Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Summary

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

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.

Introduction

When studying the interactions between plant viruses and other pathogens with their infected plant hosts, it is important to visualize the pathogens and their respective nucleic acids in situ, regardless whether they cause negative effects on their hosts. This is most important when studying pathogen movement within and between plant cells. In situ localization of gene products of the pathogen is an essential step that complements other approaches for studying the pathogenicity process. Many plant pathogens, especially viruses, are transmitted by insects, having complex and intimate interactions with their vectors. Localization of these viruses in th....

Protocol

1. General Whitefly, Plant, and Virus Preparations for FISH Analysis

1. Rear B and Q biotype whiteflies on cotton seedlings (Gossypium hirsutum L. cv. Acala) and maintain inside insect-proof cages and growth rooms under standard conditions of 25±2 °C, 60% relative humidity, and a 14-hr light/10-hr dark photoperiod.
2. Perform PCR to test infection with endosymbionts using endosymbiont-specific primers as previously described^15-19.
3. Purchase Tomato seedlings (Solanum esculentum cv. Beefsteak) from a commercial nursery or plant tomato seeds.
4. Maintain under the same reari....

Results

The system studied in this manuscript is shown in Figure 1 and includes an infected plant with TYLCV, an adult and a nymph of the whitefly B. tabaci, and the internal anatomy of the whitefly showing the path for TYLCV translocation in the insect. Figure 2 shows double FISH in an adult whitefly for the primary symbiont Portiera and the secondary symbiont Arsenophonus. Figure 3 shows double FISH for Portiera and Hamiltonella, an.......

Discussion

The protocol described here for the localization of a plant virus in its plant host and insect vector, and endosymbiotic bacteria in their specific whitefly host, can be adapted for the localization of other viruses in plants and even in animal tissues. Furthermore, the protocol can be used to localize endosymbiotic and pathogenic bacteria and other microorganisms in plant and animal systems. The described methods rely on simple concept of hybridization between a short, fluorescently-labeled oligonucleotide DNA probe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluorescently labeled  Probes	Metabion	20 bp HPLC purified	Sequence designed by customer
Toluidine blue	Sigma-Aldrich	89640
Sodium Dodecyl Sulfate	Sigma-Aldrich	L3771	Molecular Biology Grade
Formamide	Sigma-Aldrich	F9037	Molecular Biology Grade
Tris-HCl	Sigma-Aldrich	T5941	Molecular Biology Grade
Glacial Acetic Acid	Sigma-Aldrich	320099	Molecular Biology Grade
Liquid Blocker	Ted Pella Inc.	22309