Patent Application
20200181639
This application contains a Sequence Listing that has been submitted in ASCII format view EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy is named “DDPSC0081-401-PC Sequence Listing filed with Response to Correct Defects”, and is 191 kilobytes in size.
Insect pests are detrimental to crop production and human health throughout the world and insect control can in some instances consume between 10-25% of a country's gross national product (GNP). (http World Wide Web internet site “fao.org/3/a-av013e.pdf”). In the U.S., annual loss due to crop pests is estimated to exceed $120 billion USD/year. (Polaszek A. (1998) Wallingford, UK: CABI. 530 pp.).
Within crop pests, Lepidoptera are the most detrimental insect pests of cereal crop cultivation. Chemical control is often expensive, inefficient, and can be associated with negative environmental consequences. Host plant resistance is an attractive option but impeded by lack of robust Lepidoptera resistant germplasm (http World Wide Web internet site “cnbc.com/2015/05/08/insects-feast-on-plants-endangering-crops-and-costing-billions.html”).
Since 1996, commercialization of crop plants genetically engineered to produce Bacillus thuringiensis (Bt) insecticidal proteins have resulted in efficient pest control, increased yield, reduced insecticidal use, and enhanced farmer profits. (Khan Z R, et al. (2014). Philos. Trans. R Soc. Lond Biol Sci. 369: 1639).
Consequently, the cumulative area planted with Bt crops worldwide reached greater than 1 billion acres during 2011. Within the U.S., Bt Corn, Bt Soybean, and Bt Cotton accounted for 90% of all the total corn, soybean and cotton acres during 2013 (Tabashnik B E, et al. (2013). Nat. Biotech. 31: 510-521). However, evolution of field resistance against Bt in lepidopteran pests raises potential concerns about the sustainability of this approach. (Campagne P., et al. (2013) PLoS ONE 8(7): e69675. Doi:10.1371). That is further exacerbated by loss of resistance against pyramided Bt traits as well (https World Wide Web internet site dt npf.com/agriculture/web/ag/news/article/2016/08/10/rootworm-resistance-pyramided-bt.html).
Provided herein is an isolated double stranded RNA (dsRNA) molecule comprising a nucleic acid sequence complementary to about 200 to 1000 contiguous nucleotides of a target gene sequence—wherein the target gene is a MIGGS-IRTG as defined herein—involved in gut microbe clearance and/or containment induced by microbes ingested during feeding and/or active feeding. In certain aspects, the target gene is critical for insect immune responses and certain aspects provide that it is abundantly expressed in the midgut. In certain aspects, the target gene sequence includes at least one of the protein coding region, the 5′ UTR region, the 3′ UTR region, and any combination thereof, of a target gene. Further, certain aspects provide that the dsRNA molecule silences the target gene when ingested by an insect. In certain aspects, the target gene is a type 1 MIGGS RNAi target or a type 2 MIGGS RNAi target as defined elsewhere herein. In certain aspects, the target gene is a pattern recognition receptor (PRR) class gene or an insect midgut structural component gene. In certain aspects, the target gene is expressed abundantly in a midgut specific manner during active feeding.
In certain aspects, a dsRNA molecule disclosed anywhere herein comprises two annealed complementary RNA strands. In certain aspects, said dsRNA molecule comprises a single RNA strand comprising an inversely repeated sequence with a spacer in between, wherein the single RNA strand can anneal to itself to form a hairpin loop structure.
In certain aspects, a dsRNA molecule disclosed anywhere herein comprises a nucleic acid sequence complementary to about 200 to 1000 contiguous nucleotides of the protein coding region of the target gene sequence. In certain aspects, said dsRNA molecule comprises a nucleic acid sequence complementary to about 200 to 1000 contiguous nucleotides of the 5′ UTR region or the 3′ UTR region of the target gene sequence. In certain aspects, said dsRNA molecule comprises a nucleic acid sequence complementary to a contiguous region comprising at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the length of the target gene sequence protein coding region, 5′ UTR region, or 3′ UTR region. In certain aspects, said dsRNA molecule comprises a nucleic acid sequence complementary to about 200 to 650 contiguous nucleotides of a target gene sequence.
Certain aspects of this disclosure are drawn to a target gene selected from the group consisting of M. sexta-Hemolin (MsHEM), M. sexta-Serine proteinase homolog 3 (MsSPH-3), M. sexta-Peptidoglycan recognition protein 2 (MsPGRP2), M. sexta-Beta-1, 3-glycan-recognition protein 2 (MsβGRP2), M. sexta-Relish family protein 2A (MsREL2A), M. sexta-Dorsal (MsDor), M. sexta-Spätzle (MsSPZ1A), M. sexta-Toll receptor (MsTOLL), M. sexta-Scolexin A (MsSCA1), M. sexta-Hemolymph proteinase 18 (MsHP18), M. sexta-Transferrin (MsTRN), M. sexta-Arylphorin beta subunit (MsARP), M. sexta-Chymotrypsinogen-like protein 1 (MsCTL1), M. sexta-Valine Rich Midgut Protein (MsVMP1), M. sexta-Imd (MsImd), M. sexta-FADD (MsFADD), M. sexta-Dredd (MsDRD), M. sexta-Relish F (MsReIF), M. sexta-Cdc42 (MsCdc42), M. sexta-Dsor1 (MsDsor1), M. sexta-Fos (MsFos), M. sexta-Jra (MsJra), M. sexta-Caudal (MsCAD1), M. sexta-Atg8 (MsAtg8), M. sexta-Atg13 (MsAtg13), M. sexta-IAP1 (MsIAP1), M. sexta-Chitin synthase 2 (MsChs2), M. sexta-Beta-1 tubulin (MsβTub), M. sexta-Beta fructofuranosidase 1 (MsSuc1), and orthologs thereof. In certain aspects, the target gene is selected from the group consisting of M. sexta-Hemolin (MsHEM), M. sexta-Serine proteinase homolog 3 (MsSPH-3), M. sexta-Peptidoglycan recognition protein 2 (MsPGRP2), M. sexta-Beta-1, 3-glycan-recognition protein 2 (MsβGRP2), M. sexta-Relish family protein 2A (MsREL2A), M. sexta-Dorsal (MsDor), M. sexta-Spätzle (MsSPZ1A), M. sexta-Toll receptor (MsTOLL), M. sexta-Scolexin A (MsSCA1), M. sexta-Hemolymph proteinase 18 (MsHP18), M. sexta-Transferrin (MsTRN), M. sexta-Arylphorin beta subunit (MsARP), M. sexta-Chymotrypsinogen-like protein 1 (MsCTL1), M. sexta-Valine Rich Midgut Protein (MsVMP1), M. sexta-Imd (MsImd), M. sexta-FADD (MsFADD), M. sexta-Dredd (MsDRD), M. sexta-Relish F (MsReIF), M. sexta-Cdc42 (MsCdc42), M. sexta-Dsor1 (MsDsor1), M. sexta-Fos (MsFos), M. sexta-Jra (MsJra), M. sexta-Caudal (MsCAD1), M. sexta-Atg8 (MsAtg8), M. sexta-Atg13 (MsAtg13), M. sexta-IAP1 (MsIAP1), M. sexta-Chitin synthase 2 (MsChs2), M. sexta-Beta-1 tubulin (MsβTub) and M. sexta-Beta fructofuranosidase 1 (MsSuc1).
Also provided herein is an isolated double stranded RNA (dsRNA) molecule comprising a nucleic acid sequence complementary to about 200 to 1000 contiguous nucleotides of a target gene sequence, wherein the target gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-70, 71-75, 76-88, 89-105, and 106-110. In certain aspects, the target gene sequence includes at least one of the protein coding region, the 5′ UTR region, the 3′ UTR region, and any combination thereof, of a target gene. Further, certain aspects provide that the dsRNA molecule silences the target gene when ingested by an insect.
In certain aspects disclosed herein, the target gene is sequence selected from the group consisting of: i) SEQ ID NOs: 1-9, 11, 14, 31, 39, 43, 44, and 71-75.
In certain aspects disclosed herein, the target gene is sequence selected from the group consisting of: ii) SEQ ID NOs: 3, 4, and 43. In certain aspects, the dsRNA molecule causes impeded growth, developmental progression, and/or mortality and the like of TH, DMB, and FAW in an orthologous manner.
In certain aspects disclosed herein, the target gene is sequence selected from the group consisting of: iii) SEQ ID NOs: 76, 77, 80, 81, 85, 87, and 88. In certain aspects, the dsRNA molecule causes impeded growth, developmental progression, and/or mortality and the like of DBM. Further, in certain aspects, the DBM is a Bt resistant strain.
In certain aspects disclosed herein, the target gene is sequence selected from the group consisting of: iv) SEQ ID NOs: 89, 92, 96, 101, 103, and 105. In certain aspects, the dsRNA molecule causes impeded growth, developmental progression, and/or mortality and the like of FAW.
In certain aspects disclosed herein, the target gene is sequence selected from the group consisting of: v) SEQ ID NOs: 107-110. In certain aspects, the dsRNA molecule caused impeded growth, developmental progression, and/or mortality and the like of RFB.
In certain aspects of the dsRNA molecule disclosed above, the dsRNA molecule comprises two annealed complementary RNA stands. In certain aspects, the dsRNA molecule comprises a single RNA strand comprising an inversely repeated sequence with a spacer in between and where the single RNA strand can anneal to itself to form a hairpin loop structure.
In certain of the dsRNA molecule disclosed above, the dsRNA molecule comprises a nucleic acid sequence complementary to about 200 to 1000 contiguous nucleotides of the protein coding region of the target gene sequence. In certain aspects, the dsRNA molecule comprises a nucleic acid sequence complementary to a contiguous region comprising at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the length of a sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-70, 71-75, 76-88, 89-105, and 106-110. In certain aspects, the dsRNA molecule comprises a nucleic acid sequence complementary to a contiguous region comprising at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the length of the target gene sequence protein coding region, 5′ UTR region, or 3′ UTR region.
In certain aspects disclosed herein the dsRNA molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 111-119, 120-126, 127-135, and 136-139. In certain aspects, the dsRNA is a fragment of at least about 200 nucleotides thereof. In certain aspects, i) the isolated dsRNA molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 110-119, or the fragment thereof, causes impeded growth, developmental progression, and/or mortality and the like of TH; ii) the isolated dsRNA molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 120-126, or the fragment thereof, causes impeded growth, developmental progression, and/or mortality and the like of DBM; iii) the isolated dsRNA molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 127-135, or the fragment thereof, causes impeded growth, developmental progression, and/or mortality and the like of FAW; or iv) the isolated dsRNA molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 136-139, or the fragment thereof, causes impeded growth, developmental progression, and/or mortality and the like of RFB.
In certain of any of the above aspects, the dsRNA molecule can form siRNA. Certain aspects provide for an isolated siRNA molecule derived from the processing of said dsRNA molecule.
