Research in the Ghanim lab was supported by research grant no. 908-42.12/2006 from the German-Israeli Foundation (GIF), grant no. IS-4062-07 from the United States-Israel Binational Agricultural Research and Development Fund (BARD), and research grant no. 884/07 from Israel Science Foundation (ISF) to M.G.

1. General Whitefly, Plant, and Virus Preparations for FISH Analysis
<ol>
	<li>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&#177;2 &#176;C, 60% relative humidity, and a 14-hr light/10-hr dark photoperiod.</li>
	<li>Perform PCR to test infection with endosymbionts using endosymbiont-specific primers as previously described15-19.</li>
	<li>Purchase Tomato s...

<ol>
	<li>Czosnek, H., Laterrot, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+worldwide+survey+of+Tomato+yellow+leaf+curl+viruses.">A worldwide survey of Tomato yellow leaf curl viruses.</a> Arch. Virol. 142, 1391-1406 (1997).</li><li>Ling, K. S., Simmons, A. M., Hassell, R. L., Keinath, A. P., Polston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+report+of+Tomato+Yellow+Leaf+Curl+Virus+in+South+Carolina.">First report of Tomato Yellow Leaf Curl Virus in South Carolina.</a> Plant Dis. 90, 379 (2006).</li><li>Polston, J. E., McGovern, R. J., Brown, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+Tomato+yellow+leaf+curl+virus+in+Florida+and+implications+for+the+spread+of+this+and+other+geminiviruses+of+tomato.">Introduction of Tomato yellow leaf curl virus in Florida and implications for the spread of this and other geminiviruses of tomato.</a> Plant Dis. 83, 984-988 (1999).</li><li>Polston, J. E., Rosebrock, T. R., Sherwood, T., Creswell, T., Shoemaker, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+Tomato+yellow+leaf+curl+virus+in+North+Carolina.">Appearance of Tomato yellow leaf curl virus in North Carolina.</a> Plant Dis. 86, 73 (2002).</li><li>Frohlich, D. R., Torres-Jerez, I., Bedford, I. D., Markham, P. G., Brown, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phylogeographical+analysis+of+the+Bemisia+tabaci+species+complex+based+on+mitochondrial+DNA+markers.">A phylogeographical analysis of the Bemisia tabaci species complex based on mitochondrial DNA markers.</a> Mol. Ecol. 8, 1683-1691 (2002).</li><li>Ghanim, M., Morin, S., 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=Rate+of+Tomato+yellow+leaf+curl+virus+translocation+in+the+circulative+transmission+pathway+of+its+vector,+the+whitefly+Bemisia+tabaci.">Rate of Tomato yellow leaf curl virus translocation in the circulative transmission pathway of its vector, the whitefly Bemisia tabaci.</a> Phytopathology. 91, 188-196 (2001).</li><li>Ghanim, M., Rosell, R. C., Campbell, L. R., Czosnek, H., Brown, J. K., Ullman, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digestive++salivary+and+reproductive+organs+of+Bemisia+tabaci+(Gennadius)+Hemiptera:+Aleyrodidae)+B+type.">Digestive salivary and reproductive organs of Bemisia tabaci (Gennadius) Hemiptera: Aleyrodidae) B type.</a> J. Morphol. 248, 22-40 (2001).</li><li>Hunter, W. B., Hiebert, E., Webb, S. E., Tsai, J. K., Polston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+geminiviruses+in+the+whitefly+Bemisia+tabaci+(Homoptera:+Aleyrodidae).">Location of geminiviruses in the whitefly Bemisia tabaci (Homoptera: Aleyrodidae).</a> Plant Dis. 82, 1147-1151 (1998).</li><li>Rosell, R. C., Torres-Jerez, I., Brown, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+pathway+of+geminivirus+in+whitefly+extracts,+saliva,+hemolymph+and+honeydew.">Temporal pathway of geminivirus in whitefly extracts, saliva, hemolymph and honeydew.