Three MAPK Kinases, MEK1, SIPKK, and NPK2, are not Involved in Activation of SIPK after Wounding and Herbivore Feeding but Important for Accumulation of Trypsin Proteinase Inhibitors

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We identified three Nicotiana attenuata mitogen-activated protein kinase (MAPK) kinases (MAPKKs), NaMEK1, NaSIPKK, and NaNPK2, whose transcript levels were up-regulated in a wild tobacco plant, N. attenuata, after wounding and simulated herbivore attack. A virus-induced gene silencing approach was used to analyze the functions of these three MAPKKs in plant defense against the tobacco hornworm Manduca sexta. NaMEK1 and NaSIPKK influenced the accumulation of the precursor of jasmonic acid (JA), 12-oxo-phytodienoic acid, and silencing NaSIPKK enhanced the levels of wounding- and herbivory-induced JA. In-gel kinase assays indicated that all three MAPKKs were not required for the activation of NaSIPK, an important MAPK in plant responses to wounding and herbivory. However, NaMEK1, NaSIPKK, and NaNPK2 appeared to regulate the levels of trypsin proteinase inhibitor activity. Bioassays revealed that M. sexta larval growth was not impaired on N. attenuata plants silenced in NaMEK1, NaSIPKK, or NaNPK2 expression. Our findings suggest that NaSIPKK is involved in JA biosynthesis after herbivore attack without activating NaSIPK, suggesting that the network of MAPK signaling in N. attenuata's defense responses against herbivore attack is more complicated than previously thought.