DEWAX Transcription Factor Is Involved in Resistance to Botrytis cinerea in Arabidopsis thaliana and Camelina sativa

The cuticle of land plants is the first physical barrier to protect their aerial parts from biotic and abiotic stresses. DEWAX, an AP2/ERF-type transcription factor, negatively regulates cuticular wax biosynthesis. In this study, we investigated the resistance to Botrytis cinerea in Arabidopsis thaliana and Camelina sativa overexpressing DEWAX and in Arabidopsis dewax mutant. Compared to wild type (WT) leaves, Arabidopsis DEWAX OX and dewax leaves were more and less permeable to toluidine blue dye, respectively. The ROS levels increased in DEWAX OX leaves, but decreased in dewax relative to WT leaves. Compared to WT, DEWAX OX was more resistant, while dewax was more sensitive to B. cinerea; however, defense responses to Pseudomonas syringae pv. tomato DC3000:GFP were inversely modulated. Microarray and RT-PCR analyses indicated that the expression of defense-related genes was upregulated in DEWAX OX, but downregulated in dewax relative to WT. Transactivation assay showed that DEWAX upregulated the expression of PDF1.2a, IGMT1, and PRX37. Chromatin immunoprecipitation assay revealed that DEWAX directly interacts with the GCC-box motifs of PDF1.2a promoter. In addition, ectopic expression of DEWAX increased the tolerance to B. cinerea in C. sativa. Taken together, we suggest that increased ROS accumulation and DEWAX-mediated upregulation of defense-related genes are closely associated with enhanced resistance to B. cinerea in Arabidopsis and C. sativa.

Camelina sativa, which belongs to the Brassicaceae family, has recently gained increasing interest for its potential non-food usages, including in cosmetics, lubricants, and as biofuels (Moser, 2010;Waraich et al., 2013). C. sativa has a short life cycle (100-120 days), and can be grown on marginal agricultural lands owing to its characteristics, such as tolerance to cold stress, relatively good growth under nutrient-poor soil conditions, and high levels of polyunsaturated fatty acids in the seed oils (Putnam et al., 1993;Zubr, 1997;Enjalbert et al., 2013). To date, transgenic C. sativa plants with high seed-oil content or enhanced drought resistance have been developed via Agrobacterium-mediated transformation (Liu et al., 2012;Lee et al., 2014;An and Suh, 2015;Kim et al., 2016). However, only few reports on genetically improved C. sativa with enhanced tolerance to pathogens are available (Zakharchenko et al., 2013).
In this study, we observed that DEWAX OX plants were more resistant to the fungal pathogen, B. cinerea, but were more susceptible to the bacterial pathogen, Pto DC3000 compared with wild type (WT). To understand the molecular mechanisms underlying the defense responses of Arabidopsis and C. sativa plants overexpressing DEWAX to B. cinerea, further analyses were carried out. Finally, we revealed that DEWAX plays as a transcriptional activator that upregulates the expression of the defense-related genes such as PDF1.2a, IGMT1, and PRX37 via binding to their gene promoters. The results suggest that DEWAX-mediated upregulation of defense-related genes in addition to an increase in ROS levels is important in the enhanced tolerance to B. cinerea in Arabidopsis and C. sativa overexpressing DEWAX.