</a> Phytopathology. 89, 239-246 (1999).</li><li>Czosnek, H., Ghanim, M., 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+circulative+pathway+of+begomoviruses+in+the+whitefly+vector+Bemisia+tabaci&#8212;insights+from+studies+with+Tomato+yellow+leaf+curl+virus.">The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci&#8212;insights from studies with Tomato yellow leaf curl virus.</a> Ann. Appl. Biol. 140, 215-231 (2002).</li><li>Gottlieb, 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=The+transmission+efficiency+of+Tomato+yellow+leaf+curl+virusby+the+whitefly+Bemisiatabaciis+correlated+with+the+presence+of+aspecificsymbiotic+bacterium+species.">The transmission efficiency of Tomato yellow leaf curl virusby the whitefly Bemisiatabaciis correlated with the presence of aspecificsymbiotic bacterium species.</a> J. Virol. 84, 9310-9317 (2010).</li><li>Brumin, M., Levy, M., 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=Transovarial+transmission+of+Rickettsia+spp.+and+organ-specific+infection+of+the+whitefly+Bemisia+tabaci.">Transovarial transmission of Rickettsia spp. and organ-specific infection of the whitefly Bemisia tabaci.</a> Appl. Environ. Microbiol. 78, 5565-5574 (2012).</li><li>Ghanim, M., Medina, V., 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=.">.</a> Localization of Tomato yellow leaf curl virus in its whitefly vector Bemisia tabaci. In Tomato Yellow Leaf Curl Virus Disease, Management, Molecular Biology, Breeding for Resistance. , 175-187 (2007).</li><li>Ghanim, M., Brumin, M., Popovski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+rapid+and+inexpensive+method+for+localization+of+Tomato+yellow+leaf+curl+virus+and+Potato+leafroll+virus+in+plant+and+insect+vectors.">A simple, rapid and inexpensive method for localization of Tomato yellow leaf curl virus and Potato leafroll virus in plant and insect vectors.</a> J. Virol. Methods. 159, 311-314 (2009).</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> J. Virol. 86, 13241-13252 (2012).</li><li>Nirgianaki, 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=Wolbachia+infections+of+the+whitefly+Bemisia+tabaci.">Wolbachia infections of the whitefly Bemisia tabaci.</a> Curr. Microbiol. 47, 93-101 (2003).</li><li>Baumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+bacteriocyte-associated+endosymbionts+of+plant+sap-sucking+insects.">Biology of bacteriocyte-associated endosymbionts of plant sap-sucking insects.</a> Ann. Rev. Microbiol. 59, 155-189 (2005).</li><li>Gottlieb, 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=Identification+and+localization+of+a+Rickettsia+sp.+in+Bemisia+tabaci+(Homoptera)+Aleyrodidae).">Identification and localization of a Rickettsia sp. in Bemisia tabaci (Homoptera) Aleyrodidae).</a> App. Environ. Microbiol. 72, 3646-3652 (2006).</li><li>Li, Z. X., Lin, H. Z., Guo, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+Wolbachia+infection+in+Bemisia+tabaci.">Prevalence of Wolbachia infection in Bemisia tabaci.</a> Curr. Microbiol. 54, 467-471 (2007).</li><li>Skaljac, M., Zanic, K., Hrncic, S., Radonjic, S., Perovic, T., 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=Diversity+and+localization+of+bacterial+symbionts+in+three+whitefly+species+(Hemiptera:+Aleyrodidae)+from+the+east+coast+of+the+Adriatic.">Diversity and localization of bacterial symbionts in three whitefly species (Hemiptera: Aleyrodidae) from the east coast of the Adriatic.</a> Bull. Entomol. Res. 103, 48-59 (2013).</li></ol>

The authors have nothing to disclose.

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&#16...

