Figure 4.

Expression of NtMEK2^DD activates SIPK and WIPK in tobacco. (A) Induction of NtMEK2 and its mutants by steroid in Agrobacterium-mediated transient transformation. Tobacco leaves were infiltrated with Agrobacterium carrying pTA7002 constructs. DEX (30 μM) was infiltrated 40 h later, and samples were taken at indicated times. The expression of transgenes was monitored by immunoblot analysis by using anti-Flag antibody (Top). The kinase activities of Flag-tagged NtMEK2 and its mutants were determined by immune complex (IC)-kinase assay with HisSIPK^R (Middle) or HisWIPK^R (Bottom) as a substrate. (B) Induction of NtMEK2^DD expression activates SIPK and WIPK in vivo. The MAPK activities in cells after DEX treatment were determined by an in-gel kinase assay with MBP substrate (Top). The identities of the two kinases were confirmed by immune complex–kinase assays by using SIPK-specific (Ab-p48N; Middle) or WIPK-specific (Ab-p44N; Bottom) antibody. (C) Expression of NtMEK7^DD does not activate SIPK or WIPK. The levels of NtMEK7 transgene expression were determined by immunoblot analysis (Upper). Endogenous SIPK and WIPK activities were detected by an in-gel kinase assay with MBP as a substrate (Lower).