Plant Mol Biol DOI 10.1007/s11103-015-0330-1
Arabidopsis abscisic acid receptors play an important role in disease resistance Chae Woo Lim1 · Sung Chul Lee1
Received: 21 August 2014 / Accepted: 30 April 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Stomata are natural pores of plants and constitute the entry points for water during transpiration. However, they also facilitate the ingress of potentially harmful bacterial pathogens. The phytohormone abscisic acid (ABA) plays a pivotal role in protecting plants against biotic stress, by regulating stomatal closure. In the present study, we investigated the mechanism whereby ABA influences plant defense responses to Pseudomonas syringae pv. tomato (Pst) DC3000, which is a virulent bacterial pathogen of Arabidopsis, at the pre-invasive stage. We found that overexpression of two ABA receptors, namely, RCAR4/ PYL10-OX and RCAR5/PYL11-OX (hereafter referred to as RCARs), resulted in ABA-hypersensitive phenotypes being exhibited during the seed germination and seedling growth stages. Sensitivity to ABA enhanced the resistance of RCAR4-OX and RCAR5-OX plants to Pst DC3000, through promoting stomatal closure leading to the development of resistance to this bacterial pathogen. Protein phosphatase HAB1 is an important component that is responsible for ABA signaling and which interacts with ABA receptors. We found that hab1 mutants exhibited enhanced resistance to Pst DC3000; moreover, similar to RCAR4-OX and RCAR5-OX plants, this enhanced resistance was correlated with stomatal closure. Taken together, our findings demonstrate that alteration of RCAR4- or RCAR5-HAB1
Electronic supplementary material The online version of this article (doi:10.1007/s11103-015-0330-1) contains supplementary material, which is available to authorized users. * Sung Chul Lee [email protected] 1
Department of Life Science, Chung-Ang University, Seoul 156‑756, Korea
mediated ABA signaling influences resistance to bacterial pathogens via stomatal regulation. Keywords Abscisic acid · ABA receptor · Protein phosphatase · Stomatal immunity Abbreviations ABA Abscisic acid ABI ABA-insensitive AHG ABA-hypersensitive germination AIP AKT1 interacting protein phosphatase AIPH AIP1 homologue BiFC Bimolecular fluorescence complementation cfu Colony forming units HAB Hypersensitive to ABA hpi Hours post-inoculation PAMPs Pathogen-associated molecular patterns PP2C Protein phosphatase type 2 C PRR Pattern recognition receptors Pst Pseudomonas syringae pv. tomato PYL PYR-like PYR Pyrabactin resistance qRT-PCR Quantitative reverse transcription-polymerase chain reaction RCAR Regulatory component of ABA receptor SOS Stomatal opening solution WT Wild-type
Introduction Plants are constantly challenged by biotic and abiotic stresses such as pathogens, drought, and high salinity. In response to pathogen attacks, plants have developed various resistance mechanisms; this process has resulted in
13
the local and systemic induction of a broad spectrum of defense mechanisms (Jones and Dangl 2006). Plants have innate and induced mechanical barriers that protect them from various pathogens such as bacteria and fungi. On the other hand, most pathogens have mechanisms that enable them to overcome physical and chemical plant barriers, in order successfully to infect the host plants. For successful establishment of infection, pathogens must initially gain entry into the plant tissues. Fungal pathogens can directly penetrate the plant tissues by applying mechanical forces or by utilizing cell wall degrading enzymes; on the other hand, bacterial pathogens require natural openings such as stomata, hydathodes, and lenticels (Melotto et al. 2008) or wound sites to enter plant tissues. Among these, stomatal pores constitute the main entry route into leaf tissues. The plant hormone abscisic acid (ABA) plays diverse roles in responses to biotic and abiotic stresses. This hormone triggers changes in a number of plant physiological processes, thereby resulting in adaptation to various stresses. In the past decade, several studies have investigated the role of ABA in plant defense responses and in determining the outcome of plant–bacterial pathogen interactions (Melotto et al. 2008; Ton et al. 2009). Stomatal closure leads to drought stress tolerance and serves as a defense mechanism for preventing bacterial pathogen invasions (Melotto et al. 2006, 2008). Recently, the mechanism of ABA-induced stomatal closure in response to biotic and abiotic stresses has been extensively studied (Santiago et al. 2009; Cao et al. 2011; Desclos-Theveniau et al. 2012; Lee et al. 2013; Lim et al. 2014). Stomatal defense is associated with stomatal closure in response to pathogen-associated molecular patterns (PAMPs), including flg22 and lipopolysaccharides (Melotto et al. 2006; Amborabe et al. 2008; Cho et al. 2008; Zeng and He 2010). Plant recognition of PAMPs via pattern recognition receptors (PRRs) activates ABA signaling in guard cells, thereby leading to stomatal closure (Melotto et al. 2008). The ABA signaling pathway has several key components, each of which serves as a positive and negative regulator at every step. SnRK2 type kinases and group A protein phosphatases (PP2Cs) act as positive and negative regulators, respectively, of ABA signaling (Lee and Luan 2012). Phenotypic analysis has revealed that the triple mutant (snrk2.2/snrk2.3/snrk2.6) displays an ABA-insensitive phenotype by opening stomata during water-deficient conditions; on the other hand, mutants of pp2cs exhibit a more profound hypersensitive ABA phenotype (Merlot et al. 2001; Fujii et al. 2007; Rubio et al. 2009). To initiate ABA signaling, it is necessary to perceive ABA via ABA specific receptors in plant cells. Several ABA receptors have been identified; however, the functions of these receptors remain to be elucidated (Liu et al. 2007; Christmann and Grill 2009; Park et al. 2009; Umezawa et al. 2009).
13
Plant Mol Biol
Pyrabactin resistance (PYR)/PYR-like (PYL) or regulatory component of ABA receptor (RCAR) comprises a 14-member family and acts as an ABA receptor (Park et al. 2009; Umezawa et al. 2009). Under stress condition, ABA levels increase and ABA receptors interact directly with the active site of PP2Cs; this process inhibits phosphatase activities, thereby leading to downstream activation of ABA signaling (Lee and Luan 2012). When the ABA levels decrease, PP2Cs interact with and inactivate SnRKs, thereby leading to suppression of ABA signaling (Lee et al. 2009, 2013). The relationship between ABA and pathogen resistance has been extensively investigated; however, the roles of the ABA components remain to be clarified. Recently, we reported that alteration of the gene expressions of RCAR2/PYL7 and RCAR3/PYL8, and their interacting partner PP2CA affects disease resistance and ABA sensitivity. Additionally, ABA sensitivity is positively correlated with stomatal immunity (Lim et al. 2014). Many studies have revealed that individual RCAR members have different combination of interaction with PP2C members and different requirement of ABA for the interaction with different PP2Cs (Park et al. 2009; Ma et al. 2009; Santiago et al. 2009; Hao et al. 2011). In addition, RCAR members exhibited functional difference, in spite of their functional redundancy (Antoni et al. 2012; Hao et al. 2011). In this aspect, functional role of other RCAR members in plant–microbe interaction need to be studied. In the present study, we investigated the functional role of two other homologous ABA receptors, namely RCAR4 and RCAR5, in stomatal immunity. We previously showed that similar to RCAR2 (Lee et al. 2009, 2013) and RCAR3 (Lim et al. 2014), overexpression of RCAR4 and RCAR5 leads to enhanced ABA sensitivity. Therefore, in the present study, we used dip-inoculation methods to examine the influence of the stomatal aperture on plant defense responses to Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we analyzed the roles of HOMOLOGY TO ABI1 (HAB1), interacting with RCAR4 and RCAR5 and functioning as a negative regulator in the ABA signaling pathway, in stomatal defense against Pst DC3000. Our findings indicate that RCARs and HAB1 play pivotal roles in plant defense responses during the pre-invasive stage, and that ABA sensitivity is important in stomatal defense.
Materials and methods Plant material and growth conditions Arabidopsis thaliana (ecotype Col-0) plants were routinely grown at a 9:1:1 ratio of peat moss, perlite, and vermiculite. The plants were maintained at 24 °C and 60 % humidity under fluorescent light (130 μmol photons m−2 s−1)
Plant Mol Biol
with a 16-h light/8-h dark cycle. Prior to in vitro culture, seeds of A. thaliana (ecotype Col-0) were surface sterilized with 70 % ethanol for 1 min and treated with 2 % sodium hydroxide for 10 min. The seeds were then washed 10 times with sterile distilled water and sown on Petri dishes containing Murashige and Skoog (1962) (MS) agar medium (Sigma, St. Louis, MO, USA) supplemented with 1 % sucrose. The plates were sealed and incubated at 24 °C in a chamber under fluorescent light (130 μmol photons m−2 s−1) with a 16-h light/8-h dark cycle. Seeds with hab1 insertion lines, i.e., SALK_002104 (Saez et al. 2004, 2006), were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA).
and 35S-SPYCE(M) vectors via XbaI/XhoI, to generate the bimolecular fluorescence complementation (BiFC) constructs (Waadt et al. 2008). For transient expression, A. tumefaciens strain GV3101 harboring each construct was mixed with p19 strain to avoid gene silencing and it was then infiltrated into the abaxial side of 5-week-old Nicotiana benthamiana leaves by using a 1-mL needleless syringe. At 3 days after infiltration, leaf discs were cut and the lower epidermal cells were examined under a confocal microscope (510 UV/Vis Meta; Zeiss, Oberkochen, Germany) equipped with LSM Image Browser software.
Yeast two‑hybrid assay
For germination tests, 100 seeds per genotype were sown on plates containing MS agar medium supplemented with various concentrations of ABA. After 7 days, the number of seeds showing radicle emergence were counted. For root growth assays during the post-germinative stage, 4-day-old seedlings from wild-type (WT) and RCAR4 and RCAR5OX transgenic Arabidopsis lines were transferred to plates containing MS agar medium supplemented with 10 μM of ABA. After 7 days, the root lengths of the seedlings were measured.
Yeast two-hybrid (Y2H) assays were conducted as described previously (Lee et al. 2007). The cDNA fragments of RCAR4 and RCAR5 as bait and HAB1 as prey were subcloned into the pGBKT7 and pGADGH vectors, respectively. These constructs were introduced into the yeast strain AH109, according to the lithium acetate-mediated transformation method (Ito et al. 1983). After selection on SCleucine–tryptophan medium, transformant candidates were transferred to SC-adenine–histidine–leucine–tryptophan medium for growth evaluation; this evaluation provided an indication of protein–protein interactions. Next, 5 μL of yeast cell culture (OD600 = 0.5) was spotted onto SC–leucine–tryptophan medium or SC-adenine–histidine–leucine– tryptophan medium containing 0 or 10 μM of ABA (Sigma). Generation of transgenic RCAR4 and RCAR5 overexpressing mutants Full-length RCAR4 and RCAR5 cDNA were cloned into pENTR/D-TOPO vectors (Invitrogen, Carlsbad, CA, USA). After sequencing, the cloned genes were integrated into pK2GW7 by using the LR reaction to induce constitutive expression of the RCAR4 and RCAR5 genes under the control of the CaMV 35S promoter (Karimi et al. 2002). The correct construct was introduced into Agrobacterium tumefaciens strain GV3101 via electroporation. Arabidopsis transformation with the RCAR4 and RCAR5 genes was performed by using the floral dip method (Clough and Bent 1998). For the selection of transgenic lines, seeds harvested from putative transformed plants were sown on plates containing MS agar medium supplemented with 50 μg mL−1 of kanamycin. Bimolecular fluorescence complementation assay Full-length cDNAs of RCAR4, RCAR5, and HAB1 without stop codons were subcloned into 35S-SPYNE(R)173
ABA treatment and phenotypic analyses
Bacterial growth assay We used the virulent pathogen, Pseudomonas syringae pv. tomato (Pst) DC3000, in this study. Bacterial infection was performed as described previously (Lim et al. 2014). Briefly, the leaves of 5-week-old plants were syringe-infiltrated or dipped in a bacterial suspension of Pst DC3000 [1 × 106 or 1 × 108 colony forming units (cfu) per mL] in 10 mM MgCl2 containing 0.02 % Silwet L-77 (Lehle Seeds). The inoculated plants were maintained at 24 °C and 100 % humidity for 24 h and were then transferred to a growth chamber and cultured at 100 % humidity for 2–3 days. The amount of bacterial growth in the infected leaves of each plant was determined according to Katagiri et al. (2002). Stomatal aperture bioassay The stomatal aperture bioassay was conducted as described previously, but with some modifications (Lee et al. 2013). Briefly, leaf peels were collected from the rosette leaves of 5-week-old plants and were floated in a stomatal opening solution (SOS; 50 mM KCl, 10 mM MES-KOH, 10 μM CaCl2, pH 6.15) under light. The peels were incubated for a further 3 h to obtain 80 % stomatal opening in A. thaliana Col-0 plants under laboratory conditions. The buffer was replaced with SOS containing various concentrations of ABA or bacterial cells (1 × 108 cells mL−1) resuspended
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in water. For the control, the buffer was replaced with new SOS without ABA or water alone. Leaf peels were then incubated for various periods. In each individual sample, 100 stomata were randomly observed under a Nikon Eclipse 80i microscope. The widths of individual stomata were recorded by using Image J 1.46r software (http:// imagej.nih.gov/ij). Each experiment was performed in triplicate. RNA isolation and semi‑quantitative and quantitative reverse transcription‑polymerase chain reaction Total RNA was isolated from dehydrated or infected Arabidopsis leaf tissues, by using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). To remove genomic DNA, all the RNA samples were digested with RNA-free DNase. After quantification with a spectrophotometer, 1 μg of total RNA was used to synthesize cDNA, by using a Transcript First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. Simultaneously, cDNAs were synthesized without reverse transcriptase and were subjected to semi-quantitative RT-PCR to eliminate the possibility of contamination by genomic DNA in the cDNA samples. Semi-quantitative RT-PCR analysis was conducted by using Extaq DNA polymerase with the following specific primers: RCAR4 (forward, 5′-ATGAACGGT GACGAAACAAAG-3′; reverse, 5′-TCATATCTTCTTCTC CATAGATTCTG-3′); RCAR5 (forward, 5′-ATGGAAACT TCTCAAAAATATCATAC-3′; reverse, 5′-TTACAACTTTA GATGAGCCACCCT-3′); HAB1 (forward, 5′-GACTACC TCTCAATGCTTGCTCTAC-3′; reverse, 5′-CCTTCTAAC AATATTGAGTAATTAAC-3′); and UBQ1 as an internal control (forward, 5′-GTAGTGCTAAGAAGAGGAAGA-3′; reverse, 5′-TCAAGCTTCAACTCCTTCTT-3′). For quantitative RT-PCR analysis, the synthesized cDNA was amplified in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) with iQ™SYBR Green Supermix and the following specific primers: RCAR4 (forward, 5′-CAAAGAAGGTG GAGAGCGAGTACAT-3′; reverse, 5′-CCACTTCTCTTAC GCTACCAACCTC-3′); RCAR5 (forward, 5′-ATGGAAA CTTCTCAAAAATATCATACG-3′; reverse, 5′-CTCTCTCG GCTGAACTCCGCTG-3′); HAB1 (forward, 5′-GACTAC CTCTCAATGCTTGCTCTAC-3′; reverse, 5′-CCCTTCTAA CAATATTGAGTAATTAAC-3′); RD29B (forward, 5′-GTT GAAGAGTCTCCACAATCACTTG-3′; reverse, 5′-ATTAA CCCAATCTCTTTTTCACACA-3′); RD20 (forward, 5′-TG GTTTCCTATCTAAAGAAGCTGTG-3′; reverse, 5′-ATAC AAATCCCCAAACTGAATAACA-3′); PR1 (forward, 5′-T GTGGTCACTACACTCAAGTTGTTT-3′; reverse, 5′-GTG TGTATGCATGATCACATCATTA-3′); and Actin 8 as an internal control (forward, 5′-CAACTATGTTCTCAGGTA TTGCAGA-3′; reverse, 5′-GTCATGGAAACGATGTCTCT TTAGT-3′).
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Plant Mol Biol
Results and discussion Ectopic expression of RCAR4 and RCAR5 leads to ABA hypersensitivity in planta In our previous studies, we showed that overexpression of RCAR2 and RCAR3 confers ABA hypersensitivity and enhanced disease resistance (Lee et al. 2013; Lim et al. 2013, 2014). Therefore, in the present study, we examined the functional role of other homologous ABA receptors, namely, RCAR4 and RCAR5, in response to ABA treatment and pathogen infection. Although RCAR2-5 are monomeric receptors, in particular RCAR4 inhibits protein phosphatase activity in the absence of ABA (Hao et al. 2011) and functional role of RCAR5 remained elusive. The results of multiple alignment analysis revealed that RCAR4 shares higher sequence homology with RCAR2 (54.7 % identity, 71.1 % similarity) and RCAR3 (71.4 % identity, 86.7 % homology) than does RCAR5 (39.6 % identity, 58.5 % similarity and 42.3 % identity, 58.5 % similarity, respectively). Based on these, we postulated that RCAR4 and RCAR5 show different gene function relative to RCAR2 and RCAR3. We initially generated transgenic plants overexpressing the coding sequence of RCAR4 and RCAR5. We found that the expression levels of these genes were undetectable in wild-type plants under the conditions used in the present study. However, the overexpressing lines of RCAR4 and RCAR5 showed significant accumulation of each transcript (Fig. 1a). Under normal growth conditions, the phenotype of transgenic lines was indistinguishable from that of wild-type plants (data not shown). Analysis of the ABA responses showed that all the transgenic lines were ABA-hypersensitive during seed germination (Fig. 1b) and early seedling growth (Supplementary Fig. S1). In the germination assay, the germination rate of plants grown in the absence of ABA did not differ significantly between the wild-type and the RCAR4-OX and RCAR5OX transgenic lines. On the other hand, the RCAR4-OX and RCAR5-OX plants were more sensitive to 0.5 μM and 1.0 μM ABA than those of the wild-types during seed germination (Fig. 1b). RCAR4-OX and RCAR5-OX plants germinated and grown in the presence of 0.5 μM ABA exhibited hypersensitivity to ABA (Supplementary Fig. S1). In comparison with wild-type plants, the root lengths of RCAR4-OX and RCAR5-OX plants after 7 days were 8–52 and 24–66 % shorter, respectively. To confirm that the reduced root growth in RCAR4-OX and RCAR5-OX plants was not caused by a developmental defect, but was solely derived from the inhibitory effect of ABA, 4-day-old seedlings of each plant were transferred and grown in the presence of 10 μM ABA. In comparison with wild-type plants, the primary root lengths of RCAR4-OX and RCAR5OX plants after 7 days were 12–27 and 38–54 % shorter,
Plant Mol Biol
RCAR4-OX WT #1 #2 (30) RCAR4
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Fig. 1 ABA-hypersensitivity of RCAR4-OX and RCAR5-OX mutants. a RT-PCR analysis of RCAR4 and RCAR5 expression from the leaves of WT (Col-0) plants and transgenic lines (T3). The UBQ1 gene served as an internal control. The numbers in parentheses indicates the cycles of PCR. b Germination rate of RCAR4-OX (left) and RCAR5-OX mutants and WT seeds on ×0.5 MS medium supplemented with various concentrations of ABA. The number of
seeds with an emerged radicle was counted every day up to day 7. c Inhibition of primary root growth in RCAR4-OX (upper) and RCAR5OX (bottom) mutants and WT seedlings. Four-day-old seedlings were transferred in ×0.5 MS containing 0 or 10 μM ABA and were grown vertically. After 7 days, representative images were taken. Data are presented as mean ± standard deviation from three independent experiments
respectively. Our results suggest that RCAR4 and RCAR5 have different functions in ABA signaling. Additionally, they indicate that ectopic expression of RCAR4 and RCAR5 confers enhanced ABA sensitivity during germination and root elongation.
ABA-induced stomatal closure. To evaluate stomatal closure, we measured the pore sizes of stomata in RCAR4OX, RCAR5-OX and wild-type plants after treatment with ABA (Fig. 2). The results of our previous studies suggested that ABA promotes stomatal closure via the functioning of ABA receptors as positive regulators (Lee and Luan 2012; Lee et al. 2013). In the present study, we observed no clear differences in the stomatal aperture of wild-type, RCAR4OX, and RCAR5-OX plants grown in the absence of ABA. After treatment with 10 or 20 μM ABA for 2.5 h, the degree of stomatal closure was higher in RCAR4-OX and
RCAR4‑OX and RCAR5‑OX mutants show ABA‑hypersensitive stomatal closure Next, we examined whether RCAR4-OX and RCAR5-OX plants have other ABA-associated phenotypes such as
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Plant Mol Biol
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Overexpression of RCAR4 and RCAR5 regulates disease resistance to P. syringae pv. tomato DC3000 infection
1 0
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ABA (μM) Fig. 2 ABA-hypersensitive stomatal closing in RCAR4-OX and RCAR5-OX mutants. Leaf peels of WT plants and RCAR4-OX and RCAR5-OX mutants were incubated with 10 or 20 μM ABA for 2.5 h. The pictures show representative stomata at each ABA concentration (a). The stomatal apertures were measured under the microscope in WT plants and RCAR4-OX and RCAR5-OX mutants (b). Data are presented as mean ± standard error (n = 100). Different letters indicate significant differences at p
Arabidopsis abscisic acid receptors play an important role in disease resistance Chae Woo Lim1 · Sung Chul Lee1
Received: 21 August 2014 / Accepted: 30 April 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Stomata are natural pores of plants and constitute the entry points for water during transpiration. However, they also facilitate the ingress of potentially harmful bacterial pathogens. The phytohormone abscisic acid (ABA) plays a pivotal role in protecting plants against biotic stress, by regulating stomatal closure. In the present study, we investigated the mechanism whereby ABA influences plant defense responses to Pseudomonas syringae pv. tomato (Pst) DC3000, which is a virulent bacterial pathogen of Arabidopsis, at the pre-invasive stage. We found that overexpression of two ABA receptors, namely, RCAR4/ PYL10-OX and RCAR5/PYL11-OX (hereafter referred to as RCARs), resulted in ABA-hypersensitive phenotypes being exhibited during the seed germination and seedling growth stages. Sensitivity to ABA enhanced the resistance of RCAR4-OX and RCAR5-OX plants to Pst DC3000, through promoting stomatal closure leading to the development of resistance to this bacterial pathogen. Protein phosphatase HAB1 is an important component that is responsible for ABA signaling and which interacts with ABA receptors. We found that hab1 mutants exhibited enhanced resistance to Pst DC3000; moreover, similar to RCAR4-OX and RCAR5-OX plants, this enhanced resistance was correlated with stomatal closure. Taken together, our findings demonstrate that alteration of RCAR4- or RCAR5-HAB1
Electronic supplementary material The online version of this article (doi:10.1007/s11103-015-0330-1) contains supplementary material, which is available to authorized users. * Sung Chul Lee [email protected] 1
Department of Life Science, Chung-Ang University, Seoul 156‑756, Korea
mediated ABA signaling influences resistance to bacterial pathogens via stomatal regulation. Keywords Abscisic acid · ABA receptor · Protein phosphatase · Stomatal immunity Abbreviations ABA Abscisic acid ABI ABA-insensitive AHG ABA-hypersensitive germination AIP AKT1 interacting protein phosphatase AIPH AIP1 homologue BiFC Bimolecular fluorescence complementation cfu Colony forming units HAB Hypersensitive to ABA hpi Hours post-inoculation PAMPs Pathogen-associated molecular patterns PP2C Protein phosphatase type 2 C PRR Pattern recognition receptors Pst Pseudomonas syringae pv. tomato PYL PYR-like PYR Pyrabactin resistance qRT-PCR Quantitative reverse transcription-polymerase chain reaction RCAR Regulatory component of ABA receptor SOS Stomatal opening solution WT Wild-type
Introduction Plants are constantly challenged by biotic and abiotic stresses such as pathogens, drought, and high salinity. In response to pathogen attacks, plants have developed various resistance mechanisms; this process has resulted in
13
the local and systemic induction of a broad spectrum of defense mechanisms (Jones and Dangl 2006). Plants have innate and induced mechanical barriers that protect them from various pathogens such as bacteria and fungi. On the other hand, most pathogens have mechanisms that enable them to overcome physical and chemical plant barriers, in order successfully to infect the host plants. For successful establishment of infection, pathogens must initially gain entry into the plant tissues. Fungal pathogens can directly penetrate the plant tissues by applying mechanical forces or by utilizing cell wall degrading enzymes; on the other hand, bacterial pathogens require natural openings such as stomata, hydathodes, and lenticels (Melotto et al. 2008) or wound sites to enter plant tissues. Among these, stomatal pores constitute the main entry route into leaf tissues. The plant hormone abscisic acid (ABA) plays diverse roles in responses to biotic and abiotic stresses. This hormone triggers changes in a number of plant physiological processes, thereby resulting in adaptation to various stresses. In the past decade, several studies have investigated the role of ABA in plant defense responses and in determining the outcome of plant–bacterial pathogen interactions (Melotto et al. 2008; Ton et al. 2009). Stomatal closure leads to drought stress tolerance and serves as a defense mechanism for preventing bacterial pathogen invasions (Melotto et al. 2006, 2008). Recently, the mechanism of ABA-induced stomatal closure in response to biotic and abiotic stresses has been extensively studied (Santiago et al. 2009; Cao et al. 2011; Desclos-Theveniau et al. 2012; Lee et al. 2013; Lim et al. 2014). Stomatal defense is associated with stomatal closure in response to pathogen-associated molecular patterns (PAMPs), including flg22 and lipopolysaccharides (Melotto et al. 2006; Amborabe et al. 2008; Cho et al. 2008; Zeng and He 2010). Plant recognition of PAMPs via pattern recognition receptors (PRRs) activates ABA signaling in guard cells, thereby leading to stomatal closure (Melotto et al. 2008). The ABA signaling pathway has several key components, each of which serves as a positive and negative regulator at every step. SnRK2 type kinases and group A protein phosphatases (PP2Cs) act as positive and negative regulators, respectively, of ABA signaling (Lee and Luan 2012). Phenotypic analysis has revealed that the triple mutant (snrk2.2/snrk2.3/snrk2.6) displays an ABA-insensitive phenotype by opening stomata during water-deficient conditions; on the other hand, mutants of pp2cs exhibit a more profound hypersensitive ABA phenotype (Merlot et al. 2001; Fujii et al. 2007; Rubio et al. 2009). To initiate ABA signaling, it is necessary to perceive ABA via ABA specific receptors in plant cells. Several ABA receptors have been identified; however, the functions of these receptors remain to be elucidated (Liu et al. 2007; Christmann and Grill 2009; Park et al. 2009; Umezawa et al. 2009).
