Characterization of Disease Resistance Induced by a Pyrazolecarboxylic Acid Derivative in Arabidopsis thaliana

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid (SA)-mediated signaling pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in Arabidopsis. The soil drench application of CMPA enhanced a broad range of disease resistance against the bacterial pathogen Pseudomonas syringae and fungal pathogens Colletotrichum higginsianum and Botrytis cinerea in Arabidopsis, whereas CMPA did not show antibacterial activity. Foliar spraying with CMPA induced the expression of SA-responsible genes such as PR1, PR2 and PR5. The effects of CMPA on resistance against the bacterial pathogen and the expression of PR genes were observed in the SA biosynthesis mutant, however, while they were not observed in the SA-receptor-deficient npr1 mutant. Thus, these findings indicate that CMPA induces SAR by triggering the downstream signaling of SA biosynthesis in the SA-mediated signaling pathway.


Introduction
Systemic acquired resistance (SAR) is a defense mechanism that is induced in the whole plant body after a hypersensitive response, including tissue necrosis caused by pathogens, through the salicylic acid (SA)-mediated signaling pathway [1][2][3]. As SAR is effective against a broad range of pathogens, it plays an important role in defending plants from pathogen attacks. SAR in Arabidopsis thaliana has been well characterized, and a set of pathogenesis-related (PR) genes have been identified as SAR marker genes [4][5][6][7]. Additionally, various types of SAR-deficient Arabidopsis mutants have been identified and used to investigate the mechanism of SAR. The SA biosynthesis-deficient mutant sid2 contains a mutation in the Isochorismate Synthase 1 (ICS1) gene [8]. The isochorismate generated in chloroplasts is transferred to the cytosol by Enhanced Disease Susceptibility 5 (EDS5) and converted into isochorismate-9-glutamate by AvrPphB Susceptible 3 (PBS3), which is then converted into SA by Enhanced Pseudomonas Susceptibility 1 (EPS1) [9,10]. Other mutant plants include members of the SA signal-deficient Nonexpressor of PR Genes 1 (npr1) family, which have a mutation in the SA receptor protein [11][12][13][14], and NahG transgenic plants, which are unable to accumulate SA because they express the SA-degrading enzyme salicylate hydroxylase [6,7]. During SAR induction, the NPR1 proteins are reduced and transferred from the cytoplasm to the nucleus, in which an increase in the total glutathione (GSH) level takes part [15,16].
We previously reported that a pyrazolecarboxylic acid derivative, 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA), induced disease resistance in rice against rice blast and bacterial blight [32,33], and it induced PR gene expression and disease resistance without SA accumulation in tobacco [34]. However, as the SA receptor-deficient mutant is not available in tobacco, the issue of whether CMPA activates the SA-mediated signaling pathway for SAR induction remains unelucidated. As the physicochemical properties of water-soluble CMPA are different from those of water-insoluble BTH, INA, and NCI, CMPA may activate pathways other than the SA-NPR1-mediated signaling pathway. Therefore, in this study, we aimed to determine how CMPA activates disease resistance using Arabidopsis plants.

Induction of a Broad Range of Disease Resistance in Arabidopsis by CMPA
The ability of CMPA to enhance disease resistance in wild-type Arabidopsis (Col-0) was assessed using several virulent pathogens, including the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst), which causes bacterial speck; the hemibiotrophic fungal pathogen Colletotrichum higginsianum (Ch), which causes anthracnose; the necrotrophic fungal pathogen Botrytis cinerea (Bc), which causes gray mold; and the viral pathogen cucumber mosaic virus (CMV). Soil drenching treatment with 0.25 mg/pot CMPA and foliar treatment with 1.25 mM did not cause any morphological changes or growth inhibition in plants, whereas foliar treatment with more than 6.25 mM CMPA resulted in tissue necrosis similar to a hypersensitive reaction (Supplementary Figure S1).
Susceptibility to Pst was estimated by measuring the bacterial growth in leaf tissues three days after the challenge inoculation. Treatment with 0.25 mg/pot CMPA by means of soil drenching five days prior to the challenge inoculation inhibited bacterial growth in the infected tissues relative to that in the water-treated control plants (96% reduction from the control) ( Figure 1A). CMPA at concentrations of up to 200 µg/mL did not affect the growth rate of Pst in the liquid culture (Supplementary Table S1). These results indicated that CMPA enhanced resistance against Pst by activating the plant immune system. Susceptibility to Ch was estimated using the area of necrotrophic lesions that appeared on the detached leaves two days after drop inoculation. Foliar treatment with 1.25 mM CMPA five days prior to the challenge inoculation reduced the ratio of the lesion area to infected leaf area compared to the untreated control plants (37% reduction from the control) ( Figure 1B), indicating that CMPA enhanced resistance against Ch in Arabidopsis.
Disease resistance against Bc was estimated by measuring the size of the necrotrophic lesions that appeared on the infected leaves three days after drop inoculation. Foliar treatment with 1.25 mM CMPA was performed five days prior to the challenge inoculation. The ratio of spreading lesions to the whole infected leaves was significantly smaller in the CMPA-treated plants than in the untreated control plants (34% reduction from the control) ( Figure 1C), indicating that CMPA enhanced resistance against Bc in Arabidopsis.
CMPA-treated plants than in the untreated control plants (34% reduction from the control) ( Figure 1C), indicating that CMPA enhanced resistance against Bc in Arabidopsis.
The effects of CMPA on CMV were estimated by detecting the viral coat protein in the plant tissues. Plants were foliar treated with 0.625 mM CMPA five days prior to the CMV inoculation. The results indicated that treatment with CMPA did not affect the viral multiplication and spread in the infected plants (Supplementary Figure S2).

