A Novel WRKY Transcription Factor HmoWRKY40 Associated with Betalain Biosynthesis in Pitaya (Hylocereus monacanthus) through Regulating HmoCYP76AD1

Betalains are water-soluble nitrogen-containing pigments with multiple bioactivities. Pitaya is the only large-scale commercially grown fruit containing abundant betalains for consumers. However, the upstream regulators in betalain biosynthesis are still not clear. In this study, HmoWRKY40, a novel WRKY transcription factor, was obtained from the transcriptome data of pitaya (Hylocereus monacanthus). HmoWRKY40 is a member of the Group IIa WRKY family, containing a conserved WRKY motif, and it is located in the nucleus. The betalain contents and expression levels of HmoWRKY40 increased rapidly during the coloration of pitaya and reached their maximums on the 23rd day after artificial pollination (DAAP). Yeast one-hybrid and transient expression assays showed that HmoWRKY40 could bind and activate the promoter of HmoCYP76AD1. Silencing the HmoWRKY40 gene resulted in a significant reduction of betacyanin contents. These results indicate that HmoWRKY40 transcriptionally activates HmoCYP76AD, which is involved in the regulation of pitaya betalain biosynthesis. The results of the present study provide new regulatory networks related to betalain biosynthesis in pitaya.


Introduction
Betalains are water-soluble and nitrogen-containing pigments with antioxidant properties, which were first extracted from the common beet (Beta vulgaris). Betalains can be divided into betacyanins and betaxanthins according to their chemical structures [1]. Betalains are restricted to 13 families of the order Caryophyllales, except thatMolluginaceae and Caryophyllaceae produce anthocyanins instead of betalain pigments. In nature, betalains and anthocyanins are mutually exclusive and cannot co-exist in one plant at the same time. Therefore, betalains can be used as additional help for classification in the plant kingdom [2].
Betalains not only can present different colors but also play fundamental roles in plant responses and adaptation to biotic and abiotic stresses, such as disease, drought, high salinity and low-and high-temperature stresses [3][4][5]. Betalains have a high nutritional value and are widely used as natural colorants of foods, drugs and cosmetics due to their high antioxidative and free radical scavenging activities [6][7][8]. Therefore, understanding the biosynthetic and regulatory pathways for betalain biosynthesis is essential to the improvement of betalain production in plants through biotechnological approaches.

Changes in Betalain Contents at All Coloration Stages of Pitaya Pulp
The contents of betacyanins and betaxanthins were detected in the pulps of the Guanhuahong pitaya during fruit maturation. As shown in Figure 1, the contents of betacyanins gradually increased at all stages of pulp coloration in the Guanhuahong pitaya. Higher contents of betacyanins were detected compared with betaxanthins. Betacyanins increased during fruit maturation and the highest content of betacyanins was detected at the fully mature stage, while betaxanthins kept lower contents at all stages in the Guanhuahong pitaya ( Figure 1B).

Promoter Analyses of HmoCYP76AD1
CYP76AD1, encoding a novel cytochrome P450, is a key gene for the conversion of L-DOPA to cyclo-DOPA, which produces the red betacyanin pigment in beets [9]. Our previous studies showed that HmoCYP76AD1 is likely involved in betalain biosynthesis [10]. Putative cis-acting elements in the promoter regions of HmoCYP76AD1 (the sequence is listed in the Supplementary Text S1) were analyzed using the Plant-CARE database ( Table 1). The locations of motifs are shown based on "+1" being the transcription start site. The core promoter element of "TATA-box" motifs is around −30 of the transcription start, which was proven for most of the promoters (plants and other eukaryotic nuclear genes). "TATA-box" motifs in the promoter of the HmoCYP76AD1 ranged from 130 bp downstream (+) to 730 bp upstream (−). These results indicate that the promoter of HmoCYP76AD1 is a variant promoter. In addition to the core cis-acting elements, such as a TATA-box and a CAAT-box, a typical W-box motif with a core sequence of (C/T)TGAC(C/T) was identified in the HmoCYP76AD1 promoter ( Table 1). The W-box motif of HmoCYP76AD1

