Figure 2.
An RRR motif in the N-terminal region of JAZ10.4 is required for interaction with MYC2. A, Site-directed mutagenesis of the RRR motif within the CMID of JAZ10.4. The indicated R→A substitution mutants of JAZ10.4 were tested for interaction with MYC2 or JAZ10.4 as a positive control. Y2H assays were performed as described in Figure 1. B, In vitro pull-down assay of the JAZ10.4-MYC2 interaction. Assays were performed using the indicated JAZ10.4-His recombinant proteins and crude extracts from leaves of wild-type (WT) or 35S:cMyc-MYC2 transgenic (T) plants. Protein bound to JAZ-His was separated by SDS-PAGE and analyzed by immunoblotting (anti-cMyc antibody) for the presence of cMyc-MYC2. The Coomassie blue-stained gel shows total input protein as a loading control. C, The CMID of JAZ10.4 is sufficient for MYC2 binding. In vitro pull-down assays were performed as described in B using JAZ3, JAZ3ΔJas, or a chimeric JAZ (JAZ3ΔJas-J101-78) in which the CMID of JAZ10.4 was fused to the C terminus of JAZ3ΔJas. [See online article for color version of this figure.]