www.sciencemag.org/cgi/content/full/332/6025/103/DC1

Supporting Online Material for

Perception of UV-B by the Arabidopsis UVR8 Protein Luca Rizzini, Jean-Jacques Favory, Catherine Cloix, Davide Faggionato, Andrew O’Hara,2 Eirini Kaiserli, Ralf Baumeister, Eberhard Schäfer, Ferenc Nagy, Gareth I. Jenkins, Roman

Ulm*

*To whom correspondence should be addressed. E-mail: roman.ulm@unige.ch

Published 1 April 2011, Science 332, 103 (2011) DOI: 10.1126/science.1200660

This PDF file includes:

Materials and Methods Figs. S1 to S7 References

1

SUPPORTING ONLINE MATERIAL

MATERIALS AND METHODS

Plant material and growth conditions. cop1-4 and uvr8-6 are in the Columbia ecotype

(Col) (1, 2). The cop1-4/Pro35S:YFP-COP1, cop1-4 uvr8-6/Pro35S:YFP-COP1 and Col/

Pro35S:CFP-UVR8 lines were described before (2, 3).

UV-B irradiation. Plants were grown and irradiated exactly as described previously (2, 4).

Irradiation of cell-free protein extracts was carried out on ice. Longer-term irradiations

(>1h) in experiments with HEK293T cell extracts and yeast two-hybrid analysis used

narrowband UV-B tubes (Philips TL20W/01RS, 1.5 mol m-2 s-1) (see spectrum in fig. S7).

Short-term irradiations (<15 min) of plant and yeast extracts were performed under

broadband UV-B lamps with WG305 cut-off filters (Philips TL40W/12RS, 21 mol m-2 s-1)

(see spectrum in fig. S7). These UV-B treatments are identical to the ones used for

photomorphogenic plant responses described in (refs. 2, 3) and (ref. 4), respectively. Note

that 15 min irradiation of Arabidopsis seedlings with broadband UV-B under WG305 cut-

off filters resulted in the identification of the uvr8 mutant in a luciferase reporter-based

genetic screen (2) and that under these conditions only one hundred genes are upregulated

at a genome-wide level in wild type at 1h postirradiation, and only one gene at 6h

postirradiation (4) (no sustained cellular effect). Therefore, both irradiation conditions are

2

equally capable of eliciting physiological changes in gene expression with negligible

damage (2, 4).

Yeast two-hybrid. Gateway-based cloning was used to insert the UVR8 and COP1 coding

regions in frame to the LexA DNA binding domain (BD) in pBTM116-D9 (5) (vector

kindly provided by Ulrich Stelzl, Max Delbrück Center for Molecular Medicine, Berlin-

Buch) and to the GAL4 activation domain (AD) in pGADT7_GW vector (6) (kindly

provided by Thomas Kretsch, University of Freiburg). The empty vectors used as negative

controls were generated as described before (7). Transformation of lithium acetate-treated

L40 yeast cells was carried out according to Gietz and Woods (2002) (8). The genotype of

the S. cerevisiae reporter strain L40 is MATa trp1 leu2 his3 ade2 LYS2::lexA-HIS3

URA3::lexA-lacZ GAL4 (9). For the interaction assays, transformed yeast colonies were

grown in a white light field supplemented by narrowband UV-B (1.5 mol m-2 s-1) at 30°C

overnight either under a WG305 (+UV-B) or a WG345 (-UV-B control) cut-off filter (light

field and filters as described before (2)). Ten colonies were then combined and resuspended

in YPD media, OD600 was measured and cells were spun down and washed. The

quantitative interaction assay was carried out with chlorophenol red-β-D-galactopyranoside

(CPRG; Roche Applied Science) as substrate, according to the Yeast Protocols Handbook

(Clontech, Version PR973283). The lacZ β-galactosidase activity is expressed as Miller

units.

