Yamamoto S, Suzuki K, Shinshi H
Plant Molecular Biology Laboratory, Molecular Biology Department, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Plant J. 1999 Dec;20(5):571-9. doi: 10.1046/j.1365-313x.1999.00634.x.
In cultured XD6S tobacco cells, xylanase from Trichoderma viride (TvX) induced the expression of a luciferase reporter gene that was under the control of a GCC box, which is an 11 bp sequence (TAAGAGCCGCC) that is found in the 5'-upstream region of pathogen-responsive defence genes that include genes for class I basic chitinase. TvX-induced biosynthesis of ethylene was not required for the TvX-activated transcription. The TvX-induced, GCC box-mediated transcription of the reporter gene was completely blocked not only by staurosporine, an inhibitor of serine/threonine protein kinases, at 1 microM, but also by calyculin A, an inhibitor of protein phosphatases 1 and 2A, at 0.2 microM. It appeared also that protein synthesis de novo was required for the GCC box-mediated transcription of the reporter gene. Accumulation of mRNAs for various ERFs (ethylene-responsive transcription factors), which have been shown to bind specifically to the GCC box, was also induced by TvX prior to increases in the level of mRNA for a class I basic chitinase. In particular, the level of mRNA for EFR2 reached a maximum from 3 to 6 h, whereas levels of mRNAs for ERF3 and ERF4 were highest 0.5 h after the start of treatment of TvX and decreased thereafter. Moreover, induction of accumulation of the mRNA for ERF2 was inhibited by staurosporine and calyculin A. These results suggest that ERF2 might play a major role in TvX-induced, GCC box-mediated transcription of genes and that both protein kinase(s) and protein phosphatase(s) might be involved, as positive regulators, in the signal transduction pathway that leads to expression of ERF2 and subsequent GCC box-mediated transcription of genes.
在培养的XD6S烟草细胞中，来自绿色木霉的木聚糖酶（TvX）诱导了荧光素酶报告基因的表达，该报告基因受GCC框控制，GCC框是一个11 bp的序列（TAAGAGCCGCC），存在于病原体响应防御基因的5'上游区域，包括I类碱性几丁质酶基因。TvX激活的转录不需要TvX诱导的乙烯生物合成。TvX诱导的、GCC框介导的报告基因转录不仅被1 μM的丝氨酸/苏氨酸蛋白激酶抑制剂星形孢菌素完全阻断，也被0.2 μM的蛋白磷酸酶1和2A抑制剂花萼海绵诱癌素A完全阻断。似乎报告基因的GCC框介导的转录也需要从头合成蛋白质。各种乙烯响应转录因子（ERF）的mRNA积累也被TvX诱导，这些转录因子已被证明能特异性结合GCC框，且发生在I类碱性几丁质酶mRNA水平升高之前。特别是，EFR2的mRNA水平在3至6小时达到峰值，而ERF3和ERF4的mRNA水平在TvX处理开始后0.5小时最高，此后下降。此外，星形孢菌素和花萼海绵诱癌素A抑制了ERF2 mRNA积累的诱导。这些结果表明，ERF2可能在TvX诱导的、GCC框介导的基因转录中起主要作用，并且蛋白激酶和蛋白磷酸酶可能作为正调节因子参与导致ERF2表达及随后GCC框介导的基因转录的信号转导途径。
