**1. Introduction**

Pathogen recognition triggers the altered accumulation of three major defense hormones: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). SA is essential for establishing resistance to many virulent biotrophic pathogens, especially as a component of systemic acquired resistance (SAR) [1,2], while JA and ET tend to be associated with resistance to fungal necrotrophic pathogens [3,4]. While JA and ET interact synergistically to activate certain disease responses, the JA and ET pathways act at least independently or even antagonistically with respect to the SA-dependent pathway [4,5]. Antagonistic interactions between SA and JA hormone signaling networks have been characterized [6–8]. JA levels decline soon after SA begins to accumulate [9]; this, therefore, suggests that, in response to a pathogen that can induce synthesis of both SA and JA, cross-talk is used by the plant to adjust the response in favor of the more effective pathway (i.e., the SA-mediated pathway). Similarly, SA acts antagonistically with ET [10–13], and their biosynthesis pathways can be mutually repressed [14,15]. More recently, Huang et al. [16] revealed a mechanism by which SA antagonizes ET signaling: the direct interaction of NPR1 (the core component of SA signaling) with EIN3 (the transcription factor mediating ET-responses) blocks transcription of EIN3-induced genes, and this interaction is further enhanced by SA. Therefore, tradeoffs between plant defenses against pathogens with different lifestyles must be

strictly regulated [4,17], implying the fine-tuned deployment of conserved defense signals in different plant-pathogen interactions.

ABA is another major phytohormone involved in the regulation of a great variety of abiotic stress responses in plants. In addition, ABA assists in controlling many developmental and growth characteristics of plants, including seed germination and dormancy, leaf abscission, closure of stomata, or inhibition of fruit ripening [18]. ABA also controls the responses of plants to biotic stresses caused by a broad range of plant pathogens [19–22]. However, the ABA effect varies in different pathosystems, being the outcome influenced by the infection biology. ABA biosynthesis is required for effective disease resistance against necrotrophic fungal pathogens [23–25], whereas ABA has been shown to be involved in conferring susceptibility against bacterial diseases, with ABA-deficient mutants showing resistance enhancement [21,26,27]. In fact, some bacteria have acquired new virulence strategies for exploiting their host through the secretion of type III virulence effectors that promote enhancement of ABA levels in the infected plant [28–30]. Therefore, endogenous ABA synergizes with JA and exhibits a complex antagonistic relationship with SA during disease development [6,7,29]. Likewise, antagonistic interactions between components of the ABA and ET signaling pathways seem to modulate gene expression in response to biotic and abiotic stress (Fujimoto et al., 2000; Chen et al., 2002; Anderson et al., 2004; Yang et al., 2005; Broekaert et al., 2006) [5,31–34], but it remains unknown whether a convergent point exists between these two signaling pathways or whether they operate in parallel. Despite all these evidences, the specific components of the ABA signaling apparatus, which exploit the positive and negative responses of ABA during immune responses, remain unknown. Therefore, understanding the regulatory system of ABA-mediated responses to pathogens is critical for improving agricultural issues related to disease resistance. In contrast, specific components of ABA perception have been recently identified for stomatal closure signal integration [35]. Thus, PYL2 is sufficient for guard-cell ABA-induced response, and PYL4/5 are essential receptors for a guard-cell response to CO<sup>2</sup> [35].

Three major protein families form the core ABA signaling pathway; (i) the soluble ABA receptors, which are 14 members of pyrabactin resistance 1 (PYR1) and PYR1-like (PYL) proteins, also known as regulatory component of ABA receptors (RCAR) family and collectively referred to as PYR/PYL/RCAR, (ii) group A of type 2C protein phosphatases (PP2Cs), and (iii) SNF1-related protein kinases (SnRKs) subfamily 2 (SnRK2s), namely SnRK2.2, 2.3 and 2.6 (Cutler et al., 2010; Hubbard et al., 2010; Klingler et al., 2010; Raghavendra et al., 2010) [18,36–38]. In the absence of ABA, PP2Cs dephosphorylate and inactivate SnRK2s, repressing ABA-dependent responses [39,40]. When ABA concentration increases in response to stress conditions or developmental cues, ABA binds to receptors of the PYR/PYL/RCAR family, which leads to the formation of ternary complexes with PP2Cs, thereby inactivating them [41–43]. This results in the activation of SnRK2s, which subsequently phosphorylate a myriad of substrate proteins [44].

The PYR/PYL/RCAR ABA receptor family is unusually large, comprising 14 members in Arabidopsis and even more in crops, such as tomato, maize, or soybean (Gonzalez-Guzman et al., 2012; Gonzalez-Guzman et al., 2014; Helander et al., 2016) [45–47]. However, the biological roles of the individual PYR/PYL/RCAR members are still being established, which is complicated by functional redundancy. At least 13 PYR/PYL/RCAR members are able to perceive ABA, and the generation of quadruple, pentuple, and sextuple mutants is required to obtain robust ABA-insensitive phenotypes [41,43,45]. Moreover, the analysis of combined *pyr*/*pyl* mutants shows quantitative regulation of both stomatal aperture and transcriptional response to ABA [45]. Inactivation of six highly transcribed members, *PYR1*, *PYL1*, *PYL2*, *PYL4*, *PYL5*, and *PYL8*, generates a mutant that is practically blind to ABA in the classical assays that measure ABA sensitivity [45]. However, in spite of the receptor gene expression patterns and biochemical analyses of different receptor-phosphatase complexes suggesting that the function of ABA receptors is not completely redundant [45,48,49], only the single *pyl8* mutant has been reported to show a non-redundant role in root sensitivity to ABA [50]. In contrast, *pyr1* shows wild-type sensitivity to ABA and only shows a conditional

phenotype -pyrabactin resistance in germination assays in medium supplemented with the ABA agonist pyrabactin [43]. In eukaryotes, functional diversification follows the evolutionary expansion of a gene family. Identification of specific roles for members of a multigene family is usually limited by laboratory conditions, whereas the plethora of conditions found in complex biological contexts offers chances to identify specific roles. Here, we were able to unveil a non-redundant role in plant immunity for PYR1, one of the 13 members of the multigene ABA receptor family, and revealed that the PYR1 receptor is pivotal in modulating the cross-talk between the SA and ET signaling pathways during the defense.

