This work was supported by grants from the National Natural Science Foundation of China (31630058 to X.W. and 31772134 to W.L.).

1. Nonviruliferous insect rearing
<ol>
	<li>Collect WBPHs from rice fields and rear with rice seedlings in 1 L glass beakers covered with insect-proof net in an incubator at 28 &#176;C with 16 h light and 8 h dark. Because SRBSDV is not transmitted via eggs, newly hatched nymphs are not viruliferous.</li>
	<li>With a brush pen, gently brush insects from the beaker rearing insects into a new beaker of fresh rice seedlings each week until WBPH nymphs have hatched. Continue rearing these hatched nonviruliferous nymphs to 2- or 3-instar. 
	&#8203;NOTE: Brush carefully to avoid WBPHs flying from the beaker or damaging them.</li>
</ol>
2. Virus acquisition and collection of viruliferous insects
<ol>
	<li>Transfer nonviruliferous insects from the glass beakers onto fresh SRBSDV-infected rice plants covered with an insect-proof net for a 2 d virus-acquisition access period (AAP) by feeding on plants. Then, collect the insects in glass beakers containing fresh rice seedlings.</li>
	<li>After 2 d, collect the insects from glass beakers with a manual aspirator for dissection and excision of the gut. 
	&#8203;NOTE:The minimum AAP of SRBSDV is 5 min for both WBPH nymphs and adults, but the insects should be allowed to feed on fresh SRBSDV-infected rice plants for 2 d to achieve the acquisition efficiency of up to 80%.</li>
</ol>
3. Reagent preparation
<ol>
	<li>Dissolve 8.5 g of NaCl, 3.5 g of Na2HPO4&#183;12H2O, and 0.25 g of NaH2PO4 in 1 mL of ddH2O to prepare 0.01 M solution of phosphate-buffered saline (PBS).</li>
	<li>Add 4 g of paraformaldehyde to 100 mL of PBS to prepare 4% (m/v) paraformaldehyde in PBS.</li>
	<li>Add 2 mL of Triton X-100 into 98 mL of PBS to prepare 2% (v/v) Triton X-100.</li>
</ol>
4. Dissection of adults and excision of guts
<ol>
	<li>Use a pipettor to place 100 &#181;L of PBS on a glass slide. Place the slide on the stage of an optical microscope.</li>
	<li>Collect the SRBSDV-infected adults from glass beakers with a manual aspirator and place them in 1.5 mL tubes. Place the tubes on ice to paralyze insects, and then transfer a paralyzed adult into the 100 &#181;L of PBS on the slide with the abdomen up. 
	NOTE: The insects will be thoroughly paralyzed after 5 min on ice.</li>
	<li>Use tweezers in one hand to clamp the body, and then remove the head with another set of tweezers in the other hand.</li>
	<li>Clamp the sides of the abdomen with one set of tweezers and clamp the ovipositor or the copulatory organ of the tail with the other set. Then pull away the intersegmental membrane of one abdominal segment carefully and slowly expose the gut in the abdomen.</li>
	<li>Continue tearing away the membrane and gradually pull out the complete gut from the abdomen. Gently pull off the tail, which is connected to the end of the gut, to remove the complete gut. 
	NOTE: Pull very carefully or the gut will be damaged.</li>
	<li>Place excised guts into a 200 &#181;L centrifuge tube, add 200 &#181;L of PBS to the tube, and gently suck-release the solution with a pipette to wash the guts thoroughly.</li>
</ol>
5. Dissection of nymphs and excision of guts
NOTE: Nymph bodies are more fragile than adult bodies, and the gut is easily damaged when pulled from the tail. Therefore, the most reliable method to excise the nymph gut is by pulling from the head.
<ol>
	<li>Use a pipettor to place 100 &#181;L of PBS on a glass slide. Place the slide on the stage of an optical microscope.</li>
	<li>Collect the SRBSDV-infected nymphs from glass beakers with a manual aspirator and place them into 1.5 mL tubes. Place the tubes on ice to paralyze insects. Then, transfer a paralyzed nymph into the 100 &#181;L of buffer on the slide with the abdomen facing up.</li>
