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

Summary

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

Abstract

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.

Introduction

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 saliva^1,2,3,4. 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 others^{5,6,7,8,9,10,11,12,13}. 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 responses^14,15,16,17. 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 autophagy^18,19. 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 manner^22,23. RDV was first reported to be transmitted by an insect vector and causes a severe disease of rice in Asia^24,25. 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 days^{26,27,28,29,30}. 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 insects^31,32,33. 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 (<50 kDa) fragments before entering the ovary^18,19. 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.

Protocol

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

1. 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.
   1. Keep the cages with leafhoppers in an incubator that contains an in-built humidity controller at 26 °C with a relative humidity of 60-75% under a photoperiod of 16 h light and 8 h dark.
2. Use an aspirator to gently transfer all the adults from their cage into a new cage that contains fresh rice seedlings each week.
   1. Let more than 200 adults mate and lay eggs in the rice.
   2. Retain the old rice seedlings for the nymphs to emerge. Rear these new nonviruliferous nymphs to the 2-instar stage.

2. Virus acquisition and the collection of viruliferous insects

1. 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.
   1. Release the nymphs to a cage that contains an RDV-infected rice plant grown in a pot.
   2. 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.
2. 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.

3. Collection of salivary proteins using a feeding cage

1. 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.
2. Confine 15-20 leafhoppers in each feeding cage, and then cover the other end of the cage with a thin foam mat.
   1. 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.
3. Immerse the seedling roots in water so that the rice plant will remain alive. Allow the leafhoppers to feed on them for 2 days.
4. 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.
   ​NOTE: This sample can be stored at -80 °C for 3 months at the most, if it is not instantly used for detection.

4. Reagent preparation

1. 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).
2. 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 °C for 15 min.
3. Dissolve 8 g of SDS, 4 mL of ß-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.
4. Mix 800 mL Tris-glycine buffer with 200 mL methanol to prepare the transfer buffer.
5. 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.

5. Western blotting to detect the saliva and viral proteins

1. Grind 0.1 g of the rice samples with liquid nitrogen until the tissue becomes a powder. Add 200 µL 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.
   1. Remove the supernatant and place it in a new vial. Load 10 µL 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.
2. Put a 0.45 µm 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.
3. Sandwich the gel and transfer it for 90-120 min at 100 V in the Transfer buffer.
4. 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.
5. Wash the membrane with TBST solution three times, with 5 min washing each time.
6. 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.
7. Wash the membrane with TBST solution three times for 5 min each time.
8. 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.
9. Drain the excess reagent and take a colorimetric picture of the chemiluminescent picture. Combine them to see the ladder with protein bands.

Representative Results

Figure 1 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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Buffer	Composition	Comments/Description
5x Tris-glycine buffer	15.1 g Tris base 94 g glycine  5 g SDS in 1 L sterile water	Stock solution
1x Tris-glycine buffer	200 mL of 5x Tris-glycine buffer 800 mL sterile water	Work solution, for SDS-PAGE
10x Tris-buffered saline (TBS) buffer	80 g NaCl 30 g Tris base 2 g KCl in 1 L sterile water	Stock solution
TBS with Tween 20 (TBST) solution	100 mL 10x TBS solution 3 mL Tween 20 900 mL sterile water	Work solution
4x protein sample buffer	8 g SDS 4 mL β-mercaptoethanol 0.02 g bromophenol blue 40 mL glycerol in 40 mL 0.1 M Tris-HCl (pH 6.8)	For protein extraction
Transfer buffer	800 mL Tris-glycine buffer 200 mL methanol	For protein transfer

Table 1: Buffers, solutions, and reagents used in the study. The composition of the buffers and the solutions, along with their usage, are listed.

Discussion

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' cross-kingdom biological factors into the hosts^1,3,4. The cross-kingdom biological factors, including elicitors, effectors, and small RNA, are critical for insect-host communication^14,15,16. 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.

Materials

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