ARTICLES https://doi.org/10.1038/s41477-017-0099-0 UVR8 interacts with WRKY36 to regulate HY5 transcription and hypocotyl elongation in Arabidopsis Yu Yang1,2, Tong Liang1,2, Libo Zhang1, Kai Shao1,2, Xingxing Gu1,2, Ruixin Shang1,2, Nan Shi1, Xu Li1, Peng Zhang1 and Hongtao Liu 1* UV RESISTANCE LOCUS 8 (UVR8) is an ultraviolet-B (UVB) radiation photoreceptor that mediates light responses in plants. How plant UVR8 acts in response to UVB light is not well understood. Here, we report the identification and characteriza- tion of the Arabidopsis WRKY DNA-BINDING PROTEIN 36 (WRKY36) protein. WRKY36 interacts with UVR8 in yeast and Arabidopsis cells and it promotes hypocotyl elongation by inhibiting HY5 transcription. Inhibition of hypocotyl elongation under UVB requires the inhibition of WRKY36. WRKY36 binds to the W-box motif of the HY5 promoter to inhibit its transcription, while nuclear localized UVR8 directly interacts with WRKY36 to inhibit WRKY36–DNA binding both in vitro and in vivo, leading to the release of inhibition of HY5 transcription. These results indicate that WRKY36 is a negative regulator of HY5 and that UVB represses WRKY36 via UVR8 to promote the transcription of HY5 and photomorphogenesis. The UVR8–WRKY36 interac- tion in the nucleus represents a novel mechanism of early UVR8 signal transduction in Arabidopsis. ltraviolet-B light is an inherent part of sunlight, which has ner1,6,16,17,22. The central role of HY5 in the UVB acclimation response significant biological effects on plants. Low-level, non-dam- is further confirmed by the UVB stress hypersensitive phenotype of aging UVB serves as a photomorphogenic signal to regu- the hy5 mutant1,16,21. In darkness, HY5 is a target of COP1 and gets U 23 late photomorphogenesis. For example, UVB inhibits hypocotyl degraded via the proteasome . Under UVB stimulation, however, growth, as well as biosynthesis and accumulation of ‘sunscreen’ pig- COP1 is required for the induction of HY5 expression. The UVB- ments1,2. UV RESISTANCE LOCUS 8 (UVR8) is the long-sought- inducible HY5 stabilization is probably a consequence of the UVR8– after UVB photoreceptor that is required for UVB responses3–5. The COP1 interaction1,17 and HY5 is involved in a positive feedback loop UVR8 protein is localized in both the cytoplasm and the nucleus, promoting COP1 expression by binding the COP1 promoter21. HY5 while its main activity is assumed to be nuclear. UVR8’s protein and HYH interact directly with a T/G-box cis-acting element of the abundance is not affected by UVB, but UVB irradiation induces HY5 promoter, mediating the UVB-activated HY5 transcription24. the nuclear accumulation of UVR8 (refs 1,6). Direct interaction Blue light photoreceptor cryptochromes (CRYs) interact with the between photoreceptors and their respective target proteins has TFs cryptochrome-interacting basic-helix-loop-helix 1 (CIB1) and been recognized as a fundamental mechanism underlying the sig- PHYTOCHROME-INTERACTING FACTOR 4/5 (PIF4/PIF5) to nal transduction of plant photoreceptors. Only a few proteins have regulate transcription25–29, while red/far red light photoreceptor pho- been reported to physically interact with UVR8 to mediate UVB tochromes (PHYs) interact with PIFs to regulate transcription30,31. signal transduction. The E3 ubiquitin ligase CONSTITUTIVELY The mechanism by which UVR8 triggers UVB photomophogenic PHOTOMORPHOGENIC 1 (COP1) is a central regulator of light responses in the nucleus and whether UVR8 interacts with TF to signalling7 that interacts with UVR8 in a UVB-dependent man- directly regulate transcription are still unknown. Whether other ner1,3,8,9. COP1 is required for UVB-induced nuclear accumulation TFs are involved in the UVB-induced HY5 transcription is also of UVR8 and also UVR8-mediated UVB signalling10,11. The WD40- unknown. Here, we identify and characterize the Arabidopsis WRKY repeat proteins REPRESSOR OF UVB PHOTOMOPHOGENESIS 1 DNA-BINDING PROTEIN 36 (WRKY36), which physically inter- (RUP1) and RUP2 are negative regulators of UVB signalling12. acts with UVR8 in vivo, and find that the UVR8–WRKY36 complex RUP1 and RUP2 directly interact with UVR8 to mediate UVR8 accumulates in nuclei in response to a photomorphogenic UVB light redimerization so as to disrupt the UVR8–COP1 interaction13. stimulus. WRKY36 promotes hypocotyl elongation by repressing the The basic leucine-zipper transcription factor (TF) ELONGATED transcription of HY5, and WRKY36 is involved in UVB responses HYPOCOTYL 5 (HY5) plays a very important role in de-etiolation14. downstream of UVR8. Nucleus-localized UVR8 that is activated HY5 and its homologue HYH mediate UVB-induced gene expression under UVB represses the DNA-binding activity of WRKY36. These changes downstream of UVR8 (refs 15–21). HY5 was proposed to be results demonstrate that UVR8 can regulate gene expression in involved in UVB signalling when its transcription was identified as response to UVB light by directly interacting with the TF WRKY36. UVB induced15. Both UVR8 and COP1 are required for UVB-induced HY5 transcription. Treatment with UVB induces the transcription UVR8 physically interacts with WRKY36. Whether UVR8 inter- and translation levels of HY5 in a UVR8- and COP1-dependent man- acts with TFs to regulate transcription and UVB responses is still 1National Key Laboratory of Plant Molecular Genetics, Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China. 2Shanghai College of Life Science, University of Chinese Academy of Sciences, Shanghai, China. *e-mail: [email protected] 98 NATURE PLAnts | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PLANTS ARTICLES unknown. To address this question, we performed a yeast-two WRKY36 messenger RNA increased by about twofold within the first hybrid screen with a library of Arabidopsis thaliana TF open read- hour of UVB light irradiation and then decreased (Supplementary ing frames32 to identify TFs that interact with A. thaliana UVR8. Fig. 1c). Interestingly, the UVB-induced WRKY36 transcription was WRKY36 was identified in this screen. In yeast cells, WRKY36 not UVR8 dependent, since UVB light induced the transcription of interacted with UVR8 in both darkness and UVB (Supplementary WRKY36 in the uvr8 mutant (Supplementary Fig. 1c). Fig. 1a). WRKY36 is a novel WRKY protein (Supplementary Fig. 1b) Next, we examined the interaction between UVR8 and whose function has not been reported previously. The Arabidopsis WRKY36 using an in vitro pull-down assay. UVR8 was expressed genome encodes more than 70 WRKY proteins33–35. WRKY36 and purified as reported before36. Dimeric UVR8 changed to belongs to the subfamily IIb33. The messenger RNA expression of monomeric UVR8 after UVB treatment (Supplementary Fig. 1d). WRKY36 is regulated by UVB light, as shown by our quantitative The Escherichia coli-expressed UVR8 interacted with the E. coli- polymerase chain reaction (qPCR) analyses. When continuous- expressed WRKY36 in a UVB-independent manner in the in white-light-grown seedlings were exposed to UVB light, the level of vitro pull-down assay (Fig. 1a), indicating that both dimeric and a Input UVR8-IP d WRKY36 DUF WRKY His–WRKY36 ++ + ++ + WRKY36N UVR8–His – ++ – ++ WRKY36C UVB ––+ ––+ WRKY36 UVR8 UVR8N UVR8 UVR8C beGFP BF Merge nYFP BF Merge cCFP – cCFP 20 μm 20 m UVR8C WRKY36–nYFP μ WRKY36C–nYFP BF Merge P cCFP nYFP UVR8–cCF WRKY36C–nYFP BF Merge P cCFP – C UVR8 UVR8–cCF WRKY36–nYFP cfInput Myc–IP Input GST-IP GFPUVR8 GFPUVR8 His–WRKY36C ++ + ++ + WRKY36TAP WRKY36TAP GFP– GFP– GST–UVR8N –+ – –+ – UVR8 UVB– UVB+ UVR8 UVB– UVB+ GST–UVR8C –– + –– + WRKY36C UVR8 UVR8N WRKY36 UVR8C Fig. 1 | UVR8 physically interacts with WRKY36. a, UVR8 interacts with WRKY36, as shown by in vitro pull-down assays. His-tagged UVR8 bound to anti-UVR8 beads were mixed with His-tagged WRKY36 purified from E. coli. b, In BiFC assays, UVR8 interacts with WRKY36 in vivo under white light conditions (without UVB treatment). N. benthamiana were co-transformed with WRKY36–nYFP and cCFP, nYFP and UVR8–cCFP or WRKY36–nYFP and UVR8–cCFP. c, Co-immunoprecipitation assays using 14-day-old transgenic seedlings expressing 35 S::UVR8–GFP or 35 S::UVR8–GFP and 35 S::WRKY36– TAP treated with or without UVB for 20 min. Input: immunoblots showing the level of GFP–UVR8, WRKY36–TAP in the total protein extract. Myc-IP: immunoprecipitation products precipitated by the anti-Myc antibody. Total proteins (input) or immunoprecipitation products were probed in immunoblots with antibodies to GFP or Myc. d, Schematic representation of WRKY36 or UVR8 used in this work. WRKY36 contains an unknown domain (DUF) and a DNA-binding domain (WRKY). UVR8 contains seven repeats of a β -propeller fragment and a C terminus (including a C27 domain). e, BiFC assays of the in vivo protein interaction under white light conditions (without UVB treatment). Epidermal cells of the N. benthamiana leaf were co-transformed with WRKY36C–nYFP and cCFP or nYFP and UVR8C–cCFP or WRKY36C–nYFP or UVR8C–cCFP. f, By in vitro pull-down assays, only UVR8C interacts with WRKY36C. GST-tagged UVR8N or UVR8C bound to GST beads were mixed with His-tagged WRKY36C purified from E. coli. BF, bright field; IP, immunoprecipitation; Merge, overlay of the YFP and bright field images. NATURE PLAnts | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants