Two-hybrid protein-protein interaction analysis in Arabidopsis protoplasts: establishment of a heterodimerization map of group C and group S bZIP transcription factors.

In vivo protein-protein interactions are frequently studied by means of yeast two-hybrid analysis. However, interactions detected in yeast might differ considerably in the plant system. Based on GAL4 DNA-binding (BD) and activation domains (AD) we established an Arabidopsis protoplast two-hybrid (P2H) system. The use of Gateway-compatible vectors enables the high-throughput screening of protein-protein interactions in plant cells. The efficiency of the system was tested by examining the homo- and heterodimerization properties of basic leucine zipper (bZIP) transcription factors. A comprehensive heterodimerization matrix of Arabidopsis thaliana group C and group S bZIP transcription factors was generated by comparing the results of yeast and protoplast two-hybrid experiments. Surprisingly, almost no homodimerization but rather specific and selective heterodimerization was detected. Heterodimers were preferentially formed between group C members (AtbZIP9, -10, -25, -63) and members of group S1 (AtbZIP1, -2, -11, -44, -53). In addition, significant but low-affinity interactions were detected inside group S1, S2 or C AtbZIPs, respectively. As a quantitative approach, P2H identified weak heterodimerization events which were not detected in the yeast system. Thus, in addition to cell biological techniques, P2H is a valuable tool for studying protein-protein interaction in living plant cells.