#### **2. Results**

#### *2.1. The SnRK2s Protein Kinases are Engaged in Disease Resistance to Fungal Infection*

Liquid chromatography-mass spectrometry (LC-MS) showed marked accumulation of ABA in full expanded leaves of Arabidopsis plants at 72 h after drop inoculation with a spore suspension of the fungal necrotroph *Plectosphaerella cucumerina* (Figure 1A). ABA enhancement supported the upregulation of *ABI4* gene expression, an ABA-responsive gene encoding a transcription factor [23] (Figure 1B). Therefore, ABA biosynthesis and signaling were triggered by *P. cucumerina* infection. The ABA-mediated activation of three monomeric SnRK2s (i.e., SnRK2.2, −2.3, and −2.6) is central to ABA signaling [51], so we investigated whether SnRK2s were engaged in the defense responses to this pathogen. Transgenic lines overexpressing HA-tagged SnRK2.6 (SnRK2.6-HA/OE) and SnRK2.2 (SnRK2.2-HA/OE) were inoculated with *P. cucumerina* or mock-treated, and leaf samples were collected at 0, 24, and 48 h post-inoculation (h.p.i.). Immunoprecipitation of SnRK2.2-HA and SnRK2.6-HA and the subsequent kinase assay of the immunoprecipitate were performed by determining the incorporation of <sup>32</sup>P to purified ABF2 protein fragment substrate (amino acids Gly-73 to Gln-119) [52] in gel-kinase assays. Results revealed two- and three-fold enhancement for SnRK2.6 and SnRK2.2 kinase activity, respectively, following fungal inoculation (Figure 1C,D). For both kinases, enhanced activity occurred at 24 h.p.i., and the activation was sustained at 48 h.p.i. Therefore, ABA-activated SnRK2s were actively engaged in response to this fungal pathogen.

We then investigated whether gain-of-function or loss-of-function in SnRK2s altered disease resistance to *P. cucumerina*. Symptoms of the fungal disease appear in the form of necrotic lesions, which are measured to quantify the degree of plant susceptibility [25,53,54]. Inoculation of transgenic plants individually overexpressing (OE) SnRK2.2, −2.3, and −2.6 revealed no significant variation in disease susceptibility towards *P. cucumerina* when compared to Col-0 plants (Figure 1E); thus, either endogenous SnRK2s levels are sufficient to achieve pathogen-triggered ABA signaling or overexpression of SnRK2s additionally requires increased ABA levels to enhance their activity. Although functional redundancy between SnRK2.2 and SnRK2.3 exists, functional segregation between SnRK2.6 and SnRK2.2/2.3 has been described [52]. Therefore, we inoculated an *snrk2.2*/*2.3* double mutant and the single *snrk2.6* mutant with *P. cucumerina* and recorded disease resistance. The triple *snrk2.2*/*2.3*/*2.6* mutant, which is drastically affected in plant growth [51], was not compatible with the pathogenic assay and was, therefore, not used in the present study. Figure 1F–G show that *snrk2.2*/*2.3* and *snrk2.6* plant resistance to *P. cucumerina* was severely compromised. Moreover, an ABA deficient mutant (i.e., *aba2*) was similarly affected in disease resistance to this pathogen (Figure 2A). In summary, our results indicated that pathogen-induced ABA accumulation and concurrent activation of SnRK2s positively regulated disease resistance to *P. cucumerina*.

