CmWRKY15 Facilitates Alternaria tenuissima Infection of Chrysanthemum

Abscisic acid (ABA) has an important role in the responses of plants to pathogens due to its ability to induce stomatal closure and interact with salicylic acid (SA) and jasmonic acid (JA). WRKY transcription factors serve as antagonistic or synergistic regulators in the response of plants to a variety of pathogens. Here, we demonstrated that CmWRKY15, a group IIa WRKY family member, was not transcriptionally activated in yeast cells. Subcellular localization experiments in which onion epidermal cells were transiently transfected with CmWRKY15 indicated that CmWRKY15 localized to the nucleus in vivo. The expression of CmWRKY15 could be markedly induced by the presence of Alternaria tenuissima inoculum in chrysanthemum. Furthermore, the disease severity index (DSI) data of CmWRKY15-overexpressing plants indicated that CmWRKY15 overexpression enhanced the susceptibility of chrysanthemum to A. tenuissima infection compared to controls. To illustrate the mechanisms by which CmWRKY15 regulates the response to A. tenuissima inoculation, the expression levels of ABA-responsive and ABA signaling genes, such as ABF4, ABI4, ABI5, MYB2, RAB18, DREB1A, DREB2A, PYL2, PP2C, RCAR1, SnRK2.2, SnRK2.3, NCED3A, NCED3B, GTG1, AKT1, AKT2, KAT1, KAT2, and KC1were compared between transgenic plants and controls. In summary, our data suggest that CmWRKY15 might facilitate A. tenuissima infection by antagonistically regulating the expression of ABA-responsive genes and genes involved in ABA signaling, either directly or indirectly.


Introduction
Recently, the complex mechanism by which abscisic acid (ABA) responds to pathogens has been extensively studied and reviewed [1,2].Stomatal closure is commonly regarded as a defense mechanism that can prevent bacterial pathogen infection [3].As a result, ABA can have a positive effect on disease resistance through its regulation of stomatal movements.Furthermore, ABA has emerged as an important regulator of interactions with other hormones involved in plant defense mechanisms [1].
Plants must defend themselves against diverse types of pathogens and must be capable of enduring pathogen-induced stress conditions.A number of plant hormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), are associated with pathogen defense mechanisms [4].It is generally believed that SA behaves as a critical regulator of defense responses against biotrophic pathogens such as Oidium neolycopersici and Hyaloperonospora parasitica [5].JA-and ET-associated defense mechanisms yield resistance against necrotrophic pathogens, such as Botrytis cinerea, Alternaria brassicicola and Plectosphaerella cucumerina [6,7].Several studies have suggested that ABA can interact antagonistically or synergistically with pathogen infection-related signaling pathways involving SA, JA and ET.Although ABA negatively regulates SA, it can enhance resistance to necrotrophic pathogen attacks by increasing JA biosynthesis [6].Thus, these results demonstrate that ABA can function in plant immunity by promoting one defense pathway while impairing another signaling pathway, allowing plants to integrate and fine-tune their defense mechanisms against diverse types of pathogens and colonization attempts [8].
Moreover, an ever-growing body of evidence has revealed that ABA can act either synergistically or antagonistically with host defense mechanisms against pathogens.Mounting evidence supports the notion that ABA represses resistance to pathogens.For example, ABA accumulation was found to increase the susceptibility of barley to the hemi-biotroph Magnaporthe oryzae [9].In addition, three Arabidopsis ABA mutants, aba2-12, aao3-2, and abi4, were found to exhibit enhanced stress-adaptive responses to the necrotroph B. cinerea; ABA biosynthesis was impaired in both aba2-12 and aao3-2, whereas abi4 was insensitive to ABA.These three ABA mutants exhibited increased susceptibility to the necrotroph Alternaria brassicicola [7].Thus, ABA fine-tuned and enhanced the immune response to pathogen attacks.Similarly, exogenous ABA application was found to provide rice (Oryza sativa) with the capability to resist brown spot disease caused by Cochliobolus miyabeanus [2].Interestingly, exogenously administered ABA also enhanced the basal defense of tomato (Solanum lycopersicon L.) against Alternaria solani [10].Taken together, these data indicate that the role of ABA in regulating pathogenassociated pathways is multifaceted.Moreover, the mechanism of interaction between WRKY transcription factors and ABA in response to pathogens remains an open question.
The WRKY family was named based on the WRKY domain, consisting of a conserved WRKYGQK heptapeptide at the N-terminus along with a C 2 H 2 -or C 2 HC-type zinc finger motif [11].Additional research has shown that WRKY transcription factors can bind to the W box (TTGACY) sequence, thus allowing interaction with the promoters of target genes.The WRKY transcription factor family has been identified in many species, such as A. thaliana, Glycine max, Brachypodium distachyon and Chrysanthemum morifolium [11,12].In Arabidopsis, the WRKY transcription factor family consists of over 74 members, and 15 WRKY genes were isolated from chrysanthemum [13].Five group II WRKY genes have been recently cloned from chrysanthemum, one of which inhibits aphid population growth [14].In this study, we focused on CmWRKY15, which is homologous to AtWRKY40 in Arabidopsis [13].
There are three functionally and structurally homologous WRKY transcription factors in A. thaliana: WRKY18, WRKY40 and WRKY60.These transcription factors trigger a complex pattern of activity that is implicated in pathogen infection and ABA-associated signaling [15].WRKY40 acts as a negative or positive regulator of plant defense mechanisms against pathogens.For example, WRKY18 and WRKY40, which are closely related, worsened biotrophic powdery mildew infection of Arabidopsis due to their negative effects on pre-infection mechanisms of host defense [15].In contrast, WRKY18 and WRKY40 were found to act synergistically in effector-triggered immunity, as the wrky18wrky40 double mutant showed increased susceptibility to the bacterial pathogen Pseudomonas syringae DC3000 releasing the effector AvrRPS4 [16].
Chrysanthemum (Chrysanthemum morifolium), which is one of the most famous cut flowers globally, has high ornamental value and occupies an irreplaceable position in international flower commerce [19].Black spot disease, one of the most harmful diseases of chrysanthemum, is caused by the necrotrophic fungus Alternaria.High humidity and warm conditions result in serious disease, which causes year-round reductions in greenhouse-based yields.Spraying broad-spectrum fungicide not only leads to increases in cost and energy consumption but also causes severe environmental contamination and alters pesticide resistance.Hence, to alleviate pathogen damage, more research is needed on the molecular mechanisms of Alternaria tenuissima infection of chrysanthemum.Previous research suggests that WRKY genes act as central regulatory factors in plant disease immunity [20,21].In this study, we investigated the mechanisms by which CmWRKY15 regulates the ABA response to A. tenuissima in chrysanthemum.

Plant materials and growth conditions
We used the chrysanthemum cultivar 'Jinba', which was obtained from the Chrysanthemum Germplasm Resource Conservation Center, Nanjing Agricultural University, China.Uniform cuttings were propagated in pots using a 1:1 (v/v) mixture of soil and vermiculite and cultivated in a greenhouse (day/night temperature of 25/18°C, a light/dark photoperiod of 14/10 h, a light intensity of 50 μmol m -2 s -1 and a relative humidity of 70%).

Phylogenetic analysis of homologous CmWRKY15 sequences
The CmWRKY15 amino acid sequence was aligned with its homologues using DNAman 5.2.2 software and BLAST (http://www.ncbi.nlm.gov/blast).The conserved WRKY domains of CmWRKY15 homologues were acquired from the NCBI database (http://www.ncbi.nlm.nih.gov).We performed various sequence alignments of the WRKY domains from different species using ClustalW [22].To obtain better classifications of the multiple branches, we generated phylogenetic trees containing eighteen representative orthologs from the CmWRKY15 alignment using the MEGA5 program [23] following the neighbor-joining method with 1000 bootstrap replicates.

