Escobar M A, Civerolo E L, Summerfelt K R, Dandekar A M
Department of Pomology, University of California, Davis, CA 95616, USA.
Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):13437-42. doi: 10.1073/pnas.241276898. Epub 2001 Oct 30.
Crown gall disease, caused by the soil bacterium Agrobacterium tumefaciens, results in significant economic losses in perennial crops worldwide. A. tumefaciens is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate de novo auxin and cytokinin biosynthesis to generate tumors. Specifically, the iaaM and ipt oncogenes, which show approximately 90% DNA sequence identity across studied A. tumefaciens strains, are required for tumor formation. By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of iaaM and ipt, we generated transgenic Arabidopsis thaliana and Lycopersicon esculentum plants that are highly resistant to crown gall disease development. In in vitro root inoculation bioassays with two biovar I strains of A. tumefaciens, transgenic Arabidopsis lines averaged 0.0-1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis. Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of A. tumefaciens displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis. This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor-ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable. If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks.
冠瘿病由土壤细菌根癌农杆菌引起，在全球多年生作物中造成了重大经济损失。根癌农杆菌是少数具有特征明确的水平基因转移系统的生物体之一，拥有一套致癌基因，这些基因整合到植物基因组中后，会协调生长素和细胞分裂素的从头生物合成以产生肿瘤。具体而言，iaaM和ipt致癌基因在所有研究的根癌农杆菌菌株中显示出约90%的DNA序列同一性，是肿瘤形成所必需的。通过表达两种旨在启动iaaM和ipt的RNA干扰（RNAi）的自互补RNA构建体，我们培育出了对冠瘿病高度抗性的转基因拟南芥和番茄植株。在用两种根癌农杆菌生物变种I菌株进行的体外根接种生物测定中，转基因拟南芥品系的肿瘤发生率平均为0.0 - 1.5%，而野生型对照的平均肿瘤发生率为97.5%。同样，几个经转化的番茄品系在通过茎接种三种根癌农杆菌生物变种I菌株、一种生物变种II菌株和一种生物变种III菌株进行挑战时，肿瘤发生率在0.0%至24.2%之间，而对照的平均肿瘤发生率为100%。这种抗性机制基于mRNA序列同源性，而非传统植物抗性基因所特有的高度特异性受体 - 配体结合相互作用，应该具有高度持久性。如果在田间条件下成功且持久，RNAi介导的致癌基因沉默可能在木本作物和观赏植物砧木的改良中具有广泛的适用性。