RNA Isolation and Quantitative RT-PCR Analysis
Total RNA was isolated from 4-week-old Col-0, dewax, DEWAX lines (OX1 and OX2), and 6-week-old transgenic C. sativa lines FIGURE 1 | Overexpression of DEWAX increased cuticle permeability and ROS accumulation in Arabidopsis. (A) Cuticle permeability analysis. Leaves of WT, dewax, and DEWAX overexpression lines were stained with 0.05% toluidine blue for 8 min. (B) In situ detection of hydrogen peroxide. Leaves of WT, dewax, and DEWAX overexpression lines were incubated in 10 mM MES (pH 6.5) containing 0.1% DAB for 30 min, de-stained by boiling in ethanol-lactophenol (2:1) for 5 min, washed with 50% ethanol, rinsed with distilled water, and then photographed. (C) Measurements of ROS levels. Leaves of WT, dewax, and DEWAX overexpression lines were incubated in 10% MS salt solution containing 20 µM H 2 DCFDA and 0.1% Tween-20 for 30 min. DCF fluorescence in aliquots taken from the extracts was measured at 488 and 525 nm. Leaf fresh weights were used to normalize the values. Data were statistically analyzed using Student's t-test ( * P < 0.05). Error bars indicate ± SD from triplicate experiments. (D) Measurements of hydrogen peroxide content. Five-week-old WT, dewax, OX1, and OX2 leaves (200 mg) were incubated in 1.5 mL of 5 mM MES buffer (pH 6.7) for 7 h. Peroxidase reaction was started by adding DCHBS, 4-AAP, and peroxidase (Megazyme) and the reaction mixture was incubated for 1 h at 25 • C. Absorbance for the quinoneimine dye was spectrophotometrically measured at 520 nm. H 2 O 2 content was estimated using a standard curve. Data were statistically analyzed using Student's t-test ( * P < 0.01). Error bars indicate ± SD from triplicate experiments.
(T 1 generation) using the NucleoSpin RNA Plant Extraction Kit (Macherey-Nagel) according to the instructions of the manufacturer. The total RNA was reverse-transcribed with GoScript TM Reverse Transcription System (Promega), and the cDNA was used in RT-PCR analysis. RT-PCR was performed using the Access Quick TM RT-PCR system (Promega, Madison, WI, United States) with gene-specific primers (Supplementary  Table S1). Arabidopsis EIF4 (At3g13920) and C. sativa ACTIN11 genes (Hutcheon et al., 2010) were used in RT-PCR analysis as a control for cDNA quality and quantity. Quantitative RT-PCR (qRT-PCR) was executed using SYBR R FAST Universal 2x qPCR Master Mix (KAPA Biosystems, Wilmington, MA, United States) in a final volume of 20 µL. PCR was performed according to the manufacturer's protocols using gene specific primers described in Supplementary Table S1. PP2A (At1g13320) and CsACT11 genes were used to determine the RNA quality and quantity.

Toluidine Blue Staining
Four-week-old Arabidopsis rosette leaves were stained at room temperature for 8 min without shaking using freshly prepared staining solution containing 0.05% toluidine blue and 0.01% Tween-20. After staining, leaves were rinsed five times with distilled water and photographed.

Pathogen Inoculation
Botrytis cinerea strain B05.10 was kindly provided by Dr. Mengiste (Purdue University, United States) and was cultured :GFP in WT, dewax and DEWAX overexpression lines were measured at 3 dpi. Colony forming unit (CFU) was normalized as CFU/mg using total weights of the inoculated leaves. The results are representative of three independent experiments. Error bars indicate SE. Asterisks indicate a statistically significant difference compared with wild type (WT) using Student's t-test ( * * P < 0.01, * * * P < 0.001).
on 2× V8 tomato agar (36% V8 juice, 0.2% CaCO 3 , and 2% Bacto-agar). Harvesting and inoculation of conidia were carried out according to Veronese et al. (2006). Detached Arabidopsis leaves were laid out on 3MM paper with water and inoculated with 5-µl droplets of conidial suspension (5 × 10 5 spores/mL), kept under a sealed transparent cover to maintain high humidity and transferred to a growth chamber (21 ± 2 • C, 16 h light/8 h dark). C. sativa plants were sprayed with B. cinerea conidial suspension (2 × 10 5 spores/mL). After inoculation, the plants were transferred to growth chambers (21 ± 2 • C, 16 h light/8 h dark) and covered with a sealed clear lid to maintain low light intensity and high humidity. Quantification of B. cinerea biomass in C. sativa was carried out according to Gachon and Saindrenan (2004).
A Pto DC3000 strain harboring pDSK-GFPuv (Pto DC3000:GFP, Wang et al., 2007) was used for dip inoculation (Katagiri et al., 2002). Briefly, 3-week-old Arabidopsis plants grown in meshed pots were dipped in 50 ml of bacterial suspensions (optical density at 600 nm of 0.1) containing 0.05% Silwet L-77 (Lehle Seeds, Round Rock, TX, United States) for few seconds. After inoculation, the plants were covered with a clear lid and transferred to a growth room (23 ± 2 • C, 16 h light/8 h dark). Inoculated plants were photographed under ultraviolet light to determine PtoDC3000:GFP proliferation using GFP signals and bacterial populations were measured by serial dilution assays (Katagiri et al., 2002). Colony forming unit (CFU) was normalized as CFU/mg using total weights of the inoculated leaves.

