Figure 3.

Activation of NtMEK2 during HR-like cell death initiated by fungal elicitin. (A) Cryptogein (Cry), a fungal elicitin from Phytophthora cryptogea, induces HR-like cell death in tobacco suspension cells. Cells were treated with cryptogein at a final concentration of 25 nM, and cell viability was monitored by the fluorescein diacetate method (20) at the indicated times. Data represent the mean of three replicates ±SE. (B) Ab-NtMEK2 specifically recognized NtMEK2 protein. Two nanograms each of the purified recombinant NtMEK1, NtMEK2, NtMEK7, and NtMEK8 were subjected to immunoblot analysis with Ab-NtMEK2. (C) Activation of NtMEK2 in cells treated with cryptogein. Protein extracts (75 μg) from cryptogein-treated cells were immunoprecipitated with purified Ab-NtMEK2 (4 μg) in the absence (−) or the presence of the N-terminal portion of NtMEK2 (ΔNtMEK2), NtMEK7 (ΔNtMEK7), or NtMEK1 (ΔNtMEK1) as competitor. Kinase activity of the immune complex was subsequently assayed with HisSIPK^R or HisWIPK^R as a substrate.