Name: immunofluorescent labeling of plant virus and insect vector proteins in hemipteran guts
Uploaded: 2021-05-14T15:00:00
Description: Watch this Scientific Journal Video about Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts at JoVE.com

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 ...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteroptera+as+vectors+of+plant+pathogens.">Heteroptera as vectors of plant pathogens.</a> Neotropical Entomology. 88 (3), 519-545 (2004).</li><li>Gautam, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-virus+interactions+in+a+plant+host+and+in+a+hemipteran+vector:+Implications+for+vector+fitness+and+virus+epidemics.">Virus-virus interactions in a plant host and in a hemipteran vector: Implications for vector fitness and virus epidemics.</a> Virus Research. 286, 198069 (2020).</li><li>Ghanim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+mechanisms+and+components+that+determine+the+transmission+efficiency+of+Tomato+yellow+leaf+curl+virus+(Geminiviridae;+Begomovirus)+by+its+whitefly+vector.">A review of the mechanisms and components that determine the transmission efficiency of Tomato yellow leaf curl virus (Geminiviridae; Begomovirus) by its whitefly vector.</a> Virus Research. 186, 47-54 (2014).</li><li>Hogenhout, S. 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U., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+on+the+transmission+mechanism+of+tenuiviruses+by+their+vector+insects.">New insights on the transmission mechanism of tenuiviruses by their vector insects.</a> Current Opinion in Virology. 33, 13-17 (2018).</li><li>Qin, F., 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=Invasion+of+midgut+epithelial+cells+by+a+persistently+transmitted+virus+is+mediated+by+sugar+transporter+in+its+insect+vector.">Invasion of midgut epithelial cells by a persistently transmitted virus is mediated by sugar transporter in its insect vector.</a> PLOS Pathogens. 14, 1007201 (2018).</li><li>Coons, A. H., Creech, H. J., Jones, R. N., Berliner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+demonstration+of+pneumococcal+antigen+in+tissues+by+the+use+of+fluorescent+antibody.">The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody.</a> Journal of Immunology. 45, 159-170 (1942).</li><li>Barnard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+fluorescence+immunoassays.">The development of fluorescence immunoassays.</a> Progress in Clinical and Biological Research. 285, 15-37 (1988).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+c-Jun+N-terminal+kinase+pathway+of+a+vector+insect+is+activated+by+virus+capsid+protein+and+promotes+viral+replication.">The c-Jun N-terminal kinase pathway of a vector insect is activated by virus capsid protein and promotes viral replication.</a> eLife. 6, 26591 (2017).</li><li>Huo, 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=Insect+tissue-specific+vitellogenin+facilitates+transmission+of+plant+virus.">Insect tissue-specific vitellogenin facilitates transmission of plant virus.</a> PLoS Pathogens. 14 (2), 1006909 (2018).</li><li>Zhang, 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=TurboID-Based+proximity+labeling+for+in+planta+identification+of+protein-protein+interaction+networks.">TurboID-Based proximity labeling for in planta identification of protein-protein interaction networks.</a> Journal of Visualized Experiments: JoVE. (159), e60728 (2020).</li><li>Than, W., Qin, F. L., Liu, W. W., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Sogatella+furcifera+proteome+that+interact+with+P10+protein+of+southern+rice+black-streaked+dwarf+virus.">Analysis of Sogatella furcifera proteome that interact with P10 protein of southern rice black-streaked dwarf virus.</a> Scientific Reports. 6, 32445 (2016).</li><li>Pu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+characteristics+of+Southern+rice+black-streaked+dwarf+virus+by+rice+planthoppers.">Transmission characteristics of Southern rice black-streaked dwarf virus by rice planthoppers.</a> Crop Protection. 41, 71-76 (2012).</li><li>Jia, D., Chen, H., Mao, Q., Liu, Q., Wei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+of+viral+dissemination+from+the+midgut+determines+incompetence+of+small+brown+planthopper+as+a+vector+of+southern+rice+black-streaked+dwarf+virus.">Restriction of viral dissemination from the midgut determines incompetence of small brown planthopper as a vector of southern rice black-streaked dwarf virus.</a> Virus Research. 167, 404-408 (2012).</li><li>Zhang, X., Zhang, L., Liu, W., Li, L., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+application+of+the+antibodies+of+Sogatella+furcifera+VAMP7+and+Vti1a+proteins+in+expressed+in+Escherichia+coli.">Preparation and application of the antibodies of Sogatella furcifera VAMP7 and Vti1a proteins in expressed in Escherichia coli.</a> Plant Protection. 47, 55-60 (2021).</li><li>Ammar, E. D., Nault, L. R., Rodriquez, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Internal morphology and ultrastructure of leafhoppers and planthoppers. , 1 (1985).</li><li>Tsai, J., Perrier, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+the+digestive+and+reproductive+systems+of+Dalbulus+maidis+and+Graminella+nigrifrons+(Homoptera:+Cicadellidae).">Morphology of the digestive and reproductive systems of Dalbulus maidis and Graminella nigrifrons (Homoptera: Cicadellidae).</a> Fla Entomology. 79, 563 (1996).</li><li>Wei, T., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rice+reoviruses+in+insect+vectors.">Rice reoviruses in insect vectors.</a> Annual Review of Phytopathology. 54, 99-120 (2016).</li><li>Kruse, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining'omics+and+microscopy+to+visualize+interactions+between+the+Asian+citrus+psyllid+vector+and+the+Huanglongbing+pathogen+Candidatus+Liberibacter+asiaticus+in+the+insect+gut.">Combining'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut.</a> PLoS ONE. 12, 0179531 (2017).</li><li>Koga, R., Tsuchida, T., Fukatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quenching+autofluorescence+of+insect+tissues+for+in+situ+detection+of+endosymbionts.">Quenching autofluorescence of insect tissues for in situ detection of endosymbionts.</a> Applied Entomology and Zoology. 44, 281-291 (2009).</li><li>King, R. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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>&#160;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 ...

This protocol can be used to study various movement in insects, functions of insect proteins, and interactions between virus and vector insect in vivo. This method is efficient and informative. The structures of the insect gut and the auxiliary cells are clearly visualized when viewed with the laser scanning confocal microscopy.Demonstrating the procedure will be Lu Zhang, PhD from our laboratory. To begin, transfer non-viruliferous insects from glass beakers onto fresh southern rice black streaked dwarf virus, SRBSDV, infected rice plants covered with an insect proof net for a two-day virus acquisition access period. Then collect the insects in glass beakers containing fresh rice seedlings.After two days, collect the insects from the glass beakers using a manual aspirator for dissection and excision of the gut. Prepare 0.01 molar PBS, 4%paraformaldehyde, and 2%Triton X-100 as described in the text manuscript. Use a pipetter to place 100 microliters of PBS on a glass slide and place the slide on the stage of an optical microscope.Use manual aspirator to collect the SRBSDV infected adults from the glass beakers and place them in a 1.5 milliliter tube on ice to paralyze the insects. Transfer a paralyzed adult into the 100 microliters of PBS on the slide with the abdomen up. Use tweezers to clamp the body and remove the head with another set of tweezers.With one set of tweezers, clamp the sides of the abdomen and clamp the ovipositor or the copulatory organ of the tail with the other set, then carefully pull the intersegmental membrane of one abdominal segment to slowly expose the gut in the abdomen. 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.Place excised guts into a 200 microliter centrifuge tube. Add 200 microliters of PBS to the tube and use a pipette to thoroughly wash the guts. Use a pipetter to place 100 microliters of PBS on a glass slide and place the slide on the stage of an optical microscope.Use manual aspirator to collect the SRBSDV infected nymphs from glass beakers and place them in 1.5 milliliter tube placed on ice to paralyze the insects. Transfer a paralyzed nymph into the 100 microliters of buffer on the slide with the abdomen facing up. Detach the tail of the nymph, then clamp the insect body to fix it gently and use the other pair 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. With the tweezers still clamping the body, use the other pair to move the head carefully and gradually pull out the gut. Detach the gut from the head to obtain an intact gut without damaging the body.Place the excised guts into a tube. Add PBS to the tube and gently suck release the solution with a pipette to wash the guts thoroughly. Prepare the antibodies and mounting medium as described in the text manuscript.Place the freshly excised and PBS washed WBPH guts in 100 microliters of 4%paraformaldehyde in a 200 microliter centrifuge tube at room temperature. After two hours, replace the paraformaldehyde solution with 200 microliters of PBS. After 10 minutes, remove the PBS to eliminate any paraformaldehyde and repeat the PBS wash step twice.After two PBS washes, add 200 microliters of non-ionic detergent Triton X-100 to permeabilize the samples at room temperature. After 30 minutes, remove the Triton X-100 and wash away any remaining detergent with three 10-minute washes with PBS. Dilute the labeled antibodies one to 50 with 50 microliters of bull serum albumin.Add the diluted antibodies to the tube and incubate the samples overnight at four degrees Celsius. On the morning after removing the antibody, wash away the remaining antibody diluent with three 10-minute washes with PBS. Dilute one microliter of DyLight 633-Phalloidin with 50 microliters of PBS and add 50 microliters of diluted phalloidin to the tube for a two-hour incubation at room temperature.After removing phalloidin, thoroughly wash away the remaining phalloidin with three 10-minute washes with PBS. Place a drop of mounting medium containing DAPI on a microscope slide. Transfer the guts to the medium and gently place the cover glass over the sample without creating bubbles.The representative laser scanning confocal micrograph of excised WBPH guts from adults after labeling with phalloidin showed three sections:the foregut, midgut, and hindgut. Among these, midgut is the initial infection site of SRBSDV. The monolayer epithelial cell structure of the gut facilitates the study of the cellular localization of insect proteins and colocalization of viral and insect proteins.Excised WBPH guts with DyLight 488 labeled anti-VAMP-7 and SRBSDV virions with DyLight 549 labeled anti-SRBSDV antibody are shown here. VAMP-7 and SRBSDV virions were shown to co-localize in the cytoplasm suggesting that VAMP-7 may play a role in virus transmission in vivo. Permeabilized guts should be cleaned thoroughly after incubation with luciferin conjugated antibodies to reduce the background and non-specific binding.This is a reliable method to view the localization of virus, other pathogens, and insect proteins in insects. It provides the foundation for further studies of the relationship between pathogens and insects.

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Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

This protocol aims to visualize heterochromatin aggregates in Drosophila polytene cells.

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.