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 4 of 22

**Figure 1.** Participation of SnRK2s kinases in the response of Arabidopsis plants to infection by the fungal pathogen *P. cucumerina*. (**A**) ABA accumulation determined in mock and *P*. *cucumerina*-infected Col-0 plants. (**B**) RT-qPCR of *ABI4* in mock and in *P. cucumerina*-infected Col-0. (**C**,**D**) *P. cucumerina*mediated activation of SnRK2.6 (**C**) and SnRK2.2 (**D**). Transgenic Arabidopsis plants expressing HAtagged versions of the kinases were inoculated with *P. cucumerina*, or were mocked, and leaf samples were taken at 0, 24, and 48 h.p.i., and the protein extracts were immunoprecipitated with anti-HA antibodies. The immunoprecipitates were incubated with a His-ABF2 fragment (Gly73 to Gln 119; ΔABF2) in the presence of [γ-32P]ATP, and the proteins were resolved by SDS-PAGE. Bands corresponding to ΔABF2 fragments and to SnRK2.6 and SnRK2.2 kinases are indicated. Radioactivities of ΔABF2 fragment bands were measured with a phosphoimager, and the values were plotted on the graphs shown at the right of the figures. Error bars indicate S.E.M.; *n* = 3. (**E**) Disease resistance towards *P. cucumerina* of transgenic plants overexpressing SnRK2.6, SnRK2.2, and SnRK2.3 in comparison to Col-0. (**F**) Disease resistance towards *P. cucumerina* in the double *snrk2.2 snrk2.3* mutant and in *snrk2.6* mutant plants. (**G**) Representative leaves from each genotype at 12 days following inoculation with *P. cucumerina*. (**H**) Disease resistance towards *P. cucumerina* in the triple PP2C mutants *pp2ca1 1hab1 1abi1-2* and *abi2-2 hab1 abi1-2.* For the bioassays with *P. cucumerina,* lesion diameter of 25 plants per genotype and four leaves per plant were determined 12 d following inoculation with *P. cucumerina*. Data points represent the average lesion size ± SE of measurements. An ANOVA was conducted to assess significant differences in the activation of SnRKs, ABA **Figure 1.** Participation of SnRK2s kinases in the response of Arabidopsis plants to infection by the fungal pathogen *P. cucumerina*. (**A**) ABA accumulation determined in mock and *P*. *cucumerina*-infected Col-0 plants. (**B**) RT-qPCR of *ABI4* in mock and in *P. cucumerina*-infected Col-0. (**C**,**D**) *P. cucumerina*-mediated activation of SnRK2.6 (**C**) and SnRK2.2 (**D**). Transgenic Arabidopsis plants expressing HA-tagged versions of the kinases were inoculated with *P. cucumerina*, or were mocked, and leaf samples were taken at 0, 24, and 48 h.p.i., and the protein extracts were immunoprecipitated with anti-HA antibodies. The immunoprecipitates were incubated with a His-ABF2 fragment (Gly73 to Gln 119; ∆ABF2) in the presence of [γ-<sup>32</sup>P]ATP, and the proteins were resolved by SDS-PAGE. Bands corresponding to ∆ABF2 fragments and to SnRK2.6 and SnRK2.2 kinases are indicated. Radioactivities of ∆ABF2 fragment bands were measured with a phosphoimager, and the values were plotted on the graphs shown at the right of the figures. Error bars indicate S.E.M.; *n* = 3. (**E**) Disease resistance towards *P. cucumerina* of transgenic plants overexpressing SnRK2.6, SnRK2.2, and SnRK2.3 in comparison to Col-0. (**F**) Disease resistance towards *P. cucumerina* in the double *snrk2.2 snrk2.3* mutant and in *snrk2.6* mutant plants. (**G**) Representative leaves from each genotype at 12 days following inoculation with *P. cucumerina*. (**H**) Disease resistance towards *P. cucumerina* in the triple PP2C mutants *pp2ca1 1hab1 1abi1-2* and *abi2-2 hab1 abi1-2*. For the bioassays with *P. cucumerina*, lesion diameter of 25 plants per genotype and four leaves per plant were determined 12 d following inoculation with *P. cucumerina*. Data points represent the average lesion size ± SE of measurements. An ANOVA was conducted to assess significant differences in the activation of SnRKs, ABA accumulation, ABI4 transcript accumulation, and disease symptoms, with a priori *p* < 0.05 level of significance; the asterisks \* above the bars indicate statistically significant differences regarding mock treatments or Col-0 plants. Asterisks above the bars indicate different homogeneous groups with statistically significant differences.

statistically significant differences.

activation of SnRK2s positively regulated disease resistance to *P. cucumerina*.

accumulation, ABI4 transcript accumulation, and disease symptoms, with a priori *p* < 0.05 level of significance; the asterisks **\*** above the bars indicate statistically significant differences regarding mock treatments or Col-0 plants. Asterisks above the bars indicate different homogeneous groups with

We then investigated whether gain-of-function or loss-of-function in SnRK2s altered disease resistance to *P. cucumerina*. Symptoms of the fungal disease appear in the form of necrotic lesions, which are measured to quantify the degree of plant susceptibility [25,53,54]. Inoculation of transgenic plants individually overexpressing (OE) SnRK2.2, -2.3, and -2.6 revealed no significant variation in disease susceptibility towards *P. cucumerina* when compared to Col-0 plants (Figure 1E); thus, either endogenous SnRK2s levels are sufficient to achieve pathogen-triggered ABA signaling or overexpression of SnRK2s additionally requires increased ABA levels to enhance their activity. Although functional redundancy between SnRK2.2 and SnRK2.3 exists, functional segregation between SnRK2.6 and SnRK2.2/2.3 has been described [52]. Therefore, we inoculated an *snrk2.2/2.3* double mutant and the single *snrk2.6* mutant with *P. cucumerina* and recorded disease resistance. The triple *snrk2.2/2.3/2.6* mutant, which is drastically affected in plant growth [51], was not compatible with the pathogenic assay and was, therefore, not used in the present study. Figure 1F–G show that *snrk2.2/2.3* and *snrk2.6* plant resistance to *P. cucumerina* was severely compromised. Moreover, an ABA deficient mutant (i.e., *aba2*) was similarly affected in disease resistance to this pathogen (Figure

