HbWRKY27, a group IIe WRKY transcription factor, positively regulates HbFPS1 expression in Hevea brasiliensis

Farnesyl pyrophosphate synthase (FPS) is a key enzyme that catalyzes the formation of farnesyl pyrophosphate, the main initiator for rubber chain initiation in Hevea brasiliensis Muell. Arg. The transcriptional regulatory mechanisms of the FPS gene still not well understood. Here, a WRKY transcription factor designated HbWRKY27 was obtained by screening the latex cDNA library applied the HbFPS1 promoter as bait. HbWRKY27 interacted with the HbFPS1 promoter was further identified by individual Y1H and EMSA assays. HbWRKY27 belongs to group IIe WRKY subfamily which contains a typical WRKY domain and C-X5-CX23-HXH motif. HbWRKY27 was localized to the nucleus. HbWRKY27 predominantly accumulated in latex. HbWRKY27 was up-regulated in latex by ethrel, salicylic acid, abscisic acid, and methyl jasmonate treatment. Transient expression of HbWRKY27 led to increasing the activity of the HbFPS1 promoter in tobacco plant, suggesting that HbWRKY27 positively regulates the HbFPS1 expression. Taken together, an upstream transcription factor of the key natural rubber biosynthesis gene HbFPS1 was identified and this study will provide novel transcriptional regulatory mechanisms of the FPS gene in Hevea brasiliensis.

www.nature.com/scientificreports/ and Wu' method 14 . Five groups (10 trees in each group) were employed in each treatment, in which the plant hormone was applied at 3, 6, 9, 12, and 24 h before tapping. The other group was not treated with hormone as control. After the treatments at all time points, latex from all the tested trees were collected. Latex from the same group was mixed together thoroughly. The resulting solution was then divided into five equal volumes for RNA extraction. N. benthamiana seeds were sowed on moist filter paper in a glass garden, and then incubate them in a growth chamber maintained at a relative humidity of 60-70%, a temperature of 28 °C, and 14 h day/10 h night cycle. After a week, the seedlings were potted in soil and placed in a greenhouse maintained at 26-28 °C, a relative humidity of 60-70%, and 14 h day/10 h night cycle. Two months old seedlings were used to test.
Isolation of DNA and RNA. Genomic DNA isolated from young leaves of CATAS 7-33-97 using the Plant Genomic DNA Extraction Kit (TaKaRa, Dalin, China). Isolation of total RNA from different samples was carried out in accordance with the method of Wang et al. 15 .
Yeast one-hybrid (Y1H) assay. The HbFPS1 promoter fragment (1066 bp) was cloned by PCR with primers (Table 1) using the Genomic DNA as the template in accordance with described method 10 . Then the HbFPS1 promoter fragment was inserted in bait vector pHIS2.1, generate the pHIS-pHbFPS1 construct. Latex cDNA library was constructed in accordance with the user manual of Matchmaker Gold Yeast One-Hybrid Library Screening System Kit (Clontech, CA, USA). The screening was performed according to the protocol of Matchmaker Gold Y1H System (Clontech, CA, USA). More than 1 × 10 6 clones were screened and the positive clones were sequenced and analyzed. 35 transformants were obtained and 22 positive colonies were further obtained after re-streaking the primary positive colonies on the same selective medium. These colonies were further analyzed by plasmid rescue followed by sequence analysis. The interaction between HbWRY27 and the promoter of HbFPS1 was further confirmed by individual Y1H assays. HbWRKY27 was amplified by PCR (see Table 1 for PCR primers) and cloned into the prey vector pGADT7-Rec2 to generate pGADT7-HbWRKY27. pHIS-pHbFPS1 and pGADT7-HbWRKY27 were cotransformed into the yeast Y187 strain. p53-HIS2, pGAD-Rec2-53, as well as pHIS-pHbFPS1 were employed as control. The introduced cells was examined on SD/-Leu-His plates and triple dropout (TDO) plates (SD/-Trp-His-Leu ) supplemented with 80 mM 3-amino-1,2,4-triazole (3-AT) for 5 d at 28 °C.
Electrophoretic mobility shift assay (EMSA). The full length cDNA of HbWRY27 was cloned by PCR (primers see Table 1). The cDNA fragment was cloned into the vector pET-28a, and then introduced into Escherichia coli strain BL21 to product the HbWRKY27 recombinant proteins according to the user manual (Novagen, Madison, WI, USA). The DNA-protein binding reaction was performed by incubating double-stranded DNA of the HbFPS1 promoter or the mutated promoter with purified recombinant protein at room temperature. The W-box in the HbFPS1 promoter was mutated (changing TTGAC to TTGAA) by single-tube 'megaprimer' PCR method 16 . EMSA was performed with SYBR Green and SYPRO Ruby EMSA stains as described manufacturer's protocol of EMSA kits (Invitrogen, Carisbad, CA, USA). Phylogenetic analysis. The homologous protein sequences of HbWRKY27 were obtained from GenBank and phylogenetic analysis was carried out based on the neighbor-joining method using MEGA 5.0 software 16 . Subcellular localization. The cDNAs of HbWRKY27 was amplified by PCR with the primers (Table 1) and inserted into the pCAMBIA1302 vector containing the green fluorescent protein (GFP) gene, thereby generating pHbWRKY27-GFP. pHbWRKY27-GFP and pCAMBIA1302 vector were transformed into A. tumefaciens strain GV3101 via electroporation. Then A. tumefaciens harboring pCAMBIA1302 or pHbWRKY27-GFP were transformed into onion epidermis by infiltration as previously described 17 . Introduced onion epidermal cells was analyzed at 2 days after cultured on MS medium. Fluorescence and 4′, 6′-diamidino-2-phenylindole hydrochloride (DAPI) staining were monitored under a confocal microscope (Leica, Wetzlar, Germany). www.nature.com/scientificreports/ Expression analyses of HbWRKY27. Expression of HbWRKY27 was analyzed by real-time qPCRs in accordance with the manufacturer's instruction of SYBR Premix Taq Kit (TaKaRa, Dalin, China). HbACTIN7 was used as a control gene as described previously 17 . The relative expression level of HbWRKY27 was calculated using the 2 −ΔΔCT method 19 . Three biological repeats were carried out. Data are presented as mean ± SE (n = 3).
Transient expression assay. The HbFPS1 promoter was cloned by PCR (primers see Table 1) and the HbFPS1 promoter and the mutated promoter fragment was inserted into the pGreenII 0800-LUC vector, generating a reporter construct pGreenII-pHbFPS1-LUC and pGreenII-pHbFPS1M-LUC. To generate effector gene, HbWRKY27 was also cloned through PCR primers (Table 1) and inserted into pCAMBIA1301. The generated HbFPS1-LUC HbFPS1M-LUC construct and the effector construct, was introduced into tobacco leaves as previously described 20 . The dual-luciferase (LUC) assay was performed according to the manufacturer's protocol of a dual-luciferase reporter assay system (Promega, Fitchburg, WI, USA). More than three biological repeats were carried out. Difference was accepted as significant at P ≤ 0.05.

