*2.7. SA-Mediated Defense Genes are Poised for Enhanced Activation through Chromatin Remodeling in pyr1 Plants*

We then asked whether other markers diagnostic of an immune status were also activated in *pyr1* plants. The expression of the extracellular subtilase *SBT3.3* gene has been recently described to be a switch for poising SA-related gene expression and immune priming [62]. Moreover, constitutive *SBT3.3* expression, MPK activation, and readied SA-related genes convey in plants defective in the RNA-directed DNA methylation (RdDM) pathway, which negatively regulates immune priming [54]. *Plants* 

Consequently, the expression level of genes encoding SBT3.3 and either of the two subunits of RNA Pol V (i.e., NRPD2 and NRPE1) controlling RdDM were determined by RT-qPCR. Figure 7A shows the constitutive upregulation of *SBT3.3* and concurrent downregulation of *NRPD2* in *pyr1* plants compared to Col-0, congruent with the activation of immune priming in the mutant. The downregulation was specific for *NRPD2*, encoding the second large subunit of Pol V, because the expression of the gene encoding the large NRPE1 subunit exhibited a minimal variation in *pyr1* plants (Figure 7A). [54]. Consequently, the expression level of genes encoding SBT3.3 and either of the two subunits of RNA Pol V (i.e., NRPD2 and NRPE1) controlling RdDM were determined by RT-qPCR. Figure 7A shows the constitutive upregulation of *SBT3.3* and concurrent downregulation of *NRPD2* in *pyr1* plants compared to Col-0, congruent with the activation of immune priming in the mutant. The downregulation was specific for *NRPD2*, encoding the second large subunit of Pol V, because the expression of the gene encoding the large NRPE1 subunit exhibited a minimal variation in *pyr1* plants (Figure 7A).

RNA-directed DNA methylation (RdDM) pathway, which negatively regulates immune priming

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 12 of 22

*2.7. SA-Mediated Defense Genes are Poised for Enhanced Activation through Chromatin Remodeling in pyr1* 

We then asked whether other markers diagnostic of an immune status were also activated in *pyr1* plants. The expression of the extracellular subtilase *SBT3.3* gene has been recently described to

**Figure 7.** Loss of PYR1 function provokes the setting of hallmarks characteristic of primed immunity. (**A**) Comparative RT-qPCR of *SBT3.3*, *NRPD2*, and *NRPE1* transcript levels between healthy Col-0 and *pyr1-2* plants. The expression was normalized to the constitutive *ACT2*/*8* gene and then to the expression in Col-0 plants. (**B**) Chromatin immunoprecipitation (ChIP) and comparison between Col-0 and *pyr1-2* plants of the level of histone H3 Lys4 trimethylation (H3K4me3) and histone H3 Lys9 acetylation (H3K9ac) on the *SBT3.3*, *PR-1*, *WRKY6*, and *WRKY53* gene promoters as present in leaf samples. The setting of histone marks in *ACTIN2* was used as an internal control. Data are standardized for Col-0 histone modification levels. Data represent the mean ± SD; *n* = 3 biological replicates. An ANOVA was conducted to assess significant differences between MPKs activation and PR1 accumulation (*p* < 0.05); the asterisks \* above the bars indicate statistically significant differences regarding Col-0 plants.