**Figure 2.** PYR1 is required for disease resistance towards *P*. *cucumerina*. (**A**). Disease resistance towards *P. cucumerina* in Col-0, the resistant *ocp3-1 mutant*, the susceptible *aba2-1,* and the triple and quadruple multi-locus mutants *pyl4 pyl5 pyl8*, *pyr1 pyl4 pyl8, pyr1 pyl4 pyl5*, and *pyr1 pyl1 pyl2 pyl4.* (**B**) Disease resistance in single *pyl1*, *pyr1,* and *pyl4* mutants, in a transgenic line overexpressing PYR1 (PYR1-OE), and in Col-0. Below the graph, the representative leaves from each genotype are shown at 12 days following inoculation with *P. cucumerina*. (**C**) Comparative disease resistance towards *P. cucumerina* among the allelic *pyr1-1*, *pyr1-2,* and *pyr1-8* mutants. 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. **Figure 2.** PYR1 is required for disease resistance towards *P. cucumerina*. (**A**). Disease resistance towards *P. cucumerina* in Col-0, the resistant *ocp3-1 mutant*, the susceptible *aba2-1*, and the triple and quadruple multi-locus mutants *pyl4 pyl5 pyl8*, *pyr1 pyl4 pyl8*, *pyr1 pyl4 pyl5*, and *pyr1 pyl1 pyl2 pyl4*. (**B**) Disease resistance in single *pyl1*, *pyr1*, and *pyl4* mutants, in a transgenic line overexpressing PYR1 (PYR1-OE), and in Col-0. Below the graph, the representative leaves from each genotype are shown at 12 days following inoculation with *P. cucumerina*. (**C**) Comparative disease resistance towards *P. cucumerina* among the allelic *pyr1-1*, *pyr1-2*, and *pyr1-8* mutants. 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.

ABA signaling through SnRK2s is negatively regulated by clade A protein phosphatase type 2C

(PP2C), particularly by ABI1, ABI2, PP2CA/AHG3, AHG1, HAB1, and HAB2 (see [55] and references ABA signaling through SnRK2s is negatively regulated by clade A protein phosphatase type 2C (PP2C), particularly by ABI1, ABI2, PP2CA/AHG3, AHG1, HAB1, and HAB2 (see [55] and references therein). Therefore, clade A PP2Cs might negatively regulate ABA-mediated disease resistance to *P. cucumerina*. Because of the demonstrated redundancy existing for these PP2Cs, combined inactivation of selected groups of these phosphatases is required to determine functionality. We combined loss-of-function mutations in ABI1, ABI2, HAB1, and PP2CA genes to determine their contribution to ABA-mediated disease resistance. Different combinations of mutations were used with two triple mutants, *pp2ca1-1;hab1-1;abi1-2* and *abi2-2;hab1-1;abi1-2*, which represent four of the nine closely related group A PP2Cs. Both multi-locus mutants showed an extreme response to exogenous ABA, partial constitutive response to endogenous ABA, and partial constitutive activation of SnRK2s in *pp2ca1-1;hab1-1;abi1-2* [51,55]. Inoculation of both triple mutants with *P. cucumerina* showed no defective disease resistance (Figure 1H). This result suggests that the demonstrated redundancy of PP2Cs masks the manifestation of a clear phenotype upon pathogen inoculation. Additionally, ABA response in triple *pp2c* mutants was partially equivalent to that of lines OE SnRK2s, which did not show altered disease resistance to the pathogen (Figure 1E). It is also possible that other members of the large PP2C family, represented by 76 homologous genes [56], are key for resistance to *P. cucumerina*. This interpretation is supported by previous studies showing that a distinct PP2C member (i.e., AtDBP1) is required for other aspects of plant immunity [57,58].

#### *2.2. The Requirement of the PYR1 Receptor for Antifungal Resistance*

We next investigated which one of 14 soluble PYR/PYL/RCAR receptors perceived the ABA produced during *P. cucumerina* infection. Partial functional redundancy of ABA receptors has been demonstrated by genetic analysis; however, PYL8 plays a non-redundant role to regulate root sensitivity to ABA [45,46]. Additionally, both transcriptional and physiological ABA responses and

signaling of environmental cues in guard cells mediated by individual receptors are starting to be elucidated [35]. We characterized disease resistance to *P. cucumerina* in a series of multi-locus mutants from different PYR/PYL receptors. The triple *pyl4;pyl5;pyl8*, *pyr1;pyl4;pyl8,* and *pyr1;pyl4;pyl5* mutants, and the quadruple *pyr1;pyl1;pyl2;pyl4* mutant, representing the highest genetic impairment in PYR/PYL function without affecting plant growth [45], were inoculated with *P. cucumerina*, and their impact on disease resistance was compared to *aba2-1* (which enhances susceptibility [25]), to *ocp3-1* (which enhances resistance [53]), and Col-0 plants. The two triple mutants incorporating the *pyr1* mutation (i.e., *pyr1;pyl4;pyl8* and *pyr1;pyl4;pyl5*) exhibited noticeably enhanced disease susceptibility (Figure 2A), which was of a magnitude similar to that observed in *aba2-1* plants. Conversely, the disease resistance of the triple *pyl4;pyl5;pyl8* mutant was unaltered compared to Col-0 plants. The quadruple mutant (also containing the *pyr1* mutation) enhanced disease susceptibility to *P. cucumerina*. The results showed that the PYR1 receptor was pivotal for eliciting ABA-mediated defense responses towards *P. cucumerina*.

The specificity of PYR1 at eliciting plant immune responses was further tested by assaying the single *pyr1-1* mutant. The individual *pyr1-1* mutant had a compromised disease resistance phenotype (Figure 2B), contrasting to other single *pyl* mutants (e.g., *pyl1*, *pyl4*) for which resistance to the fungus remained intact. Moreover, the overexpression of the PYR1 receptor (PYR1-OE line) conferred significant enhancement of resistance to the fungus (Figure 2B). Other mutant alleles of the PYR1 receptor, predicted to produce a variety of defects in PYR1 (i.e., *pyr1-2* and *pyr1-8* [43]), consistently compromised disease resistance to *P. cucumerina*, showing *pyr1-2* mutant allele as the strongest phenotype (Figure 2C). These results supported that the PYR1 receptor positively promoted ABA-dependent plant immunity against *P. cucumerina*. Interestingly, these results also indicated that other major receptors for ABA response, i.e., PYL1, PYL4, PYL5, PYL8, were not recruited in plant response against *P. cucumerina*. Furthermore, PYR1 appeared similarly to be required for the immune activation to *Alternaria brassicicola*, another fungal necrotroph and the causal agent of black spot disease in Brassica species. Results shown in Supplemental Figure S1 indicate that upon inoculation with *A. brassicicola*, both *aba2* and *pyr1* plants, compared to Col-0, *pyl1*, and *pyl4* plants, showed remarkable enhancement in disease susceptibility to this pathogen. The enhancement of necrosis in *A. brassicicola*-inoculated leaves of *pyr1* plants gave further support to the importance of PYR1-mediated perception of ABA for mounting effective defense responses towards necrotrophs.