Results
HbWRY27 interacts with HbFPS1 promoter. The HbFPS1 promoter had been cloned in previous study 21 . To understand the transcriptional regulatory of HbFPS1, the HbFPS1 promoter was employed as the bait to screen transcription factors that interact with the HbFPS1 promoter from Y1H-based latex cDNA library. Twenty-two colonies were obtained and sequenced. Eight candidates encoding transcription factors were obtained (Supplementary Information Table S1). Among of candidates, one cDNA encoding WRKY transcription factor, named HbWRKY27 according to its homologs in Genbank, was obtained.
HbWRKY27 had an open read frame of 1266 bp in length. The molecular mass of the deduced HbWRKY27 protein was 46.9 kDa. HbWRKY27 had a typical WRKY domain and C-X5-CX23-HXH motif (Fig. 1A). HbWRKY27 was classified into group IIe WRKY subfamily (Fig. 1B). A binding site (W-box) for WRKYs in the promoter of HbFPS1 was predicted 21 . HbWRKY27 interacted with HbFPS1 promoter was further identified by individual Y1H assays ( Fig. 2A). To further determine that HbWRKY27 physically bound with the HbFPS1 promoter, EMSA was used to confirm the binding affinity of HbWRKY27 to the HbFPS1 promoter. The recombinant HbWRKY27 protein was obtained by heterologous expressing of HbWRKY27 in E. coli (Fig. 2B). In addition, the W-box in the HbFPS1 promoter was mutated (changing TTGAC to TTGAA) by PCR method. The DNA-protein binding signal was detected with the recombinant HbWRKY27 protein incubated with the HbFPS1 promoter. No binding signal was detected with the mutated HbFPS1 promoter (Fig. 2C). The result of EMSA also displayed HbWRKY27 interacted with HbFPS1 promoter and the TTGAC is necessary for binding of HbWRKY27 protein to the HbFPS1 promoter.

Subcellular localization of HbWRKY27.
To elucidate the subcellular localization of HbWRKY27, the green fluorescent protein (GFP) gene was employed as a marker to fuse HbWRKY27 in-frame, generating the HbWRKY27-GFP construct. Compared with the fluorescence was clearly visible in the cytoplasm and nucleus of the cell transformed with 35S-GFP, the fluorescence was restricted to the nucleus of the cell transformed with HbWRKY27-GFP (Fig. 2D), suggesting that HbWRKY27 was a nuclear-localized protein.
Expression profile of HbWRKY27. Expression of HbWRKY27 was analyzed by qPCR. The result of qPCR showed that HbWRKY27 predominantly accumulated in latex, but little expression was detected in the leaves, flowers, roots, and barks (Fig. 3A). To investigate HbWRKY27 expression in response to ABA, SA, ET, and JA treatment in latex, qPCR analysis of HbWRKY27 expression were carried out. The expression of HbWRKY27 was up-regulated by ABA, SA, ET, and JA treatment. The expression of HbWRKY27 reached its maximum level after 9 h of SA, ET, and JA treatment, while the expression of HbWRKY27 reached its maximum level after 24 h of ABA treatment (Fig. 3B).

HbWRKY27 activates the transcription of HbFPS1. To further study the regulatory relationship of
HbWRKY27 and the transcription of HbFPS1, the luciferase (LUC) was employed as a report gene to fuse with the HbFPS1 promoter fragment to generate the pHbFPS1:LUC construct, and the effector p35S-HbWRKY27 was constructed (Fig. 4A). pHbFPS1:LUC was introduced into tobacco leaves along with p35S-GUS or p35S-HbWRKY27. Dual-luciferase assays indicated that HbWRKY27 had significant activation effect on transcription from the HbFPS1 promoter and had no activation effect on transcription from the mutated HbFPS1 promoter (Fig. 4B), indicating that HbWRKY27 could bind the HbFPS1 promoter and activate the HbFPS1 promoter in the transcription level.