Promoter Analyses of HmoCYP76AD1
CYP76AD1, encoding a novel cytochrome P450, is a key gene for the conversion of L-DOPA to cyclo-DOPA, which produces the red betacyanin pigment in beets [9]. Our previous studies showed that HmoCYP76AD1 is likely involved in betalain biosynthesis [10]. Putative cis-acting elements in the promoter regions of HmoCYP76AD1 (the sequence is listed in the Supplementary Text S1) were analyzed using the Plant-CARE database ( Table  1). The locations of motifs are shown based on "+1" being the transcription start site. The core promoter element of "TATA-box" motifs is around −30 of the transcription start, which was proven for most of the promoters (plants and other eukaryotic nuclear genes). "TATA-box" motifs in the promoter of the HmoCYP76AD1 ranged from 130 bp downstream (+) to 730 bp upstream (−). These results indicate that the promoter of HmoCYP76AD1 is a variant promoter. In addition to the core cis-acting elements, such as a TATA-box and a CAAT-box, a typical W-box motif with a core sequence of (C/T)TGAC(C/T) was identified in the HmoCYP76AD1 promoter ( Table 1). The W-box motif of HmoCYP76AD1 was predicted to be at 120 bp (−) upstream. The W-box is a cognate binding site for WRKY TFs, suggesting the possible involvement of WRKY TFs in the regulation of HmoCYP76AD1.

Cloning and Sequence Analyses of HmoWRKY40
A full-length WRKY gene that was up-regulated during the color development of the pitaya fruit pulp was obtained from the RNA-Seq database of the Guanhuahong pitaya. The WRKY gene shows high similarity (50%) to BvWRKY40 from B. vulgaris and therefore was named HmoWRKY40 (the sequence is listed in Supplementary Text S2). The open reading frame (ORF) of the HmoWRKY40 was 1065 bp in length and encoded a polypeptide of 354 amino acids, with a calculated molecular weight of 38.78 kDa and a theoretical isoelectric point (pI) of 6.91. HmoWRKY40 had one highly conserved amino acid sequence of WRKYGQK at the N-terminal end, which is a WRKY domain and a defining characteristic of WRKY TFs. The HmoWRKY40 also contained a putative zinc-finger motif (C-X 4-5 -C-X 22-23 -H-X 1 -H) (Figure 2A). A phylogenetic tree was constructed using the amino acid sequence of HmoWRKY40 and WRKYs from Arabidopsis thaliana, Oryza sativa, B. vulgaris and Solanum lycopersicum. HmoWRKY40 was clustered with Group IIa WRKYs, along with the BvWRKY40 from B. vulgaris and the AtWRKY40, AtWRKY18 and AtWRKY60 from A. thaliana ( Figure 2B).

Expression Analyses of HmoWRKY40 and HmoCYP76AD1
Expression levels of HmoWRKY40 and HmoCYP76AD1 were analyzed by RT-qPCR to explore their relationship to betalain biosynthesis during the different developmental stages of the Guanhuahong pitaya pulps (sequences are listed in Supplementary Text S2). Expression levels of HmoWRKY40 and HmoCYP76AD1 increased significantly during the coloration period (23rd to 25th DAAP) of the Guanhuahong pitaya pulps (Figure 3), which is consistent with the betalain accumulation and the color development of the pulps ( Figure 1).

Nuclear Localization of HmoWRKY40
The full-length coding sequence of HmoWRKY40 was fused with the GFP gene to analyze its subcellular localization. Transient expression of constructs in epidermal cells of Nicotiana benthamiana showed that the fluorescence of HmoWRKY40 was detected exclusively in the nucleus, whilst for the positive control, the GFP signal was observed around the cytoplasm and the nucleus (Figure 4). of WRKYGQK at the N-terminal end, which is a WRKY domain and a defining characteristic of WRKY TFs. The HmoWRKY40 also contained a putative zinc-finger motif (C-X4-5-C-X22-23-H-X1-H) (Figure 2A). A phylogenetic tree was constructed using the amino acid sequence of HmoWRKY40 and WRKYs from Arabidopsis thaliana, Oryza sativa, B. vulgaris and Solanum lycopersicum. HmoWRKY40 was clustered with Group IIa WRKYs, along with the BvWRKY40 from B. vulgaris and the AtWRKY40, AtWRKY18 and AtWRKY60 from A. thaliana ( Figure 2B). The same and similar amino acids are indicated by blue and red shades, respectively. The WRKY motif and the zinc-finger structure are indicated by the red box and the black asterisks, respectively, and (B) The phylogenetic tree of HmoWRKY40 and WRKYs from Arabidopsis thaliana, Oryza sativa, Beta vulgaris and Solanum lycopersicum. HmoWRKY40 protein is indicated by a red asterisk. The phylogenetic tree was constructed using the maximum likelihood method by MEGA7.0.

