Patent Application
20170211082
Sequence Listing
The Sequence Listing submitted via EFS-Web as ASCII compliant text file format (.txt) filed on Jan. 25, 2017, named “SequenceListing_ST25”, (created on Jan. 22, 2016, 24 KB), is incorporated herein by reference. This Sequence Listing serves as paper copy of the Sequence Listing required by 37 C.F.R. §1.821(c) and the Sequence Listing in computer-readable form (CRF) required by 37 C.F.R. §1.821(e). A statement under 37 C.F.R. §1.821(f) is not necessary.
Field of the Invention
This invention relates to double stranded RNA and compositions containing double stranded RNA (dsRNA) to reduce the fitness and/or increase mortality of Diaphorina citri. (Asian citrus psyllid, Hemiptera: Liviidae) thereby reducing infestation of D. citri on citrus plants, and thereby reducing transmission of plant pathogens for which D. citri is a vector. One example of such microorganisms are Candidatus Liberibacter species. The invention also relates to methods of reducing the fitness and/or increasing mortality of D. citri by applying dsRNA as topical sprays, soil treatment, or other delivery methods to plants on which D. citri feeds. The invention also relates to genetically altered plants that express the dsRNA described herein; and to genetically altered microorganisms that express the dsRNA described herein.
Description of Related Art
Huanglongbing (HLB) (also called “citrus greening disease”) is the most serious disease that threatens citrus crops worldwide. It is especially devastating to the U.S. and Florida citrus industries. The causative agents of HLB are Candidatus Liberibacter species of bacteria. This includes Candidatus Liberibacter africanus (CLaf), Candidatus Liberibacter asiaticus (CLas), and Candidatus Liberibacter americanus (CLam), however, until 2015 only CLas was found in U.S. The CLas and CLam bacteria are transmitted from plant to plant via Asian citrus psyllid (ACP; D. citri). The CLaf bacteria is transmitted from plant to plant via the psyllid Trioza enytreae. This disease is devastating the citrus industry in Florida because no effective treatments currently exists. Furthermore, both the causative pathogen and the insect vector have spread to other parts of the U.S. (California, Texas, Arizona) as well as to other citrus-producing countries/regions (Brazil, Asia, Middle East, China). Further, Murraya paniculata and other plants also host CLam and/or CLas. D. citri feed on these infected plants and carry the bacteria to non-infected plants. A list of these host plants could be found in freshfromflorida.com/content/download/24041/486974/hostlist.pdf. As such, the need exists for a composition and methods to stop the transmission of the disease. One approach to preventing disease transmission is to increase the mortality of ACP, the vector for the bacteria. Another approach is to reduce the fitness of ACP. Reducing ACP populations, by increasing mortality or reducing fitness, is the goal, thereby reducing transmission of the bacteria and the disease. One approach for reducing ACP populations, while not reducing beneficial insects, is to use RNAi technology.
Fire, et al. (U.S. Pat. No. 6,506,559) disclose a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. This cellular mechanism was named RNA interference, or RNAi. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559) disclose a method to inhibit expression of a target gene in a cell, the method involves introducing a double-stranded ribonucleic acid, dsRNA, into the cell in an amount sufficient to trigger the RNAi process which leads to inhibition of specific protein translation of the target gene's messenger RNA (mRNA). One strand of the dsRNA trigger has a sequence which corresponds to the nucleotide sequence of the target mRNA. The dsRNA triggers the cell's natural defense mechanism, defined as RNA interference (RNAi), which uses the dsRNA trigger (designed dsRNA herein), to produce the guide strand small interfering RNA (siRNA) which is incorporated into the RNA-induced silencing complex (RISC) which is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a double-stranded RNA (dsRNA) fragment. The RISC siRNAA— complex causes degradation of the targeted mRNA, thereby preventing translation into a protein. As used herein, a “trigger” is any dsRNA molecule that causes RNAi activity against a specific gene.
One mechanism of action involves a long trigger (i.e., a long dsRNA) being cleaved by an enzyme called “dicer” to produce several siRNA that are shorter in length than the dsRNA. The size of these smaller siRNA is believed to range from about 19 base pairs to about 25 base pairs, but the most common classes of siRNA contain 21 base pairs or 24 base pairs (Hamilton, et al., 2002 EMBO J., 21:4671-4679). However, others have determined that the siRNA can be shorter than 19 base pairs. See, e.g., Guleda, et al., Geno. Proteomic Bioinfo., 184(6)183-199 (2011). The siRNA molecules are each then incorporated into RISC. The duplex RNA is unwound leaving the anti-sense strand to guide RISC to complementary mRNA for subsequent endonucleolytic cleavage. This results in the reduction of the corresponding protein that would have been made from the targeted and degraded mRNA. Thus, this is commonly referred to as gene-silencing or downregulation.
A few RNAi sequences have been identified and are currently being evaluated for (i) reducing ACP vector capacity to host CLas, and (ii) nymph and/or adult survival. See, e.g., El-Shesheny, et al. (2013) PLoS ONE 8(5): e65392 (doi.org/10.1.3711journal.pone.0065392); and Killiny, et al. (2014) PLoS ONE 9(10): e110536 (doi.org/10.1371/journal.pone.0110536). Li, et al., Insect Sci., 20:31-39 (2013) reviews attempts to use RNAi to control Hemiptera and the overall lack of success of killing Hemiptera via this technique.
One goal of using RNAi is that if one selects a RNA sequence that is relatively unique to the target pest (in this case, D. citri) and which is effective in reducing expression of the target gene such that increased mortality of the target pest occurs, then the treatments can avoid harming beneficial insects, such as pollinators, predators, and parasitoid species. This invention successfully meets these goals in that the dsRNA sequence for ACP trehalase mRNA target (described herein) appears to be unique to ACP and does not harm bees or other pollinators. Trehalase hydrolyzes the disaccharide α,α-trehalose to two molecules of D-glucose. α,α-trehalose is the main sugar found in insect hemolymph (insect blood), and it has a critical role in energy production and biosynthesis of macromolecules including chitin. Trehalase has been purified and characterized from several different insects where it is found in soluble (Tre-1) and membrane-bound (Tre-2) forms encoded by distinct genes. The soluble form is found in hemolymph and the bound form is found in many different insect tissues See, Lee, et al., (2007) Biosci Biotechnol Biochem 71(9);2256-65.
The coding sequence of ACP trehalase (hereinafter “trehalase”) was obtained by automated computational analysis from a D. citri genomic sequence (NW_007377674.1), annotated using the gene prediction method Gnomon (NCBI, Rockville, Md.). See also, Reese, et al. (2013) J. Genomics 2:54-58; doi: 10,7150/jgen.7692. Supporting evidence includes similarity to 16 proteins, and 100% coverage of the annotated genomic feature by RNAseq alignments, including 5 samples with support for all annotated introns. Comparative analysis of the coding sequence of trehalase with other insects genomic sequences indicate that it corresponds to Tre-1 (soluble form of trehalase), as it shares higher homology to other known insects soluble trehalase homologs (Aphis glycines, GenBank: mRNA: JQ246351.1/protein: AFJ00065.1, Nilaparvata lugens, mRNA: H790319.1/protein: ACN85420.1; Locusta migratoria, mRNA: H795020.1/protein: ACP28173,1). See, Reese, et al. (2013) J. Genomics 2:54-58 (doi: 10.715/gen.7692); Hunter, et al. (2009) Open Entomol. 3:18-29; Hunter, et al. (2008) Florida Entomology Society meeting, abstracts, p.11 (flaentsoc.org/2008annmeetabstracts.pdf); and Hunter, et al. (2014) J. Citrus Pathology 1:4.7 (Proc. 3rd Int'l Res. Conf, Huanglongbing, Orlando, Fla.). The cDNA sequence of trehalase is in SEQ ID NO: 26.
A need exists for reducing transmission of CLas and/or CLam to non-infected plants, and in particular, citrus plants. A method of reducing fitness and survival of D. citri (and thereby reducing D. citri infestation) reduces the number of infected D. citri that can transmit HLB pathogens to uninfected plants, thereby slowing down or stopping pathogen transmission and disease spread. Furthermore, any solution to this problem which does not reduce beneficial insects, such as predators and parasitoids of psyllids, as well as beneficial pollinators, will have the added benefits of biological control pressures that will help suppress psyllid populations.
It is an object of this invention to have a dsRNA that reduces the fitness and/or increases the mortality (or reduces the survival) of D. citri after the D. citri ingests the dsRNA. This dsRNA contains a sense region containing between approximately sixteen nucleotides and 1908 nucleotides, and an anti-sense region complementary to the sense region. The anti-sense region is also complementary to D. citri trehalase cDNA or a fragment of D. citri trehalase cDNA. This D. citri trehalase cDNA has the sequence of SEQ ID NO: 26 or encodes D. citri trehalase having the amino acid sequence of SEQ ID NO: 29. The fragment of D. citri trehalase cDNA has the sequence of SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO. 11. It is another object of this invention that the sense region of this dsRNA has a sequence of between 16 nt and 469 nt of SEQ ID NO: 4, between 16 nt and 438 nt of SEQ ID NO: 9, between 16 nt and 730 nt of SEQ ID NO: 14, or between 16 nt and 1908 nt of the RNA equivalent of SEQ ID NO: 26.
