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>
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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. 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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 ...

This protocol provides a technique for detecting the salivary proteins of leafhoppers and plant hosts to further investigate the function of these proteins in the plant hosts. This technique is easy to perform and the system is stable and replicable. This method could also be helpful in the detection of salivary proteins of other hemiptera and plant hosts.To begin, rear the adult leafhoppers on rice seedlings in a 40 by 35 by 20 centimeter cube cage. Keep one side of the cage covered with an insect-proof net for ventilation. Place the cages with the leafhoppers in an incubator containing an inbuilt humidity controller at 26 degrees Celsius with a relative humidity of 60 to 75%under a photo period of 16 hours light and eight hours dark.Once a week, use an aspirator to gently transfer all adults from their cage into a new cage containing fresh rice seedlings. Allow more than 200 adults to mate and lay eggs in the rice. Retain the old rice seedlings for the nymphs to emerge and rear these new non-viruliferous nymphs to the second instar stage.Using the aspirator, carefully transfer the second instar non-viruliferous nymphs to a glass culture tube for one to two hours for starvation. Then release the nymphs into a cage containing a rice dwarf virus infected rice plant grown in a pot and allow the nymphs to feed on the infected rice plant for two days. After two days, carefully transfer these nymphs to a new cage that contains fresh virus-free rice seedlings, and allow the nymphs to feed on the virus-free rice seedlings for 12 days to complete the circulative transmission period of the rice dwarf virus.To collect the salivary proteins, prepare five small pipe-like feeding cages covered with insect-proof netting on one end. After confining 15 to 20 leafhoppers in each feeding cage, cover the other end of the cage with a thin foam mat. Fix one rice seedling between the end of the cage and a foam mat with tapes, ensuring that the leafhoppers in the feeding cage can feed on the rice seedlings exposed to the interior of the cage.Immerse the seedling roots in water so that the rice plant will remain alive, and allow the leafhoppers to feed on them for two days. After two days, remove the leafhoppers from their feeding cages and collect the rice seedlings on which the leafhoppers fed. Cut the seedlings outside the cage and recover the feeding regions of the seedlings.Prepare 1x Tris-Glycine buffer by diluting 200 milliliters of the prepared 5x buffer with 800 milliliters of sterile water. Then prepare 10x TBS buffer and autoclave the solution at 121 degrees Celsius for 15 minutes. Next, prepare the 4x protein sample buffer.Then prepare the transfer buffer by mixing 800 milliliters of the Tris-Glycine buffer with 200 milliliters of methanol. Finally, prepare the TBST solution by adding 100 milliliters of 10x TBS and three milliliters of Tween 20 to 900 milliliters of sterile water. To detect the salivary and viral proteins, grind 0.1 grams of the rice samples with liquid nitrogen until the tissue becomes a powder.Then add 200 microliters of the 4x protein sample buffer to the sample and boil for 10 minutes. Centrifuge the samples and transfer the supernatant into a new vial. Load 10 microliters of the sample onto an SDS-PAGE gel and run it in Tris-Glycine buffer at 150 volts for 45 to 60 minutes.While the gel is running, place a 0.45 micron nitrocellulose membrane and other sandwich supplies in the transfer buffer for 30 minutes. After the run, sandwich the gel and transfer it for 90 to 120 minutes at 100 volts in the transfer buffer. After the blotting is complete, place the membrane in a 7%non-fat dry milk blocking solution in TBST and incubate for 20 minutes.After that, add an antibody against the rice dwarf virus P8 or vitellogenin and incubate the membrane for two hours or overnight. After the incubation, wash the membrane with TBST three times with a five minute incubation between each wash. Then add Goat anti-Rabbit IgG as a secondary antibody and incubate the membrane for 60 to 90 minutes.Use the ECL Western kit for chemiluminescent detection in mixed detection reagents one and two in the kit at a ratio of one-to-one. Put the membrane onto the mixed reagent and incubate the blot for five minutes. Drain the excess reagent and take a chemiluminescent and colorimetric picture of the membrane.Combine the pictures to see the latter with protein bands. Western blot analysis for detection of vitellogenin showed specific and expected bands of approximately 220 kilodaltons in feeding rice plants and salivary glands of the insects. In contrast, no band was observed in the non-feeding plants, indicating that vitellogenin was released in the plant host as a salivary protein.Western blot analysis for detection of the rice dwarf virus P8 protein showed a specific and expected band of approximately 46 kilodaltons in feeding plants and viruliferous insect bodies. In contrast, no band was observed in non-feeding plants and non-viruliferous leafhopper bodies, proving that the viral proteins could also be detected in the feeding plants. The viral loading in the leafhoppers, the detection timing, and replicates should be taken into account while performing this protocol.This method can also be applied for studying mosquitoes that transmit arboviruses.

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