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| Interference of the Expression of Non-structural Protein Pns11 of Rice dwarf virus (RDV) Inhibits Viral Replication in Leafhopper (Nephotettix cincticeps) Vector |
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Abstract Rice dwarf virus (RDV) is mainly transmitted by leafhopper (Nephotettix cincticeps) in a persistent-propagative manner. The genome of RDV consists of 12 dsRNA segments, encoding at least 7 structural proteins (P1, P2, P3, P5, P7, P8 and P9) and 5 non-structural proteins (Pns4, Pns6, Pns10, Pns11 and Pns12). Pns11 of RDV is known to be a gene silencing suppressor in plant, and participates in the formation of viroplasm for viral replication and assembly of progeny virions in the cultural cells of N. cincticeps. However, the definite function of Pns11 in viroplasm in insect vector is still unknown yet. In the present study, RNA interference (RNAi) was firstly applied via delivering the synthesized dsRNAs in vitro from Pns11 gene into cultural cells of N. cincticeps. Immuno-fluorescence assay showed that when Pns11 was knocked down, the antigens of Pns11 were significantly reduced, and RDV was restricted within 1~2 cultural cells. The infectivity evaluated with the fluorescent antibody focus counting method showed the level of the viral infection decreased about 5 times. These results indicated that RNAi targeting on Pns11 gene significantly reduced the expression of Pns11, leading the RDV accumulation and infection, was blocked in the cultural cells. Thus, Pns11 was assumed to associate with viral replication. Then, microinjection of dsRNAs from Pns11 into the body of N. cincticeps was performed. qRT-PCR test showed the knockdown of Pns11 significantly decreased the viruliferous rate of insect more than 60%. And the RNAi effect steadily lasted for more than 12 d, which was nearly a circulative transmission period. qRT-PCR assay showed that knockdown of Pns11 caused more than 80% reduction in the relative expression levels of P8 and Pns11 genes. It was suggested that RNAi targeting on Pns11 gene inhibited the viral accumulation in body of N. cincticeps, and decreased the number of viruliferous insect. Immuno-fluorescence assay displayed that at 6 d after microinjection, RDV and Pns11 were detected in the epithelia cells of filter chamber and midgut of N. cincticeps in control, while blocked in 1~2 epithelia cells of filter chamber in treatment of Pns11 knockdown. At 12 d after microinjection, RDV and Pns11 were detected in the whole of intestines and salivary gland in control, while still blocked in 2~3 epithelia cells of filter chamber in the treatment of Pns11 knockdown. These observations indicated that knockdown of Pns11 inhibited the spread of RDV in insect vector via blocking viral replication. Finally, capability of viral transmission by insect was tested. The result showed that when Pns11 was knocked down, insect vectors were unable to transmit RDV in 14 d after microinjection. These results confirmed that Pns11 was a viral replication factor, which was necessary for RDV infection and multiplication. It also verified Pns11 was an effective target to block viral replication and transmission. This study theoretically bases for virus disease control via blocking RDV multiplication and circulation in insect vector.
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Received: 19 June 2015
Published: 05 October 2015
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