Transcriptional activity analysis of CmWRKY15
The transcriptional activation of CmWRKY15 was evaluated using a yeast assay system [24].The ORF of CmWRKY15 without the stop codon was amplified using the Phusion 1 High Fidelity PCR Kit (New England Biolabs, Ipswich, MA, USA) with the primer pair CmWRKY15-GATE-SAL-F/CmWRKY15-GATE-NOT-R (S1 Table ).The PCR products were cloned into the pENTR™1A vector (Invitrogen, Carlsbad, CA, USA) by Sal I/Not I double digestion and ligation.Sequencing was carried out to confirm the presence of CmWRKY15 in the construct.pEN-TR™1A-CmWRKY15 and pDEST-GBKT7 were recombined to form pDEST-GBKT7-CmWRKY15 using LR Clonase™ II enzyme mix (Invitrogen).pCL1 and pDEST-GBKT7 plasmids were used to create positive and negative control strains, respectively.pDEST-GBKT7-CmWRKY15, pCL1 and pGBKT7 were introduced into Saccharomyces cerevisiae strain Y2HGold (Clontech) according to the manufacturer's instructions.Selection of transformants carrying either pGBKT7-CmWRKY15 or pGBKT7 was performed using SD/-Trp medium, whereas pCL1 was selected using SD/-Leu medium.Y2H cells containing pCL1 (positive control) can persist on SD/-His-Ade medium.Conversely, Y2H cells containing pGBKT7 (negative control) cannot grow on this medium.We utilized a luminescence assay to assess the transcriptional activity of CmWRKY15.The CmWRKY15 ORF was amplified by PCR using the primer set CmWRKY15-GATE-F/R (S1 Table ) containing Bam HI and Not I sites to obtain the 35S:: GAL4DB-CmWRKY15 fusion construct.Further, the amplified DNA fragment was inserted into the pENTRTM1A dual selection vector (Invitrogen) to obtain pENTRTM1A-CmWRKY15, which was confirmed by sequencing.We recombined this plasmid with 35S::GAL4DB to generate 35S::GAL4DB-CmWRKY15 via the LR reaction (Invitrogen).Arabidopsis protoplasts were obtained and transfected according to the protocol previously described by Yoo et al [25].We transfected 7.5 μg of 35S::GAL4DB-AtARF5, 35S::GAL4DB or 35S::GAL4DB-CmWRKY15 with 7.5 μg of 5X GAL4-LUC, which contains the luciferase reporter gene driven by five copies of the GAL4 binding element.The luciferase activity was assessed as described previously [26].Three independent experiments were performed.

Subcellular localization of CmWRKY15
To assess the subcellular localization of CmWRKY15, we performed transient transfection of onion epidermal cells [27].To generate the p35S::GFP-CmWRKY plasmid, we first generated a green fluorescent protein (GFP)-CmWRKY15 fusion construct using the plasmid pENTR™1A-CmWRKY15, which is based on pMDC43, and LR Clonase™ II enzyme mix (Invitrogen).The p35S::GFP-CmWRKY construct and the empty pMDC43 vector, which was used as a marker for transgene expression, were transiently transfected into onion epidermal cells.Confocal laser microscopy was used to monitor GFP expression.

Transformation of chrysanthemum
We acquired CmWRKY15-overexpressing chrysanthemum transformants to further analyze the function of CmWRKY15.The Agrobacterium tumefaciens strain EHA105 was transformed with the plasmid 35S::CmWRKY15 using the freeze-thaw method.Transformation of chrysanthemum was performed as described above [14].Leaf discs (5 mm in diameter) obtained from mature plants of 'Jinba' cultured in vitro were used as explants.Initially, we selected transformants by cultivating them on a medium containing 8 mg L -1 hygromycin.After regeneration, RNA was extracted from the putative transgenic chrysanthemum and wild type (WT) plants using the RNAiso reagent (TaKaRa).Extracted RNA was digested with RNase-free DNase I (TaKaRa) and reverse transcribed using M-MLV reverse transcriptase (TaKaRa).The transcript level of CmWRKY15 was assessed with quantitative real-time PCR (qPCR) analysis using SYBR 1 Green (TaKaRa) and the primer pair CmWRKY15-DL-F/R (S1 Table ).The primer pair CmEF1α-F/R was used to amplify the reference gene CmEF1α.All qPCRs were run on a Mastercycler ep realplex device (Eppendorf, Hamburg, Germany).Fold changes in expression were calculated using the 2 −ΔΔCt method [28].

