Identification of three MAPKKKs forming a linear signaling pathway leading to programmed cell death in Nicotiana benthamiana.

ABSTRACT: BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells. In plants, MAPK cascades are associated with responses to various abiotic and biotic stresses such as plant pathogens. MAPK cascades function through sequential phosphorylation: MAPK kinase kinases (MAPKKKs) phosphorylate MAPK kinases (MAPKKs), and phosphorylated MAPKKs phosphorylate MAPKs. Of these three types of kinase, the MAPKKKs exhibit the most divergence in the plant genome. Their great diversity is assumed to allow MAPKKKs to regulate many specific signaling pathways in plants despite the relatively limited number of MAPKKs and MAPKs. Although some plant MAPKKKs, including the MAPKKK? of Nicotiana benthamiana (NbMAPKKK?), are known to play crucial roles in plant defense responses, the functional relationship among MAPKKK genes is poorly understood. Here, we performed a comparative functional analysis of MAPKKKs to investigate the signaling pathway leading to the defense response. RESULTS: We cloned three novel MAPKKK genes from N. benthamiana: NbMAPKKK?, NbMAPKKK?, and NbMAPKKK?2. Transient overexpression of full-length NbMAPKKK? or NbMAPKKK? or their kinase domains in N. benthamiana leaves induced hypersensitive response (HR)-like cell death associated with hydrogen peroxide production. This activity was dependent on the kinase activity of the overexpressed MAPKKK. In addition, virus-induced silencing of NbMAPKKK? or NbMAPKKK? expression significantly suppressed the induction of programmed cell death (PCD) by viral infection. Furthermore, in epistasis analysis of the functional relationships among NbMAPKKK?, NbMAPKKK?, and NbMAPKKK? (previously shown to be involved in plant defense responses) conducted by combining transient overexpression analysis and virus-induced gene silencing, silencing of NbMAPKKK? suppressed cell death induced by the overexpression of the NbMAPKKK? kinase domain or of NbMAPKKK?, but silencing of NbMAPKKK? failed to suppress cell death induced by the overexpression of NbMAPKKK? or NbMAPKKK?. Silencing of NbMAPKKK? suppressed cell death induced by the NbMAPKKK? kinase domain but not that induced by NbMAPKKK?. CONCLUSIONS: These results demonstrate that in addition to NbMAPKKK?, NbMAPKKK? and NbMAPKKK? also function as positive regulators of PCD. Furthermore, these three MAPKKKs form a linear signaling pathway leading to PCD; this pathway proceeds from NbMAPKKK? to NbMAPKKK? to NbMAPKKK?.

SUBMITTER: Hashimoto M 

PROVIDER: S-EPMC3507812 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature