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

BMC Plant Biology (Impact Factor: 3.81). 07/2012; 12(1):103. DOI: 10.1186/1471-2229-12-103
Source: PubMed


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 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.

We 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.

These 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.

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    • "Plant MAPK cascade genes were first reported in Arabidopsis. Up to now, MAPK cascade family genes have been identified in many other plant species, including poplar, rice, soybean, maize, tomato and N. benthamiana [3]–[12]. "
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    ABSTRACT: Mitogen-activated protein kinase (MAPK) cascades have important functions in plant growth, development, and response to various stresses. The MAPKK and MAPKKK gene families in tomato have never been systematically analyzed. In this study, we performed a genome-wide analysis of the MAPKK and MAPKKK gene families in tomato and identified 5 MAPKK genes and 89 MAPKKK genes. Phylogenetic analyses of the MAPKK and MAPKKK gene families showed that all the MAPKK genes formed four groups (groups A, B, C, and D), whereas all the MAPKKK genes were classified into three subfamilies, namely, MEKK, RAF, and ZIK. Evolutionary analysis showed that whole genome or chromosomal segment duplications were the main factors responsible for the expansion of the MAPKK and MAPKKK gene families in tomato. Quantitative real-time RT-PCR analysis showed that the majority of MAPKK and MAPKKK genes were expressed in all tested organs with considerable differences in transcript levels indicating that they might be constitutively expressed. However, the expression level of most of these genes changed significantly under heat, cold, drought, salt, and Pseudomonas syringae treatment. Furthermore, their expression levels exhibited significant changes in response to salicylic acid and indole-3-acetic acid treatment, implying that these genes might have important roles in the plant hormone network. Our comparative analysis of the MAPKK and MAPKKK families would improve our understanding of the evolution and functional characterization of MAPK cascades in tomato.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "It has, for instance, been used to produce the haemagglutinin antigen from avian influenza virus A (Kanagarajan et al. 2012) and for the production of monoclonal antibodies (Castilho et al. 2011). Furthermore, it should be mentioned that transient expression, especially in N. benthamiana, is also intensively used to analyse the function of proteins (for instance, Hashimoto et al. 2012). "
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    ABSTRACT: Production of recombinant proteins in plants is of increasing importance for practical applications. However, the production of stable transformed transgenic plants is a lengthy procedure. Transient expression, on the other hand, can deliver recombinant proteins within a week, and many viral vectors have been constructed for that purpose. Each of them is reported to be highly efficient, robust and cost-effective. Here, a variety of expression vectors which were designed for transient and stable plant transformation, including pPZP3425, pPZP5025, pPZPTRBO, pJLTRBO, pEAQ-HT and pBY030-2R, was compared for the expression of green fluorescent protein and β-glucuronidase in Nicotiana benthamiana by Agrobacterium-mediated transient expression. Our results show that pPZPTRBO, pJLTRBO and pEAQ-HT had comparable expression levels without co-infiltration of a RNA-silencing inhibitor. The other vectors, including the non-viral vectors pPZP5025 and pPZP3425, needed co-infiltration of the RNA-silencing inhibitor P19 to give good expression levels. Electronic supplementary material The online version of this article (doi:10.1007/s11105-013-0614-z) contains supplementary material, which is available to authorized users.
    Full-text · Article · Jul 2013 · Plant Molecular Biology Reporter
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    • "In addition to ETI, transient overexpression of a number of R genes or defense regulators can also induce pathogen-independent cell death reminiscent of HR in Nicotiana benthamiana (N. benthamiana) [7]–[11]. "
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    ABSTRACT: Programmed cell death has been associated with plant immunity and senescence. The receptor kinase XA21 confers resistance to bacterial blight disease of rice (Oryza sativa) caused by Xanthomonas oryzae pv. oryzae (Xoo). Here we show that the XA21 binding protein 3 (XB3) is capable of inducing cell death when overexpressed in Nicotiana benthamiana. XB3 is a RING finger-containing E3 ubiquitin ligase that has been positively implicated in XA21-mediated resistance. Mutation abolishing the XB3 E3 activity also eliminates its ability to induce cell death. Phylogenetic analysis of XB3-related sequences suggests a family of proteins (XB3 family) with members from diverse plant species. We further demonstrate that members of the XB3 family from rice, Arabidopsis and citrus all trigger a similar cell death response in Nicotiana benthamiana, suggesting an evolutionarily conserved role for these proteins in regulating programmed cell death in the plant kingdom.
    Full-text · Article · May 2013 · PLoS ONE
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