Transactivation Assay in Yeast
For the transcactivation assay in yeast, DEWAX-F (603 bp for full-length), DEWAX-N (312 bp for N-terminal region), and DEWAX-C (291 bp for C-terminal region) DNA fragments were amplified via PCR using DEWAX cDNA and DEWAX-specific primers (Supplementary Table S1). SmaI and SacI-digested PCR fragments were cloned into the pGBKT7 vector (BD Biosciences Clontech). The recombinant vector (pAtDEWAX-F, pAtDEWAX-N, and pAtDEWAX-C) were transformed into List of genes that were upregulated in stems of DEWAX OX2 relative to WT. Total RNA was isolated from stems of DEWAX OX2 and WT plants and subjected to microarray analysis using Arabidopsis ATH1 gene chips. FC, fold change. (B) Quantitative RT-PCR (qRT-PCR) analysis of genes upregulated in leaves of DEWAX OX2 as compared to WT. Total RNA was isolated from leaves of 4-week-old WT, dewax, and DEWAX overexpression lines (OX1 and OX2) and subjected to qRT-PCR analysis. The PP2A (At1g13320) gene was used as a reference to determine the RNA quality and quantity. Data were statistically analyzed using Student's t-test ( * P < 0.01). Error bars indicate ± SD from triplicate experiments.

Transcriptional Activation Assay
To generate the reporter constructs, DNA fragments corresponding to the PDF1.2a (∼1.3 kb), IGMT (∼1.5 kb), PRX37 (∼1.2 kb) promoter regions were amplified using genespecific primers and cloned into the SalI and SpeI sites of the GAL4-LUC binary vector. The recombinant pPZP212 vector (Hajdukiewicz et al., 1994) harboring Arabidopsis DEWAX driven by the CaMV35S promoter, reported by Go et al. (2014), was used as an effector construct. Protoplast isolation was carried out according to Yoo et al. (2007).
The reporter and effector constructs were isolated using a Qiagen Plasmid Midi Kit and co-introduced into tobacco protoplasts (Miura et al., 2007). After incubation of the transfected tobacco protoplasts in the dark at 23 • C for 16 h, the protoplasts were subjected to enzymatic assays to quantify luciferase and β-glucuronidase activities using a dualluciferase assay system (Promega). Luciferase gene expression was normalized to that of the β-glucuronidase gene.

Construction of a Binary Vector and Transformation of C. sativa
To generate transgenic C. sativa plants overexpressing Arabidopsis DEWAX, the full-length DEWAX gene was amplified from cDNA converted from RNA isolated from 3-week-old leaves with DEWAX F1 and DEWAX R1 primers (Supplementary Table S1). The PCR product was digested with Sac1 and Sma1 and cloned into the binary vector pBA002. The binary vectors were transformed into Agrobacterium strain GV3101 by the freeze-thaw method (An, 1987).
Agrobacterium harboring the 35S:MYC-DEWAX construct (Go et al., 2014) was inoculated in YEP medium (containing 50 µg/mL rifampicin and 50 µg/mL spectinomycin) and cultured at 30 • C overnight with shaking. The culture was centrifuged at 3,000 rpm for 20 min and the pellet was resuspended in transformation solution (5% sucrose and 0.05% Silwet L-77) at an OD 600 of 0.8. Five-to six-week-old C. sativa plants were transformed with Agrobacterium by a modified floral dip method (Lu and Kang, 2008;Liu et al., 2012). T 1 transgenic seeds were sowed on mixed soil and selected by spraying of 0.03% (v/v) BASTA herbicide.

