Journal of

Plant Biotechnology

Fig. 2. The MtMKK5-MtMPK3/6 module directly interacts with MtNIN. (A, B) MtMPK3 is a Rhizobium-response gene, and its induction is required for MAPK signaling activation. After incubation for 1 hr with/without 5 μM U0126, M. truncatula roots were infected with S. meliloti for the indicated time durations. The levels of the transcripts of MtMPK3 (A) and MtMPK6 (B) were assessed by qRT-PCR. The error bars indicate the S.E. (n = 3); a Student’s t-test was performed (**, P < 0.01; n.s.: no significant). (C) MtMPK3 and 6 are phosphorylated by MaMKK5a. Myc-tagged MtMPK3 and 6 were transfected with/without MtMKK5a-HA into Arabidopsis protoplasts. The MtMPK3 and 6 proteins were immunoprecipitated using anti-myc antibodies and subjected to SDS-PAGE. The dual TEY motif phosphorylation of MtMPK3 and 6 were detected using anti-pTEpY monoclonal antibodies. The expression levels of MtMPKs and MtMKK5a were determined via immunoblotting. (D) MtNIN and MtMPK3/6 interact physically, as demonstrated via a yeast two hybrid assay. The yeasts were selected on synthetic medium lacking Leu, Trp, and His (-LTH) containing 5 mM 3-AT or medium lacking Leu and Trp (-LT)

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