Virus-specific resistance in transgenic plants expressing viral movement protein

苹果褪绿叶斑病病毒（ACLSV） ORF2编码的运动蛋白50KDa蛋白（50KP）在转基因西烟叶植株中得到了表达。表达50kp的植株（50KP-plants）补充了ACLSV感染性cDNA克隆（pCLSF）的50kp缺陷突变体的合成传播。与非转基因对照植物（NT-plants）相比，接种ACLSV的50kp植株的症状严重程度大大增强，上部叶片的病毒积累量增加。然而，大多数50kp植株接种了另一种毛状病毒属（Grapevine berry inner necrosis virus， GINV）后，既没有出现明显的症状，也不支持病毒在接种叶片或上部的积累。相比之下，接种了GINV的NT-和cp -植株出现了全身性症状和病毒积累。接种苹果茎沟病毒（ASGV）和苹果茎蚀病毒（nt）后，50KP-、CP-和nt -植株在症状发展和病毒积累方面无显著差异。ACLSV-50KP和gvi - mp （39KP）与绿色、黄色或青色荧光蛋白（GFP、YFP或CFP）融合，在50KP-和NT-植物叶片细胞中短暂表达。50KP-植物的叶片表皮和原生质体中，39KP的细胞内和细胞间运输及小管诱导活性受到特异性干扰，而50KP和ASGV-36KP在50KP-植物的细胞中不受干扰。当39KP-YFP与50KP-CFP在nt植物的叶表皮共表达时，这两种MPs都被限制在单个细胞中，这表明50KP-CFP干扰了39KP-YFP在细胞间的运输。使用50KP缺失突变体的突变分析表明，39KP的细胞间运输受到了保留上述活性的缺失蛋白的干扰，而不受功能失调的50KP的干扰。表达50KP缺失蛋白的转基因植株对GINV表现出特异性抗性，而表达功能失调的50KP的转基因植株对GINV没有任何抗性。以上结果表明，50KP植物对GINV的特异性抗性是由于50KP干扰39KP在细胞内和细胞间的转运。少

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