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. 2016 Apr 13;171(2):1366–1377. doi: 10.1104/pp.15.01741

The GSK3/Shaggy-Like Kinase ASKα Contributes to Pattern-Triggered Immunity1,[OPEN]

Hansjörg Stampfl 1,2, Marion Fritz 1,2, Silvia Dal Santo 1,2,2, Claudia Jonak 1,2,*
PMCID: PMC4902580  PMID: 27208232

The GSK3/Shaggy-like kinase ASKα and its direct substrate glucose-6-phosphate dehydrogenase constitute a signaling module contributing to innate immunity in Arabidopsis thaliana.

Abstract

The first layer of immunity against pathogenic microbes relies on the detection of conserved pathogen-associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs) to activate pattern-triggered immunity (PTI). Despite the increasing knowledge of early PTI signaling mediated by PRRs and their associated proteins, many downstream signaling components remain elusive. Here, we identify the Arabidopsis (Arabidopsis thaliana) GLYCOGEN SYNTHASE KINASE3 (GSK3)/Shaggy-like kinase ASKα as a positive regulator of plant immune signaling. The perception of several unrelated PAMPs rapidly induced ASKα kinase activity. Loss of ASKα attenuated, whereas its overexpression enhanced, diverse PTI responses, ultimately affecting susceptibility to the bacterial pathogen Pseudomonas syringae. Glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the oxidative pentose phosphate pathway, provides reducing equivalents important for defense responses and is a direct target of ASKα. ASKα phosphorylates cytosolic G6PD6 on an evolutionarily conserved threonine residue, thereby stimulating its activity. Plants deficient for or overexpressing G6PD6 showed a modified immune response, and the insensitivity of g6pd6 mutant plants to PAMP-induced growth inhibition was complemented by a phosphomimetic but not by a phosphonegative G6PD6 version. Overall, our data provide evidence that ASKα and G6PD6 constitute an immune signaling module downstream of PRRs, linking protein phosphorylation cascades to metabolic regulation.

Metazoans and plants rely on complex signaling networks to defend against pathogenic microbes. Pathogen-associated molecular patterns (PAMPs), molecules associated with groups of pathogens, and damage-associated molecular patterns (DAMPs), released from host tissues as a consequence of pathogen attack, are recognized by cell surface-localized pattern recognition receptors (PRRs) to initiate a signaling cascade that activates pattern-triggered immunity (PTI; Schwessinger and Zipfel, 2008; Kawai and Akira, 2010; Kumar et al., 2011). In Arabidopsis (Arabidopsis thaliana), the bacterial PAMP flagellin and the fungal PAMP chitin are directly recognized by the receptor kinase FLAGELLIN SENSING2 (FLS2) and the chitin elicitor receptor kinase CERK1, respectively, while the DAMP pep1 is perceived by two redundant receptor-like kinases, PEPR1 and PEPR2 (for Pep1 receptors 1 and 2; Chinchilla et al., 2006; Miya et al., 2007; Krol et al., 2010; Yamaguchi et al., 2010). Upon flagellin perception, FLS2 and its coreceptor BRI1-ASSOCIATED KINASE1 (BAK1)/SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 heteromerize (Chinchilla et al., 2007; Heese et al., 2007) and initiate a cascade of phosphorylation events that coordinate a wide range of defense responses. Responses triggered by PTI signaling include an apoplastic burst of reactive oxygen species (ROS), influx of calcium into the cytosol, activation of Ca2+-dependent protein kinases (CDPKs) and mitogen-activated protein kinases (MAPKs), deposition of callose at the cell wall, closure of stomata, and transcriptional reprogramming, ultimately leading to a broad resistance to pathogens (Boller and Felix, 2009; Segonzac et al., 2011; Tena et al., 2011; Bigeard et al., 2015).

The rapid production of ROS is a conserved immune response. ROS can act directly as antimicrobial compounds and cross‐link cell wall compounds to block pathogen ingress and indirectly as systemic secondary messengers to trigger additional immune responses (Lamb and Dixon, 1997). In Arabidopsis, ROS production during PTI is mediated by plasma membrane-localized NADPH oxidases (RBOHs; Torres et al., 2002; Nühse et al., 2007; Zhang et al., 2009). NADPH oxidases require NADPH to generate superoxide, which can be dismutated subsequently to hydrogen peroxide.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the oxidative pentose phosphate pathway, which generates NADPH and metabolic intermediates for biosynthetic pathways. G6PD activity was shown to be induced in tobacco (Nicotiana tabacum) by infection with Phytophthora nicotianae (Scharte et al., 2009) and to be necessary for elicitor- and pathogen-induced ROS production in rice (Oryza sativa) and tobacco (Pugin et al., 1997; Scharte et al., 2009; Asai et al., 2011; Kano et al., 2013). However, the mechanisms enhancing G6PD activity during the immune response remain unknown. Likewise, little is known about G6PD-regulated PTI responses.

