One of the early pieces of evidence implicating the involvement of plant MAPKs in stress signalling is the purification and identification of SIPK from tobacco (Zhang and Klessig, 1997). In-gel kinase assays revealed a second smaller MAPK that was activated by tobacco mosaic virus (TMV) infection and elicitin treatment. This MAPK was determined to be WIPK using a member-specific antibody (Seo et al., 1995; Zhang et al., 1998; Zhang and Klessig, 1998a). Recently, a third MAPK, Ntf4 (where Nt denotes Nicotiana tabacum), which shares high homology with SIPK, was identified from tobacco (Ren et al., 2006).
MEK2-SIPK/Ntf4/WIPK module
SIPK and WIPK can be induced not only by various pathogenic signals, but also by wounding and various abiotic stresses, indicating that these MAPKs integrate different abiotic and biotic stress responses. SIPK activation by stress/
PAMPs precedes the induction of SA (Zhang and Klessig, 1997, 1998a, b;
Zhang et al., 1998). Inhibition of SIPK and WIPK activation by staurosporine and K-252a suppresses HR-like cell death in tobacco suspension cells treated with elicitin from oomycetic pathogens (Zhang et al., 2000). The activation of SIPK and WIPK by TMV is gene-for-gene specific, implying a role(s) in disease resistance and possibly HR (Zhang and Klessig, 1998a; Romeis et al., 1999).
Potato virus X (PVX)-mediated silencing of endogenous SIPK/WIPK attenuates N gene-mediated resistance against TMV (Jin et al., 2003; Liu et al., 2003).
The kinetics of the WIPK activation upon elicitor treatment coincides with the onset of HR-like cell death (Zhang et al., 2000). This was further confirmed by the observations that transient expression of SIPK/WIPK and transgenic Ntf4
44 F. Song et al.
plants with elevated levels of Ntf4 protein accelerate HR-like cell death after treatment with elicitors (Zhang and Liu, 2001; Liu et al., 2003; Samuel et al., 2005; Ren et al., 2006). Generally, the activation of SIPK/Ntf4/WIPK is very rapid, which potentially allows them to control multiple defence responses either directly or indirectly. Therefore, it is likely that SIPK, Ntf4 and WIPK are convergent points in the signalling pathway of defence responses.
Based on both the in vitro and the in vivo evidence, it was concluded that NtMEK2 is a shared common upstream MAPKK of SIPK, Ntf4 and WIPK (Yang et al., 2001; Ren et al., 2006). More direct evidence for the roles of SIPK, Ntf4 and WIPK in regulating defence response and HR-like cell death came from gain-of-function studies using a constitutively active mutant of NtMEK2 (Yang et al., 2001; Ren et al., 2006). Expression in tobacco of NtMEK2DD under the control of a steroid-inducible promoter activates endogenous SIPK, Ntf4 and WIPK, which leads to HR-like cell death in the absence of pathogen (Yang et al., 2001; Jin et al., 2003; Yoshioka et al., 2003; Ren et al., 2006). The magnitude and the kinetics of SIPK/Ntf4/WIPK activation by NtMEK2DD are similar to those induced by pathogens or pathogen-derived elicitors that induce HR-like cell death (Zhang and Klessig, 1998b;
Zhang et al., 2000). Using a similar approach, activation of SIPK or Ntf4 alone was shown to be sufficient to induce HR-like cell death (Zhang and Liu, 2001; Ren et al., 2006). However, the HR-like phenotype is delayed in the absence of WIPK activity, consistent with a role for WIPK as a positive feed-forward regulator in the MAPK cascade, accelerating the cell death process (Liu et al., 2003).
Based on these analyses, the NtMEK2-SIPK/Ntf4/WIPK module plays important roles in regulating tobacco innate immunity. Potential MAPKKKs upstream of NtMEK2-SIPK/Ntf4/WIPK include orthologues of Arabidopsis MEKK1 and tomato MAPKKKα (Asai et al., 2002; del Pozo et al., 2004).
However, the identity of the MAPKKK that acts upstream of the NtMEK2-SIPK/Ntf4/WIPK module still needs to be identified. Another MAPKKK that functions in tobacco innate immunity is Nicotiana protein kinase 1 (NPK1), which was previously shown to play a role in cell-plate formation during cytokinesis. It was found that silencing NPK1 expression interferes with the function of the disease resistance genes N, Bs2 and Rx, but does not affect Pto- and Cf4-mediated resistance (Jin et al., 2002). Furthermore, tobacco rattle virus (TRV)-mediated silencing of NQK1 and NRK1 in the cascade NPK1-NQK1-NRK1, a MAPK cascade that is required in cytokinesis, also attenuates N-mediated resistance to TMV (Liu et al., 2004), indicating that the NPK1-NQK1-NRK1 cascade might constitute a complete MAPK cascade playing an important role in N-mediated resistance to TMV. However, NPK1 is not an upstream kinase of the NtMEK2-SIPK/Ntf4/WIPK module. Therefore, it appears that at least two different MAPK cascades function in N-mediated resistance. It is also possible that their function in TMV resistance is secondary.
The lack of complete cell plate formation when NQK1 or NRK1 is silenced will allow the virus to spread more readily.
MAPKs in Plant Defence Responses 45
Downstream events of the NtMEK2-SIPK/Ntf4/WIPK module
Biosynthesis of stress ethylene
ET is involved in regulating plant responses to both biotic and abiotic stresses, in addition to its functions in plant growth and development. Increase in ethylene biosynthesis occurs in plants under a wide variety of stresses. The involvement of MAPK cascade in biosynthesis of stress ET was established by the findings from studies on the NtMEK2-SIPK/WIPK module. In a conditional gain-of-function transgenic system, the activation of SIPK by NtMEK2DD results in a dramatic increase in ET production. The increase in ET after the activation of SIPK coincides with a dramatic increase in ACS activity, which is followed by the activation of a subgroup of genes encoding key enzymes in the ET biosynthetic pathway. After ET production in NtMEK2DD plants, expression of ETHYLENE-RESPONSE FACTOR genes is strongly activated, similar to the effect in tobacco plants with the genotype NN infected with TMV (Kim, C.Y. et al., 2003). Thus, the induction of ET biosynthesis is involved in defence responses mediated by the NtMEK2-SIPK/WIPK module. This finding led to the identification of the first plant MAPK substrate Arabidopsis ACS6, which is phosphorylated by MPK3 and MPK6, and to the discovery of a new mechanism that modulates the biosynthesis of ET by MAPK cascade (Liu and Zhang, 2004).
Involvement of ROS
Several studies have shown that generation of ROS is related to the activation of stress-responsive MAPKs in plants under stresses. High concentrations of H2O2, when exogenously applied, activate SIPK/WIPK. It was also reported that NADPH oxidase is a downstream target of SIPK/WIPK in the induction of H2O2 generation. Activation of SIPK/WIPK by the active NtMEK2DD induces NbrbohB expression (Yoshioka et al., 2003). However, Avr9-induced SIPK/
WIPK activation is not dependent on a burst of ROS from membrane-associated NADPH oxidases (Romeis et al., 1999). The rapid oxidative burst induced by elicitin is also not required for SIPK activation by elicitin either. No rapid H2O2 burst in NtMEK2DD plants after dexamethasone (DEX) treatment was detected (Yang et al., 2001; Ren et al., 2002). These results suggest that SIPK/Ntf4/
WIPK activation is not involved in the early ROS burst from NADPH oxidase in pathogen-infected plants. Recently, it was found that chloroplast-generated ROS are involved in HR-like cell death after SIPK/Ntf4/WIPK activation (Liu et al. 2007). Therefore, the MAPK activation after the perception of patho-gens/elicitors is independent of ROS burst. However, ROS generation is associated with cell death induced by activation of the NtMEK2-SIPK/WIPK module, similar to the pathogen-induced HR cell death (Ren et al., 2002).
Furthermore, ROS-mediated mitochondrial dysfunction precedes HR cell death induced by the activation of the NtMEK2-SIPK/WIPK module (Takahashi et al., 2003; Liu et al. 2007; Takabatake et al., 2007). It is therefore most likely that the activation of the NtMEK2-SIPK/WIPK module and ROS generation
46 F. Song et al.
represent two parallel, interconnected downstream events after the sensing step, in which the ROS burst may positively feed into MAPK activation.
WRKY transcription factors
In the conditional gain-of-function NtMEK2DD transgenic tobacco plants, the activation of endogenous SIPK and WIPK by NtMEK2DD induces expression of several groups of defence genes. Gel-mobility shift assays revealed a strong increase in the binding activity to the W box in nuclear extracts from the NtMEK2DD plants, suggesting a direct phosphorylation regulation of WRKY transcription factors by the NtMEK2-SIPK/WIPK module. A rapid increase in the expression of several WRKY genes in the NtMEK2DD plants was also observed. These results placed the NtMEK2-SIPK/WIPK module upstream of the WRKY family proteins (Kim and Zhang, 2004). Yeast two-hybrid screens for direct downstream components have identified two WRKY transcription factors that can be phosphorylated by SIPK or WIPK. SIPK phosphorylates NtWRKY1, resulting in enhanced DNA-binding activity of WRKY1 to a W box sequence from CHN50, a defence gene encoding a chitinase. Coexpression of SIPK and NtWRKY1 in Nicotiana benthamiana led to more rapid cell death than expression of SIPK alone, suggesting that WRKY1 is a putative substrate of SIPK (Menke et al., 2005). Another WRKY transcription factor that acts downstream of WIPK is NtWIPK-interacting factor (NtWIF), which directly interacts with WIPK. In vitro phosphorylation assays demonstrated that WIPK efficiently phosphorylates NtWIF. Coexpression of NtWIF with WIPK in tobacco cells increases its transcriptional activity (Yap et al., 2005). Over-expression of NtWIF in tobacco plants enhances HR upon TMV infection and cryptogein treatment, while its silencing by RNAi suppresses such HR (Chung and Sano, 2007). Transgenic tobacco plants overexpressing NtWIF exhibit constitutive accumulation of transcripts for defence genes including PR-1a and PR-2 and elevated levels of SA (Waller et al., 2006). Therefore, NtWIF is a transcription factor that is directly phosphorylated by WIPK and regulates defence response through influencing SA biosynthesis.