Virus-specific resistance in transgenic plants expressing viral movement protein

苹果褪绿叶斑病毒（ACLSV）ORF2编码的50KDa蛋白（50KP）是一种运动蛋白，在转基因西烟草植物中表达。表达 50KP 的植物（50KP 植物）补充了 ACLSV 感染性 cDNA 克隆（pCLSF）的 50KP 缺陷突变体的同系传播。与非转基因对照植物（NT-植物）相比，接种ACLSV的50KP植物中症状严重程度大大增强，上部叶中病毒的积累增加。然而，大多数接种葡萄浆果内坏死病毒（GINV）（毛病毒属的另一种物种）的 50KP 植物既没有出现明显的症状，也不支持病毒在接种叶或上部叶中积累。相反，在接种 GINV 的 NT 和 CP 植物中，出现全身症状并且病毒积累相同。接种苹果茎纹病毒（ASGV）或苹果茎凹陷病毒后，50KP-、CP-和NT-植物的症状发展和病毒积累没有差异。与绿色、黄色或青色荧光蛋白（GFP、YFP 或 CFP）融合的 ACLSV-50KP 和 GINV-MP (39KP) 在 50KP- 和 NT- 植物的叶细胞中瞬时表达。 39KP 的细胞内和细胞间运输以及小管诱导活性在来自 50KP- 植物叶子的叶表皮和原生质体中受到特异性干扰，相比之下，50KP 和 ASGV-36KP 的那些在来自 50KP- 植物的细胞中从未受到干扰。当 39KP-YFP 与 50KP-CFP 在 NT 植物的叶表皮中共表达时，两种 MP 都局限于单细胞，表明 50KP-CFP 干扰 39KP-YFP 的细胞间运输。使用 50KP 缺失突变体进行的突变分析表明，39KP 的细胞间运输受到干扰，删除的蛋白质保留了上述活性，但功能障碍的 50KP 则不然。表达50KP缺失蛋白的转基因植物保留了对GINV表现出特异性抗性的活性，而表达功能失调的50KP的转基因植物则没有表现出对病毒的任何抗性。从这些结果可以得出结论，50KP-植物对GINV的特异性抗性是由50KP干扰39KP的细胞内和细胞间运输引起的。较少的

The 50KDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV), a movement protein, was expressed in transgenic Nicotiana occidentalis plants. The 50KP-expressing plants (50KP-plants) complemented the syntemic spread of the 50KP-defective mutants of an infectious cDNA clone of ACLSV (pCLSF). Severity of symptoms was greatly enhanced and the accumulation of virus in upper leaves was increased in 50KP-plants inoculated with ACLSV compared with nontransgenic control plants (NT-plants). However, most 50KP-plants inoculated with Grapevine berry inner necrosis virus (GINV), another species of the genus Trichovirus, neither developed obvious symptoms nor supported virus accumulation in inoculated or upper leaves. In contrast, systemic symptoms developed and virus accumulated equally in NT- and CP-plants inoculated with GINV. After inoculation with Apple stem grooving virus (ASGV) or Apple stem pitting virus, there was no difference in symptom development and virus accumul … More ation among 50KP-, CP-, and NT-plants. ACLSV-50KP and GINV-MP (39KP) fused to Green, Yellow or Cyan fluorescent proteins (GFP, YFP or CFP) were expressed transiently in leaf cells of 50KP- and NT- plants. The intracellular and intercellular trafficking and tubule-inducing activity of 39KP were specifically interfered in leaf epidermis and protoplasts from 50KP- Plant leaves, in contrast that those of 50KP and ASGV-36KP were never interfered in cells from 50KP- plants. When 39KP-YFP was co-expressed with 50KP-CFP in leaf epidermis of NT-plants, both MPs were confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP. Mutational analyses using 50KP deletion mutants showed that the cell-to-cell trafficking of 39KP was interfered with the deleted proteins retained the activities described above, but not with the dyslunctional 50KP. Transgenic plants expressing the 50KP-deleted proteins retained the activities showed specific resistance against GINV, in contrast with transgenic plants expressing dysfunctional 50KP did not show any resistance to the virus. From these results, it is concluded that the specific resistance of 50KP-plants against GINV is caused by the interference of the intracellular and intercellular trafficking of 39KP with 50KP. Less