In plants defective in RdDM-mediated epigenetic control, immune priming is activated concurrently with chromatin histone activation marks being enriched in SA-related genes, including the *SBT3.3* gene itself [54,62]. Thus, we hypothesized that SA-related defense genes and *SBT3.3* in *pyr1-2* plants are poised for enhanced expression by differential histone modification. We used chromatin immunoprecipitation (ChIP) to analyze H3K4me3 and H3K9ac activation marks on the *SBT3.3* and *PR-1* gene promoter regions in *pyr1* and Col-0 plants. We also examined the genes encoding WRKY6 and WRKY53, transcriptional regulators of SA-defense genes. Figure 7B shows that H3K4me3 marks in the *SBT3.3* promoter region notably increased in *pyr1* plants compared to Col-0 plants, while H3K9ac marks remained invariant (Figure 7B), supporting previous descriptions of plants constitutively expressing primed immunity [54,62,63]. On the *PR1* promoter, both H3K4me3 and H3K9ac activation marks increased three- and two-fold, respectively, in *pyr1* plants compared to Col-0 (Figure 7B). Likewise, histone activation marks also moderately increased in the *WRKY6* and *WRKY53* promoters of *pyr1* plants compared to Col-0 plants (Figure 7B). The setting of histone marks in *pyr1* plants remained unchanged in the *ACTIN2* gene promoter, which was used as the control (Figure 7B). Therefore, chromatin activation marks proliferated in the promoter regions of the priming regulatory gene *SBT3.3* and the SA-responsive genes in *pyr1* plants and would explain why the PR-1 protein showed accelerated and enhanced accumulation in *pyr1* plants following pathogen inoculation (Figure 6). Our results indicated that ABA and its PYR1-mediated perception represented novel integral components of a signaling process, repressing SA-mediated immunity.

#### *2.8. NahG Plants Abrogate the Altered Disease Resistance Response of pyr1 Plants*

To evaluate the role of SA for *pyr1*-altered resistance, we generated a *pyr1;NahG* double mutant. In plants carrying the *NahG* transgene, salicylate hydroxylase depletes the plant of this defense hormone [64]. Compared to Col-0, *NahG* plants showed an anticipated increase in susceptibility to *P. syringae* DC3000 due to SA depletion (Figure 8A). Interestingly, in *pyr1;NahG* plants, the *pyr1*-mediated enhanced resistance was abrogated, and instead enhanced susceptibility to *P. syringae* DC3000 emerged (Figure 8A). Moreover, when assayed against the fungal pathogen *P. cucumerina*, *NahG* plants behaved like Col-0, both showing the same degree of susceptibility (Figure 8B), suggesting normal metabolic levels of SA played no major role in the resistance towards this pathogen. Surprisingly, in *pyr1;NahG* plants, the *pyr1*-mediated-enhanced susceptibility to *P. cucumerina* was abrogated (Figure 8B), with *pyr1;NahG* plants to be behaving as Col-0 or *NahG* plants. This suggested that the PYR1-mediated perception of ABA negatively regulated the SA pathway. When this negative regulation failed, such as in *pyr1* plants, the SA levels increased, and the resistance to *P. syringae* DC3000 was activated. As a trade-off effect, the elevated SA levels presumably interfered with JA or ET signaling pathways required for mounting a resistance response to fungal pathogens.

#### *2.9. pyr1-Mediated Enhanced SA Content Blocks ET Perception*

The SA and JA signal pathways are under an antagonistic equilibrium. Therefore, we wondered if the enhanced SA levels of *pyr1* plants could be affecting JA signaling in this mutant. We studied *pyr1* plants for altered responses to JA using the widely applied root growth inhibition assay. In the absence of JA, primary root length of *pyr1* seedlings was comparable to that of Col-0 plants (Supplemental Figure S2), and in the presence of JA, root growth reduction in the mutant was also similar to that observed in Col-0 plants (Supplemental Figure S2), providing evidence that JA perception was not impaired in the mutant. In addition, comparison of the expression level of different JA-responsive genes at different times following *P. cucumerina* inoculation in Col-0 and *pyr1* plants revealed that JA signaling appeared to be not affected in the mutant (Supplemental Figure S3). Instead, for some of the genes analyzed, a higher induction was recorded in *pyr1* plants. Therefore, JA signaling was not compromised in *pyr1* plants.

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 14 of 22

**Figure 8.** Effect of *NahG* on disease resistance and insensitivity to ACC of *pyr1* plants and seedlings. (**A**,**B**) Comparative disease resistance towards *P.s.* DC3000 and *P. cucumerina* among Col-0, *pyr1-2*, *NahG,* and *pyr1-2NahG* plants. Growth of *P. syringae* DC3000 was measured at 3 d.p.i. Error bars represent standard deviation (*n* = 12). For *P. cucumerina*, data points represent the average lesion size ± SE of measurements. An ANOVA was conducted to assess significant differences in disease symptoms (*p* < 0.05); the letters above the bars indicate different homogeneous groups with statistically significant differences. (**C**) Apical hook region of the indicated seedlings germinated and grown on MS/2 in the dark for 4 d in the presence of the indicated concentration of ACC. (**D**) Hypocotyl length of seedlings germinated and grown in the dark for 4 d on MS/2 medium supplemented with the denoted concentrations of ACC. Error bars represent standard deviation (*n* = 50). An ANOVA was conducted, and no significant differences were observed in hypocotyl length (*p* < 0.05). (**E**) Apical hook region of Col-0 seedlings germinated and grown on MS/2 in the dark for 4 d in the presence of the indicated concentration of ACC and SA. **Figure 8.** Effect of *NahG* on disease resistance and insensitivity to ACC of *pyr1* plants and seedlings. (**A**,**B**) Comparative disease resistance towards *P.s.* DC3000 and *P. cucumerina* among Col-0, *pyr1-2*, *NahG*, and *pyr1-2NahG* plants. Growth of *P. syringae* DC3000 was measured at 3 d.p.i. Error bars represent standard deviation (*n* = 12). For *P. cucumerina*, data points represent the average lesion size ± SE of measurements. An ANOVA was conducted to assess significant differences in disease symptoms (*p* < 0.05); the letters above the bars indicate different homogeneous groups with statistically significant differences. (**C**) Apical hook region of the indicated seedlings germinated and grown on MS/2 in the dark for 4 d in the presence of the indicated concentration of ACC. (**D**) Hypocotyl length of seedlings germinated and grown in the dark for 4 d on MS/2 medium supplemented with the denoted concentrations of ACC. Error bars represent standard deviation (*n* = 50). An ANOVA was conducted, and no significant differences were observed in hypocotyl length (*p* < 0.05). (**E**) Apical hook region of Col-0 seedlings germinated and grown on MS/2 in the dark for 4 d in the presence of the indicated concentration of ACC and SA.

We next asked whether ET signaling, which is also pivotal for resistance to fungal pathogens [10,12,13], could be the one impaired in *pyr1* plants due to the elevated levels of SA. This hypothesis gained even more relevance in view of the recently described mechanism explaining the antagonism between SA and ET in the suppression of apical hook formation and early seedling establishment via NPR1-mediated

repression of EIN3 and EIL1 [16]. We, therefore, assayed Col-0 and *pyr1* seedlings, grown in the dark in the presence or absence of a low concentration of the ethylene precursor ACC (5 µM), for the induction of the ET-mediated triple response. The triple response in Arabidopsis consists of shortening and thickening of hypocotyls and roots and exaggeration of the curvature of apical hooks. Compared to Col-0, the assay revealed that *pyr1* seedlings showed no curvature of the apical hook (Figure 8C) and also showed less shortened hypocotyls (Figure 8D) when grown in the presence of ACC. Thus, the *pyr1* mutant was impaired in ET perception. The enhanced SA content in *pyr1* seedlings was the causal link mediating insensitivity to ET since in *pyr1;NahG* double mutant, normal sensitivity to ET was re-established (Figure 8C,D). Moreover, when Col-0 seedlings were assayed in the presence of high amounts of SA (100 µM), the ACC-induced triple response was abrogated (Figure 8E), further sustaining that the elevated levels of SA in *pyr1* plants blunted ET perception. Thus, our results suggested that perception of ABA through PYR1 acted primarily as a module negatively controlling the SA pathway. When ABA/PYR1 failed, the SA pathway was released, and the resistance to *P. syringae* DC3000 was activated. As a trade-off effect, the enhanced accumulation of the SA pathway blocked the ET pathway, the later required for resistance to fungal necrotrophic pathogens.

