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 seedlings (Solanum esculentum cv. Beefsteak) from a commercial nursery or plant tomato seeds.</li>
	<li>Maintain under the same rearing conditions described above until the plants reach the four true-leaf stage, the age most suitable for whitefly-mediated inoculation of TYLCV15 (see section 4.1 below).</li>
</ol>
2. Insect Handling, Fixation, Probe Design, Hybridization, and Visualization for Endosymbiont FISH
<ol>
	<li>Collect 10 adult whiteflies by aspiration from cotton plants, or pick 10 3rd or 4th instar nymphs from cotton leaves.</li>
	<li>Immediately immerse in Carnoy&#8217;s fixative (chloroform-ethanol-glacial acetic acid, 6:3:1, vol/vol) inside Eppendorf tubes. Fix for 2 hr to overnight (preferably overnight).</li>
	<li>Completely remove the fixative and decolorize in 6% H2O2 in ethanol for 2 hr.</li>
	<li>Preserve the samples in absolute ethanol for several days to several weeks at room temperature, or proceed to the next step.</li>
	<li>Design probes as complementary reverse DNA primers based on the 16s ribosomal RNA genes of each bacterium. For designing the probes, take into the account the same considerations for designing PCR primers. Use the oligonucleotide probes BTP1 5&#39;-Cy5-TGTCAGTGTCAGCCCAGAAG-3&#39;, Rb1 5&#39;-Cy3-TCCACGTCGCCGTCTTGC-3&#39;, BTH 5&#39;-Cy3-CCAGATTCCCAGACTTTACTCA-3&#39; and Ars2 5&#39;-cy3-TCATGACCACAACCTCCAAA-3&#39; for specific targeting of Portiera, Rickettsia, Hamilonella,&#160;and Arsenophonus, respectively.</li>
	<li>Hybridize samples overnight in the dark in hybridization buffer (20 mM Tris-HCl, pH 8.0, 0.9 M NaCl, 0.01% [wt/vol] sodium dodecyl sulfate, 30% [vol/vol] formamide) containing 10 pmol fluorescent probe per ml. Combine more than one probe with different fluorescent dyes if necessary. Use noninfected whiteflies with the targeted bacterium and no-probe samples as negative controls.</li>
	<li>Mount the samples whole, on a microscope slide, in hybridization buffer contained with a liquid blocker, cover with a cover slip, seal with nail polish, and view under a fluorescence or confocal microscope.</li>
</ol>
3. Whitefly Organ Dissection, Fixation, Hybridization, and Visualization for TYLCV FISH
<ol>
	<li>Collect adult whiteflies&#160;continuously reared on TYLCV-infected tomato plants by aspiration and anesthetize by exposure to paper towel impregnated with acetone. Expose whiteflies only for 1-2 min as too long exposure to acetone can fix whiteflies tissues. Use nonviruliferous whiteflies reared on noninfected tomato plants and no probe samples for controls to confirm specific hybridization.</li>
	<li>Mount whiteflies on depression microscope slides for organ dissection using a stereo microscope.</li>
	<li>Pull away the insect head from the prothorax and dissect salivary glands in 1x PBS supplemented with 1% toluidine blue stain for better visualization, due to their small size. Allow absorption of the stain for 2-3 min.</li>
	<li>Pull the insect apart at the juncture between the thorax and the abdomen to observe and expel out the midgut. Expel the contents of the abdomen by pushing with forceps on the abdomen tip.</li>
	<li>Gently remove the PBS and toluidine blue from successful dissections and gently add 300 &#181;l of Carnoy&#8217;s fixative to fix the organs for 5 min.</li>
	<li>Add the fixative from one side of the depressed well and not from the top to prevent uncontrolled movement of the dissected organs. Once touching the fixative, the organs will adhere to the glass, and throughout the process they will not move.</li>
	<li>Remove fixative and add 500 &#181;l of hybridization buffer supplemented with 10 pmol of the fluorescent DNA probe Cy5-5&#39;-GGAACATCAGGGCTTCGATA-3&#39;, which is complementary to a sequence in TYLCV coat protein gene.</li>
	<li>Hybridize overnight at room temperature in the dark by incubating the slide with the specimen inside a small humid chamber composed of small plastic box with wet towel paper at the bottom.</li>
	<li>Following the hybridization, pick up the organs with a fine dissection tool and rapidly move them to a fresh microscope slide containing 30 &#181;l of new hybridization buffer supplemented with DAPI (0.1 mg/ml&#160;in 1x PBS) and contained with liquid blocker.</li>
	<li>Cover the specimens with a cover slip, seal with nail polish and view under a fluorescence or confocal microscope.&#160;</li>
</ol>
4. Plant Handling, Hand-sectioning, Fixation, and Hybridization for TYLCV FISH
<ol>
	<li>Inoculate tomato seedlings with TYLCV using viruliferous whitefly-mediated inoculation. The virus can be detected using this FISH method in symptomless plants one week after inoculation. Typical disease symptoms are visible three weeks post inoculation. Use noninfected tomato plants and no probe samples for controls to confirm specific hybridization.</li>
	<li>Use a sharp histological razor blade for preparing hand-cut 2-4 cm long longitudinal or cross sections of tomato stems and leaves.</li>
	<li>In Eppendorf tubes, fix the sections in Carnoy&#8217;s fixative for 2 hr&#160;to overnight at room temperature.</li>
	<li>Remove fixative and preserve the sections in absolute ethanol for several days to several weeks at room temperature, or proceed to the next step.</li>
	<li>Wash the sections three times, 1 min each, in hybridization buffer.</li>
	<li>Hybridize the sections with 500 &#181;l of hybridization buffer supplemented with 10 pmol of the fluorescent oligonucleotide probe Cy3-5&#39;-GGAACATCAGGGCTTCGATA-3&#39; which is complementary to a sequence in TYLCV coat protein gene.</li>
	<li>Wash the sections three times, 1 min each, in hybridization buffer.</li>
	<li>Mount sections whole in hybridization buffer supplemented with DAPI (0.1 mg/ml&#160;in 1x PBS) and contained with liquid blocker, cover with a coverslip, seal with nail polish and view under a fluorescence or confocal microscope.</li>
</ol>

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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. 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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. 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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&#160;and the target DNA or RNA molecule in the cell. The outcome is a specific hybridization, with minimum background, and a signal detected using fluorescence or confocal microscopes. Several probes with different fluorescent dyes, that target more than one gene, can be used simultaneously. This enables visualizing more than one target in a single cell or tissue. The procedures described in these protocols are simple and the sample processing time is minimal. Problems associated with other protocols such as high background signal, long processing time for specimens and the use of numerous and costly materials for the analysis, are not constraints in the protocol described here.
The B and Q biotypes of B. tabaci whiteflies were used to describe the protocols in this manuscript; however, rearing conditions apply to any whitefly biotype, with their respective host plant. The whitefly completes its life cycle in three weeks under the indicated conditions. Each individual of the B biotype population used was infected with the primary endosymbiont Portiera, and the secondary endosymbiotic bacteria Hamiltonella and Rickettsia. The Q biotype individuals were infected with Portiera and Arsenophonus. Other whitefly populations around the world were shown to be infected with these or other endosymbiotic bacterial species, and with different spatial localization patterns in the body16-20.
TYLCV infected plants show typical disease symptoms three weeks following the inoculation. For TYLCV localization, symptomatic plants can be used for virus acquisition by whiteflies for 24 hr and the virus can be detected in infected plants and insects using the FISH protocol described above. While in insects the probes used can be conjugated with many fluorophore which can excised with different wavelengths, including Cy3 and Cy5, in plants Cy3 is the most suitable and does not fluoresce at similar wavelengths as chloroplasts, the most abundant organelles in the plant cell.
Successful implementation of the described protocols for the localization of bacteria and viruses in plants and insects depends on good fixation of the samples for better visualization and probe penetration, good probe design for specificity and clearing with H2O2 in the case of whole insects for better signal visualization. In the case of FISH in plants, hand-made sections must be as thin as possible for good probe penetration and better visualization of the signal. This can be improved by putting the leaf between two Styrofoam pieces to help with cutting and by using professional razor blades which can be purchased for this purpose from several vendors.
The protocol is not suitable for subcellular localization of targets, however it has the potential to be developed, using the probe format describe here, into a suitable method for subcellular localization. For example, the short probes described here can be conjugated to molecules such as biotin and not fluorescent molecules, which in turn can be targeted using gold particles conjugated to streptavidin that can be visualized under TEM. This later modification can be suitable for subcellular localization of several targets including gene transcripts and microorganisms. Finally, the described protocols are suitable for hybridization with nucleic acids such as DNA and RNA but not proteins.
For obtaining the best results in the FISH protocols described here, it is best to use fresh insect and plant material, as well as fresh fixative and hybridization buffers. The probes should always be kept in -20 &#186;C in small aliquots to avoid repeated freezing and thawing. The processing time of each described step can be calibrated depending on the organisms studied. As mentioned in the protocol, following fixation and decolorization, whitefly and plant samples can be preserved for long time in absolute ethanol at room temperature. This step is particularly useful when sending samples for processing from one location to another, or when performing long time course experiments. The quality of the hybridization signal was not affected following this long time preservation.

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.

<table><tbody><tr><td>Fluorescently labeled&amp;nbsp; Probes</td><td>Metabion</td><td>20 bp HPLC purified</td><td>Sequence designed by customer</td></tr><tr><td>Toluidine blue</td><td>Sigma-Aldrich</td><td>89640</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td>Molecular Biology Grade</td></tr><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>F9037</td><td>Molecular Biology Grade</td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T5941</td><td>Molecular Biology Grade</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma-Aldrich</td><td>320099</td><td>Molecular Biology Grade</td></tr><tr><td>Liquid Blocker</td><td>Ted Pella Inc.</td><td>22309</td><td></td></tr></tbody></table>

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, and Figure 4 shows double FISH for Portiera and Rickettsia, both figures in B. tabaci 4th instar nymph. Figure 5 shows FISH for TYLCV and DAPI staining in a whitefly dissected midgut, and Figure 6 shows a FISH for TYLCV and DAPI staining in a dissected whitefly primary salivary glands. Figure 7 shows FISH for TYLCV and DAPI staining in hand-cut TYLCV-infected plant sections.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig1highres.jpg" src="/files/ftp_upload/51030/51030fig1.jpg" /> 
Figure 1.&#160;General overview of the system studied in this manuscript. A) Typical disease symptoms caused by infection with Tomato yellow leaf curl virus (TYLCV). B) External view of an adult whitefly. C) External view of the 4th nymphal stage in the whitefly developmental life cycle. Other nymphal stages are similar in the general external view but smaller in size. D) Schematic view of the internal anatomy of an adult B. tabaci showing the path of TYLCV acquisition from the plant sieve elements, circulation and transmission (black arrows). Red particles refer to TYLCV virions. p: phloem; s: stylet; e: esophagus; fc: filter chamber; dm: descending midgut; am: ascending midgut; ca: caeca; hg: hindgut; bc: bacteriocytes; psg: primary salivary gland. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51030/51030fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig2highres.jpg" src="/files/ftp_upload/51030/51030fig2.jpg" /> 
Figure 2.&#160;Double FISH of Portiera and Arsenphonus on B. tabaci Q biotype adult (A) and zoom on the labeled area (B) both under bright field channel and combined optical sections detecting the fluorescent signals emitted by each probe. Portiera-specific probe (red) conjugated to Cy5 and Arsenphonus-specific probe (yellow) conjugated to Cy3 were used. e: egg. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51030/51030fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig3highres.jpg" src="/files/ftp_upload/51030/51030fig3.jpg" /> 
Figure 3.&#160;Double FISH of Portiera and Hamiltonella on B. tabaci nymph. Portiera-specific probe (red) conjugated to Cy5 and Hamiltonella-specific probe (green) conjugated to Cy3 were used. A) Hamiltonella hybridization signal obtained from combined optical sections and viewed under dark field. B) Portiera hybridization signal obtained from combined optical sections and viewed under dark field. C) Portiera and Hamiltonella merged signals under bright field. D) Portiera and Hamiltonella combined signals viewed under dark field. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51030/51030fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig4highres.jpg" src="/files/ftp_upload/51030/51030fig4.jpg" /> 
Figure 4.&#160;Double FISH of Portiera and Rickettsia on B. tabaci nymph. Portiera-specific probe (red) conjugated to Cy5 and Rickettsia-specific probe (blue) conjugated to Cy3 were used. A) Rickettsia hybridization signal obtained from combined optical sections and viewed under dark field. B) Portiera hybridization signal obtained from combined optical sections and viewed under dark field. C) Portiera and Rickettsia combined signals under bright field. D) Portiera and Rickettsia combined signals under dark field. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51030/51030fig4highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig5highres.jpg" src="/files/ftp_upload/51030/51030fig5.jpg" /> 
Figure 5.&#160;FISH of TYLCV on dissected midgut of an adult B. tabaci female that had acquired the virus for 48 hr from an infected tomato plant. TYLCV-specific probe (red) conjugated to Cy3 was used, and the midgut was stained with DAPI before mounting and visualization. A) The dissected midgut as seen from combined optical sections under bright field. B) TYLCV FISH signal (red) and DAPI stained nuclei (blue) as seen from combined optical sections under dark field. fc: filter chamber; dm: descending midgut; am: ascending midgut; ca: caeca; hg: hindgut. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51030/51030fig5highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig6highres.jpg" src="/files/ftp_upload/51030/51030fig6.jpg" /> 
Figure 6.&#160;FISH of TYLCV on dissected primary salivary gland of an adult B. tabaci female that had acquired the virus for 48 hr from an infected tomato plant. TYLCV-specific probe (red) conjugated to Cy3 was used, and the salivary gland stained with DAPI before mounting and visualization. A) The dissected and DAPI stained primary salivary gland as seen from combined sections under bright field. B) TYLCV FISH signal (red) and DAPI stained nuclei (blue) as seen from combined sections under dark field. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51030/51030fig6highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/51030/51030fig7highres.jpg" src="/files/ftp_upload/51030/51030fig7.jpg" /> 
Figure 7.&#160;FISH of TYLCV on hand-cut longitudinal section through a stem of TYLCV-infected plant. TYLCV-specific probe (red) conjugated to Cy3 was used and combined with DAPI staining before mounting and visualization. A) One section shown with red TYLCV signal in a phloem sieve element, and DAPI stained nuclei seen from combined sections under dark field. B) The same combined sections shown in (A) under bright field for orientation. ph: phloem; xy: xylem. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51030/51030fig7highres.jpg" target="_blank">Click here to view larger image.</a>

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 | Immunology and Infection | Video

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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23.4K Views.  Volcani Center.  We beschrijven hier een eenvoudige fluorescentie in situ hybridisatie (FISH) werkwijze voor de lokalisatie van virussen en bacteriën in insecten en plantenweefsels. Dit protocol kan worden verlengd voor de visualisatie van mRNA in ganse berg en preparaten. 

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

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Neuroscience

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

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.

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. ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

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

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