Properties Of DsRNA Degrading Nucleases And Their Contribution To RNAi Efficacy In Different Insect Species

Double-stranded RNA(dsRNA) mediated gene silencing has been a powerful tool used for reverse genetic research in insect science and supposed to be a very promising technique that could be widely used for pest management in crop protection against insect pests.RNA interference(RNAi)was achieved by administration of dsRNA to the target site and processing to small interfering RNA(siRNA)by Dicer enzyme and then coupled with Argonaute protein to form RNA-induced silencing complex(RISC)further combined to the complementary mRNA to induce target RNA degradation.It is a fact that the elements involved in RNAi were indispensable for successful gene silencing.Widely distribution of core RNAi machineries contributes to robust RNAi response were founded in various insect species.However,systemic RNAi efficiency varied among insects has been a barrage for the application of RNAi as an effective tool for functional genomic research and biological control of pests by dsRNA expressed transgenic plants or gene specific dsRNA insecticides.Systemic RNAi requires systematic spread of dsRNA throughout the whole body which rely on the stable transition and uptake of dsRNA.The most important issue is that the dsRNA molecules must avoid from degradation by nucleases to keep long term stability before it gets to the target sites and induce sustainable RNAi response.Widely distributed gut specific dsRNase has been confirmed to contribute strong cleavage activity against dsRNA molecules in different insects.Different degrees of dsRNA degradation in the gut and the hemolymph were consistent with varied RNAi efficacy among different insects.In the present study,we aim to explore the negative roles of dsRNA degrading nucleases regarding to the poor RNAi response.Revealing the biochemical and molecular properties of dsRNA degrading nuclease and comparing the dsRNA degrading activities among different insect species and tissues could help us to create a more comprehensive understanding about the major factors that affect the persistence of dsRNA,which is helpful to provide efficient strategies to improve dsRNA stability i.e.removal of dsRNA degrading activity.The results of this study are summarized as follows:1 Development of a method for enzymatic analysis of dsRNA degrading nucleasesIn order to have a better understanding on the enzymatic properties of dsRNA degrading nucleases,the fluorescence method developed for Dicer cleavage assay was adopted and modified by selecting suitable combinations of fluorophores and quenchers for monitoring dsRNA degrading activity.The fluorescence method was then validated using traditional gel electrophoresis and a well-accepted micro-quantitative PCR method.The results found 5-FAM fluorescent donor-and 3-BHQ1 quencher-labeled 24 bp dsRNA to be the most sensitive substrate,the fluorescence intensity in the reaction with 0.5 ?M 5-FAM/3-BHQ1 labeled substrate continuously increased about 6-fold from 215.33 to a maximum of 1232.15 in 25 min,it showed long-lasting continuous changes in fluorescence and a high fluorescence yield and was thus the best candidate for further experiments.The enzyme activity from different sources checked by the fluorescence method not match so perfect but have the same degrading tendency compared with gel electrophoresis method.The enzyme activity of serial diluted solutions checked by the fluorescence method produced similar curves with a correlation coefficient of 0.976 compared with micro-quantitative PCR method.The fluorescence method with 5-FAM/3-BHQ1 labeled substrate gave reliable results was suitable for analyzing dsRNA degrading activity.Furthermore,our method is easier to execute than the qPCR method,and more accurate than gel electrophoresis.It was a good candidate could replace the the conventional methods for high throughput quantification of dsRNA degrading activity.2 Biochemical properties of dsRNA degrading nuclease in different insectsThe RNAi sensitivity usually varied in different insect species.It has been reported that the variation of RNAi sensitivity among different insect species was attributable to dsRNA degradation.Thus,its of great significance to conduct in-depth study on the dsRNA degrading nucleases from different insect species.Here the biochemical properties and activity distribution of dsRNA degrading nucleases in different insects with varied RNAi sensitivity from different orders were characterized with a modified fluorescence assay method.The results revealed that there are certain commonalities and differences in the biochemical characteristics of dsRNA degrading enzymes in different insects.All the insect dsRNA degrading nucleases tested showed high activity in alkali pH environment.The activities were promoted with proper mg2+ in mosct insects except inhibited activity with addition of Mg2+in T.castaneum and D.melanogaster.Most of insect showed highly activity in higher temperature except coleoptera insect T.castaneum and Z.atratus showed reduced activity sharply at 47?.The enzymes from different insects had various optimal reaction conditions and kinetic parameters.The whole body enzyme activity was dramatically different among insect species though the enzymes with higher affinity(lower Km)were usually balanced by a smaller Vmax to maintain a proper degradation capacity.The activity among tissues of different insect species was also found not proportional.But all the insects tested showed hundred times more dsRNA degrading activity in their gut than in other tissues.Reaction environment analysis demonstrated that the physiological condition in gut fluid and serum of different insects was all not the optimal and became a limitation for dsRNA degrading activity,activity could be depressed as much as 378.2-fold in L.migratoria.When compared the tissue activity with RNAi sensitivity,the order of gut activity was a slightly better indicator of their ingestion RNAi and hemolymph activity was comparable to their injection RNAi sensitivity.Discussions came to the conclusion that physiological environments in insects could function to regulate dsRNA degrading activity,and the activity in the tissue which first contact delivered dsRNA could indicate RNAi efficacy better than the activity in whole body.The quick degradation of dsRNA and tolerance to ingestion RNAi in caterpillars was resulted from not only the high expression level of dsRNA degrading nucleases,but also the higher pH in their gut fluid.3 Characterization and mRNA expression analysis of the DNA/RNA non-specific endonuclease from different insectsDNA/RNA non-specific endonuclease family is the major enzyme could function in exogenous nucleic acids in different organisms.There were two subfamilies including dsRNase and Endonuclease G were reported in insects.In this study,in order to further study the ability of degrade exogenous dsRNA among different insects,we selected different insect species with varied RNAi sensitivity as target to identify genes belonging to this family by searching the genomic and transcriptome database based on the sequence features of dsRNase and Endonuclease G.We have identified 6,5,5 and 2 genes encoding the DNA/RNA non-specific endonuclease family from S.litura,C.suppressalis,T.castaneum and L.striatellus,respectively.All these genes were confirmed the existence of DNA/RNA non-specific endonuclease(NUC)or Endonuclease NS domain(PF01223).Multiple amino acid sequence alignment revealed that key amino acid residues corresponding to the active site,substrate binding site and magnesium ion binding site are conserved among insect NUC proteins.Phylogenetic analysis revealed that the DNA/RNA non-specific endonuclease superfamily could be divided into different subfamilies.Each insect species identified only one gene for the Endonuclease G single gene subfamily.Except for one gene from T.castaneum belong to Deoxyribonuclease I subfamily,all the rest are dsRNase subfamily.Tissue distribution analysis found that there were 3,2 and 2 genes over-expressed in the gut of S.litura,C.suppressalis and T.castaneum.The only dsRNase of L.striatellus was also highly expressed in the gut with minor differences between other tissues when compared with other gut specific dsRNases.The transcript level of SldsRNases and the relative dsRNA degrading activity were bouth found to be higher in older larvae(3rd-6th instar)than younger larvae(1st-2nd).4 Functional analysis of dsRNases by heteroglous expressionMultiple dsRNases were distributed in different insects as reported in previous study.However,we have little knowledge about their contributions in dsRNA degradation in different insects as they were not proved possese dsRNA degrading activity yet.In order to uncover the specific role of individual dsRNases in dsRNA degradation,here we applied the Bac-to-bac baculovirus expression system and sf9 cells to generate the recombinant proteins of dsRNases from S.litura and T.castaneum,dsRNase and EndoG from L.striatellus.The correct insertion of constructed plasmid to the bacmid DNA were verified by PCR analysis.The successful expression of target recombinant proteins was further verified by western blot analysis.There were four,one and one dsRNases from S.litura(SldsRNase-1,SldsRNase-2,SldsRNase-3 and SldsRNase-4),T.castaneum(TcdsRNase-1)and L.striatellus(LsdsRNase)have the ability to degrade dsRNA as detected by fluorencesce method,respectively.Which means the rapidly degradation of dsRNA in S.litura was contributed by multiple dsRNases' function and leading to a more complicated dsRNA degradation mechanism.5 Functional analysis of TcdsRNases by RNAiIn order to investigate if enhanced tolorence to RNAi was contributed by degradation of dsRNA by dsRNase in vivo.We select the red flour beetle Tribolium castaneum as target.The dsRNA degrading activity were measured after injection dsRNA targeting different TcdsRNases,the RNAi efficiency was then evaluated subsequently.The results found that the dsRNA degrading activity could be suppressed 95.7%followed by knockdown of TcdsRNase-1.Only 7.9%depletion of enzyme acitivity with silenced TcdsRNase-2.While the dsRNA degrading activity were increased 12.7%and 44.4%with silencing of TcdsRNase-3 and TcdsRNase-4,respectively.These results further confirmed the dsRNA degrading activity was predominantly driven by TcdsRNase-1.The effect of TcdsRNase-1 to RNAi efficacy was further verified by RNAi-of-RNAi strategy.Removal of nuclease activity with 88.9%suppressed transcript level of TcdsRNase-1,the efficiency of RNAi could be improved by injection or ingestion dsRNA targeting TcLac2a and TcCYP6BK13.For the injection RNAi assay,the transcript levels of TcLac2a were reduced by 58.2%and 58.9%with injected 0.2 ng and 2 ng dsRNA targeting TcLac2a compared with that of the control larvae injected with dsEGFP.The transcript levels of TcCYP6BK13 were reduced by 38.6%and 46.1%with injected 2 ng and 20 ng dsRNA targeting TcCYP6BK13 compared with that of the control larvae injected with dsEGFP.For the ingestion RNAi assay,the transcript levels of TcLac2a and TcCYP6BK13 were reduced by 54.0%and 20.9%with feeding dsRNA targeting TcLac2a and TcCYP6BK13 compared with that of the control larvae feeding with dsEGFP,respectively.All of these provided a good instruction for improving RNAi efficiency with removal of nuclease activity and the minimized dsRNA dose used for pest control could be a good strategy for cost savings.6 Functional analysis of SldsRNases by CRISPR/Cas9As a kind of lepidoptera insect,the existence of multiple dsRNase gene copies has been found in Spodoptera litua,and most of them were confirmed their ability to degrade dsRNA.Two gut specific dsRNases SldsRNase-1 and SldsRNase-2 were confirmed their predominant role in dsRNA degradation by comparing the regulation of dsRNA degrading activity combined with the transcript level of SldsRNases in different feeding diets.Then we disrupted these two gut specific dsRNases by using CRISPR/Cas9 technique.The homozygous progeny larvae with disruption of either two dsRNases(SL1KO and SL2KO)showed 79.3%and 40.4%significant inhibited dsRNA degrading activity and the double knockout strain with disruption of both two dsRNases(SLISL2KO)showed 96.3%significant inhibited dsRNA degrading activity.The nuclease activity was significantly inhibited 83.2%in the gut fluid,whereas just inhibited 23.3%in the hemolymph of SL1SL2KO strain compared to the WT strain.Accordingly,the peak level of dsRNA content was almost 20 times in the SL1SL2KO compared to the WT strain with a similar dsRNA persistence time in the hemolymph.Comparative analysis of RNAi effect induced by dsRNA target six endogenous gene revealed that only dsRNA target CYP gene could efficiently suppress target mRNA level.Then comparison of injection and feeding RNAi effect between WT and SL1SL2KO strain was carried out by using dsRNA target CYP.For the injection RNAi,target mRNA level was inhibited slightly higher in SL1SL2KO strain than WT strain,but there is no significant difference.For the feeding RNAi,target mRNA level was not changed in the WT strain,but significant inhibited 23.2%in the SL1SL2KO strain.All of these suggest that the dsRNA degrading activity in S.litura is predominantly driven by SldsRNase-1 and SldsRNase-2,and their expression could be affected by feeding diets.The enhanced feeding RNAi response in SL1SL2KO strain further implies that the expression of SldsRNase-1 and SldsRNase-2 was a negative factor for successful RNAi.But the effect was somewhat limited because the efficiency did not dramatically improved.All of this might imply there were other negative regulators like unknown nucleases could also involve and contribute residue nuclease activities might contribute to poor RNAi response in S.litura.Furthermore,the number of egg laying adults,egg masses and larvae hatched eggs were significantly reduced in the SL1SL2KO strain compared to the WT strain.But no differences in growth and development observed between them,which means the fertility was severely affected with disruption of SldsRNase-1 and SldsRNase-2.