Off-Target Effect Of DsRNA And Feasibility For Its Application In Control Of Rice Plant-Hoppers

RNA interference based pest control has developed as a new modern plant protection technology.The application of RNAi in pest control has significant advantages and broad application prospects.However,its practical application still faces many problems,such as the needsof selecting effective application ways for different pests and the suitable functional genes as effective targets in order to improve the control effect of dsRNA.It is also necessary to study the occurrence rules of off-target effect of RNAi,so that the impact on non-target organisms could be minimized and the pest control spectrum of dsRNA could be expanded.Therefore,in this study the chitin synthase gene（CHS）,which is only found in fungi and arthropodsand has been proved to have good lethal interference effect,was taken as RNAi target gene,rice plant-hoppers as a pest complex were selected as the target pests,and the technical problems related to dsRNA control of rice plant-hoppers were studied by performing gene interference experiments and simulated field application experiments.Firstly,the off-target effect of dsRNA was verified.Then the effective application method of dsRNA against rice planthopper was screened.On these bases,the feasibility of using dsRNA off-target effect to expand pest control spectrum was explored.Now the results are summarized as follows:The chitin synthase genes（CHS）in different insects were searched online.After homology analysis,the relatively conserved CHS1 was selected as the target gene for RNAi,which has been studied as the target gene in various insect RNAi experiments.First,the homologous genes of CHS1 from Nilaparvata lugens（NlCHS1）,Laodelphgax striatellus（LsCHS1）,Sogatella furcifera（Horváth）（SfCHS1）and Tribolium castaneum（TcCHS1）were cloned and verified.Based on base sequence alignment,a highly conserved segment of 113bp was selected as the dsRNA target site,and the sequence identities of target site between NlCHS1 and others were calculated to be 93%,93%and 83%,respectively.According to the reported off-target occurrence rules,it predicted that dsRNA（dsNlCHS1）synthesized with the sequence of NlCHS1 target site as templet could produce off-target RNAi effect in all the other three insect species.Then,T7 promoter was used to synthesize the corresponding dsNlCHS1.To verify the off-target rule,dsNlCHS1 was used to inject T.castaneum the insect with lowest gene identity for triggering off-target RNAi,and dsTcCHS1 was also synthesized and used as the positive control to trigger target RNAi.The results showed that dsNlCHS1could effectively interfere with the expression of homologous genes in T.castaneum.The off-target knockdown efficiency was 88.1%,slightly lower than the targeted knockdown efficiency of dsTcCHS1,which was 93.0%.Further observation of the phenotypical lethal effect found that dsTcCHS1 targeted interference caused 95.5%mortality in 10 days after treatment,and correspondingly the dsNlCHS1 off-target interfere lead to only 59.1%mortality.Discussions came to the conclusions,these results were consistent with the reported dsRNA off-target law,and indicated that the expected off-target effect of dsRNA could be achieved by selecting the conserved sequences of homologous genes of pests,but the efficiency of off-target interference was lower than targeted interference.The dsRNA targeting the chitin synthase gene of L.striatellus（dsLsCHS1）and N.lugens（dsNlCHS1）were synthesized by the same method above,and field trail imitations were performed to screen the efficient method for control of pests with dsRNA.Considering dsRNA are water-soluble macromolecules and easy to be degraded in natural water and soil environment,two feasible methodsspraying and seedling dipping were selected in this study to compare their application efficiency.The results showed that the survival rate of N.lugens and L.striatellus treated either by dsRNA spraying or by seedling dipping all decreased gradually after treatments.Ten days later,the mortality of N.lugens and L.striatellus treated by spraying with 100 ng/μL targeted dsRNA were calculated as 54.7%and 52.8%,respectively.Whereas the mortality of N.lugens and L.striatellus inoculated on the rice seedlings treated by dipping with 100 ng/μL targeted dsRNA were only 24.1%and 26.8%,respectively.As compared with the blank control,all treatments caused significant mortality.However,for the same pest with the same dsRNA the mortality of spraying treatment was obviously higher than that of rice seedling dipping treatment.The LC₅₀ for dsLsCHS1 to spray L.striatellus was 69.63 ng/μL,which was much lower than its seedling dipping LC₅₀that is evaluated as 273.84 ng/μL.The LC₅₀ for dsNlCHS1 to spray N.lugens was 83.01ng/μL,which was also much lower than that of seedling dipping treatment（257.19 ng/μL）.These results indicate that spraying is a relatively efficient method for field application of pesticide dsRNA.dsNlCHS1 and dsLsCHS1 were synthesized by the same method above,and both N.lugens and L.striatellus were treated by spraying with each of these two dsRNA to observe the control effects of targeted interference and off-target interference.The results showed that spraying dsLsCHS1 and dsNlCHS1 with 100ng/μL concentration both could produce lethal effect on L.striatellus.The off target interference of dsNlCHS1 caused 45.3%mortality in10 days,which was significantly lower than that of targeted interference of dsLsCHS1,treated in the same way（52.4%）.Both dsLsCHS1 and dsNlCHS1 spraying at 100 ng/μL were also lethal to N.lugens.The mortality of dsNlCHS1 targeted interference was 56.7%after 10days of treatment,which was significantly higher than the off-target interference of dsLsCHS1 treated in the same way（29.7%）.These results showed that the off-target effect of dsRNA could expand the insecticidal spectrum of dsRNA,but the control effect of off-target interference was lower than that of targeted interference under the same conditions,so it could be applied as a concurrent treatment.