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. S., Navas-Castillo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+begomoviruses+and+other+whitefly-borne+viruses:+dependence+on+the+vector+species.">Transmission of begomoviruses and other whitefly-borne viruses: dependence on the vector species.</a> Phytopathology. , (2019).</li><li>Cohen, S., Nitzany, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+and+host+range+of+the+tomato+yellow+leaf+curl+virus.">Transmission and host range of the tomato yellow leaf curl virus.</a> Phytopathology. 56, 1127-1131 (1966).</li><li>Moriones, E., Navas-Castillo, J. <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,+an+emerging+virus+complex+causing+epidemics+worldwide.">Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide.</a> Virus Research. 71 (1-2), 123-134 (2000).</li><li>Ghanim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+mechanisms+and+components+that+determine+the+transmission+efficiency+of+tomato+yellow+leaf+curl+virus+(Geminiviridae;+Begomovirus)+by+its+whitefly+vector.">A review of the mechanisms and components that determine the transmission efficiency of tomato yellow leaf curl virus (Geminiviridae; Begomovirus) by its whitefly vector.</a> Virus Research. 186, 47-54 (2014).</li><li>Navot, N., Pichersky, E., Zeidan, M., Zamir, D., Czosnek, H. <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+-+a+whitefly-transmitted+geminivirus+with+a+single+genomic+component.">Tomato yellow leaf curl virus - a whitefly-transmitted geminivirus with a single genomic component.</a> Virology. 185 (1), 151-161 (1991).</li><li>Sanchez-Campos, S., 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:+No+evidence+for+replication+in+the+insect+vector+Bemisia+tabaci.">Tomato yellow leaf curl virus: No evidence for replication in the insect vector Bemisia tabaci.</a> Scientific Reports. 6, 30942 (2016).</li><li>Pakkianathan, B. C., 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=Replication+of+tomato+yellow+leaf+curl+virus+in+its+whitefly+vector,+Bemisia+tabaci.">Replication of tomato yellow leaf curl virus in its whitefly vector, Bemisia tabaci.</a> Journal of Virology. 89 (19), 9791-9803 (2015).</li><li>Rodriguez-Negrete, E. 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. S. <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+whitefly+vesicle+associated+membrane+protein-associated+protein+B+in+the+transmission+of+Tomato+yellow+leaf+curl+virus.">Implication of whitefly vesicle associated membrane protein-associated protein B in the transmission of Tomato yellow leaf curl virus.</a> Virology. 535, 210-217 (2019).</li><li>Maluta, N. K., Garzo, E., Moreno, A., Lopes, J. R., Fereres, A. <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+benefits+population+growth+of+the+Q+biotype+of+Bemisia+tabaci+(Gennadius)+(Hemiptera:+Aleyrodidae).">Tomato yellow leaf curl virus benefits population growth of the Q biotype of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae).</a> Neotropical Entomology. 43 (4), 385-392 (2014).</li><li>Czosnek, H., Hariton-Shalev, A., Sobol, I., Gorovits, R., Ghanim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incredible+journey+of+begomoviruses+in+their+whitefly+vector.">The incredible journey of begomoviruses in their whitefly vector.</a> Viruses. 9 (10), 273 (2017).</li><li>Wei, 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=Vector+development+and+vitellogenin+determine+the+transovarial+transmission+of+begomoviruses.">Vector development and vitellogenin determine the transovarial transmission of begomoviruses.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (26), 6746-6751 (2017).</li><li>Ghanim, M., Morin, S., Zeidan, M., Czosnek, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+transovarial+transmission+of+tomato+yellow+leaf+curl+virus+by+its+vector,+the+whitefly+Bemisia+tabaci.">Evidence for transovarial transmission of tomato yellow leaf curl virus by its vector, the whitefly Bemisia tabaci.</a> Virology. 240 (2), 295-303 (1998).</li><li>Xie, 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=Highly+sensitive+serological+methods+for+detecting+tomato+yellow+leaf+curl+virus+in+tomato+plants+and+whiteflies.">Highly sensitive serological methods for detecting tomato yellow leaf curl virus in tomato plants and whiteflies.</a> Virology Journal. 10, 142 (2013).</li><li>Arya, 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=Basic+principles+of+real-time+quantitative+PCR.">Basic principles of real-time quantitative PCR.</a> Expert Review of Molecular Diagnostics. 5 (2), 209-219 (2005).</li><li>Wei, 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=Specific+cells+in+the+primary+salivary+glands+of+the+whitefly+Bemisia+tabaci+control+retention+and+transmission+of+begomoviruses.">Specific cells in the primary salivary glands of the whitefly Bemisia tabaci control retention and transmission of begomoviruses.</a> Journal of Virology. 88 (22), 13460-13468 (2014).</li><li>Wang, L. 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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. ...

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