It is another object of this invention to have a D. citri trehalase dsRNA solution that contains an agriculturally acceptable carrier and D. citri trehalase dsRNA. It is another object of this invention that one strand of the D. citri trehalase dsRNA has one of the following sequences: between 16 nt and 469 nt of SEQ ID NO: 4, between 16 nt and 438 nt of SEQ ID NO: 9, between 16 nt and 730 nt of SEQ ID NO: 14, between 16 nt and 1908 nt of the RNA equivalent of SEQ ID NO: 26, and between 16 nt and 1908 nt of the RNA equivalent of a cDNA encoding D. citri trehalase having an amino acid sequence of SEQ ID NO: 29. It is a further object of this invention that the agriculturally acceptable carrier of the dsRNA solution can be water, surfactant, liposome, lipid, protein, peptide, nanotube, chitin, inactivated microorganism, or a combination thereof. This dsRNA solution can also contain a compound that prevents dsRNA degradation, a translaminar chemical, a mineral, a clay, a fertilizer, a sugar, or a combination thereof.
It is an object of this invention to have a method of reducing D. citri infestation on a treated plant (the number of D. citri that feed of the treated plant) compared to the D. citri infestation on an untreated plant (the number of D. citri that feed of the untreated plant) by administering the D. citri trehalase dsRNA solution described supra to an untreated plant in an amount effective to kill D. citri that ingest or absorb the dsRNA solution to generate a treated plant, and allowing the D. citri to ingest or absorb the dsRNA solution and thereby killing the D. citri that ingested or absorbed the dsRNA solution. This dsRNA solution contains an agriculturally acceptable carrier and D. citri trehalase dsRNA which has insecticidal activity against D. citri, thereby reducing the D. citri infestation on the treated plant compared the D. citri infestation on the untreated plant. In one embodiment of this invention, the administering step involves spraying the D. citri trehalase dsRNA solution onto the untreated plant to generate the treated plant. In another embodiment of this invention, the administering step involves applying the D. citri trehalase dsRNA solution to the soil surrounding the untreated plant to allow for the roots of the untreated plant to absorb the D. citri trehalase dsRNA solution and/or the D. citri dsRNA, thereby generating the treated plant. An alternative embodiment of this invention, the administering step involves applying the D. citri trehalase dsRNA solution to one or more roots of the untreated plant and the roots absorbs the D. citri trehalase dsRNA solution and/or the D. citri dsRNA to generate the treated plant.
It is an object of this invention to have a method for reducing the fitness or survival of D. citri that feed on an altered plant that contains a D. citri trehalase dsRNA by introducing the D. citri trehalase dsRNA into a wild-type plant upon which the D. citri feeds, thereby producing the altered plant containing the D. citri trehalase dsRNA, and allowing D. citri to feed on the altered plant containing the D. citri trehalase dsRNA and ingest the D. citri trehalase dsRNA, such that the D. citri trehalase dsRNA reduces the fitness or survival of D. citri that ingest the D. citri trehalase dsRNA. In one embodiment of this invention, the D. citri trehalase dsRNA has one of the following sequences: between 16 nt and 469 nt of SEQ II) NO: 4, between 16 nt and 438 nt of SEQ ID NO: 9, between 16 nt and 730 nt of SEQ ID NO: 14, between 16 nt and 1908 nt of the RNA equivalent of SEQ ID NO: 26, and between 16 nt and 1908 nt of the RNA equivalent of a cDNA encoding D. citri trehalase having amino acid sequence of SEQ ID NO: 29. The introducing of the D. citri trehalase dsRNA into the wild-type plant can occur by spraying a dsRNA solution containing the D. citri trehalase dsRNA onto the wild-type plant; by applying the dsRNA solution containing the D. citri trehalase dsRNA to the roots of the wild-type plant, or by applying the dsRNA solution containing the D. citri trehalase dsRNA to the soil around the wild-type plant so that the roots of the wild-type plant absorb the D. citri trehalase dsRNA solution and/or the D. citri trehalase dsRNA. This dsRNA solution can also contain an agriculturally acceptable carrier (such as water, surfactant, liposome, lipid, protein, peptide, nanotube, chitin, inactivated microorganism, or a combination thereof) or other compounds (such as, a compound that prevent dsRNA degradation, a translaminar chemical, a mineral, a clay, a fertilizer, a sugar, or a combination thereof).
It is another object of this invention to have a method for reducing the fitness or survival of D. citri that feed on an altered plant that contains a D. citri trehalase dsRNA by introducing an expression vector encoding D. citri trehalase dsRNA into a wild-type plant cell upon which the D. citri feeds, thereby producing an altered plant cell, selecting an altered plant cell that produces the D. citri trehalase dsRNA, inducing the altered plant cell that produces the D. citri trehalase dsRNA to grow into an altered plant that produces and contains the D. citri trehalase dsRNA, and allowing D. citri to feed on the altered plant containing the D. citri trehalase dsRNA and ingest the D. citri trehalase dsRNA, such that the D. citri trehalase dsRNA reduces the fitness or survival of D. citri that ingest the D. citri trehalase dsRNA. In this embodiment, the expression vector has at least one heterologous promoter operably linked a polynucleotide that contains a sense region and an anti-sense region. It is a further object of this invention that the sequence of the sense region is between 16 nt and 469 nt of SEQ ID NO: 1, between 16 nt and 438 nt of SEQ ID NO: 6, between 16 nt and 730 nt of SEQ ID NO: 11, between 16 nt and 1908 nt of the RNA equivalent of SEQ ID NO: 26, and between 16 nt and 1908 nt of the RNA equivalent of a cDNA encoding D. citri trehalase having amino acid sequence of SEQ ID NO: 29. Furthermore, the anti-sense region has a sequence complementary to the sequence of the sense region. In one embodiment of the invention, one promoter controls transcription of the sense region and the anti-sense region. In another embodiment, one promoter controls transcription of the sense region, and a second promoter controls transcription of the anti-sense region.
It is another object of this invention to have a method of reducing transmission by D. citri of a disease-causing microorganism from a treated plant to an untreated plant by applying the dsRNA solution described supra to a wild-type plant to produce the treated plant, and allowing D. citri to feed on the treated plant and ingest or absorb the dsRNA solution, such that the dsRNA solution contains an agriculturally acceptable carrier and a D. citri trehalase dsRNA, and such that the D. citri trehalase dsRNA kills the D. citri that ingests or absorbs the D. citri trehalase dsRNA, because dead D. citri are unable to transmit the disease-causing microorganism to an untreated plant. It is a further object of this invention that the D. citri trehalase dsRNA has one of the sequences described above. The step of applying the dsRNA solution to the wild-type plant occurs by (i) spraying the dsRNA solution onto the wild-type plant, (ii) applying the dsRNA solution to the roots of the wild-type plant, and/or (iii) applying the dsRNA solution to the soil around the wild-type plant so that the roots of the wild-type plant absorb the dsRNA solution containing the D. citri trehalase dsRNA.
It is an object of this invention to have a genetically altered plant, and parts thereof (i.e., leaves, flowers, stems, roots, cell, pollen, protoplast, etc.), that contain an expression vector which has a first promoter operably linked to a D. citri trehalase sense polynucleotide and a second promoter operably linked to a D. citri trehalase anti-sense polynucleotide, such that the D. citri trehalase sense polynucleotide and the D. citri trehalase anti-sense polynucleotide are complementary to each other. The D. citri trehalase sense polynucleotide has a sequence of between 16 nt and 469 nt of SEQ ID NO: 1, between 16 nt and 438 nt of SEQ ID NO: 6, between 16 nt and 730 nt of SEQ ID NO: 11, between 16 nt and 1908 nt of SEQ ID NO: 26, and/or between 16 nt and 1908 nt of a cDNA encoding D. citri trehalase having the amino acid sequence of SEQ ID NO: 29. In one embodiment, the first promoter and the second promoter are the same promoters. In another embodiment, the first promoter and the second promoter are different promoters.
It is another object of this invention to have a genetically altered plant, and parts thereof (i.e., leaves, flowers, stems, roots, cell, pollen, protoplast, etc.), that contains an expression vector having a promoter operably linked to a polynucleotide which has a D. citri trehalase sense region and a D. citri trehalase anti-sense region, such that the D. citri trehalase anti-sense region is complementary to the D. citri trehalase sense region. The D. citri trehalase sense polynucleotide has a sequence of between 16 nt and 469 nt of SEQ ID NO: 1, between 16 nt and 438 nt of SEQ ID NO: 6, between 16 nt and 730 nt of SEQ ID NO: 11, between 16 nt and 1908 nt of SEQ ID NO: 26, and/or between 16 nt and 1908 nt of a cDNA encoding D. citri trehalase having the amino acid sequence of SEQ ID NO: 29.
It is an object of this invention to have a method for making a genetically altered plant that produces a D. citri trehalase dsRNA by transforming a wild-type plant cell with a D. citri trehalase dsRNA producing expression vector to generate a genetically altered plant cell that produces the D. citri trehalase dsRNA, and growing the genetically altered plant cell that produces the D. citri trehalase dsRNA to produce the genetically altered plant that produces the D. citri trehalase dsRNA. Two D. citri trehalase dsRNA producing expression vectors are described supra. It is another object of this invention to have a genetically altered plant, and parts thereof (i.e., leaves, flowers, stems, roots, cell, pollen, protoplast, etc.), produced by this method and that produce D. citri trehalase dsRNA. It is a further object of this invention to have a genetically altered plant cell of this genetically altered plant.
It is another object of this invention to have a cDNA encoding D. citri trehalase with the sequence of SEQ ID NO: 26. It is another object of this invention to have an expression vector containing a heterologous promoter operably linked to a polynucleotide that encodes D. citri trehalase having the amino acid sequence of SEQ ID NO: 29.
