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 preference^5,6. Furthermore, the transmission efficiency of a given begomovirus species/strain varies for different whitefly cryptic species even under the same experimental conditions^7,8,9,10, indicating that there is a complex interaction between begomoviruses and whiteflies. To better understand the mechanisms underlying whitefly-begomovirus interactions, localization and quantification of the virus in whitefly tissues are essential.

Tomato yellow leaf curl virus (TYLCV) is a begomovirus that was first reported in Israel but nowadays causes serious damage to tomato production worldwide^11,12. Due to its economic importance, it is one of the best-studied begomoviruses¹³. Like other monopartite begomoviruses, TYLCV is a single-strand circular DNA virus with a genome size of about 2,800 nucleotides¹⁴. While still under debate, several lines of evidence support the replication of TYLCV in whiteflies^15,16,17. Moreover, the interaction of TYLCV particles and whitefly proteins has been reported^6,18,19,20. For virus transmission, whiteflies acquire TYLCV by feeding on virus-infected plants, virions pass along the food canal to reach the esophagus, penetrate the midgut wall to reach the hemolymph, and then translocate into the primary salivary glands (PSGs). Finally, virions are egested with saliva along the salivary duct into plant phloem²¹. Furthermore, several studies show that TYLCV is able to be transovarially transmitted from female whiteflies to their offspring^22,23. In other words, to achieve productive transmission, the virus has to overcome cellular barriers within the whitefly to translocate from one tissue to another. During the crossing of these barriers, interactions between whitefly and virus proteins are likely to occur, probably determining the efficiency with which the viruses are transmitted.

Immunofluorescence is a commonly used technique for protein distribution analysis. The specificity of antibodies binding to their antigen form the basis of immunofluorescence. Due to the economic significance of TYLCV, monoclonal antibodies against the TYLCV coat protein have been developed, offering a highly sensitive way to localize the virus²⁴. Quantitative PCR (qPCR) allows sensitive and specific quantification of nucleic acids. This technique is most frequently based on the use of hydrolysis probe (e.g., TaqMan) or fluorescent dye (e.g., SYBR Green) detection. For hydrolysis probe-based qPCR, specific probes are needed, which consequently increase the cost. Fluorescent dye-based qPCR is simpler and more cost-effective, because labeled amplicon-specific hybridization probes are not required²⁵. So far, several studies have used immunofluorescence and qPCR along with other methods to investigate the complex begomovirus-whitefly interactions. For example, Pan et al. performed qPCR and immunofluorescence analysis of the virus in whitefly tissues and found that the difference in ability to transmit tobacco curly shoot virus (TbCSV) between whitefly species AsiaII 1 and Middle East Asia Minor 1 (MEAM1) was due to the virus being able to efficiently cross the midgut wall of AsiaII1 but not MEAM1⁸. Similarly, while Mediterranean (MED) whiteflies can readily transmit TYLCV, they fail to transmit tomato yellow leaf curl China virus (TYLCCNV). Selective transmission was investigated using immunofluorescence detection of virus in the PSGs, which showed that TYLCCNV do not easily cross the PSGs of MED whiteflies²⁶. Immunofluorescence colocalization of TYLCV CP and the autophagy marker protein ATG8-II in whitefly midguts shows that autophagy plays a critical role in repressing the infection of TYLCV in the whitefly²⁷.

Here, using TYLCV as an example, we describe a protocol for the localization of begomoviruses in whitefly midguts, PSGs, and ovaries by an immunofluorescence technique. The technique includes dissection, fixation, and incubation with primary and dye-labeled secondary antibodies. Fluorescence signals showing the location of viral proteins in the whitefly tissues can then be detected under a confocal microscope. More importantly, this protocol can be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV using SYBR Green-based qPCR in whitefly midguts, PSGs, hemolymph, and ovaries, that can be used to compare the amount of virus in different whitefly tissue samples.