Genomic DNA Isolation and PCR Analysis
Genomic DNA was isolated from leaves of 3-week old non-transgenic and transgenic C. sativa plants (T 1 generation) using extraction buffer (200 mM Tris-HCl, pH 7.5, 250 mM NaCl, 25 mM EDTA, and 0.5% sodium dodecyl sulfate) and subjected to genomic DNA PCR analysis with CaMV35S promoter F and DEWAX CR1 primers (Supplementary Table S1). Thermal cycles included 32 cycles of denaturation at 94 • C for 30 s, annealing at 60 • C for 30 s, and elongation at 72 • C for 60 s.

Overexpression of DEWAX Increased Cuticle Permeability and ROS Accumulation in Arabidopsis
To investigate the cuticle permeability of WT, dewax, and two DEWAX OX lines, rosette leaves of 4-week-old plants were stained with 0.05% toluidine blue. As shown in Figure 1A, compared to WT, Arabidopsis DEWAX OX and dewax leaves were more and less permeable to toluidine blue dye, respectively. Based on the previous reports that Arabidopsis bdg and lacs2 mutants with a permeable cuticle showed increased ROS levels (L'Haridon et al., 2011), ROS assay was performed in the leaves of WT, dewax, and DEWAX OX lines. Both in situ assay of hydrogen peroxide and fluorometric measurements of ROS levels showed that the levels of ROS were lower in dewax leaves, but higher in DEWAX OX1 and OX2 leaves than in WT leaves (Figures 1B,C). When hydrogen peroxide content was spectrophotometrically measured after leaves of WT, dewax, and DEWAX OX lines were preadapted in incubation buffer, the levels of hydrogen peroxide were approximately twofold higher in DEWAX OX1 and OX2 leaves than in WT leaves, but no remarkable differences were detected between WT and dewax leaves ( Figure 1D).

Overexpression of DEWAX Enhanced Resistance, While Disruption of DEWAX Increased Susceptibility to B. cinerea
To investigate the response of WT, dewax, and two DEWAX OX lines to the necrotrophic pathogen B. cinerea and the hemibiotrophic bacterial pathogen Pto DC3000, first, we examined visible disease symptoms 72 h after inoculation of B. cinerea on the adaxial surface of the leaves. In a spot inoculation assay, significantly larger lesions were observed on the leaves of dewax mutant, while smaller lesions were formed on DEWAX OX as compared with WT leaves (Figures 2A,B), indicating that DEWAX OX shows enhanced resistance, and dewax enhanced susceptibility, to B. cinerea. On the other hand, compared to WT, DEWAX OX was more susceptible to Pto DC3000:GFP, while dewax was more resistant to this pathogen (Figures 2C,D). These results indicate that DEWAX is involved in the disease resistance responses of Arabidopsis and plays a positive role in the defense to fungal pathogens, but a negative role in the defense to bacterial pathogens.