It is another object of this invention to utilize the method of virus-induced gene silencing for plant gene to reduce transmission by D. citri of one or more disease-causing microorganisms from a treated plant to an untreated plant by the killing of D. citri or by reducing the fitness or survival of D. citri. It is a further object of this invention to have a recombinant virus containing a heterologous polynucleotide that encodes D. citri trehalase, a fragment thereof, and/or a sequence that is the reverse complementary thereof, such that the recombinant virus can be used to infect a plant in need of treatment and produce dsRNA based on the sequence of the heterologous polynucleotide during infection of the plant. The virus can be a DNA virus or an RNA virus. The heterologous polynucleotide can have a sense region; a sense region and an anti-sense region which is complementary to the sense region; or a sense region, an anti-sense region, and a linker. It is another object of this invention to generate an altered plant that can produce the dsRNA described hereby by infecting a wild-type plant with the recombinant virus that can produce the dsRNA described herein. In one embodiment, the recombinant virus is a recombinant Citrus tristeza virus.
The D. citri trehalase gene sequence is not identical to trehalase sequences in various beneficial insects which could be analyzed. As such, the use of D. citri trehalase dsRNA (or fragments of trehalase dsRNA) to reduce fitness and survival of D. citri provides a mechanism for reducing the spread of C. Liberibacter species and other microorganisms for which D. citri are a host from infected plants to uninfected plants without harming beneficial insects. Non-limiting example of such other microorganisms for which D. citri are carriers include reovirus. See, Marutani-Hert, et al. (2009) Florida Entomologist 92:314-320. Accordingly, the dsRNA can be administered to plants, or produced by genetically altered plants, or by microbes (bacteria/yeast/viruses/fungi etc.). The inventions described herein include the dsRNA described herein, compositions containing the dsRNA described herein, genetically altered plants or microbes that produce the dsRNA described herein, methods for generating the genetically altered plants, and methods of using the dsRNA described herein to reduce fitness and survival of D. citri populations and thereby reduce D. citri infestation, and also thereby reduce the transmission of microorganisms that infect plants, such as but not limited to C. Liberibacter species, by psyllid species, and more specifically by D. citri.
Table 1 provides an explanatory list of the sequences discussed herein and include for D. citri trehalase cds and dsRNA sequences; maternal protein exuperantia cds and dsRNA sequences; and pterin-4-alpha-carbinolamine dehydratase (PCBD1) cds and dsRNA sequences; and the primers used. Note that three cds for trehalase, each covering different sections of trehalase cds are listed in Table 1 and used in the examples, below.
Because this invention involves production of genetically altered plants and involves recombinant DNA techniques, the following definitions are provided to assist in describing this invention. The terms “isolated”, “purified”, or “biologically pure” as used herein, refer to material that is substantially or essentially free from components that normally accompany the material in its native state or when the material is produced. In an exemplary embodiment, purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid or particular bacteria that are the predominant species present in a preparation is substantially purified. In an exemplary embodiment, the term “purified” denotes that a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. Typically, isolated nucleic acids or proteins have a level of purity expressed as a range. The lower end of the range of purity for the component is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%, about 80%, about 90% or more than about 90%.
The term “gene” refers to a DNA sequence involved in producing a RNA or polypeptide or precursor thereof. The polypeptide or RNA can be encoded by a full-length coding sequence (cds) or by intron-interrupted portions of the coding sequence, such as exon sequences. In one embodiment of the invention, the gene target is the trehalase mRNA in ACP. The polynucleotide cds sequence of trehalase is SEQ ID NO: 26.
The term “nucleic acid” as used herein, refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically, “nucleic acid” polymers occur in either single- or double-stranded form, but are also known to form structures comprising three or more strands. The term “nucleic acid” includes naturally occurring nucleic acid polymers as well as nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Exemplary analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs) and bridged nucleic acid (RNA, 2′-O′-aminoethylene bridged nucleic acid. See, e.g., Rahman, et al. (2007) Nucleosides Nucleotides Nucleic Acids 26:1625-1628). “DNA”, “RNA”, “polynucleotides”, “polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein.
For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp), or nucleotides (nt). Estimates are typically derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). Because of the degeneracy of nucleic acid codons, one can use various different polynucleotides to encode identical polypeptides. Table 2. infra, contains information about which nucleic acid codons encode which amino acids.
The term “primer” refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. A primer may occur naturally, as in a purified restriction digest, or may be produced synthetically.
A primer is selected to be “substantially complementary” to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example; a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence is sufficiently complementary with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
“dsRNA” refers to double-stranded RNA that comprises a sense region and an antisense region of a selected target gene (or sequences with high sequence identity thereto so that gene silencing can occur), as well as any smaller double-stranded RNAs formed therefrom by RNAse or Dicer activity. Such dsRNA can include portions of single-stranded RNA, but contains at least 18 base pairs of dsRNA. A dsRNA after been processed by Dicer generates siRNAs (18-25 by in length) that are double-strand, and could contain ends with 2 nucleotide overhangs, which will be single-stranded. It is predicted that usually siRNA around 21 nt in length (or, alternatively, between 17 and 27 nt in length), will be incorporated into RISC. In one embodiment, the sense region and the antisense region of a dsRNA are on the same strand of RNA and are separated by a linker. In this embodiment, when the sense region and the anti sense region anneal together, the dsRNA contains a loop which is the linker. One promoter operably linked to the DNA or RNA encoding both the sense region and the antisense region is used to produce the one RNA molecule containing both the sense region and the anti-sense region. In another embodiment, the sense region and the antisense region are present on two distinct strands of RNA (a sense strand and the anti-sense strand which is complementary to the sense strand) which anneal together to form the dsRNA. In this embodiment, a promoter is operably linked to each strand of DNA or RNA; where one DNA or RNA strand encodes the RNA containing the sense region and the other strand of DNA or RNA encodes the RNA containing the anti-sense region. In this embodiment, the promoter on each strand can be the same as or different from the promoter on the other strand. After the RNAs are transcribed, two RNA strands anneal together because the sense region and the anti-sense region are complementary to each other, thus forming the dsRNA. In yet another embodiment, one strand of DNA or RNA can encode both the sense region and the anti-sense region of the dsRNA. However, the DNA or RNA coding each region are separated from each other so that two promoters are necessary to transcribe each region. That is, the DNA or RNA encoding the anti-sense region and the DNA or RNA encoding the sense region are operably linked to their own promoter. Again, the two promoters can be the same promoter or different promoters. In one embodiment, the promoter can be a T7 RNA polymerase promoter. Other promoters are well-known in the art and can be used (see discussion infra). While many embodiments of this invention use DNA to encode the sense region and/or anti-sense region, as described infra, it is possible to use a recombinant RNA virus to produce the dsRNA described herein. In such case, the genome virus is RNA which has been altered to have one of the sequences of D. citri trehalse described herein or reverse complement thereof.
Regarding the specificity, it is driven by the siRNA. There are two publications that addressed this issue in insects. One publication shows that to be specific, a siRNA should share a minimum of at least 19 nt in length with the target mRNA. See, Whyard, et al. (2009) Insect Biochem Molecul. Biol. 39:824-32. The second publication shows that it is necessary to share a contiguous sequence length of 20 nt or longer, between the dsRNA and the target mRNA for efficacy in degradation (silencing). See, Bachman, et al. (2013) Transgenic Res. 22:1207-22.
Active dsRNA molecules have worked when they were as long as 1,000 bp, and should work when even longer. For the purposes of the inventions described herein, any siRNA having at least 19 nt :length derived from SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16 and SEQ ID NO: 21 will be specific to ACP. This region is specific to ACP, because across the active trigger there are no regions of 25 nt or longer which is 100% identical to any known insect, animal, human or plant species sequence. In one embodiment the dsRNA can be any 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, or longer contiguous nucleotides of D. citri trehalase cDNA (SEQ ID NO: 26) or of SEQ ID NOs: 1, 6, and 11, up to and including all 469 nt of SEQ ID NO: 1, up to and including all 438 nt of SEQ ID NO: 6, up to and including all 730 nt of SEQ ID NO: 11, and up to and including all 1908 nt of SEQ ID NO: 26. In alternative embodiments, the dsRNA can range in length between 16 bp and 30 bp, between 16 bp and 25 bp, between 18 and 30 bp, and between 19 bp and 28 bp. In yet another embodiment, RNA forms that are created by RNAse III family (Dicer or Dicer-like ribonuclease) or Dicer activity that are longer dsRNA are within the scope of this invention.
siRNA can be synthetically made, expressed and secreted directly from a transformed cell, or microbe, or can be generated from a longer dsRNA by enzymatic activity. These siRNAs can be blunt-ended or can have 1 bp to 4 bp overlapping ends of various nucleotide combinations. Also modified microRNAs comprising a portion of the putative trehalase gene and its complementary sequence are included herein as dsRNAs. For clarification, “bp” is an abbreviation for “basepairs” and “nt” is an abbreviation for “nucleotide”.
In one embodiment of this invention, dsRNA is used to control ACP without such dsRNA being co-delivered with a transfection-promoting agent, although in some embodiments the dsRNA of the invention can be provided in a solution with a transfection-promoting agent. In one embodiment of the invention, the dsRNA is expressed in a plant to be protected, or expressed in microorganisms which can be endemic organisms of the plant (microbes, virus, phytoplasma, viroids, fungal, protists) or free-living microbes (yeasts, bacteria, protists, fungi) any of which are delivered, alive, dead or processed, via root treatments, or foliar sprayed on plants, or injected into plants, which are to be protected. Alternatively, the microorganism can be a transgenic organism endemic to the plant and deliver dsRNA to the plant. See, e.g., Subhas, et al. (2014) J. Biotech. 176:42-49 for an example of virus induced gene silencing using Citrus tristeza virus. See, also, Tenllado, et at (2003) BMC Biotechnol 3:3 for an example of a crude extract of a bacterial cell culture containing dsRNA that protects plants against viral infections.
