Name: detecting virus and salivary proteins of a leafhopper vector in the plant host
Uploaded: 2021-09-14T14:00:00
Description: Watch this Scientific Journal Video about Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host at JoVE.com

This work was supported by grants from the National Natural Science Foundation of China (31772124 and 31972239) and Fujian Agriculture and Forestry University (Grant KSYLX014).

The non-viruliferous adult leafhoppers were propagated in the Vector-borne Virus Research Center in Fujian Agriculture and Forestry University, China.
1. Nonviruliferous insect rearing
<ol>
	<li>Rear the adults on rice seedlings in a cube cage that is 40 cm x 35 cm x 20 cm (length x width x height). Keep one side of the cage covered with an insect-proof net for ventilation.
	<ol>
		<li>Keep the cages with leafhoppers in an incubator that contains an in-built humidity controller at 26 &#176;C...

<ol>
	<li>Hogenhout, S. A., Ammar el, D., Whitfield, A. E., Redinbaugh, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+vector+interactions+with+persistently+transmitted+viruses.">Insect vector interactions with persistently transmitted viruses.</a> Annual Review of Phytopathology. 46, 327-359 (2008).</li><li>Cranston, P. S., Gullan, P. J., Resh, V. H., Carde, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+of+insects.">Phylogeny of insects.</a> Encyclopedia of Insects. , (2003).</li><li>Ammar el, D., Tsai, C. W., Whitfield, A. E., Redinbaugh, M. G., Hogenhout, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+aspects+of+rhabdovirus+interactions+with+insect+and+plant+hosts.">Cellular and molecular aspects of rhabdovirus interactions with insect and plant hosts.</a> Annual Review of Entomology. 54, 447-468 (2009).</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>Hattori, M., Konishi, H., Tamura, Y., Konno, K., Sogawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laccase-type+phenoloxidase+in+salivary+glands+and+watery+saliva+of+the+green+rice+leafhopper,+Nephotettix+cincticeps.">Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps.</a> Journal of Insect Physiology. 51 (12), 1359-1365 (2005).</li><li>Ma, R., Reese, J. C., William, I. V., Bramel-Cox, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+pectinesterase+and+polygalacturonase+from+salivary+secretions+of+living+greenbugs,+schizaphis+graminum+(Homoptera:+aphididae).">Detection of pectinesterase and polygalacturonase from salivary secretions of living greenbugs, schizaphis graminum (Homoptera: aphididae).</a> Journal of Insect Physiology. 36 (7), 507-512 (1990).</li><li>Miles, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+aspects+of+the+chemical+relation+between+the+rose+aphid+and+rose+buds.">Dynamic aspects of the chemical relation between the rose aphid and rose buds.</a> Entomologia Experimentalis et Applicata. 37 (2), 129-135 (2011).</li><li>Urbanska, A., Tjallingii, W. F., Dixon, A., Leszczynski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenol+oxidising+enzymes+in+the+grain+aphid's+saliva.">Phenol oxidising enzymes in the grain aphid's saliva.</a> Entomologia Experimentalis et Applicata. 86 (2), 197-203 (1998).</li><li>Miles, P. W., Peng, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+salivary+physiology+of+plant+bugs:+detoxification+of+phytochemicals+by+the+salivary+peroxidase+of+aphids.">Studies on the salivary physiology of plant bugs: detoxification of phytochemicals by the salivary peroxidase of aphids.</a> Journal of Insect Physiology. 35 (11), 865-872 (1989).</li><li>Will, T., van Bel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+interactions+between+aphids+and+plants.">Physical and chemical interactions between aphids and plants.</a> Journal of Experimental Botany. 57 (4), 729-737 (2006).</li><li>Ma, R. Z., Reese, J. C., Black, W. C., Bramel-Cox, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+loss+in+a+greenbug-susceptible+sorghum+due+to+pectinases+and+pectin+fragments.">Chlorophyll loss in a greenbug-susceptible sorghum due to pectinases and pectin fragments.</a> Journal of the Kansas Entomological Society. 71 (1), 51-60 (1998).</li><li>Madhusudhan, V. V., Miles, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+salivary+components+as+a+possible+reason+for+differences+in+the+responses+of+alfalfa+to+the+spotted+alfalfa+aphid+and+pea+aphid.">Mobility of salivary components as a possible reason for differences in the responses of alfalfa to the spotted alfalfa aphid and pea aphid.</a> Entomologia Experimentalis et Applicata. 86 (1), 25-39 (1998).</li><li>Funk, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alkaline+phosphatase+activity+in+whitefly+salivary+glands+and+saliva.">Alkaline phosphatase activity in whitefly salivary glands and saliva.</a> Archives of Insect Biochemistry &amp; Physiology. 46 (4), 165-174 (2010).</li><li>Hogenhout, S. A., Bos, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+proteins+that+modulate+plant-insect+interactions.">Effector proteins that modulate plant-insect interactions.</a> Current Opinion in Plant Biology. 14 (4), 422-428 (2011).</li><li>Tomkins, M., Kliot, A., Maree, A. F., Hogenhout, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-layered+mechanistic+modelling+approach+to+understand+how+effector+genes+extend+beyond+phytoplasma+to+modulate+plant+hosts,+insect+vectors+and+the+environment.">A multi-layered mechanistic modelling approach to understand how effector genes extend beyond phytoplasma to modulate plant hosts, insect vectors and the environment.</a> Current Opinion in Plant Biology. 44, 39-48 (2018).</li><li>Huang, H. J., Lu, J. B., Li, Q., Bao, Y. Y., Zhang, C. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+transcriptomic/proteomic+analysis+of+salivary+gland+and+secreted+saliva+in+three+planthopper+species.">Combined transcriptomic/proteomic analysis of salivary gland and secreted saliva in three planthopper species.</a> Journal of Proteomics. , (2018).</li><li>Hogenhout, S. A., Bos, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+proteins+that+modulate+plant--insect+interactions.">Effector proteins that modulate plant--insect interactions.</a> Current Opinion in Plant Biology. 14 (4), 422-428 (2011).</li><li>Sun, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mosquito+salivary+protein+promotes+flavivirus+transmission+by+activation+of+autophagy.">A mosquito salivary protein promotes flavivirus transmission by activation of autophagy.</a> Nature Communications. 11 (1), 260 (2020).</li><li>Sri-In, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+salivary+protein+of+Aedes+aegypti+promotes+dengue-2+virus+replication+and+transmission.">A salivary protein of Aedes aegypti promotes dengue-2 virus replication and transmission.</a> Insect Biochemistry and Molecular Biology. 111, 103181 (2019).</li><li>Conway, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aedes+aegypti+D7+saliva+protein+inhibits+dengue+virus+infection.">Aedes aegypti D7 saliva protein inhibits dengue virus infection.</a> Plos Neglected Tropical Diseases. 10 (9), 0004941 (2016).</li><li>Wang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whitefly+effector+Bsp9+targets+host+immunity+regulator+WRKY33+to+promote+performance.">A whitefly effector Bsp9 targets host immunity regulator WRKY33 to promote performance.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 374 (1767), 20180313 (2019).</li><li>Omura, T., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+outer+capsid+proteins+in+transmission+of+phytoreovirus+by+insect+vectors.">Role of outer capsid proteins in transmission of phytoreovirus by insect vectors.</a> Advances in Virus Research. 54, 15-43 (1999).</li><li>Chen, Q., Liu, Y., Long, Z., Yang, H., 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=Viral+release+threshold+in+the+salivary+gland+of+leafhopper+vector+mediates+the+intermittent+transmission+of+rice+dwarf+virus.">Viral release threshold in the salivary gland of leafhopper vector mediates the intermittent transmission of rice dwarf virus.</a> Frontiers in Microbiology. 12, 639445 (2021).</li><li>Fukushi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+studies+on+the+dwarf+disease+of+rice+plant.">Further studies on the dwarf disease of rice plant.</a> Journal of the Faculty of Agriculture, Hokkaido Imperial University. 45 (3), 83-154 (1940).</li><li>Miyazaki, N., 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+functional+organization+of+the+internal+components+of+rice+dwarf+virus.">The functional organization of the internal components of rice dwarf virus.</a> Journal of Biochemistry. 147, 843-850 (2010).</li><li>Wei, T., Shimizu, T., Hagiwara, K., Kikuchi, A., Omura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pns12+protein+of+rice+dwarf+virus+is+essential+for+formation+of+viroplasms+and+nucleation+of+viral-assembly+complexes.">Pns12 protein of rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes.</a> Journal of General Virology. 87, 429-438 (2006).</li><li>Chen, Q., Zhang, L., Chen, H., Xie, L., 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=Nonstructural+protein+Pns4+of+rice+dwarf+virus+is+essential+for+viral+infection+in+its+insect+vector.">Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector.</a> Virology Journal. 12, 211 (2015).</li><li>Chen, Q., 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=Nonstructural+protein+Pns12+of+rice+dwarf+virus+is+a+principal+regulator+for+viral+replication+and+infection+in+its+insect+vector.">Nonstructural protein Pns12 of rice dwarf virus is a principal regulator for viral replication and infection in its insect vector.</a> Virus Research. 210, 54-61 (2015).</li><li>Chen, Q., Zhang, L., Zhang, Y., Mao, 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=Tubules+of+plant+reoviruses+exploit+tropomodulin+to+regulate+actin-based+tubule+motility+in+insect+vector.">Tubules of plant reoviruses exploit tropomodulin to regulate actin-based tubule motility in insect vector.</a> Scientific Reports. 7, 38563 (2017).</li><li>Wei, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spread+of+Rice+dwarf+virus+among+cells+of+its+insect+vector+exploits+virus-induced+tubular+structures.">The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures.</a> Journal of Virology. 80 (17), 8593-8602 (2006).</li><li>Mao, Q., 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+bacterial+symbiont-mediated+vitellogenin+uptake+into+oocytes+to+support+egg+development.">Insect bacterial symbiont-mediated vitellogenin uptake into oocytes to support egg development.</a> mBio. 11 (6), 01142 (2020).</li><li>Tufail, M., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characteristics+of+insect+vitellogenins.">Molecular characteristics of insect vitellogenins.</a> Journal of Insect Physiology. 54 (12), 1447-1458 (2008).</li><li>Sappington, T. W., Raikhel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characteristics+of+insect+vitellogenins+and+vitellogenin+receptors.">Molecular characteristics of insect vitellogenins and vitellogenin receptors.</a> Insect Biochemistry and Molecular Biology. 28 (5-6), 277-300 (1998).</li><li>Ji, R., 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=Vitellogenin+from+planthopper+oral+secretion+acts+as+a+novel+effector+to+impair+plant+defenses.">Vitellogenin from planthopper oral secretion acts as a novel effector to impair plant defenses.</a> New Phytologist. , (2021).</li></ol>