13
Plant Mol Biol
Pyrabactin resistance (PYR)/PYR-like (PYL) or regulatory component of ABA receptor (RCAR) comprises a 14-member family and acts as an ABA receptor (Park et al. 2009; Umezawa et al. 2009). Under stress condition, ABA levels increase and ABA receptors interact directly with the active site of PP2Cs; this process inhibits phosphatase activities, thereby leading to downstream activation of ABA signaling (Lee and Luan 2012). When the ABA levels decrease, PP2Cs interact with and inactivate SnRKs, thereby leading to suppression of ABA signaling (Lee et al. 2009, 2013). The relationship between ABA and pathogen resistance has been extensively investigated; however, the roles of the ABA components remain to be clarified. Recently, we reported that alteration of the gene expressions of RCAR2/PYL7 and RCAR3/PYL8, and their interacting partner PP2CA affects disease resistance and ABA sensitivity. Additionally, ABA sensitivity is positively correlated with stomatal immunity (Lim et al. 2014). Many studies have revealed that individual RCAR members have different combination of interaction with PP2C members and different requirement of ABA for the interaction with different PP2Cs (Park et al. 2009; Ma et al. 2009; Santiago et al. 2009; Hao et al. 2011). In addition, RCAR members exhibited functional difference, in spite of their functional redundancy (Antoni et al. 2012; Hao et al. 2011). In this aspect, functional role of other RCAR members in plant–microbe interaction need to be studied. In the present study, we investigated the functional role of two other homologous ABA receptors, namely RCAR4 and RCAR5, in stomatal immunity. We previously showed that similar to RCAR2 (Lee et al. 2009, 2013) and RCAR3 (Lim et al. 2014), overexpression of RCAR4 and RCAR5 leads to enhanced ABA sensitivity. Therefore, in the present study, we used dip-inoculation methods to examine the influence of the stomatal aperture on plant defense responses to Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we analyzed the roles of HOMOLOGY TO ABI1 (HAB1), interacting with RCAR4 and RCAR5 and functioning as a negative regulator in the ABA signaling pathway, in stomatal defense against Pst DC3000. Our findings indicate that RCARs and HAB1 play pivotal roles in plant defense responses during the pre-invasive stage, and that ABA sensitivity is important in stomatal defense.
Materials and methods Plant material and growth conditions Arabidopsis thaliana (ecotype Col-0) plants were routinely grown at a 9:1:1 ratio of peat moss, perlite, and vermiculite. The plants were maintained at 24 °C and 60 % humidity under fluorescent light (130 μmol photons m−2 s−1)
Plant Mol Biol
with a 16-h light/8-h dark cycle. Prior to in vitro culture, seeds of A. thaliana (ecotype Col-0) were surface sterilized with 70 % ethanol for 1 min and treated with 2 % sodium hydroxide for 10 min. The seeds were then washed 10 times with sterile distilled water and sown on Petri dishes containing Murashige and Skoog (1962) (MS) agar medium (Sigma, St. Louis, MO, USA) supplemented with 1 % sucrose. The plates were sealed and incubated at 24 °C in a chamber under fluorescent light (130 μmol photons m−2 s−1) with a 16-h light/8-h dark cycle. Seeds with hab1 insertion lines, i.e., SALK_002104 (Saez et al. 2004, 2006), were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA).
and 35S-SPYCE(M) vectors via XbaI/XhoI, to generate the bimolecular fluorescence complementation (BiFC) constructs (Waadt et al. 2008). For transient expression, A. tumefaciens strain GV3101 harboring each construct was mixed with p19 strain to avoid gene silencing and it was then infiltrated into the abaxial side of 5-week-old Nicotiana benthamiana leaves by using a 1-mL needleless syringe. At 3 days after infiltration, leaf discs were cut and the lower epidermal cells were examined under a confocal microscope (510 UV/Vis Meta; Zeiss, Oberkochen, Germany) equipped with LSM Image Browser software.
Yeast two‑hybrid assay
For germination tests, 100 seeds per genotype were sown on plates containing MS agar medium supplemented with various concentrations of ABA. After 7 days, the number of seeds showing radicle emergence were counted. For root growth assays during the post-germinative stage, 4-day-old seedlings from wild-type (WT) and RCAR4 and RCAR5OX transgenic Arabidopsis lines were transferred to plates containing MS agar medium supplemented with 10 μM of ABA. After 7 days, the root lengths of the seedlings were measured.