Certain further aspects provide of an insecticidal composition comprising an isolated dsRNA molecule or an siRNA molecule disclosed anywhere herein, and a synthetic carrier or microbial conduit. In certain aspects, a microorganism has a natural capacity or is engineered to produce and/or deliver dsRNA to increase its bioavailability and/or biostability for causing RNA interference including but not restricted to plant growth promoting organisms, normal commensal and/or symbiotic microorganisms associated with the target insect pest or parasites and/or natural enemies of the target pest or pest target host or host cultivation range etc. from an insect or parasite and/or natural enemies of the target pest engineered or identified from natural populations containing microbial conduit to produce and/or deliver dsRNA and/or drive the transmission of such microbial conduits into natural populations of insect pests as a control option. In certain aspects of an insecticidal composition disclosed herein, the dsRNA molecule is conjugated with the synthetic carrier.
Certain aspects are also drawn to a recombinant DNA construct encoding a dsRNA molecule disclose anywhere herein. In certain aspects, the recombinant DNA construct comprising a gene silencing sequence comprising about 200 to 1000 contiguous nucleotides of a target gene sequence. In certain aspects, the target gene is a MIGGS-IRTG, as defined herein, involved in gut microbe clearance and/or containment induced by microbes ingested during feeding and/or active feeding. In certain aspects, the target gene is critical for insect immune responses. In certain aspects, the target gene is abundantly expressed in the midgut. In certain aspects, said target gene sequence includes at least one of the protein coding region, the 5′ UTR region, the 3′ UTR region, and any combination thereof, of a target gene. In certain aspects, the target gene is a type I MIGGS RNAi target or a type 2 MIGGS RNAi target as described elsewhere herein. In certain aspects, the target gene is a pattern recognition receptor (PRR) class gene or an insect midgut structural component gene. In certain aspects, the target gene is expressed abundantly in a midgut specific manner during active feeding.
In any of the above aspects of a recombinant DNA construct, the gene silencing sequence comprises about 200 to 1000 contiguous nucleotides of the protein coding region of the target gene sequence. In certain aspects, the gene silencing sequence comprises about 200 to 1000 contiguous nucleotides of the 5′ UTR region or the 3′ UTR region of the target gene sequence. In certain aspects, the gene silencing sequence comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% contiguously of the length of target gene sequence protein coding region, 5′ UTR region, or 3′ UTR region. In certain aspects, the gene silencing sequence comprises about 200 to 650 contiguous nucleotides of the target gene sequence.
In any of the above aspects of a recombinant DNA construct, and as noted throughout this disclosure, in certain aspects, a target gene can be selected from the group consisting of M. sexta-Hemolin (MsHEM), M. sexta-Serine proteinase homolog 3 (MsSPH-3), M. sexta-Peptidoglycan recognition protein 2 (MsPGRP2), M. sexta-Beta-1, 3-glycan-recognition protein 2 (MsβGRP2), M. sexta-Relish family protein 2A (MsREL2A), M. sexta-Dorsal (MsDor), M. sexta-Spätzle (MsSPZ1A), M. sexta-Toll receptor (MsTOLL), M. sexta-Scolexin A (MsSCA1), M. sexta-Hemolymph proteinase 18 (MsHP18), M. sexta-Transferrin (MsTRN), M. sexta-Arylphorin beta subunit (MsARP), M. sexta-Chymotrypsinogen-like protein 1 (MsCTL1), M. sexta-Valine Rich Midgut Protein (MsVMP1), M. sexta-Imd (MsImd), M. sexta-FADD (MsFADD), M. sexta-Dredd (MsDRD), M. sexta-Relish F (MsReIF), M. sexta-Cdc42 (MsCdc42), M. sexta-Dsor1 (MsDsor1), M. sexta-Fos (MsFos), M. sexta-Jra (MsJra), M. sexta-Caudal (MsCAD1), M. sexta-Atg8 (MsAtg8), M. sexta-Atg13 (MsAtg13), M. sexta-IAP1 (MsIAP1), M. sexta-Chitin synthase 2 (MsChs2), M. sexta-Beta-1 tubulin (MsβTub), M. sexta-Beta fructofuranosidase 1 (MsSuc1), and orthologs thereof. In certain aspects a target gene can be selected from the group consisting of M. sexta-Hemolin (MsHEM), M. sexta-Serine proteinase homolog 3 (MsSPH-3), M. sexta-Peptidoglycan recognition protein 2 (MsPGRP2), M. sexta-Beta-1, 3-glycan-recognition protein 2 (MsβGRP2), M. sexta-Relish family protein 2A (MsREL2A), M. sexta-Dorsal (MsDor), M. sexta-Spätzle (MsSPZ1A), M. sexta-Toll receptor (MsTOLL), M. sexta-Scolexin A (MsSCA1), M. sexta-Hemolymph proteinase 18 (MsHP18), M. sexta-Transferrin (MsTRN), M. sexta-Arylphorin beta subunit (MsARP), M. sexta-Chymotrypsinogen-like protein 1 (MsCTL1), M. sexta-Valine Rich Midgut Protein (MsVMP1), M. sexta-Imd (MsImd), M. sexta-FADD (MsFADD), M. sexta-Dredd (MsDRD), M. sexta-Relish F (MsReIF), M. sexta-Cdc42 (MsCdc42), M. sexta-Dsor1 (MsDsor1), M. sexta-Fos (MsFos), M. sexta-Jra (MsJra), M. sexta-Caudal (MsCAD1), M. sexta-Atg8 (MsAtg8), M. sexta-Atg13 (MsAtg13), M. sexta-IAP1 (MsIAP1), M. sexta-Chitin synthase 2 (MsChs2), M. sexta-Beta-1 tubulin (MsβTub) and M. sexta-Beta fructofuranosidase 1 (MsSuc1).
Further aspects provide for a recombinant DNA construct comprising a gene silencing sequence comprising about 200 to 1000 contiguous nucleotides of a target gene sequence, wherein the target gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-70, 71-75, 76-88, 89-105, and 106-110. In certain aspects, the target gene sequence includes at least one of the protein coding region, the 5′ UTR region, the 3′ UTR region, and any combination thereof, of a target gene.
In any of the above aspects of a recombinant DNA construct, the target gene sequence is selected from the group consisting of: i) SEQ ID NOs: 1-9, 11, 14, 31, 39, 43, 44, and 71-75; ii) SEQ ID NOs: 3, 4, and 43; iii) SEQ ID NOs: 76, 77, 80, 81, 85, 87, and 88; iv) SEQ ID NOs: 89, 92, 96, 101, 103, and 105; and v) SEQ ID NOs: 107-109, and 110. In certain aspects, the gene silencing sequence comprises about 200 to 1000 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-70, 71-75, 76-88, 89-105, and 106-110. In certain aspects, the gene silencing sequence comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% contiguously of a sequence selected from the group consisting of SEQ ID Nos: 1-14, 16-29, 31-70, 71-75, 76-88, 89-105, and 106-110. In certain aspects, the gene silencing sequence comprises about 200 to 650 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-70, 71-75, 76-88, 89-105, and 106-110. In certain aspects, the gene silencing sequence is operably linked to one or more promoters for the expression of a dsRNA molecule that silences the target gene when ingested by an insect. In certain aspects, the construct is an expression vector. And, in certain aspects, the expression vector can target single or multiple insect RNAi target genes or chimeric RNAi target genes.
Certain aspects of the disclosure also provide for a host cell comprising the dsRNA molecule, the siRNA molecule, a polynucleotide encoding a dsRNA molecule, and/or the construct or a dsRNA encoding segment thereof disclose anywhere herein. In certain aspects, the host cell is a bacterial or plant cell or organelle. In certain aspects, the organelle is a plastid. In certain aspects, the host cell is a transgenic and/or transplastomic plant cell. In certain aspects, the hose cell expresses a dsRNA and/or produces an siRNA disclosed anywhere herein.
Certain aspects also provide for a transgenic and/or transplastomic plant comprising a dsRNA molecule, an siRNA molecule, a polynucleotide encoding the dsRNA, and/or a construct or a dsRNA encoding segment disclosed anywhere herein. In certain aspects, at least one cell of the plant expresses the dsRNA molecule and/or produces the siRNA molecule. Further, certain aspects provide for a seed, part, tissue, cell, or organelle of the above transgenic and/or transplastomic plant. In certain aspects, the seed, part, tissue, cell, or organelle comprises the dsRNA molecule and/or the siRNA molecule. In certain aspects, the organelle is a plastid.
Certain aspects provide for a method of silencing: (i) an insect immune response gene and/or (ii) an insect gene encoding for structural components of the insect midgut. In certain aspects, the method comprises providing for ingesting an isolated dsRNA molecule, an siRNA molecule, an insecticidal composition, a host cell, a transgenic and/or transplastomic plant, transplastomic plant and/or a seed, part, tissue, cell, or organelle as disclosed anywhere herein, to an insect.
Certain aspects provide for a method of protecting a plant from an insect pest of the plant. In certain aspects, the method comprises topically applying to a plant an isolated dsRNA molecule, an siRNA molecule, and/or an insecticidal composition disclosed anywhere herein, and providing the plant in the diet of the insect pest. In certain aspects, the dsRNA is topically applied by expressing the dsRNA in a microbe and topically applying the microbe onto the plant.
Certain aspects provide for a method of producing a plant resistance to a pest insect of said plant. In certain aspects, the method comprises transforming a plant with a polynucleotide encoding the dsRNA molecule and/or a construct or a dsRNA encoding segment thereof as disclosed anywhere herein, wherein the plant expresses a dsRNA molecule and/or produces an siRNA disclose anywhere herein.
Certain aspects provide for a method of improving crop yield. In certain aspects, the method comprises growing a population of crop plants transformed with a polynucleotide encoding a dsRNA molecule and/or a construct or a dsRNA encoding segment thereof wherein the plant expresses a dsRNA molecule and/or produces an siRNA molecule as discloses anywhere herein. In certain aspects, the population of transformed plants produces higher yields in the presence of pest insect infestation than a control population of untransformed plants.
Certain aspects provide for a method for producing a plant resistant against a pest insect of said plant. In certain aspects, the method comprises: a) transforming a plant cell and/or organelle with a polynucleotide encoding a dsRNA molecule and/or a construct or a dsRNA encoding segment thereof as disclosed anywhere herein; b) regenerating a plant from the transformed plant cell and/or organelle; and c) growing the transformed plant under conditions suitable for the expression of said double stranded RNA molecule, wherein said transformed plant of (c) is resistant to the plant pest insect compared to an untransformed plant.
In certain of any of the aforementioned aspects, the method the dsRNA is ingested by an actively feeding stage of the insect. In certain aspects, the ingestion of the dsRNA induces a melanotic response in the insect larvae. In certain aspects, the ingestion of the dsRNA results in perturbation of gut microbial homeostasis. In certain aspects, the ingestion of the dsRNA results in defective clearance of opportunistic microbes. In certain aspects, the ingestion of the dsRNA results in defective containment of gut microbes.
