Transcriptomic analysis of useful genes applicable for RNAi-based control of the small brown planthopper and rice stripe virus

Abstract

The small brown planthopper (SBPH), Laodelphax striatellus (Fallén), is an economically important phytophagous species in the family of Delphacidae of Hemiptera. It has a wide distribution range from south-east Asia to Siberia and Europe, and attacks several important agricultural crops including rice, corn, wheat, oat and barley. SBPH is one of the most serious pest insects of rice plants because it transmits the rice stripe virus (RSV) in a circulative-propagative manner which typical symptoms include pale and discontinuous yellow stripes, blotches and dead tissue streaks on the leaves. Chemical insecticides have been used to this economic pest for several decades. Buprofezin is an insect growth regulator (IGR) pesticide which is active against larval stages to cause cuticular lesions that result in the disruption of chitin synthesis. Because this insecticide has generally been considered to have a good efficacy against the target pests while being harmless to beneficial insects, it has been used widely in integrated pest management programs especially used in the control of homopteran pests. As buprofezin used to control L. striatellus for more than a decade, the buprofezin resistant occurrence of L. striatellus was reported recently. To survey the responses of SBPH to buprofezin, transcriptome analysis of buprofezin treated SBPH had been investigated. 200ppm buprofezin was exposed to 4th instar SBPH by the dipping method, and extracted total RNA for RNA-seq by Illumina platform. Total of 2 x 26,848,684 and 2 x 27,310,742 of 101 base paired-end raw reads were obtained from buprofezin treated SBPH and control samples, respectively. The cDNA library containing 23,817 contigs was constructed by Trinity de novo assembler and TransDecoder ORF finder. The quality filtered raw reads from experimental and control SBPH were subjected to the library with Bowtie2 and eXpress computer programs to obtain the differential gene expression profile. Following analysis revealed that 170 and 144 contigs were down- and up-regulated over five-folds in the buprofezin treated samples, respectively. GO (gene ontology) enrichment analysis were subjected to study the changes of gene expression profile. Total of 9 GO terms showed differential expression under buprofezin treatment. These transcriptome and gene expression profiling date will provide important information for future studies of molecular biology and physiology of L. striatellus. To develop an alternative pest control strategy, six buprofezin-specific genes selected by differential expressed genes from RNA-seq of buprofezin-treated L. striatellus were synthesized to dsRNAs, and applied to L. striatellus to assess the insecticidal efficacy. Two and three of those dsRNAs showed moderated and substantial insecticidal activity up to 60% of mortality in 7 days post treatment, respectively. These results demonstrated the potential of gene screening strategy for the development of RNAi-based pest management program. RNA interference (RNAi) was not only suggested as a promising strategy for controlling insect pests, but also affects the transmission of plant pathogens of insects. To disturb transmission of the RSV in SBPH by using RNAi pathway, we chose nine genes highly expressed in RSV-viruliferous SBPH by transcriptome sequencing. These SBPH-derived dsRNAs were applied to the insects indirectly through xylem of rice leaves by irrigation. qPCR result demonstrated that three out of eight SBPH-derived dsRNAs successfully reduced the replication of RSV in viruliferous SBPH in dose-dependent manner, suggesting that these three dsRNAs could suppress replication of RSV and provide a new tool for RSV control strategy. In summary, an effective high-throughput NGS method largely broadens the target selection of specific RNAi genes from the non-model pest L.striatellus. It may lead to new strategy in designing the RNAi-based technology against SBPH damage and rice stripe virus transmission by L.striatellus.