	<li>Use tweezers to detach the tail of the nymph. Then clamp the insect body to fix gently and use the other set to clamp the head. Gently pull the head away from the body while still maintaining its attachment to the gut so that the head is detached from the body, but the gut is still attached to the thorax and abdomen. 
	NOTE: After the head is detached, the corpus adiposum of the nymph will flow out, making the PBS turbid. Remove the turbid PBS solution and change with 100 &#181;L of fresh PBS.</li>
	<li>With the tweezers still clamping the body, use the other set to move the head carefully, and gradually pull out the gut.</li>
	<li>Gently detach the gut from the head with tweezers, and eventually obtain an intact gut without damaging the body of the planthopper.</li>
	<li>Place excised guts into a 200 &#181;L centrifuge tube, add 200 &#181;L of PBS to the tube, and gently suck-release the solution with a pipette to wash the guts thoroughly. 
	&#8203;NOTE: The excised guts should be cleaned well with PBS to remove any contaminating fat bodies from the abdominal cavity; they can interfere with the staining protocol.</li>
</ol>
6. Labeling protocols for SRBSDV virions and an insect protein
<ol>
	<li>Prepare the antibodies and mounting medium used in this assay. Antibodies are anti-SRBSDV antibody labeled with Dylight 549 (red) against SRBSDV virions18, anti-VAMP7 antibody labeled with Dylight 488 (green) against the insect protein VAMP716,19, Dylight 633 phalloidin (blue), and mounting medium containing 4,6-diamidino-2-phenylindole (DAPI, blue).</li>
	<li>Place the freshly excised and PBS-washed WBPH guts immediately in 100 &#181;L of 4% (m/v) paraformaldehyde in a 200 &#181;L centrifuge tube and hold for 2 h at room temperature. 
	NOTE: The freshly excised WBPH guts should not be soaked in PBS for a longer duration, or the epithelial cells will be damaged.</li>
	<li>Remove 4% (m/v) paraformaldehyde with a pipettor, and then add 200 &#181;L of PBS into the 200 &#181;L centrifuge tube. After 10 min, remove PBS using a pipettor to eliminate any paraformaldehyde.</li>
	<li>Repeat this PBS wash step twice.</li>
	<li>Remove the PBS and add 200 &#181;L of nonionic detergent Triton X-100 (2%, v/v). Permeabilize the samples in the nonionic detergent for 30 min at room temperature.</li>
	<li>Remove 2% (v/v) Triton X-100 with a pipettor, and then wash away any remaining detergent with three 10 min washes with 200 &#181;L of PBS (see steps 6.3 and 6.4).</li>
	<li>Dilute anti-SRBSDV antibody labeled by Dylight 549 (red) and anti-VAMP7 antibody labeled by Dylight 488 (green) 1:50 with 50 &#181;L of bull serum albumin (3%, m/v).</li>
	<li>Add the diluted antibodies to the tube and incubate samples overnight at 4 &#176;C.</li>
	<li>Remove the antibody diluent with a pipettor, and then wash away the remaining antibody diluent with three 10 min washes with 200 &#181;L of PBS.</li>
	<li>Dilute 1 &#181;L of Dylight 633 phalloidin with 50 &#181;L of PBS.</li>
	<li>Add 50 &#181;L of diluted phalloidin to the tube and incubate samples for 2 h at room temperature.</li>
	<li>Remove the phalloidin diluent with a pipettor, and then wash away the remaining phalloidin with three 10 min washes with 200 &#181;L of PBS. 
	NOTE: Thorough washes are critical to reduce the background and nonspecific binding.</li>
	<li>Place a drop of mounting medium containing DAPI on a microscope slide and transfer the guts to the medium. 
	NOTE: Gently unfold each gut with tweezers and avoid creating bubbles. There are about 15 guts per slide.</li>
	<li>Gently place a coverglass over the samples without creating bubbles. 
	NOTE: The slide should be held at 4 &#176;C in the dark to inhibit fluorescence quenching before observation with a laser scanning confocal microscope.</li>
	<li>View all samples with a laser scanning confocal microscope. Capture the images using blue light and save the files on a computer.</li>
</ol>

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The authors have nothing to disclose.