In certain of any of the aforementioned aspects, the silencing of the target gene results in reduced appetite and/or developmental defects resulting in incomplete development and/or mortality and/or decrease the reproductive success of the insect. In certain aspects, the reduced appetite and/or developmental defects and/or mortality and/or reduced reproductive fitness of the insect is observed after sustained feeding for at least 72 hours.
In certain of any of the aforementioned aspects, the insect is of the order Lepidoptera, Coleoptera, Hemiptera, Blattodea, or Diptera. In certain aspects, the insect is Manduca sexta (M. sexta) (tobacco hornworm), Spodoptera frugiperda (fall armyworm), Ostrinia nubilalis (European corn borer), Plutella xylostella (Diamondback moth), Leptinotarsa decemlineata Say (Colorado potato beetle), Diabrotica spp. (Corn rootworm complex), Tribolium castaneum (Red flour beetle), Popillia japonica (Japanese beetle), Agrilus planipennis (Emerald ash borer), Diaphorina citri (Asian citrus psyllid), Cimex lectularius (Bed bug), a cockroach or termite, or insect pests such as mosquitoes and flies.
In certain of any of the aforementioned aspects, the plant host is selected from the group consisting of Zea mays L (corn), Sorghum bicolor (sorghum), Setaria italica (fox tail millet), Pennisetum glaucum (Pearl millet), Solanum tuberosum (potato), Oryza sativa (rice), Lycopersicon esculentum (tomato), Solanum melongena (eggplant), cultivars of the Brassica oleracea family, Citrus sinensis (Orange), trees of the Oleaceae family, and crops of Rosaceae.
The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.
RNAi-mediated gene-silencing offers a sustainable alternative approach to insect control. Most of the successful RNAi-based pest control strategies thus far employ homology dependent silencing of essential gene functions. Despite this, effective RNAi-based crop protection is lacking for Lepidopteran pests, due to their variable sensitivity to ingested double stranded RNA (dsRNA). (Terenius O, et al. (2011). J. Insect Physiol. 57(2): 231-245).
Plant pests are in constant contact with, and ingest significant amount of microbes during herbivory. (Gayatri Priya N. et al. (2012). 7(1), PLos ONE. E30768; Peñuelas and Terradas (2014). 19(5): Trends Plant Sci. 278-280; Engel and Moran (2013). FEMS microbiol. rev. 37(5): 699-735). This interaction between ingested microbes and insect midgut is often considered passive. Recent studies suggest, however, an active role of midgut specific immune responses in reducing variation of core microbial communities during insect herbivory through the activation of pattern recognition receptors (PRR). (Casanova-Torres and Goodrich-Blair (2013). Insects. 4: 320-338; Tang X, et al. (2012) 7(7) PLoS ONE:e36978; Ryu J H. et al. (2008). Science. 37(5): 777-82; Shrestha S. et al. (2009). J. Asia Pac. Entomol. 12: 277-283; Buchon, N. et al. (2013). Front. Cell. Infect. Microbiol. 11: 615-626). Further, maintenance of core gut microbial communities via active immune responses and/or their containment in the midgut is key to successful herbivory.
Although, the core innate immune response pathways are conserved, their specific components are under strong selection for diversification. (Casanova-Torres and Goodrich-Blair (2013). Insects. 4: 320-338). Therefore, it is contemplated herein that these pathways provide novel and specific targets for devising sustainable pesticidal RNAi biotechnologies against insect pests. Although gut immune responses have been studied from an immunological perspective, their active manipulation via genetic engineering for pest protection is currently lacking.
Provided herein is the identification of “insect RNAi target genes” (IRTGs) involved in gut microbial clearance and/or containment induced by microbes ingested during feeding and/or active feeding (referred to herein as microbe-induced gut specific genes (MIGGS)) and examples of a novel biotechnology for insect protection via inter-specific silencing of MIGGS-IRTGs. In certain aspects, the MIGGS-IRTGs are Lepidoptera-specific. For example, in certain aspects detailed below, the insect is Manduca sexta (M. sexta; Lepidoptera) (tobacco hornworm (TH)). For example, in certain aspects detailed below, the insect is Spodoptera frugiperda (fall armyworm (FAW)). For example, in certain aspects detailed below, the insect is Plutella xylostella (Diamondback moth (DBM). For example, in certain aspects detailed below, the insect is Ostrinia nubilalis (European corn borer). Still, it is also considered understood that successful, feeding-induced loss of appetite, developmental defects, and/or lethality has the potential to provide protection beyond the order Lepidoptera in an orthologous manner. For example, protection against coleopteran pests such as Leptinotarsa decemlineata (Say) (Colorado potato beetle), Diabrotica spp. (Corn rootworm complex), and Tribolium castaneum (Red Flour Beetle (RFB)). Additionally, this MIGGS-RNAi technique may allow containment of disease transmitting insect vectors and/or enable further manipulation of the plant-microbe-insect interactions for devising pesticidal RNAi for crop protection.
In certain aspects detailed below, silencing of a target gene can result in reduced appetite and/or developmental defects and/or mortality and/or reduced fitness of the insect. In certain aspects these effects are observed after sustained feeding for at least about 24, 36, 48, or 72 hours, or any time inbetween.
To the extent necessary to provide descriptive support, the subject matter and/or text of the appended claims is incorporated herein by reference in their entirety.
It will be understood by all readers of this written description that the exemplary embodiments described and claimed herein may be suitably practiced in the absence of any recited feature, element or step that is, or is not, specifically disclosed herein.
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a dsRNA molecule,” is understood to represent one or more dsRNA molecules. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. Numeric ranges are inclusive of the numbers defining the range. Even when not explicitly identified by “and any range in between,” or the like, where a list of values is recited, e.g., 1, 2, 3, or 4, unless otherwise stated, the disclosure specifically includes any range in between the values, e.g., 1 to 3, 1 to 4, 2 to 4, etc.
The headings provided herein are solely for ease of reference and are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole.
As used herein, the term “non-naturally occurring” condition, substance, polypeptide, polynucleotide, composition, entity, plant, organism, individual, and/or any combination thereof, or any grammatical variants thereof and the like, is a conditional term that explicitly excludes, but only excludes, those forms that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or might be at any time, determined or interpreted by a judge or an administrative or judicial body to be, “naturally-occurring.”
As used herein, the term “identity,” e.g., “percent identity” to an amino acid sequence or to a nucleotide sequence disclosed herein refers to a relationship between two or more amino acid sequences or between two or more nucleotide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid in the corresponding position of the comparator sequence, the sequences are said to be “identical” at that position. The percentage of “sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid occurs in both sequences to yield the number of “identical” positions. The number of “identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of “sequence identity.” Percentage of “sequence identity” is determined by comparing two optimally aligned sequences over a comparison window. In order to optimally align sequences for comparison, the portion of a nucleotide or amino acid sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant. An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of “identical” positions between the reference and comparator sequences. Percentage “sequence identity” between two sequences can be determined using, e.g., the program “BLAST” which is available from the National Center for Biotechnology Information, and which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for amino acid sequence comparison), which programs are based on the algorithm of Karlin and Altschul ((1993). Proc. Natl. Acad. Sci. USA. 90(12): 5873-5877).
As used herein, the term “complementary” refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. When using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However; when a U is denoted in the context of the present invention, the ability to substitute a T is implied, unless otherwise stated.
As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by peptide bonds (also known as amide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of “polypeptide,” and unless specifically stated otherwise the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. Thus, it can be generated in any manner, including by chemical synthesis.
As used herein, the term “protein” refers to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein.
As used herein, the term “nucleotide” refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs. Further, the term nucleotide also includes those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide.
As used herein, the term “polynucleotide” refers to polymers of nucleotides, and includes but is not limited to DNA, RNA, DNA/RNA hybrids including polynucleotide chains of regularly and/or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an —OH, then and —H, then an —OH, then an —H, and so on at the 2′ position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included. The term “polynucleotide” is also intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). A polynucleotide can be single stranded or double stranded.
As used herein, the term “nucleic acid” refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated” nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
As used herein, a “coding region” is a portion of nucleic acid comprising codons translatable into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example 5′ untranslated regions (5′ UTRs; also known as a leader sequence), 3′ untranslated regions (3′ UTRs; also known as a trailer sequence), promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode a selection marker gene and a gene of interest. In addition, a vector, polynucleotide, or nucleic acid can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a polypeptide subunit or fusion protein as provided herein. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
A variety of transcription regulatory regions are known to those skilled in the art. These include, without limitation, transcription regulatory regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription regulatory regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription regulatory regions include tissue-specific promoters and enhancers.
Similarly, a variety of translation regulatory elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES).
As used herein, the term “vector” is nucleic acid molecule as introduced into a host cell or organelle, thereby producing a transformed host cell or organelle. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker gene and other genetic elements known in the art. Illustrative types of vectors include plasmids, phages, viruses and retroviruses.
As used herein, the term “transformed” cell or organelle, or a “host” cell organelle, is a cell or organelle into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses those techniques by which a nucleic acid molecule can be introduced into such a cell or organelle, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transformed cell or a host cell can be a bacterial cell or a eukaryotic cell.
As used herein, the term “expression” refers to a process by which a gene produces a biochemical, for example, a polynucleotide or a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). It also includes without limitation transcription of the gene into an RNA molecule that is not translated into a polypeptide but is capable of being processed by cellular RNAi mechanisms. If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., an RNA produced by transcription of a gene or a polypeptide that is translated from a mRNA transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
As used herein the term “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).
As used herein, the term “hpRNA” refers to hairpin RNA comprising a single-stranded loop region and a base-paired stem of an inversely repeated sequence. hpRNA can be generated from an hpRNA construct (or vector) and/or an hpRNA transgene comprising an inversely-repeated sequence of the RNAi target gene with a spacer region between the repeats. The RNA transcribed from such a sequence self-hybridizes to form a hairpin structure. The stem can be used as a substrate for the generation of siRNAs, but few or none are generated from the loop. Since a spacer region is needed for the stability of the transgene construct, but is not involved in siRNA production, an intron sequence is often used in this position. (Watson J M, et al. (2005). FEBS Letters. 579: 5982-8987).
As used herein, the term “siRNA” refers to small (or short) interfering RNA (or alternatively, silencing RNA) duplexes that are capable of inducing the RNA interference (RNAi) pathway. These molecules can vary in length (generally between 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5′ or 3′ end of the sense strand and/or the antisense strand. The term “siRNA” includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.
As used herein, the phrase “duplex region” refers to the region in two complementary or substantially complementary polynucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between polynucleotide strands that are complementary or substantially complementary. For example, a polynucleotide strand having 21 nucleotide units can base pair with another polynucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the “duplex region” has 19 base pairs. The remaining bases may, for example, exist as 5′ and 3′ overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity as used herein refers to 79% or greater complementarity. For example, a mismatch in a duplex region consisting of 19 base pairs results in 94.7% complementarity, rendering the duplex region substantially complementary.