#### **3. Discussion**

Despite the demonstrated role of ABA on the final outcome of immune responses, the specific components of the ABA signaling apparatus and the specific mechanisms that exploit ABA to positively and negatively influence immune responses to specific plant-pathogen interactions have remained largely unknown. Here, we showed that SnRK2s kinases were actively engaged in activating resistance towards *P. cucumerina,* whereas the loss-of-function of any of the three individual SnRK2s compromised this resistance. Furthermore, we demonstrated that PYR1 was pivotal and played a positive role in disease resistance to *P. cucumerina* since overexpression of PYR1 (i.e., *PYR1-OE* transgenic line) conferred significantly enhanced resistance. Conversely, in PYR1 loss-of-function mutants, the resistance was compromised. Therefore, the PYR1 receptor had functional specificity in perceiving ABA produced in response to fungal infection to activate plant immunity. This study provided novel information about a specific ABA receptor-mediating specific plant immune responses and pinpointed ABA-activated SnRK2s as cardinal components for plant resistance. This information helps construct a functional classification scheme of the different members of the PYR/PYL receptor family with respect to their downstream signaling pathways in a true biological context. Thus, specific non-redundant roles for PYR1 and PYL8 have been reported in plant immunity (this work) and root ABA sensitivity [50], respectively. An explanation for the specific role of PYR1 in pathogen response could be the selective and highly localized pathogen-induced expression of *PYR1* in vascular bundles (Figure 3A). This expression pattern mirrors the expression of genes encoding ABA-biosynthetic enzymes [65–68]. Therefore, the synthesis of ABA and the pathogen-induced expression of *PYR1* spatially concur in the vasculature, supporting the hypothesis that vascular tissues function as an integrating node, triggering stress signaling that sets in motion the local and systemic immune responses in the plant [67,69,70].

This study showed that resistance to *A. brassicicola* was also dependent on ABA and PYR1, reinforcing the importance of this signal pathway for activating immunity against necrotrophs. This further reconciled with results shown above and also with previous studies showing that ABA promotes enhanced resistance to the necrotroph *P. cucumerina* [23,25,71]. Moreover, when a fungal necrotroph is a shift to a biotrophic lifestyle by changing the inoculation method and also the developmental stage of the plant [72], as reported for *P. cucumerina* [22], then ABA exerts an opposite effect, and the resistance to this same pathogen is suppressed. This contradictory role of ABA at controlling the disease resistance has also been observed for biotrophic pathogens (e.g., *P. syringae* DC3000), with resistance appearing negatively regulated by ABA, whereas resistance is enhanced in ABA-deficient mutants (Mohr & Cahill, 2007; Jensen et al., 2008; Fan et al., 2009; Verhage et al., 2010) [4,21,26,27]. In fact, we showed that the growth of *P. syringae* DC3000 was severely restricted in *pyr1* plants, as documented for *aba2 aao3* plants or the ABA-insensitive *abi1-1* and *abi2-1* mutants [8,28]. These results demonstrated the Janus functions of PYR1 in disease resistance, mediating repression of immunity against biotrophic pathogens, whereas activation against necrotrophs. Consequently, PYR1 may regulate which of these two plant immune programs prevails. This hypothesis supports previous observations of ABA as a hormone that interacts antagonistically or synergistically with the SA-JA-ET backbone of the plant immune signaling network, redirecting defense outputs [4,28,73–75]. Yet, how does the ABA/PYR1 module interfere with immunity to drive simultaneously the repression and activation of the SA and JA/ET defense pathways, respectively? Hormone cross-talk allows different hormone signaling pathways to act antagonistically or synergistically, providing the powerful regulatory potential to flexibly tailor the plant's adaptive response to a range of environmental cues [4]. Our results showed basal activation of the SA-dependent pathway in *pyr1* mutants, and that *pyr1* was insensitive to the damping effect of ABA on SA signaling. This finding supported previous work demonstrating the negative role of ABA on disease resistance to biotrophs, and that *P. syringae*-induced ABA levels in Col-0 suppress SA biosynthesis and action, enhancing susceptibility to this pathogen [21,28,73,75,76]. Interestingly, our finding that JA perception remained intact in *pyr1* plant but ET perception became compromised added a degree of specificity for the understanding of the disease resistance phenotype of the mutant. The observation that in *pyr1;NahG* plants, the *pyr1*-mediated ET-insensitivity was reversed, and that SA *per se* could block ET perception in Col-0 plants (Figure 8 and Huang et al., 2020), pointed towards SA-mediated repression of ET signaling modulated by ABA and PYR1 during pathogenesis. The positive effect of ABA at promoting ET-dependent resistance to fungal pathogens may be indirect: perception of pathogenic ABA by PYR1 dampens SA signaling, which, in turn, stops ET pathway repression by SA. This ABA and PYR1-modulated cross-talk regulation of SA and ET pathways may provide the plant with a powerful regulatory potential to boost its defenses according to the lifestyle of the attacker. This phenomenon may also explain why disease-promoting biotrophic pathogens (e.g., *P. syringae* DC3000) have developed strategies to alter the host ABA physiology as part of the infection strategy [28,76].

