Patent Application
20190059364
The invention relates generally to methods of molecular biology and gene silencing to control insect pests in agriculture.
RNA interference (RNAi) has been gaining attention as a loss-of-function research tool. It shows great potential as a novel biological control mechanism for the protection of crops against different pests. Its intracellular mode of action is highly conserved and well described. The inherent sequence-specific nature of the mechanism allows for selectively targeting organisms, such as insect pests, by optimizing dsRNA fragments corresponding to species-specific gene sequences. RNAi technology, allowing for in vivo post-transcriptional silencing of essential genes, thereby causing mortality with little effect on nontarget species, has gained significant interest in pest management research over the past years.
RNA interference (RNAi) holds great promise as a novel strategy against insect pests of agricultural crops. This is because, in principle, the only information needed to target an essential insect gene with exquisite specificity is the insect gene sequence; and the RNAi molecules can be delivered via transgenesis of the crop plant. Specifically, the plant is engineered to express double-stranded RNA (dsRNA) against the insect gene of interest. On ingestion by the insect, the dsRNA is internalized into cells, where it is cleaved by an insect dsRNA-specific enzyme, Dicer-2, into small interfering RNA (siRNA, ca. 21 nt) that guides the Argonaute protein of the RNA induced silencing complex (RISC) to degrade complementary mRNAs. In planta RNAi has yielded significant plant protection against the western corn rootworm Diabrotica virgifera virgifera, cotton bollworm Helicoverpa armigera, and Colorado potato beetle Leptinotarsa decemlineata, and insecticidal RNAi in transgenic crops are reported to be near commercial release.
Despite these substantial advances, many RNAi studies on insects have yielded moderate or variable knock-down of gene expression, with limited effects on insect phenotype and performance. These problems apply particularly to plant sap-feeding insects, such as whiteflies, aphids, psyllids and planthoppers, including major pests and vectors of plant viruses, where RNAi against essential genes often reduces growth or reproduction, but has small or no effect on survivorship. A possible cause of the limited efficacy of in planta RNAi against many insects is that the plant RNAi molecules can be degraded by non-specific nucleases in the insect saliva, gut lumen or hemolymph. Accordingly, there is a need in the art for a method of increasing the efficacy of RNAi in plant sap-feeding insects.
The present invention, in general, features methods and compositions for controlling plant infestations by repressing, delaying, silencing, or otherwise reducing gene expression within a particular pest.
In one aspect, the invention features a method including administering to a phloem sap-feeding insect one or more dsRNAs capable of suppressing activity of one or more RNAi-suppressing nuclease genes expressed in the gut of the insect and one or more dsRNAs capable of suppressing one or more osmoregulatory genes expressed by the insect. In some embodiments, the phloem sap-feeding insect is an aphid, a whitefly, a psyllid, a mealybug, a planthopper, or a leafhopper. In some preferred embodiments, the whitefly is Bemisia tabaci. In other preferred embodiments, the aphid is the pea aphid, Acyrthosiphon pisum.
In some embodiments of the previous aspect, the nuclease is a dsRNAse. In some embodiments, the osmoregulatory gene is an aquaporin or a glucohydrolase of family GH-13. In some embodiments in which the osmoregulatory gene is a glucohydrolase, the glucohydrolase is a sucrase. In some embodiments, the osmoregulatory gene is an aquaporin.
In preferred embodiments of the previous aspect, the method includes administration of two dsRNAs that suppress the activity of two dsRNAses, a dsRNA that suppresses an aquaporin, and a dsRNA that suppresses a sucrase.
In some embodiments of the previous aspect, the administration of dsRNAs is performed in planta. In yet other embodiments, the administration of dsRNA is performed in an artificial diet.
In another aspect, the invention features a plant that is resistant to a phloem sap-feeding insect, wherein the plant includes one or more dsRNAs capable of suppressing activity of one or more RNAi-suppressing nuclease genes expressed in the gut of the insect and one or more dsRNAs capable of suppressing one or more osmoregulatory genes expressed by the insect. In some embodiments, the insect that feeds on the plant is an aphid, a whitefly, a psyllid, a mealybug, a planthopper, or a leafhopper. In some preferred embodiments, the whitefly is Bemisia tabaci. In other preferred embodiments, the aphid is the pea aphid, Acyrthosiphon pisum.
