Because plants lack an adaptive immune system, appropriate perceptions and responses of individual cells to various environmental stimuli, such as the biotic stress caused by phytopathogenic microorganisms, are critically important. 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 . 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. The HR is frequently accompanied by programmed cell death (PCD), which plays a particularly important role in the defense against biotrophic pathogens but is also an essential function in normal plant development and differentiation . Although many plant components required for the PCD-associated HR have been identified, the entire signaling pathway leading to PCD has not been elucidated.
The mitogen-activated protein kinase (MAPK) cascade is a highly evolutionarily conserved signal transduction mechanism found in eukaryotic cells. Subsequent to activation of the cascade by various extracellular stimuli, the signal is transduced intracellularly by sequential phosphorylation. In plants, MAPK cascades are associated with developmental and hormonal responses and with stress responses to abiotic and biotic factors . A MAPK cascade consists of three functionally linked protein kinases: a MAPK is phosphorylated and activated by a MAPK kinase (MAPKK), which is in turn activated by an upstream MAPK kinase kinase (MAPKKK). Typical MAPK substrates are cytoplasmic or nuclear proteins, such as transcription factors . MAPKKKs are the most divergent of these three types of kinases in plants; the Arabidopsis thaliana genome contains approximately 60 MAPKKKs, 10 MAPKKs, and 20 MAPKs .
Based on phylogenetic analysis of the amino acid sequences of their catalytic kinase domains, plant MAPKKKs have been classified into three groups: A, B, and C . Group A contains many MAPKKKs involved in PCD and stress and defense responses; e.g., A. thaliana AtMEKK1 is involved in the signaling pathway of basal defense induced by PAMPs , and Medicago sativa MsOMTK1  is involved in that of oxidative stress-induced cell death. Group A also includes MAPKKKs that have important functions in HR induction. Silencing of the genes encoding Nicotiana tabacum NPK1 (NtNPK1) and Nicotiana benthamiana MAPKKKα (NbMAPKKKα) suppresses the N gene-mediated HR induced by the helicase domain of tobacco mosaic virus (TMV) replicase and Pto-mediated HR induced by Pseudomonas syringae pv. tomato (Pst) effector avrPto, respectively [7, 8]. Recently, N. benthamiana NbMAPKKKε and its tomato (Solanum lycopersicum) ortholog SlMAPKKKε have been implicated in PCD induction in the HR against Gram-negative bacterial pathogens . In addition, silencing of the genes encoding the MAPKK MEK2 and the MAPK SIPK, both of which act downstream of NbMAPKKKα, also attenuates the N gene-mediated HR against TMV . Conversely, silencing of the tomato orthologs of MAPKK MEK1 and MAPK NTF6, both of whose tobacco orthologs act downstream of NtNPK1in tobacco, leads to loss of the Pto-mediated HR in tomato . Therefore, the NtNPK1- and NbMAPKKKα-initiated MAPK cascades are essential for both the N gene-mediated and the Pto-mediated HR, suggesting that at least two distinct MAPK cascades are involved in the regulation of a single HR event . Furthermore, it is now becoming apparent that two distinct MAPK cascades are involved in non-HR environmental responses .
Plants generally appear to use the same MAPKK/MAPK sets in different responses to environmental stimuli. The A. thaliana MAPKKs AtMKK4 and AtMKK5 and/or their downstream component MPK6 are involved not only in the signaling pathway for basal defense downstream of AtMEKK1 but also in ethylene production and stomata formation [5, 13, 14]. Given the relatively limited number of MAPKKs and MAPKs in plants, the diversity of these responses (functions) is assumed to be possible due to the great diversity of MAPKKKs [15, 16]. Therefore, comparative functional analysis among MAPKKKs is needed to reveal the molecular mechanisms underlying a variety of responses to environmental stresses.
We previously showed that systemic necrosis, the disease symptom caused by plantago asiatica mosaic virus Li1 (PlAMV-Li1), was accompanied by resistance traits similar to HR. Using tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) , we demonstrated that NbSGT1 and NbRAR1, which are important in the HR, and the MAPK cascade including NbMAPKKKα/NbMEK2, are essential for the induction of PCD-associated systemic necrosis induced by PlAMV-Li1 [18, 19]. This result and those described above led us to hypothesize that other MAPKKK genes in addition to NbMAPKKKα are involved in the systemic necrosis induced by PlAMV-Li1.
In the present study, we isolated three novel group A MAPKKK genes from N. benthamiana, a model plant of the family Solanaceae, using an expressed sequence-tag (EST) database. The three cloned genes were designated NbMAPKKKβ, NbMAPKKKγ, and NbMAPKKKε2. Further study revealed that NbMAPKKKβ and NbMAPKKKγ are positive regulators of PCD. In addition, the results of epistasis analysis performed using VIGS and agroinfiltration suggest that two of these MAPKKKs (NbMAPKKKβ and NbMAPKKKγ), together with NbMAPKKKα, comprise a linear signaling pathway important in the induction of PCD.