Organic chemial approach to the elucidation of the mechanism of plant transformation induced by Agrobacterium tumefaciens

根癌农杆菌对植物的转化已经得到了极大的发展，以创造获得新特性的植物，例如，抗除草剂或抗病毒。许多科学家一直在探索植物转化的机制，尽管整个机制还不完全清楚。由于植物转化包括几个复杂的步骤，需要一个影响每个步骤的特定化学探针来阐明其机理。转化调节剂的发现和根据它们的作用模式对这些探针进行分类都是有帮助的。我们设计了一种利用内含子-GUS双元载体来替代耗时的马铃薯块茎圆盘实验的方法，用于快速、定量地检测根癌农杆菌转化植物的调节因子。在供试的几种寄主植物中，烟草BY-2悬浮细胞最适合GUS的表达。此外，由于受试材料的植物毒性(如果有的话)是从对BY-2悬浮细胞的生长抑制中明显表现出来的，该方法可以排除植物毒性化合物。在我们用马铃薯块茎圆盘法筛选冠瘤形成抑制剂的过程中，分离到了恶唑霉素。恶唑霉素对根癌农杆菌具有较强的抗菌活性，从而抑制了冠瘤的形成。有趣的是，恶唑霉素酯虽然能抑制冠瘤的形成，但没有抗菌活性。恶唑霉素类似物Curromycins被报道为抗生素。我们从红霉素产生菌中分离得到红霉素B，并制备了红霉素B二乙酸酯。柯罗霉素B对根癌农杆菌具有抑制冠瘤形成和抑菌活性。柯罗霉素B二乙酸酯虽然能抑制冠瘤的形成，但没有显示出任何抗菌活性。这些结果表明，红霉素B二乙酸酯是研究根癌农杆菌转化植物机理的一种很有前途的化学探针。

Plant transformation by Agrobacterium tumefaciens has been dramatically developed to create plants acquiring new characters, e.g., herbicide- or virus-resistance. Many scientists have been exploring the mechanism for the plant transformation, although the entire mechanism is not yet fully understood. As the plant transformation consists of several complex steps, a specific chemical probe affecting each step is required to elucidate its mechanism. The discovery of transformation regulators and the classification of these probes based on their mode of action are both helpful. We devised a rapid and quantitative bioassay method for regulators of plant tranformation by A.tumefaciens with an intron-GUS binary vector as an alternative method to the time-consuming potato tuber disk assay. Among several host plants tested, tobacco BY-2 suspension cells were the most preferable for this GUS expression assay. Furthermore, as the phytotoxicity of the test material, if any, is apparent from the growth inhibition of BY-2 suspension cells, this method can exclude phytotoxic compounds.In the course of our screening by the potato tuber disk assay for inhibitors of crown gall formation, oxazolomycin was isolated. Oxazolomycin inhibited crown gall formation owing to strong anitbacterial activity to the pathogen, A.tumefaciens. Interestingly, oxazolomycin esters had no antibacterial activity, although they inhibited crown gall formation. Curromycins, oxazolomycin analogs, were reproted as antibiotics. We isolated curromycin B from the curromycin-producing strain and prepared curromycin B diacetate. Curromycin B showed both crown gall formation inhibitory and antibacterial activities against A.tumefaciens. Curromycin B diacetate did not show any antibacterial activity, although it inhibited crown gall fromation. These results indicated that curromycin B diacetate was a promising chemical probe for studying the mechanism of plant transformation by A.tumefaciens.