As used herein, the phrase “gene silencing” refers to a process by which the expression of a specific gene product is lessened or attenuated. Silencing of a gene does not require that the expression or presence of the gene product is completely absent, but that in the context (e.g., comparing expression of a target gene in a plant expressing a gene silencing nucleic acid compared to a control plant or the health of an insect feeding on a gene silencing nucleic acid compared to a control insect), an observable effect in comparison to a control is observed. While gene silencing can take place by a variety of pathways, unless specified otherwise, as used herein, gene silencing refers to decreases in gene product expression that results from RNA interference (RNAi) as understood by one of ordinary skill in the art. The level of gene silencing can be measured by a variety of means, including, but not limited to, measurement of transcript levels by Reverse transcription polymerase chain reaction (PCR), Northern Blot Analysis, B-DNA techniques, transcription-sensitive reporter constructs, expression profiling (e.g. DNA chips), and related technologies. Alternatively, the level of silencing can be measured by assessing the level of the protein encoded by a specific gene. This can be accomplished by performing a number of studies including Western Analysis, measuring the levels of expression of a reporter protein that has e.g. fluorescent properties (e.g. GFP) or enzymatic activity (e.g. alkaline phosphatases), or several other well-known procedures. Further, gene silencing can be assessed by its effect on a pest insect such as resulting in reduced appetite and/or developmental defects and/or mortality of an insect.
As used herein, the term “control” is consistent with its well-established scientific use that refers to a standard of comparison recognized by one of ordinary skill in the art as having a representative level of expression, phenotype, resistance, feeding, mortality, development, etc. Further, one of ordinary skill in the art will recognize, for example, that a statistical outlier and/or non-representative result produced by chance, abnormal environmental condition, manipulation, or other reason, that varies from a standard representation, would not be an appropriate control.
As used herein, “microbe-induced gut specific genes (MIGGS)” refers to a gene or group of genes expressed in the insect midgut in response to microbes ingested during normal process of insect feeding and primarily functioning to clear or respond to the ingested microbes and/or contain the microbes to insect gut via maintenance of midgut structural integrity.
As used herein, “actively feeding stage of the insect” refers to all feeding stages of insects with both complete and incomplete metamorphosis.
Provided herein are nucleic acid molecules for use in, among other things, crop protection from insect pests. In certain aspects disclosed herein, the nucleic acid molecules are isolated. The nucleic acid molecules specifically target certain insect genes (referred to herein interchangeably as “target genes,” “RNAi target genes,” “insect RNAi target genes,” and “IRTGs”), in insects for gene silencing. For example, in certain aspects, the nucleic acid molecules target certain insect microbe-induced gut gene (MIGGS) RNAi targets. In certain aspects, the silencing of a target gene occurs when a nucleic acid molecule of this disclosure is ingested by an insect. In certain aspects, the target gene is an insect gene that is implicated in insect immune responses (type 1 MIGGS RNAi target). A critical immune response gene is a genetically tractable nuclear or cytoplasmic loci that is important for providing cellular and/or humoral defense in insects against internal microorganisms, external microorganisms, and/or other insect parasites. In certain aspects, the immune response genes (type 1 MIGGS RNAi target) can also be a pattern recognition receptor (PRR) gene (Casanova-Torres and Goodrich-Blair (2013). Insects. 4:320-338). A PPR gene is a genetically tractable loci of an insect that encodes soluble or membrane bound proteins that recognize signatures associated with and/or released by microorganisms. PRR genes can activate or be activated by the immune response pathways to minimize microbial infection and can be co-regulated by the immune deficiency (IMD) pathway (Tang X, et al. (2012) 7(7) PLoS ONE:e36978; Ryu J H. et al. (2008). Science. 37(5): 777-82; Shrestha S. et al. (2009). In certain aspects the PRR type genes are co-regulated by the immune deficiency (IMD) pathway in TH were identified, these genes having been recently summarized. (Casanova-Torres and Goodrich-Blair (2013). Insects (4): 320-338; Zhong X, et al. (2012). Insect Biochem. Mol. Biol. 42(7): 514-524); Zhang X, et al. (2015). Insect Biochem. Mol. Biol. 62:38-50; Cao X, et al. (2015). Insect Biochem. Mol. Biol. 62:64-74; Kanost M R, et al. (2016). Insect Biochem. Mol. Biol 76:118-147). In certain aspects, the target gene is an insect gene that is necessary for structural integrity of insect organs including the mid-gut and also facilitates the containment of the ingested microbes to the insect gut. (type 2 MIGGS RNAi target). In certain aspects, the target gene is an insect midgut structural component gene (type 2) (Odman-Naresh et al. (2013). PLoS ONE 8:e82015. 10.1371/journal.pone.0082015). A midgut structural component gene is a genetically tractable loci in an insect that encodes chitin fibrils, proteins, or glycoproteins that form a protective sac-like structure called peritrophic matrix enveloping the insect food bolus/midgut also functioning to contain the ingested microbes in the gut (Engel and Moran (2013). FEMS Microbiol Rev. 37 699-735). In certain aspects, the target genes (type 1 and 2 MIGGS RNAi targets) are predominantly expressed in the insect midgut, for example, abundantly and/or exclusively expressed in the larval and/or adult insect midgut in response to active feeding and/or microbial infection and/or responding to microbes ingested during feeding. In certain aspects, the target gene is induced predominantly in a midgut specific manner during active feeding (type 1 and type 2 MIGGS RNAi targets). The midgut abundance of both type 1 and 2 MIGGS RNAi target genes may mitigate problems associated with reduced amounts of bioavailability.
Representative examples of insect MIGGS RNAi target genes and their nucleic acid sequences identified from published literature are provided herein. In certain aspects, the target gene is one or more of M. sexta-Hemolin (MsHEM), M. sexta-Serine proteinase homolog 3 (MsSPH-3), M. sexta-Peptidoglycan recognition protein 2 (MsPGRP2), M. sexta-Beta-1, 3-glycan-recognition protein 2 (MsβGRP2), M. sexta-Relish family protein 2A (MsREL2A), M. sexta-Dorsal (MsDor), M. sexta-Spätzle (MsSPZ1A), M. sexta-Toll receptor (MsTOLL), M. sexta-Scolexin A (MsSCA1), M. sexta-Hemolymph proteinase 18 (MsHP18), M. sexta-Transferrin (MsTRN), M. sexta-Arylphorin beta subunit (MsARP), M. sexta-Chymotrypsinogen-like protein 1 (MsCTL1), M. sexta-Valine Rich Midgut Protein (MsVMP1), M. sexta-Imd (MsImd), M. sexta-FADD (MsFADD), M. sexta-Dredd (MsDRD), M. sexta-Relish F (MsReIF), M. sexta-Cdc42 (MsCdc42), M. sexta-Dsor1 (MsDsor1), M. sexta-Fos (MsFos), M. sexta-Jra (MsJra), M. sexta-Caudal (MsCAD1), M. sexta-Atg8 (MsAtg8), M. sexta-Atg13 (MsAtg13), M. sexta-IAP1 (MsIAP1), M. sexta-Chitin synthase 2 (MsChs2), M. sexta-Beta fructofuranosidase 1 (MsSuc1), and orthologs thereof.
In certain aspects, the target gene is one or more of M. sexta-Hemolin (MsHEM), M. sexta-Serine proteinase homolog 3 (MsSPH-3), M. sexta-Peptidoglycan recognition protein 2 (MsPGRP2), M. sexta-Beta-1, 3-glycan-recognition protein 2 (MsβGRP2), M. sexta-Relish family protein 2A (MsREL2A), M. sexta-Dorsal (MsDor), M. sexta-Spätzle (MsSPZ1A), M. sexta-Toll receptor (MsTOLL), M. sexta-Scolexin A (MsSCA1), M. sexta-Hemolymph proteinase 18 (MsHP18), M. sexta-Transferrin (MsTRN), M. sexta-Arylphorin beta subunit (MsARP), M. sexta-Chymotrypsinogen-like protein 1 (MsCTL1), M. sexta-Valine Rich Midgut Protein (MsVMP1), M. sexta-Imd (MsImd), M. sexta-FADD (MsFADD), M. sexta-Dredd (MsDRD), M. sexta-Relish F (MsReIF), M. sexta-Cdc42 (MsCdc42), M. sexta-Dsor1 (MsDsor1), M. sexta-Fos (MsFos), M. sexta-Jra (MsJra), M. sexta-Caudal (MsCAD1), M. sexta-Atg8 (MsAtg8), M. sexta-Atg13 (MsAtg13), M. sexta-IAP1 (MsIAP1), M. sexta-Chitin synthase 2 (MsChs2), and M. sexta-Beta fructofuranosidase 1 (MsSuc1), M. sexta-Sickie (MsSck), M. sexta-Akirin (MsAki), M. sexta-Cactus (MsCac), M. sexta-Gloverin (MsGlv) and M. sexta-Beta-1-tubulin (MsβTub).
In certain aspects, the target gene is an ortholog of one or more of M. sexta-Hemolin (MsHEM), M. sexta-Serine proteinase homolog 3 (MsSPH-3), M. sexta-Peptidoglycan recognition protein 2 (MsPGRP2), M. sexta-Beta-1, 3-glycan-recognition protein 2 (MsβGRP2), M. sexta-Relish family protein 2A (MsREL2A), M. sexta-Dorsal (MsDor), M. sexta-Spätzle (MsSPZ1A), M. sexta-Toll receptor (MsTOLL), M. sexta-Scolexin A (MsSCA1), M. sexta-Hemolymph proteinase 18 (MsHP18), M. sexta-Transferrin (MsTRN), M. sexta-Arylphorin beta subunit (MsARP), M. sexta-Chymotrypsinogen-like protein 1 (MsCTL1), M. sexta-Valine Rich Midgut Protein (MsVMP1), M. sexta-Imd (MsImd), M. sexta-FADD (MsFADD), M. sexta-Dredd (MsDRD), M. sexta-Relish F (MsReIF), M. sexta-Cdc42 (MsCdc42), M. sexta-Dsor1 (MsDsor1), M. sexta-Fos (MsFos), M. sexta-Jra (MsJra), M. sexta-Caudal (MsCAD1), M. sexta-Atg8 (MsAtg8), M. sexta-Atg13 (MsAtg13), M. sexta-IAP1 (MsIAP1), M. sexta-Chitin synthase 2 (MsChs2), M. sexta-Beta fructofuranosidase 1 (MsSuc1), and other IMD pathway or structural integrity genes.