In some embodiments of the previous aspect, the nuclease is a dsRNAse. In some embodiments, the osmoregulatory gene is an aquaporin or a glucohydrolase of family GH-13. In some embodiments in which the osmoregulatory gene is a glucohydrolase, the glucohydrolase is a sucrase. In some embodiments, the osmoregulatory gene is an aquaporin.
In preferred embodiments of the previous aspect, the plant includes two dsRNAs that suppress the activity of two dsRNAses, a dsRNA that suppresses an aquaporin, and a dsRNA that suppresses a sucrase.
In another aspect, the invention features a composition including one or more dsRNAs capable of suppressing activity of one or more RNAi-suppressing nuclease genes expressed in the gut of the insect and one or more dsRNAs capable of suppressing one or more osmoregulatory genes expressed by the insect.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.
In general, the present technology provides a system/method for the use of RNAi suppression of RNAi suppressors. This dramatically increases the efficacy of RNAi against essential genes, so achieving high mortality of an insect pest. Our methods described herein also provide in planta RNAi against sucking insect pests of agriculture. The technology can be stacked with transgenes (e.g. Bacillus thuringiensis [Bt]) that are effective against chewing pests (beetles, caterpillars etc.).
It is demonstrated herein that the dsRNA (the RNAi molecule) is degraded nonspecifically in the gut of the whitefly Bemisia tabaci and the pea aphid Acyrthosiphon pisum, and we identify gut nucleases in both the whitefly and aphid that are suppressors of RNAi. We also show both reduced dsRNA degradation and increased efficacy of RNAi against osmoregulation genes in whiteflies that are co-administered dsRNA against the nucleases and osmoregulation genes.
The purpose of our study was to identify the factors in the plant and insect that limit the efficacy of RNAi against phloem-feeding insects, and to use this information for improved design of RNAi. Our experiments were conducted on the whitefly Bemisia tabaci, which is a globally-important pest of many crops.
We hypothesized that whitefly RNAi may be limited by processing of dsRNA to small RNAs (sRNAs) in the plant and, additionally or alternatively, by nonspecific degradation of RNAi molecules in the insect. We tested these hypotheses by following the fate of dsRNA constructed against a 370 nt fragment of the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria, administered to the insects via artificial diets and plant transgenesis. These experiments led us to identify non-specific degradation of dsRNA by B. tabaci, which we then reduced by RNAi against two candidate B. tabaci nuclease genes. In our final experiments, we tested the efficacy of stacking RNAi against the nuclease genes with RNAi against candidate essential genes of B. tabaci. Our genes of choice were an aquaporin and a glucohydrolase of family GH-13, which are candidate osmoregulation genes that protect the insect from rapid dehydration and death. We call these genes BtAQP1 and BtSUC1, respectively. Our results provide new insights into the fate of RNAi molecules administered to phloem-feeding insects by in planta RNAi, and the value of this technology as a novel insect pest control strategy.
Furthermore, as described herein, we have obtained information about phloem-mobile RNA molecules from the RNA content of both wild-type tissue grafted onto transgenic plants and phloem-feeding insects. When these two methods were applied in this study to stable transgenic tomato containing ds-GFP under two alternative phloem-specific promoters, they yielded the full-length 370 nt dsRNA, but not sRNA in the 20-25 nt range. This result cannot be attributed to technical difficulties in detecting sRNA because both full-length dsRNA and sRNA were detected in bulk transgenic leaf samples of the same total RNA content.
Further insight into the delivery of in planta RNAi to phloem-feeding insects was obtained from our comparison of the ds-GFP products in whiteflies feeding from ds-GFP transgenic plants and ds-GFP-supplemented artificial diet. Specifically, sRNA was detected in insects fed on ds-GFP via the diet but not the plant. These data evidence that non-specific nuclease activity in the insect gut degraded the dsRNA in the phloem sap, quantitatively preventing dsRNA delivery to gut cells, where siRNA is generated by the cytoplasmic RNAi machinery; but that the high concentrations of dsRNA in the artificial diets saturated the gut nuclease activity, such that a proportion of the ingested dsRNA was translocated to gut cells, yielding detectable sRNA in the insect.