Physiological Changes Associated with CMPA-Induced Resistance
To characterize the disease resistance induced by CMPA, we examined the physiological changes in the CMPA-treated wild-type plants. A gene expression analysis of SAR marker genes in the plants after two days of CMPA treatment was performed using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of PR1, PR2, and PR5 were upregulated in the CMPA-treated plants compared with those in the untreated control plants (Figure 2A). The upregulation was similar to treatment with the SAR-inducing chemicals BIT and BTH (Figure 2A), indicating that CMPA activated the SA-mediated signaling pathway. The expression of the SA biosynthetic enzyme ICS1 was unchanged with the CMPA and BTH treatments, whereas BIT upregulated its expression ( Figure 2A). The expression levels of the JA-related genes VSP2 and PDF1.2 and the ET-related gene ERF1 were not significantly different between the CMPAtreated plants and untreated control plants ( Figure 2B), indicating that CMPA did not activate the JA-and ET-mediated signaling pathways. Endogenous SA accumulation in the leaves was examined two and five days after treatment with CMPA. The levels of free and total SA (free SA + SA glucoside) were not significantly different from those of the untreated control plants ( Figure 3A). Increased endogenous GSH levels are important for The effects of CMPA on CMV were estimated by detecting the viral coat protein in the plant tissues. Plants were foliar treated with 0.625 mM CMPA five days prior to the CMV inoculation. The results indicated that treatment with CMPA did not affect the viral multiplication and spread in the infected plants (Supplementary Figure S2).

Physiological Changes Associated with CMPA-Induced Resistance
To characterize the disease resistance induced by CMPA, we examined the physiological changes in the CMPA-treated wild-type plants. A gene expression analysis of SAR marker genes in the plants after two days of CMPA treatment was performed using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of PR1, PR2, and PR5 were upregulated in the CMPA-treated plants compared with those in the untreated control plants ( Figure 2A). The upregulation was similar to treatment with the SAR-inducing chemicals BIT and BTH (Figure 2A), indicating that CMPA activated the SA-mediated signaling pathway. The expression of the SA biosynthetic enzyme ICS1 was unchanged with the CMPA and BTH treatments, whereas BIT upregulated its expression (Figure 2A). The expression levels of the JA-related genes VSP2 and PDF1.2 and the ET-related gene ERF1 were not significantly different between the CMPA-treated plants and untreated control plants ( Figure 2B), indicating that CMPA did not activate the JA-and ET-mediated signaling pathways. Endogenous SA accumulation in the leaves was examined two and five days after treatment with CMPA. The levels of free and total SA (free SA + SA glucoside) were not significantly different from those of the untreated control plants ( Figure 3A). Increased endogenous GSH levels are important for SAR induction, and the level of GSH increased more than two-fold after two days of CMPA treatment ( Figure 3B). These results indicated that CMPA induced SAR in Arabidopsis without SA accumulation. SAR induction, and the level of GSH increased more than two-fold after two days of CMPA treatment ( Figure 3B). These results indicated that CMPA induced SAR in Arabidopsis without SA accumulation.  SAR induction, and the level of GSH increased more than two-fold after two days of CMPA treatment ( Figure 3B). These results indicated that CMPA induced SAR in Arabidopsis without SA accumulation.