3

Immunoprecipitation and protein gel blot analysis. Protein extracts were incubated with

monoclonal anti-GFP antibodies (Invitrogen A11120) and protein A-agarose (Roche

Applied Science) in extraction buffer EB (50 mM Tris pH 7.5, 150 mM NaCl, 10%

glycerol, 5 mM MgCl2, 0.1% Igepal, 2 mM benzamidine, 1 mM PMSF, 10 mM leupeptine,

10 mM dichloroisocumarin, 1% (v/v) protease inhibitor cocktail for plant extracts (Sigma),

10 mM each of the proteasome inhibitors MG132, MG115, ALLN, PSI) for 2 h at 4°C, and

beads were washed three times in buffer EB containing 2 mM benzamidine. For protein gel

blot analysis, total cellular proteins or immunoprecipitates were separated by

electrophoresis in 8% SDS-PAGE and electrophoretically transferred to PVDF membranes

according to the manufacturer’s instructions (Bio-Rad). We used polyclonal anti-UVR8 (2),

anti-actin (Sigma), anti-LexA (Millipore) and monoclonal anti-GFP (Clontech) as primary

antibodies, with horseradish peroxidase-conjugated protein A (Pierce), or anti-rabbit and

anti-mouse immunoglobulins (Dako A/S) as secondary antibodies, as required. Signals

were detected using the ECL Plus Western detection kit (GE Healthcare) or SuperSignal

West Femto Maximum Sensitivity Substrate (Thermo Scientific) for blots containing

immunoprecipitates.

Crosslinking of proteins. Proteins were extracted from Arabidopsis seedlings at 4°C in

PBS containing 0.1% Igepal, 1 mM PMSF, 10 mM leupeptine, 10 mM dichloroisocumarin,

1% (v/v) protease inhibitor cocktail for plant extracts (Sigma), 10 mM each of MG132,

MG115, ALLN, PSI). After elimination of cell debris by centrifugation (10 min, 4°C,

12,000 g), supernatants were irradiated with UV-B in small Petri dishes for the indicated

times. Dithiobis (succinimidyl propionate) (2 mM final concentration) (DSP; Thermo

4

Scientific) was then added to the extract, which was kept at 4°C with shaking for 30 min.

The crosslinker was then quenched by 50 mM Tris pH 7.6 for 15 min at room temperature.

Protein sample buffer containing -ME (5% final) (reversal of crosslink) or without

reducing agent (no reversal of crosslink) was added, and samples boiled for 10 min before

loading.

UV-B treatment of protein gels. After gel electrophoresis, the protein gel was transferred

to a thin layer of SDS-PAGE running buffer to avoid drying of the gel. The gel was then

irradiated for 10 min under broadband UV-B before the proteins were electrophoretically

transferred onto a PVDF membrane according to standard procedures using a Mini Trans-

Blot Cell (Bio-Rad Laboratories).

HEK293T cell culture and transfection. In order to express UVR8 in HEK293T cells, the

UVR8 coding region including the stop codon was cloned into the expression vectors

pDEST27 (incl. N-terminal GST-tag) and pcDNA-DEST40 (expression of UVR8 without a

tag) (Invitrogen, Karlsruhe, Germany). HEK293T cells were cultured in MEM with

Glutamax supplemented with 10% FBS. For transfection experiments, cells were grown

until 60–80% confluence and transfected with plasmid DNA using GeneJuice Transfection

Reagent (Novagen) as described previously (10). Cell lysis was performed using

CytoBuster extraction buffer (Novagen) as described before (10).

5

UVR8 multiple alignment and tertiary structure prediction. The multiple alignment

was edited with Jalview (11). The tertiary structure prediction was done with the automated

homology modeling server (PS)2 using RCC1, PDB entry 1A12, chain A, as template (12).

The predicted model was edited with PyMOL v1.1 (The PyMOL Molecular Graphics

System, Version 1.1, Schrödinger, LLC).