The saliva directly secreted by the salivary glands of the piercing-sucking insects plays a pivotal role because it predigests and detoxifies the host tissues and vectors&#39; cross-kingdom biological factors into the hosts1,3,4. The cross-kingdom biological factors, including elicitors, effectors, and small RNA, are critical for insect-host communication14,15,</sup...

The vector-host transmission mode of arboviruses, a fundamental problem, is at the frontier of biological science. Many plant viruses of agricultural importance are horizontally transmitted by insect vectors1. More than one-half of plant viruses are vectored by hemipteran insects, including aphids, whiteflies, leafhoppers, planthoppers, and thrips. These insects have distinct features that enable them to efficiently transmit plant viruses1. They possess piercing-sucking mouthparts and feed on the sap from phloem and xylem, and secrete their saliva1,2,3,4. With the development and improvement of techniques, the identification and functional analyses of salivary components are becoming a new focus of intensive research. The known salivary proteins in saliva include numerous enzymes, such as pectinesterase, cellulase, peroxidase, alkaline phosphatase, polyphenol oxidase, and sucrase, among others5,6,7,8,9,10,11,12,13. The proteins in saliva also include elicitors that trigger the host defense response, thereby altering the performance of insects, and effectors that suppress the host defense, which enhances insect fitness and components that induce host pathological responses14,15,16,17. Therefore, saliva proteins are vital materials for communication between insects and hosts. During the transmission of viruses, the saliva secreted by the salivary glands of piercing-sucking viruliferous insects also contains viral proteins. Viral components utilize the flow of saliva to release them from the insect to the plant host. Therefore, the insect saliva bridges the virus-vector-host tritrophic interaction. Investigating the biological function of saliva proteins secreted by viruliferous insects helps to understand the relationship of virus-vector-host.
For animal viruses, it is reported that the saliva of mosquitoes mediates the transmission and pathogenicity of West Nile virus (WNV) and Dengue virus (DENV). The saliva protein AaSG34 promotes dengue-2 virus replication and transmission, while the saliva protein AaVA-1 promotes DENV and Zika virus (ZIKV) transmission by activating autophagy18,19. The saliva protein D7 of mosquitoes can inhibit DENV infection in vitro and in vivo via direct interaction with the DENV virions and recombinant DENV envelope protein20. In plant viruses, the begomovirus tomato yellow leaf curl virus (TYLCV) induces the whitefly salivary protein Bsp9, which suppresses the WRKY33-mediated immunity of plant host, to increase the preference and performance of whiteflies, eventually increasing the transmission of viruses21. Because studies of the role that insect salivary proteins play in plant hosts have lagged behind those of animal hosts, a stable and reliable system to detect the salivary proteins in plant hosts is urgently required.
The plant virus known as rice dwarf virus (RDV) is transmitted by the leafhopper Nephotettix cincticeps (Hemiptera: Cicadellidae) with high efficiency and in a persistently propagative manner22,23. RDV was first reported to be transmitted by an insect vector and causes a severe disease of rice in Asia24,25. The virion is icosahedral and double-layered spherical, and the outer layer contains the P8 outer capsid protein22. The circulative transmission period of RDV in N. cincticeps is 14 days26,27,28,29,30. When the RDV arrives at salivary glands, virions are released into saliva-stored cavities in the salivary glands via an exocytosis-like mechanism23. The vitellogenin (Vg) is the yolk protein precursor essential for oocyte development in female insects31,32,33. Most insect species have at least one Vg transcript of 6-7 kb, which encodes a precursor protein of approximately 220 kDa. The protein precursors of Vg can usually be cleaved into large (140 to 190 kDa) and small (&#60;50 kDa) fragments before entering the ovary18,19. Previous proteomic analysis revealed the presence of the peptides derived from Vg in the secreted saliva of the leafhopper Recilia dorsalis, although their function is unknown (unpublished data). It is newly reported that Vg, which is orally secreted from planthoppers, functions as an effector to damage the defenses of plants34. It is unknown whether the Vg of N. cincticeps could also be released to the plant host with salivary flow, and then could play a role in the plant to interfere with plant defenses. To address whether N. cincticeps exploits salivary proteins, such as Vg, to inhibit or activate plant defenses, the first step is identifying proteins released to the plant during feeding. Understanding the method to identify the salivary proteins present in the plant is potentially essential to explain the function of saliva proteins and the interactions between Hemiptera and plants.
In the protocol presented here, N. cincticeps is used as an example to provide a method to examine the presence of salivary proteins in the plant host introduced through insect feeding. The protocol primarily details the collection and detection of salivary proteins and is helpful for further investigation on most hemipterans.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roche</td>
			<td>D609K69032</td>
			<td>For 5&#215;Tris-glycine buffer and 10&#215;TBS buffer preparation</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Sigma-Aldrich</td>
			<td>WXBD0677V</td>
			<td>For 5&#215;Tris-glycine buffer preparation</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>SLCB4394</td>
			<td>For 5&#215;Tris-glycine buffer preparation</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019318</td>
			<td>For 10&#215;TBS buffer preparation</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10016318</td>
			<td>For 10&#215;TBS buffer preparation</td>
		</tr>
		<tr>
			<td>&#223;-mercaptoethanol</td>
			<td>Xiya Reagent</td>
			<td>B14492</td>
			<td>For 4&#215; protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>SHBL3668</td>
			<td>For 4&#215; protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010618</td>
			<td>For 4&#215; protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10014118</td>
			<td>For transfer buffer preparation</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Coolaber SCIENCE&#38;TeCHNoLoGY</td>
			<td>CT30111220</td>
			<td>For TBST preparation</td>
		</tr>
		<tr>
			<td>non-fat dry milk</td>
			<td>Becton.Dickinso and company</td>
			<td>252038</td>
			<td>For membrane blocking, antibodies dilution</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG</td>
			<td>Sangon Biotech</td>
			<td>D110058-0001</td>
			<td>Recognization of the primary andtibody</td>
		</tr>
		<tr>
			<td>ECL Western kit</td>
			<td>ThermoFisher Scientific</td>
			<td>32209</td>
			<td>Chemiluminescent substrate</td>
		</tr>
		<tr>
			<td>nitrocellulose membrane</td>
			<td>Pall Corporation</td>
			<td>25312915</td>
			<td>For proteins transfer</td>
		</tr>
		<tr>
			<td>Buffers and Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td>Composition</td>
			<td></td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>&#160;5&#215;Tris-glycine buffer</td>
			<td>15.1 g Tris base 
			94 g glycine 
			&#160;5 g SDS in 1 L sterile water</td>
			<td></td>
			<td>&#160;Stock solution</td>
		</tr>
		<tr>
			<td>1&#215;Tris-glycine buffer</td>
			<td>200 mL of 5&#215;Tris-glycine buffer 
			800 mL sterile water</td>
			<td></td>
			<td>Work solution, for SDS-PAGE</td>
		</tr>
		<tr>
			<td>10&#215;Tris-buffered saline (TBS) buffer</td>
			<td>80 g NaCl 
			30 g Tris base 
			2 g KCl 
			in 1 L sterile water</td>
			<td></td>
			<td>Stock solution</td>
		</tr>
		<tr>
			<td>TBS with Tween 20 (TBST) solution</td>
			<td>100 mL 10&#215;TBS solution 
			3 mL Tween 20 
			900 mL sterile water</td>
			<td></td>
			<td>Work solution</td>
		</tr>
		<tr>
			<td>4&#215; protein sample buffer</td>
			<td>8 g SDS 
			4 mL &#223;-mercaptoethanol 
			0.02 g bromophenol blue 
			40 mL glycerol 
			in 40 mL 0.1 M Tris-HCl (pH 6.8)</td>
			<td></td>
			<td>For protein extraction</td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>800 mL Tris-glycine buffer 
			200 mL methanol</td>
			<td></td>
			<td>For protein transfer</td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>SHBL3668</td>
			<td>For 4x protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>Constant temperature incubator</td>
			<td>Ningbo Saifu Experimental Instrument Co., Ltd.</td>
			<td>PRX-1200B</td>
			<td>For rearing leafhoppers</td>
		</tr>
		<tr>
			<td>Electrophoresis</td>
			<td>Tanon Science &#38; Technology Co.,Ltd.</td>
			<td>Tanon EP300</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>Electrophoretic transfer core module</td>
			<td>BIO-RAD</td>
			<td>1703935</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010618</td>
			<td>For 4x protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Sigma-Aldrich</td>
			<td>WXBD0677V</td>
			<td>For 5x Tris-glycine buffer preparation</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG</td>
			<td>Sangon Biotech</td>
			<td>D110058-0001</td>
			<td>Recognization of the primary andtibody</td>
		</tr>
		<tr>
			<td>High-pass tissue grinding instrument</td>
			<td>Shanghai Jingxin Industrial Development Co., Ltd.</td>
			<td>JXFSIPRP-24</td>
			<td>For grinding plant tissues</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10016318</td>
			<td>For 10x TBS buffer preparation</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10014118</td>
			<td>For transfer buffer preparation</td>
		</tr>
		<tr>
			<td>Mini wet heat transfer trough</td>
			<td>BIO-RAD</td>
			<td>1703930</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019318</td>
			<td>For 10x TBS buffer preparation</td>
		</tr>
		<tr>
			<td>nitrocellulose membrane</td>
			<td>Pall Corporation</td>
			<td>25312915</td>
			<td>For proteins transfer</td>
		</tr>
		<tr>
			<td>non-fat dry milk</td>
			<td>Becton.Dickinso and company</td>
			<td>252038</td>
			<td>For membrane blocking, antibodies dilution</td>
		</tr>
		<tr>
			<td>Pierce ECL Western kit</td>
			<td>ThermoFisher Scientific</td>
			<td>32209</td>
			<td>Chemiluminescent substrate</td>
		</tr>
		<tr>
			<td>Protein color instrument</td>
			<td>GE Healthcare bio-sciences AB</td>
			<td>Amersham lmager 600</td>
			<td>For detecting proteins</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>SLCB4394</td>
			<td>For 5x Tris-glycine buffer preparation</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roche</td>
			<td>D609K69032</td>
			<td>For 5x Tris-glycine buffer and 10&#215;TBS buffer preparation</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Coolaber SCIENCE&#38;TeCHNoLoGY</td>
			<td>CT30111220</td>
			<td>For TBST preparation</td>
		</tr>
		<tr>
			<td>Vertical plate electrophoresis tank</td>
			<td>BIO-RAD</td>
			<td>1658001</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Shanghai Jinghong Experimental equipment Co., Ltd.</td>
			<td>XMTD-8222</td>
			<td>For boil the protein samples</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Xiya Reagent</td>
			<td>B14492</td>
			<td>For 4x protein sample buffer preparation</td>
		</tr>
	</tbody>
</table>

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.

Figure 1&#160;illustrates all of the steps in this protocol: insect rearing, virus acquisition, the collection of salivary proteins via rice feeding, and the western blot. The western blots results showed that specific and expected bands of approximately 220 kDa were observed in the samples of feeding rice and salivary glands of insects on the membrane incubated with antibodies against Vg. In contrast, no band was observed in the non-feeding rice sample. The result in </stro...

Watch this Scientific Journal Video about Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host at JoVE.com

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

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 ...

Research

JoVE Journal

Biology

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

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

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 ...

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 ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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 ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...