G6PD activity can be regulated by the cellular redox status, the NADPH-NADP+ ratio, and protein phosphorylation (Wendt et al., 2000; Debnam et al., 2004; Schürmann and Buchanan, 2008; Dal Santo et al., 2012). Under high-salinity conditions, G6PD activity is regulated by the Arabidopsis protein kinase ASKα. ASKα phosphorylates a cytosolic G6PD isoform (G6PD6) on an evolutionarily conserved Thr residue, thereby enhancing G6PD activity and salt stress tolerance. ASKα is a member of the evolutionarily conserved GLYCOGEN SYNTHASE KINASE3 (GSK3) class. GSK3 was originally identified as a regulator of glycogen metabolism in mammals but is now recognized as a central multifunctional kinase that acts as a regulator of numerous signaling pathways, including cell fate determination, cell cycle regulation, apoptosis, cancer, and innate immunity in mammals (Jope and Johnson, 2004; Wang et al., 2014). In Arabidopsis, there are 10 GSK3 family members that can be grouped into four classes (Jonak and Hirt, 2002). ASKα belongs to group 1 of plant GSK3s. In addition to regulating abiotic stress responses, other Arabidopsis GSK3 family members play an important regulatory role in brassinosteroid (BR) signaling (Li and Nam, 2002; Vert and Chory, 2006; Rozhon et al., 2010) and development (Gudesblat et al., 2012; Kim et al., 2012; Khan et al., 2013; Cho et al., 2014; Kondo et al., 2014).

The discovery of numerous players acting downstream of PAMP perception has significantly advanced our knowledge; however, little is known about the regulation of metabolic processes necessary for a successful PTI response. In this work, we link PAMP-induced kinase signaling with metabolic regulation. We identified and characterized the role of the Arabidopsis GSK3 ASKα in regulating PTI responses. Furthermore, we connect ASKα with G6PD activity in flagellin-triggered immune responses and provide genetic and biochemical evidence that the cytosolic isoform G6PD6, and thereby the oxidative pentose phosphate pathway, contributes to a successful PTI response and resistance to the bacterial pathogen Pseudomonas syringae in Arabidopsis.

RESULTS

ASKα Regulates Flagellin-Induced Immune Responses

To investigate whether ASKα might be involved in immune signaling, we first analyzed the in vivo kinase activity of ASKα in response to flagellin using the 22-amino acid epitope flg22. Immunokinase assays with ASKα-specific antibodies (Dal Santo et al., 2012) and myelin basic protein (MBP) as a general substrate showed basal ASKα activity in nontreated and mock-treated Arabidopsis seedlings. flg22 treatment induced ASKα activity 15 min after elicitation, which persisted over the experimental period of 3 h (Fig. 1A). flg22 also induced ASKα expression levels (Supplemental Fig. S1A).

Figure 1.

Figure 1.

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ASKα regulates flg22-triggered immune responses. A, ASKα kinase activity upon flg22 treatment. Twelve-day-old wild-type seedlings were treated with 1 µm flg22 or water as a mock control for the indicated periods of time. ASKα immunokinase activity was assayed with [γ-32P]ATP and MBP as a general substrate. CBB, Coomassie Brilliant Blue. B, flg22-induced ROS burst in wild-type (WT), askα, and ASKα-overexpressing plants elicited with 100 nm flg22. Results are expressed as relative luminescence units (RLU). Values are means ± se (n = 20). C, Expression of PTI marker genes in wild-type, askα, and ASKα-overexpressing lines elicited with 100 nm flg22 for the indicated periods of time. EF1A was used as a reference gene. Relative gene expression was normalized to the steady-state control. Data represent means ± sd (n = 3). D, Callose deposition in wild-type, askα, and ASKα-overexpressing plants syringe infiltrated with 1 µm flg22. Results are presented as the relative callose intensity per field of view detected by Aniline Blue staining normalized to the wild type. Values are means ± sd (n = 6). The asterisk indicates a significant difference (Student’s t test compared with the wild type; *, P < 0.05). E, MAPK activation in wild-type, askα, and ASKα-overexpressing seedlings after treatment with 100 nm flg22. MAPK activity was detected by western-blot analyses using anti-phospho-p44/42 MAPK antibodies. A western blot with anti-actin antibodies was used to ensure equal loading. F, flg22-induced seedling growth inhibition of wild-type, askα, and ASKα-overexpressing lines. Results are expressed as the fresh weight ratio of untreated (NT) to treated (T) seedlings, and average values ± relative se (n = 12) are indicated. Different letters indicate significant differences in fresh weight change according to genotypes and flg22 concentrations (two-way ANOVA; P ≤ 0.05).

The rapid induction of ASKα activity by flg22 prompted us to further analyze the role of ASKα in PTI. PAMP-triggered signaling leads to RBHOD-dependent ROS production, resulting in an early oxidative burst (Nühse et al., 2007; Zhang et al., 2007). In response to flg22, plants with enhanced ASKα activity (Dal Santo et al., 2012) produced more ROS than wild-type plants, whereas askα knockout plants showed a reduced early oxidative burst (Fig. 1B; Supplemental Fig. S1D).

PTI is associated with transcriptional reprogramming (Navarro et al., 2004; Zipfel et al., 2004). To investigate whether ASKα plays a role in the PAMP-triggered transcriptional response, we analyzed the expression of flg22-responsive genes by quantitative reverse transcription-PCR. In ASKα overexpressors, expression levels of FRK1, PHI1, WRKY33, and CYP81F were elevated, whereas transcript levels were moderately reduced in askα mutants, when compared with wild-type plants (Fig. 1C).

PRR activation triggers MAPK cascades (Meng and Zhang, 2013; Bigeard et al., 2015). To investigate PAMP-induced MAPK activation in ASKα mutants, wild-type, askα, and ASKα-overexpressing seedlings were inoculated with flg22 and MAPK activation was visualized by immunoblotting using an anti-pTEpY antibody, which recognizes phosphorylated, active forms of MAPKs. No significant difference in MAPK activation was observed in plants deficient for or overexpressing ASKα as compared with the wild type, indicating that ASKα functions either independently or downstream of MAPK activation (Fig. 1E).

In addition to these early flg22-induced responses, we also analyzed the involvement of ASKα in two late PTI outputs. PAMP treatment induces ROS- and GSL5/PMR4-dependent deposition of callose at the cell wall to strengthen cell wall integrity and hinder pathogen entry (Gómez-Gómez et al., 1999; Kim et al., 2005; Zhang et al., 2007; Luna et al., 2011). Aniline Blue staining was used to detect callose deposition in flg22-infiltrated leaves of the wild type and askα mutants. Wild-type and ASKα-overexpressing plants showed a substantial deposition of callose after flg22 treatment, but reduced numbers of callose deposits were detected in askα leaf tissue (Fig. 1D; Supplemental Fig. S1C), indicating that functional ASKα is necessary for proper flg22-induced callose deposition and cell wall defense.

Prolonged exposure to flg22 is linked to growth inhibition in wild-type seedlings (Gómez-Gómez et al., 1999). Using different concentrations of flg22, askα seedlings showed a reduced rate of growth inhibition, whereas ASKα overexpression led to enhanced growth inhibition when compared with wild-type seedlings (Fig. 1F; Supplemental Table S1). Taken together, these data provide evidence that flg22 stimulates ASKα activity and that ASKα mediates various flg22-induced defense responses.

ASKα Modulates PTI Responses to Chitin and pep1

The regulation of flg22-induced responses by ASKα prompted us to investigate whether ASKα is involved in mediating the responses to unrelated immunogenic compounds perceived by other receptor complexes. First, we analyzed ASKα activity in plants exposed to the fungal PAMP chitin and to the endogenous Arabidopsis DAMP pep1. Like flg22, chitin and pep1 induced ASKα activity (Fig. 2A), suggesting that ASKα plays a role in signaling diverse molecular patterns. In line with this notion, plants deficient in ASKα showed a reduced oxidative burst upon treatment with chitin and pep1, whereas ROS production was increased in ASKα-overexpressing plants (Fig. 2, C and D; Supplemental Fig. S1, E and F). Furthermore, pep1-induced seedling growth inhibition was reduced in askα seedlings and enhanced in ASKα overexpressors (Fig. 2B; Supplemental Table S1), indicating a role for ASKα in the response to diverse molecular patterns.

Figure 2.

Figure 2.

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ASKα is involved in mediating chitin- and pep1-induced responses. A, ASKα in vivo kinase activity 15 min after treatment with 1 µm flg22, 1 mg mL−1 chitin, or 1 μm pep1. CBB, Coomassie Brilliant Blue. B, Pep1-induced seedling growth inhibition of wild-type (WT), askα, and ASKα-overexpressing lines. Results are expressed as the fresh weight ratio of untreated (NT) to treated (T) seedlings, and average values ± relative se (n = 12) are indicated. Different letters indicate significant differences in fresh weight change according to genotypes and pep1 concentrations (assessed by two-way ANOVA; P ≤ 0.05). C and D, ROS formation upon treatment with 1 μm pep1 (C) or 200 μg mL−1 chitin (D) in the wild type and plants deficient for or overexpressing ASKα. Results are expressed as relative luminescence units (RLU). Values are means ± se (n = 20).

G6PD Activity Is Required for the Enhanced ROS Burst in ASKα Overexpressors

G6PD activity has been implicated in immune responses in different organisms (Pugin et al., 1997; Roos et al., 1999; Chao et al., 2008; Scharte et al., 2009). Previously, we have shown that G6PD6 is an in vivo target of ASKα under salt stress conditions (Dal Santo et al., 2012). Thus, we hypothesized that ASKα might regulate G6PD6 activity upon PAMP perception. First, we investigated G6PD activity in the wild type and plants with altered ASKα activity in response to flg22 treatment. Exposure of wild-type plants to flg22 moderately enhanced G6PD activity. In contrast, G6PD activity was reduced in askα, while plants overexpressing ASKα showed a flg22-induced increase in G6PD activity that was higher than that in the wild type (Fig. 3A).

Figure 3.

Figure 3.

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G6PD6 activity contributes to PTI responses. A, G6PD activity upon flg22 treatment. Total G6PD activity was determined in seedlings of wild-type (WT), askα, and ASKα-overexpressing lines OE4 and OE5 15 min after mock (ctrl) and flg22 (100 nm) treatment. Results are expressed as the x-fold G6PD activity change compared with the control. Different letters indicate significant differences in G6PD activity change upon treatment (two-way ANOVA; P ≤ 0.05). B, G6PD-dependent ROS formation in wild-type and ASKα-overexpressing lines OE4 and OE5 elicited with 100 nm flg22 with and without preincubation with 10 mm glucosamine-6-phosphate (GN6P) for 1 h. C, ROS formation in the wild type and plants deficient for or overexpressing G6PD6 (lines OE4 and OE8) elicited with 100 nm flg22, 1 μm pep1, or 200 μg mL−1 chitin. Results are expressed as relative luminescence units (RLU). Values are means ± se (n = 20). D, flg22- and pep1-induced seedling growth inhibition in the wild type, g6pd6, and two G6PD6 overexpressor lines. Results are expressed as the fresh weight ratio of untreated (NT) to treated (T) seedlings, and average values ± relative se (n = 18) are indicated. Different letters indicate significant differences in fresh weight change according to genotypes and flg22 or pep1 concentrations (two-way ANOVA; P ≤ 0.05).

Next, we investigated whether the enhanced oxidative burst observed in ASKα-overexpressing plants might be mediated by G6PD. Inhibition of G6PD activity by the competitive G6PD inhibitor glucosamine-6-phosphate (Glaser and Brown, 1955) suppressed the flg22-induced oxidative burst in the wild type (Fig. 3B; Supplemental Fig. S2A). The increased flg22-triggered oxidative burst in ASKα overexpressors also was suppressed to levels similar to those in wild-type plants, providing evidence that G6PD activity is necessary for PAMP-induced ROS production and the ASKα-mediated oxidative burst.

G6PD6 Contributes to PAMP-Triggered Responses

The suppression of the flg22-induced oxidative burst in ASKα overexpressors by biochemical G6PD inhibition prompted us to investigate whether G6PD6 contributes to PTI responses. First, we analyzed the PAMP-triggered oxidative burst in g6pd6 mutants (Dal Santo et al., 2012) and G6PD6-overexpressing plants (Supplemental Fig. S2B) to diverse immunogenic peptides. Treatment of leaf discs with flg22, chitin, or pep1 induced an enhanced early oxidative burst in plants overexpressing G6PD6, whereas ROS production was lower or similar to wild-type levels in g6pd6 mutants (Fig. 3C; Supplemental Fig. S2C).

Seedling growth inhibition assays showed that G6PD6 overexpressors are hypersensitive to flg22, whereas g6pd6 knockout plants showed a reduced response (Fig. 3D; Supplemental Table S1). A similar result was observed in assays with pep1 (Fig. 3D; Supplemental Table S1), indicating that, like ASKα, G6PD6 can modify PAMP- and DAMP-triggered responses.

Thr-467 Is Necessary for G6PD6 Function in PTI Responses

Previously, we showed that G6PD6 can be phosphorylated in vivo by ASKα on the evolutionarily conserved Thr-467, thereby enhancing G6PD activity (Dal Santo et al., 2012). To assess whether G6PD6 phosphorylation is important during the immune response, we used g6pd6 knockout plants (which show an impaired PTI response) expressing a Thr-467 phosphomimetic G6PD6 variant (G6PD6 T467E) or a phosphonegative G6PD6 variant (G6PD6 T467A; Supplemental Fig. S3A). In seedling growth inhibition assays, the reduced sensitivity of g6pd6 to flg22 and pep1 was restored by the expression of G6PD6 and the phosphomimetic mutant G6PD6 T467E, whereas expression of the phosphonegative G6PD6 T467A in g6pd6 did not complement the phenotype (Fig. 4A; Supplemental Table S1). Furthermore, to assess whether the effect of ASKα on PTI responses is indeed mediated by the phosphorylation of G6PD6, we used askα knockout plants (which show reduced flg22- and pep1-induced seeding growth inhibition) expressing G6PD6 T467E (Dal Santo et al., 2012). Expression of G6PD6 T467E in askα rendered seedlings hypersensitive to flg22- and pep1-induced growth inhibition (Fig. 4B; Supplemental Table S1), indicating that ASKα-induced phosphorylation of G6PD6 on Thr-467 is important for PTI signaling.

Figure 4.

Figure 4.

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ASKα and G6PD6 function as a module to regulate PTI responses. A, flg22- and pep1-induced seedling growth inhibition of wild-type (WT), g6pd6, and g6pd6 plants expressing wild-type G6PD6, G6PD6 T/E, or G6PD6 T/A. B, flg22- and pep1-induced seedling growth inhibition of wild-type, askα, and askα plants expressing either wild-type G6PD6 or G6PD6 T/E. Results are expressed as the fresh weight ratio of untreated (NT) to treated (T) seedlings, and average values ± relative se (n = 18) are indicated. Different letters indicate significant differences in fresh weight change according to genotypes and flg22 or pep1 concentrations (two-way ANOVA; P ≤ 0.05). C, ROS formation in wild-type, askα, g6pd6, and askα/g6pd6 plants elicited with 100 nm flg22. Results are expressed as relative luminescence units (RLU). Values are means ± se (n = 20). D, flg22- or pep1-induced seedling growth inhibition in wild-type, askα, g6pd6, and askα/g6pd6 plants. Results are expressed as the fresh weight ratio of untreated to treated seedlings, and average values ± relative se (n = 18) are indicated. Different letters indicate significant differences in fresh weight change according to genotypes and flg22 or pep1 concentrations (two-way ANOVA; P ≤ 0.05). E, Working model. ASKα has a functional role in PRR-dependent signaling, which initiates innate immunity. PAMP/DAMP perception activates ASKα, which in turn regulates diverse PTI signaling outputs. ASKα phosphorylates G6PD6 and thereby stimulates its activity. G6PD provides NADPH for RBOH-dependent ROS production.