As is shown herein, the application of RNAi in whiteflies to test whether RNAi-mediated suppression of dsRNase genes resulted in enhanced efficacy of RNAi against other insect genes, specifically the two predicted osmoregulation genes, AQP1 and SUC1. As predicted, orally-delivered ds-dsRNase1&2 both protected ds-GFP from non-specific degradation and increased the efficacy of RNAi against osmoregulation genes, as quantified by gene expression and survivorship of insects administered RNAi via artificial diet. These data demonstrate that the efficacy of RNAi in various insects may be significantly improved by RNAi-mediated suppression of dsRNase genes, and potentially other RNAi suppressors.
As is shown herein, our results provide proof of principle that the efficacy of RNAi against the whitefly B. tabaci can be enhanced by the dual strategies of, first, stacking RNAi against multiple genes with related physiological roles but distinct molecular functions and, second, using RNAi to suppress suppressors of RNAi. These approaches create wider opportunities for in planta RNAi as a control strategy against B. tabaci, which is a globally important crop pest, and offer a template for comparable strategies against other insect pests with poor or variable RNAi efficacy.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Plants and Insects
Tomato plants (Solanum lycopersicum cv. Florida Lanai) were grown in compost supplemented with Miracle-Gro® Water Soluble All Purpose Plant Food in climate-controlled chambers at 25±2 ° C. with a 14L:10D light cycle at 400 μmol/m2/s PAR.
The Bemisia tabaci MEAM1 culture (mtCO1 GenBank accession no. KM507785) was derived from a collection from poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) in Ithaca, N.Y., USA in 1989. The insects were maintained on 5-6-week-old tomato plants at 25±2° C. with a 14L:10D light cycle at 400 μmol/m2/s PAR. One-day-old adult males and female insects were caged to test plant leaves using BugDorm-1 Insect Cages (Bio Quip, Rancho Dominguez, Calif.) or custom clip-cages, and fed on a sterile artificial liquid diet containing 0.5 M sucrose and 0.15 M amino acids in Parafilm sachets. Some experiments used isolated guts, dissected with fine pins from adult female and male insects into phosphate-buffered saline.
RNA Extractions
For total RNA isolation, leaves or whiteflies were homogenized in TRIzol® Reagent (Cat# 15596-026, Thermo Fisher Scientific, Waltham, USA) with 1:1 (vol) Lysing Matrix D Bulk beads (Cat# 116540434, MP Biomedicals, Santa Ana, USA) on a MP FastPrep-24™ homogenizer (Cat# 116004500, MP Biomedicals, Santa Ana, USA) with 5.5 M/S for 2×30 seconds. The lysed samples were centrifuged at 13,000 rpm for 3 min to remove the beads and RNA was isolated from the supernatant following the manufacturer's instructions. RNA samples used for qRT-PCR were further processed to remove genomic DNA by incubation with 10 μl DNase 1, using the reagents and protocol in the RNase-free DNase set (Cat# 79254, Qiagen, Valencia, USA). To collect dsRNA from diets, samples of the liquid diet were combined with isopropanol (1:1, by vol) and 3 M sodium acetate pH 5.2 (1:10 by volume). The concentration of RNA was determined spectrophotometrically with a Nanodrop-2000 (Thermo Fisher Scientific, Waltham, USA), and RNA integrity was verified by denaturing formaldehyde gel-electrophoresis.
Synthesis of cDNA and dsRNA
cDNA libraries for amplification of whitefly genes were prepared with oligo (dT) primers using the Reverse Transcription System (Cat# A3500, Promega, Madison, USA) following the manufacturer's instructions. Candidate osmoregulatory genes, comprising aquaporin AQP1 (NCBI Accession KX390870), sucrase SUC1 (also known as a-glucohydrolase GH13-1) (NCBI Accession KX390871), and candidate nuclease genes dsRNase1 (NCBI Accession KX390872) and dsRNase2 (NCBI Accession KX390873) identified in this study, were amplified from the cDNA template in a reaction mix containing 0.4 μM primers (Table 1), 2 U Invitrogen Platinum Taq DNA polymerase (Cat# 10966018, Thermo Fisher Scientific, Waltham, USA), 1.5 mM MgCl2 and 200 ng cDNA template in 25 μl volume, using a Techne thermal cycler. The thermal profile comprised 2 min at 94° C. for initial denaturation, 30 cycles with 95° C. for 30 sec, 55° C. for 30 sec, 72° C. for 1 min (depending on products length, 1 kb/min) and final extension cycle of 72° C. for 5 min. Amplicon sequences were verified by Sanger sequencing, then introduced into PGEM-T vector (Cat# A1360, Promega, Madison, USA) and transformed into DH5α™ competent cells. The plasmid was extracted and, following confirmation by sequencing, used as template for dsRNA synthesis with primers listed in Table 1.
The dsRNA was synthesized using the AmpliScribe TM T7-Flash Transcription Kit (Cat# ASF3257, Epicentre Biotechnologies, Madison, USA), according to the manufacturer's instructions. The templates were the EGFP-pBAD plasmid (plasmid #54762, AddGene Plasmid
Repository) for ds-GFP, and plasmids obtained for the whitefly genes identified above. The dsRNA product was quantified by Nanodrop, and run on a gel with 1 kb plus molecular weight ladder (Cat# 10787026, Thermo Fisher Scientific, Waltham, USA) to confirm the predicted size.
Construction of dsRNA Expression Cassette
The dsRNA expression cassette was constructed in the pHANNIBAL vector. A 370 nt GFP sequence was amplified and inserted in inverted orientation into pHANNIBAL using the PDK intron as a spacer and different restriction sites, XhoI and EcoRI for sense GFP and HindIII and XbaI for the inverted GFP sequence. Two phloem-companion cell-specific promoters, Galactinol Synthase from melon Cucumis melo (CmGAS) and sucrose-H+ symporter from Arabidopsis thaliana (AtSUC2) were cloned and inserted into the binary vector pER8 using XhoI/SpeI and Xhol restriction sites separately. The GFP-intron-rGFP cassette was assembled and inserted downstream of the promoter in pER8 vector using a Xhol restriction site for AtSUC2 and SpeI site for CmGAS1. For RNAi constructs with multiple targets, ca. 150 bp of the different gene fragments were, first, fused together through Gibson assembly method into XhoI and EcoRI sites in pHANNIBAL vector, or reversed fused sequence into HindIII and XbaI sites (e.g. XhoI−RNase1+RNase2+AQP1−EcoRI for forward sequence cloning and HindIII−AQP1+RNase2+RNase1−XbaI for inverted orientation cloning). The final dsRNA expression cassettes were assembled downstream of AtSUC2 promoter in pER8 at Xho I site. All vectors were verified by sequencing.
Generation of Tomato Transgenics with dsRNA Gene Construct
The binary vector pER8 was introduced via electroporation into Agrobacterium tumefaciens strain LBA4404. Kanamycin selection at 500 μg/ml was used to select for transformants. Transformation of the tomato plants was performed. Putative transgenic plants were transferred to soil and maintained in an incubator at 25±2° C. with a relative humidity of 60-70% and with a 14L:10D light cycle at 400 μmol/m2/s PAR. Total DNA was extracted from leaves of 5-6-week-old transgenic plants and verified for transformation by PCR with sequence specific primers (Table 1). Eight transgenic lines were confirmed for each promoter.
Administration of dsRNA to Whiteflies
dsRNA was administered to adult insects either via chemically-defined diets (at 0.1-1 μg/μl, varying with experiment) or via transgenic tomato lines. For analysis of the fate of ds-GFP, 100 whiteflies were administered to each diet cage and ca. 250 whiteflies were caged to each plant. For insect performance experiments on artificial diets, 10 replicate groups of 40 adult whiteflies (one day post-emergence) were applied to each diet treatment and mortality was monitored daily over 6 days, with insects transferred to fresh diet containing dsRNA every two days. At the end of each experiment, all live insects were transferred to 500 μl TRIzol® Reagent (Cat# 15596-026, Thermo Fisher Scientific, Waltham, USA) and stored at −80° C. prior to isolation of total RNA (as above). Transgenic plants at the 4-5 leaf stage were used for performance assays of insects on plants. Ten 2-day-old adult insects (1:1 sex ratio) were transferred in a clip-cage to the abaxial surface of each of the second and third leaf of each plant, with 4 replicate plants for all treatments, apart from the ds-dsRNase plants with three replicates. Eight days after infestation, the number of dead insects was scored, and surviving insects were flash-frozen and stored at −80° C. for RNA extraction.