A. tenuissima infection of transgenic chrysanthemum and wildtype plants
Groups of 50 cuttings each were taken from transgenic chrysanthemum (W15) and non-transgenic chrysanthemum ('Jinba', WT) plants for A. tenuissima resistance detection.Groups of 10 cuttings each were used as controls with watering.Cuttings were maintained at 22 ± 3°C under a 14/10 h light/dark photoperiod in aerated water for 20 d. A. tenuissima conidia were isolated from naturally infected diseased chrysanthemum plants and cultured on potato dextrose agar medium at 25°C in the dark [29].Host resistance was assessed using the method described by Deng et al [30].After a 15-d culture, the conidia were suspended in sterile distilled water, and an aqueous suspension of 10 6 spores per ml was prepared with a few drops of Triton X-100 added as a wetting agent.This mixture was sprayed over the seedlings produced from 20-dayold root cuttings.Each treatment comprised 50 seedlings of each of transgenic chrysanthemum plant and wild type plants.Mock treatments were carried out with 10-μl droplets of sterile distilled water.After inoculation, the seedlings produced from 20-day-old root cuttings were maintained at 100% relative humidity in the dark at a temperature of 25°C for 48 h and then moved to a chamber held at a constant temperature of 25°C with a 16/8 h photoperiod, a light intensity of 50 μmol m −2 s −1 and a relative humidity of 90%.Three seedlings each of W15-1, W15-2 and WT were used for leaf sampling under each treatment at 0 h, 6 h, 24 h, 48 h, 72 h, 96 h and 120 h after exposure to A. tenuissima.We removed the third true leaf from each of the three seedlings of the same line.Each experiment included three biological replicates.Samples collected at defined time points for each treatment were pooled for RNA extraction.The proportion of plants showing disease symptoms (PPD) was measured 14 d after inoculation.The mean lesion size (MLS) at this time point was used to determine the resistance level according to Xu [29].Fourteen days after inoculation, plant disease severity was assessed on a 0-5 scale based on the percentage of leaf area that was symptomatic, such that 0 represented 0%, 1 represented up to 10%, 2 represented 11-25%, 3 represented 26-50%, 4 represented 51-75%, and 5 represented more than 75% or an abscised leaf.Chlorotic portions of leaves were considered infected.The disease severity index (DSI) for each plant was calculated using the following formula: (no.leaves in class × severity class)/(no.leaves examined × maximum severity class) × 100.The host plant response was classified based on the mean DSI such that 0 was immune (I), 1-10 was highly resistant (HR), 11-20 was resistant (R), 21-30 was moderately resistant (MR), 31-45 was moderately susceptible (MS), 46-70 was susceptible (S), and over 70 was highly susceptible (HS).

Measurement of ABA content
Uniform cuttings were propagated in pots using a 1:1 (v/v) mixture of soil and vermiculite and cultivated in a greenhouse (day/night temperature of 25/18°C, a light/dark photoperiod of 14/ 10 h, a light intensity of 50 μmol m -2 s -1 and a relative humidity of 70%).Each treatment comprised 10 seedlings of each of transgenic chrysanthemum line and wild type plants.Mock treatments were carried out with 10-μl droplets of sterile distilled water.After inoculation, seedlings produced from 20-day-old root cuttings were maintained at 100% relative humidity in the dark at a temperature of 25°C for 48 h and then moved to a chamber held at a constant temperature of 25°C with a 16/8 h photoperiod, a light intensity of 50 μmol m −2 s −1 and a relative humidity of 90%.The leaves of three seedlings from each treatment were sampled at 0 h and 24 h after exposure to A. tenuissima.We removed the third true leaf from each of the three plants of the same line.Each experiment included three biological replicates.Leaves from two transgenic lines and wildtype 'Jinba' were frozen in liquid nitrogen and then stored at -80°C until they were used in experiments.The ABA content was analyzed by ultra-performance liquid chromatography (UPLC).The extraction was performed based on a previously published protocol [31].In brief, 1 g of lyophilized plant material was immersed in 10 ml of cold 80% (v/v) methanol with constant shaking at 4°C for 12 h.After mechanical homogenization, the extract was clarified by centrifugation (10,000 g for 15 min) followed by incubation at 4°C with constant shaking for 1 h with the addition of 0.2 g of PVPP; the extract was then centrifuged as described previously.The supernatant was collected and passed through a C 18 Sep-Pak cartridge (Waters Corp., Milford, MA, USA).An aliquot of the eluate was dried under a stream of nitrogen gas, and the residue was dissolved in 1 ml of methanol.ABA was quantified using an Agilent 1290 Infinity UPLC system.