One of ordinary skill in the art would understand that nucleic acid molecules can be, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In certain aspects of any target gene silencing nucleic acid molecule described anywhere herein, the nucleic acid molecule is a DNA molecule. In certain aspects of any target gene silencing nucleic acid molecule described anywhere herein, the nucleic acid molecule is a RNA molecule. In certain aspects of any target gene silencing nucleic acid molecule described anywhere herein, the RNA molecule is a double stranded molecule (dsRNA), for example, for use in the RNA interference (RNAi) process. As used herein, a dsRNA molecule is a RNA molecule comprising at least one annealed, double stranded region. In certain aspects, the double stranded region comprises two separate RNA strands annealed together. In certain aspects, the double stranded region comprises one RNA strand annealed to itself, for example, as can be formed when a single RNA strand contains an inversely repeated sequences with a spacer in between. One of ordinary skill in the art will understand that complementary nucleic acid sequences are able to anneal to each other but that two sequences need not be 100% complementary to anneal. The amount of complementarity needed for annealing can be influenced by the annealing conditions such as temperature, pH, and ionic condition. In certain aspects, the annealed RNA sequences are 100% complementary across the annealed region. In certain aspects, the annealed RNA sequences are less than 100% complementary across the annealed region but have enough complementarity to anneal within their environment, such as in a host cell or the gut of an insect. In certain aspects, the annealed RNA sequences are substantial complementarity as defined elsewhere herein.
It is contemplated that the nucleic acid molecules disclosed anywhere herein for the silencing of target genes derive their specificity from comprising a nucleic acid sequence that is complementary or substantially complementary to at least a portion of a target gene sequence. Substantially complementary sequences, however, may be more likely to have reduced specificity and produce off-target effects. As referred to anywhere herein, a target gene sequence can include at least the target gene protein coding region, the 5′ untranslated region (5′ UTR), and/or the 3′ untranslated region (3′ UTR) and any portion or combination thereof. For example, predicted UTR regions can be identified using previously established criteria (Siepel, et al. (2005). Genome Res. 15: 1034-1050) when corresponding genomic sequences are available.
In certain aspects, an isolated double stranded RNA (dsRNA) molecule comprises a nucleic acid sequence complementary to about 21 to 2000 contiguous nucleotides of a target gene sequence discloses anywhere herein. For example, in certain aspects, an isolated double stranded RNA (dsRNA) molecule comprises a nucleic acid sequence complementary to about any of 21, 22, 23, 24, 25, 30, 40, 50, 60, 100, 120, 200, 240, 300, 400, 500, 600, 650, 750, 1000 to about any of 23, 24, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 650, 750, 1000, or 2000 contiguous nucleotides of a target gene sequence. For example, in certain aspects, an isolated dsRNA molecule comprises a nucleic acid sequence complementary to about 100 to 1000 or about 200 to 1000 contiguous nucleotides of a target gene sequence. For example, in certain aspects, an isolated dsRNA molecule comprises a nucleic acid sequence complementary to about 100 to 1000 or about 200 to 1000 contiguous nucleotides of the protein coding region of a target gene sequence. For example, in certain aspects, an isolated dsRNA molecule comprises a nucleic acid sequence complementary to about 100 to 1000 or about 200 to 1000 contiguous nucleotides of the 5′ UTR region or the 3′ UTR region of a target gene sequence. In certain aspects, the isolated dsRNA molecule comprises a nucleic acid sequence complementary to a contiguous region comprising at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the length of the target gene sequence protein coding region, the target gene sequence 5′ UTR region, the target gene 3′ UTR region, and/or any combination thereof. For example, if a target gene sequence protein coding region is determined to be 200 nucleotides long, then an isolated dsRNA molecule comprising a nucleic acid sequence complementary to a contiguous region comprising 95% of the length of the target gene sequence protein coding region would be complementary to a contiguous region 190 nucleotides long.
In certain aspects of any target gene silencing nucleic acid molecule described anywhere herein, including dsRNA molecules for RNAi, the target gene comprises one or more of the nucleic acid sequence of SEQ ID NO: 1-14, 16-29, 31-69, 70-75, 76-88, 89-105, and 106-110. In certain aspects, the target gene comprises the nucleic acid sequence of SEQ ID NO: 3 or 14. Thus, in certain aspects, the isolated dsRNA molecule comprises a nucleic acid sequence complementary to about any of 21, 22, 23, 24, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 650, 750, 1000 to about any of 23, 24, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 650, 750, 1000, or 2000 contiguous nucleotides of a target gene sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-69, 70-75, 76-88, 89-105, and 106-110. For example, in certain aspects, the isolated dsRNA molecule comprises a nucleic acid sequence complementary to about 100 to 1000 or about 200 to 1000 contiguous nucleotides of a target gene sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-69, 70-75, 76-88, 89-105, and 106-110. In certain aspects, the isolated dsRNA comprises a nucleic acid sequence complementary to about 200 to 1000 contiguous nucleotides of the protein coding region of a target gene sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-69, 70-75, 76-88, 89-105, and 106-110. In certain aspects, the isolated dsRNA molecule comprises a nucleic acid sequence complementary to a contiguous region comprising at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the length of the target gene sequence protein coding region, the target gene 5′ UTR region, and/or the target gene 3′ UTR region. In certain aspects, the isolated dsRNA molecule comprises a nucleic acid sequence complementary to a contiguous region comprising at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the length of a sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-69, 70-75, 76-88, 89-105, and 106-110.
In certain aspects of any target gene silencing nucleic acid molecule described anywhere herein, the nucleic acid molecule can form siRNA. Thus, certain aspects provide for an siRNA molecule derived from the processing of a dsRNA molecule for silencing a target gene disclosed herein.
Certain aspects of the disclosure provide for an insecticidal composition comprising a nucleic acid molecule disclosed anywhere herein for silencing a target gene, including long dsRNA, hpRNA, and siRNA. In certain aspects, the insecticidal composition also comprises a synthetic carrier or a microbial conduit. For example, a microbial conduit can be a microorganism that has a natural capacity or is engineered to produce and/or deliver dsRNA to increase its bioavailability and/or biostability for causing RNA interference. Representative examples include plant growth promoting organisms, normal commensal and/or symbiotic microorganisms associated with the target insect pest or pest target host or host cultivation range etc. from an insect engineered or identified from natural populations to produce and/or deliver dsRNA. In certain aspects a microbial conduit can be used as a direct topical application on a whole plant or coated onto a seed or mixed with growth media or transmitted through fertilizer or irrigation, etc. In certain aspects, the nucleic acid molecule of the insecticidal composition is conjugated to the synthetic carrier. For example, a synthetic carrier can be an inert chemical compound with a natural or engineered affinity to bind (conjugate) a dsRNA molecule to increase its biostability and/or bioavailability for causing RNA interference. In certain aspects, a synthetic carrier comprises a combination of inert chemicals or nanoparticles that upon combining and/or individually have a net positive charge or general affinity to bind to negatively charged dsRNA. Representative examples include chitosan, liposomes, carbon quantum dots, biodegradable particles of plant (e.g. coconut coir or grain flour, etc.) or soil (e.g. calcified clay) origin etc. In certain aspects, the dsRNA conjugated with a synthetic carrier can be used as a direct topical application directly and/or after aerosolization on a whole plant or coated onto a seed or mixed with growth media or transmitted through fertilizer or irrigation, etc. In certain aspects, dsRNA or a composition comprising dsRNA can be used as a direct topical spray on application to whole plant, coated onto a seed or mixed with growth media or transmitted through fertilizer or irrigation or combined with plant growth promoting microbes etc.
Certain aspects of this disclosure provide for a recombinant nucleic acid construct, such as a DNA vector, comprising and/or encoding a nucleic acid molecule disclosed anywhere herein for silencing a target gene, including long dsRNA, hpRNA, and siRNA. Certain aspects provide for recombinant nucleic acid constructs comprising and/or encoding an RNAi precursor of a nucleic acid molecule disclosed anywhere herein for silencing a target gene, including long dsRNA, hpRNA, and siRNA.
Certain aspects of this disclosure provide for a recombinant nucleic acid construct, such as a DNA vector, comprising a target gene silencing sequence for silencing a target gene described anywhere herein. In certain aspects, a recombinant DNA construct comprises a gene silencing sequence comprising about any of 21, 22, 23, 24, 25, 30, 40, 50, 60, 100, 120, 200, 240, 300, 400, 500, 600, 650, 750, 1000 to about any of 23, 24, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 650, 750, 1000, or 2000 contiguous nucleotides of a target gene sequence disclosed anywhere herein. In certain aspects, a recombinant DNA construct comprises a gene silencing sequence comprising about 100 to 1000 or about 200 to 1000 contiguous nucleotides of a target gene sequence. In certain aspects, the gene silencing sequence comprises about 100 to 1000 or about 200 to 1000 contiguous nucleotides of the protein coding region of the target gene sequence. In certain aspects, the gene silencing sequence comprises about 100 to 1000 or about 200 to 1000 contiguous nucleotides of the 5′ UTR region or the 3′ UTR region of the target gene sequence. In certain aspects, the gene silencing sequence comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% contiguously of the length of target gene sequence protein coding region, the target gene sequence 5′ UTR region, target gene sequence 3′ UTR region and/or any combination thereof.
In certain aspects, the target gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-69, 70-75, 76-88, 89-105, and 106-110. In certain aspects, the gene silencing sequence comprises about 100 to 1000 or about 200 to 1000 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-69, 70-75, 76-88, 89-105, and 106-110. In certain aspects, the gene silencing sequence comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% contiguously of the length of a sequence selected from the group consisting of SEQ ID NOs: 1-14, 16-29, 31-69, 70-75, 76-88, 89-105, and 106-110.
In certain aspects, the recombinant DNA construct has a gene silencing sequence operably linked to one or more promoters for the expression of a dsRNA molecule that silences the target gene when ingested by an insect. Thus, in certain aspects, the construct is an expression vector. Representative promoters for use in expressing a dsRNA molecule include, but are not limited to, CaMV35S or ZmUbi1 promoters etc. In certain aspects, the expression vector can target single or multiple insect RNAi target genes, for example, the vector could comprise one or more gene silencing sequences or could employ multiple vectors to target multiple insect RNAi target genes or chimeric dsRNA molecules.
Provided herein are host cells, plants, and plants parts comprising, expressing, processing, and the like a dsRNA as described anywhere herein for inducing RNAi in an insect. In certain aspects, a host cell comprises a dsRNA molecule, siRNA molecule, a polynucleotide encoding a dsRNA molecule, and/or a construct or a dsRNA encoding segment thereof described anywhere herein. Representative examples of host cells include bacterial cells, fungal cells, yeast cells, plant cells, plant organelles (e.g., including plastids), and mammalian cells. In certain aspects, the host cell is a bacterial or plant cell. In certain aspects, the host cell is a transgenic and/or transplastomic plant cell. One of ordinary skill will understand that there are many well-known methods for introducing a nucleic acid, such as a vector, into a host cell including well-known methods for generating transgenic and/or transplastomic plant cells. In certain aspects, the hose cell expresses a dsRNA molecules and/or produces siRNA to silence a target gene. In certain aspects, a transgenic and/or transplastomic plant can comprise a dsRNA molecule, siRNA, a polynucleotide encoding a dsRNA, and/or a construct or a dsRNA encoding segment thereof. In certain aspects, at least one cell of a transgenic and/or transplastomic plant expresses a dsRNA molecule and/or produces a siRNA for silencing a target gene. Certain aspects provide for a seed, part, tissue, cell, or organelle of a plant described herein, wherein said seed, part, tissue, cell, or organelle comprises a dsRNA molecule and/or the siRNA for silencing a target gene.
