Resistance Evaluation Of RNAi-based Insect-resistant Rice Csu260-16 Against Chilo Suppressalis

Rice is one of the most important food crops.Chilo suppressalis is an important destructive rice pest occurring in major Chinese rice-growing areas,which causes severe economic losses on rice production every year.RNA interference(RNAi)is a biological process in which small RNA(s RNA)molecules sequence-specifically silence gene expression.With the continuous development of RNAi technology,RNAi-based insect-resistant genetically engineered(IRGE)crops provide a promising approach for pest management by suppressing gene expression or translation.The microRNA(miRNA)pathway is one of the main three pathways of RNAi mechanism in insect.Because of its high efficiency and precise target,it is considered to be a new strategy with great potential for control of pests.A miRNA-mediated IRGE rice line expressing endogenous C.suppressalis miRNA Csu-novel-260,showing significant resistance against C.suppressalis,provides an attractive control strategy for C.suppressalis by suppressing the disembodied(dib)gene expression.The efficacy of RNAibased insect-resistant transgenic crops against the target pests should be systematically evaluated,before its large-scale commercial application.In this study,the evaluation of resistance of the transgenic event,Csu260-16,against the target pest,C.suppressalis,was conducted.The results provide a new viewpoint for the application of RNAi-based plants and inspiration for environmental risk assessment.The main findings are as follows:1.Expression level of Csu-novel-260 in transgenic rice.A spatiotemporal expression analysis of Csu-novel-260 in transgenic rice line was performed by qRT-PCR to determine the paddy field pest exposure dose.The qRT-PCR analysis showed that the Csu-novel-260 level in the leaves of transgenic rice Csu260-16 was the highest in the seedling stage,decreased significantly at the tillering stage and booting stage,and increased again at the maturity stage,showing a trend of first decline and then rise.The mean content of Csu-novel-260 in the terminal leaves of transgenic rice was 233.70 ± 68.39fmol/g fresh weight.2.C.suppressalis diet feeding assays,detached rice tissue feeding assays and Csdib expression levels.The stability study of the chemically synthesized Csu-novel-260 agomir in the artificial diet of C.suppressalis found that the expression of Csu-novel-260 decreased significantly at 36 h,so we know that the diet was replaced every 24 h.In diet feeding assays,Csu-novel-260 agomir at 200 fmol/g significantly inhibited C.suppressalis pupation.However,larval survival and pupal weight were not significantly affected.Additionally,the transgenic line significantly decreased Cry1C-resistant and Cry1C-susceptible C.suppressalis larval survival in detached rice tissue feeding assays,indicating that Cry1C-resistant C.suppressalis was not cross-resistant to Csu-novel-260 expressed in miRNA-mediated IRGE rice.The qRT-PCR showed that Csdib expression levels were significantly suppressed when C.suppressalis fed on the transgenic line.A miRNA-mediated transgenic line pyramided with the cry1 C gene,showing diverse modes of action,provides an optimal C.suppressalis resistance management strategy.