#### *2.3. Local Induction of PYR1 Gene Expression by P. cucumerina*

A reasonable explanation for the specific role of PYR1 in plant immunity might be the specific upregulation of *PYR1* expression in response to the pathogen. Therefore, we next investigated whether transcriptional reprogramming occurred to enhance *PYR1* expression upon pathogen inoculation. Transgenic plants expressing the promoter of the *PYR1* gene fused to the β-glucuronidase GUS reporter gene (*pPYR1::GUS*) [45] were used to detect potential *P. cucumerina*-mediated activation of *PYR1*. Transgenic lines carrying the *pPYL1::GUS* and *pPYL4::GUS* gene constructs were also assayed to determine specificity. Local infection, i.e., by drop inoculation on the upper leaf surface with a *P. cucumerina* spore suspension, of transgenic *pPYR1::GUS* plants revealed early transcriptional activation of *PYR1* triggered by the pathogen (Figure 3A). *PYR1* induction mostly occurred within the vascular bundles of the primary and secondary veins of the *P. cucumerina*-inoculated leaf sectors. This highly localized induced expression pattern was specific to *PYR1* because neither *PYL1* nor *PYL4* genes were transcriptionally activated under similar circumstances (Figure 3A). The local induction of *pPYR1::GUS* concurred with local synthesis and deposition of callose (Figure 3B) and later on with cell death (Figure 3C). These microscopy markers demarcated inoculated tissue sectors in advance to the appearance of visible necrosis and served to delimit local transcriptional responses. Moreover, callose deposition was compromised in *pyr1-1* and *aba2-1* mutants following fungal infection (Figure 3D), thus supporting the participation of ABA and PYR1 in this local process.

in this local process.

*2.3. Local Induction of PYR1 Gene Expression by P. cucumerina* 

A reasonable explanation for the specific role of PYR1 in plant immunity might be the specific upregulation of *PYR1* expression in response to the pathogen. Therefore, we next investigated whether transcriptional reprogramming occurred to enhance *PYR1* expression upon pathogen inoculation. Transgenic plants expressing the promoter of the *PYR1* gene fused to the βglucuronidase GUS reporter gene (*pPYR1::GUS*) [45] were used to detect potential *P. cucumerina*mediated activation of *PYR1.* Transgenic lines carrying the *pPYL1::GUS* and *pPYL4::GUS* gene constructs were also assayed to determine specificity. Local infection, i.e., by drop inoculation on the upper leaf surface with a *P. cucumerina* spore suspension, of transgenic *pPYR1*::*GUS* plants revealed early transcriptional activation of *PYR1* triggered by the pathogen (Figure 3A). *PYR1* induction mostly occurred within the vascular bundles of the primary and secondary veins of the *P. cucumerina*inoculated leaf sectors. This highly localized induced expression pattern was specific to *PYR1* because neither *PYL1* nor *PYL4* genes were transcriptionally activated under similar circumstances (Figure 3A). The local induction of *pPYR1::GUS* concurred with local synthesis and deposition of callose (Figure 3B) and later on with cell death (Figure 3C). These microscopy markers demarcated inoculated tissue sectors in advance to the appearance of visible necrosis and served to delimit local transcriptional responses. Moreover, callose deposition was compromised in *pyr1-1* and *aba2-1* 

**Figure 3.** Local activation of *PYR1* gene expression at pathogen inoculation sites, and the requirement of PYR1 for pathogen-induced callose deposition. (**A**) Comparative histochemical analysis of GUS activity in rosette leaves from transgenic plants carrying *pPYR1::GUS*, *pPYL1::GUS*, and *pPYL4::GUS* **Figure 3.** Local activation of *PYR1* gene expression at pathogen inoculation sites, and the requirement of PYR1 for pathogen-induced callose deposition. (**A**) Comparative histochemical analysis of GUS activity in rosette leaves from transgenic plants carrying *pPYR1::GUS*, *pPYL1::GUS*, and *pPYL4::GUS* gene constructs and those were either mocked or inoculated *P. cucumerina*. Leaves were stained for GUS activity at 36 h.p.i. The left panel corresponds to mocked plants. The central and right panels correspond to enlargements of the inoculated leaf sectors. Black arrow points towards leaf tissues proximal to the inoculation point, and white arrows denote tissues that directly received the spore inoculum. Note that *pPYR1::GUS* is heavily induced in leaf veins within the inoculated sector. (**B**) Characteristic spore-inoculated leaf sector, similar to those shown in A, stained with aniline blue to detect pathogen-induced callose deposition (top panel), or with trypan blue (lower panel) to identify incipient cell deterioration due to fungal infection at 36 h.p.i. (**C**) Aniline blue staining and epifluorescence microscopy were applied to visualize callose accumulation. Micrographs indicate *P. cucumerina* inoculation and infection site in the different Arabidopsis genotypes at 0 h.p.i (right panel), at 24 h.p.i. (central panel), and at 48 h.p.i. (right panel). (**D**) The number of yellows pixels (corresponding to pathogen-induced callose) per million on digital photographs of infected leaves were used as a means to express arbitrary units (i.e., to quantify the image) at the indicated times. Bars represent mean ± SD, *n* = 15 independent replicates. An ANOVA was conducted to assess significant differences in callose deposition (*p* < 0.05); the asterisks \* above the bars indicate statistically significant differences regarding Col-0 plants.

