Cloning And Interaction Analysis Of MAP Kinases In Arabidopsis Thaliana

MAPK cascades play a central role in many signaling pathways. The MAP K members are dephosphorylated and inactivated without external stimuli. In response to external cues such as rays, drought and pathogens, sequential phosphorylation of MAP KKKs, MAP KKs and MAP Ks activates the expression of downstream target genes to regulate plant growth and development.In this study, coding sequences of 27 MAPKKKs, 10 MAPKKs and 13 MAPKs were cloned using gateway in vitro DNA recombination technology. Yeast two-hybrid was used to investigate interactions between MAP KKs and MAPKKKs, and a total of 21 interactions were identif ied. MKK5 strongly interacts with Raf23, Raf25 and Raf47, and weakly interacts with MAP KKK9 and Raf27. Strong interaction between MKK7 and Raf48, and weak interactions between MKK7 and Raf22/41 were also found. Interactions between MKK8 and Raf23/25/47 are strong, but the interaction between MKK8 and Raf27 is weak. Interactions between Raf28 and MKK1/2/3/4 6/9/10 were also found with strong signals with MKK9/10. No downstream MAPKKs were identified to interact with MAP KKK12. BiFC was further used to confirm that Raf23 and Raf47 interact with MKK5, while Raf22 and Raf48 showed no interaction with MKK5.The subcellular localizations of these MAPK related proteins were examined. It was found that they mainly located in the cytoplasm and on the plasma membrane. Some members are also localizated in the nucleus, such as Raf25, Raf28, MKK3, MKK5 and MKK6.Taken together, we investigated the interactions between MAPKKKs and MAP KKs, and the subcellular localization of these MAP K proteins, which facilitates the further study of MAPK signal pathways.