Plant photoreceptors and their signaling components compete for COP1 binding via VP-peptide motifs (2019)
Figure 3

Figure 3 - Chimeras betwen the UV-B-sensing UVR8 core and various VP motifs are functional in the UV-B signaling pathway.

A. Sequence alignment of the N- and C- termini of UVR8, chimera UVR8HY5VP (replacing the UVR8 VP motif with the corresponding sequence from HY5), chimera UVR8HY5C44 (replacing the C44 domain of UVR8 with the corresponding sequence from HY5), and chimera UVR8TRIB1VP (replacing the UVR8 VP motif with the TRIB1 VP motif and a truncation of the rest of the UVR8 C-terminus). The black box indicates the core VP motif of UVR8. The VP is colored in red, the anchor residues in orange, the divergent residues of the HY5 VP core sequence in green, the HY5 sequence replacing the UVR8 C-terminal 44 amino acids (C44) in cyan, and the divergent residues of the TRIB1 VP core sequence in blue. Asterisks represent amino acids identical in all constructs, amino acids 21-390 of UVR8 are not shown. The previously crystallized UVR8 core domain (PDB: 4D9S) is highlighted with a gray bar.

B. Representative image showing the phenotype of wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 seedlings grown for 4 days under white light (- UV-B) or white light supplemented with UV-B (+ UV-B). The scale bar represents 5 mm.

C. Quantification of hypocotyl length data shown in B. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median.

D. Anthocyanin accumulation in seedlings shown in B. Average and SEM are shown (n = 3).

E. Quantitative real-time PCR analysis of RUP2, CHS, HY5 and ELIP2 expression in wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 4-day old seedlings grown under white light in response to 2h of UV-B (+UV-B vs. -UV-B). Error bars represent SEM of 3 biological replicates. Note: the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera.

F. Yeast 3-hybrid analysis of the COP1-HY5 interaction in the presence of the UVR8HY5C44 chimera. (Left pane) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Right panel) Representative filter lift assays of the yeast analyzed in left panel. AD, activation domain; BD, DNA binding domain; Met, methionine.

G. Binding kinetics of the UVR8HY5C44 chimera pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8HY5C44 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8HY5C44 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.

H. Size-exclusion chromatography assay of purified, recombinant chimeric proteins expressed in Sf9 insect cells in the presence and absence of UV-B.