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 K).
   2. Add 5–7 ceramic beads (2 mm in diameter) into each PCR tube and grind the samples to perform the tissue lysis.
   3. Incubate all samples at 65 °C for 1 h followed by 10 min at 100 °C, and then centrifuge briefly. Use this supernatant as the template for PCR amplification.
      NOTE: The incubation time at 65 °C can be increased if necessary.
   4. Prepare the PCR reaction as follows: 1 U of Taq DNA polymerase, 2 µL of 10x buffer (with Mg²⁺), 1.6 µL of dNTP mixture (2.5 mM), 0.5 µL of each primer (10 μM each), 2 µL of whitefly tissue lysate supernatant, and double distilled water to a total volume of 20 µL per reaction. The forward primer sequence is 5'-TTGATTTTTTGGTCATCCAGAAGT-3' and the reverse primer sequence is 5'-TAATATGGCAGATTAGTGCATTGGA-3'.
      NOTE: A negative control where the DNA is substituted with nuclease-free water and a positive control with previously verified MEAM1 whitefly DNA should be included.
   5. Perform PCR using the following cycling parameters: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 50–55 °C for 30 s, and extension at 72 °C for 1 min, followed by 72 °C for 10 min.
   6. Digest the amplified product with restriction enzyme Taq I. To do so, prepare the digestion mixture with 0.1 µL (1 U) of enzyme,1 μL of 10x Taq I Buffer, 1 μL of BSA, 5 μL of amplified product, and double distilled water to a total volume of 10 μL. Incubate at 65 °C for 1 h in a thermocycler.
   7. Perform agarose gel electrophoresis of the samples by loading 5–7 µL of each digested product and a DNA ladder (100-2,000 bp) into the wells of a 1% agarose gel. Then, observe the DNA band sizes by gel-red staining in a gel documentation machine using UV light. Compare the band sizes of the digested products to the positive control to determine the purity of the whitefly population.
3. Agro-inoculate the infectious clone of TYLCV (GenBank accession number AM282874.1) (pBINPLUS-SH2-1.4A) into 3–4 true leaf stage tomato plants (Solanum lycopersicum L. cv. Hezuo 903).
   1. Inoculate 10 mL of Luria–Bertani (LB) medium containing kanamycin (50 μg/mL) and rifampicin (50 μg/mL) with a single colony. Incubate at 28 °C with shaking at 200 rpm until the OD₆₀₀ reaches ~1.5.
   2. Harvest agrobacteria by centrifugation at 4,000 x g for 10 min. Discard the supernatant.
   3. Resuspend pellets in 10 mL of infiltration buffer, consisting of 200 μM acetosyringone, 10 mM 2-(N-morpholino) ethane sulfonic acid (MES), and 10 mM MgCl₂. Incubate at room temperature (RT) for 1–2 h.
   4. Infiltrate 2–3 plant leaves using the mixture from step 1.3.3 with a 1 mL needleless syringe. Maintain plants in a greenhouse at 26 ± 1 °C.
4. About 1 month later, perform a visual inspection and choose plants showing yellow curl leaf and stunt symptoms.
5. Transfer non-viruliferous whitefly adults onto TYLCV-infected tomato plants for 48 h for virus acquisition.

2. Dissection and Collection of Midguts, Primary Salivary Glands (PSG), Ovaries, and Hemolymph of Female Whiteflies

1. Collect whiteflies using aspiration, and then place them on ice to put them in a coma. Add 1x PBS (phosphate-buffered saline) buffer onto a microscope slide, and then place whiteflies in the 1x PBS buffer for dissection under a stereo microscope.
   1. For the midgut dissection, break the abdomen of the whitefly and pull out the midgut using a fine acupuncture needle (0.25 mm in diameter). Transfer the midgut to clean, 1x PBS.
   2. For PSG dissection, break the body of the whitefly in the thorax near the head from the dorsal side. Next, insert a needle into the thorax to fix the whitefly, and use another needle to shake the whitefly until the two salivary glands are separated from the body. Then, transfer the PSGs to clean, 1x PBS.
   3. For the ovary dissection, completely break the abdomen of the whitefly, and use a needle to separate the ovaries from other tissues. Then, remove excess tissues by pipetting.
   4. For the hemolymph collection, add 10 μL of 1x PBS buffer onto a microscope slide, and then place a whitefly in the buffer. To obtain the hemolymph, gently tear a hole in the abdomen using a fine acupuncture needle, and slightly press the abdomen to release the hemolymph out into the buffer. Collect 8 μL of the liquid as the hemolymph sample.

3. Localization of TYLCV in Whitefly Midgut, Primary Salivary Glands, and Ovaries by Immunofluorescence

NOTE: The procedure for the localization of TYLCV in the midguts is presented as an example. The method can be used for PSGs and ovaries.