Also provided for herein are various methods of using a dsRNA molecule or vector encoding such dsRNA described anywhere herein for inducing RNAi in an insect and/or silencing a target gene. In certain aspects, this provides for control of insect pests.
Certain aspects provide for a method of silencing an insect immune response gene and/or an insect gene encoding for structural components of the insect midgut. In certain aspects, a method provides for the silencing an insect MIGGS-IRTG. Such method comprises providing for ingestion through spray, drenches, granules, seed coating or plant-incorporated protectant, or the like, to an insect an isolated dsRNA (pure or crude extract), siRNA, insecticidal composition, host cell, transgenic and/or transplastomic plant, and/or the seed, part, tissue, cell, or organelle thereof described anywhere herein.
Certain aspects provide for a method of silencing an insect immune response gene and/or an insect gene encoding for structural components of the insect midgut. In certain aspects, a method provides for the silencing an insect MIGGS-IRTG. Such method comprises providing for ingestion through spray, drenches, granules, seed coating or plant-incorporated protectant, or the like, to an insect an isolated dsRNA, siRNA, insecticidal composition, host cell, transgenic and/or transplastomic plant, and/or the seed, part, tissue, cell, or organelle thereof described anywhere herein.
Certain aspects provide for protecting a plant, such as a crop plant, from an insect pest including but not limited to pests of the order Lepidoptera like Manduca sexta (tobacco hornworm), Spodoptera frugiperda (fall armyworm), Ostrinia nubilalis (European corn borer), Plutella xylostella (Diamondback moth) or pests of the order Coleoptera like Leptinotarsa decemlineata Say (Colorado potato beetle), Diabrotica spp. (Corn rootworm complex), Tribolium castaneum (Red flour beetle), Popillia japonica (Japanese beetle), Agrilus planipennis (Emerald ash borer) or pests of the order Hemiptera like Diaphorina citri (Asian citrus psyllid), Cimex lectularius (Bed bug) or pests of the order Blattodea like all species of cockroaches and termites or insect pests of the order Diptera like all species of Mosquitoes and flies etc. Representative examples of plant hosts include, but are not restricted to, Zea mays L (corn), Sorghum bicolor (sorghum), Setaria italica (fox tail millet), Pennisetum glaucum (Pearl millet), Solanum tuberosum (potato), Oryza sativa (rice), Lycopersicon esculentum (tomato), Solanum melongena (eggplant), all cultivars of Brassica oleracea family, Citrus sinensis (Orange), trees of Oleaceae family and crops of Rosaceae etc. Such methods comprise, for example, topically applying to the plant the isolated dsRNA (pure or crude extract), the siRNA, and/or the insecticidal composition described anywhere herein, and providing the plant in the diet of the insect pest. In certain aspects the dsRNA molecule is topically applied by expressing the dsRNA in a microbe followed by topically applying the microbe onto the plant and/or seed.
Certain aspects provide for producing a plant resistance to a pest insect of said plant. Such methods comprises transforming the plant with a polynucleotide encoding a dsRNA and/or a construct or a dsRNA encoding segment describe anywhere herein, wherein the plant expresses a dsRNA and/or siRNA and/or the plant comprises a dsRNA and/or siRNA containing insecticidal compositions described anywhere herein, for silencing a target gene. In certain aspects, the transformed plant is more resistant to a pest insect of said plant than untransformed plants.
Certain aspects provide for improving crop yield. Such methods comprise growing a population of crop plants transformed with a polynucleotide encoding a dsRNA and/or the construct or a dsRNA encoding segment thereof described anywhere herein, wherein the plant expresses a dsRNA and/or siRNA and/or the plant comprises a dsRNA and/or siRNA containing insecticidal compositions described anywhere herein, for silencing a target gene. In certain aspects, a population of transformed plants produces higher yields in the presence of pest insect infestation than a control population of untransformed plants.
Certain aspects of the disclosure provide for an insecticidal composition comprising a nucleic acid molecule disclosed anywhere herein for silencing a target gene, including long dsRNA, hpRNA, and siRNA. In certain aspects, the insecticidal composition also comprises a synthetic carrier or a microbial conduit. For example, a microbial conduit can be a microorganism that has a natural capacity or is engineered to produce and/or deliver dsRNA to increase its bioavailability and/or biostability for causing RNA interference. Representative examples include plant growth promoting organisms, normal commensal and/or symbiotic microorganisms associated with the target insect pest or parasites and/or natural enemies of the target pest or pest target host or host cultivation range etc. from an insect or parasite and/or natural enemies of the target pest engineered or identified from natural populations containing microbial conduit to produce and/or deliver dsRNA and/or drive the transmission of such microbial conduits into natural populations of insect pests as a control option. In certain aspects a microbial conduit can be used as a direct topical application on a whole plant or coated onto a seed or mixed with growth media or transmitted through fertilizer or irrigation, etc. In certain aspects, the nucleic acid molecule of the insecticidal composition is conjugated to the synthetic carrier. For example, a synthetic carrier can be an inert chemical compound with a natural or engineered affinity to bind (conjugate) a dsRNA molecule to increase its biostability and/or bioavailability for causing RNA interference. In certain aspects, a synthetic carrier comprises a combination of inert chemicals or nanoparticles that upon combining and/or individually have a net positive charge or general affinity to bind to negatively charged dsRNA. Representative examples include chitosan, liposomes, carbon quantum dots, biodegradable particles of plant (e.g. coconut coir or grain flour, etc.) or soil (e.g. calcified clay) origin etc. In certain aspects, the dsRNA conjugated with a synthetic carrier can be used as a direct topical application directly and/or after aerosolization on a whole plant or coated onto a seed or mixed with growth media or transmitted through fertilizer or irrigation, etc. In certain aspects dsRNA or composition comprising the dsRNA can be used as a direct topical spray on application to whole plant, coated onto a seed or mixed with growth media or transmitted through fertilizer or irrigation or combined with plant growth promoting microbes etc.
Certain aspects provide for producing a plant resistant against a pest insect of said plant. Such methods comprise first transforming a plant cell with a polynucleotide encoding the dsRNA and/or the construct or a dsRNA encoding segment described anywhere herein. Next, a plant is regenerated from the transformed plant cell. The plant is then grown under conditions suitable for the expression of the dsRNA. In certain aspects, the transformed plant confers genetically tractable (maternal and/or paternal inherited) gain of function phenotypically manifested as an ability to impair the normal feeding and/or growth and/or development and/or reproductive success of the target plant pest and is consequently resistant to the plant pest insect compared to a control untransformed plant.
In certain aspects of any of the aforementioned methods, the insect larvae ingest the dsRNA. In certain aspects of any of the aforementioned methods, ingestion of the dsRNA induces a melanotic response in the insect larvae. In certain aspects of any of the aforementioned methods, ingestion of the dsRNA results in perturbation of gut microbial homeostasis. In certain aspects of any of the aforementioned methods, ingestion of the dsRNA results in defective clearance of opportunistic microbes. In certain aspects of any of the aforementioned methods, ingestion of the dsRNA results in defective containment of gut microbes.
One of ordinary skill in the art will recognize that, the inventors have demonstrated midgut specific expression of a representative number of MIGGS-IRTGS in TH in response to their feeding on a lab strain of Escherichia coli (E. coli) bacteria. For example, a set of 20 MIGGS-IRTGS (SEQ ID NOs: 1-9, 11, 14, 31, 39, 43, 44, 71-75) that are induced in TH larvae feeding on an induction medium (Wang et al. (2006). J. Biol. Chem. 281(14): 9271-9278) is disclosed herein. It was previously reported that these twenty genes are expressed abundantly in the gut (Table 1). The gut specific expression of 2/20 genes were tested and validated the same (
Orthologs of the representative TH MIGGS-IRTG (SEQ ID NOs: 76-88) set were identified from transcriptomic resources of an economically important Bt. resistant lepidopteran pest DBM using a combination of reciprocal best Blast analysis (Ward et al. (2014). PLoS ONE 9(7): e101850) and literature curation. Most of these MIGGS-IRTGS were induced in response to the feeding of DBM larvae on induction medium (Wang et al. (2006). J. Biol. Chem. 281(14): 9271-9278), similar to observations with TH larvae.
It was also demonstrated that most of the MIGGS-IRTGS were induced in another economically important lepidopteran pest, FAW, feeding on plants grown on representative field soil, using a RNA-Seq approach.
The RNA-Seq approach also identified additional RNAi candidates (SEQ ID NOs: 89-105) belonging to the MIGGS category. Further, orthologs of the expanded representative TH MIGGS-IRTG set (SEQ ID NO: 106-110) were identified from transcriptomic resources of an economically important Coleopteran pest RFB. It was demonstrated that most of the MIGGS-IRTGS were induced in response to the feeding of RFB beetles on induction medium (Wang et al. (2006). J. Biol. Chem. 281(14): 9271-9278), similar to observations with the order lepidoptera.
It was demonstrated that the targeted silencing of 9 out of 20 MIGGS-IRTGS employing bacterially expressed dsRNA protocol (Timmons L. et al. (2001). Gene. 263:103-112.) is insecticidal to TH larvae. Insecticidal activity against TH larvae correlated with the down regulation of target transcripts. It was demonstrated that the targeted silencing of 7 out of 14 MIGGS-IRTGS using bacterially expressed dsRNA protocol (Timmons L. et al. (2001). Gene. 263:103-112.) is insecticidal to DBM larvae. It was demonstrated that targeted silencing of 9 MIGGS-IRTGS are insecticidal to FAW larvae using bacterially expressed dsRNA protocol (Timmons L. et al. (2001). Gene. 263:103-112.). A core set of three MIGGS-IRTGS (SEQ ID NO: 3, 4, and 43) was identified that are efficacious against all three lepidopteran pests TH, DBM and FAW in an orthologous manner and that leaf discs coated with dsRNA against the core MIGGS-IRTGS are insecticidal against TH, DBM and FAW larvae. Further, plastidal expressed dsRNA against the core MIGGS-IRTGS impacted larval growth and survival.
The following examples are included to demonstrate certain embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
In certain aspects, work was performed towards the identification, induction, isolation and cloning of the selected M. sexta (tobacco hornworm (TH)) MIGGS-IRTGS into a bacterial expression system capable of enabling the cloned genes to produce dsRNA (Timmons L. et al. (2001). Gene. 263:103-112.). Upon ingestion by M. sexta larvae, the bacterially expressed dsRNA is intended to silence, or at least knock-down, reduce, and the like the corresponding MIGGS-IRTG in order to curtail the feeding behavior and/or cause lethal effects in the insect pests. Based upon these results, additional testing was done on other representative insect species to demonstrate and establish for purposes of support wide applicability of the compositions and approaches of the disclosure.
For MIGGS-IRTG selection, PRR type genes co-regulated by the immune deficiency (IMD) pathway in TH were identified, these genes having been recently summarized (Casanova-Torres and Goodrich-Blair (2013). Insects (4): 320-338; Zhong X, et al. (2012). Insect Biochem. Mol. Biol. 42(7): 514-524); Zhang X, et al. (2015). Insect Biochem. Mol. Biol. 62:38-50; Cao X, et al. (2015). Insect Biochem. Mol. Biol. 62:64-74; Kanost M R, et al. (2016). Insect Biochem. Mol. Biol 76:118-147). Notably, most of the PRR genes selected for this study are abundantly induced or predicted to express in a midgut specific manner (Pauchet Y, et al. (2010). Insect. Mol. Biol. 19: 61-75; Kim and Lee (2014). Front. Cell. Infect. Microbiol. 3: 116; Lee and Hase (2014). Nat. Chem. Biol., 10: 416-424).
The TH-Transferrin, Arylphorin β subunit, chymotrypsinogen-like protein 1 and few other immunity related genes do not belong to the conventional PRR type immune responsive genes. However, these genes were included as they potentially contribute to the midgut microbial homeostasis through IMD co-regulation (Pauchet Y, et al. (2010). Insect. Mol. Biol. 19: 61-75). Additionally identified were a TH gene indicated to be a valine rich midgut protein critical for the formation of midgut peritrophic matrix and related genes critical for maintaining the structural integrity of the midgut, which are possibly involved, in the gut microbial containment. (Odman-Naresh et al. (2013). PLoS ONE 8:e82015. 10.1371/journal.pone.0082015; Engel and Moran (2013). FEMS Microbiol Rev. 37 699-735). A non-insect gene that encodes for catalase 1 from cassava (Manihot esculanta) was used as a control.
TH larvae required for isolation of IRTG genes and subsequent bioassays were reared as follows: Eggs were procured from Carolina Biological Sciences (Burlington, N.C., USA). The eggs were not surface sterilized and used directly for conventional rearing (CR). The larval colony establishment and maintenance was performed employing a Phytatray II (Sigma, MO, USA) unit containing Gypsy Moth diet. (Gunaratna R T and Jiang H (2013). Dev. Comp. Immunol. 39: 388-398).
Bt.-resistant DBM eggs were procured from Benzon Research (Carlisle, Pa., USA) and reared conventionally, as described above.
FAW eggs were procured from Benzon Research (Carlisle, Pa., USA) and reared conventionally, as described above.
For germ free (GF) rearing procedure, all eggs were surface sterilized with a solution of Tween-80 (polyoxyethylene sorbitan monooleate), bleach, and distilled water as described previously. (Broderick N A, et al. (2009). Environ. Entomol. 29:101-107). The surface sterilized eggs were transferred to Phytatray II (Sigma, MO, USA) unit containing Gypsy Moth diet augmented with antibiotics (500 mg/l each of penicillin, gentamicin, rifampicin, streptomycin). (Broderick N A, et al. (2009). Environ. Entomol. 29:101-107; Gregory R. Richards (2008). Journal of Bacteriology. 190, 4870-4879).
Both CR and GF larvae were reared in an environmental chamber with a 16:8 hours (light:dark) photoperiod at 25° C., until use. For the induction of a representative set of MIGGS-IRTGS of PRR category, 75 colony forming units (CFU) of DH5a competent cells (Invitrogen, CA, USA) re-suspended in PBS buffer were injected into healthy 2-3 instar M. sexta larvae. The larvae were snap frozen in liquid nitrogen and processed for RNA isolation and cDNA synthesis described herein.
For testing the up-regulation of MIGGS-IRTGS by oral feeding on induction media, first instar larvae were reared on Luria broth agar media plated with a mixture of live E. coli (3×107 cells), M. luteus (30 μg), and curdlan (30 μg) in 50 μl of H2O (Wang et al. (2006). J. Biol. Chem. 281(14): 9271-9278).
Total RNA was isolated using RNeasy Mini Kit reagent (QIAGEN, NY, USA) and treated with TURBO DNase (Ambion-Life Technologies, NY, USA) using manufacturer's protocols. One μg of DNase treated RNA was used for cDNA synthesis using iScript cDNA synthesis kit (Bio-Rad, CA, USA). The cDNA was used as a template for amplifying near full-length transcripts of the IRTG. Similarly, a control gene from cassava was also amplified. For tissue specific cDNA synthesis the control and treatment larvae were squeezed to isolate hemolymph fraction (HL), dissect midgut (MDG) to obtain rest of the body as described in Pauchet et al. (2010). Insect. Mol. Biol. 19: 61-75). The cDNA template was used for RT-PCR reactions were appropriate using the SuperScript III One-Step RT-PCR system following manufacturers protocol (Thermo Scientific; USA).
Transcripts of TH, DBM, and FAW MIGGS-IRTGS and non-insect control genes were PCR amplified using PrimeSTAR GXL DNA Polymerase (Clontech Laboratories, CA, USA). The PCR reactions were conducted using the following conditions: denaturation at 98° C. for 30 s, annealing at 55/60° C. for 30 s and elongation at 72° C. for 45 s, for 35 cycles. The PCR products were resolved by agarose gel electrophoresis and stained with ethidium bromide. The transcripts were gel eluted using QIAquick gel extraction kit (QIAGEN, NY, USA).
Sequence confirmed transcripts were cloned into pCR8/GW vector (Invitrogen, CA, USA) using manufacturer's protocol. The sequence confirmed recombinant pCR8 clones were cloned into L4440gtwy using LR clonase enzyme (Inivtrogen, CA, USA). The L4440gtwy is a modified version of Timmons and Fire feeding Vector and was a kind gift from Guy Caldwell (Addgene plasmid #11344). (Timmons & Fire (1998). Nature, 395: 854).
For ingestible RNAi bioassays, sequence confirmed MIGGS-IRTG were cloned into an L4440 feeding vector between two T7 promoters in inverted orientation and transformed into an E. coli bacterial strain carrying IPTG-inducible expression of T7 polymerase, HT115 (DE3). (Timmons & Fire (1998). Nature, 395: 854). Modification of IRTG in this manner was previously demonstrated to induce the expression of dsRNA. (Timmons L, et al. (2001). Gene, 263, 103-112; Kamath R S, et al. (2000). Genome Biol. 2: 1-10.).
The HT115 (DE3) strain is an RNase III-deficient E. coli strain whose T7 polymerase activity is IPTG-inducible. The HT115 (DE3) genotype is as follows: F-, mcrA, mcrB, IN (rrnD-rrnE) 1, lambda -, rnc14::Tn10 (DE3 lysogen: lavUV5 promoter—T7 polymerase) (IPTG-inducible T7 polymerase) (RNase III minus), with tetracycline as a selectable marker. (Kamath R S, et al. (2000). Genome Biol. 2:1-10). The standard heat shock protocol for transformation of L4440::IRTG and control construct was used.
Single colonies of HT115 bacteria containing cloned L4440 plasmids were picked and grown in a 5 mL LB culture with 50 mg/ml ampicillin (Amp) One mL of the liquid culture was saved for plasmid isolation followed by sequence confirmation of the L4440::IRTG clone. The recombinant bacterial clones were grown for 8 hours in liquid culture and were seeded directly on LB plates containing 1 mM IPTG and 50 mg/ml Amp for inducing the dsRNA. (Kamath R S, et al. (2000). Genome Biol. 2: 1-10). Seeded plates were allowed to dry under laminar airflow chamber and incubated at 37° C. temperature overnight.
For larval bioassay three to five 1-2-instar TH, DBM, and FAW larvae were placed on induced plates containing HT115 (DE3) cells containing the desired L4440::IRTG. Bioassays were conducted testing the TH larvae against MIGGS-IRTGS and controls listed in Table 1.
Phenotypic differences in the larval development on L4440::IRTG containing HT115 (DE3) plates were documented and compared with the larval growth on negative and positive controls containing HT115 (DE3) plates. The larval phenotypes for a given treatment with appropriate controls were only considered true and documented if they were reproducibly observed in 2/3 or 4/5 larvae, in at least two independent feeding experiments.
For sprayable RNAi, a 24-well plate-based bioassay system was developed using a modified cetyl trimethylammonium bromide method of MEGAscript RNAi kit following manufacturer's protocol (Thermo Scientific, USA) for large-scale purification of dsRNA against a given MIGGS-IRTG. Integrity of the dsRNA was determined by electrophoresis on 1% agarose gel and its concentration determined using NanoDrop UV-VIS spectrometer. Leaf discs of 1 cm2 diameter were detached from Nicotiana benthamiana (for TH) or Arabidopsis Col-WT (for DBM) or wheat cultivar Bobwhite (for FAW) plants grown on field soil were drop inoculated with 0, 4, 8 or 16 μg of purified dsRNA in TE buffer. Air-dried dsRNA coated leaf discs were placed in the bioassay plate containing 1 mL of 1% Murashige and Skoog agar medium per well. Each well contained one leaf disc and was infested with conventionally reared three first instar TH or DBM or FAW larvae. dsRNA coated leaf discs were replaced once every 24 hours and insecticidal activity of each dsRNA measured as a function of larval mortality after five days continuous feeding. The RFB bioassays were conducted using a previously published flour disc assay protocol (Cao et al. (2018). Int. J. Mol. Sci. 19, 1079) with adult beetles.
For RNA-Seq analysis to test if the MIGGS pathway genes are induced by soil microbiome in FAW, wheat (Triticum aestivum) seeds were surface sterilized and planted in 4.5″ pots containing field soil or filled with 4:1 sterile turface:sand mix. Seedlings were grown a growth chamber at for 20 days and infested with ten first-instar FAW larvae per pot. Vigorous larval feeding activity was confirmed and larval samples collected for RNA-Seq analysis. A “pooled RNA-Seq” approach (Rajkumar et al. (2015). BMC Genomics. 16(1): 548) was used to obtain a snap shot of differential FAW gene expression in response to feeding on plants grown on microbe rich (field soil) and microbe depleted (sterile surface) substrate.
In order to demonstrate additional dsRNA delivery methods a plastidal dsRNA expression system was employed. Since high concentrations of long dsRNAs can be stably produced in plastids (Zhang et al. (2015). Science. 347(6225): 991-994), three of the most potent insecticidal TH MIGGS-IRTGS (SEQ ID NO: 3, 4, and 43) dsRNAs (dsMsPGRP2; dsMsβGRP2 and dsMsCHS2) were expressed by plastid transformation in tobacco plants in collaboration with Plastomics Inc. following a previously published protocol (Zhang et al. (2015). Science. 347(6225): 991-994). Detached leaves of stable transplastomic lines expressing dsRNA against MIGGS targets were fed to TH larvae and assessed for insecticidal activity.
Bioassays (
The representative phenotypes of TH larvae at 192 hours post exposure (HPE) to larvae exposed to bacterially (HT115 (DE3)) expressed dsRNA against MIGGS RNAi targets MsPGRP2 (
Consistent with above, the TH larvae exposed to bacterially expressed negative control MeCAT1 dsRNA displayed vigorous feeding (area between the arrowheads
In general, melanization is a highly conserved immune response and is often associated with microbial infection of insects. (Kim S R, et al. (2005). Insect molecular biology, 14(2): 185-194. doi:10.1111/j.1365-2583.2004.00547). The intensification of melanotic response in TH larvae upon continued exposure to bacterially expressed dsRNA against the MIGGS RNAi targets MsPGRP2 and MsVMP1 containing HT115 (DE3) plates strongly indicates an infection, possibly due to the defective clearance of opportunistic microbes ingested during feeding. Such defective clearance has been previously associated with the perturbation of gut microbial homeostasis. (Packey and Sartor (2009). Curr. Opin. Infect. Dis. 22(3): 292-301).
Closer observation indicated that the CR larvae feeding on bacterially expressed dsRNA against the MIGGS RNAi targets MsPGRP2 and MsVMP1 displayed discernable mortality starting at day 5, reaching up to 100% and 80% mortality respectively, by day 8 (
To test if both the incidence and intensity of the observed phenotype could be delayed by clearing gut microbiotas, a GF set of TH larvae were also subjected to the above treatment with appropriate controls, in parallel. Interestingly, that the incidence of larval mortality was not only delayed, but also lower in GF larvae in comparison to CR larvae (
The incidence of larval mortality on bacterially expressed dsRNA against MIGGS RNAi targets MsPGRP2 and MsVMP1 plates also correlated with the development of melanotic reaction (
Although MsVMP1 is not directly involved in immune responses, down regulation may abrogate microbial containment, resulting in an infectious phenotype (
Most of the TH MIGGS RNAi targets disclosed herein (e.g., Table 1) are inducible and have been identified from open access midgut specific immunotranscriptome and/or other datasets (Pauchet Y, et al. (2010) Insect. Mol. Biol. 19:61-75; Odman-Naresh et al. (2013) PLoS ONE 8:e82015; Kanost M R, et al. (2016) Insect Biochem. Mol. Biol 76:118-147; Brummett et al. (2017) Insect Biochem Mol Biol. 81: 1-9; Cao X, et al. (2015) Insect Biochem. Mol. Biol. 62:64-74); Zhong X, et al. (2012) Insect Biochem. Mol. Biol. 42(7): 514-524; Xia Xu et al (2012). Dev Comp Immunol. 38(2): 275-284). A validation study targeting two MIGGS RNAi targets MsHEM and MsSPH3 confirmed their preferential midgut specific manner (
Oral feeding of TH larvae on induction media containing a mixture of live E. coli and lyophilized cell wall signatures from gram positive bacteria and fungi (
High-throughput screening of microbe induced MIGGS RNAi targets (
Additionally, a combination of reciprocal best BLAST analysis and literature curation was used to identify the orthologs of TH MIGGS RNAi targets from the DBM transcriptomic resources (Table 2).
It was demonstrated that 14 MIGGS RNAi target genes identified could also be induced in Bt resistant strain of DBM feeding on the induction media (
High throughput screening of microbe induced DBM MIGGS RNAi target genes (
In order to test if our MIGGS RNAi technology could work using multiple dsRNA delivery platforms we tested if dsRNA against the four TH MIGGS insecticidal targets could work in a sprayable format. We used dsRNA against MsPGRP2, MsβGRP2, MsCHS2 and MsVMP1 for sprayable RNAi assays (
Similarly, in order to test an additional delivery platform, the core insecticidal MIGGS RNAi targets MsPGRP2; MsβGRP2 and MsCHS2 were expressed by plastid transformation in tobacco plants in collaboration with Plastomics Inc. Detached leaves of stable transplastomic lines (
Next, we tested if our MIGGS RNAi technology can also work in a sprayable format against Bt resistant strain of DBM. We used the DBM orthologs of insecticidal TH MIGGS core set (PxPGRP2, PxβGRP2 and PxCHS2) and two newly discovered insecticidal DBM MIGGS targets (PxCAC and PxIMD1) for sprayable RNAi assays at a concentration of 0.1, 0.5 and 0.25 μg. We observed lethality ranging from 53-67%, with PxPGRP2 being the most effective target for killing DBM larvae (
Most of our MIGGS RNAi target gene induction procedures thus far relied upon either direct injection or oral feeding of extraneously supplied microbial signatures. To determine if our MIGGS RNAI target genes are induced under field conditions, we exposed the larvae of economically important lepidopteran pest FAW to wheat seedlings grown on microbe rich and microbe depleted plants (
Most notably, FAW orthologs of TH insecticidal targets including PGRP2, βGRP2 and IMD were captured in the data set. This discovery indicated that MIGGS pathway genes are up regulated in response to insect feeding on plants exposed to microbes in the field soil.
Preliminary experiments were performed to screen the insecticidal activity of 17 FAW MIGGS RNAi targets discovered during RNA-Seq (Table 3, above) in which dsRNA against the FAW orthologs of insecticidal TH MIGGS core set (SfPGRP2 (SEQ ID NO. 89), SfβGRP2 (SEQ ID NO. 101) and SfCHS2 (SEQ ID NO. 105) and three newly discovered MIGGS targets SfCTL (SEQ ID NO. 96), SfRC (SEQ ID NO. 103) and SfGAL (SEQ ID NO. 92) from RNA-Seq were fed to FAW larvae at 0, 4, 8 or 16 μg-purified dsRNA in TE buffer. FAW 1st instar larvae were allowed to feed on dsRNA coated leaves following the bioassay described for TH above. Data indicated that FAW larvae (
Experiments with an economically important coleopteran pest RFB were also conducted to test if MIGGS-IRTGS targets identified by a combination of reciprocal best BLAST analysis and literature curation (Table 4) are insecticidal against the order coleoptera.
Preliminary feeding trails with 1 μg of purified indicated that dsRNA against RFB MIGG RNAi targets TcPGRP2 (SEQ ID NO. 107), TcβGRP2 (SEQ ID NO. 108), TcMDGP (SEQ ID NO. 109) and TcCHS2 (SEQ ID NO. 110) is insecticidal to the adult RFB beetles. Significant rates of RFB mortalities were observed (Figure. 25) when scored on a 0-3 scale were 0, 1, 2 and 3 indicated ≤0, 25, 50 or ≥50 mortality respectively in comparison to the negative control treatment. Data is average of 3 replicates/treatment ±SEM at p≤0.001(***); p≤0.01(**) and p≤0.05(*).
One possible reason for larval mortality could involve down regulation of MIGGS-IRTGS transcripts upon feeding on exogenously supplied dsRNA against the target genes. Preliminary RT-PCR data indicated that the larval phenotypes (
Importantly RNA-Seq analysis indicated that the MIGGS-IRTG pathway targets are induced by soil microbiome indicating that our novel RNAi approach could be effective even under field conditions.
Given the ease of identification, high specificity, and applicability to diverse pests and delivery platforms, RNAi silencing of the MIGGS-IRTG pathway genes identified, this approach offers an unprecedented potential as a novel pesticidal strategy.
The translation of these preliminary findings into a pesticidal RNAi technology against economically important pests might lead to sustainable alternatives including but not restricted to the methods described anywhere herein.
Additionally, the proposed approach will shed more light into understanding the tri-trophic interaction between plants-microbe-insect interactions as it pertains to sustainable insect pest protection.
SEQ ID NOs: 1-14 and 31-44 are representative examples of M. sexta-RNAi target gene sequences.
SEQ ID NO: 15 is non-insect gene sequence that encodes for catalase 1 from cassava (Manihot esculanta).
SEQ ID NOs: 16-29 are coding region sequences of representative M. sexta-RNAi target genes.
SEQ ID NO: 30 is the coding region sequence of catalase 1 from cassava (Manihot esculanta).
SEQ ID NOs: 45-70 are 5′UTR and 3′UTR region sequences of representative M. sexta-RNAi target genes.
SEQ ID NOs: 71-75 are the coding region sequences of additional representative M. sexta-RNAi target genes.
SEQ ID NOs: 76-88 are the coding region sequences of representative P. xylostella-RNAi target genes.
SEQ ID NOs: 89-105 are the coding region sequences of representative S. frugiperda-RNAi target genes.
SEQ ID NOs: 106-110 are the coding region sequences of representative T. castaneum-RNAi target genes.
SEQ ID NOs: 111-119 are representative examples of Manduca sexta insecticidal dsRNA sequences.
SEQ ID NOs: 120-126 are representative examples of Plutella xylostella insecticidal dsRNA sequences.
SEQ ID NOs: 127-135 are representative examples of Spodoptera frugiperda insecticidal dsRNA sequences.
SEQ ID NOs: 136-139 are representative examples of Tribolium castaneum insecticidal dsRNA sequences.
Manduca sexta Caspase-6; HM234679.1 in NCBI
S. frugiperda-Attacin (SfAtta); rep_c9395
S. frugiperda-CtypeLectin15 (SfCTL15); Joint2_rep_c488
S. frugiperda-Galectin4 (SfGlc4); rep_c2653
S. frugiperda-Lysozyme (SfLys); rep_c18992
S. frugiperda-Hemolymph proteinase 10 (SfHP10); c12881
S. frugiperda- Trypsin like serine protease (SfTSP); rep_c48453
S. frugiperda- C Type Lectin 6 (SfCTL6); Joint2_ rep_c448
S. frugiperda- Serine protease homolog 13 (SfSph13); rep_c1904
S. frugiperda- Cecropin (SfCec); rep_c42380
S. frugiperda- Relish (SfRel); c13122
S. frugiperda- Toll (SfToll); joint2_ c3284
S. frugiperda- Beta 1, 3 glucanase recognition protein (SfβGRP2); EF641300
S. frugiperda- c20042 (Sfc20042); Un-annotated
S. frugiperda- rc16438 (Sfrc16438); Un-annotated
S. frugiperda- j2rc2367 (Sfrc2367); Un-annotated
S. frugiperda- Chitin synthase B (SfChsB); AY52599)
T. castaneum- Peptidoglycan recognition protein LC (TcPGRPLC);
T. castaneum- Peptidoglycan recognition protein 2 (TcPGRP2);
T. castaneum- Beta 1-3 glucan binding protein (TcβGRP2); XM_966587.4
T. castaneum- midgut protein (TcMDGP); XM_971351
T. castaneum- Chitin synthase 2 (TcCHS2); EFA 10719.1
The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
This application claims priority to PCT Application No. PCT/US2018/030506, filed May 1, 2018, which claims the benefit of U.S. Provisional Application No. 62/492,556, filed May 1, 2017, the disclosures of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/030506 | 5/1/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62492556 | May 2017 | US |
