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 with a relative humidity of 60-75% under a photoperiod of 16 h light and 8 h dark.</li>
	</ol>
	</li>
	<li>Use an aspirator to gently transfer all the adults from their cage into a new cage that contains fresh rice seedlings each week.
	<ol>
		<li>Let more than 200 adults mate and lay eggs in the rice.</li>
		<li>Retain the old rice seedlings for the nymphs to emerge. Rear these new nonviruliferous nymphs to the 2-instar stage.</li>
	</ol>
	</li>
</ol>
2. Virus acquisition and the collection of viruliferous insects
<ol>
	<li>Carefully transfer the 2-instar nonviruliferous nymphs to a glass culture tube (2.5 cm in diameter by 15 cm high) for 1-2 h for starvation using the aspirator.
	<ol>
		<li>Release the nymphs to a cage that contains an RDV-infected rice plant grown in a pot.</li>
		<li>Allow the nymphs to feed on the infected rice plant for 2 days. 
		NOTE: Carefully water the rice plant and avoid washing away the nymphs. The 2-instar nymphs are approximately 1.6-2 mm long.</li>
	</ol>
	</li>
	<li>Carefully transfer these nymphs to a new cage that contains fresh virus-free rice seedlings with a relative humidity of 60-75% under a photoperiod of 16 h light and 8 h dark. Allow the nymphs to feed on the infected rice plant for 12 days to complete the circulative transmission period of RDV.</li>
</ol>
3. Collection of salivary proteins using a feeding cage
<ol>
	<li>Prepare five small pipe-like feeding cages (2.5 cm in diameter by 4 cm high) in which one end is covered with insect-proof netting.</li>
	<li>Confine 15-20 leafhoppers in each feeding cage, and then cover the other end of the cage with a thin foam mat.
	<ol>
		<li>Fix one rice seedling (5-6 cm high) between the end of cage and a foam mat with tapes. Ensure that the leafhoppers in the feeding cage can feed on the rice seedlings exposed to the interior of the cage.</li>
	</ol>
	</li>
	<li>Immerse the seedling roots in water so that the rice plant will remain alive. Allow the leafhoppers to feed on them for 2 days.</li>
	<li>Remove the leafhoppers from their feeding cages and collect the rice seedlings on which they fed. Cut the parts of seedlings outside of the cage and recover the feeding regions of seedlings. 
	&#8203;NOTE: This sample can be stored at -80 &#176;C for 3 months at the most, if it is not instantly used for detection.</li>
</ol>
4. Reagent preparation
<ol>
	<li>Dissolve 15.1 g of Tris-base, 94 g of glycine, and 5 g of SDS in 1 L of sterile water to prepare 5xTris-glycine buffer. Dilute 200 mL of 5xTris-glycine buffer with 800 mL of sterile water to prepare 1xTris-glycine buffer (see Table 1 for buffer composition).</li>
	<li>Dissolve 80 g of NaCl, 30 g of Tris-base, and 2 g of KCl in 1 L of sterile water to prepare 10xTris-buffered saline (TBS) buffer. Autoclave the solution at 121 &#176;C for 15 min.</li>
	<li>Dissolve 8 g of SDS, 4 mL of &#223;-mercaptoethanol, 0.02 g of bromophenol blue, and 40 mL of glycerol in 40 mL of 0.1 M Tris-HCl (pH 6.8) to prepare 4x protein sample buffer.</li>
	<li>Mix 800 mL Tris-glycine buffer with 200 mL methanol to prepare the transfer buffer.</li>
	<li>Add 100 mL 10xTBS solution and 3 mL Tween 20 to 900 mL sterile water to prepare the TBS buffer with Tween 20 (TBST) solution.</li>
</ol>
5. Western blotting to detect the saliva and viral proteins
<ol>
	<li>Grind 0.1 g of the rice samples with liquid nitrogen until the tissue becomes a powder. Add 200 &#181;L of 4x protein sample buffer to the sample and boil it for 10 min. Centrifuge the samples at 12,000 x g for 10 min at room temperature.
	<ol>
		<li>Remove the supernatant and place it in a new vial. Load 10 &#181;L of the sample into an SDS-PAGE gel, and run it in Tris-glycine buffer at 150 V for 45-60 min. 
		NOTE: The residue after centrifugation can be discarded.</li>
	</ol>
	</li>
	<li>Put a 0.45 &#181;m nitrocellulose membrane and other sandwich supplies in the Transfer buffer for 30 min. 
	NOTE: This step can be done before the gel has completed its run.</li>
	<li>Sandwich the gel and transfer it for 90-120 min at 100 V in the Transfer buffer.</li>
	<li>Take the membrane and place it in 7% non-fat dry milk blocking solution in TBST solution for 20 min. Add the specific antibody against RDV P8 or Vg to a 7% solution of non-fat dry milk in TBST. Incubate with the antibody for staining the membrane for 2 h or overnight.</li>
	<li>Wash the membrane with TBST solution three times, with 5 min washing each time.</li>
	<li>Add the goat anti-rabbit IgG as a secondary antibody to 7% non-fat dry milk with TBST. Incubate with the antibody for 60-90 min at room temperature.</li>
	<li>Wash the membrane with TBST solution three times for 5 min each time.</li>
	<li>Use the ECL Western kit for the chemiluminescent method. Mix Detection Reagents 1 and 2 in the kit at a ratio of 1:1 in a tube. Put the mixed reagent onto the membrane and incubate the blot for 5 min.</li>
	<li>Drain the excess reagent and take a colorimetric picture of the chemiluminescent picture. Combine them to see the ladder with protein bands.</li>
</ol>