Statistical analysis
One-way analysis of variance was used to identify significant differences among treatments.Multiple comparisons were performed using Tukey's multiple range test (p = 0.05).All statistical analyses were carried out using SPSS v17.0 (SPSS Inc., Chicago, IL, USA).

Subcellular localization of CmWRKY15
The 35S::GFP-CmWRKY15 construct and a positive vector harboring only 35S::GFP were introduced into onion epidermal cells via particle bombardment.Onion epidermal cells expressing 35S::GFP showed GFP fluorescence throughout the cells (Fig 2a).In contrast, GFP fluorescence was localized solely in the nuclei of the onion epidermal cells transformed with the 35S::GFP-CmWRKY15 fusion protein (Fig 2a).These results indicated that CmWRKY15 localized to the nucleus in vivo.

Transrepression assay of CmWRKY15
Transcriptional activation of CmWRKY15 was evaluated using a yeast one-hybrid assay.The negative control pGBKT7 and the pGBKT7-CmWRKY15 construct were both incapable of growing on SD/-His-Ade medium, whereas the pCL1 positive control grew normally (Fig 2b).These results suggest that CmWRKY15 was not transcriptionally active in yeast cells.
To further understand the transactivation function of CmWRKY15, Arabidopsis protoplasts were transfected with the plasmid encoding CmWRKY15 together with a reporter plasmid.The results (Fig 2c) indicate that 35S::GAL4DB-AtARF5 showed a significant increase in relative LUC units (RLUs) compared to 35S::GAL4DB-CmWRKY15 in Arabidopsis protoplasts (P<0.01),whereas 35S::GAL4DB-CmWRKY15 showed a decrease in RLUs compared with

CmWRKY15 overexpression enhanced the susceptibility of chrysanthemum to A. tenuissima
We used transgenic chrysanthemum lines overexpressing CmWRKY15, the relative expression levels of which were determined using quantitative real time PCR (qPCR).In the overexpressing plants (W15-1, W15-2, W15-5 and W15-6), the expression levels of CmWRKY15 were markedly higher than those in the nontransformed controls (Fig 3).

Analysis of ABA concentrations in transgenic chrysanthemum and wildtype 'Jinba'
Endogenous ABA content was measured in WT, W15-1 and W15-2 plants under normal conditions and 24 h after exposure to A. tenuissima.The results show that WT plants had a higher ABA content than transgenic lines overexpressing CmWRKY15 (W15-1 and W15-2) (Fig 5) under normal conditions and after exposure to A. tenuissima.These data indicate that the ABA

