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.
Highlights
Planthoppers (Hemiptera: Fulgoromorpha) and leafhoppers (Hemiptera: Cicadellidae) are the most destructive insect pests of rice in the temperate and tropical regions of East and Southeast Asia
Abundant fluorescent foci appeared in rice yellow stunt virus (RYSV)-infected cells treated with dsGFP (60%) or with Double-stranded RNAs of the 3′ untranslated region (ds3′UTR) (55%) (Figure 6A). These results suggested that the small RNA produced from ds3′UTR did not affect the accumulation of RYSV genomic RNA and that the P6 protein plays an important role in the establishment of the viroplasm
Cultured leafhopper and planthopper cell lines have played an important role in the investigation of insect-transmitted rice viruses, especially reoviruses and rhabdoviruses
Summary
Planthoppers (Hemiptera: Fulgoromorpha) and leafhoppers (Hemiptera: Cicadellidae) are the most destructive insect pests of rice in the temperate and tropical regions of East and Southeast Asia They cause direct detrimental effects on plant growth, such as plant wilting and leaf chlorosis and transmit several rice pathogens, including phytoplasma and plant viruses, that cause enormous economic loss every year (Oya, 1980; Sōgawa, 1982). Rice-infecting rhabdoviruses are transmitted in a persistent manner by leafhoppers (Jackson et al, 2005; Wei and Li, 2016; Yang et al, 2017; Wang et al, 2018b) These viruses possess complex genomes comprising a long negative-strand singlestranded RNA (-ssRNA) and have complicated infection strategies (Dietzgen et al, 2017; Lefkowitz et al, 2018). The limited number of technical innovations in insect systems far has seriously hampered research progress
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