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The authors have nothing to disclose.

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 communication14,15,16. Therefore, uncovering more varieties and functions of salivary components will promote understanding the relationship between insects and hosts. Here, a system of detection for salivary proteins in the plant host was provided, which will enable further investigation on the function of salivary protein in plant hosts.
This protocol provides techniques to detect the salivary proteins of leafhoppers with piercing-sucking mouthparts by collecting the feeding plants. Some remarkable points should be noted to obtain the best and reliable results. (1) The viral loading in the infected rice plants is critical. The viral titers in rice plants directly affect the acquisition of insects. When the insect colony is highly viruliferous, the probability of collecting viral proteins in saliva in vitro will be increased. Therefore, the viral proteins released from the saliva to the plant host will be much easier to detect. Choosing infected plants that display significant symptoms is recommended to serve as the source for viruses. (2) Detection timing. It is believed that some of the salivary proteins are transient in the plant host because they are subjected to degradation by the plant host or diluted in the plant. Instant detection of the feeding plants after the 2-day feeding is recommended. It is also hypothesized that a longer retention time would be better for some specific salivary proteins in the plant. Therefore, the detection timing of some salivary proteins could be determined in further studies. (3) Ensure that there are enough replicates. The number of insects that survive will decrease because the confined insects in the feeding cage have limited activity and ability to feed. Using enough replicates will help to enrich the salivary proteins. Three to five replicates are typically enough. If there is only one replicate, it would be better to confine 15-20 leafhoppers to feed on one rice seedling.
These representative results showed the presence of viral protein P8 in the feeding plant of viruliferous leafhoppers. It was revealed that the virus mixed with the flow of saliva is released from the salivary glands. It was then released from the insect to the plant host, ultimately finishing the viral horizontal transmission. However, it is still unknown whether Vg plays the role of elicitor or effector in the process of insect feeding and whether or not it triggers or suppresses the immune response of the plant host. Previously, the saliva of more than 10,000 R. dorsalis was collected via membrane feeding and analyzed using LC-MS/MS (unpublished data). The presence of Vg in the saliva was verified, although its function is unknown. Here, it has been proven that Vg also exists in the saliva of N. cincticeps and is even released to the plant. Combined with the studies on planthoppers16, it is presumed that the presence of Vg in hemipteran saliva is universal. A new finding reports that the Vg of planthopper saliva is an effector to damage the plant defense system34. More studies are required to address whether the Vg in most hemipteran saliva functions as an effector. It is believed that this protocol provides a stable and reliable methodology to examine the presence of salivary proteins secreted by leafhoppers in plants. This protocol is expected to be applicable for the detection of salivary proteins of most hemipterans.