A putative mechanism by which CmWRKY15 regulates ABA-responsive genes in response to A. tenuissima treatment
To identify the mechanisms by which CmWRKY15 responds to A. tenuissima-induced stress, the expression levels of a set of ABA-related genes, including ABF4, ABI4, ABI5, RAB18, DREB1A, DREB2A, PP2C, SnRK2.2,SnRK2.3,RCAR1, MYB2, PYL2, NCED3A, NCED3B, GTG1, AKT1, AKT2, KAT1, KAT2 and KC1, were compared between transgenic and WT plants ( Fig 6,S1 Fig).Under non-stress conditions, the expression of most ABA-related genes was similar between transgenic lines and WT plants.Following A. tenuissima exposure, the transcription of the ABA-upregulated genes was impaired to varying degrees in CmWRKY15overexpressing lines compared to the WT plants, while the ABA-downregulated genes were significantly upregulated in CmWRKY15-overexpressing lines.ABF4, ABI4, ABI5, DREB1A, DREB2A, MYB2, and RAB18 were all downregulated significantly in the transgenic lines (Fig 6a -6g), and the expression level of DREB1A was induced nearly ten-fold in WT plants compared with that in the transgenic lines within 24 h of exposure to A. tenuissima.Moreover, the relative expression of ABF4 was 7.49 in WT plants within 24 h of exposure, whereas this value was only 1.39 in W15-1 and 1.33 in W15-2.At their peaks, the expression levels of ABI4, ABI5 and MYB2 were upregulated 2-3-fold in the control compared with the transgenic lines.Before A. tenuissima inoculation, the expression of most ABA-related genes was not significantly different between transgenic chrysanthemum and WT plants.In contrast, after A. tenuissima infection, most genes were upregulated (Fig 6) or downregulated (S1 Fig) in WT plants compared with W15-1 and W15-2 plants.In addition to these genes, we evaluated the expression of two variants of NCED3 in chrysanthemum, NCED3A and NCED3B.The expression of these two genes was robustly induced by A. tenuissima infection, and their expression was significantly reduced in transgenic plants but not in WT plants, consistent with the observation of an increased ABA level in WT plants compared with transgenic lines overexpressing CmWRKY15 (W15-1 and W15-2).Collectively, these results suggest that CmWRKY15 might function as a transcriptional repressor that can facilitate A. tenuissima infection by directly or indirectly inhibiting the expression of ABA-responsive genes and genes related to ABA signaling, which can increase the expression of genes that are downregulated by ABA.

Analysis of CmWRKY15 homologues
Three WRKY transcription factor homologues in Arabidopsis, WRKY18, WRKY40 and WRKY60, can interfere with pathogen defense.Several studies have demonstrated that CmWRKY15 homologues have a role in the pathogen response in a wide variety of species.For example, a gene involved in diverse stress-response pathways, GhWRKY40 (isolated from cotton (Gossypium hirsutum)), negatively responded to invasion by the bacterial pathogen Ralstonia solanacearum by downregulating most defense-related genes [32].However, VpWRKY3 from Vitis pseudoreticulata was used to generate transgenic tobacco plants with enhanced resistance to R. solanacearum [33].In addition, the expression of both BnWRKY18 and BnWRKY40 was significantly induced after treatment with the two necrotrophic fungal pathogens Sclerotinia sclerotiorum and Alternaria brassicae [34], which is consistent with earlier findings indicating that A. tenuissima induced CmWRKY15 expression in chrysanthemum [13].Moreover, a fungal pathogen-activated elicitor was found to immediately and transiently induce PcWRKY4 expression in parsley cells (Petroselinum crispum) [35].Recent research clearly shows that WRKY transcription factors homologous to CmWRKY15 participate in either biotic or abiotic stress-related responses.For example, VpWRKY3 participates in the ABA signaling pathway and the salt stress response [33].Similarly, GarWRKY28 and GarWRKY51 are salt-responsive genes from the salt-tolerant wild cotton species Gossypium aridum [36].Moreover, CrWRKY18 responds to JA in Catharanthus roseus [37].Previous research clearly demonstrates that GhWRKY40, VpWRKY3 and PcWRKY4 are localized in the nucleus [32,33,35], in agreement with the finding that the CmWRKY15 protein is targeted to the nucleus; additionally, GarWRKY28 and GarWRKY51 proteins are predicted to reside in the nucleus [36].CmWRKY15 was not found to exhibit transcriptional activation, unlike GhWRKY40 and VpWRKY3, which were found to function as transcriptional activators [32,33].Together, these results suggest that the homologues of CmWRKY15 in different species appear to have multiple roles in the response to pathogens.They also have diverse roles in controlling tolerance to biotic and abiotic stresses.Most homologous genes exhibit the same nuclear localization pattern, whereas their transcriptional activation patterns tend to differ.
Our results demonstrating the response of CmWRKY15 to A. tenuissima and its association with ABA-related genes represent fundamental data that will be useful in future research.
Analysis of CmWRKY15-dependent regulation of ABA-responsive genes in response to A. tenuissima treatment Quantitative real-time PCR analysis suggested that the expression of a set of ABA-related genes, including ABF4, ABI4, ABI5, RAB18, DREB1A, DREB2A, PP2C, SnRK2.2,SnRK2.3,RCAR1, MYB2, PYL2, NCED3A, NCED3B, GTG1, AKT1, AKT2, KAT1, KAT2 and KC1, was altered to varying degrees in plants overexpressing CmWRKY15 compared to control (WT) plants ( Fig 6,S1 Fig).Among these genes, ABF4, ABI4, ABI5, RAB18, DREB1A, DREB2A, PYL2, PP2C, SnRK2.2,SnRK2.3,RCAR1, MYB2, NCED3A, NCED3B and GTG1 exhibited responses that were synergistic with ABA-associated pathways or positively regulated ABA-associated pathways.Together with the observation that these genes were upregulated during exposure to A. tenuissima, we can conclude that ABA increases resistance to A. tenuissima by fine-tuning a set of ABA-related genes.Moreover, AKT1, AKT2, KAT1, KAT2, and KC1 were downregulated by ABA, but they were upregulated in transgenic lines during exposure to A. tenuissima.Before A. tenuissima inoculation, the expression of most ABA-associated genes did not significantly differ between CmWRKY15-overexpressing plants and WT plants.In contrast, most genes were upregulated or downregulated in WT plants compared to W15-1 and W15-2 plants following exposure to A. tenuissima.A previous study showed that CmWRKY15 was markedly upregulated in the presence of A. tenuissima inoculum [13].Therefore, our results show that CmWRKY15 in the transgenic overexpression lines has minimal effects under normal conditions.Conversely, CmWRKY15 has a predominant role in regulating ABA-associated signaling in response to black spot disease.
Analysis of ABF4, ABI4, ABI5, MYB2, RAB18, DREB1A and DREB2A transcript levels In this study, the transcript levels of ABF4, ABI4, ABI5, MYB2, RAB18, DREB1A and DREB2A were significantly upregulated in WT plants compared to W15-1 and W15-2 plants following exposure to A. tenuissima.A chromatin co-immunoprecipitation (ChIP) assay using an antibody against WRKY40 demonstrated that AtWRKY40 directly targets several of these genes [18].Note that ABI4, DREB1A, and DREB2A belong to a clade of Apetala-2 domain transcription factors, MYB2 is a MYB-related transcription factor, RAB18 is a rab-related protein, and ABI5 and ABF4 belong to a class of basic leucine zipper transcription factors.These four transcription factor families have been recognized as important players with positive roles in the ABA signaling network [18,38].Our finding that these genes are upregulated in WT plants (Fig 6a -6g) suggests that CmWRKY15 downregulates ABA-responsive genes and genes involved in ABA signaling in response to A. tenuissima infection.Moreover, CmWRKY15 might indirectly regulate some ABA-responsive and ABA signaling genes via another pathway that represses NCED3A and NCED3B, leading to a decrease in ABA content.
Given that CmWRKY15 suppressed the expression of ABF4/ABI5, which function downstream of SnRK2s, we would expect the expression of PYL2, PP2C, RCAR1, SnRK2.2 and SnRK2.3 to be upregulated or unaltered in the CmWRKY15-overexpressing plants compared to the controls.Based on the evidence above, we can conclude that the crosstalk between CmWRKY15 and ABA-mediated signaling pathways in plants during A. tenuissima-induced stress is complicated.Further research on the rudimentary mechanism underlying this crosstalk is necessary.