In one embodiment, a dsRNA solution is administered to the plant on which D. citri feed. A dsRNA solution contains one or more of the dsRNAs discussed herein and an agriculturally acceptable carrier. An agriculturally acceptable carrier can be water, one or more liposomes, one or more lipids, one or more surfactants, one or more proteins, one or more peptides, one or more nanotubes, chitin, and/or one or more inactivated microorganisms that encapsulate the dsRNA. See WO 2003/004644 for examples of other agriculturally acceptable carriers. The dsRNA solution can also contain one or more sugars, compounds that assist in preventing dsRNA degradation, translaminar chemicals, chemical brighteners, clays, minerals, and/or fertilizers. One can spray the dsRNA solution on plants (leaves, branches, trunk, exposed roots, etc.) which the D. citri will ingest or absorb when on the plant. One can apply the dsRNA solution to the soil around the plant so that the plant's roots absorb the dsRNA solution and transport it to other parts of the plants. Then when D. citri feed on the plant, they will ingest the dsRNA (and perhaps other components of the dsRNA solution) in the dsRNA solution when feeding on the plant. Alternatively, one or more roots can be placed in a container which contains the dsRNA solution so that those roots absorb the dsRNA solution. The dsRNA solution can also be injected into the plant. As such, the dsRNA solution can be in a spray dsRNA solution, a drenching dsRNA solution, or an injectable dsRNA solution. Other types of solutions are known in the art.
The term “chimeric” and “fusion” when referring to a gene or polynucleotide are used to refer to a gene or polynucleotide containing at least two functionally relevant DNA/RNA polynucleotides (such as promoter, 5′UTR, coding region, 3′UTR, and/or intron) that are not naturally associated with each other, such as a fusion of functionally relevant polynucleotides from different sources to form a plant-expressible chimeric polynucleotide that generates a dsRNA targeting AC P′s trehalase transcript.
Oligonucleotides and polynucleotides that are not commercially available can be chemically synthesized e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Other methods for synthesizing oligonucleotides and polynucleotides are known in the art. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
The terms “identical” or percent “identity”, in the context of two or more polynucleotides or polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of nucleotides or amino acids (respectively) that are the same (e.g., 80%, 85% identity, 90% identity, 99%, or 100% identity), when compared and aligned for maximum correspondence over a designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
The phrase “high percent identical ” or “high percent identity”, in the context of two polynucleotides or polypeptides, refers to two or more sequences or sub-sequences that have at least about 80%, identity, at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide or amino acid identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In an exemplary embodiment, a high percent identity exists over a region of the sequences that is at least about 16 nucleotides or amino acids in length. In another exemplary embodiment, a high percent identity exists over a region of the sequences that is at least about 50 nucleotides or amino acids in length. In still another exemplary embodiment, a high percent identity exists over a region of the sequences that is at least about 100 nucleotides or amino acids or more in length. In one exemplary embodiment, the sequences are high percent identical over the entire length of the polynucleotide or polypeptide sequences.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algodthm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of aligninent of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math, 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of various algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al. (eds), Current Protocols in Molecular Biology, 1995 supplement).
The “complement” of a particular polynucleotide sequence is that nucleotide sequence which would be capable of forming a double-stranded DNA or RNA molecule with the represented nucleotide sequence, and which can be derived from the represented nucleotide sequence by replacing the nucleotides by their complementary nucleotide according to Chargaff's rules (A<>T; G<>C) and reading in the 5′ to 3′ direction, i.e., in opposite direction of the represented nucleotide sequence.
In one embodiment of the invention, sense and antisense RNAs and dsRNA can be separately expressed in-vitro or in-vivo. In-vivo production of sense and anti sense RNAs can use different chimeric polynucleotide constructs using the same or different promoters or using an expression vector containing two convergent promoters in opposite orientation. These sense and anti sense RNAs which are formed, e.g., in the same host cells, or synthesized, and can then combine to form dsRNA. It is clear that whenever reference is made herein to a dsRNA chimeric or fusion polynucleotide or a dsRNA molecule, that such dsRNA formed, e.g., in plant cells, from sense and antisense RNA produced separately is also included. Also synthetically made dsRNA annealing RNA strands are included herein when the sense and antisense strands are present together.
As used herein, the term “promoter” refers to a polynucleotide that in its native state is located upstream or 5′ to a translational start codon of an open reading frame (or protein-coding region) and that is involved in recognition and binding of RNA polymerase and other proteins (trans-acting transcription factors) to initiate transcription. A “plant promoter” is a native or non-native promoter that is functional in plant cells, even if the promoter is present in a microorganism that infects plants or a microorganism that does not infect plants. The promoters that are predominately functional in a specific tissue or set of tissues are considered “tissue-specific promoters”. A plant promoter can be used as a 5′ regulatory element for modulating expression of a particular desired polynucleotide (heterologous polynucleotide) operably linked thereto. When operably linked to a transcribeable polynucleotide, a promoter typically causes the transcribeable polynucleotide to be transcribed in a manner that is similar to that of which the promoter is normally associated.
Plant promoters can include promoters produced through the manipulation of known promoters to produce artificial, chimeric, or hybrid promoters. Such promoters can also combine cis-elements from one or more promoters, for example, by adding a heterologous regulatory element to an active promoter with its own partial or complete regulatory elements. The term “cis-element” refers to a cis-acting transcriptional regulatory element that confers an aspect of the overall control of gene expression. A cis-element may function to bind transcription factors, trans-acting protein factors that regulate transcription. Some cis-elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one cis-element.
The term “vector” refers to DNA, RNA, a protein, or polypeptide that are to be introduced into a host cell or organism. The polynucleotides, protein, and polypeptide which are to be introduced into a host can be therapeutic or prophylactic in nature; can encode or be an antigen; can be regulatory in nature; etc. There are various types of vectors including viruses, viroids, plasmids, bacteriophages, cosmids, and bacteria.
An expression vector is nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome. Thus, an expression vector are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an “expression cassette”. In contrast, as described in the examples herein, a “cassette” is a polynucleotide containing a section of an expression vector. The use of the cassettes assists in the assembly of the expression vectors. An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s).
A heterologous polynucleotide sequence is operably linked to one or more transcription regulatory elements (e.g., promoter, terminator and, optionally, enhancer) such that the transcription regulatory elements control and regulate the transcription and/or translation of that heterologous polynucleotide sequence. A cassette can have the heterologous polynucleotide operably linked to one or more transcription regulatory elements. As used herein, the term “operably linked” refers to a first polynucleotide, such as a promoter, connected with a second transcribeable polynucleotide, such as a gene of interest, where the polynucleotides are arranged such that the first polynucleotide affects the transcription of the second polynucleotide. In some embodiments, the two polynucleotide molecules are part of a single contiguous polynucleotide. In other embodiments, the two polynucleotides are adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell. Similarly a terminator is operably linked to the polynucleotide of interest if the terminator regulates or mediates transcription of the polynucleotide of interest, and in particular, the termination of transcription. Constructs of the present invention would typically contain a promoter operably linked to a transcribeable polynucleotide operably linked to a terminator.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, organism, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells may express genes/polynucleotides that are not found within the native (non-recombinant or wild-type) form of the cell or express native genes in an otherwise abnormal amount over-expressed, under-expressed or not expressed at all compared to the non-recombinant or wild-type cell or organism. In particular, one can alter the genomic DNA of a wild-type plant by molecular biology techniques that are well-known to one of ordinary skill in the art and generate a recombinant plant.
The terms “transgenic”, “transformed”, “transformation”, and “transfection” are similar in meaning to “recombinant”. “Transformation”, “transgenic”, and “transfection” refer to the transfer of a polynucleotide into a host organism or into a cell. Such a transfer of polynucleotides can result in genetically stable inheritance of the polynucleotides or in the polynucleotides remaining extra-chromosomally (not integrated into the chromosome of the cell). Genetically stable inheritance may potentially require the transgenic organism or cell to be subjected for a period of time to one or more conditions which require the transcription of some or all of the transferred polynucleotide in order for the transgenic organism or cell to live and/or grow. Polynucleotides that are transformed into a cell but are not integrated into the host's chromosome remain as an expression vector within the cell. One may need to grow the cell under certain growth or environmental conditions in order for the expression vector to remain in the cell or the cell's progeny. Further, for expression to occur the organism or cell may need to be kept under certain conditions. Genetically altered organisms or cells containing the recombinant polynucleotide can be referred to as “transgenic” or “transformed” organisms or cells or simply as “transformants”, as well as recombinant organisms or cells.
A genetically altered organism is any organism with any changes to its genetic material, whether in the nucleus or cytoplasm (organelle). As such, a genetically altered organism can be a recombinant or transformed organism. A genetically altered organism can also be an organism that was subjected to one or more mutagens or the progeny of an organism that was subjected to one or more mutagens and has mutations in its DNA caused by the one or more mutagens, as compared to the wild-type organism (Le, organism not subjected to the mutagens). Also, an organism that has been bred to incorporate a mutation into its genetic material is a genetically altered organism.
Transformation and generation of genetically altered monocotyledonous and dicotyledonous plant cells is well known in the art. See, e.g., Weising, et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Pat. No. 5,679,558; Agrobacterium Protocols, ed: Gartland, Humana Press Inc. (1995); and Wang, et al. Acta Hort. 461:401-408 (1998). The choice of method varies with the type of plant to be transformed, the particular application and/or the desired result. The appropriate transformation technique is readily chosen by the skilled practitioner.
The polynucleotide encoding the putative trehalase (SEQ ID NO: 1) or a portion thereof, operably linked to one or two appropriate promoters, can be stably inserted in a conventional manner into the genome (cytoplasmic genome or nucleic genome) of a single plant cell, and the genetically altered plant cell can be used in a conventional manner to produce a genetically altered plant that produces the dsRNA of this invention which can reduce fitness and survival of ACP. In this regard, a disarmed Ti-plasmid, containing the polynucleotide of this invention, in Agrobacterium tumefaciens can be used to genetically alter the plant cell, and thereafter, a genetically altered plant can be regenerated from the genetically altered plant cell using the procedures described in the art, for example, in EP 0 116 718, EP 0 270 822, WO 84/02913 and EP 0 242 246. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, in Handbook of Plant Cell Culture, pp. 124-176, MacMillan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, in Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee, et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).
Preferred Ti-plasmid vectors each contain the polynucleotide encoding trehalase or a portion of trehalase between the border sequences; or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 233 247), pollen mediated transformation (as described, for example in EP 0 270 356, WO 85/01856, and U.S. Pat. No. 4.684,611), plant RNA virus-mediated transformation (as described, for example in EP 0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (as described, for example in U.S. Pat. No. 4,536,475), and other methods such as the methods for transforming certain lines of corn (e.g., U.S. Pat. No. 6,140,553; Fromm, et al., Bio/Technology 8:833-839 (1990); Gordon-Kamm, et al., The Plant Cell 2:603-618 (1990) and rice (Shimamoto, et al., Nature 338:274-276 (1989); Datta et al., Bioilechnology 8:736-740 (1990)) and the method for transforming monocots generally (WO 92/09696). For cotton transformation, the method described in WO 00/71733 can be used. For soybean transformation, reference is made to methods known in the art, e.g., Hinchee, et al. (Bio/Technology 6:915 (1988)) and Christou, et al. (Trends Biotechnology 8:145 (1990)) or the method of WO 00/42207.
The resulting genetically altered plant can be used in a conventional plant breeding scheme to produce more genetically altered plants with the same characteristics or to introduce the polynucleotide encoding dsRNA trehalase in other varieties of the same or related plant species. Seeds, which are obtained from the genetically altered plants, contain the dsRNA trehalase gene as a stable genomic insert. Plants containing a dsRNA in accordance with the invention include plants having or derived from root stocks of plants containing the dsRNA trehalase gene of the invention, e.g., fruit trees or ornamental plants. Hence, any wild-type grafted plant parts inserted on a genetically altered plant or plant part are included in the invention because the RNAi activity is transported to these grafted parts, and any insects feeding on such grafted plant are similarly affected by the dsRNA or siRNA of the invention.
For a genetically altered plant that produces dsRNA, one constructs an expression vector or cassette (made from DNA) that encodes, at a minimum, a first promoter and the dsRNA sequence of interest such that the promoter sequence is 5′ (upstream) to and operably linked to the dsRNA sequence. The expression vector or cassette may optionally contain a second promoter (same as or different from the first promoter) upstream and operably linked to the reverse complementary sequence of the dsRNA sequence such that two strands of RNA that are complementary to each other can be produced. Alternatively, the expression vector or cassette can contain one promoter operably linked to both the dsRNA sequence (sense strand) in question and the complement or reverse complement of the dsRNA sequence (anti-sense strand) in question, such that the transcribed RNA can bend on itself and the two desires sequences can anneal. Alternatively, a second expression vector or cassette (made from DNA) can encode, at a minimum, a second promoter (same as or different from the promoter) operably linked to the reverse complementary sequence of the dsRNA such that two strands of complementary RNA can be produced in the plant. The expression vector(s) or cassette(s) is/are inserted in a plant cell genome (nuclear or cytoplasmic). The promoter(s) used should be a promoter(s) that is/are active in a plant. Of course, the expression vector or cassette can have other transcription regulatory elements, such as enhancers, terminators, etc.
Promoters that are active in plants are well-known in the field. Such promoters can be constitutive, inducible, and/or tissue-specific. Non-limiting examples of constitutive plant promoters include 35S promoters of the cauliflower mosaic virus (CaMV) (e.g., of isolates CM 1841 (Gardner, et al., Nucleic Acids Research 9:2871-2887 (1981)), CabbB-S (Franck, et al., Cell 21:285-294 (1980)) and CabbB-JI (Hull and Howell, Virology 86:482-493 (1987))), ubiquitin promoter (e.g., the maize ubiquitin promoter of Christensen, et al., Plant Mol. 18:675-689 (1992)), gos2 promoter (de Pater, et al., The Plant 2:834-844 (1992)), emu promoter (Last, et al., Theor. Appl. Genet. 81:581-588 (1990)), actin promoter (see, e.g., An, et al., The Plant 110:107 (1996)) and Zhang, et al., The Plant Cell 3:1155-1165 (1991)); Cassava vein mosaic virus promoters (see, e.g., WO 97/48819 and Verdaguer, et al., Plant Mol. Biol. 37:1055-1067 (1998)), the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S4 or S7 promoter), alcohol dehydrogenase promoter (e.g., pAdh1S (GenBank accession numbers X04049, X00581)), and the TRI′ promoter and the TR2′ promoter which drive the expression of the 1′ and 2′ genes, respectively, of the T-DNA (Velten, et al., EMBO J. 3:2723-2730 (1984)). Tissue-specific promoters are promoters that direct a higher level of transcriptional expression in some cells or tissues of the plant than in other cells or tissue. Non-limiting examples of tissue-specific promoters include the phosphoenolpyruvate carboxylase (PEP or PPC1) promoter (Pathirana, et al., Plant J. 12:293-304 (1997), and Kausch, et al., Plant Mol. Biol. 45(1):1-15 (2001)), chlorophyll A/B binding protein (CAB) promoter (Bansal, et al., Prot. Natl. Acad. Sci. USA 89(8):3654-8 (1992)), small subunit of ribulose-1,5-bisphosphate carboxylase (ssRBCS) promoter (Bonsai, et al., Proc. Natl. Acad. Sci. USA 89(8):3654-8 (1992)), senescence activated promoter (SEE1) (Robson, et al., Plant Biotechnol. J. 2(2):101-12 (2004)), and sorghum leaf primoridia specific promoter (RS2) (GenBank Accession No. EI979305.1). These promoters (PPC1, CAB, ssRBCS, SSE1, and RS2) are all active in the aerial part of a plant. Further, the PPC1 promoter is a strong promoter for expression in vascular tissue and is one potential useful embodiment of the current invention. Furthermore, phloem specific promoters derived from citrus can be used to generate transgenic plants to drive the expression of dsRNA into the phloem. Some examples are the sucrose synthase-1 promoter (CsSUS1p and CsSUS1p-2) (Singer et al., Planta 234:623-637 (2011)) and the phloem protein-2 promoter (CsPP2) (Miyata et al., Plant Cell Report 31(11):2005-2013 (2012)) from Citrus sinensis. Alternatively, a plant-expressible promoter can also be a wound-inducible promoter, such as the promoter of the pea cell wall invertase gene (Zhang, et al., Plant Physiol. 112:1111-1117 (1996)).
Other types of RNA polymerase promoters that can be used are promoters from microorganisms, such as, but not limited to the bacteriophage T7 RNA polymerase promoter, yeast Galactose ((GLA) promoter, yeast glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, yeast Alcohol Oxidase (AOX) promoter.
Other elements which can be used to increase transcription expression in plant cells include, but are not limited to, an intron (e.g., hsp70 intron) at the 5′ end or 3′ end of the chimeric gene, or in the coding sequence of the chimeric dsRNA gene (such as, between the region encoding the sense and antisense portion of the dsRNA), promoter enhancer elements, duplicated or triplicated promoter regions, 5′ leader sequences different from the chimeric gene or different from an endogenous (plant host) gene leader sequence, 3′ untranslated sequences different from the chimeric gene or different from an endogenous (plant host) 3′ untranslated sequence.
The expression vector or cassette could contain suitable 3′ untranslated transcription regulation sequences (i.e., transcript formation and polyadenylation sequences). Potential polyadenylation and transcript formation sequences include those sequences in the nopaline synthase gene (Depicker, et al., J. Molec. Appl. Genetics 1:561-573 (1982)), the octopine synthase gene (Gielen, et al., EMBO J. 3:835-845 (1984)), the SCSNI or the Mali c enzyme terminators (Schunmann, et al., Plant Functional Biology 30:453-460 (2003)), and the T-DNA gene 7 (Velten and Schell, Nucleic Acids Research 13:6981-6998 (1985)).
In another embodiment of this invention, one can infect plants with either an RNA virus or DNA virus which can produce virus-related the siRNAs described herein in the plant's cells during replication of the virus which kills D. citri that feed on the infected plant. The dsRNA, either derived from a replication intermediate or secondary-structure characters of some single-stranded viral RNA region, can accumulate to high levels in virus-infected plant cells. In the case of plant DNA viruses, the dsRNA may be formed by annealing of overlapping complementary transcripts. See, Baulcombe (2004) Nature 431:356-363. This method, referred to as virus-induced gene silencing for plant gene (VIGS), offers an attractive alternative to genetically altered plants that produce dsRNA because VIGS allows the investigation of gene function without requiring plant transformation. See, e.g., Ruiz, et al. (1998) Plant Cell 10:937-946; and Burch-Smith, et al. (2004) Plant Physiol. 142:21-27. Recombinant plant viruses can be constructed to carry an inserted partial or whole sequence of a candidate gene (a heterologous polynucleotide) operably linked to a viral promoter. Such recombinant plant viruses can infect and move systemically in plants, producing dsRNA (which are then processed into siRNA by the plant's cells) or produce anti-sense RNA. The dsRNA and/or siRNA and/or anti-sense RNA can be ingested by an insect pest during its feeding, mediating suppression of the insect's endogenous gene transcripts. A VIGS derived from the Citrus tristeza virus (ON) expressing a fragment of the Abnormal wing disc (Awg) gene from D. citri was shown to induce RNAi effects on D. citri (Hajeri, et al. (2014) J. of Biotech. 176:42-49, doi:10.1016/j.jbiotec.2014.02.010). Insects that developed on Citrus macrophylla plants infected with the CTV-Awd had lower levels of corresponding mRNA, resulting malformed-wing phenotype in adults and increased adult mortality. In a similar approach, a recombinant CTV containing SEQ ID NO: 4 or SEQ ID NO: 5; SEQ ID NO: 9 or SEQ ID NO: 10; SEQ ID NO: 14 or SEQ ID NO: 15; the RNA equivalent of SEQ ID NO: 26 (as the candidate gene); the reverse complement thereof; and/or fragments thereof can be generated using reverse genetics or other techniques that are well-known in the art field. The recombinant CTV are then allowed to infect citrus plants and replicate, thereby producing RNA containing the anti-sense sequence and sense sequence of the candidate gene which form dsRNA. When D. citri feed on the infected plant, the psyllid will ingest the dsRNA, causing psyllid death.
In another embodiment, a recombinant virus contains the sense region and anti-sense region of trehalase gene (or fragments thereof) on one polynucleotide (can be an RNA virus or DNA virus). Then, during viral replication, the sense region and the anti-sense region anneal to each other, forming dsRNA. Again, when D. citri feed on the infected plant, the psyllid ingests the dsRNA causing psyllid death. In this embodiment the sense region and anti-sense region can optionally be separated by a linking region that separates the sense region and anti-sense such that a hair-pin loop can be formed upon annealing. In this embodiment, when the recombinant virus is an RNA virus (such as CTV), it contains RNA having the sequence of SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 14 and SEQ ID NO: 15; the RNA equivalent of SEQ ID NO: 26 and its reverse complement; and/or fragments thereof. In this embodiment, when the recombinant virus is a DNA virus, it contains the DNA sequence of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 26, and/or fragments thereof, and the complementary sequence thereof. See, also, U.S. Patent Pub. 2013/0125254 Dawson et al.; and U.S. Patent Pub. 2015/0067918 Kress for more information regarding CTV VIGS. See, also, EP 2,730,657 A1 for additional information about VIGS.
“Insecticidal activity” of a dsRNA, as used herein, refers to the capacity to reduce fitness and/or survival of insects when the insects ingest or feed on the dsRNA, such reduction in fitness and/or survival is greater than the level of fitness and/or survival of insects that are not exposed to the dsRNA. “Insect-control” of a dsRNA, as used herein, refers to the capacity to inhibit the insect development, fertility, inhibition of pheromone production, or growth in such a manner that the insect population causes less damage to a plant, produces fewer offspring, are less fit, more susceptible to insect pathogens, or are more susceptible to predator attack, or are deterred from feeding on such plant, compared to the insect population that is untreated or to an insect population that feed on an untreated plant.
The term “plant” includes whole plants, plant organs, progeny of whole plants or plant organs, embryos, somatic embryos, embryo-like structures, protocorms, protocorm-like bodies (PLBs), and suspensions of plant cells. Plant organs comprise, e,g., shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like). The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to the molecular biology and plant breeding techniques described herein, specifically gymnosperms and angiosperms (monocotyledonous (monocots) and dicotyledonous (dicots) plants). It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous. The plants described herein are citrus plants, such as but not limited to, orange, lemon, lime, tangerine, Persian lime, and grapefruit. Plants also include citrons and near relatives of citrus or citron plants. Other plants on which D. citri teed are included in this invention. Non-limiting examples of such other plants include Aegle marmelos, Aeglopsis chevaliers, Afraegle gabonensis, Afraegle paniculata, Atalantia missionis, Atalantia monophylla, Balsamocitrus dawei, Citropsis gslletsaila Citropsis schweinfurthii, Clausena anisum-olens, Clausena excavate, Clausena indica, Clausena lansium, Eremocitrus glauca, Fortunella crassifolia, Fortunella margarita, Fortunella polyandra, Limonia acidissima, caloxylon, Microcitrus australasica, Microcitronella, Murraya exotica, Murraya koenigii, Murraya paniculata, Naringi crenulata, Pamburus missionis, Poncirus trifoliate, Severinia buxlfolia, Swinglea ghninosa, Toddalia asiatica, Triphasia trifolia, Vepris lanceolata, Zanthoxylum Agara.
An “effective amount” is an amount sufficient to effect desired beneficial or deleterious results. An effective amount can be administered in one or more administrations. In terms of treatment, an “effective amount” is that amount sufficient to make the target pest non-functional by causing an adverse effect on that pest, including, but not limited to, physiological damage to the pest; inhibition or modulation of pest growth; inhibition or modulation of pest reproduction; or death of the pest. In one embodiment of the invention, the target insect ingests the dsRNA containing solution which disrupts production of critical protein(s) that are necessary for developmental, feeding, and/or reproductive functions of the insect.
The term “a nucleic acid consisting essentially of”, “a dsRNA consisting essentially of”, “a polynucleotide consisting essentially of”, and grammatical variations thereof mean nucleic acids that differ from a reference nucleic acid sequence by 20 or fewer nucleotides and also perform the function of the reference nucleic acid sequence. Such variants include sequences which are shorter or longer than the reference nucleic acid sequence, have different residues at particular positions, or a combination thereof.
The terms “approximately” and “about” refer to a quantity, level, value or amount that varies by as much as 30% in one embodiment, or in another embodiment by as much as 20%, and in a third embodiment by as much as 10% to a reference quantity, level, value or amount. As used herein, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a bacterium” includes both a single bacterium and a plurality of bacteria.
Many techniques involving molecular biology discussed herein are well-known to one of ordinary skill in the art and are described in, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual 4th ed. 2012, Cold Spring Harbor Laboratory; Ausubel et al. (eds.), Current Protocols in Molecular Biology. 1994—current, John Wiley & Sons; and Kriegler, Gene Transfer and Expression: A Laboratory Manual (1993). Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology maybe found in e.g., Benjamin Lewin, Genes IX, Oxford University Press, 2007 (ISBN 0763740632); Krebs, et al. (eds), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, NTH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Having now generally described this invention, the same will be better understood by reference to certain specific examples and the accompanying drawings, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims. The examples and drawings describe at least one, but not all embodiments, of the inventions claimed. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.
D. citri trehalase sequence was obtained by automated computational analysis from a D. citri genomic sequence (GenBank NW_007377674.1), annotated using gene prediction method: Gnomon. Supporting evidence includes similarity to: 16 Proteins, and 100% coverage of the annotated genomic feature by RNAseq alignments, including 5 samples with support for all annotated introns. All sequences annotated as trehalase in the transcriptomic library were selected, aligned in order to check for possible sequencing errors, and finally the correct sequence was identified. The final sequence was submitted to another round of comparative analysis, which included: (1) sequencing and comparative analysis of nucleotide sequence using BLAST® analyses tools (NCBI website, blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm that the sequence is from trehalase; (2) in silico translation to find the putative trehalase protein amino acid sequence; (3) followed by comparative analysis of the amino acid sequence with sequences contained on GenBank, Pfam, and Prosite websites, using various protein analyses tools to confirm the identity of the sequence. This analysis also shows that the amino acid sequence contains functional domains associated with other trehalase proteins.
With reference to D. citri trehalase gene, SEQ ID NO: 4, SEQ ID NO: 9 and SEQ ID NO: 14 are the sense strand sequence of double-stranded RNA polynucleotides designed with one strand that is capable of hybridizing to the mRNA transcribed from the D. citri trehalase gene at positions 168-605 (SEQ ID NO: 6), 809-1277 (SEQ ID NO: 1), and 1157-1886 (SEQ ID NO: 11). The three designed long dsRNAs were made using an Ambion MEGAscript RNAi. Kit (ThermoFisher Scientific, Waltham, Mass.).
With reference to D. citri maternal protein exuperantia gene, SEQ ID NO: 19 is the sense strand sequence of double-stranded RNA polynucleotides designed with one strand that is capable of hybridizing to the mRNA transcribed from the D. citri exuperantia transcript at positions 1-447 (SEQ ID NO: 16). The designed long dsRNAs were made using an Ambion MEGAscript RNAi Kit (ThermoFisher Scientific, Waltham, Mass.).
With reference to D. citri pterin-4-alpha-carbinolamine dehydratase transcript (PCBD1), SEQ ID NO: 24 is the sense strand sequence of double-stranded RNA polynucleotides designed with one strand that is capable of hybridizing to the snRNA transcribed from the D. citri ptetin-4-alpha-carbinolamine dehydratase transcript at positions 3-268 (SEQ ID NO: 21). The designed long dsRNAs were made using an Ambion MEGAscript RNAi Kit (ThermoFisher Scientific, Waltham, Mass.).
A cDNA encoding trehalase (SEQ ID NO: 26) was synthesized from D. citri total RNA extracted using Trizol (Invitrogen, Waltham, Mass.). Initially, cDNA was reversed transcribed from 5 μg of total RNA using SuperScript® III First-Strand Synthesis System (Invitrogen, Waltham, Mass.), using the random hexamers provided by the manufacturer. The region encoding nucleotides 809-1277 of trehalase (SEQ ID NO: 1) was amplified by PCR by combining the cDNA, the 5′ primer (SEQ ID NO: 2) and the 3′ primer (SEQ ID NO: 3) with DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.) to generate an amplicon containing nucleotides 809-1277 of trehalase flanked by T7 RNA polymerase recognition sequence.