Yeast two-hybrid (Y2H) assays were conducted as described previously (Lee et al. 2007). The cDNA fragments of RCAR4 and RCAR5 as bait and HAB1 as prey were subcloned into the pGBKT7 and pGADGH vectors, respectively. These constructs were introduced into the yeast strain AH109, according to the lithium acetate-mediated transformation method (Ito et al. 1983). After selection on SCleucine–tryptophan medium, transformant candidates were transferred to SC-adenine–histidine–leucine–tryptophan medium for growth evaluation; this evaluation provided an indication of protein–protein interactions. Next, 5 μL of yeast cell culture (OD600 = 0.5) was spotted onto SC–leucine–tryptophan medium or SC-adenine–histidine–leucine– tryptophan medium containing 0 or 10 μM of ABA (Sigma). Generation of transgenic RCAR4 and RCAR5 overexpressing mutants Full-length RCAR4 and RCAR5 cDNA were cloned into pENTR/D-TOPO vectors (Invitrogen, Carlsbad, CA, USA). After sequencing, the cloned genes were integrated into pK2GW7 by using the LR reaction to induce constitutive expression of the RCAR4 and RCAR5 genes under the control of the CaMV 35S promoter (Karimi et al. 2002). The correct construct was introduced into Agrobacterium tumefaciens strain GV3101 via electroporation. Arabidopsis transformation with the RCAR4 and RCAR5 genes was performed by using the floral dip method (Clough and Bent 1998). For the selection of transgenic lines, seeds harvested from putative transformed plants were sown on plates containing MS agar medium supplemented with 50 μg mL−1 of kanamycin. Bimolecular fluorescence complementation assay Full-length cDNAs of RCAR4, RCAR5, and HAB1 without stop codons were subcloned into 35S-SPYNE(R)173
ABA treatment and phenotypic analyses
Bacterial growth assay We used the virulent pathogen, Pseudomonas syringae pv. tomato (Pst) DC3000, in this study. Bacterial infection was performed as described previously (Lim et al. 2014). Briefly, the leaves of 5-week-old plants were syringe-infiltrated or dipped in a bacterial suspension of Pst DC3000 [1 × 106 or 1 × 108 colony forming units (cfu) per mL] in 10 mM MgCl2 containing 0.02 % Silwet L-77 (Lehle Seeds). The inoculated plants were maintained at 24 °C and 100 % humidity for 24 h and were then transferred to a growth chamber and cultured at 100 % humidity for 2–3 days. The amount of bacterial growth in the infected leaves of each plant was determined according to Katagiri et al. (2002). Stomatal aperture bioassay The stomatal aperture bioassay was conducted as described previously, but with some modifications (Lee et al. 2013). Briefly, leaf peels were collected from the rosette leaves of 5-week-old plants and were floated in a stomatal opening solution (SOS; 50 mM KCl, 10 mM MES-KOH, 10 μM CaCl2, pH 6.15) under light. The peels were incubated for a further 3 h to obtain 80 % stomatal opening in A. thaliana Col-0 plants under laboratory conditions. The buffer was replaced with SOS containing various concentrations of ABA or bacterial cells (1 × 108 cells mL−1) resuspended
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in water. For the control, the buffer was replaced with new SOS without ABA or water alone. Leaf peels were then incubated for various periods. In each individual sample, 100 stomata were randomly observed under a Nikon Eclipse 80i microscope. The widths of individual stomata were recorded by using Image J 1.46r software (http:// imagej.nih.gov/ij). Each experiment was performed in triplicate. RNA isolation and semi‑quantitative and quantitative reverse transcription‑polymerase chain reaction Total RNA was isolated from dehydrated or infected Arabidopsis leaf tissues, by using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). To remove genomic DNA, all the RNA samples were digested with RNA-free DNase. After quantification with a spectrophotometer, 1 μg of total RNA was used to synthesize cDNA, by using a Transcript First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. Simultaneously, cDNAs were synthesized without reverse transcriptase and were subjected to semi-quantitative RT-PCR to eliminate the possibility of contamination by genomic DNA in the cDNA samples. Semi-quantitative RT-PCR analysis was conducted by using Extaq DNA polymerase with the following specific primers: RCAR4 (forward, 5′-ATGAACGGT GACGAAACAAAG-3′; reverse, 5′-TCATATCTTCTTCTC CATAGATTCTG-3′); RCAR5 (forward, 5′-ATGGAAACT TCTCAAAAATATCATAC-3′; reverse, 5′-TTACAACTTTA GATGAGCCACCCT-3′); HAB1 (forward, 5′-GACTACC TCTCAATGCTTGCTCTAC-3′; reverse, 5′-CCTTCTAAC AATATTGAGTAATTAAC-3′); and UBQ1 as an internal control (forward, 5′-GTAGTGCTAAGAAGAGGAAGA-3′; reverse, 5′-TCAAGCTTCAACTCCTTCTT-3′). For quantitative RT-PCR analysis, the synthesized cDNA was amplified in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) with iQ™SYBR Green Supermix and the following specific primers: RCAR4 (forward, 5′-CAAAGAAGGTG GAGAGCGAGTACAT-3′; reverse, 5′-CCACTTCTCTTAC GCTACCAACCTC-3′); RCAR5 (forward, 5′-ATGGAAA CTTCTCAAAAATATCATACG-3′; reverse, 5′-CTCTCTCG GCTGAACTCCGCTG-3′); HAB1 (forward, 5′-GACTAC CTCTCAATGCTTGCTCTAC-3′; reverse, 5′-CCCTTCTAA CAATATTGAGTAATTAAC-3′); RD29B (forward, 5′-GTT GAAGAGTCTCCACAATCACTTG-3′; reverse, 5′-ATTAA CCCAATCTCTTTTTCACACA-3′); RD20 (forward, 5′-TG GTTTCCTATCTAAAGAAGCTGTG-3′; reverse, 5′-ATAC AAATCCCCAAACTGAATAACA-3′); PR1 (forward, 5′-T GTGGTCACTACACTCAAGTTGTTT-3′; reverse, 5′-GTG TGTATGCATGATCACATCATTA-3′); and Actin 8 as an internal control (forward, 5′-CAACTATGTTCTCAGGTA TTGCAGA-3′; reverse, 5′-GTCATGGAAACGATGTCTCT TTAGT-3′).
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Plant Mol Biol
Results and discussion Ectopic expression of RCAR4 and RCAR5 leads to ABA hypersensitivity in planta In our previous studies, we showed that overexpression of RCAR2 and RCAR3 confers ABA hypersensitivity and enhanced disease resistance (Lee et al. 2013; Lim et al. 2013, 2014). Therefore, in the present study, we examined the functional role of other homologous ABA receptors, namely, RCAR4 and RCAR5, in response to ABA treatment and pathogen infection. Although RCAR2-5 are monomeric receptors, in particular RCAR4 inhibits protein phosphatase activity in the absence of ABA (Hao et al. 2011) and functional role of RCAR5 remained elusive. The results of multiple alignment analysis revealed that RCAR4 shares higher sequence homology with RCAR2 (54.7 % identity, 71.1 % similarity) and RCAR3 (71.4 % identity, 86.7 % homology) than does RCAR5 (39.6 % identity, 58.5 % similarity and 42.3 % identity, 58.5 % similarity, respectively). Based on these, we postulated that RCAR4 and RCAR5 show different gene function relative to RCAR2 and RCAR3. We initially generated transgenic plants overexpressing the coding sequence of RCAR4 and RCAR5. We found that the expression levels of these genes were undetectable in wild-type plants under the conditions used in the present study. However, the overexpressing lines of RCAR4 and RCAR5 showed significant accumulation of each transcript (Fig. 1a). Under normal growth conditions, the phenotype of transgenic lines was indistinguishable from that of wild-type plants (data not shown). Analysis of the ABA responses showed that all the transgenic lines were ABA-hypersensitive during seed germination (Fig. 1b) and early seedling growth (Supplementary Fig. S1). In the germination assay, the germination rate of plants grown in the absence of ABA did not differ significantly between the wild-type and the RCAR4-OX and RCAR5OX transgenic lines. On the other hand, the RCAR4-OX and RCAR5-OX plants were more sensitive to 0.5 μM and 1.0 μM ABA than those of the wild-types during seed germination (Fig. 1b). RCAR4-OX and RCAR5-OX plants germinated and grown in the presence of 0.5 μM ABA exhibited hypersensitivity to ABA (Supplementary Fig. S1). In comparison with wild-type plants, the root lengths of RCAR4-OX and RCAR5-OX plants after 7 days were 8–52 and 24–66 % shorter, respectively. To confirm that the reduced root growth in RCAR4-OX and RCAR5-OX plants was not caused by a developmental defect, but was solely derived from the inhibitory effect of ABA, 4-day-old seedlings of each plant were transferred and grown in the presence of 10 μM ABA. In comparison with wild-type plants, the primary root lengths of RCAR4-OX and RCAR5OX plants after 7 days were 12–27 and 38–54 % shorter,
Plant Mol Biol
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Fig. 1 ABA-hypersensitivity of RCAR4-OX and RCAR5-OX mutants. a RT-PCR analysis of RCAR4 and RCAR5 expression from the leaves of WT (Col-0) plants and transgenic lines (T3). The UBQ1 gene served as an internal control. The numbers in parentheses indicates the cycles of PCR. b Germination rate of RCAR4-OX (left) and RCAR5-OX mutants and WT seeds on ×0.5 MS medium supplemented with various concentrations of ABA. The number of
seeds with an emerged radicle was counted every day up to day 7. c Inhibition of primary root growth in RCAR4-OX (upper) and RCAR5OX (bottom) mutants and WT seedlings. Four-day-old seedlings were transferred in ×0.5 MS containing 0 or 10 μM ABA and were grown vertically. After 7 days, representative images were taken. Data are presented as mean ± standard deviation from three independent experiments
respectively. Our results suggest that RCAR4 and RCAR5 have different functions in ABA signaling. Additionally, they indicate that ectopic expression of RCAR4 and RCAR5 confers enhanced ABA sensitivity during germination and root elongation.
ABA-induced stomatal closure. To evaluate stomatal closure, we measured the pore sizes of stomata in RCAR4OX, RCAR5-OX and wild-type plants after treatment with ABA (Fig. 2). The results of our previous studies suggested that ABA promotes stomatal closure via the functioning of ABA receptors as positive regulators (Lee and Luan 2012; Lee et al. 2013). In the present study, we observed no clear differences in the stomatal aperture of wild-type, RCAR4OX, and RCAR5-OX plants grown in the absence of ABA. After treatment with 10 or 20 μM ABA for 2.5 h, the degree of stomatal closure was higher in RCAR4-OX and
RCAR4‑OX and RCAR5‑OX mutants show ABA‑hypersensitive stomatal closure Next, we examined whether RCAR4-OX and RCAR5-OX plants have other ABA-associated phenotypes such as
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Plant Mol Biol
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Overexpression of RCAR4 and RCAR5 regulates disease resistance to P. syringae pv. tomato DC3000 infection
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ABA (μM) Fig. 2 ABA-hypersensitive stomatal closing in RCAR4-OX and RCAR5-OX mutants. Leaf peels of WT plants and RCAR4-OX and RCAR5-OX mutants were incubated with 10 or 20 μM ABA for 2.5 h. The pictures show representative stomata at each ABA concentration (a). The stomatal apertures were measured under the microscope in WT plants and RCAR4-OX and RCAR5-OX mutants (b). Data are presented as mean ± standard error (n = 100). Different letters indicate significant differences at p