How does then ABA/PYR1-mediated signaling control SA-mediated defenses? *pyr1* plants bear constitutive activation of *ICS* expression and moderate enhanced level of SA. Besides, *pyr1* plants carry the hallmarks of immune priming, including (1) basal activation of MPKs; (2) repression of *NRPD2* and, therefore, the RdDM mechanisms that negatively control the onset of defense; (3) activation of the SBT3.3 subtilase; and (4) readying of SA-related genes for enhanced expression by pertinent chromatin modifications. Therefore, *pyr1* plants mirror the phenotypes of RdDM defective mutants, which exhibit simultaneous enhanced susceptibility and resistance to necrotrophs and biotrophs, respectively [54], supporting SA signaling activation in *pyr1* plants. The fact that immune priming and SA-mediated resistance are negatively regulated by the RdDM, and that in *pyr1* plants, the ABA repression of SA pathway is relieved, both observations unveil the importance of ABA/PYR1 as new element participating in an epigenetic mechanism of control of gene expression in plant immunity.

### **4. Materials and Methods**

#### *4.1. Plants Growth Conditions*

Arabidopsis thaliana plants were grown in a growth chamber (19–23 ◦C, 85% relative humidity, 100 mEm−<sup>2</sup> s <sup>−</sup><sup>1</sup> fluorescent illumination) on a 10-h-light and 14-hr-dark cycle. All mutants and transgenic plants are in Col-0 background; SnRK2.6-HA/OE and SnRK2.2-HA/OE were previously described [77,78]; snrk2.2 snrk2.3 and snrk2.6 were described in [44,45,51]; the triple mutants pp2ca1-1 hab1-1 abi2-2 and abi2-2 hab1-1 abi1-2 were described in [22]; ocp3-1 and aba2-1 mutants described in [25,53], and the triple pyl4 pyl5 pyl8, pyr1 pyl4 pyl8, and pyr1 pyl4 pyl5 mutants, along with the quadruple pyr1 pyl1 pyl2 pyl4 and single pyl1, pyl4, pyr1-1, pyr1-2 and pyr1-8 mutants were described in [41,45]. Transgenic lines carrying pPYR1::GUS, pPYL1::GUS, pPYL4::GUS were described previously [45]. NahG plants were described previously [64].

#### *4.2. Gene Expression Analysis*

Total RNA was extracted from plant tissues using TRIzol (Invitrogen, Waltham, MA, USA) and purified by lithium chloride precipitation. Reverse transcription was done using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas Life Sciences, Waltham, MA, USA). Quantitative PCR (qPCR) was performed using an ABI PRISM 7000 sequence detection system and SYBR-Green (Perkin-Elmer Applied Biosystems, Foster, CA, USA). *ACTIN2* and *ACTIN8* were the reference genes. The primers used for RT-qPCR experiments are provided in Table S1. RT-qPCR analyses were performed at least three times using sets of cDNA samples from independent experiments.

