Virus-specific resistance in transgenic plants expressing viral movement protein

苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus，ACLSV）ORF 2编码的50 KDa蛋白（50 KP）在烟草中得到表达。50 KP表达植物（50 KP植物）补充了ACLSV的感染性cDNA克隆（pCLSF）的50 KP缺陷突变体的同系传播。与非转基因对照植株（NT-植株）相比，ACLSV接种的50 KP-植株的症状严重程度大大增强，上部叶片中病毒的积累增加。然而，大多数50 KP-植物接种葡萄藤浆果内坏死病毒（GINV），另一种属Trichovirus，既不发生明显的症状，也不支持接种或上部叶片中的病毒积累。与此相反，全身症状的发展和病毒积累同样的NT和CP植物接种GINV。接种苹果茎沟病毒（ASGV）和苹果茎痘病毒（APTT）后，症状的发展和病毒的传播没有差异。 关于我们 在50个KP-、CP-和NT-植物之间的转化。将与绿色、黄色或青色荧光蛋白（GFP、YFP或CFP）融合的ACLSV-50 KP和GINV-MP（39 KP）在50 KP-和NT-植物的叶细胞中瞬时表达。39 KP的细胞内和细胞间运输和微管诱导活性在50 KP-植物叶片的叶表皮和原生质体中被特异性地干扰，而50 KP和ASGV-36 KP的细胞内和细胞间运输和微管诱导活性在50 KP-植物的细胞中从未被干扰。当39 KP-YFP与50 KP-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50 KP-CFP干扰39 KP-YFP的细胞间运输。使用50 KP缺失突变体的突变分析表明，39 KP的细胞间运输受到保留上述活性的缺失蛋白质的干扰，但不受功能障碍的50 KP的干扰。表达50 KP缺失蛋白的转基因植物保留了活性，表现出对GINV的特异性抗性，而表达功能失调的50 KP的转基因植物则没有表现出对病毒的任何抗性。这些结果表明，50 KP-植物对GINV的特异性抗性是由于50 KP干扰了39 KP在细胞内和细胞间的运输而引起的。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