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 their vectors is important for studying the path for transmission, and the possible interaction sites inside the vector. The transmission of some insect-vectored plant viruses is aided by endosymbiotic bacteria that reside within these insects. To better study the transmission of these plant viruses by their vectors, it is also essential to visualize the endosymbiotic bacteria in general, and those involved in virus transmission, in particular. Colocalization of the virus and endosymbiotic bacteria is thus desired for investigating the possible relationships between these organisms within their insect host. Aside from virus transmission, endosymbiotic bacteria influence several aspects of the biology of insect vectors, thus spatial localization of these bacteria within insects is of high interest and importance.
Tomato yellow leaf curl virus (TYLCV) (Begomovirus, Geminiviridae) is the most important viral disease complex of cultivated tomato worldwide1-4. TYLCV is a phloem-limited virus and is exclusively vectored by the whitefly Bemisia tabaci 5,6. A model describing the translocation of begomoviruses in their whitefly vectors has been proposed6-9. Two specific barriers are actively crossed during this circulative transmission: the midgut/hemolymph and the hemolymph/salivary gland barriers. This transmission process is hypothesized to be mediated by unknown receptors which recognize the virus capsid. TYLCV is thought to cross B. tabaci midgut6,10, and is absorbed through the primary salivary gland6&#160;before it is injected into the plant. In the whitefly hemolymph, TYLCV interacts with a GroEL protein produced by the insect secondary endosymbiotic bacterium Hamiltonella. This interaction ensures safe transport of TYLCV in the hemolymph, and protects it from attack by the insect immune system11. Hamiltonella and Portiera, the primary endosymbiont of whiteflies, are housed in bacteriocytes, insect cells found in the hemolymph and house endosymbiotic bacteria11. B. tabaci harbors additional endosymbiotic bacteria including Rickettsia, Arsenophonus, Wolbachia,&#160;and Fritschea, which can be localized inside or outside the bacteriocytes, and have diverse effects on the insect&#8217;s biology12.
Several reports have attempted to study the localization of TYLCV in plants and whiteflies by using time-consuming and costly protocols such as Transmission Electron Microscopy (TEM), antibodies and RNA in situ on microscopic thick section10,13, a recent study described the localization of plant viruses inside their plant and vector hosts by using a simple protocol14. Here we describe a simple protocol for the localization of TYLCV in B. tabaci dissected midguts and salivary glands, and in sections prepared from TYLCV-infected plants. We further describe the localization of Portiera, the primary endosymbiont of B. tabaci, and its secondary endosymbionts Hamitonella, Rickettsia and Arsenophonus. This protocol is based on using short DNA probes, that are fluorescently labeled on their 5&#39;&#160;end, and specifically hybridize to complementary sequences in viral or bacterial gene sequences. The sample processing is relatively easy and the signal obtained is highly specific. The described protocol can be used to localize viruses, bacteria and other pathogens in their plant, animal and insect hosts, and can further be used to localize mRNA in any given tissue.

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			<td height="21" style="height:21px;width:219px;">Fluorescently labeled&#160; Probes</td>
			<td style="width:95px;">Metabion</td>
			<td style="width:129px;">20 bp HPLC purified</td>
			<td style="width:212px;">Sequence designed by customer</td>
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			<td height="21" style="height:21px;width:219px;">Toluidine blue</td>
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			<td height="21" style="height:21px;width:219px;">sodium dodecyl sulfate</td>
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			<td style="width:129px;">L3771</td>
			<td style="width:212px;">Molecular Biology Grade</td>
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			<td style="width:129px;">F9037</td>
			<td style="width:212px;">Molecular Biology Grade</td>
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			<td height="21" style="height:21px;width:219px;">Tris-HCl</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">T5941</td>
			<td style="width:212px;">Molecular Biology Grade</td>
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			<td height="21" style="height:21px;width:219px;">Glacial Acetic Acid</td>
			<td style="width:95px;">Sigma-Aldrich</td>
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			<td style="width:212px;">Molecular Biology Grade</td>
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fluorescence in situ hybridizations (fish) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues

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&#160;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&#39; or 3&#39; 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.

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...

Watch this Scientific Journal Video about Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues at JoVE.com

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

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.

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

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

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23.2K visualizações.  Volcani Center.  Descrevemos aqui um simples fluorescência de hibridação in situ (FISH), o método para a localização de vírus e bactérias, em tecidos de plantas e de insectos. Este protocolo pode ser estendido para a visualização de mRNA em toda montagem e cortes microscópicos. 