Analysis of GTG1 transcript levels
There was little variation in GTG1 expression levels between CmWRKY15-overexpressing plants and WT plants after challenge with A. tenuissima; however, at 48 h, the expression of GTG1 in the control plants was nearly 3-fold higher than that in the CmWRKY15-overexpressing plants (Fig 6o).GPCR-type G proteins are candidate plasma membrane-type ABA receptors, which are found on the cell surface and are required for ABA-induced promotion of stomatal closure [39].Based on our data, we hypothesize that GTG1 is likely to interact with ABA to induce stomatal closure, thus protecting plants from A. tenuissima infection.At 48 h, CmWRKY15 may have a negative effect on GTG1 cooperation with ABA to limit A. tenuissima infection.
Analysis of NCED3A and NCED3B transcript levels NCED (9-cis-epoxycarotenoid dioxygenase) is an essential enzyme for the biosynthesis of ABA, which may be produced using two substrates: 9-cis-violaxanthin and/or 9'-cis-neoxanthin [41].In Arabidopsis, NCEDs are predominantly localized in vascular tissues, which suggests that the ABA-activated stress pathway begins with a signal from the vascular tissues that induces stomatal closure [40].NCED genes have been investigated to determine their roles in drought and salt stress.Additionally, a previous study revealed that AtNCED3 is involved in infections involving pathogenic organisms, such as Pseudomonas syringae pv.Tomato DC3000 and B. cinerea [42].Here, using chrysanthemum, we evaluated the expression of two NCED3 variants, NCED3A and NCED3B.We found that the concentration of endogenous ABA was higher in WT plants compared to transgenic lines overexpressing CmWRKY15 (W15-1 and W15-2) (Fig 5).In addition, the expression of NCED3A and NCED3B was robustly induced by A. tenuissima infection and was significantly inhibited in transgenic plants but not in WT plants (Fig 6m and 6n).These results suggest that CmWRKY15 may regulate ABA biosynthesis to a certain extent and interfere with ABA-induced stomatal closure to facilitate A. tenuissima infection.However, the complexity of the relationship between CmWRKY15 and NCED3 remains to be determined.
Overexpression of CmWRKY15 in chrysanthemum facilitated A. tenuissima infection Based on the DSI data (Table 1) and the A. tenuissima treatment results ( of CmWRKY15 [13], which supports a role for ABA in the regulation of CmWRKY15 expression.However, the basal mechanisms that fine-tune the interactions between CmWRKY15 and ABA-responsive genes that modulate the A. tenuissima defense response remain unclear.Additional research on the integrative mechanisms of the signaling network will increase our understanding of the complicated crosstalk between CmWRKY15 ABA-mediated responses to A. tenuissima infection in chrysanthemum.

Conclusions
CmWRKY15 was not transcriptionally activated in yeast cells.The results of subcellular localization experiments indicated that CmWRKY15 localized to the nucleus in vivo.A comparison of the disease severity index (DSI) data of CmWRKY15-overexpressing plants with the DSI data of controls indicated that CmWRKY15 overexpression enhanced the susceptibility of chrysanthemum to A. tenuissima infection.Furthermore, an analysis of CmWRKY15-dependent regulation of ABA-responsive genes associated with A. tenuissima infection suggested that CmWRKY15 might facilitate A. tenuissima infection by antagonistically regulating the expression of ABA-responsive genes and ABA signaling genes, either directly or indirectly, which could lead to the upregulation of genes that are downregulated by ABA.

Fig 2 .
Fig 2. Subcellular localization and transactivation analysis of CmWRKY15.a Subcellular localization of CmWRKY15.b The analysis of CmWRKY15 transcriptional activity was carried out using a yeast assay system.The positive control cells (Y2H cells containing pCL1) were able to grow on SD/-His-Ade medium.Conversely, the negative control cells (Y2H cells containing pGBKT7) could not grow on this medium.c Relative luciferase activities in Arabidopsis mesophyll protoplasts after transfection with 35S::GAL4DB-CmWRKY15. doi:10.1371/journal.pone.0143349.g002

Table 1 .Fig 5 .Fig 6 .
Fig 5. ABA concentrations in transgenic chrysanthemum and wild type 'Jinba' chrysanthemum.ABA concentrations of the control were assessed at 0 h, and ABA concentrations of plants exposed to A. tenuissima were assessed at 24 h.doi:10.1371/journal.pone.0143349.g005

Fig 4 )
Fig 7. A putative model describing the effects of the interaction between CmWRKY15 and ABA-associated genes on the defense response to A. tenuissima infection.doi:10.1371/journal.pone.0143349.g007