1. Dissect the midguts in TBS buffer (10 mM Tris-HCl, 150 mM sodium chloride, pH = 7.5) as described in step 2.2.1. Collect 15–20 whitefly midguts, transfer them into a glass Petri dish (3.5 cm in diameter), and then remove excess TBS buffer.
2. Add 1 mL of 4% paraformaldehyde to fix midguts for 2 h at room temperature, and then remove the fixative solution. Pipette slowly to wash the midguts 3x in TBS for 2 min each.
3. Incubate the midguts in 1 mL of 0.5% Triton X-100 for 30 min to permeabilize. Then remove the permeabilization solution and wash the midguts 3x with TBST (1x TBS with 0.05% Tween 20) as described in step 3.2.
4. Add 1 mL of 1% BSA (bovine serum albumin prepared in TBST) to block the midguts. Perform the blocking step at RT for 2 h, and then remove the blocking solution and wash the midguts 3x in TBST.
5. Dilute the primary antibodies (MAb 1C4 against TYLCV coat protein) at 1:400 with TBST²⁴ and add the solution into the Petri dish to immerse the midguts. Incubate at 4 °C overnight in the dark. Discard the primary antibody solution and wash 3x in TBST.
6. Dilute the secondary antibodies (anti-mouse) at 1:400 with TBST and add the solution into the Petri dish to immerse the midguts. Incubate the dish for 2 h at RT in the dark. Discard the secondary antibody solution and wash the midguts 3x in TBST.
7. Transfer the midguts to a clear microscope slide. Aspirate as much TBST off the slide as possible. Add 1 drop of DAPI (4', 6-diamidino-2-phenylindole, dihydrochloride) to stain the nucleus, and then place the coverslip and seal with nail polish. Examine under a confocal microscope.

4. Quantification of TYLCV in Whitefly Midgut, Primary Salivary Glands, Hemolymph, and Ovaries with qPCR

1. Dissect the whitefly midguts, PSGs, hemolymph, and ovaries in 1x PBS as described above.
2. Extract the DNA of the midguts, PSGs, hemolymph, and ovaries.
   1. After dissection, wash the midguts, PSGs, and ovaries in clean, 1x PBS buffer 2–3x.
   2. Collect 20 midguts or 20 PSGs in 5 μL of 1x PBS and transfer them into 25 μL lysis solution. Transfer each ovary in 5 μL of 1x PBS and then transfer into 25 μL lysis solution individually.
   3. Grind midguts, PSGs, and ovaries samples as described in step 1.2.2.
   4. Collect 8 μL of hemolymph with 1x PBS into a PCR tube, and then add 10 μL of lysis solution. Mix thoroughly.
3. Extract DNA from all samples as described in 1.2.3.
4. Prepare two separate qPCR master mixes (18 μL each) for TYLCV and the internal control (β-actin gene) as follows: 10 μL of SYBR Green mix, 0.8 μL of each primer (10 μM each), and 6.4 μL of double distilled water per reaction. For TYLCV, the forward primer sequence is 5′-GAAGCGACCAGGCGATATAA-3′ and the reverse primer sequence is 5′-GGAACATCAGGGCTTCGATA-3′. For β-actin, the forward primer sequence is 5′-TCTTCCAGCCATCCTTCTTG-3′ and the reverse primer sequence is 5′-CGGTGATTTCCTTCTGCATT-3′.
5. Dispense 18 µL of the master mix into vials of qPCR strip tubes or a 96 well plate. Then, add 2 µL of the DNA samples into each vial. Perform all reactions with at least three technical replicates and three biological replicates.
   NOTE: Step 4.5 should be performed on ice.
6. Close the qPCR tubes or seal the 96 well plates with adhesive plate seal.
7. Centrifuge to collect the liquid at the bottom of the qPCR tubes or 96 well plate.
8. Place the tubes or plate into the real-time thermal cycler and perform qPCR.
   1. Perform the qPCR using the following cycling parameters: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s for primer annealing and elongation, 1 cycle at 95 °C for 15 s, ending with a melting curve step of 60 °C to 95 °C with an increment of 0.5 °C per 15 s.
   2. Choose SYBR as the fluorophore dye and unknown as sample type.
9. Export the quantitative data to a spreadsheet format and analyze the relative quantity of viral DNA by the 2^−ΔΔCT method²⁸.

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