#### *4.3. Immunoprecipitation of HA–SnRKs and In Vitro Phosphorylation*

HA-tagged SnRK2.2 and SnRK2.6 were immunoprecipitated and used for in vitro kinase assay, as described previously [78].

#### *4.4. Chromatin Immunoprecipitation*

Chromatin isolation and immunoprecipitation were performed, as described [54,62]. Chip samples, derived from three biological replicates, were amplified in triplicate and measured by quantitative PCR using primers for *SBT3.3*, *PR-1*, *WRKY6*, *WRKY53*, and *Actin2*, as reported [54,62]. All ChIP experiments were performed in three independent biological replicates. The antibodies used for the immunoprecipitation of modified histones from 2 g of leaf material were antiH3K4m3 (#07-473 Millipore) and antiH3K9ac (#07-352 Millipore).

#### *4.5. Western Blot*

Protein crude extracts were prepared by homogenizing ground frozen leaf material with Tris-buffered saline (TBS) supplemented with 5 mM DTT, protease inhibitor cocktail (Sigma-Aldrich), and protein phosphatase inhibitors (PhosStop, Roche). Protein concentration was measured using Bradford reagent; 25 µg of total protein was separated by SDS-PAGE (12% acrylamide *w*/*v*) and transferred to nitrocellulose filters. The filter was stained with Ponceau-S after transfer and used as a loading control.

#### *4.6. Pathogen Assays*

*Pseudomonas syringae* DC3000 was grown for two days, and a culture with O.D. 2 <sup>×</sup> <sup>10</sup>−<sup>4</sup> was used to infect 5-week-old *Arabidopsis* leaves by infiltration, and the bacterial growth was determined following [54,62]. Twelve samples were used for each data point and represented as the mean ± SD of log c.f.u./cm<sup>2</sup> . For *Plectosphaerella cucumerina* and *Alternaria brassicicola* bioassays, 5-week-old plants were inoculated, as described [23,24], with a suspension of fungal spores of 2.5 <sup>×</sup> <sup>10</sup><sup>4</sup> , 5 <sup>×</sup> <sup>10</sup><sup>6</sup> , and 5 <sup>×</sup> <sup>10</sup><sup>6</sup> spores/mL, respectively. The challenged plants were maintained at 100% relative humidity. Disease symptoms were evaluated by determining the lesion diameter of at least 100 lesions (25 plants per genotype and four leaves per plant) at 3, 12, and 8 days after inoculation with *P. cucumerina* and *A. brassicicola*, respectively. For pathogen-induced callose deposition analyses, infected leaves were stained at 24, 48, and 72 h.p.i. with aniline blue, and callose deposition quantifications were performed, as described by [53].

#### *4.7. Determination of Plant Hormones and Metabolites*

ABA, JA, SA levels were determined, as described previously [25,71].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/21/16/5852/s1, Figure S1: Disease resistance towards *Alternaria brassicicola* in Col-0, *aba2-1*, *pyr1*, *pyl2* and *pyl4* plants and comparison of Col-0 and *pyr1-2* plants upon inoculation with *Altenaria brassicicola*. Figure S2: Response of Col-0 and *pyr1-2* seedlings to JA. Figure S3: Comparative RT-qPCR analysis for the expression of different JA-responsive and biosynthesis genes in either mocked or *P. cucumerina*-inoculated Col-0 and *pyr1* plants. Table S1: primer sequences.

**Author Contributions:** M.G.-G. and P.L.R. performed SnRKs kinase assays and provided genetic resources. J.G.-A. and B.G. performed the rest of the experiment shown in the manuscript. P.V. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was founded by the Spanish AEI agency by grant BIO2017-82503-R to P.L.R. and by grant RTI2018-098501-B-I00 to P.V.

**Acknowledgments:** We acknowledge V. Flors for the technical assistance in the analysis of plant metabolites and hormones.

**Conflicts of Interest:** Authors declare no competing financial interest.