Expression Analyses of HmoWRKY40 and HmoCYP76AD1
Expression levels of HmoWRKY40 and HmoCYP76AD1 were analyzed by RT-qPCR to explore their relationship to betalain biosynthesis during the different developmental stages of the Guanhuahong pitaya pulps (sequences are listed in Supplementary Text S2).

Nuclear Localization of HmoWRKY40
The full-length coding sequence of HmoWRKY40 was fused with the GFP gene to analyze its subcellular localization. Transient expression of constructs in epidermal cells as 1). The data represent the mean values from three biological replicates (±S.D.).

Nuclear Localization of HmoWRKY40
The full-length coding sequence of HmoWRKY40 was fused with the GFP gene to analyze its subcellular localization. Transient expression of constructs in epidermal cells of Nicotiana benthamiana showed that the fluorescence of HmoWRKY40 was detected exclusively in the nucleus, whilst for the positive control, the GFP signal was observed around the cytoplasm and the nucleus (Figure 4).

Analyses of Transcriptional Activities of HmoWRKY40
The transcriptional activities of HmoWRKY40 were determined according to the changes in the yeast color after staining. As shown in Figure 5, Y2H Gold yeast strains containing pGBKT7-HmoWRKY40, a positive control (pGBKT7-p53+pGADT7-T) and a negative control (pGBKT7) could all grow normally on the SD/-Trp medium. On the auxotrophic selection medium SD/-Trp-Ade-His, the yeast strains harboring both pGBKT7-HmoWRKY40 and the positive control all grew normally at 30 • C after 3 days of culturing, and the colonies turned blue with the addition of X-α-gal. However, the yeast strains harboring the empty pGBKT7 vector (the negative control) could not grow on the deficient medium SD/-Trp-Ade-His. These results suggest that HmoWRKY40 has a transcriptional activation function in yeast.

Virus-Induced Gene Silencing (VIGS) Analyses of HmoWRKY40
A gene silencing assay was performed to further explore the function of HmoWRKY40. As shown in Figure 6A, when compared to the control, the silencing of HmoWRKY40 exhibited green and yellow patches in the scales of the Hongguan No. 1 pitaya (H. monacanthus) and resulted in the reduction of betacyanins and betaxanthins ( Figure 6B). Results from the RT-qPCR analyses showed that the HmoWRKY40 and HmoCYP76AD1 were silenced ( Figure 6C,D). These results indicate that HmoWRKY40 plays an important role in the betalain biosynthetic pathway of pitayas.
negative control (pGBKT7) could all grow normally on the SD/-Trp medium. On the auxotrophic selection medium SD/-Trp-Ade-His, the yeast strains harboring both pGBKT7-HmoWRKY40 and the positive control all grew normally at 30 °C after 3 days of culturing, and the colonies turned blue with the addition of X-α-gal. However, the yeast strains harboring the empty pGBKT7 vector (the negative control) could not grow on the deficient medium SD/-Trp-Ade-His. These results suggest that HmoWRKY40 has a transcriptional activation function in yeast.

Virus-Induced Gene Silencing (VIGS) Analyses of HmoWRKY40
A gene silencing assay was performed to further explore the function of HmoWRKY40. As shown in Figure 6A, when compared to the control, the silencing of HmoWRKY40 exhibited green and yellow patches in the scales of the Hongguan No. 1 pitaya (H. monacanthus) and resulted in the reduction of betacyanins and betaxanthins ( Figure 6B). Results from the RT-qPCR analyses showed that the HmoWRKY40 and HmoCYP76AD1 were silenced ( Figure 6C,D). These results indicate that HmoWRKY40 plays an important role in the betalain biosynthetic pathway of pitayas. The WRKY TF can recognize and bind to the W-box element in the promoter region of target genes. The promoter region of HmoCYP76AD1 had the W-box element (Table 1). Therefore, the interaction between HmoWRKY40 and the W-box in the promoter region of HmoCYP76AD1 was analyzed using a yeast one-hybrid assay. Yeast cells harboring  1). The data represent mean values from three biological replicates (±S.D.). ** indicates significant differences at p value < 0.01 using a two-tailed t-test and (D) RT-qPCR analyses of HmoCYP76AD1 in VIGS treatment scales. The expression level of pTRV2-HmoCYP76AD1 was used as the calibrator (set as 1).The data represent mean values from three biological replicates (±S.D.). ** indicates significant differences at p value < 0.01 using a two-tailed t-test.

Interaction of HmoWRKY40 with the W-Box in the Promoter of HmoCYP76AD1
The WRKY TF can recognize and bind to the W-box element in the promoter region of target genes. The promoter region of HmoCYP76AD1 had the W-box element (Table 1). Therefore, the interaction between HmoWRKY40 and the W-box in the promoter region of HmoCYP76AD1 was analyzed using a yeast one-hybrid assay. Yeast cells harboring pABAi-HmoCYP76AD1-pro could not grow on the SD/-Ura medium supplemented with 100 or 200 ng/mL AbA ( Figure 7A). Yeast cells containing pABAi-HmoCYP76AD1-pro+pGADT7-HmoWRKY40 grew on the SD/-Leu medium with the addition of 100 or 200 ng/mL AbA ( Figure 7B). These results indicate that the HmoWRKY40 protein has a DNA binding activity and could directly bind to the promoter of HmoCYP76AD1, which is possibly involved in the betalain biosynthesis of pitaya.

HmoWRKY40 Activates the Transcription of Promoter of HmoCYP76AD1
The ability of HmoWRKY40 to activate the transcription of HmoCYP76AD1 was performed using the transient dual-luciferase assays in tobacco leaves. As shown in Figure 8, the overexpression of HmoWRKY40 significantly increased the LUC/REN ratio. The LUC/REN ratio of CaMV35S-HmoWRKY40 co-transduced with CaMV35S-REN/HmoCYP76AD1-pro and was 2-fold higher than that of the control (Figure 8). These results demonstrate that HmoWRKY40 activates the transcription of the HmoCYP76AD1 promoter, suggesting that HmoWRKY40 has a functional role in the regulation of HmoCYP76AD1.

HmoWRKY40 Activates the Transcription of Promoter of HmoCYP76AD1
The ability of HmoWRKY40 to activate the transcription of HmoCYP76AD1 was performed using the transient dual-luciferase assays in tobacco leaves. As shown in Figure 8, the overexpression of HmoWRKY40 significantly increased the LUC/REN ratio. The LUC/REN ratio of CaMV35S-HmoWRKY40 co-transduced with CaMV35S-REN/HmoCYP76AD1-pro and was 2-fold higher than that of the control (Figure 8). These results demonstrate that HmoWRKY40 activates the transcription of the HmoCYP76AD1 promoter, suggesting that HmoWRKY40 has a functional role in the regulation of Hmo-CYP76AD1.

Discussion and Conclusions
Betalains are one of the major plant pigments found only in some plants in the order of Caryophyllales, with the exception of the Caryophyllaceae and Molluginaceae families [2]. CYP76AD1 is one of the two precursors of betacyanin, which catalyzes the conversion of L-DOPA to cylco-DOPA [9,18]. Silencing of the CYP76AD1 gene results in the production of only yellow betaxanthins and the loss of red pigment. Introduction of CYP76AD1 into the yellow betalain mutants (an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation) produces both yellow and red pigmentation [9]. The expression of CYP76AD1 in yeast confirms its role in betalain biosynthesis [9,11]. Moreover, heterologous expression of BvCYP76AD1 or CqCYP76AD1 in tobacco results in an accumulation of betalains [11]. In our previous study, comp16058_c0_seq1 (named HmoCYP76AD1), an important candidate gene of cyt-P450 genes involved in the betalain biosynthetic pathway, was identified from pitaya by RNA-Seq and its amino acid sequence was 74% homologous with BvCYP76AD1 [10]. In this study, the expression and promoter of HmoCYP76AD1 were analyzed. Expression levels of HmoCYP76AD1 increased gradually during pulp coloration and decreased at the full maturation stage (Figure 3). A typical W-box motif with a core sequence of (C/T)TGAC(C/T) was found in the HmoCYP76AD1 promoters (Table 1). These results indicate that HmoCYP76AD1 is possibly involved in the betalain biosynthesis of pitaya.
The WRKY proteins are plant-specific proteins involved in multiple biological processes in plants. WRKYs not only play important roles in biotic and abiotic stresses in many plant processes [19][20][21], but are also involved in the regulation of plant growth and developmental processes including trichome [22] and seed development [23], germination [24], leaf senescence [25] and the synthesis of secondary metabolites including tryptamine [26], alkaloids [27], terpenes [28] and betalains [16]. WRKY proteins are clustered into three major groups (I, II and III) and group II can be further subdivided into five subgroups (IIa, IIb, IIc, IId and IIe). WRKY proteins can activate or inhibit the expression of downstream genes by binding the W-box ((C/T)TGAC(C/T)) elements of its target gene promoter [16,29,30]. In our previous study, a HmoWRKY44 TF belonging to a member of Figure 8. HmoWRKY40 activated the transcriptions of HmoCYP76AD1. The activation was measured as a ratio of LUC to REN. The ratio of LUC/REN of the empty vector plus promoter was used as the calibrator (set as 1). Three independent experiments were conducted (n = 3). The error bars indicate one standard error. Significant differences from CaMV35S-Empty were determined according to Student's t-test (** indicates p < 0.01).

Discussion and Conclusions
Betalains are one of the major plant pigments found only in some plants in the order of Caryophyllales, with the exception of the Caryophyllaceae and Molluginaceae families [2]. CYP76AD1 is one of the two precursors of betacyanin, which catalyzes the conversion of L-DOPA to cylco-DOPA [9,18]. Silencing of the CYP76AD1 gene results in the production of only yellow betaxanthins and the loss of red pigment. Introduction of CYP76AD1 into the yellow betalain mutants (an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation) produces both yellow and red pigmentation [9]. The expression of CYP76AD1 in yeast confirms its role in betalain biosynthesis [9,11]. Moreover, heterologous expression of BvCYP76AD1 or CqCYP76AD1 in tobacco results in an accumulation of betalains [11]. In our previous study, comp16058_c0_seq1 (named HmoCYP76AD1), an important candidate gene of cyt-P450 genes involved in the betalain biosynthetic pathway, was identified from pitaya by RNA-Seq and its amino acid sequence was 74% homologous with BvCYP76AD1 [10]. In this study, the expression and promoter of HmoCYP76AD1 were analyzed. Expression levels of HmoCYP76AD1 increased gradually during pulp coloration and decreased at the full maturation stage (Figure 3). A typical W-box motif with a core sequence of (C/T)TGAC(C/T) was found in the HmoCYP76AD1 promoters (Table 1). These results indicate that HmoCYP76AD1 is possibly involved in the betalain biosynthesis of pitaya.
The WRKY proteins are plant-specific proteins involved in multiple biological processes in plants. WRKYs not only play important roles in biotic and abiotic stresses in many plant processes [19][20][21], but are also involved in the regulation of plant growth and developmental processes including trichome [22] and seed development [23], germination [24], leaf senescence [25] and the synthesis of secondary metabolites including tryptamine [26], alkaloids [27], terpenes [28] and betalains [16]. WRKY proteins are clustered into three major groups (I, II and III) and group II can be further subdivided into five subgroups (IIa, IIb, IIc, IId and IIe). WRKY proteins can activate or inhibit the expression of downstream genes by binding the W-box ((C/T)TGAC(C/T)) elements of its target gene promoter [16,29,30]. In our previous study, a HmoWRKY44 TF belonging to a member of the Group I; WRKY family was obtained from pitaya fruit. The W-box motif of HmoWRKY44 is predicted to be from 581 bp downstream (+) to 1351 bp upstream (−) of the transcription start site [16]. HmoWRKY44 could activate the HmoCYP76AD1 expression by binding to its promoter, which is responsible for the betalain biosynthesis of pitaya [16]. In the present work, HmoWRKY40, a novel WRKY TF, was obtained from pitaya transcriptome data. The HmoWRKY40 had one WRKY domain at 120 bp (−) upstream and belonged to the Group IIa WRKY family (Figure 2). Expression levels of HmoWRKY40 increased significantly during the coloration period (23rd to 25th DAAP) of the Guanhuahong pitaya pulps (Figure 3), which was consistent with the expression trend of HmoCYP76AD1 and the betalain accumulation of the pulp (Figure 1). These results suggest that HmoWRKY40 is involved in the betalain biosynthesis of pitaya.
The WRKY proteins are typically nuclear-localized proteins [30][31][32][33]. In our study, HmoWRKY40 was located in the nucleus (Figure 4). Silencing of the HmoWRKY40 resulted in a decrease in betalain contents in the pitaya scale ( Figure 6). Yeast single-hybrid and dual luciferase experiments showed that HmoWRKY40 can activate the expression of the HmoCYP76AD1 gene by binding to its promoter, which participates in the regulation of betalain biosynthesis (Figures 7 and 8). The LUC/REN ratio of CaMV35S-HmoWRKY40 cotransduced with CaMV35S-REN/HmoCYP76AD1-pro had a relatively higher expression than that of the control (Figure 8), which is consistent with HmoWRKY44 [16]. These results indicate that HmoWRKY40 plays a key role in pitaya betalain biosynthesis through regulating the expression of the HmoCYP76AD1 gene.
In summary, a novel WRKY transcription factor HmoWRKY40, belonging to a member of the Group IIa WRKY family, was obtained from pitaya. HmoWRKY40 activated the Hmo-CYP76AD1 expression by binding to its promoter. Silencing of HmoWRKY40 resulted in a significant reduction in betalain contents. These results suggest that HmoWRKY40 plays a key role in the betalain biosynthesis of pitaya. The present study provides new insights into the transcriptional regulation of genes related to betalain biosynthesis in pitaya.