#### *2.4. Resistance Enhancement of pyr1 Plants to Pseudomonas syringae DC3000*

In marked contrast to the results shown above, the role of ABA in repressing plant immunity against the (hemi) biotrophic pathogens *P. syringae* DC3000 has been previously documented [6,19–21]. Therefore, we asked whether the negative role of ABA in plant immunity against *P. syringae* DC3000 could similarly be funneled through PYR1. If so, we would expect resistance enhancement in *pyr1* plants. *pyr1* plants were inoculated by leaf infiltration with *P. syringae* DC3000, and the rate of bacterial growth in the inoculated leaves was determined at 3 days post-inoculation in comparison to *aba2*, *pyl1*, *pyl4*, and Col-0 plants. Figure 4A shows that bacterial growth was reduced 10-fold in both *aba2* and *pyr1*

mutants compared to Col-0, *pyl1*, and *pyl4*. This result confirmed the negative role of ABA in resistance towards *P. syringae* DC3000 and demonstrated the specific requirement of PYR1 for the negative role of ABA during this plant-pathogen interaction. Moreover, pre-treatment of Col-0 with 150 µM ABA, applied by drenching, predictably provoked disease susceptibility enhancement to *P. syringae* DC3000 (Figure 4B), denoting a damping effect of ABA on SA signaling. This ABA-mediated enhancement in susceptibility to *P. syringae* DC3000 did not occur in *pyr1-2* plants whose enhanced resistance was not altered by the hormone (Figure 4B). to *aba2*, *pyl1*, *pyl4,* and Col-0 plants. Figure 4A shows that bacterial growth was reduced 10-fold in both *aba2* and *pyr1* mutants compared to Col-0, *pyl1,* and *pyl4*. This result confirmed the negative role of ABA in resistance towards *P. syringae* DC3000 and demonstrated the specific requirement of PYR1 for the negative role of ABA during this plant-pathogen interaction. Moreover, pre-treatment of Col-0 with 150 μM ABA, applied by drenching, predictably provoked disease susceptibility enhancement to *P. syringae* DC3000 (Figure 4B), denoting a damping effect of ABA on SA signaling. This ABAmediated enhancement in susceptibility to *P. syringae* DC3000 did not occur in *pyr1-2* plants whose enhanced resistance was not altered by the hormone (Figure 4B).

of bacterial growth in the inoculated leaves was determined at 3 days post-inoculation in comparison

In marked contrast to the results shown above, the role of ABA in repressing plant immunity against the (hemi) biotrophic pathogens *P. syringae* DC3000 has been previously documented [6,19– 21]. Therefore, we asked whether the negative role of ABA in plant immunity against *P. syringae*

statistically significant differences regarding Col-0 plants.

*2.4. Resistance Enhancement of pyr1 Plants to Pseudomonas syringae DC3000* 

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 8 of 22

gene constructs and those were either mocked or inoculated *P. cucumerina*. Leaves were stained for GUS activity at 36 h.p.i. The left panel corresponds to mocked plants. The central and right panels correspond to enlargements of the inoculated leaf sectors. Black arrow points towards leaf tissues proximal to the inoculation point, and white arrows denote tissues that directly received the spore inoculum. Note that *pPYR1::GUS* is heavily induced in leaf veins within the inoculated sector. (**B**) Characteristic spore-inoculated leaf sector, similar to those shown in A, stained with aniline blue to detect pathogen-induced callose deposition (top panel), or with trypan blue (lower panel) to identify incipient cell deterioration due to fungal infection at 36 h.p.i. (**C**) Aniline blue staining and epifluorescence microscopy were applied to visualize callose accumulation. Micrographs indicate *P. cucumerina* inoculation and infection site in the different Arabidopsis genotypes at 0 h.p.i (right panel), at 24 h.p.i. (central panel), and at 48 h.p.i. (right panel). (**D**) The number of yellows pixels (corresponding to pathogen-induced callose) per million on digital photographs of infected leaves were used as a means to express arbitrary units (i.e., to quantify the image) at the indicated times. Bars represent mean ± SD, *n* = 15 independent replicates. An ANOVA was conducted to assess significant differences in callose deposition (*p* < 0.05); the asterisks **\*** above the bars indicate

**Figure 4.** Response of *pyr1* plants to infection by *P. syringae DC3000*. (**A**) Col-0, *aba2-1*, *pyr1-2*, *pyl1,*  and *pyl4* mutants were inoculated with *P. syringae* DC3000, and their disease responses were recorded. (**B**) Col-0 and *pyr1* plants were pre-treated with 150 μM ABA, applied by drenching, before inoculation with *P. syringae* DC3000, and the growth of the bacteria was recorded in comparison to mocked plants. Growth of *P. syringae* DC3000 was measured at 3 d.p.i. Error bars represent standard **Figure 4.** Response of *pyr1* plants to infection by *P. syringae DC3000*. (**A**) Col-0, *aba2-1*, *pyr1-2*, *pyl1*, and *pyl4* mutants were inoculated with *P. syringae* DC3000, and their disease responses were recorded. (**B**) Col-0 and *pyr1* plants were pre-treated with 150 µM ABA, applied by drenching, before inoculation with *P. syringae* DC3000, and the growth of the bacteria was recorded in comparison to mocked plants. Growth of *P. syringae* DC3000 was measured at 3 d.p.i. Error bars represent standard deviation (*n* = 12). An ANOVA was conducted to assess significant differences in disease symptoms, with a priori *p* < 0.05 level of significance; the asterisks \*, \*\* above the bars indicate different homogeneous groups with statistically significant differences.

