Krenz Bjoern, Bronikowski Agathe, Lu Xiaoyun, Ziebell Heiko, Thompson Jeremy R, Perry Keith L
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, 334 Plant Science Building, Ithaca, NY 14853-5904, USA.
Lehrstuhl Biochemie, Department Biologie - Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
J Gen Virol. 2015 Sep;96(9):2904-2912. doi: 10.1099/vir.0.000185. Epub 2015 May 15.
The single-stranded, positive-sense and tripartite RNA virus Cucumber mosaic virus (CMV) was used in this study as a method for monitoring the initial stages of virus infection following aphid transmission. The RNA2 of CMV was modified to incorporate, in a variety of arrangements, an open reading frame (ORF) encoding an enhanced green fluorescent protein (eGFP). The phenotypes of five engineered RNA2s were tested in Nicotiana tabacum, Nicotiana clevelandii and Nicotiana benthamiana. Only one construct (F4), in which the 2b ORF was truncated at the 3' end and fused in-frame with the eGFP ORF, was able to systemically infect N. benthamiana plants, express eGFP and be transmitted by the aphid Myzus persicae. The utility of this construct was demonstrated following infection as early as one day post-transmission (dpt) continuing through to systemic infection. Comparisons of the inoculation sites in different petiole sections one to three dpt clearly showed that the onset of infection and eGFP expression always occurred in the epidermal or collenchymatous tissue just below the epidermis; an observation consistent with the rapid time frame characteristic of the non-persistent mode of aphid transmission.
本研究使用单链、正义和三分体RNA病毒黄瓜花叶病毒（CMV）作为监测蚜虫传播后病毒感染初始阶段的一种方法。CMV的RNA2经过修饰，以多种排列方式并入一个编码增强型绿色荧光蛋白（eGFP）的开放阅读框（ORF）。在烟草、克利夫兰烟草和本氏烟草中测试了五种工程化RNA2的表型。只有一种构建体（F4），其中2b ORF在3'端被截断并与eGFP ORF框内融合，能够系统感染本氏烟草植株，表达eGFP并由桃蚜传播。早在传播后一天（dpt）直至系统感染，该构建体的效用在感染后都得到了证明。对1至3 dpt不同叶柄切片接种部位的比较清楚地表明，感染和eGFP表达的起始总是发生在表皮或紧挨着表皮的厚角组织中；这一观察结果与蚜虫非持久性传播模式的快速时间框架特征一致。
