Fig. 6.

Interaction of CBL2/3 and CIPK3/9/23/26 at the tonoplast. (A) Yeast two-hybrid assay of the interactions between CBL2/3 and different CIPKs. Yeast AH109 cells were cotransformed with various combinations of BD- and AD-fusion constructs as indicated above and on the left of each panel, respectively. CIPK26ΔF represents CIPK26 lacking NAF domain. Serial decimal dilutions of corresponding yeast cells were spotted onto selective SD medium without leucine and tryptophan as a control or onto the selective SD medium lacking leucine, tryptophan, and histidine for monitoring growth. Photographs were taken after cultivation for 3 d at 30 °C. (B) BiFC analysis of CBL2/3 and CIPK3/9/23/26 interaction in plant protoplasts. The CBL2-YC or CBL3-YC fusion construct, in combination with various YN-fusion constructs as indicated in the left column, was transformed into Arabidopsis mesophyll protoplasts. The YFP signal and chloroplast autofluorescence (Chl) were imaged under a Zeiss confocal microscope as green and red, respectively. (Scale bar: 10 μm.)