Materials
Two pitaya cultivars, Guanhuahong (red peel and green scales with red pulp, H. monacanthus) and Hongguan No. 1 (red peel and red scales with red pulp, H. monacanthus) from the same orchard in Jinsuinong (Zhongluotan Village, Guangzhou City, Guangdong Province, China), were used as materials. Fruits of Guanhuahong pitayas were collected on the 19th, 23rd, 25th, 27th and 29th day after artificial pollination (DAAP) ( Figure 1A). Scales of the Hongguan No. 1 pitaya on the 23rd DAAP were used for virus-induced gene silencing (VIGS). All samples were frozen in liquid nitrogen immediately and stored at −80 • C for future analysis.

Measurement of Betalain Contents
Freeze-dried pitaya pulps were ground into powder in liquid nitrogen. Betalain contents were extracted and measured following the procedure of Hua et al. (2016) [10]. All measurements were repeated three times. A two-tailed t-test was used to determine the significance at p < 0.05 and p < 0.01 using the SPSS 17.0 software.

Gene Cloning and Sequence Analyses
Total RNA was extracted using the EASYspin Plus polysaccharide polyphenol complex plant RNA rapid extraction kit (RN53) (Aidlab, Beijing, China). The RNA quality and concentration were assessed respectively by 1.2% agarose gel and spectrophotometry. RNA samples with an OD 260 /OD 280 ratio of 1.9-2.1 were reverse-transcribed into cDNA using the PrimeScript TM RT reagent Kit with the gDNA Eraser (Perfect Real Time) according to the manufacturer's instructions (TaKaRa, Shiga, Japan).
HmoWRKY40 was obtained from the RNA-Seq database [10]. The open reading frame (ORF) of the HmoWRKY40 sequence was cloned by gene-specific primer pairs (Supplementary Table S1) and the NCBI database (http://www.ncbi.nlm.nih.gov/) was used to find similar sequences for homology alignment. Sequences were aligned using the ClustalX and GeneDoc software. The phylogenetic tree was constructed using the Maximum likelihood method in MEGA7.0. The theoretical isoelectric point (pI) and relative molecular mass of HmoWRKY40 were predicted at http://web.expasy.org/compute_pi/.