Therefore, our results indicated that the dual antagonistic role of ABA in plant immunity was mediated through the PYR1 receptor, which reciprocally activates and represses immune responses towards necrotrophic and biotrophic pathogens, respectively.

#### *2.5. SA–Responsive Defense Genes are Activated in PYR1 Defective Mutants*

We investigated whether *pyr1* and *aba2* plants carried constitutive elevated expression of SA-responsive genes, which might explain the observed enhanced resistance to *P. syringae* DC3000 (Figure 4). The accumulation of *PR-1* and *PR-2* transcript, which are SA- and pathogen-responsive genes, was examined by RT-qPCR. In addition, we examined *PR-4* and *PR-5*, which are also pathogen-responsive genes but are simultaneously influenced by SA and ET [59]. Transcript accumulation was also evaluated in *pyl1* and *pyl4* mutants, which served as additional controls. Figure 5A shows that *pyr1* and *aba2* plants carried constitutive elevated levels of SA-dependent *PR-1* and *PR-2* transcripts compared to Col-0, *pyl1*, or *pyl4* plants. Conversely, the constitutive levels of transcript accumulation for *PR-4* and *PR-5* occurring in Col-0 were repressed in *pyr1* and *aba2* plants, and only partially enhanced in *pyl1* plants (Figure 5A). The enhanced expression of *PR-1* and *PR-2* and the concerted repression of *PR-4* and *PR-5* were corroborated by the *pyr1* allelic series, with the *pyr1-2* allele showing the strongest differences (Figure 5B). Thus, the ABA/PYR1 module might function as an integration node regulating distinct branches of defenses. The constitutive activation of *PR1*- and *-2* in *pyr1* plants supported the enhanced accumulation of both free and conjugated SA observed in the mutant, which concurred also with elevated expression of *ICS1*, encoding isochorismate synthase, a pivotal enzyme controlling SA biosynthesis [60,61] (Figure 5C,D). On the other hand, *pyr1* plants only

showed a moderate reduction, less than two-fold, of JA content in comparison to Col-0 (Figure 5E). The conspicuous enhancement in SA content in healthy *pyr1* plants, therefore, explained the resistance phenotypes of the mutant when confronted with *P. syringae* DC3000. However, the notorious enhanced susceptibility of *pyr1* plants to the fungal necrotrophs *P. cucumerina* and *A. brassicicola* remained unsolved, as it could not simply be explained by the moderate reduction of JA levels as attained in the mutant. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 10 of 22

**Figure 5.** Expression of SA-responsive and ET-responsive genes in *pyr1* and *aba2* mutants. (A,B) RTqPCR analysis showing constitutive expression levels of *PR-1*, *PR-2*, *PR-4,* and *PR-5* genes in (**A**) Col-0, *aba2-1*, *pyr1-1*, *pyl1,* and *pyl4* plants, and (**B**) their comparative expression levels in the allelic *pyr1- 1*, *pyr1-2,* and *pyr1-8* mutants. Data represent mean ± SD; *n* = 3 replicates. The expression was normalized to the constitutive *ACT2* and *ACT8* genes and then to the expression in Col-0 plants. (**C**– **E**) Accumulation of free SA, total SA, and total JA in Col-0 and *pyr1-2* plants. Data represent the average of three biological replicates. An ANOVA was conducted to assess significant differences in RT-qPCR and hormone analysis, with a priori *p* < 0.05 level of significance; the asterisks **\*** above the bars indicate statistically significant differences regarding Col-0 plants. *2.6. Enhanced Activation of MAPK Kinases in pyr1 Plants*  **Figure 5.** Expression of SA-responsive and ET-responsive genes in *pyr1* and *aba2* mutants. (**A**,**B**) RT-qPCR analysis showing constitutive expression levels of *PR-1*, *PR-2*, *PR-4*, and *PR-5* genes in (**A**) Col-0, *aba2-1*, *pyr1-1*, *pyl1*, and *pyl4* plants, and (**B**) their comparative expression levels in the allelic *pyr1-1*, *pyr1-2*, and *pyr1-8* mutants. Data represent mean ± SD; *n* = 3 replicates. The expression was normalized to the constitutive *ACT2* and *ACT8* genes and then to the expression in Col-0 plants. (**C**–**E**) Accumulation of free SA, total SA, and total JA in Col-0 and *pyr1-2* plants. Data represent the average of three biological replicates. An ANOVA was conducted to assess significant differences in RT-qPCR and hormone analysis, with a priori *p* < 0.05 level of significance; the asterisks \* above the bars indicate statistically significant differences regarding Col-0 plants.

