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.
