TAGGING OF PLANT POTYVIRUS REPLICATION AND MOVEMENT BY INSERTION OF BETA-GLUCURONIDASE INTO THE VIRAL POLYPROTEIN
TAGGING OF PLANT POTYVIRUS REPLICATION AND MOVEMENT BY INSERTION OF BETA-GLUCURONIDASE INTO THE VIRAL POLYPROTEIN
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DOI：
10.1073/pnas.89.21.10208
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发表时间：
1992-11-01
影响因子：
11.1
通讯作者：
CARRINGTON, JC
中科院分区：
文献类型：
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作者：
DOLJA, VV;MCBRIDE, HJ;CARRINGTON, JC
从烟草蚀刻马铃薯Y病毒基因组的全长cDNA克隆中获得了侵染性RNA转录本，其中含有细菌β-葡萄糖苷酸酶(GUS)基因插入N-末端35-kDa蛋白酶的多蛋白编码序列和辅助成分-蛋白酶之间。重组病毒能够在植物中系统传播，并积累到与野生型烟草蚀刻马铃薯Y病毒相当的水平。在35 kDa的蛋白水解酶和辅助组分-蛋白水解酶的作用下，产生了具有酶活性的GUS-辅助组分-蛋白酶融合蛋白。恢复了在多次植物间转移后保留GUS插入的病毒传代系，以及持续GUS序列缺失的病毒传代系。在时间进程实验中使用原位组织化学GUS分析，可以显示单个机械接种的叶表皮细胞、邻近的表皮细胞和叶肉细胞、长距离运输后的韧皮部伴生细胞以及接种部位上下器官的维管组织周围的细胞中的病毒活性。该系统是研究植物病毒复制、短距离和长距离病毒移动以及病毒与宿主相互作用的有力工具。此外，我们还表明，马铃薯Y病毒可以作为高效的、自主复制的载体，在植物中表达外源基因。
Infectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial beta-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper component-proteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper component-proteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sustained a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-distance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short- and long-distance virus movement, and virus-host interactions. Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants.