title: Fluorescence in situ hybridizations (FISH) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues.
abstract: Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5' or 3' ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.
authors.affiliation: Department of Entomology, Volcani Center; Institute of Plant Sciences and Genetics in Agriculture, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem.
authors.forename: Adi
authors.initials: A
authors.lastname: Kliot
authors.affiliation: Department of Entomology, Volcani Center.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Kontsedalov
authors.affiliation: Department of Entomology, Volcani Center.
authors.forename: Galina
authors.initials: G
authors.lastname: Lebedev
authors.affiliation: Department of Entomology, Volcani Center.
authors.forename: Marina
authors.initials: M
authors.lastname: Brumin
authors.affiliation: Department of Entomology, Volcani Center.
authors.forename: Pakkianathan Britto
authors.initials: PB
authors.lastname: Cathrin
authors.affiliation: Department of Entomology, Volcani Center.
authors.forename: Julio Massaharu
authors.initials: JM
authors.lastname: Marubayashi
authors.affiliation: Department of Entomology, Volcani Center; Department of Applied Sciences, Institute for Adriatic Crops and Karst Reclamation.
authors.forename: Marisa
authors.initials: M
authors.lastname: Skaljac
authors.affiliation: The Institute of Plant Sciences, Volcani Center.
authors.forename: Eduard
authors.initials: E
authors.lastname: Belausov
authors.affiliation: Institute of Plant Sciences and Genetics in Agriculture, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem.
authors.forename: Henryk
authors.initials: H
authors.lastname: Czosnek
authors.affiliation: Department of Entomology, Volcani Center; ghanim@volcani.agri.gov.il.
authors.forename: Murad
authors.initials: M
authors.lastname: Ghanim
mesh_terms: D000818:Animals; D053839:Begomovirus / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D006430:Hemiptera / Q000382:microbiology* / Q000821:virology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018551:Solanum lycopersicum / Q000382:microbiology / Q000821:virology; D010935:Plant Diseases / Q000382:microbiology* / Q000821:virology; D010944:Plants / Q000382:microbiology* / Q000821:virology; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D013559:Symbiosis
chemical_list: D004269:DNA, Bacterial; D004279:DNA, Viral; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012367:RNA, Viral