+ UV-B- UV-BADBD UV BUV B

Antigen T

ADBD

p53

COP1

--

UVR8 COP1

COP1

UVR8

UVR8N400

C341

COP1N282UVR8

COP1C341UVR8

Figure S1. Yeast two-hybrid interaction of UVR8 and COP1 specifically under a low fluence rate of narrowband UV-B (0.1 mol m-2 s-1) As a control mammalian p53 and antigen T were cloned into the binding domain (BD) and activation domainmol m 2 s 1). As a control, mammalian p53 and antigen T were cloned into the binding domain (BD) and activation domain (AD) vectors, respectively. The mutation in UVR8N400 (deletion of the C-terminal 40 aa in uvr8-2) impaired the interaction with COP1. A mutation in COP1 representing cop1-4 (COP1N282; N-terminal 282 aa without WD40 domain) similarly impaired the i t ti ith ild t UVR8 Th COP1 C t i l 341 i id d th WD40 d i l (COP1C341)interaction with wild-type UVR8. The COP1 C-terminal 341 amino acids encompassed the WD40 domain only (COP1C341) and were sufficient for the interaction with wild-type UVR8.

A

- UVR8dimer

*- UVR8

0 0 1.5 2.6 5.25 2110.5

broad-narrowband

UV-B [mol m-2 s-1]

-6

band

Col uvr8

-

B

8-6

21 μmol m-2 s-1 10.5 μmol m-2 s-1

uvr8

- UVR8dimer

*- UVR8

0 10 20 40 80 20 40 16080 UV B [ ]1600 10 20 40 80 20 40 16080 UV-B [s]160

6C5 μmol m-2 s-1 2.5 μmol m-2 s-1

uvr8

-6C

- UVR8dimer

*- UVR8

0 40 80 160 320 80 160 640320 UV-B [s]640

Fig S2 (A) Analysis of dose dependence of UVR8 monomerization in Arabidopsis protein extracts

[ ]

Fig. S2. (A) Analysis of dose-dependence of UVR8 monomerization in Arabidopsis protein extracts. Protein extracts were exposed to the indicated UV-B fluence rates for one minute on ice. (B and C) Reciprocal relationship between treatment duration and fluence rate in stimulating UVR8

i i P i d i h i di d UV B fl fmonomerization. Protein extracts were exposed on ice to the indicated UV-B fluence rates of broadband (B) and narrowband UV-B (C) for the indicated times. (A-C) Samples were non-heat-denatured and the protein gels were irradiated by UV-B after the gel run (15 min; 21 mol m-2 s-1) p g y g ( ; )and before transfer to the membrane. The protein gel blots were probed with an anti-UVR8 antibody.

A BA B

Figure S3. The 14 tryptophans of UVR8 are all located at the top of the predicted UVR8 -propeller structure. View from the side (A) and from the top (B). The 14 tryptophans are highlighted and shown in yellow.

UVR8 UVR8W233F UVR8W337A

+ ++ ++ - -- - -UVR8W337FUVR8

UVBUVB- LexA-UVR8dimer

*- LexA-UVR8*

*

Fig. S4. Analysis of UVR8W233F, UVR8W337A and UVR8W337F for UV-B-mediated UVR8 monomerization in yeast.Fig. S4. Analysis of UVR8 , UVR8 and UVR8 for UV B mediated UVR8 monomerization in yeast. Proteins were electrophoretically separated without previous heat denaturation. The protein gel blot was probed with an anti-LexA antibody.

A

175

83

47 5

175

62- UVR847.5

32

- UVR8

Root StemB

Leaf epidermis Leaf epidermal guard cell

Sepal Petal

C

Fig. S5. UVR8 is expressed throughout the plant from early in development and subcellularly localised in the nucleus and cytosol. (A) Western blot analysis of UVR8 in different plant organs. (B) Confocalimages of GFP fluorescence in different plant tissues of uvr8 1/Pro :GFP UVR8 grown in white lightimages of GFP fluorescence in different plant tissues of uvr8-1/ProUVR8:GFP-UVR8 grown in white light (20 mol m-2 s-1) and exposed to UV-B (3 mol m-2 s-1) for 4 hours. Scale bars = 20 m. (C) Confocalimages of GFP fluorescence in epidermal tissues of uvr8-1/ProUVR8:GFP-UVR8 young seedlings grown in white light (100 mol m-2 s-1). Scale bars = 0.5 mm. The uvr8-1/ProUVR8:GFP-UVR8 line 6-2 and confocal microscopy were described before (13).

