GFP激发光源用于花叶病毒的研究

2022年10月，南京师范大学生命科学学院在《Biology》期刊上发表文献《The Characterization of the Tobacco-Derived Wild Tomato Mosaic Virus by Employing Its Infectious DNA Clone》，文献中使用了上海路阳仪器有限公司销售的LUYOR-3415RG手持式双波长荧光蛋白激发光源用于观察绿色荧光蛋白和红色荧光蛋白在烟草上的表达。

文献摘要：

Simple Summary:

 Wild tomato mosaic virus (WTMV, genus Potyvirus, family Potyviridae) is an emerging viral pathogen that endangers Nicotiana tabacum production. The field survey conducted in this study shows that WTMV is becoming an epidemic in China. An infectious DNA clone of the tobacco-derived WTMV is constructed. It can infect wild eggplant, black nightshade, and tobacco plants but can not infect various local pepper varieties. WTMV evolves into three groups that coincide with their original hosts, tobacco, pepper, or wild eggplant. Thus, the tobacco-derived WTMV might divergently evolves to adapt to tobacco other than peppers. We show that WTMV is compatible with the coinfection of cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) in tobacco but not other potyviruses. Specifically, WTMV can interfere with the infection of other potyvirus species in tobacco, a phenomenon known as superinfection exclusion previously observed within the same potyviral species. This study contributes essential knowledge on the evolution, infectivity, and recent epidemics of WTMV, and provides the key tool for further disease-resistance and field management studies.

Abstract: 

Viral diseases of cultivated crops are often caused by virus spillover from wild plants. Tobacco (N. tabacum) is an important economic crop grown globally. The viral pathogens of tobacco are traditional major subjects in virology studies and key considerations in tobacco breeding practices.

A positive-strand RNA virus, wild tomato mosaic virus (WTMV), belonging to the genus potyvirus in the family potyviridae was recently found to infect tobacco in China. In this study, diseased tobacco leaf samples were collected in the Henan Province of China during 2020–2021. Several samples from different locations were identified as WTMV positive. An infectious DNA clone was constructed based on one of the WTMV isolates. By using this clone, we found that WTMV from tobacco could establish infections on natural reservoir hosts, demonstrating a possible route of WTMV spillover and overwintering in the tobacco field. Furthermore, the WTMV infection was found to be accompanied by other tobacco viruses in the field. The co-inoculation experiments indicate the superinfection exclusion (SIE) between WTMV and other potyvirus species that infect tobacco. Overall, our work reveals novel aspects of WTMV evolution and infection in tobacco and provides an important tool for further studies of WTMV.

Plant Inoculation and Phenotyping

Two methods were used for the inoculation experiments depending on plant species. The agroinfiltration method was used to inoculate N. benthamiana, N. tobaccum, C. annuum, S. lycopersicum, and S. melongena. Agrobacterium tumefaciens EHA105 harboring an infectious clone was suspended in an infiltration solution (10 mM MES [pH 5.6], 10 mM MgCl2, and 150 mM acetosyringone), and the concentration was adjusted to OD600 = 1.0. The A. tumefaciens EHA105 harboring empty vector pCB301-304-CEN was used as a negative control. The suspensions were infiltrated into the plant leaves with needleless syringes. 

For the inoculation on the plants of S. nigrum and S. torvum, the rub inoculation method was adopted. The virus-infected N. benthamiana leaves were ground in phosphate-buffered solution (PBS, 0.01 M, pH 7.4) with mortars in a 10:1 (volume of PBS to weight of leaves) ratio to produce the inoculums. Oil paint brushes were used for rub inoculation on the leaves. The plants inoculated with PBS were used as a negative control. About three-week-old seedlings were adopted for inoculation by each method.

Photographs were taken about one-month post-inoculation for tobacco plants and about two-weeks post-inoculation for other species. The infection of fluorescent proteintagged viruses was tracked with a hand-held lamp LUYOR-3415RG (Luyor,Shanghai, China), and the photographs were taken with an LP510 filter for GFP or a BP590 filter (LUV-590A, Luyor) for RFP. In order to merge the photographs of the plants co-infected by different viruses, Photoshop CC (v14.0) was used to extract the RGB signals of fluorescent proteins.

文献地址：https://www.mdpi.com/2079-7737/11/10/1467   

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