Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Summary

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

Abstract

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.

Introduction

In the last decades, begomoviruses (genus Begomovirus, family Geminiviridae) have caused serious damage to the production of many vegetable, fiber, and ornamental crops worldwide¹. Begomoviruses are transmitted in a persistent manner by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), which is a complex species containing over 35 cryptic species^2,3. Begomoviruses may directly or indirectly affect whitefly physiology and behavior, such as fecundity⁴, longevity⁴, and host preference⁵

Protocol

1. Whitefly, Virus, Plants, and Acquisition of the Virus

1. Rear whiteflies (MEAM1) on cotton (Gossypium hirsutum cv. Zhemian 1793) in insect-proof cages in a greenhouse at 26 ± 1 °C with a 14:10 light:dark cycle and 60 ± 10% relative humidity.
2. Perform conventional PCR based on whitefly mitochondrial cytochrome oxidase I gene to determine the purity of the whitefly population.
   1. Collect 20 adult whiteflies and transfer them individually to a PCR tube containing 30 μL of lysis buffer (10 mM Tris, pH = 8.4, 50 mM KCl, 0.45% [wt/vol] Tween-20, 0.2% [wt/vol] gelatin, 0.45% [vol/vol] Nonidet P 40, 60 g/mL Proteinase....

Results

The MEAM1 whiteflies of the B. tabaci complex and TYLCV were used as an example here to describe the procedures. The overview of the immunofluorescence and viral quantification procedures described in this manuscript is shown in Figure 1. Figure 2 shows representative results of immunofluorescence detection of TYLCV and DAPI staining in PSGs, midguts, and ovaries, indicating that TYLCV accumulated more in the PSGs and midguts, and less in the ovaries. <.......

Discussion

Here we describe a protocol for the localization and quantification of a begomovirus in the tissues of its whitefly vector by immunofluorescence and qPCR. Dissection represents the first step to localize and quantify the virus in whitefly tissues. The whitefly body is about 1 mm in length, which means that the tissues are extremely small, and it is difficult to dissect them. Besides, there are strong connections between the tissues. For example, the ovaries are tightly connected with the bacteriocytes, making them diffic.......

Materials

Name	Company	Catalog Number	Comments
4% Paraformaldehyde	MultiSciences	F0001
4',6-diamidino-2-phénylindole (DAPI)	Abcam	ab104139
Bovine Serum Albumin (BSA)	MultiSciences	A3828
CFX Connect Real-Time PCR Detection System	Bio-RAD	185-5201
Confocal microscopy	Zeiss	LSM800
Dylight 549-goat anti-mouse	Earthox	E032310-02	Secondary antibody
Monoclonal antibody (MAb 1C4)			Primary antibody
Phosphate Buffered Saline (PBS)	Sangon Biotech	B548119-0500
Stereo microscope	Zeiss	Stemi 2000-C
TB green premix Ex Taq (Tli RNase H Plus)	TaKaRa	RR820A	qPCR master mix
Thermocycler	Thermofisher	A41182
Tissuelyzer	Shaghai jingxin	Tissuelyser-48
Triton-X-100	BBI life sciences	9002-93-1
Tween 20	BBI life sciences	9005-64-5