The Interaction Between The Nonstructural Protein Pns10 Of Rice Dwarf Virus And Actin Of Leafhopper

Rice dwarf virus (RDV), a member of genus Phytoreovirus in the family Reoviridae, infects rice plants in Japan, Korea, China and Southeast Asian countries.In natural conditions, RDV is transmitted by Nephotettix cincticeps, Nephotettix apicalis, Nephotettix virescens and Recilia dorsalis in a persistent-propagative manner. N. cincticeps is the major vector with a infection rate of over 50%, R. dorsalis is the minor vector with a infection rate of less than 10%. The mechanism of affinity difference between RDV and N. cincticeps and R. dorsalis is unclear up to now. The nonstructural protein Pns10, a key factor of RDV transmission, encoded by RDV forms virus-containing tubules in the main vector N. cincticeps. The tubules pass through the microvilli of epithelial cells which consist of actin to help virus fulfill the intercellular transport. In order to analysis the mechanism of affinity difference between RDV and N. cincticeps and R. dorsalis, we have to find out the vector factors related to the spread of the virus. We screened first the library of N. cincticeps by yeast two-hybrid technique and the result showed that a number of proteins, such as NADH dehydrogenase and actin, interacted with Pns10. According to sequences of actin gene in NCBI, we designed the primes to amplify the cytoplasmic actin and muscle actin of N. cincticeps. The findings from yeast two-hybrid indicated that Pns10 of RDV didn’t interact with muscle actin of N. dncticeps but interacted with cytoplasmic actin of N. cincticeps. For the sake of detecting whether the specific interaction exist in R. dorsalis, we amplified the cytoplasmic actin and muscle actin of R. dorsalis. The yeast two-hybrid experiments showed that the cytoplasmic of R. dorsalis didn’t interact with Pns10 as well as muscle actin. In addition, the specific interaction only between Pns10 of RDV and cytoplasmic actin of leafhopper was verified by GST pull-down technology.We analysis the amino acid sequence of cytoplasmic actin of N. cincticeps and R. dorsalis and only three amino acids were different between the two proteins. In order to clarify which amino acids played an important role in the interaction, we constructed the cytoplasmic actin mutant of R. dorsalis. After a methionine at codon 262 was instead of a leucine, cytoplasmic actin of R. dorsalis can interact with Pns10 by yeast two-hybrid technique. There were no effects of the interactoins between Pns10 and cytoplasmic actin of R, dorsalis after an isoleucine at codon 11 was instead of a valine and an alanine at codon 348 was instead of a valine.The Pns10 tubules passed though microvilli which consist of cytoplasmic actin in N. cincticeps. In order to clarify whether the interaction between Pns 10 and cytoplasmic actin can impact on the virus transmission by vector or not, we dissected the N. cincticeps and R. dorsalis feed with virus-infected rice plant. The virus non-structural proteins Pns10, Pns 12 and actin was labeled by fluorescein cross-linked antibody. When compared with the R. dorsalis, the infection rate of the virus observed by confocal microscopy was higher in the N. cincticeps. It also showed that a large number of Pns10 tubules can pass through the microvilli in the digestive tract, while only a small amount of Pns10 tubules can pass through the microvilli in the digestive tract of R. dorsalis.In summary, the Pns10 of RDV interacted with cytoplasmic actin of N. cincticeps but didn’t interact with cytoplasmic actin of R. dorsalis. There was an impact on the interaction between Pns10 and cytoplasmic actin by using a leucine to instead of a methionine at codon 262 of R. dorsalis. The capability of Pns10 tubules passing through the microvilli which consisting of cytoplasmic actin was affected because cytoplasmic actin of R, dorsalis didn’t interact with Pns10 of RDV. This is a molecular biological explanation for R. dorsalis as the minor vector in RDV transmission.