Effects of CMPA on SA-Deficient Mutant Plants
To confirm whether SA biosynthesis was required for SAR induction by CMPA, we examined the effects of CMPA on the gene expression and disease resistance in sid2 mutants defective in SA biosynthesis and NahG transgenic plants constitutively expressing SA hydroxylase, an SA-degrading enzyme. The expression levels of PR1, PR2, and PR5 were significantly upregulated in the sid2 mutant by CMPA and BTH but not by BIT ( Figure 4A). CMPA treatment induced disease resistance against Pst in both the sid2 and NahG plants ( Figure 4B). These results indicated that SA biosynthesis was not required for the disease resistance and enhanced expression of SAR marker genes induced by CMPA. mM CMPA, and the GSH levels were quantified by means of HPLC. Values are shown as the means ± SE. Significant difference between the control and chemical-treated plants are indicated by asterisks. (Unpaired student's t-test, ** p < 0.01.)

Effects of CMPA on SA-Deficient Mutant Plants
To confirm whether SA biosynthesis was required for SAR induction by CMPA, we examined the effects of CMPA on the gene expression and disease resistance in sid2 mutants defective in SA biosynthesis and NahG transgenic plants constitutively expressing SA hydroxylase, an SA-degrading enzyme. The expression levels of PR1, PR2, and PR5 were significantly upregulated in the sid2 mutant by CMPA and BTH but not by BIT (Figure 4A). CMPA treatment induced disease resistance against Pst in both the sid2 and NahG plants ( Figure 4B). These results indicated that SA biosynthesis was not required for the disease resistance and enhanced expression of SAR marker genes induced by CMPA.

Effects of CMPA on SA Receptor-Deficient Mutant Plants
Analyses using wild-type and SA-deficient plants indicated that CMPA induced disease resistance and expression of SAR marker genes in an SA biosynthesis-independent manner. To determine whether SA-mediated signaling for PR gene expression was required for the CMPA-induced disease resistance, we examined the effects of CMPA on defense gene expression and disease resistance in npr1 mutants defective in the SA receptor protein.
The expression of PR1, PR2, and PR5 did not increase in the CMPA-, BIT-, or BTH-treated plants, indicating that NPR1 was required for PR gene expression by CMPA ( Figure 5A). The Pst inoculation assay showed that CMPA and the other SAR inducers did not enhance disease resistance in the npr1 mutants ( Figure 5B). These results indicated that SA-mediated signaling through the NPR1 protein was required for SAR induction by CMPA. ease resistance and expression of SAR marker genes in an SA biosynthesis-independent manner. To determine whether SA-mediated signaling for PR gene expression was required for the CMPA-induced disease resistance, we examined the effects of CMPA on defense gene expression and disease resistance in npr1 mutants defective in the SA receptor protein. The expression of PR1, PR2, and PR5 did not increase in the CMPA-, BIT,-or BTH-treated plants, indicating that NPR1 was required for PR gene expression by CMPA ( Figure 5A). The Pst inoculation assay showed that CMPA and the other SAR inducers did not enhance disease resistance in the npr1 mutants ( Figure 5B). These results indicated that SA-mediated signaling through the NPR1 protein was required for SAR induction by CMPA.  Three-week-old npr1 plants were treated with water (control), BIT (0.5 mg/mL), BTH (0.1 mg/pot), or CMPA (0.25 mg/pot) via the soil drenching method five days prior to inoculation with Pst. The bacterial population in the leaves was estimated three days after inoculation. Each experiment was performed with 6 samples that were prepared from 3 leaves via homogenizing leaf disks in 10 mM MgCl 2 . Values are shown as the means ± SE (n = 6).

Discussion
CMPA, without antimicrobial activity, enhanced disease resistance to a broad range of pathogens, including bacterial and fungal pathogens, and induced the expression of SAR marker genes in wild-type Arabidopsis plants, indicating that CMPA fulfills the criteria for a plant activator [18]. CMPA induced disease resistance and PR gene expression in the SAdeficient sid2 mutant and NahG transgenic plant but not in the npr1 mutant, indicating that CMPA induced SAR by activating SA-mediated signaling downstream of SA biosynthesis, as well as by BTH. This observation was supported by the ICS1, VSP2, PDF1.2, and ERF1 expression or SA accumulation in the wild-type plants not being affected by CMPA.
Gray mold is an important disease requiring control because Bc causes disease in more than 200 plant species. Although Bc is a necrotrophic pathogen that does not induce SA-mediated signaling upon infection [35], the induction of resistance against Bc by exoge-nous SA or BTH has been reported in several plants, including Arabidopsis, tomato, and tobacco [36][37][38][39]. As CMPA is effective against Bc in Arabidopsis, it could also be effective in controlling gray mold in other plants, such as tomatoes and tobacco.
Although the physicochemical properties of water-soluble CMPA are different from those of water-insoluble BTH, INA, and NCI, their modes of action for resistance induction to Pst are similar. Whether CMPA acts directly on the NPR1 protein is unknown; however, this water-soluble plant activator might enable us to clarify the interaction between the plant activator and the NPR1 protein [13,14].
Treatment with CMPA did not result in resistance against CMV infection, whereas SAR was reportedly effective against CMV [41], suggesting that the mode of action of CMPA is different from that of other plant activators. When CMPA was applied to Arabidopsis leaves at high concentrations (>6.25 mM), tissue necrosis, similar to cell death during a hypersensitive response, was observed. This side effect was not observed at low CMPA treatment concentrations. Moreover, low CMPA concentrations did not affect the induction of resistance against bacterial and fungal pathogens. However, the combination of the invisible side effects, even at low concentrations, and the damage to the foliar surface due to rub inoculation with carborundum may explain why CMPA is not effective against CMV. As such a high-concentration solution cannot be prepared for water-insoluble BTH, INA or NCI, leaf necrosis is a specific characteristic of CMPA. Thus, this study revealed that CMPA is a novel type of plant activator, which stimulates the downstream of SA in the SA-NPR1-mediated pathway for SAR induction but induces a different spectrum of disease resistance.