Expression of DEWAX-Regulated Genes in DEWAX OX Lines and the dewax Mutant
To obtain further insight in the DEWAX-mediated defense responses of Arabidopsis, comparative transcriptome analysis of WT and DEWAX OX was conducted (Go et al., 2014; E-MEXP-3781 at http://www.ebi.ac.uk/arrayexpress). Interestingly, the expression of genes involved in pathogen defense and ROS production was clearly upregulated in the stems of DEWAX OX as compared to those of WT (Penninckx et al., 2003;Almagro et al., 2009;Pedreira et al., 2011). Genes that were upregulated 10-fold and more in DEWAX OX2 than in WT are listed in Figure 3A. RT-PCR was carried out to confirm their transcript levels in 4-week-old WT, dewax, and DEWAX OX1 and OX2 leaves. The expression levels of PDF1.2a and PDF1.2b, involved in plant defense, were remarkably induced in DEWAX OX1 and OX2 leaves, but reduced in dewax as compared to WT leaves. The transcript levels of indole glucosinolate methyltransferase (IGMT1) and peroxidase C2 precursor (PRX37 and PRX38) were increased in DEWAX OX1 and OX2 leaves, while no significant downregulation was observed in dewax relative to WT. No noticeable differences in the expression of At1g67810 and At4g16260, encoding SULFUR (Stimulate CpNifS Cysteine Desulfurase Activity) and a β-1,3glucanase precursor, respectively, were observed in dewax, and DEWAX OX1 and OX2 leaves (Figure 3B and Supplementary Figure S1).

DEWAX Functions as a Transcriptional Activator
DEWAX has been reported to function as a transcriptional repressor in cuticular wax biosynthesis (Go et al., 2014). However, upregulation of defense-related genes in DEWAX OX and their downregulation in dewax prompted us to examine whether DEWAX acts as a transcriptional activator in tobacco protoplasts and yeast cells. A reporter construct, GAL4-LUC, harbors a luciferase gene driven by a promoter including a GAL4 motif. DEWAX was translationally ligated to the C-terminus of the GAL4 DNA-binding domain under the control of CaMV35S promoter in pBI221 vector  and used as an effector construct, GAL4DB:DEWAX. Tobacco protoplasts were transformed with a GAL4-LUC reporter construct only or co-transformed with a GAL4-LUC construct and an effector construct, GAL4DB or GAL4DB:DEWAX. Strong luciferase activities were observed in protoplasts transformed with GAL4-LUC reporter and a GAL4DB:DEWAX effector constructs as compared to those transformed with GAL4-LUC reporter and GAL4DB effector constructs ( Figure 4A).
In addition, the full-length (F, amino acids 1 to 201), N-terminal (N, amino acids 1 to 106), and C-terminal (C, amino acids 107 to 201) regions of DEWAX were cloned downstream of the GAL4-binding domain in the pGBKT7 vector. Yeast Y190 cells, which possess the HIS3 and lacZ genes driven by the promoter including GAL4-responsive elements, were transformed with the constructed vectors. Yeast cells that were able to survived on tryptophan-deficient selective medium (SD/-Trp) were further selected on tryptophan-and histidine-deficient medium (SD/-Trp-His) supplemented with 10 mM 3-amino-1, 2, 4-aminotriazole (3-AT) and subsequently subjected to an X-gal filter-lifting assay. Yeast cells transformed with the F or N construct survived on SD/-Trp-His medium supplemented with 10 mM 3-AT, and generated blue colonies by the β-galactosidase activities in X-gal lift assay. However, yeast cells transformed with the pGBKT7 vector or the C construct did not survive on the selective medium, and showed no β-galactosidase activity. These results indicate that DEWAX is able to act as a transcriptional activator, and amino acid residues 1 to 106 of DEWAX are involved in transcriptional activation ( Figure 4B).