For double-strand RNA synthesis, each of the DNA polynucleotides encoding trehalase (nucleotides 809-1277, SEQ ID NO: 1; nucleotides 168-605, SEQ ID NO: 6; and nucleotides 1157-1886, SEQ ID NO: 11) was synthesized in separate reactions. For this purpose, the plasmid pACP-Tr_1 containing nucleotides 809-1277 of trehalase (SEQ ID NO: 1) flanked by T7 RNA polymerase recognition sequences was combined with the 5’ primer (SEQ ID NO: 2) and the 3′ primer (SEQ ID NO: 3), DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.) to generate an amplicon containing nucleotides 809-1277 of trehalase flanked by T7 RNA polymerase recognition sequences. The plasmid pACP-Tr_2 containing nucleotides 168-605 of trehalase (SEQ ID NO: 6) flanked by T7 RNA polymerase recognition sequences was combined with the 5′ primer (SEQ II) NO: 7) and the 3′ primer (SEQ ID NO: 8), DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.) to generate an amplicon containing nucleotides 168-605 of trehalase flanked by T7 RNA polymerase recognition sequences. The plasmid pACP-Tr_3 containing nucleotides 1157-1886 of trehalase (SEQ II) NO: 11) flanked by T7 RNA polymerase recognition sequences was combined with the 5′ primer (SEQ ID NO: 12) and the 3′ primer (SEQ ID NO: 13), DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.) to generate an amplicon containing nucleotides 1157-1886 of trehalase flanked by T7 RNA polymerase recognition sequences. Each of these amplicons were submitted to electrophoresis to check for presence of a single band of the correct size. Each amplicon was separately purified. Each of the purified amplicons were separately combined with the T7 RNA polymerase, nucleotides, and the appropriate buffer per MEGAscript kit (Life Technologies, Waltham, Mass.) to produce sense and anti-sense single-stranded RNA having the sequences of SEQ ID NOs: 4 and 5 (nucleotides 809-1277of trehalase); the sequence of SEQ ID NOs: 9 and 10 (nucleotides 168-605 of trehalase); and the sequence of SEQ ID NOs: 14 and 15 (nucleotides 1157-1886 of trehalase), respectively. The sense and anti-sense single-stranded RNAs are then allowed to anneal to produce double-stranded RNA. Each set of dsRNAs produced were digested with RNAse A and DNAse to remove single-strand RNA and DNA, respectively, purified and quantified.
A cDNA encoding the maternal protein exuperantia (SEQ ID NO: 16) was synthesized from D. citri total RNA extracted using Trizol (Invitrogen, Waltham, Mass.). cDNA was transcribed from 5 μg of total RNA using SuperScript® III First-Strand Synthesis System Cat. No. (Invitrogen, Waltham, Mass.), using random hexamers provided by the manufacturer. The region nucleotides 1-447 of exuperantia (SEQ ID NO: 16) was amplified by PCR by combining the cDNA, the 5′ primer (SEQ ID NO: 17) and the 3′ primer (SEQ ID NO: 18) with DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.) to generate an amplicon containing nucleotides 1-447 of exuperantia flanked by T7 RNA polymerase recognition sequence. This amplicon was cloned into the pCR™2.1-TOPO® plasmid using the TOPO® TA cloning Kit, Cat. No. K4500.110 (Invitrogen, Waltham, Mass.). The plasmid (hereafter named pACP-Exu_1) is then transfected into One Shot TOP 10 competent E. coli. For double-strand RNA synthesis, first DNA polynucleotide encoding nucleotides 1-447 of exuperantia (SEQ ID NO: 16) was synthesized by combining the plasmid p-ACP-Exu_1 containing nucleotides 1-447 of exuperantia (SEQ ID NO: 16) flanked by T7 RNA polymerase recognition sequences, the 5′ primer (SEQ ID NO: 17), and the 3′ primer (SEQ ID NO: 18), DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.) to produce an amplicon containing nucleotides 1-447 of exuperantia flanked by T7 RNA polymerase recognition sequences. The generated amplicon was submitted to electrophoresis to check for presence of a single band of the correct size, and then the amplicon was purified. The purified amplicon was combined with the T7 RNA polymerase, nucleotides, and the appropriate buffer per MEGAscript kit (Life Technologies, Waltham, Mass.) to produce sense and anti-sense single-stranded RNA having the sequences of SEQ ID NOs: 19 and 20, respectively. The sense and anti-sense single-stranded RNAs are then allowed to anneal to produce double-stranded RNA. The produced dsRNA was digested with RNAse A and DNAse to remove single-strand RNA and DNA, respectively, purified and quantified.
A cDNA encoding nucleotides 3-268 of pterin-4-alpha-carbinolamine dehydratase (PCBD1) (SEQ ID NO: 21) was synthesized from D. citri total RNA extracted using Trizol (Invitrogen, Waltham, Mass.). cDNA was transcribed from 5 μg of total RNA using SuperScript® III First-Strand Synthesis System Cat. No. (Invitrogen, Waltham, Mass.), using random hexamers provided by the manufacturer. The region encoding nucleotides 3-268 of PCBD1 (SEQ ID NO: 21) was amplified by PCR combining the cDNA, the 5′ primer (SEQ ID NO: 22) and the 3′ primer (SEQ ID NO: 23) with DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.) to produce an amplicon containing nucleotides 3-268 of PCBD1 flanked by T7 RNA polymerase recognition sequences. This amplicon was cloned into the pCR™2.1-TOPO® plasmid using the TOPO® TA cloning Kit, Cat. No. K4500J10 (Invitrogen, Waltham, Mass.). The plasmid (hereafter named pACP-Pcd_1) is then transfected into One Shot TOP 10® competent E. coli. For double-strand RNA synthesis, a DNA polynucleotide encoding nucleotides 3-268 of PCBD1 (SEQ ID NO: 21) was synthesized by combining the plasmid pACP-Pcbd1_1 containing nucleotides 3-268 of PCBD1 (SEQ ID NO: 21) flanked by T7 RNA polymerase recognition sequences, the 5′ primer (SEQ ID NO: 22) and the 3′ primer (SEQ ID NO: 23), DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.) to generate an amplicon. This amplicon was submitted to electrophoresis to check for presence of a single band of the correct size and then purified. The purified amplicon encoding nucleotides 3-268 of PCBDI (SEQ ID NO: 21) flanked by T7 RNA polymerase recognition sequences was combined with the T7 RNA polymerase, nucleotides, and the appropriate buffer per MEGAscript kit (Life Technologies, Waltham, Mass.) to produce sense and anti-sense single-stranded RNA (SEQ NOs: 24 and 25, respectively) which were allowed to anneal to form double-stranded RNA. The dsRNA was digested with RNAse A and DNAse to remove single-strand RNA and DNA, respectively, purified and quantified.
In general, the RNAi feeding bioassay using plant cuttings has the following protocol. In citrus, the “flush”, which are new foliar shoots growth, were collected from potted citrus seedlings grown in a glasshouse to produce small cuttings. The flush are about 7-8 cm long. The plant flush was washed in 0.2% bleach water, for 10 min. Then the base of each stem of each flush was cut at a 45 degree angle while submerged in filtered water. The flush was then placed into a 1.5 mL tube containing 0.5 mL water. A dsRNA solution (100 ng of dsRNA in 300 μL of water) was added to the water in the tube, the tube top was wrapped with plastic or Parafilm™ (Bemis NA, Neenah, Wis.) and placed under artificial lighting to stimulate absorption of the dsRNA solution. After the flush absorbs the entire dsRNA solution (approximately 8 hours), the tube was filled with water using a 26 gauge syringe needle and syringed filtered (0.45 μ). The treated cuttings were then placed in a cage and approximately 15 adult insects of D. citri (actual number of insects per cage per experiment ranges from 13 to 18 insects) were added to the cage to feed on the treated cutting. Mortality of adult D. citri were scored daily for up to 15 days after start of feeding access period.
Using this RNAi feeding bioassay, dsRNA based on SEQ ID NO: 1 (SEQ ID NOs: 4 and 5, sense and anti-sense strands respectively), SEQ ID NO: 6 (SEQ ID NOs: 9 and 10, sense and anti-sense strands respectively), and SEQ ID NO: 11 (SEQ ID NOs: 14 and 15, sense and anti-sense strands respectively), were evaluated for their capacity to cause mortality on D. citri. Treatments were (1) cuttings with dsRNA based on SEQ ID NO: 1; (2) cuttings with dsRNA based on SEQ ID NO: 6; (3) cuttings with dsRNA based on SEQ ID NO: 11; (4) negative control (cuttings that just receive water). Each treatment group were composed of three replicates (a replicate means one cage containing one cutting and approximately fifteen adult D. citri).
Insect mortality scored after 15 days of feeding on treated and control cuttings, showed that Treatment # 1 (dsRNA based on SEQ ID NO: 1) increased average mortality to 95.92% of the insects among replicates (47 of 49 adult insects). Treatment 42 (dsRNA based on SEQ ID NO: 6) increased average mortality to 86.36% of the insects among replicates (38 of 44 adult insects). Treatment #3 (dsRNA based on SEQ ID NO: 11) increased average mortality to 55.56% of the insects among replicates (25 of 45 adult insects). Treatment #4 (negative control) increased average mortality to 6.67% of the insects among replicates (3 of 45 adult insects).
Using this RNAi feeding bioassay, dsRNA based on SEQ ID NO: 16 (SEQ ID NOs: 19 and 20, sense and anti-sense strands respectively), was evaluated for its capacity to cause mortality on D. citri. Treatments were (1) cuttings with dsRNA based on SEQ ID NO: 16; and (2) negative control (cuttings that just received water). Each treatment were composed of three replicates. In this experiment, each cutting, treated with either dsRNA based on SEQ ID NO: 16 or the negative control, was caged with about 12 adult D. citri (number of D. citri per cage ranged from 11 to 13 insects). Treatment #1 was represented by four replicates, and Treatment #2 was represented by three replicates (a replicate means one cage containing one cutting and about twelve adult D. citri).
