Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR
| Autor: | Qi Guo, Tian-Yan Yin, Xiao-Wei Wang, Li-Long Pan, Fei-Xue Ban, Yin-Quan Liu |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
General Chemical Engineering Fluorescent Antibody Technique Whitefly Immunofluorescence Real-Time Polymerase Chain Reaction 010603 evolutionary biology 01 natural sciences General Biochemistry Genetics and Molecular Biology Virus Salivary Glands Hemiptera Hemolymph medicine Animals Tomato yellow leaf curl virus General Immunology and Microbiology biology medicine.diagnostic_test General Neuroscience fungi Begomovirus Ovary food and beverages biology.organism_classification Primary and secondary antibodies Virology Gastrointestinal Tract 010602 entomology Real-time polymerase chain reaction Vector (epidemiology) biology.protein Capsid Proteins Female |
| Zdroj: | Journal of visualized experiments : JoVE. (156) |
| ISSN: | 1940-087X |
| Popis: | Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions. |
| Databáze: | OpenAIRE |
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