DEWAX Promotes the Expression of PDF1.2 via Direct Binding to Conserved Sequence Motifs in its Promoter
To examine whether DEWAX is able to activate the expression of the putative target genes, PDF1.2a, IGMT1, and PRX37, their promoter regions were transcriptionally ligated to the luciferase gene (LUC) in the Gal4-LUC binary vector, and used as reporter constructs (Figure 5A). p35S and p35S-DEWAX as effector constructs were used in transactivation assay of tobacco protoplasts (Go et al., 2014). After tobacco protoplasts were co-transformed with each reporter construct containing LUC, the effector construct, and the internal control construct harboring the GUS gene, luciferase (LUC) and β-glucuronidase (GUS) activities were measured, and the LUC activity was normalized to the GUS activity. When DEWAX was expressed, LUC activity was upregulated in the protoplasts transformed with PDF1.2a, IGMT1, and PRX37 reporter constructs by approximately 2.3, 4.2, and 6.3-fold, respectively, but was downregulated in the protoplasts transformed with CER1 by approximately twofold relative to the control co-transformed with p35S ( Figure 5B).
We further investigated whether DEWAX directly interacts with the consensus GCC-box motifs of PDF1.2a promoter region using 35S:MYC-DEWAX transgenic plants (Go et al., 2014). Quantitative real-time ChIP-PCR assay showed that DEWAX binds directly to the promoters of the PDF1.2a gene in planta ( Figure 5C). This observation indicates that DEWAX activates the expression of PDF1.2a gene by direct interaction with its promoter region.
Overexpression of DEWAX Enhanced the Resistance to B. cinerea in C. sativa As DEWAX overexpression in Arabidopsis led to strong resistance to B. cinerea, we next evaluated how overexpression of DEWAX FIGURE 5 | DEWAX upregulates the expression of the PDF1.2 gene by direct binding to conserved sequence motifs in its gene promoter. (A) Schematic diagrams of reporter and effector constructs used for the transcriptional activation assay. In the reporter constructs, the promoter regions of DEWAX-activated genes were fused to the luciferase gene. In the effector constructs, DEWAX was cloned between the CaMV35S promoter and the terminator of the nopaline synthase gene (Nos-T). (B) Transcriptional activation assay of DEWAX-activated genes in tobacco protoplasts. The LUC activity was normalized to the GUS activity, which was from the internal control construct harboring the GUS gene. Mean fold change in relative luciferase activity was calculated by dividing the normalized luciferase activity obtained from the protoplasts transformed with the control effector construct. Data were statistically analyzed using Student's t-test ( * P < 0.01). Error bars indicate ± SD of the mean from triplicate experiments. -, P35S; +, P35S-DEWAX. (C) Description of the promoter region of the PDF1.2a gene and chromatin immunoprecipitation (ChIP) assay. a and b indicate regions including consensus GCC-box motifs that were used in ChIP assay. In each qRT-PCR measurement, the value for WT was set to 1 after normalization against actin7. Data were statistically analyzed using Student's t-test ( * P < 0.01). Error bars indicate ± SD of the mean from triplicate samples. in a Brassica crop, C. sativa, affects disease resistance. The 35SP-DEWAX construct harboring DEWAX gene under the control of CaMV 35S promoter was used in the transformation of C. sativa (Figure 6A). Transgenic plants were screened through spraying of 0.03% BASTA herbicide ( Figure 6B). Genomic DNA PCR analysis of non-transgenic (NT) and 16 transgenic plants (TO) using gene-specific primers (Supplementary Table S1) showed that Arabidopsis DEWAX was integrated in all transgenic plants tested (Supplementary Figure S2A). RT-PCR analysis revealed that the DEWAX gene was overexpressed in the leaves of all transgenic plants tested (Supplementary Figure S2B and  Table S1).
Next, we examined the defense responses of NT and TO plants to B. cinerea. Visible disease symptoms, B. cinerea biomass, and the expression of Arabidopsis DEWAX were investigated in leaves of NT and TO-2, TO-4, TO-17, and TO-20 lines 4 days after inoculation with B. cinerea. Yellowing or chlorosis occurred more quickly in NT than in TO leaves ( Figure 6C). To measure B. cinerea biomass, genomic DNA was isolated from the leaves 4 days after inoculation with B. cinerea and subjected to real-time quantitative PCR using B. cinerea-specific cutinase Aspecific gene primers (Supplementary Table S1), while C. sativa GSK3/shaggy-like (iASK) gene was used for normalization. Compared to NT plants, all TO lines tested had lower B. cinerea biomass (Figure 6D). Subsequently quantitative RT-PCR analysis showed that the levels of C. sativa PDF increased by 2-to 3.5fold in DEWAX OX lines compared with NT ( Figure 6E). These results indicated that overexpression of Arabidopsis DEWAX confers tolerance to B. cinerea in transgenic C. sativa.

