Abstract
Numerous viruses can be transmitted by their corresponding vector insects; however, the molecular mechanisms enabling virus transmission by vector insects have been poorly understood, especially the identity of vector components interacting with the virus. Here, we used the yeast two-hybrid system to study proteomic interactions of a plant virus (Rice stripe virus, RSV, genus Tenuivirus) with its vector insect, small brown planthopper (Laodelphax striatellus). Sixty-six proteins of L. striatellus that interacted with the nucleocapsid protein (pc3) of RSV were identified. A virus-insect interaction network, constructed for pc3 and 29 protein homologs of Drosophila melanogaster, suggested that nine proteins might directly interact with pc3. Of the 66 proteins, five (atlasin, a novel cuticular protein, jagunal, NAC domain protein, and vitellogenin) were most likely to be involved in viral movement, replication, and transovarial transmission. This work also provides evidence that the novel cuticular protein, CPR1, from L. striatellus is essential for RSV transmission by its vector insect. CPR1 binds the nucleocapsid protein (pc3) of RSV both in vivo and in vitro and colocalizes with RSV in the hemocytes of L. striatellus. Knockdown of CPR1 transcription using RNA interference resulted in a decrease in the concentration of RSV in the hemolymph, salivary glands and in viral transmission efficiency. These data suggest that CPR1 binds RSV in the insect and stabilizes the viral concentration in the hemolymph, perhaps to protect the virus or to help move the virus to the salivary tissues. Our studies provide direct experimental evidence that viruses can use existing vector proteins to aid their survival in the hemolymph. Identifying these putative vector molecules should lead to a better understanding of the interactions between viruses and vector insects.
Highlights
From the ‡State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; §USDA, ARS, Plant Protection Research Unit, Cornell University, Ithaca, NY; ¶State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China; Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
The yeast two-hybrid system, widely used in proteomics research of uncharacterized protein–protein interactions, is conducive to the study of insect proteins involved in viral transmission [16]
Our study focused on Rice stripe virus (RSV) pc3, the nucleocapsid protein, which plays an essential role in the retention or movement of the virus in the insect vector and is a determinant for viral transmission by the insect (39 – 41)
Summary
Insect Rearing—Nonviruliferous and viruliferous L. striatellus were reared on healthy and RSV-infected rice seedlings Full-Length Amplification of the CPR1 Gene, Protein Expression, and Antibody Production—The sequence of the CPR1 fragment identified from the yeast two-hybrid experiment was used to design primers (Table S1) to amplify the full-length gene sequence directly from total RNA extracted from L. striatellus using the standard Trizol reagent protocol (Invitrogen, Carlsbad, CA). Construction of Baculovirus Plasmids and the Transfection of Spodoptera frugiperda 9 Cells—The CPR1 and RSV pc genes amplified with the designed primers (Table S1) were inserted into pDEST8 plasmids and into E. coli DH10Bac cells for transposition into the bacmid, respectively. RNA Interference in L. striatellus—For synthesizing dsRNA, first, the full-length sequences of CPR1 and GFP were amplified by PCR using gene-specific primers with 23 bases of the T7 RNA polymerase promoter added according to the protocol of the T7 RiboMAX.
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