For best results, a few key points should be considered. First, a high ratio of viruliferous insects among the total population is necessary. Although the minimum AAP for SRBSDV by WBPH nymphs and adults is 5 min17, the insects should be allowed to feed on fresh SRBSDV-infected rice plants for 2 d to achieve an acquisition efficiency of up to 80%. Since the SRBSDV virions can be detected in 80% of the midguts18, we excised and labeled the viruliferous insects at 2 d after a...

Most described plant viruses are transmitted by insects from the order Hemiptera that includes aphids, whiteflies, leafhoppers, planthoppers, and thrips1,2. The piercing-sucking mouthparts of hemipteran insects pierce the plant tissue for feeding and secreting saliva, concomitantly efficiently transmitting the virus2. Different transmission mechanisms of plant viruses by vector insects have been described. These include nonpersistent, semipersistent and persistent. The persistent type is either non-propagative or propagative3,4, but for both of these types, the transmitted virus must move throughout the body of the insect. In the persistent-propagative mode, viruses initially infect and replicate in the epithelial cells of the insect&#39;s gut, then disseminate into different tissues, and eventually into the salivary glands, from where they can then be introduced into a plant through the saliva during insect feeding5,6. Persistent transmitted viruses move through different organs and replicate in their insect vectors, which requires specific interactions between virus and vector components at different stages7,8.
Viral proteins and insect proteins must interact to facilitate critical processes for virus recognition, infection, replication, or dissemination in the vector insects9,10. Although optical microscopy can be used to observe cellular structures in insects, it cannot show virion distribution, cellular localization or colocalization of viral proteins and insect protein, or the ultrastructure of insect tissues and cells. Immunofluorescence labeling was first performed by Coons et al. in the phagocytic cells of the mouse by means of labeling specific fluorescein antibodies, and now it is used widely11. The immunofluorescence technique, also known as the fluorescence antibody technique, is one of the earliest immunological labeling techniques developed and is based on the specific binding reaction between the antigen and the antibody11,12. The known antibody is first labeled with fluorescein, which is used as a probe to detect the corresponding antigens in the cells or tissues13,14. After the fluorescein-labeled antibody binds to the corresponding antigen in cells or tissues, the probe will emit bright fluorescence when irradiated with excitation wavelengths and viewed with a fluorescence microscope to localize the antigen15.
Most vector insects of plant viruses are hemipterans. A higher population density of vector insects that have a high transmission efficiency for the plant virus can lead to virus epidemics5. Southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), one of the most serious pathogens of rice, has rapidly spread throughout rice-growing areas in East and Southeast Asia, and caused serious yield losses since 201016,17. Adults and nymphs of the white backed planthopper (WBPH, Sogatella furcifera Horv&#225;th) transmit SRBSDV to rice in a persistent-propagative manner with high efficiency.Field studies have shown that outbreaks of SRBSDV-induced rice black streaked dwarf disease usually coincide with mass long-distance migration of WBPHs, a crucial factor in SRBSDV epidemics7,8,18. Vesicle-associated membrane protein 7 (VAMP7) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), which can mediate the transport of substances via vesicle fusion. VAMP7 interacts with the outer major capsid protein of SRBSDV in vitro, which indicates that VAMP7 might be closely associated with virus transmission16.
In the protocol presented here, we excised the gut from viruliferous WBPH as an example to label SRBSDV virions and VAMP7 in midgut epithelial cells16. As the initial invasion site of virus, the midgut epithelium plays vital roles in virus infection, replication, and transmission. First, we detailed the steps to excise the gut from nymphs and adults of&#160;WBPHs. Second, we used specific fluorescein-labeled antibodies to label SRBSDV virions and VAMP7 in gut epithelial cells. Then we observed epithelial cells and the cellular location of the virions and VAMP7 via a laser scanning confocal microscope. The results showed that SRBSDV virions and VAMP7 could colocalize in the cytoplasm of the midgut epithelial cells, suggesting that the specific function of VAMP7 might be related to dissemination of virions from midgut epithelial cells.

<table><tbody><tr><td>3% Bull serum albumin (BSA)</td><td>Coolaber</td><td>SL1331</td><td>Dilute antibodies</td></tr><tr><td>Cover glass</td><td>Solarbio</td><td>YA0771-18*18mm</td><td>For slide making</td></tr><tr><td>Dissecting microscope</td><td>Beitja</td><td>XTL-7045B1</td><td>For insect dissection</td></tr><tr><td>Laser scanning confocal microscope</td><td>Zeiss</td><td>Zeiss LSM880</td><td>Observe fluorescence signal</td></tr><tr><td>Microscope slides</td><td>Solarbio</td><td>ZBP-7105</td><td>For slide making</td></tr><tr><td>Mounting medium with 4&#039;6-diamidino-2-phenylindole (DAPI)</td><td>Abcam</td><td>AB104139</td><td>Label cell necleus</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>For tissues fixation</td></tr><tr><td>Phalloidin</td><td>&amp;nbsp;Invitrogen</td><td>A22284</td><td>Label actin of midgut epithiels</td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>0290C484</td><td>For tissues permeation</td></tr><tr><td>Tweezers (5-SA)</td><td>AsOne</td><td>6-7905-40</td><td>For insect dissection</td></tr></tbody></table>

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.

Figure 1&#160;illustrates all steps in this protocol: insect rearing, virus acquisition, excision of the gut, immunofluorescent labeling, and making the slide.
Excised WBPH guts from adults were fixed in 4% (m/v) paraformaldehyde, permeabilized with 2% (v/v) Triton X-100, and then incubated with Dylight 633 phalloidin10,18. The laser scanning confocal micrograph in Figure 2 sh...

Watch this Scientific Journal Video about Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts at JoVE.com

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

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 ...

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1.6K Views.  Chinese Academy of Agricultural Sciences. Dit protocol kan worden gebruikt om verschillende bewegingen in insecten, functies van insecteneiwitten en interacties tussen virus en vectorinsect in vivo te bestuderen. Deze methode is efficiënt en informatief. De structuren van de insectendarm en de hulpcellen worden duidelijk gevisualiseerd wanneer ze worden bekeken met de laserscannende confocale microscopie.Lu Zhang, PhD van ons laboratorium, zal de procedure demonstreren. Om te beginnen, breng niet-virulifere insecten over van ...

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

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Immunology and Infection

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Developmental Biology

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

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.

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

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. ...

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. ...

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 ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran insects that have piercing-sucking mouthparts. During viral transmission, the insect saliva bridges the virus-vector-host because the saliva vectors viruses, and the insect proteins, trigger or suppress the immune response of plants from insects into plant hosts. The identification and functional analyses of salivary proteins are becoming a new area of focus in the research field of arbovirus-host interactions. This protocol provides a system to detect proteins in the saliva of leafhoppers using the plant host. The leafhopper vector Nephotettix cincticeps infected with rice dwarf virus (RDV) serves as an example. The vitellogenin and major outer capsid protein P8 of RDV vectored by the saliva of N. cincticeps can be detected simultaneously in the rice plant that N. cincticeps feeds on. This method is applicable for testing the salivary proteins that are transiently retained in the plant host after insect feeding. It is believed that this system of detection will benefit the study of hemipteran-virus-plant or hemipteran-plant interactions.

This protocol demonstrates how to use the plant host to detect salivary proteins of leafhopper and plant viral proteins released by leafhopper vectors.

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran ...

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

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