DISCUSSION
Plant immune responses are essential for not only growth and development, but also productivity and yields of crops. In this study, we investigated a plant defense mechanism related to the cuticle, which is the first defensive barrier of the aerial parts of plants. We revealed that overexpression of DEWAX increased cuticle permeability and ROS accumulation in Arabidopsis. DEWAX is a transcriptional activator that upregulates the expression of the defense-related genes, PDF1.2a, IGMT1, and PRX37, by binding to their gene promoters. In particular, DEWAX directly interacts with the GCC motifs in the PDF1.2a promoter. Overexpression of DEWAX conferred enhanced tolerance to necrotrophic fungal pathogen, B. cinerea in Arabidopsis and C. sativa. Therefore, convergent line of evidence supports that increased ROS levels as well as upregulation of defense-related genes might be related to the resistance to B. cinerea in Arabidopsis and C. sativa overexpressing DEWAX. Go et al. (2014) reported the AP/ERF transcription factor DEWAX as a transcriptional repressor involved in cuticular wax biosynthesis during daily light/dark cycles. However, we revealed that DEWAX is also able to act as a transcriptional activator that induces defense-related genes in this study. AP2/ERF transcription factors are known to function as both negative and positive regulators (Ohta et al., 2001;Aharoni et al., 2004;Yant et al., 2010). The N-terminus of DEWAX harbors the "EDLL" motif, which is an acidic-type transcriptional activation domain of AP2/ERF transcription factors (Tiwari et al., 2012), which is consistent with our yeast transactivation assay results. Direct binding of DEWAX as well as binding of OCTADECANOID-RESPONSIVE ARABIDOPSIS (ORA)59 and ERF96 with the "EDLL" motif to the two GCC motifs in the PDF1.2 promoter region, is essential for the expression of PDF1.2 (Solano et al., 1998;Zarei et al., 2011;Catinot et al., 2015). Although it remains unclear how exactly the bifunctional DEWAX regulates the expression of its target genes positively or negatively, possibly, DEWAX interacts with a co-activator, such as MED25 of the mediator complex, followed by the recruitment The infected plants were collected at 4 days post inoculation, and genomic DNA was isolated. B. cinerea biomass was quantified by real-time quantitative PCR with B. cinerea cutinase A-specific primers and normalized to C. sativa iASK gene expression. All data represent the average of at least three plants. Error bars represent ± SD of the means. Data were statistically analyzed using Student's t-test ( * * P < 0.01, * * * P < 0.001). (E) qRT-PCR analysis of DEWAX and C. sativa PDF (CsPDF) genes. Total RNA was isolated from leaves of 5-week-old NT and DEWAX C. sativa OX plants and subjected to qRT-PCR analysis. The CsActin11 gene (Hutcheon et al., 2010) was used as a reference to determine the RNA quality and quantity. Data were statistically analyzed using Student's t-test ( * * P < 0.01). Error bars indicate ± SD from triplicate experiments. of transcriptional machinery including histone acetyltransferase, or with a co-repressor that interacts with histone deacetylase (Cevik et al., 2012;Huang and Geng, 2017).
