Exploration of an Actin Promoter-Based Transient Expression Vector to Trace the Cellular Localization of Nucleorhabdovirus Proteins in Leafhopper Cultured Cells

Abstract

Continuously cultured cell lines derived from planthopper and leafhopper have greatly facilitated the investigation of rice viruses transmitted by these insects. However, the lack of a suitable transient expression vector has limited their utility. Here, by cloning and analyzing the promoter sequence of the gene encoding cytoplasmic actin from the leafhopper Nephotettix cincticeps, we successfully developed the first efficient transient expression vector for cultured leafhopper cells, which can also be used to express exogenous proteins in other insect culture cell lines, including those derived from Recilia dorsalis leafhopper and Spodoptera frugiperda (Sf9). Furthermore, insertion of the Hr5 viral enhancer element and knockdown of the endogenous Dicer2 gene notably improved the vector’s expression efficiency in leafhopper cells. Using the optimized vector, we have for the first time traced the cellular localization of the proteins encoded by rice yellow stunt virus (RYSV) in cells of its insect vector and demonstrated that P6 protein is a component of the viroplasm.

Study on Differential Protein Expression in Natural Selenium-Enriched and Non-Selenium-Enriched Rice Based on iTRAQ Quantitative Proteomics

“…The annotation analysis revealed a down-regulated protein, sucrose synthase (SUS), with the highest proteinic expression. Sucrose synthase is the key enzyme for plant sucrose metabolism [39] and controls nitrogen-fixing biosynthesis and abiotic stress reaction processes [40,41]. It is designated that the glucose metabolism of synthetase in natural Se-enriched rice was better than that in non-Se-enriched rice.…”

“…It is designated that the glucose metabolism of synthetase in natural Se-enriched rice was better than that in non-Se-enriched rice. In addition, there were two tubulins and one actin, which was all down-regulated proteins [41,42] that played key roles in maintaining cell shape, movement, and signaling. Hence, signaling pathways and cell movement features of Se-enriched rice were better than non-Se-enriched rice.…”

A Review of Vector-Borne Rice Viruses

“…For example, RDV P2, protruding from the surface of the outer shell of virions, is essential for virions entry into insect vector cells in a receptor-mediated, clathrin-dependent endocytosis manner [ 151 , 152 , 153 , 154 ]. Nonstructural proteins such as RBSDV P9-1 [ 155 , 156 ], RDV Pns12 [ 157 ], RGDV Pns9 [ 158 ], RRSV Pns10 [ 77 , 159 ], and RYSV P6 [ 160 ] contribute to the assembly of viroplasm for viral genome replication and progeny virion assembly. After entry and replication, viruses counter two main physical barriers in the insect vectors for spread: midgut barriers and salivary gland barriers.…”