Northern Blots
RNA extracted from whiteflies and plants was separated on denatured polyacrylamide-urea gels (SequaGel—UreaGel System, cat# EC-833, National Diagnostics, Atlanta, Ga., USA) containing 8% monomers. The gel was pre-run at 250 V in 0.5× TBE buffer for 30 min, then 20 μg sample RNA was combined with an equal volume of Gel Loading Buffer II (Cat# AM8547, Thermo Fisher Scientific, Waltham, USA) and heated at 95° C. for 4 minutes to denature the RNA. Samples were loaded in urea-cleaned wells and run at 250 V in 0.5× TBE buffer until the loading dye migrated to the far end of the gel. Uniform sample loading was confirmed by staining of 5S rRNA with SYBR Gold, followed by transfer to Hybond-NX membrane (Cat# RPN 203T, GE Healthcare, Wilkes-Barre, Pa.) with the Owl™ HEP Series Semidry Electroblotting Systems at 0.4 A for 1 hour. The transferred RNA was cross-linked using a UV crosslinker at 120 kJ for 30 sec for HMW RNA and for LMW RNA.
The GFP probe used for northern blotting was generated using the MAXIscript® In Vitro Transcription Kit (Cat# AM1308, Thermo Fisher Scientific, Waltham, USA) with 3.125 μM alpha-32P UTP (10 mCi/ml, 800 Ci/mmol, Perkin Elmer, Waltham, USA), 5 μM UTP and 100 μM ADP. Unincorporated 32P-label was removed using a RNeasy mini column (Cat# 74104, Qiagen, Venlo, Limburg, USA). The 32P-labeled probe was brought to 100 μl with nuclease-free water, mixed with 350 μl buffer RLT (RNeasy mini kit, Cat# 74104, Qiagen, Venlo, Limburg, USA) and 250 μl 100% ethanol, collected onto a RNeasy mini column, washed twice with buffer RPE (from RNeasy mini kit, Cat# 74104, Qiagen, Venlo, Limburg, USA), and eluted with 40 μl nuclease-free water. For each membrane, half of the probe was heated at 95° C. for 4 min and immediately added to the pre-hybridized membrane at 2×106 cpm/ml final concentration. For HMW RNA detection, pre-hybridization and hybridization used Ambion Ultrahyb buffer (CAT# AM8670, Thermo Fisher Scientific, Waltham, USA) according to the manufacturer's protocol. The blot was washed twice in 2× SSC, 0.1% SDS buffer at 68° C., followed by two washes in 0.1× SSC, 0.1% SDS buffer at the same temperature. For LMW RNA detection, the blot was pre-hybridized in hybridization buffer (5× SSC, 20 mM Na2HPO4 (pH 7.2), 7% SDS, 2× Derhardt's solution) at 50 ° C. for at least two hours, then hybridized with a final concentration of 2.5×106 cpm/ml probe in the same buffer at 50° C. overnight. The membrane was washed four times in non-stringent wash buffer (3X SSC, 25 mM NaH2PO4 pH 7.5, 5% SDS) and once in stringent wash buffer (1X SSC, 0.1% SDS), and then exposed for autoradiography. The signal was collected on phosphor screen (Molecular Dynamics) and scanned using a Typhoon 9400 fluorescent imager. ImageJ was used for ds-GFP band density analyses.
qRT-PCR
To quantify the expression of target whitefly genes, qRT-PCR was performed with RNA extracted from three biological replicates of whiteflies. cDNA was prepared using random primers of High-Capacity cDNA Reverse Transcription Kit or SuperScript™ II Reverse Transcriptase (Cat# 4368814 and 18064014, Thermo Fisher Scientific, Waltham, USA) following the manufacturer's instructions. For qRT-PCR, the 20 μl reaction mix comprised 10 μl Master Mix (Bio-Rad, Hercules, Calif.) or Power SYBR Green PCR Master Mix (Applied Biosystems, Carlberg, Calif., USA)], precisely 1 μl cDNA template and 0.5-2 μl 10 μM primers qRT-PCR primers (Table 1) designed with Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, Calif.). Amplifications were conducted in a C1000™ Thermal cycler (Bio-Rad, Hercules, Calif.) with the following thermal profile: 95° C. for 5 min, 40 amplification cycles of 95° C. for 15 sec, 55° C. for 30 sec, and dissociation cycle of 95° C. for 15 sec, 55° C. for 15 sec then brought back to 95° C. Dissociation curves confirmed single peaks of the predicted size without primer dimerization. All assays included three technical replicates with template-free and non-RT as controls; and the relative expression was calculated using the 2−ΔΔCt method, normalized to the whitefly 60S ribosomal protein L13a (RPL13) gene. Mean Ct value of three technical replicates was calculated per sample.
Identification and Phylogenetic Analysis of Candidate Whitefly dsRNase Genes
To obtain an initial set of candidate nucleases in B. tabaci, the translated sequence of the non-specific nuclease Bombyx mori, which has been demonstrated to degrade dsRNA and suppress RNAi, was BLASTed (E value <1.0 e-10) against the translated RefSeq genes in the B. tabaci genome. The resultant B. tabaci genes were analyzed by Signalp and NCBI conserved domain database for signal peptide and conserved domain. For analysis of gene phylogenies, a neighboring-joining tree was constructed of the conserved DNA/RNA non-specific nuclease domain amino acid sequences (MEGA 6.06).
Tomato Plant Grafting
For each graft, a scion with few mature leaves from 5-6-week-old wild-type tomato was cut and inserted to a transgenic RNAi plant of the same age. The graft site was fastened with Parafilm; and the plant was covered with a plastic bag and kept in darkness at 25° C. for 48 h. Lighting was gradually increased over the next 3-4 days, and the bag was removed at day-7. Wildtype scions were grafted to two copies per GAS:ds-GFP line for four plants and copies of the two SUC2:ds-GFP lines for 11 plants. The plants were harvested 3 weeks after grafting for RNA isolation and northern blotting analysis of ds-GFP fragments.
Statistical Analysis
All data sets conformed to the expectations of normality by the Anderson Darling test and homogeneity of variance by the Levine and Bartlett tests. They were analyzed by one-way ANOVA with Fisher's LSD post hoc test. Statistical analyses were conducted with JMP software (SAS Institute, Miami, USA) and Minitab 17.
Delivery of Plant dsRNA to Whiteflies
The first experiments investigated the fate of dsRNA expressed under companion cell-specific promoters CmGAS from melon, Cucumis melo (expressed in companion cells of minor veins of the leaf) and AtSUC2 from Arabidopsis thaliana (expressed in all companion cells) in transgenic lines. The 370 bp ds-GFP construct (
Northern blotting of ds-GFP fragments expressed under the CmGAS and AtSUC2 promoters in 5-6-week-old transgenic tomato lines yielded a band at ca. 20-25 nt in all lines and at 370 nt in most lines (
We postulated two alternative explanations for the apparent absence of GFP sRNA in the whiteflies: that the sRNA, first, is not phloem-mobile and consequently not ingested by the whiteflies; and, second, is ingested but degraded in the whitefly. To test for the phloem mobility of the full-length and sRNA, control scions were grafted onto transgenic plants expressing ds-GFP under CmGAS or AtSUC2 promoters, with homografts onto wild-type (WT) plants as negative control. Three weeks after grafting, entire scion apices were excised and processed for GFP-RNA by northern blotting. The 370-nt band, but not the sRNA band, was detected (
dsRNA Degradation in the Whiteflies
To investigate the fate of RNAi-related molecules in the whiteflies further, adult insects were administered ds-GFP via artificial diet. The 370 nt ds-GFP was recovered in northern blots of both the diet on which the whiteflies had fed and diet without whiteflies (
We hypothesized that dsRNA ingested by whiteflies is subjected to non-specific degradation in the gut lumen, restricting the availability of dsRNA molecules for uptake by cells of the gut epithelium and intracellular processing by the RNAi machinery. To test this hypothesis, we applied phylogenetic methods to identify candidate nuclease genes in the B. tabaci genome.
Phylogenetic Analysis of Candidate dsRNase Genes in the Bemisia tabaci Genome
Our strategy to identify candidate nuclease(s) in the B. tabaci genome was to identify orthologs of a Bombyx mori DNA/RNA non-specific nuclease gene (BmdsRNase) that has been validated experimentally to cleave dsRNA and reduce the efficacy of RNAi. The B. mori dsRNase was BLASTed (E value<1.0 e-10) against transcriptome databases of the whole body, salivary gland and gut of B. tabaci, yielding three B. tabaci sequences (dsRNase1, dsRNase2, dsRNase3) with a single DNA/RNA non-specific nuclease domain (NCBI conserved domain database) and a predicted signal peptide (SignalP). A phylogenetic tree constructed by the neighbor-joining method using the amino acid sequence of the conserved DNA/RNA non-specific nuclease domain from multiple insect species aligned the B. tabaci dsRNase1 with aphid nucleases with moderate bootstrap support, and dsRNase2 and dsRNase3 with nuclease-1 of Tribolium castaneum, with excellent bootstrap support (
We amplified part of the predicted full-length cDNA sequences of dsRNase1 and dsRNase2 from whole body and gut cDNA libraries of adult B. tabaci, but failed to amplify dsRNase3. Complementary searches of the B. tabaci whole body, gut and salivary gland transcriptome databases yielded a few dsRNase3 reads only in the salivary gland transcriptome, suggesting that this gene is weakly expressed in the adult insects. Validating these transcriptome data, qRT-PCR analysis of the B. tabaci used in this study confirmed that dsRNase1 and dsRNase2 are expressed, with three-fold enrichment of dsRNase2 expression in the gut relative to the whole body (
Effect of RNAi Against dsRNase Genes on ds-GFP Ingested by the Whiteflies
We next asked whether inhibiting whitefly dsRNase genes could protect ds-GFP from nonspecific degradation and thus improve RNAi. Chemically synthesized dsRNA against dsRNase1 or dsRNase2 was fed to the whiteflies either synchronously or 3 days prior to adding ds-GFP, with dsRNase-free treatment as the control (
Effect of ds-dsRNase on Efficacy of RNAi Against Whitefly Osmoregulation Genes
The demonstration (
In the first RNAi experiment, the dsRNAs were delivered to adult whiteflies via artificial diet over a time-course of 6 days. The experimental treatments were dsRNA against the whitefly AQP1 and SUC1, either separately or in combination, and with or without the dsRNA against RNase1 and RNase2. The control samples comprised diets with ds-GFP or ds-dsRNase1&2, and dsRNA-free diets.
The impact of the dsRNAs administered via the artificial diet on the performance of the whiteflies was quantified as mortality of the insects over the 6-day test period. Relative to the control diets (ds-GFP and dsRNA-free), mortality was significantly elevated only in the two treatments containing both ds-AQP1 and ds-dsRNase1&2. In the absence of ds-AQP1, ds-SUC1 had no discernible effect on whitefly mortality. However, ds-SUC1 functioned synergistically with ds-AQP1 in the presence of ds-dsRNase1&2 to yield mortality approaching 50% and significantly greater than all other treatments. Surviving insects on day-6 were used for gene expression analysis by qRT-PCR. The ds-dsRNase treatments reduced expression of the cognate dsRNase genes by 30-35% (
We then investigated the response of the whiteflies to RNAi against the osmoregulation genes administered in planta (
1. A method of using dsRNA to suppress the activity of RNAi-suppressing nuclease genes in the gut of insects.
2. The method of paragraph 1 where the insect is a sap-sucking insect.
3. The method of paragraph 2 where the sap-sucking insect is an aphid, whitefly, mealybug, psyllid, planthopper and leafhopper.
4. The method of paragraph 1 where the dsRNA that suppresses the activity is to an osmoregulation gene.
5. The method of paragraph 4 where the osmoregulation gene is aquaporin AQP1 and/or sucrase SUC1 .
6. The method of paragraph 1 where the dsRNA that suppresses the activity is to a nuclease gene.
7. The method of paragraph 6 where the nuclease gene is dsRNase1 and dsRNase2.
8. The method of paragraph 1 where the dsRNA for osmoregulation and nuclease are both used.
9. The method of paragraph 1 where the dsRNA is administered in planta.
10. The method of paragraph 1 where the dsRNA is administered via an artificial diet.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference. Other embodiments are within the claims.
This application claims benefit of U.S. Provisional Application No. 62/552,781, filed Aug. 31, 2017, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62552781 | Aug 2017 | US |