Plant Materials and Treatment
Arabidopsis thaliana plants were grown in sterilized potting soil Kumiai Nippi 1 (Nihon Hiryo, Tokyo, Japan) in pots (5 × 5 × 5 cm) inside a growth chamber under a 16 h:8 h light:dark cycle at 23 • C with 60% humidity. The three-week-old Arabidopsis plants were treated with CMPA, BIT, BTH, or water via soil drenching or via foliar spraying.

Arabidopsis Pathogen Infection Assay
Pst was inoculated by dipping the plants in a bacterial solution (2 × 10 5 colony-forming units/mL in 10 mM MgCl 2 ) [24]. The leaves were harvested from the inoculated plants three days after inoculation and homogenized in 10 mM MgCl 2 . The homogenates were plated on nutrient broth agar containing rifampicin (50 mg/mL) at appropriate dilutions. After incubation for two days at 28 • C, the rifampicin-resistant bacterial colonies were counted. Bacterial growth was estimated using eight homogenates prepared from three leaves for each treatment.
Drop inoculation of the fungal pathogens was performed by placing 3 µL of spore suspension of Ch (1 × 10 6 spores/mL in water) or Bc (1 × 10 6 spores/mL in 1/2 PDB medium) onto detached leaves that were placed on wet filter paper in a plastic box [42,43]. The box was sealed to maintain high humidity and incubated in the dark for three days at 25 • C. The areas of the lesions and whole leaves were measured using ImageJ software (ver 1.53). More than 30 leaves were used for each treatment, and the average ratio of the lesions to the whole leaf area was calculated to estimate the disease susceptibility.
CMV (20 µg/mL) was inoculated on a lower leaf of the plant via the rub inoculation method using carborundum [41]. The presence of viral coat protein in the plant tissues was detected via the tissue printing method at zero, three, and seven days after inoculation. The tissue printing method was performed by press blotting all the plants on filter papers, followed by probing the filters with a polyclonal anti-CMV-Y coat protein antibody.

Analysis of Gene Expression by RT-qPCR Analysis
The leaves were harvested 2 days after the application of the chemicals. The total RNA was extracted from the frozen leaf samples using Sepasol ® -RNA I Super G (Nacalai Tesque Co., Ltd., Kyoto, Japan). The total RNA was used for the cDNA synthesis using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio Inc., Shiga, Japan). For the RT-qPCR, each cDNA sample was amplified using gene-specific primers (Supplementary Table S2) using TB Green ® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio Inc., Shiga, Japan) and a LightCycler ® 96 System (Roche Diagnostics K.K. Tokyo, Japan) [16]. Four to six biological replicates were used.

Extraction and Analysis of SA
Leaves were harvested from the treated plants two and five days post treatment with CMPA or water. The levels of SA and SAG were measured as previously described [24,44].

Extraction and Analysis of GSH
Leaf samples (approximately 100 mg for each sample) were harvested 0, 6, 12, 24 and 48 h after foliar treatment with CMPA. The extraction and measurement of GSH were performed as previously described [45,46]. Briefly, the GSH content in the leaf extract was determined via monobromobimane (Invitrogen) labeling of the thiol bases after the reduction of the extracts with dithiothreitol (Nacalai Tesque Co., Ltd., Japan). The labeled products were analyzed via HPLC with a fluorescence detector FP-920 (JASCO) using a TSKgel ODS-120T column (4.6 × 150 mm, Tosoh, Tokyo, Japan). The fluorescence of the thiol-bimane adducts was detected at 478 nm under excitation at 390 nm. The GSH standards were purchased from Nacalai Tesque.