described previously (Stone et al., 1994).

MAPK activities were determined in crude protein extracts from 6-d-old seedlings prepared as described (Romeis et al., 1999; Ludwig et al., 2005). Five-microgram protein crude extracts were separated on a 10.5% SDS gel, and the proteins were transferred onto nitrocellulose (Amersham) by wet electroblotting (Mini-Protean II system; Bio-Rad). Equal loading of protein was confirmed by Ponceau S Red staining of the membrane, and the membranes were subsequently subjected to western-blot analysis using the phospho-p44/p42 MAPK antibody according to the manufacturer's protocol (Cell Signaling Technology). Alternatively, MAPK activity was determined by in-gel kinase assays with myelin basic protein (Sigma) as artificial substrate as described previously (Romeis et al., 1999).

For immunocomplex kinase assays, 30 μg of total proteins from seedling extracts prepared as described above was incubated overnight at 4°C with 10 μL of isoform-specific MPK antibody. A total of 50 μL of protein G-Sepharose (GE Healthcare) was added and incubated for 2 h at 4°C. The protein-antibody complex on the beads was collected and washed three times in ice-cold protein extraction buffer and finally washed with kinase buffer (50 mm Tris, pH 7.5, 1 mm dithiothreitol, 10 mm MgCl2, and 50 μm ATP). Kinase reactions on the immunoprecipitated MPK3, MPK4, and MPK6 were performed for 30 min at room temperature in 20 μL of kinase buffer containing 5 μg of myelin basic protein and 2 μCi of [γ-32P]ATP. The reaction was stopped by adding SDS-PAGE loading buffer, and the phosphorylation of myelin basic protein was analyzed by autoradiography after SDS-PAGE.

Antibody Generation, Immunoprecipitation, and Western-Blot Analysis

PP2C5-specific antisera were generated in rabbits via immunizations with the N-terminal peptide N-MQLSKNPIKQTRNRE coupled to keyhole limpet hemocyanin (Eurogentec). For MPK3- and MPK6-specific rabbit antisera, the N-terminal peptides N-MNTGGGQYTDFPAVDTHGG and N-MDGGSGQPAADTEMT, respectively, were used (Ahlfors et al., 2004). All antisera were affinity purified on the corresponding immobilized oligopeptide used to generate the serum. The MPK4 antibody was obtained from Sigma. Immunoprecipitation of PP2C5-GFP and western-blot analysis were performed as described (Romeis et al., 2001). MPK antibodies were used for western-blot analysis in a 1:5,000 dilution.

Protein Interaction Studies

Amino acid substitutions K90A and R91Q in the KIM of PP2C5 were introduced by PCR-based, site-specific mutagenesis. Yeast two-hybrid experiments were performed using the Matchmaker System (Clontech). PP2C5 cDNA (1,173 bp) or mutated PP2C5-K90A/R91Q was cloned into pGBKT7, and MPK3, MPK4, and MPK6 were cloned into pGADT7 (Clontech). Plasmids were transformed into yeast strain AH109 using a lithium acetate/single-stranded carrier DNA/polyethylene glycol method (Gietz and Woods, 2002). After 4 to 5 d of growth on vector-selective medium (CSM-LT), 12 independent clones in pools of four clones each were propagated in liquid vector-selective medium and subsequently diluted to the same optical density. Of the three pooled cultures, 7.5 μL of a serial dilution was dropped on vector- and interaction-selective medium (CSM-LTA) and incubated at 28°C. At day 3, the growth of the clones was monitored.

For BiFC analysis, PP2C5 and point-mutated PP2C5-K90A/R91Q were cloned into vector pUCSpyNe (fusing PP2C5 C terminal to the N-terminal half of YFP) and MPK3, MPK4, and MPK6 were cloned into vector pUCSpyCe (fusing MPKs C terminal to the C-terminal half of YFP). pUCSpyNe and pUCSpyCe vectors expressing split YFP domains alone were used as controls (Walter et al., 2004). The subcellular localization of the interaction was visualized by fluorescence microscopy using a confocal laser-scanning microscope (TCS SP2; Leica [http://www.leica.com/]).

Stomatal Aperture and Germination Assay

To determine stomatal aperture, plants were grown without lid with constantly moist soil. Two leaves per line were harvested, and the lower leaf side was thinly covered with glue (Uhu hart). The dried glue was removed after several minutes, and abaxial epidermal imprints were analyzed with the microscope. Stomatal aperture of approximately 100 stomata per sample was measured using Scion Image Software, and data sets were analyzed with Student's t test.

The germination rate was determined from seeds that were surface sterilized and plated on half-strength MS medium supplemented with different ABA concentrations. After sowing, plates were chilled for 3 d at 4°C in darkness and subsequently incubated for 3 d at 22°C with an 8-h photoperiod. Emerging root tips were scored with the light microscope. Seeds of batches of exactly the same age were used for one experiment, but batches varied from one experiment to the next

Supplemental Data

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

  • Supplemental Figure S1. Cladogram and gene expression data for clade A and B PP2Cs.
  • Supplemental Figure S2. Generation of PP2C5-specific antibodies.
  • Supplemental Figure S3. Localization of clade B-PP2Cs.
  • Supplemental Figure S4. pp2c mutant plants show an enhanced Flg22-induced MAPK activation.
  • Supplemental Figure S5. PP2C5-overexpressing leaves are impaired in Flg22-induced MAPK activation.
  • Supplemental Figure S6. The PP2C5-T-DNA insertion line displays a normal drought stress response.
  • Supplemental Figure S7. pp2c5 plants are not affected in their resistance to bacterial infection.
  • Supplemental Figure S8. Southern-blot analysis of pp2c5 and ap2c1 mutants.
  • Supplemental Table S1. List of primers used for quantitative RT-PCR analysis.
  • Supplemental Materials and Methods S1.

Supplementary Material

[Supplemental Data]

Acknowledgments

We are grateful to Klaus Harter for providing the BiFC vectors and Georg Felix for Flg22 peptide. The Nottingham Arabidopsis Stock Centre is acknowledged for providing mutant seeds. We thank Caterina Brancato for technical assistance in protoplast preparation and transformation.

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