Figure 6.

Recognition of site II motifs by AtTCP20-modified proteins. A, Effect of transient expression of AtTCP20∷EAR or AtTCP20∷VP16 on the level of GUS activity in N. benthamiana leaves. Leaves were infiltrated with a culture of Agrobacterium strain containing the GUS gene under the control of the site II motif described by Tremousaygue et al. (2003; 1); a mix in a 1:1 ratio of the culture used for 1 with a culture from a recombinant Agrobacterium strain expressing the AtTCP20∷EAR protein (2); or a mix in a 1:1 ratio of the culture used for 1 with a culture from a recombinant Agrobacterium strain expressing the AtTCP20∷VP16 protein (3). B, Detection of AtTCP20-modified proteins by western-blot analysis of protein extracts from transfected N. benthamiana treated with (I) or without (NI) β-estradiol. Protein expression was induced at 48 h by painting leaves, 2 d after infiltration, with 5 μm β-estradiol in the presence of 0.002% Silwett. The arrows highlight the induced AtTCP20∷EAR and AtTCP20∷VP16 proteins. C, Detection of induced AtTCP20∷EAR protein by western-blot analysis of proteins extracted from seedlings cultivated for 3, 6, and 24 h on β-estradiol (5 μm). D, Quantification of PCNA-2 expression level in roots by Q-RT-PCR in response to induction of AtTCP20∷EAR protein. Plants were induced for 24 h with β-estradiol as described in “Materials and Methods.” In each independent experiment (1 and 2), the ratio of induced over noninduced values of expression was calculated as described in “Materials and Methods” using the average threshold cycle and efficiency values from two replicates obtained for PCNA-2, taking into account the housekeeping gene (At5g08290) expression level in each case. [See online article for color version of this figure.]