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

Masayoshi Hashimoto,Yukari Okano,Yusuke Takinami,Shigetou Namba,Yasuyuki Yamaji,Takuya Shiraishi,Kazuya Ishikawa,Ken Komatsu,Kensaku Maejima

doi:10.1186/1471-2229-12-103

Abstract

BackgroundThe 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 MAPKKKα of Nicotiana benthamiana (NbMAPKKKα), 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.ResultsWe cloned three novel MAPKKK genes from N. benthamiana: NbMAPKKKβ, NbMAPKKKγ, and NbMAPKKKε2. Transient overexpression of full-length NbMAPKKKβ or NbMAPKKKγ 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 NbMAPKKKβ or NbMAPKKKγ expression significantly suppressed the induction of programmed cell death (PCD) by viral infection. Furthermore, in epistasis analysis of the functional relationships among NbMAPKKKβ, NbMAPKKKγ, and NbMAPKKKα (previously shown to be involved in plant defense responses) conducted by combining transient overexpression analysis and virus-induced gene silencing, silencing of NbMAPKKKα suppressed cell death induced by the overexpression of the NbMAPKKKβ kinase domain or of NbMAPKKKγ, but silencing of NbMAPKKKβ failed to suppress cell death induced by the overexpression of NbMAPKKKα or NbMAPKKKγ. Silencing of NbMAPKKKγ suppressed cell death induced by the NbMAPKKKβ kinase domain but not that induced by NbMAPKKKα.ConclusionsThese results demonstrate that in addition to NbMAPKKKα, NbMAPKKKβ and NbMAPKKKγ also function as positive regulators of PCD. Furthermore, these three MAPKKKs form a linear signaling pathway leading to PCD; this pathway proceeds from NbMAPKKKβ to NbMAPKKKγ to NbMAPKKKα.

Highlights

The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells
Using tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) [17], we demonstrated that NbSGT1 and NbRAR1, which are important in the hypersensitive response (HR), and the MAPK cascade including NbMAPKKKα/ NbMEK2, are essential for the induction of programmed cell death (PCD)-associated systemic necrosis induced by plantago asiatica mosaic virus (PlAMV)-Li1 [18,19]
Ion leakage levels were closely associated with the intensity of cell death at all combinations of these genes. These three MAPK kinase kinase (MAPKKK) genes form a linear signaling pathway leading to PCD in which NbMAPKKKβ and NbMAPKKKα function as the furthest upstream and downstream components, respectively, of the three MAPKKK components

Summary

Introduction

The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells. 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. some plant MAPKKKs, including the MAPKKKα of Nicotiana benthamiana (NbMAPKKKα), are known to play crucial roles in plant defense responses, the functional relationship among MAPKKK genes is poorly understood. The plant defense response against biotic stress is triggered by the recognition of conserved pathogen-associated molecular patterns (PAMPs) or of pathogen strain-specific factors known as elicitors or effectors [1]. The response triggered by PAMPs is known as the basal defense response, whereas that triggered by specific elicitors is known as the hypersensitive response (HR) In the latter, an effector is recognized by a corresponding plant resistance (R) protein. Many plant components required for the PCD-associated HR have been identified, the entire signaling pathway leading to PCD has not been elucidated

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