Transactivation of wound-responsive genes containing the core sequence of the auxin-responsive element by a wound-induced protein kinase-activated transcription factor in tobacco plants
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Mitogen-activated protein kinases (MAPKs) constitute one of the most critical signaling components in plants. A typical example is wound-induced protein kinase (WIPK), which functions during pathogen responses in tobacco plants (Nicotiana tabacum). Searching for direct down-stream components, we previously isolated a novel transcription factor, which was activated upon phosphorylation by WIPK and designated as N. tabacum WIPK-interacting factor (NtWIF). Overexpression of NtWIF in tobacco plants enhanced the hypersensitive response (HR) upon tobacco mosaic virus infection and cryptogein treatment, while its silencing by RNAi suppressed such HR. NtWIF contains a specific motif similar to the B3 DNA binding domain, which recognizes the core TGTCTC motif called the auxin-responsive element (ARE). Using synthetic ARE sequences, NtWIF was also shown to recognize the ARE motifs and to transactivate the Luciferase (Luc)-reporter gene driven by such AREs in tobacco BY2 cultured cells. Subsequent microarray screening of NtWIF overexpressing tobacco identified 49 stress-responsive genes, and in silico analyses of available promoter regions of these genes revealed β-1,3-glucanase, ACS2, P-450, and WIPK itself to contain the ARE core motif consisted of either TGTCTC or TGTCCT. Gel shift assay showed NtWIF to efficiently bind to both sequences. Assays with 1.5-kb PR-Q and 1.2 kb WIPK promoter regions, each fused to the Luc-reporter gene, indicated NtWIF to exhibit a clear transactivation activity, which was increased up to 3-fold upon phosphorylation by WIPK. These results revealed that NtWIF directly regulates multiple stress-responsive genes containing the ARE motif in their promoters, thereby partly filling up the last step of the MAPK cascade.
KeywordsAuxin-responsive element B3-DNA binding domain MAP kinase Nicotiana tabacum Pathogenesis-related-Q Wounding
Aminocyclopropane carboxylic acid synthase
Auxin responsive element
Mitogen-activated protein kinase
MAPK/extra cellular signal-regulated kinase (ERK) kinase
Tobacco mosaic virus
The authors thank Drs Akira Katoh and Hiroshi Yoda (Nara Institute of Science and Technology) for instructions regarding microarray experiments and a generous gift of cryptogein, respectively, and Dr Malcolm Moore (Intermal, Nagoya) for critical reading of the manuscript. This work was partly supported by a grant from the Japan Society for the Promotion of Science and by a Grant-in-Aid for the 21st Century COE Research from the Ministry of Education, Culture, Sports, Science and Technology.
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