Lee L Y, Gelvin S B, Kado C I
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
J Bacteriol. 1999 Jan;181(1):186-96. doi: 10.1128/JB.181.1.186-196.1999.
When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF1010.
当与Ti（致瘤）质粒共存时，质粒pSa的osa基因的21 kDa产物可抑制根癌农杆菌引发的冠瘿瘤形成。细菌中osa的存在不影响T-DNA加工或vir（毒力）基因诱导。我们使用拟南芥根段和烟草叶盘证明，Osa抑制根癌农杆菌将这些植物转化为瘤形成、卡那霉素抗性和稳定的β-葡萄糖醛酸酶（GUS）表达的稳定表型。当根癌农杆菌含有osa时，感染植物组织中瞬时GUS活性缺乏表达，以及番茄斑驳病毒对本氏烟草进行农杆菌浸润后缺乏系统性病毒症状，表明Osa的致癌抑制作用发生在T-DNA进入植物细胞核之前。当osa存在于VirE2供体菌株中时，缺乏T-DNA但含有野生型virE2基因的根癌农杆菌菌株（VirE2供体菌株）对根癌农杆菌virE2突变体（T-DNA供体菌株）的细胞外互补作用被阻断，但当osa存在于T-DNA供体菌株中时则不会。这些数据表明，osa抑制VirE2蛋白，但不抑制根癌农杆菌的T-DNA输出。这些数据进一步表明，VirE2蛋白和T-DNA是分别从细菌中输出的。携带osa的根癌农杆菌virE2突变体成功感染了曼陀罗植物和表达VirE2蛋白的转基因烟草植物叶盘，证实osa的致癌抑制作用不是通过阻断T-DNA转移发生的。在根癌农杆菌中过表达virB9、virB10和virB11并不能克服osa的致癌抑制作用。osa基因自身的表达而非与pSa形成接合中间体来阻断转化这一发现表明，osa致癌抑制的机制可能与IncQ质粒RSF1010不同。
