Tubules of plant reoviruses exploit tropomodulin to regulate actin-based tubule motility in insect vector

Qian Chen,Linghua Zhang,Taiyun Wei,Yanshuang Zhang,Qianzhuo Mao

doi:10.1038/srep38563

Abstract

Plant reoviruses are known to exploit virion-packaging tubules formed by virus-encoding non-structural proteins for viral spread in insect vectors. Tubules are propelled by actin-based tubule motility (ABTM) to overcome membrane or tissue barriers in insect vectors. To further understand which insect factors mediate ABTM, we utilized yeast two-hybrid and bimolecular fluorescence complementation assays to test interactions between tubule protein Pns10 of rice dwarf virus (RDV), a plant reovirus, and proteins of its insect vector, the leafhopper Nephotettix cincticeps. Tropomodulin (Tmod), vitellogenin, and lipophorin precursor of N. cincticep displayed positive and strong interaction with Pns10, and actin-associated protein Tmod interacted with Pns10 in pull-down assay and the co-immunoprecipitation system. Further, we determined Pns10 tubules associated with Tmod in cultured cells and midgut of N. cincticep. The expression dynamic of Tmod was consistent with that of Pns10 and the fluctuation of RDV accumulation. Knockdown of Tmod inhibited the Pns10 expression and viral accumulation, thus decreasing the viruliferous rates of leafhopper. These results suggested that Tmod was involved in viral spread by directly interacting with Pns10 tubules, finally promoting RDV infection. This study provided direct evidence of plant reoviruses utilizing an actin-associated protein to manipulate ABTM in insect vectors, thus facilitating viral spread.

Highlights

Subsequently enter the epithelial cells via endocytosis[11]
This study investigated the molecular mechanisms involved in the spread of rice dwarf virus (RDV) Pns[10] tubules, by applying various molecular techniques such as yeast two-hybrid assay (YTH), bimolecular fluorescence complementation (BiFC), pull-down assay, the co-immunoprecipitation (Co-IP) and RNA interference (RNAi)
Mating yeast containing bait pGBKT7-Pns[10] and positive prey plasmid resulted in reporter gene activation, caused the colonies turn blue on Quadruple dropout media (QDO) plates containing X-α-Gal

Summary

Introduction

Subsequently enter the epithelial cells via endocytosis[11]. Replication of the virus is initiated after the nonstructural proteins Pns[6], Pns[11] and Pns[12] aggregate together to form the viroplasm matrix[12]. In the initially infected epithelial cells of the filter chamber, virus-containing Pns[10] tubules pass through actin-based microvilli into the lumen and extend to neighboring cells[2,6,16]. The RDV isolate deficient in Pns[10] protein expression fails to be transmitted by the leafhopper vector[17], suggesting Pns[10] to be responsible for viral spread and transmission by the insect vector. Recent studies suggest that the interaction between Pns[10] and cytoplasmic actin of efficient vector N. cincticeps is correlated with insect vector specificity[18], suggesting that Pns[10] tubule is capable of employing components from its vector to overcome membrane or tissue barriers for viral transmission. An actin-associated protein was identified to strongly interact with Pns[10], and served as a positive regulator for the spread of Pns[10] tubules

Methods

Results

Conclusion