In addition, askα g6pd6 double knockout plants showed flg22- and pep1-induced seedling growth inhibition and flg22-triggered oxidative burst responses similar to single knockouts (Fig. 4, C and D; Supplemental Fig. S3C; Supplemental Table S1), suggesting that ASKα and G6PD6 are indeed part of the same pathway. Thus, our results support a role for Thr-467 phosphorylation of G6PD6 by ASKα in PTI signaling.

ASKα and G6PD6 Contribute to Plant Immunity

Given that ASKα modulates PAMP-triggered responses, we analyzed whether ASKα is involved in mediating plant resistance to bacterial pathogen infection. Immunokinase assays of wild-type Arabidopsis plants inoculated with the virulent Pseudomonas syringae pv tomato (Pto) strain DC3000 showed that Pto infection stimulated ASKα in vivo kinase activity (Fig. 5A). P. syringae infection also induced ASKα expression levels (Supplemental Fig. S1B).

Figure 5.

Figure 5.

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ASKα and G6PD6 contribute to plant immunity during P. syringae infection. A, ASKα kinase activity upon infection with virulent P. syringae. Four-week-old wild-type plants were syringe infiltrated with Pto DC3000 (106 colony-forming units [cfu] mL−1), and ASKα immunokinase assays using ASKα-specific antibodies and MBP as a general substrate were performed after the indicated periods of time. B and D, Bacterial infection assays using ASKα (B) and G6PD6 (D) activity mutants spray inoculated with Pto DC3000 (108 cfu mL−1). Bacterial populations were quantified as cfu cm−2 leaf area at 0 and 3 d after inoculation. Results are averages ± se (n = 4). Asterisks indicate significant differences compared with the wild type (WT; Student’s t test; *, P < 0.05). C and E, Bacterial infection assays using wild-type, askα, and askα plants expressing wild-type G6PD6 or G6PD6 T/E (C) or wild-type, g6pd6, and g6pd6 plants expressing wild-type G6PD6, G6PD6 T/E, or G6PD6 T/A (E). Plants were spray inoculated with Pto DC3000 (108 cfu mL−1). Bacterial populations were quantified as cfu cm−2 leaf area at 0 and 3 d after inoculation. Results are averages ± se (n = 4). Different letters indicate significant differences in bacterial numbers at 0 and 3 d after inoculation (one-way ANOVA; P ≤ 0.05).

To assess the contribution of ASKα to plant pathogen resistance, wild-type, askα, and ASKα-overexpressing plants were spray inoculated with Pto DC3000 and in planta bacterial growth was analyzed. Bacterial numbers were significantly higher in askα mutants, whereas ASKα-overexpressing plants showed a reduced susceptibility to Pto compared with wild-type plants (Fig. 5B), indicating that ASKα contributes to resistance against P. syringae.

To evaluate whether G6PD6 plays a role during Pto infection, leaf bacterial numbers were determined in plants deficient for and overexpressing G6PD6 after infection with Pto DC3000. Similar to ASKα activity mutants, plants deficient for G6PD6 were more susceptible, whereas plants with enhanced G6PD6 activity showed reduced susceptibility to Pto compared with wild-type plants (Fig. 5D).

To analyze whether the phosphorylation of G6PD6 contributes to bacterial resistance against P. syringae, bacterial infection assays were performed on g6pd6 plants expressing the phosphomimetic G6PD6 variant (G6PD6 T467E) or the phosphonegative G6PD6 variant (G6PD6 T467A). The expression of wild-type G6PD6 and G6PD6 T467E in g6pd6 restored bacterial growth to levels similar to those in the wild type (Fig. 5E). However, expression of the phosphonegative G6PD6 variant G6PD6 T467A did not complement the increased susceptibility of g6pd6 to Pto DC3000 (Fig. 5E). Finally, to investigate whether the effect of ASKα on resistance to pathogen infection is mediated by G6PD6 phosphorylation, leaf bacterial numbers were determined in askα knockout plants expressing G6PD6 T467E after infection with Pto DC3000. The enhanced susceptibility of askα to Pto infection was restored to the levels of wild-type plants by the expression of the phosphomimetic G6PD6 variant in askα (Fig. 5C). Overall, these data indicate that ASKα acts together with G6PD6 to contribute to PTI signaling and plant bacterial resistance.

DISCUSSION

Protein phosphorylation plays a key role in PTI signaling. BIK1, MAPKs, and CDPKs are protein kinases that have been shown to function downstream of PRRs to phosphorylate different targets, including transcription factors and RBOHD. In this work, we identified the Arabidopsis GSK3 ASKα as a regulator of plant innate immunity that links phosphorylation-based signaling to metabolic regulation. We provide evidence that ASKα-induced PTI responses are, at least partly, mediated by the oxidative pentose phosphate pathway and show that ASKα and G6PD6 regulate the susceptibility of Arabidopsis plants to virulent P. syringae.

ASKα is a novel positive regulator of PTI signaling. ASKα activity was rapidly enhanced after PAMP recognition. Analysis of plants deficient in or overexpressing ASKα showed that ASKα positively regulates the PAMP-triggered early oxidative burst, defense gene expression, callose deposition, and seedling growth inhibition. The modest but robust phenotype of askα on the early ROS burst might be due to a redundancy of ASKα with other Arabidopsis GSK3 isoforms. In support of this idea, Arabidopsis seedlings deficient in group 2 GSK3s (ASKα is in group 1) have been shown to display an impaired flg22-induced ROS burst (Lozano-Durán et al., 2013).

Significantly, our study demonstrates that ASKα in vivo kinase activity is regulated by both PAMPs and DAMPs. Exposure of plants to flg22, chitin, and pep1 rapidly induced ASKα kinase activity, consistent with a positive regulatory role of ASKα in PTI. Recognition of flg22 by FLS2 and pep1 by PEPR1/PEPR2 involves BAK1 (Chinchilla et al., 2007; Heese et al., 2007; Krol et al., 2010; Schulze et al., 2010; Yamaguchi et al., 2010), but CERK1 perceives chitin in a BAK1-independent manner (Heese et al., 2007; Gimenez-Ibanez et al., 2009), providing evidence that ASKα activity can be stimulated by BAK1-dependent and -independent PRRs. Furthermore, both the induction of ASKα kinase activity and the altered PTI response in ASKα mutants by diverse immunogenic compounds with bacterial, fungal, and plant origins indicate that ASKα may play a central role in defense signaling, as it mediates the activation of immune responses upon the recognition of diverse molecular patterns.

Along this line, the involvement of ASKα in both chitin (a major constituent of fungal cell walls)- and flagellin (the principal component of bacterial flagella)-triggered immune signaling points toward a role for ASKα in mediating innate resistance to a wide range of pathogens. While the induction of ASKα activity by P. syringae infection and the increased susceptibility of askα to virulent P. syringae already provide evidence for a regulatory function of ASKα during bacterial infection, fungal pathogens remain to be tested.

Our data show that ASKα is a positive regulator of plant immunity. However, it has been reported that overexpression of the alfalfa (Medicago sativa) GSK3 MSK1 in Arabidopsis increased susceptibility to virulent P. syringae (Wrzaczek et al., 2007). The opposite effects described for ASKα and MSK1 on P. syringae susceptibility might be due to the different means of infection, spray inoculation or infiltration, or might suggest that different GSK3s have distinct, either positive or negative, regulatory functions in immunity. Future studies will aim to investigate these hypotheses.

Recently, evidence was provided that BR regulates the growth-immunity tradeoff (Albrecht et al., 2012; Belkhadir et al., 2012; Lozano-Durán et al., 2013). Several Arabidopsis GSK3 family members play an important regulatory role in BR signaling (Li and Nam, 2002; Vert and Chory, 2006; Rozhon et al., 2010), raising the question of whether cross talk between BR and PTI signaling might involve ASKα. The lack of obvious BR phenotypes in askα and ASKα-overexpressing lines (Supplemental Fig. S4) indicates that BR and PTI signaling can be uncoupled at the level of ASKα. This supports and extends the view that the cross talk between BR and immune signaling is mediated by transcription factors downstream of GSK3 (Lozano-Durán et al., 2013; Fan et al., 2014; Malinovsky et al., 2014).

Defense reactions are associated with an increased demand for reducing equivalents. The oxidative pentose phosphate pathway is a major source of NADPH, and G6PD activity has been positively correlated with infection by viral and fungal pathogens (Sindelár and Sindelárová, 2002; Scharte et al., 2009). Our analysis of Arabidopsis plants deficient in or overexpressing G6PD6 now provides genetic evidence that a cytosolic G6PD contributes to PAMP- and DAMP-triggered defense responses as well as resistance to the bacterial pathogen P. syringae. Consistent with these results, ectopic overexpression of a plastidic G6PD in the cytosol of tobacco enhanced resistance to P. nicotianae (Scharte et al., 2009).

Notably, the induction of G6PD activity in plants exposed to flg22 was dependent on functional ASKα, indicating that ASKα is necessary for PAMP-triggered stimulation of G6PD activity. Moreover, the enhanced ROS burst observed in ASKα overexpressors relied on G6PD activity. Thus, it is reasonable to assume that the increased need of NADPH for RBOH-dependent ROS production might be met by NADPH derived from G6PD and that ASKα phosphorylates and stimulates G6PD activity during defense. Consistent with this idea, the impaired flg22- and pathogen-induced defense responses of g6pd6 mutants were complemented by a phosphomimetic, but not by a phosphonegative, version of the Thr residue of G6PD6 that is targeted by ASKα.

Overall, our data indicate a model in which ASKα contributes to PTI signaling and, thus, pathogen resistance (Fig. 4E). PAMP and DAMP perception activates ASKα, which in turn regulates diverse PTI signaling outputs. ASKα phosphorylates G6PD6 and, thereby, stimulates its activity to supply the demand for NADPH for defense reactions.