Subcellular Localization of HmoWRKY40
The full-length coding sequence of HmoWRKY40 was inserted into the pGreen-35S-GFP. Cells of the Agrobacterium tumefaciens strain of GV3101-pSoup, carrying 35S-HmoWRKY 40-GFP and the pGreen-35S-GFP positive control, were separately co-expressed with At1g22590-RFP (for nuclear positioning) in a ratio of 1:1 to infect tobacco (Nicotiana benthamiana) leaves [35]. Transient expression of GFP and RFP signals were observed using a laser confocal microscope (Zeiss Axioskop 2 Plus, Germany) after two days of infiltration.

Gene Silencing of HmoWRKY40
The full length of the HmoWRKY40 fragment was ligated to the pTRV2 vector; pTRV1, pTRV2 and pTRV2-HmoWRKY40 were transformed into the Agrobacterium tumefaciens strain GV3101 (primers are listed in Supplementary Table S1). The bacterial cells were resuspended to an OD 600 of 0.4 using an MMA buffer (10 mM MES, 10 mM MgCl 2 , 100 µM acetosyringone) according to the proportion of 1:1. VIGS was performed as described in our previous study [13].

Promoter Analysis of HmoCYP76AD1
Total genomic DNA was extracted using a CTAB genomic DNA Extraction Kit (DN14) (Aidlab, China) and RNA was removed with Ribonuclease A (RNase A) (TaKaRa, Shiga, Japan). HmoCYP76AD1 was cloned using specific primer pairs (Supplementary Table S1). Promoter elements and the conserved binding domain of HmoCYP76AD1 were analyzed by the Plant-CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Yeast One-Hybrid Experiment
The promoter of HmoCYP76AD1 was ligated to the pAbAi yeast reporter vector to form a pPromoter-AbAi recombinant bait vector. The HmoWRKY40 was ligated into the expression vector pGADT7 to construct the pGADT7-AD recombinant vector and the pGADT7-HmoWRKY40 vector plasmid was used to transform the positively verified pHmoCYP76AD1-AbAi (primers are listed in Supplementary Table S1). The bait yeast strain was cultured at 30 • C until colonies appeared (3-5 d). The interaction between TF and the promoter was judged by the yeast growth [36].

Transcriptional Activity
The ORFs of the HmoWRKY40 were ligated into the vector pGBKT7; pGBKT7-p53 and pGADT7-T were co-transduced as the positive controls and the corresponding bait with an empty pGBKT7 vector was used as the negative control. The vector plasmid pGBKT7-HmoWRKY40 was transformed into the Y2HGold yeast competent cells. The yeast strain was grown on the SD/-Trp plate and cultured at 30 • C until single individual colonies appeared. The single colony was picked up and suspended in 0.9% NaCl solution. Then, suspended cells were coated on plates containing SD/-Trp-His-Ade/Kan and cultured at 30 • C for 3-5 d. X-α-gal (20 µg/mL) was added to a final concentration of 20 µg/mL on the SD/-Trp-His-Ade/Kan plate. The transcriptional activation ability of HmoWRKY40 was investigated according to the yeast color.

Dual-Luciferase Reporter Assay
The full length of HmoWRKY40 was ligated into the pEAQ vector as an effector. The promoter sequence of HmoCYP76AD1 was ligated to the pGReenII 0800-LUC (LUC0800) vector to construct the corresponding recombinant dual luciferase reporter vector. The pHmoCYP76AD1-pro-LUC was transformed into the A. tumefaciens strain GV3101. The effector and reporter bacteria cells were resuspended according to the proportion of 9:1 and used for injecting tobacco (N. benthamiana) leaves. Two days after infiltration, leaves were determined with a Luminoskan Ascent Microplate Luminometer (Thermo Scientific, Rockford, IL, USA) according to the method in the dual luciferase assay kits (Promega, Madison, WI, USA) [37].