Function of a Mitogen Activated Protein Kinase Pathway in N-gene Mediated Resistance in Tobacco

(Hailing Jin, UC Berkeley)
Suppression of NtMEK2, SIPK or WIPK expression via PVX virus-induced gene silencing compromised N gene mediated resistance. N. benthamiana::NN plants were infiltrated with Agrobacterium carrying binary PVX constructs with fragment of N (a positive control), NtMEK2, SIPK, WIPK or SIPKK (a negative control). Fourteen days later, the upper leaves that show the silencing of target gene were inoculated with GFP-tagged TMV. The presence/ spreading of the TMV virus in both the inoculated leaf and the upper inoculated leaf was determined 5 days later by visualizing the green fluoresces under UV light.The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. The conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous reports that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death. 

  • Jin, H., Liu, Y., Yang, K., Kim, C. Y., Baker, B., Zhang, S. (2003) Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco The Plant Journal 33:719-731