RIVET -: A tool for in vivo analysis of symbiotically relevant gene expression in Sinorhizobium meliloti
RIVET -: A tool for in vivo analysis of symbiotically relevant gene expression in Sinorhizobium meliloti
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DOI：
10.1094/mpmi-21-2-0162
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发表时间：
2008-02-01
影响因子：
3.5
通讯作者：
Teplitslci, Max
中科院分区：
文献类型：
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作者：
Gao, Mengsheng;Teplitslci, Max
尽管在发展研究豆科植物-根瘤菌共生体的遗传学和信号交换的敏感工具方面取得了重大进展，但细菌和植物信号在共生基因调控中的体内作用仍存在许多不确定性。在这项研究中，我们采用了TnpR重组酶为基础的体内表达技术（RIVET）的文件基因调控中华根瘤菌mneliloti。TnpR的底物，res 1-tet-res 1盒，在克隆到S.苜蓿基因组。构建了双顺反子无启动子的tnpR-β-葡萄糖醛酸糖苷酶（GUS）报告基因，以跟踪共生相关的S.苜蓿基因在相互作用的不同阶段。在原理实验的证明中，在暴露于微摩尔水平的nod操纵子诱导剂毛地黄黄酮的4小时内和在根际过夜孵育后检测到了cDNAC：：tnpR报告基因的分辨率。RIVET表明，细胞分裂基因ftsZ 2在根际中没有强烈表达，但在根瘤内部和琼脂表面被激活。在prequorate小菌落中，N-酰基高丝氨酸内酯（阿勒）合酶sinI-：tnpR-GUS报告基因的Rnesphere表达是适度的，然后随着时间的推移而增加。阿勒合成酶sinI和AHL调节基因expG在结节内被激活。
Despite significant advances in the development of sensitive tools for studying genetics and signal exchange in legumer-hizobium symbioses, many uncertainties remain about the in vivo role of bacterial and plant signals in symbiotic gene regulation. In this study, we adapted TnpR recombinase-based in vivo expression technology (RIVET) to document gene regulation in Sinorhizobium rneliloti. The substrate for TnpR, the res1-tet-res1 cassette, is stably inherited when cloned into a neutral site of the S. meliloti genome. Bicistronic promoterless tnpR-beta-glucuronidase (GUS) reporters were constructed to track expression ("resolution") of symbiotically relevant S. meliloti genes during different stages of the interaction. In proof of principle experiments, the resolution of the nodC:: tnpR reporter was detected within 4 h of exposure to micromolar levels of the nod operon inducer luteolin and after overnight incubation in the rhizosphere. RIVET demonstrated that cell division gene ftsZ2 was not strongly expressed in the rhizosphere but was activated inside the nodules and on agar surfaces. Rhizosphere expression of the N-acyl homoserine lactone (AHL) synthase sinI-:tnpR-GUS reporter was modest in prequorate microcolonies, and then increased with time. AHL synthase sinI and an AHL-regulated gene, expG, were activated inside the nodules.