Name: localization and quantification of begomoviruses in whitefly tissues by immunofluorescence and quantitative pcr
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Description: Watch this Scientific Journal Video about Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR at JoVE.com

This work was supported by National Key Research and Development Program (Grant number: 2017YFD0200600), the earmarked fund for China Agriculture Research System (grant number: CARS-23-D07) and the Bill &#38; Melinda Gates Foundation (Investment ID OPP1149777). We thank Prof. Jian-Xiang Wu for providing TYLCV CP antibodies.

1. Whitefly, Virus, Plants, and Acquisition of the Virus
<ol>
	<li>Rear whiteflies (MEAM1) on cotton (Gossypium hirsutum cv. Zhemian 1793) in insect-proof cages in a greenhouse at 26 &#177; 1 &#176;C with a 14:10 light:dark cycle and 60 &#177; 10% relative humidity.</li>
	<li>Perform conventional PCR based on whitefly mitochondrial cytochrome oxidase I gene to determine the purity of the whitefly population.
	<ol>
		<li>Collect 20 adult whiteflies and transfer them individually to a PCR tube containing 30 &#95...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bemisia+tabaci:+a+statement+of+species+status.">Bemisia tabaci: a statement of species status.</a> Annual Review of Entomology. 56, 1-19 (2011).</li><li>Navas-Castillo, J., Fiallo-Olive, E., Sanchez-Campos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+virus+diseases+transmitted+by+whiteflies.">Emerging virus diseases transmitted by whiteflies.</a> Annual Review of Phytopathology. 49, 219-248 (2011).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+infection+of+tobacco+plants+improves+performance+of+Bemisia+tabaci+but+more+so+for+an+invasive+than+for+an+indigenous+biotype+of+the+whitefly.">Viral infection of tobacco plants improves performance of Bemisia tabaci but more so for an invasive than for an indigenous biotype of the whitefly.</a> Journal of Zhejiang University-Science B. 11 (1), 30-40 (2010).</li><li>Legarrea, S., Barman, A., Marchant, W., Diffie, S., Srinivasan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+effects+of+a+begomovirus+infection+and+host+plant+resistance+on+the+preference+and+development+of+an+insect+vector,+Bemisia+tabaci,+and+implications+for+epidemics.">Temporal effects of a begomovirus infection and host plant resistance on the preference and development of an insect vector, Bemisia tabaci, and implications for epidemics.</a> PLoS One. 10 (11), 0142114 (2015).</li><li>Fang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+yellow+leaf+curl+virus+alters+the+host+preferences+of+its+vector+Bemisia+tabaci.">Tomato yellow leaf curl virus alters the host preferences of its vector Bemisia tabaci.</a> Scientific Reports. 3, 2876 (2013).</li><li>Guo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transmission+of+papaya+leaf+curl+China+virus+among+four+cryptic+species+of+the+whitefly+Bemisia+tabaci+complex.">Comparison of transmission of papaya leaf curl China virus among four cryptic species of the whitefly Bemisia tabaci complex.</a> Scientific Reports. 5, 15432 (2015).</li><li>Pan, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+efficiency+of+a+begomovirus+to+cross+the+midgut+of+different+species+of+whiteflies+results+in+variation+of+virus+transmission+by+the+vectors.">Differential efficiency of a begomovirus to cross the midgut of different species of whiteflies results in variation of virus transmission by the vectors.</a> Science China-Life Sciences. 61 (10), 1254-1265 (2018).</li><li>Pan, L. L., Cui, X. Y., Chen, Q. F., Wang, X. W., Liu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotton+leaf+curl+disease:+which+whitefly+is+the+vector.">Cotton leaf curl disease: which whitefly is the vector.</a> Phytopathology. 108 (10), 1172-1183 (2018).</li><li>Fiallo-Olive, E., Pan, L. L., Liu, S. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+the+quantification+of+virion-sense+and+complementary-sense+DNA+strands+of+circular+single-stranded+DNA+viruses.">A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses.</a> Scientific Reports. 4, 6438 (2014).</li><li>Gotz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+Bemisia+tabaci+heat+shock+protein+70+in+Begomovirus-whitefly+interactions.">Implication of Bemisia tabaci heat shock protein 70 in Begomovirus-whitefly interactions.</a> Journal of Virology. 86 (24), 13241-13252 (2012).</li><li>Zhao, J., Chi, Y., Zhang, X. J., Wang, X. W., Liu, S. 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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...

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 species2,3. Begomoviruses may directly or indirectly affect whitefly physiology and behavior, such as fecundity4, longevity4, and host preference5,6. Furthermore, the transmission efficiency of a given begomovirus species/strain varies for different whitefly cryptic species even under the same experimental conditions7,8,9,10, 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 worldwide11,12. 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 whiteflies15,16,17. Moreover, the interaction of TYLCV particles and whitefly proteins has been reported6,18,19,20. 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 offspring22,23. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>MultiSciences</td>
			<td>F0001</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-ph&#233;nylindole (DAPI)</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>MultiSciences</td>
			<td>A3828</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Connect Real-Time PCR Detection System</td>
			<td>Bio-RAD</td>
			<td>185-5201</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 549-goat anti-mouse</td>
			<td>Earthox</td>
			<td>E032310-02</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Monoclonal antibody (MAb 1C4)</td>
			<td></td>
			<td></td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sangon Biotech</td>
			<td>B548119-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>TB green premix Ex Taq (Tli RNase H Plus)</td>
			<td>TaKaRa</td>
			<td>RR820A</td>
			<td>qPCR master mix</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Thermofisher</td>
			<td>A41182</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissuelyzer</td>
			<td>Shaghai jingxin</td>
			<td>Tissuelyser-48</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>BBI life sciences</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BBI life sciences</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

localization and quantification of begomoviruses in whitefly tissues by immunofluorescence and quantitative pcr

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.

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. <...

Watch this Scientific Journal Video about Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR at JoVE.com

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

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.

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

Clarifying the spatiality and quantity of virus in whitefly vectors can help in understanding begomovirus-whitefly interactions and in the development of novel strategies for controlling serious begomoviral diseases. The advantages of this technique are that we can quickly isolate whitefly tissues, co-localize viral and whitefly proteins, and quantify viruses in whitefly whole bodies and virus-infected plants. At the appropriate experimental time point, after virus acquisition, place ice anesthetized female whiteflies into a drop of PBS on a microscope slide and use a fine acupuncture needle to open the abdomen of the whitefly under a stereo microscope.Use the needle to extract the midgut and place the midgut into a collection dish of PBS. To collect the PSGs, split the body at the thorax from the dorsal side near the head and fix the whitefly with a needle through the thorax. Use a second needle to shake the whitefly until the two salivary glands are separated from the body and transfer the glands to a new dish of PBS.To collect the ovaries, completely open the abdomen and use a needle to separate the ovaries from the other tissues. Use a pipette to remove the excess tissue and transfer the ovaries into a new dish of PBS. To collect a hemolymph sample, add 10 microliters of PBS onto a new microscope slide and place a new whitefly into the droplet.Use a fine acupuncture needle to gently make a hole in the abdomen. Then apply slight pressure to the abdomen to release the hemolymph into the buffer and collect an eight microliter sample of the liquid. To visualize TYLCV localization in harvested whitefly PSG, midgut, or ovarian tissues, transfer 15 to 20 specimens into a glass 3.5 centimeter Petri dish and aspirate the excess buffer.Fix the tissues in one milliliter of 4%paraformaldehyde for two hours at room temperature before carefully washing the samples three times for two minutes in one milliliter of fresh TBS per wash. After the last wash, permeabilize the samples with one milliliter of 0.5%Triton X-100 for 30 minutes before washing three times as just demonstrated with one milliliter of TBST per wash. After the last wash, block any nonspecific binding with one milliliter of 1%bovine serum albumin for two hours at room temperature followed by three washes in TBST as demonstrated.After the last wash, label the cells with the primary antibodies of interest overnight at four degrees Celsius protected from light. The next morning, wash the samples three times with TBST before adding the appropriate secondary antibodies for two hours at room temperature protected from light. At the end of the incubation, wash the midguts three times and transfer the specimens to a clean microscope slide.Remove as much buffer as possible and stain the nuclei with one drop of DAPI before mounting with a coverslip and examining the labeled tissues by confocal microscopy. For TYLCV quantification, wash the harvested whitefly tissue samples two to three times in fresh PBS and transfer 20 midguts, 20 PSGs, or one ovary in five microliters of PBS into individual PCR tubes containing 25 microliters of lysis solution. Add five to seven two millimeter diameter ceramic beads to each tube and grind the samples in a homogenizer.Add one eight microliter sample of hemolymph into a new PCR tube and add 10 microliters of lysis to the tube with thorough mixing. Incubate all of the samples at 65 degrees Celsius for one hour followed by a 10-minute incubation at 100 degrees Celsius and a brief centrifugation. Next, add 18 microliters of master mix into the vials of quantitative PCR strip tubes and add two microliters of each DNA sample to at least three vials.When all of the samples have been loaded, close the tubes and centrifuge to sediment the solution at the bottom of each vial. Load the vials onto a real-time thermal cycler for quantitative PCR, selecting cyber as the fluorophore dye and unknown as the sample type. Then export the quantitative data to a spreadsheet for analysis of the relative quantity of viral DNA in each sample.Here, representative images of TYLCV in DAPI staining in whitefly PSGs, midguts, and ovaries are shown. As observed, TVLCV demonstrates a greater accumulation within the PSGs and midguts than in the ovaries. Quantification of the virus in whitefly PSGs, midguts, hemolymph, and ovaries after feeding on TYLCV-infected tomato for 24, 48, and 72 hours indicates that the quantity of virus increases in different whitefly tissues in direct correlation to the increase in acquisition access period.It is best to use fresh whiteflies, as dead insects will increase the difficulty of dissection. Following this procedure, other analyses, like western blotting and co-immunoprecipitation, can be performed to answer additional questions about viral protein expression and viral and vector protein interactions.

Research

JoVE Journal

Immunology and Infection

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