Discussion
FPSs have been identified in a few plants 11,22,23,25 . FPSs belong to a small multigenic family which encodes at least two different isoforms in plants. The members of the FPS family have a different pattern of expression that vary among different plant species 11,22 . For example, in Arabidopsis FPS1 is predominantly expressed in roots and inflorescences, whereas FPS2 accumulates preferentially mRNA in inflorescences 22 . In Ginkgo biloba, the higher GbFPS expression level was detected in roots and leaves 23 , in which the ginkgolides and bilobalide are synthesized 24 . In Euphorbia pekinensis, EpFPS had a high transcription level in roots, in which terpenoids are synthesized 25 . In the rubber tree, HbFPS1 is expressed predominantly in the laticifers and is likely to encode the enzyme involved in natural rubber biosynthesis 11 . To our knowledge, the transcriptional regulatory mechanisms of FPS gene in plant has not been reported. www.nature.com/scientificreports/ WRKY transcription factors, a plant specific transcription factor family, play crucial roles in plant secondary metabolites [26][27][28] . For example, GaWRKY1 regulates the biosynthesis of gossypol in Gossypium spp 29 . AaWRKY1 regulates the biosynthesis of artemisinin in Artemisia annua 30 . In Vitis vinifera, VviWRKY40 modulates glycosylated monoterpenoid production 31 . The phylogenetic analyses revealed that HbWRKY27 is highly homologous with MeWRKY27, RoWRKY27, AtWRKY65, and OsWRKY14. OsWRKY14 regulates serotonin production through the up-regulation of the expression of tryptophan synthase gene and tryptophan decarboxylase gene in rice 32 . The role of other homologs of HbWRKY27 has never been reported. More than 80 WRKY proteins in rubber tree have been identified 33 . HbWRKY1 is demonstrated to repress the expression of HbSRPP, a natural rubber biosynthesis-related gene, suggesting HbWRKY1 might a negative regulator in natural rubber biosynthesis 15 . www.nature.com/scientificreports/ Over-expressing of HbWRKY40 in Arabidopsis increased resistance against Botrytis cinerea 34 . Except these, the function of few HbWRKYs had been reported. Here, Y1H and EMSA analysis displayed HbWRKY27 bound the HbFPS1 promoter. HbFPS1 is predominantly expressed in latex where natural rubber is synthesized 11,12 . Intriguingly, HbWRKY27 was also predominantly accumulated in latex, consisting with the expression profile of HbFPS1, suggesting the co-ordinate regulation of natural rubber biosynthesis by both HbWRKY27 and HbFPS1. Moreover transient expression of HbWRKY27 led to increase the activity of the HbFPS1 promoter in vivo, suggesting HbWRKY27 might a positive regulator in natural rubber biosynthesis. In rubber tree, the natural rubber biosynthesis pathway underlying enzymes have been identified 35,36 , but the transcriptional regulatory of rubber biosynthesis are poorly understood [37][38][39] . A few transcription factors except WRKYs had been identified to regulate natural rubber biosynthesis-related gene. For example, HbMADS4 has been identified to negatively regulate HbSRPP expression 17 , while HbMYC2b positively regulates HbSRPP expression 40 . HbCZF1 up-regulates 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR) gene expression 41 . HblMYB19 and HblMYB44 have been identified to up-regulate the HbFPS1 expression 13 . HbRZFP1 down-regulates rubber transferase gene (HRT2) expression. The interaction of 14-3-3 protein with HbRZFP1 led to relieve HbRZFP1-mediated HRT2 transcription inhibition 42 . Even so, the underlying transcriptional regulatory mechanisms of natural rubber biosynthesis are largely unknown. Further investigation of regulatory machinery of natural rubber biosynthesis will be important in manipulating natural rubber metabolism.
Plant hormones have crucial important roles in regulating natural rubber biosynthesis 39,43,44 . WRKY transcription factors are involved in SA, AB, ET, and JA signaling pathways and plays a vital role in the signal crosstalk of the SA, AB, ET, and JA signaling pathways 23,45,46 . The promoter of HbWRKY27 had a few cis-acting elements related to hormone responses and HbWRKY27 is simultaneously up-regulated by SA, AB, ET, and JA, suggesting that HbWRKY27 might integrate plant hormones signals and regulates natural rubber biosynthesis. Further investigation should be carried out to study the mechanisms by which HbWRKY27 integrates plant hormones signals and mediates natural rubber biosynthesis.

Conclusion
In the present study, HbWRKY27 was identified to bind the HbFPS1 promoter. HbWRKY27 had significant activation effect on transcription from the HbFPS1 promoter. HbWRK27 might a positive regulator of HbFPS1, which participates in natural rubber biosynthesis.