Remarkably, the regulatory function of ASKα and G6PD6 is not restricted to plant innate immunity but also is important for abiotic stress tolerance (Dal Santo et al., 2012). ASKα-G6PD6, therefore, might play a role in a general stress response pathway that is activated by different biotic and abiotic stresses. Yet, it is at first sight unanticipated that ASKα-G6PD6 are involved in ROS production after PAMP and DAMP perception but contribute to ROS detoxification under salt stress conditions (Dal Santo et al., 2012). During abiotic stress, excess levels of ROS can be generated and damage cells (Apel and Hirt, 2004). In plants exposed to high salinity, ASKα-mediated phosphorylation of G6PD6 was shown to contribute to ROS detoxification and the maintenance of cellular redox balance (Dal Santo et al., 2012). However, in addition to their damaging role in plants challenged by prolonged salt stress, ROS also have important signaling functions (Miller et al., 2010; Baxter et al., 2014) and RBOHD was shown to regulate signaling in response to salinity (Miller et al., 2009). Thus, it is an attractive hypothesis that ASKα-G6PD6 also might be involved in RBOH-dependent ROS production and signaling in salt-stressed plants and that the channeling of NADPH produced by the oxidative pentose phosphate pathway toward ROS production or ROS detoxification might be tissue and/or time dependent; this is an interesting subject for future research.

The dual role of G6PD in ROS scavenging and generation also is illustrated in certain patients with severe G6PD deficiency, the most common genetic disorder of metabolism in humans. While drug-stimulated oxidative stress induces the accumulation of excess levels of ROS in erythrocytes, causing hemolytic anemia in humans with G6PD deficiency, these patients show impaired ROS generation in leukocytes and increased susceptibility to infections (Roos et al., 1999; Chao et al., 2008). Similarly, the role of the multifunctional GSK3 in ROS metabolism appears complex (Salazar et al., 2006; Datta et al., 2007; Kim et al., 2010). Furthermore, in recent years, GSK3β has emerged as an important regulator of the animal immune system (Wang et al., 2014). GSK3β is involved in both innate and adaptive immunity by modulating cytokine production and the proliferation of T cells.

The ASKα phosphorylation site Thr-467 of G6PD6 is conserved in eukaryotes (Dal Santo et al., 2012), and interestingly, the suppression of human GSK3 or G6PD activity leads to the repression of proinflammatory and the enhancement of antiinflammatory cytokines in stimulated immune cells (Martin et al., 2005; Sanna et al., 2007), pointing toward the potential evolutionary conservation of the GSK3-G6PD module in innate immunity beyond the plant kingdom.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 plants were grown on soil at 21°C with a 12-h photoperiod or as seedlings on sterile one-half-strength Murashige and Skoog (MS) medium supplemented with vitamins and 1% (w/v) Suc (Duchefa) with a 16-h photoperiod for 4 to 5 d. For PAMP assays, seedlings were transferred to multiwell plates containing liquid one-half-strength MS medium and further cultivated in a 12-h photoperiod. Assays using soil-grown plants were performed 4 to 5 weeks after germination, prior to the reproductive transition, while assays using seedling cultures were performed 2 weeks after germination. The transfer DNA insertion mutants and overexpressor lines of ASKα (askα, ASKα OE4, and ASKα OE5), g6pd6, and askα/g6pd6 have been described (Dal Santo et al., 2012). g6pd6 plants expressing G6PD6-HA and its phosphomimetic (G6PD6-HA T/E) or phosphonegative (G6PD6-HA T/A) version have been established by stable transformation of g6pd6 with the respective G6PD6 versions cloned into pGWR8 (Rozhon et al., 2010). Site-directed mutagenesis was described (Dal Santo et al., 2012). askα plants expressing G6PD6-HA and the phosphomimetic (G6PD6-HA T/E) version were described by Dal Santo et al. (2012).

Chemicals and Peptides

flg22 (5′-QRLSTGSRINSAKDDAAGLQIA-3′) and pep1 (5′-ATKVKAKQRGKEKVSSGRPGQHN-3′) have been described previously (Felix et al., 1999; Huffaker et al., 2006). Peptides were synthesized in house, dissolved in water (stock solutions of 1 mm), and stored at −80°C. Chitin solution was always freshly prepared as a homogenous suspension in water (stock solution of 20 mg mL−1) using mortar and pestle. Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich or Duchefa.

PAMP Assays Using Seedling Cultures

Seedlings, grown for 4 d on one-half-strength MS agar plates followed by 8 to 10 d of growth on six-well plates in liquid one-half-strength MS medium, with 15 seedlings per well, were overlaid with the respective elicitor concentration diluted in liquid one-half-strength MS medium or one-half-strength MS medium alone as a mock control. For each treatment, triplicates of 15 seedlings were frozen in liquid nitrogen.

Protein Extraction, Immunoblot Analysis, and Immunokinase Assays

Proteins were extracted from frozen plant tissue as described previously (Dal Santo et al., 2012). For immunoblot analysis, proteins were separated by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with anti-HA (HA-probe Y-11:sc-805; Santa-Cruz), anti-actin (Actin MA1-744; Thermo Scientific), or anti-phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr-204; Cell Signaling Technologies) antibodies. Goat IRDye 800CW anti-mouse (#926-32210; LI-COR) or goat IRDye 800 CW anti-rabbit (#926-32211; LI-COR) antibodies were used as secondary antibodies. Signals were detected using the Odyssey Imagine System (LI-COR). Immunokinase assays were performed as described (Jonak et al., 2000) using anti-ASKα specific antibodies (Dal Santo et al., 2012) for immunoprecipitation of ASKα from 200 μg of protein extract, followed by the kinase reaction using MBP as a general substrate.

G6PD Activity Measurement

G6PD total activity was determined as described (Stanton et al., 1991) with some modifications. Five micrograms of total protein extract (1 μg μL−1) was analyzed in quadruplicate in a 100-μL total reaction volume on UV light-transparent 96-well plates (Corning). NADPH formation was recorded by absorbance measurement at 340 nm using a microplate reader spectrophotometer (Synergy 4 multimode plate reader; Biotek). In vivo G6PD activities were calculated in nmol NADPH min−1 μg−1 total protein extract.

Gene Expression Analysis

Total RNA was extracted using TRI Reagent (Sigma-Aldrich). RNA was quantified with a Nanodrop spectrophotometer (Peqlab). First-strand complementary DNA was synthesized from 2 μg of DNase-treated (RapidOut DNA Removal; Thermo Fisher Scientific) RNA using the iScript cDNA Synthesis Kit (Bio-Rad). Complementary DNA was used for real-time PCR using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on an iQ5 multicolor real-time PCR detection system (Bio-Rad). Relative gene expression values were determined by the comparative cycle threshold method. UBOX and EF1a were used as reference genes. Normalized gene expression was expressed relative to wild-type controls. Primers are listed in Supplemental Table S2.

Growth Inhibition Assays

Growth inhibition of seedlings was performed as described (Gómez-Gómez et al., 1999). Briefly, seedlings were germinated on one-half-strength MS plates for 4 d and then transferred to 48-well plates (Cellstar; Greiner), with a single seedling per well, prefilled with 500 μL of liquid one-half-strength MS medium as the nontreated control or supplemented with the respective PAMP at the indicated final concentration (treated). After 10 to 12 d of growth at 21°C in a 12-h photoperiod, growth inhibition was analyzed by determining the seedling fresh weight. Results were then expressed as the nontreated-treated ratio.

Measurement of ROS Generation

ROS detection was monitored by a luminol-based assay (Keppler et al., 1989). A minimum of 12 leaf discs (diameter = 4 mm) of four plants per genotype were cut with a cork borer from fully expanded leaves of 5-week-old plants and a single leaf disc per well was incubated in 150 μL of sterile water on opaque white 96-well plates (Thermo Fisher) overnight. Water was then replaced with a solution containing 17 µg mL−1 of the luminol derivative L-012 (Wako Chemicals), 10 µg mL−1 horseradish peroxidase type VI (Sigma-Aldrich), and the respective elicitor concentration or water as a mock control. Luminescence, expressed as relative light units, was recorded over 40 min using a Synergy 4 multimode plate reader (Biotek). Total relative light units were calculated as the integral of the individual relative light unit curves by the plate reader software (Biotek Gen5).

Callose Deposition Analysis

Callose staining was performed as described previously (Gómez-Gómez et al., 1999). Briefly, fully expanded leaves of soil-grown plants were syringe infiltrated with water as a mock control or with 1 μm flg22. After 16 h, discs of infiltrated leaves were harvested and cleared with 70% ethanol for 1 h followed by 100% ethanol overnight, rehydrated with 50% ethanol and 67 mm K2HPO4, and stained with 0.01% Aniline Blue. Stained material was mounted in 50% glycerol/67 mm K2HPO4 and examined using a UV epifluorescence microscope with a 4′,6-diamidino-2-phenylindole fluorescence filter set. Callose quantification was performed on microscope images with the Fiji (ImageJ; Schindelin et al., 2012) software using the Analyze Particles function after background subtraction and threshold adjustment (Huang et al., 2014).

Pathogen Assays

The bacterial strain used in this study was Pseudomonas syringae pv tomato DC3000. Syringe and spray inoculations were performed as described (Katagiri et al., 2002). Briefly, for bacterial growth assays, 5-week-old soil-grown plants were spray inoculated with 1 × 108 cfu mL−1 Pto DC3000 with 0.04% (v/v) Silwet L-77 (Lehle Seeds), and the bacterial titer was determined 2 h (0 d post inoculation) and 3 d post inoculation by serial dilutions. For this, discs from three different leaves per plant and four plants per genotype were ground in 10 mm MgCl2. After grinding of the tissue, samples were thoroughly vortexed and serially diluted at 1:10 dilution. Samples were then plated in triplicate on solid King’s B medium supplemented with the appropriate antibiotics and incubated at 28°C for 2 d, after which the cfu were counted.

For immunokinase and gene expression analyses, bacterial suspensions (1 × 106 cfu mL−1) were pressure infiltrated into leaves with a needleless syringe, and sample material was harvested and frozen in liquid nitrogen at the indicated time points.

Statistical Analysis

QtiPlot and Microsoft Excel software were used for statistical analysis. Statistically significant groups were determined by Student’s t test or by ANOVA, followed by Tukey’s honestly significant difference posthoc test.

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_171_2_1366__index.html (1.2KB, html)

Acknowledgments

We thank A. Auer and B. Dekrout for technical assistance and Y. Belkhadir for discussions and critical comments on the article.

Glossary

PAMP

pathogen-associated molecular pattern

DAMP

damage-associated molecular pattern

PRR

pattern recognition receptor

PTI

pattern-triggered immunity

ROS

reactive oxygen species

BR

brassinosteroid

Pto

Pseudomonas syringae pv tomato

MS

Murashige and Skoog

cfu

colony-forming units

Footnotes

1

This work was supported by the Austrian Science Foundation (grant no. P 24992–B21).

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