*

Figure S6. UVR8 proteins are well-conserved among plant species. Tryptophans (W) are highlighted in red (ArabidopsisUVR8 [Q9FN03 ARATH] Trp 285 is additionally marked with an asterisk) UniProt Knowledgebase (UniProtKB) identifiersUVR8 [Q9FN03_ARATH] Trp-285 is additionally marked with an asterisk). UniProt Knowledgebase (UniProtKB) identifiers are given for each protein used in the alignment. Representative genomes for monocots, dicots, lower plants and green algae were chosen: ARATH (Arabidopsis thaliana), VITVI (Vitis vinifera), RICCO (Ricinus communis), POPTR (Populustrichocarpa), ORYSI (Oryza sativa indica), PHYPA (Physcomitrella patens), SELMO (Selaginella moellendorfii), VOLCA (Volvox carteri), CHLRE (Chlamydomonas reinhardtii). The multiple sequence alignment was generated by ClustalW as an option of the multiple alignment editor Jalview version 2.4.0.b2 (http://www.jalview.org/).option of the multiple alignment editor Jalview version 2.4.0.b2 (http://www.jalview.org/).

A0.6

A

0.5

0.4

0.3

W/m

2

0.2

W

0.1

00

250 270 290 310 330 350 370 390

0.14B

0.12

0.1

0.08

/m2

0.06W/

0.04

0.02

0

250 270 290 310 330 350 370 390

Fig. S7. Spectra of the UV-B lamps used. (A) Philips TL40W/12 broadband UV fluorescent tubes. (B) Philips TL20W/01RS narrowband UV-B tubes. (A,B) Spectral irradiance was measured in 2-nm intervals using an OL 754 UV visible spectroradiometer (Optronix Laboratories Orlando FL)754 UV-visible spectroradiometer (Optronix Laboratories, Orlando, FL).

SUPPORTING REFERENCES

S1. T. W. McNellis et al., Genetic and molecular analysis of an allelic series of cop1

mutants suggests functional roles for the multiple protein domains. Plant Cell 6,

487 (1994).

S2. J. J. Favory et al., Interaction of COP1 and UVR8 regulates UV-B-induced

photomorphogenesis and stress acclimation in Arabidopsis. EMBO J. 28, 591

(2009).

S3. A. Oravecz et al., CONSTITUTIVELY PHOTOMORPHOGENIC1 is required for

the UV-B response in Arabidopsis. Plant Cell 18, 1975 (2006).

S4. R. Ulm et al., Genome-wide analysis of gene expression reveals function of the

bZIP transcription factor HY5 in the UV-B response of Arabidopsis. Proc. Natl.

Acad. Sci. U.S.A. 101, 1397 (2004).

S5. U. Stelzl et al., A human protein-protein interaction network: a resource for

annotating the proteome. Cell 122, 957 (2005).

S6. K. Marrocco et al., Functional analysis of EID1, an F-box protein involved in

phytochrome A-dependent light signal transduction. Plant J. 45, 423 (2006).

S7. S. Bartels et al., MAP KINASE PHOSPHATASE1 and PROTEIN TYROSINE

PHOSPHATASE1 are repressors of salicylic acid synthesis and SNC1-mediated

responses in Arabidopsis. Plant Cell 21, 2884 (2009).

S8. R. D. Gietz, R. A. Woods, Transformation of yeast by lithium acetate/single-

stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87

(2002).

S9. A. B. Vojtek, S. M. Hollenberg, Ras-Raf interaction: two-hybrid analysis. Methods

Enzymol. 255, 331 (1995).

S10. U. Schäffer et al., SnAvi--a new tandem tag for high-affinity protein-complex

purification. Nucleic Acids Res. 38, e91 (2010).

S11. A. M. Waterhouse, J. B. Procter, D. M. Martin, M. Clamp, G. J. Barton, Jalview

Version 2--a multiple sequence alignment editor and analysis workbench.

Bioinformatics 25, 1189 (2009).

S12. C. C. Chen, J. K. Hwang, J. M. Yang, (PS)2: protein structure prediction server.

Nucleic Acids Res. 34, W152 (2006).

S13. E. Kaiserli, G. I. Jenkins, UV-B promotes rapid nuclear translocation of the

Arabidopsis UV-B specific signaling component UVR8 and activates its function in

the nucleus. Plant Cell 19, 2662 (2007).