Insect mortality scored after 15 days of feeding on treated and negative control cuttings, indicated that Treatment #1 increased average mortality to 16.08% of the insects among replicates (8 of 49 adult insects). Treatment #2 induced an average mortality of 3.03% of the insects among replicates (1 of 34 adult insects).
Using this RNAi feeding bioassay, dsRNA based on SEQ ID NO: 21 (SEQ ID NOs: 24 and 25, sense and anti-sense strands respectively), was evaluated for its capacity to cause mortality on D. citri. Treatments consisted of (1) cuttings with dsRNA based on SEQ ID NO: 21; (2) negative control (cuttings that just received water). Each treatment were composed of three replicates. In this experiment, each cutting, treated with either dsRNA based on SEQ ID NO: 21 or the negative control, was caged with about 12 adult D. citri (number of D. citri per cage ranged from 11 to 13 insects). Treatment 41 was represented by four replicates, and Treatment 42 was represented by three replicates (a replicate means one cage containing one cutting and about twelve adult D. citri).
Insect mortality scored after 15 days of feeding on dsRNA treated and negative control cuttings, indicated that Treatment #1 induced an average mortality of 4.17% of the insects among replicates (2 of 48 adult insects). Treatment 42 induced an average mortality of 2.08% of the insects among replicates (1 of 48 adult insects)
Sweet orange seedlings were cultivated in plastic containers (2 gallon pots) containing potting soil and maintained in a glasshouse. Seedlings about 30 cm or 12 inches tall were trimmed, removing all branches, leaving just the seedling trunk. When the seedling produced new growth two weeks post-trimming, 20 adult female D. citri were caged on each plant, so they could oviposit eggs for 48 hours. The adults were then removed, and 10 μg of trehalase dsRNA (based on SEQ ID NO: 1) was mixed with 10 mL of water, and the dsRNA solution was poured onto the soil. Seedlings were watered as needed post-treatment. Three sweet orange seedlings were treated with dsRNA based on SEQ ID NO: 1 (treatment #1), and three sweet orange seedlings received just 10 mL of water (treatment #2, negative control). All three seedlings from each treatment group were caged together in a Bug Dorm 2 rearing cage, catalog #1462W (BioQuip, Rancho Dominguez, Calif.). The plants were maintained at 22° C. The development of D. citri was monitored over time. For all six seedlings, the eggs hatched, the nymphs developed successfully without any problems, and no mortality occurred (as determined by an absence of dead nymphs observed on the bottom of the cage), thus indicating that the dsRNA based on SEQ ID NO: 1 did not negatively impact nymph survival. About thirty days post-treatment, the nymphs successfully emerge as D. citri adults. When D. citri adults were approximately 10 days old, they started to die and fall on the bottom of the cage. The total number of dead adults D. citri were counted at fourteen days after emergence, post-eclosion. Treatment #1 resulted in death of 100% of D. citri adults in this assay (643 dead adult D. citri were counted). At the same time, no significant mortality were observed for D. citri adults in the negative control cage (Treatment 42).
Life cycle of D. citri from egg to adults vary with temperature: at 25° C. eggs hatch in 4 days and nymphs develop to the adult stage over a 13 day period for a total of 17 days from egg to adult (Tsai and Liu, J. Econ. Entomol. 93(6):1721-1725 (2000)). Mean development from egg to adult varies from 49.3 days at 15° C. to 14.1 days at 24° C. (Liu and Tsai, Ann. Appl. Biol. 137: 201-206 (2000)). At 24° C., adult males live an average of 21 to 25 days, and females live an average of 31 to 32 days (Nava, et al., Appl. Entomot 131:709-715 (2007)). These life parameters show that the conditions of the above experiment did not negatively influence nymph development, and that the mortality of D. citri adults that fed on the plants treated with dsRNA based on SEQ ID NO: 1 was not caused by natural causes, because the D. citri died when they were fairly young adults (10-14 days old). Thus, dsRNA based on SEQ ID NO: 1 suppressed D. citri population by reducing fitness and survival of the adult D. citri that developed from nymphs that fed on treated plants. This example demonstrates that dsRNA based SEQ ID NO: 1, nucleotides 809-1277 of trehalase, can suppress ACP population by killing the adult ACPs that feed and/or develop from nymphs on treated plants.
The generation of transgenic citrus plants that express the dsRNA based on SEQ ID NO: 1 (SEQ ID NOs: 4 and 5, sense and anti-sense strands respectively), is obtained using the protocol described in Oliveira, et al. (2015) SpringetPlus 4:264. In vitro-grown etiolated epicotyls segments of ‘Hamlin’ sweet orange [Citrus sinensis (L.) Osbeck.] is used as source of explants. The epicotyl portions of etiolated seedlings is cut transversally into 0.8-1 cm segments and is used for transformation. An Agrobacterium-mediated transformation method is used to transform citrus tissue. The plasmid pCACP-Tr_1 is generated by inserting the amplicon correspondent to SEQ ID NO: 1 (SEQ ID NOs: 4 and 5, sense and anti-sense strands respectively), into the pCambia2300. The plasmid is transformed into A. tumefaciens EHA 105 (Hood, et al. (1986) J. Bac. 168:1291-1301) using freeze-thaw method (Hofgen and Willmitzer (1988) Nucleic Acids Res. 16:9877), Epicotyl tissue of ‘Hamlin’ sweet orange is inoculated for 15 minutes with an overnight culture of Agrobacterium diluted to OD 600=0.4 The infected epicotyl tissue is co-cultivated for 3 days on MS basal medium containing 3% sucrose, 1 mg 1-1 BAP, 100 mg 1-1 acetosyringone, 8.0 g 1-1 agar and incubated at an average temperature of 24±1° C. in the dark. After 3 days of cocultivation, the explants is transferred to MS medium containing 300 mg 1-1 timentin, 250 mg HI cefotaxime, and 100 mg I-1 kanamycin. After 45 days in culture on kanamycin selection media, putatively transformed shoots is transferred to a root-induction medium (RIM), consisting of MS strength medium, 2% sucrose, 0.25% Gelrite, 2.5 mg 1-1 IBA, 0.5 mg 1-1 NAA, and 0.0025 mg 1-1 spermidine. Plants that develop root systems is transferred to sterile soil cones, covered with plastic bags and gradually exposed to ambient humidity in a growth chamber, over a period of 15 days. Acclimated plants is transferred to greenhouse for maturation. To confirm the plant contains the transgene (SEQ ID NO: 1), genomic DNA is extracted using Purelink® Genomic Plant DNA Purification Kit, Cat. Number K1830-01 (Invitrogen, Waltham, Mass.) and is used as a template in a PCR, using the 5′ primer (SEQ ID NO: 2) and the 3′ primer (SEQ ID NO: 3) with DNA polymerase, nucleotides and the appropriated buffer per AmpliTaq Gold® 360 Master Mix Cat. No. 4398881 (Applied Biosystems, Waltham, Mass.), The PCR amplified product is separated on a 1.2% agarose gel and visualized by ethidium bromide staining.
Honey bees (Apis mellifera) are beneficial insects to citrus trees, acting as a pollinator. The bees also feeds the nectar and pollen from the plant, and by chance could ingest trehalase dsRNA (based on SEQ ID NO: 1) from a treated citrus tree. To evaluate the absence of harm effects of trehalase dsRNA (based on SEQ ID NO: 1) to a non-target insect, i.e., honey bees, a feeding bioassay was conducted. A frame with mature sealed brood was taken from the colony and held overnight in 33° C. inside the emergence frame box. A group of 30 newly emerged bees were handfed with 5 μL of a 1:1 dsRNA:sucrose solution containing 10 ng of trehalase dsRNA based on SEQ ID NO: 1 (treatment 41). A second group of 30 newly emerged bees were handfed with 5 μL of a 1:1 sucrose solution (treatment 42, negative control water). Bees from each treatment were marked by paint (bees from each treatment were painted with a different color) and placed all together into the same hive. After 10 days, the bees were collected by micro vacuum, and the number of recovered marked bees counted. The percentage of insects in treatment #1 group recovered after 10 days was 73% (22 recovered from 30 treated). The percentage of insects in treatment #2 group recovered after 10 days was 70% (21 recovered from 30 treated). Additionally, the recovered individual honey bees from each treatment group were analyzed for any significant changes on the levels of some mRNAs using the RT-qPCR protocol set forth in Evans (2006) (J. Invert. Path. 93:135-139). The mRNA levels for the following genes were analyzed: vitellogenin (NCBI Reference Sequence: NM_001011578.1), eater (also called nimrodC1) (NCBI Reference Sequence: XM_006561053.1), hymenoptaecin (NCBI Reference Sequence: NM_001011615.1) and arginine kinase (NCBI Reference Sequence: NM_001011603). The ribosomal protein 5 gene (NCBI Reference Sequence: XM_006567834.1) was used as a reference gene. Primer pairs were designed for each of these genes using Printer 3 (bioinfo.ut.ee/primer3-0.4.0/primer3/). Comparative analysis of the mRNA levels of each gene from individuals honey bees from treatment 41 and treatment 42 were not significantly different by one-way ANOVA analysis. Thus, dsRNA based SEQ ID NO: 1, nucleotides 809-1277 of trehalase, did not produce a harmful effect when ingested by honey bees.
The foregoing detailed description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in the art that modifications and variations may be made therein without departing from the scope of the invention. All references cited herein are incorporated by reference.
This application is related to and claims priority to U.S. Patent Application No. 62/287,315 filed on Jan. 26, 2016, the contents of which are incorporated herein.
| Number | Date | Country | |
|---|---|---|---|
| 62287315 | Jan 2016 | US |
