Corrected figures for Zimmermann et al (2004)

The figures shown below were prepared for the paper Zimmermann et al. 2004 (in The Plant Journal). For reasons that are not really clear, the published version of the paper contained "pre-final" versions of figures 1 and 6. You will need the paper text to get the info from the figures. The published paper text refers to the figures in the versions shown here, the figures in the paper miss data regarding TTG1 (figure 1) or contain different aa number labels (figure 6).

Figure 1

a) Yeast two-hybrid analysis of MYB-BHLH interactions

BHLH proteins were fused to the GAL4 binding domain (BD) and assayed for their ability to bind TTG1 and the MYB proteins fused to the GAL4 activation domain (AD). The interaction strength indicated in the matrix was estimated by comparing the growth of yeast cells on triple dropout media supplemented with 3, 20 and 40 mM 3-aminotriazole (3-AT), respectively. pACT K was used in order to check whether the BHLH proteins contain a functional activation domain in yeast. The BHLH proteins of subgroup IIId and IIIe did not interact with TTG1 (exemplarily the test with AtBHLH013 is shown). AtMYB12 and AtMYB111, MYB proteins of subgroup 7 were tested as well but show results similar to AtMYB11 (no interaction with R/B-like BHLH proteins).++, growth at 20 mM 3-AT; +, growth at 3 mM 3-AT; -, no growth on triple dropout media containing 3 mM 3-AT.

b) Semiquantitative assay of interaction strength

a-galactosidase assays of yeast strains expressing combinations of BHLH and MYB proteins fused to the GAL4 binding and activation domain, respectively. Results shown represent the mean values of 12 independent a-galactosidase-assays.

c) In vitro pull down assay.

Binding of PAP1/AtMYB75 and PAP2/AtMYB90 to GL3/AtBHLH001, EGL3/AtBHLH002, AtBHLH012, TT8/AtBHLH042 and AtBHLH013. MYB proteins were expressed as GST fusion proteins. Fusion proteins were re-immobilized on GSH agarose beads and incubated with [35S]methionine-labelled BHLH proteins synthesized in a coupled transcription-translation system. As a negative control beads were loaded with GST. After extensive washing, bound proteins were analysed by SDS-PAGE. [35S]-labelled proteins were detected by autoradiography.

Figure 6

Model of the PAP1/AtMYB75 R2R3-MYB domain on the basis of the NMR structure of the MYB domain of c-MYB (Ogata et al., 1994).

Amino acids which are elements of the motif [DE]Lx2[RK]x3Lx6Lx3R are highlighted (light grey) and named according to their position in the PAP1/AtMYB75 sequence. The positions 12, 13, 16, 17, 20, 21, 24, 29 and 33 correspond to the amino acids D72, L73, R76, L77, L80, L81, R84, L87 and R91 of PAP1, respectively. Amino acids conserved throughout Arabidopsis thaliana MYB proteins are coloured in dark grey. Amino acids involved in interaction are solvent-exposed, forming a characteristic pattern positioned on the surface opposite to the DNA binding site.