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><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris base</td><td>Roche</td><td>D609K69032</td><td>For 5&amp;times;Tris-glycine buffer and 10&amp;times;TBS buffer preparation</td></tr><tr><td>glycine</td><td>Sigma-Aldrich</td><td>WXBD0677V</td><td>For 5&amp;times;Tris-glycine buffer preparation</td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>SLCB4394</td><td>For 5&amp;times;Tris-glycine buffer preparation</td></tr><tr><td>NaCl</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>10019318</td><td>For 10&amp;times;TBS buffer preparation</td></tr><tr><td>KCl</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>10016318</td><td>For 10&amp;times;TBS buffer preparation</td></tr><tr><td>&amp;szlig;-mercaptoethanol</td><td>Xiya Reagent</td><td>B14492</td><td>For 4&amp;times; protein sample buffer preparation</td></tr><tr><td>bromophenol blue</td><td>Sigma-Aldrich</td><td>SHBL3668</td><td>For 4&amp;times; protein sample buffer preparation</td></tr><tr><td>glycerol</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>10010618</td><td>For 4&amp;times; 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&amp;amp;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>&lt;strong&gt;Buffers and Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Buffer&lt;/strong&gt;</td><td>&lt;strong&gt;Composition&lt;/strong&gt;</td><td></td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&amp;nbsp;5&amp;times;Tris-glycine buffer</td><td>15.1 g Tris base&lt;br/&gt; 94 g glycine&lt;br/&gt; &amp;nbsp;5 g SDS in 1 L sterile water</td><td></td><td>&amp;nbsp;Stock solution</td></tr><tr><td>1&amp;times;Tris-glycine buffer</td><td>200 mL of 5&amp;times;Tris-glycine buffer&lt;br/&gt; 800 mL sterile water</td><td></td><td>Work solution, for SDS-PAGE</td></tr><tr><td>10&amp;times;Tris-buffered saline (TBS) buffer</td><td>80 g NaCl&lt;br/&gt; 30 g Tris base&lt;br/&gt; 2 g KCl&lt;br/&gt; 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&amp;times;TBS solution&lt;br/&gt; 3 mL Tween 20&lt;br/&gt; 900 mL sterile water</td><td></td><td>Work solution</td></tr><tr><td>4&amp;times; protein sample buffer</td><td>8 g SDS&lt;br/&gt; 4 mL &amp;szlig;-mercaptoethanol&lt;br/&gt; 0.02 g bromophenol blue&lt;br/&gt; 40 mL glycerol&lt;br/&gt; 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&lt;br/&gt; 200 mL methanol</td><td></td><td>For protein transfer</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</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 &amp;amp; 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&amp;times;TBS buffer preparation</td></tr><tr><td>Tween 20</td><td>Coolaber SCIENCE&amp;amp;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>&amp;beta;-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 Figure 2 indicates that Vg was released to the plant host as a salivary protein. On the membrane incubated with antibodies against RDV P8, a specific and expected band of approximately 46 kDa was also observed in the samples from rice that had been subjected to feeding and the bodies of viruliferous insect bodies. In contrast, no band was observed in the non-feeding rice sample and the nonviruliferous leafhopper bodies, as shown in Figure 3. This result proved that the viral proteins could also be detected in the feeding plants.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63020/63020fig01.jpg" /> 
Figure 1: Overview of the steps in the detection of salivary proteins. The steps include insect rearing, virus acquisition, salivary proteins collection via plant feeding, and western blotting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63020/63020fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63020/63020fig2v2.jpg" /> 
Figure 2: Western blot assay of Vg in plant and insect samples. Lane 1, non-feeding plants; Lane 2, viruliferous leafhopper-feeding plants; Lane 3, viruliferous leafhopper salivary glands; Lane M, marker. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63020/63020fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63020/63020fig3v2.jpg" /> 
Figure 3: Western blot assay of P8 in plant and insect samples. Lane 1, non-feeding plants; Lane 2, viruliferous leafhopper-feeding plants; Lane 3, viruliferous leafhopper bodies; Lane 4, nonviruliferous leafhopper bodies; M, marker. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63020/63020fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Composition</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>&#160;5x Tris-glycine buffer</td>
			<td>15.1 g Tris base 
			94 g glycine 
			&#160;5 g SDS in 1 L sterile water</td>
			<td>&#160;Stock solution</td>
		</tr>
		<tr>
			<td>1x Tris-glycine buffer</td>
			<td>200 mL of 5x Tris-glycine buffer 
			800 mL sterile water</td>
			<td>Work solution, for SDS-PAGE</td>
		</tr>
		<tr>
			<td>10x Tris-buffered saline (TBS) buffer</td>
			<td>80 g NaCl 
			30 g Tris base 
			2 g KCl 
			in 1 L sterile water</td>
			<td>Stock solution</td>
		</tr>
		<tr>
			<td>TBS with Tween 20 (TBST) solution</td>
			<td>100 mL 10x TBS solution 
			3 mL Tween 20 
			900 mL sterile water</td>
			<td>Work solution</td>
		</tr>
		<tr>
			<td>4x protein sample buffer</td>
			<td>8 g SDS 
			4 mL &#946;-mercaptoethanol 
			0.02 g bromophenol blue 
			40 mL glycerol 
			in 40 mL 0.1 M Tris-HCl (pH 6.8)</td>
			<td>For protein extraction</td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>800 mL Tris-glycine buffer 
			200 mL methanol</td>
			<td>For protein transfer</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers, solutions, and reagents used in the study. The composition of the buffers and the solutions, along with their usage, are listed.

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 ...

detecting-virus-salivary-proteins-leafhopper-vector-plant

Nanyang Technological University

Research

JoVE Journal

Biology

1.6K Views.  Fujian Agriculture and Forestry University. This protocol demonstrates how to use the plant host to detect salivary proteins of leafhopper and plant viral proteins released by leafhopper vectors.

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

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

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

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

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

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Genetics

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

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

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

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

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

Immunology and Infection

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

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

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

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

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

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

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

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

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

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

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

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

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Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

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

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection