Phosphoacceptor residue in C4 and active kinase function in AtSKs are required for C4/AtSK interactions.

<p>A. Comparative strength of yeast two-hybrid interactions between C4 protein and wild-type or kinase dead (KD) mutants of the sg2AtSK family members, and between C4 mutants (C4S49A and C4A49T) and wild-type sg2AtSKs measured using ß-galactosidase activity. ß-galactosidase activity was quantified using triplicate samples from three individual yeast colonies for each interaction studied and is expressed relative to the C4/AtSK23 positive control interaction. The interaction between C4 and Lamin C is included as a negative control. B. Confocal micrographs of the interaction assay between the C4 Ser49 mutants (CS49A-cEYFP and C4A49T-cEYFP) and sg2AtSK-nEYFPs following co-agroinfiltration into transgenic <i>N</i>. <i>benthamiana</i> CFP-H2B marker plants. C. Confocal micrograph of C4S49A fused to EYFP following agroinfiltration into transgenic <i>N</i>. <i>benthamiana</i> CFP-H2B marker plants. D. Confocal micrographs of the interaction assay between C4-cEYFP and kinase dead (AtSK21-KD or AtSK23-KD) mutants following co-agroinfiltration into transgenic <i>N</i>. <i>benthamiana</i> CFP-H2B marker plants. The nuclear reference CFP fluorescence appears in blue and the interaction assay EYFP signal appears in yellow in the merged image (B). Scale bar = 50 μm.