#### We next investigated whether enhanced resistance to *P. syringae* DC3000 in *pyr1* plants was associated with elevated MAPKs activation, which is linked to the activation of immune responses *2.6. Enhanced Activation of MAPK Kinases in pyr1 Plants*

following pathogen perception. We employed an antibody recognizing the phosphorylated residues within the MAPK activation loop (i.e., the pTEpY motif). Western blot analysis of protein extracts derived from healthy Col-0 and *pyr1* plants showed positive immunoreactive signals in two polypeptides corresponding to MPK6 and MPK3 (Beckers et al., 2009) (Figure 6), and the densitometric scanning of blots indicated that the MPK3 immunoreactive band was more intense in *pyr1* plants. Inoculation with *P. syringae* DC3000 promoted further activation-associated dual TEY phosphorylation of MPKs, which was noticeably higher for MPK3 in *pyr1* compared to Col-0 plants at 24 h.p.i. (Figure 6). At the latter stages of infection (i.e., 48 h.p.i.), the MPK activation was similar in Col-0 and *pyr1* plants. Therefore, MPK activation may be prone to activation in plants defective of ABA perception through the PYR1 receptor. Indeed, partial pre-activation of MPK was reflected in We next investigated whether enhanced resistance to *P. syringae* DC3000 in *pyr1* plants was associated with elevated MAPKs activation, which is linked to the activation of immune responses following pathogen perception. We employed an antibody recognizing the phosphorylated residues within the MAPK activation loop (i.e., the pTEpY motif). Western blot analysis of protein extracts derived from healthy Col-0 and *pyr1* plants showed positive immunoreactive signals in two polypeptides corresponding to MPK6 and MPK3 (Beckers et al., 2009) (Figure 6), and the densitometric scanning of blots indicated that the MPK3 immunoreactive band was more intense in *pyr1* plants. Inoculation with *P. syringae* DC3000 promoted further activation-associated dual TEY phosphorylation of MPKs, which was noticeably higher for MPK3 in *pyr1* compared to Col-0 plants at 24 h.p.i. (Figure 6). At the latter stages of infection (i.e., 48 h.p.i.), the MPK activation was similar in Col-0 and *pyr1* plants. Therefore, MPK activation may be prone to activation in plants defective of ABA perception through the PYR1 receptor. Indeed, partial pre-activation of MPK was reflected in detectable PR-1 protein accumulating in *pyr1* plants at time zero (Figure 6). This result was in agreement with the higher expression level of the *PR-1* gene determined by RT-qPCR (Figure 5A,B). Interestingly, inoculation with *P. syringae* DC3000 promoted the further accumulation of the PR-1 protein, which progressively increased over time to a much higher level in the *pyr1* mutant compared to Col-0 plants (Figure 6). *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 11 of 22 detectable PR-1 protein accumulating in *pyr1* plants at time zero (Figure 6). This result was in agreement with the higher expression level of the *PR-1* gene determined by RT-qPCR (Figure 5A,B). Interestingly, inoculation with *P. syringae* DC3000 promoted the further accumulation of the PR-1 protein, which progressively increased over time to a much higher level in the *pyr1* mutant compared to Col-0 plants (Figure 6).

**Figure 6.** Loss of PYR1 function confers enhanced mitogen-activated kinase activation and PR-1 protein accumulation following *P. syringae* DC3000 infection. Western blot with anti-pTEpY and anti-PR-1 antibodies of crude protein extracts derived from Col-0, *pyr1-2* plants at 0, 24, and 48 h.p.i with *P. syringae* DC3000. Equal protein loading was check by Ponceau-S staining of the nitrocellulose filter. MPK6 and MPK3 migrating bands are indicated on the right. The experiments were repeated three times with similar results. Scan quantification of protein bands corresponding to MPK3 and PR-1 is shown below the Western blot. Data represent the mean ± SD; *n* = 3 replicates. An ANOVA was conducted to assess significant differences in RT-qPCR analysis, with a priori *p* < 0.05 level of significance; the asterisks **\*** above the bars indicate statistically significant differences regarding Col-0 plants. Thus, we hypothesize that the lack of ABA perception through the PYR1 receptor de-represses **Figure 6.** Loss of PYR1 function confers enhanced mitogen-activated kinase activation and PR-1 protein accumulation following *P. syringae* DC3000 infection. Western blot with anti-pTEpY and anti-PR-1 antibodies of crude protein extracts derived from Col-0, *pyr1-2* plants at 0, 24, and 48 h.p.i with *P. syringae* DC3000. Equal protein loading was check by Ponceau-S staining of the nitrocellulose filter. MPK6 and MPK3 migrating bands are indicated on the right. The experiments were repeated three times with similar results. Scan quantification of protein bands corresponding to MPK3 and PR-1 is shown below the Western blot. Data represent the mean ± SD; *n* = 3 replicates. An ANOVA was conducted to assess significant differences in RT-qPCR analysis, with a priori *p* < 0.05 level of significance; the asterisks \* above the bars indicate statistically significant differences regarding Col-0 plants.

a pathway that allows cell sensitization through MPKs activation and downstream defense gene reprogramming, even in the absence of pathogen infection. Sensitized cells may be ready for the enhanced induction of this defense pathway following pathogen infection, which, in turn, may explain why *aba* and *pyr1* plants exhibit enhanced disease resistance to *P. syringae* DC3000. These observations support that ABA and PYR1 function as a repressor module of SA-mediated onset of resistance. Thus, we hypothesize that the lack of ABA perception through the PYR1 receptor de-represses a pathway that allows cell sensitization through MPKs activation and downstream defense gene reprogramming, even in the absence of pathogen infection. Sensitized cells may be ready for the enhanced induction of this defense pathway following pathogen infection, which, in turn, may explain why *aba* and *pyr1* plants exhibit enhanced disease resistance to *P. syringae* DC3000. These observations support that ABA and PYR1 function as a repressor module of SA-mediated onset of resistance.