Some Arabidopsis mutants with increased cuticular permeability exhibit resistance to pathogen infection (Nawrath, 2006;Serrano et al., 2014). For example, lcr/lacerate/cyp86A8, bdg, lacs-2 and -3, fdh, fec1, and myb96 showed enhanced resistance to B. cinerea, similar to our observations in DEWAX OX lines (Yephremov et al., 1999;Wellesen et al., 2001;Bessire et al., 2007Bessire et al., , 2011Chassot et al., 2007;Voisin et al., 2009;Seo et al., 2011). We also observed increased hydrogen peroxide levels in DEWAX OX leaves (Figure 1B), which is consistent with the results in the Arabidopsis mutants, bdg and lacs2 (L'Haridon et al., 2011), although we could not exclude the possibility that the higher levels of hydrogen peroxide in DEWAX OX leaves may be attributed, to some extent, to smaller DEWAX OX leaves. Transgenic lines overexpressing oxalate decarboxylase and thus, having higher levels of oxalic acid, which inhibits ROS production, showed decreased innate immune response (Cessna et al., 2000;L'Haridon et al., 2011). Therefore, elevated ROS production in Arabidopsis overexpressing DEWAX might be involved in the resistance to B. cinerea, although the molecular mechanisms underlying immune responses of mutants with permeable cuticle to B. cinerea are still largely unknown.
The expression of PDF1.2 was significantly upregulated in Arabidopsis seedlings treated with jasmonic acid (JA) and ethephon, a form of liquid ethylene (Lorenzo et al., 2003;Pre et al., 2008). The JA-and ethylene-responsive expression of PDF1.2 depended on ERF1 and ORA59 transcription factors, which act as integrators of JA and ethylene signaling pathways. Overexpression of ERF1 and ORA59 caused increased resistance, and ora59 mutant enhanced susceptibility to B. cinerea (Berrocal-Lobo et al., 2002;Pre et al., 2008). However, the transcript levels of DEWAX were elevated neither by JA nor by ethephon 1 , although DEWAX is classified with ERF1 and ORA59 into the group IX of Arabidopsis AP2/ERF gene family (Nakano et al., 2006). Based on the previous reports that Arabidopsis defense responses to herbivores and pathogens are regulated by circadian rhythms (Wang et al., 2011;Goodspeed et al., 2012), it would be interesting to examine whether upregulation of defense-related genes, including PDF1.2 by the diurnally controlled DEWAX may be important in circadian clock-mediated plant innate immunity. In addition, JA and salicylic acid are known to play a role as an antagonist in plant innate immunity (Thaler et al., 2012). The induction of the PDF1.2 gene in DEWAXoverexpressing Arabidopsis indicates that JA-associated defense responses are activated, and this may cause the suppression of SA-related signaling. This hypothesis supports that DEWAXoverexpressing plants are more resistant to the necrotropic fungal pathogen, B. cinerea, but are more susceptible to the semi-biotropic bacterial pathogen, Pto DC3000 compared with WT.
Although C. sativa is considered to be resistant to many diseases, it is reportedly sensitive to some fungal and bacterial pathogens causing wilting, root rot, clubroot, white rust, and downy mildew diseases (Séguin-Swartz et al., 2009;Zakharchenko et al., 2013). Zakharchenko et al. (2013) reported that transgenic C. sativa overexpressing the antimicrobial peptide cecropin P1 exhibits enhanced resistance to Erwinia carotovora and Fusarium sporotrichioides. In this study, the Arabidopsis dewax mutant showed increased resistance to Pto DC3000, whereas overexpression of DEWAX conferred elevated resistance to B. cinerea in Arabidopsis and C. sativa, suggesting the potential for developing Brassica lines resistant to various diseases. In conclusion, our study revealed that DEWAX is a transcriptional activator that upregulates the expression of defense-related genes, and overexpression of DEWAX enhances the resistance to B. cinerea in Arabidopsis and C. sativa. We provide that DEWAX-mediated defense mechanisms may be applicable in the improvement of crops with enhanced resistance to fungal and bacterial pathogens.

AUTHOR CONTRIBUTIONS
SJ, YG, and MS conceived and designed research. SJ, YG, and HC conducted experiments. SJ, JP, and MS analyzed data. SJ, JP, and MS wrote the manuscript. All authors read and approved the manuscript.