# Patent

## Title
Formulations and methods for control of weedy species

## Abstract

A formulation is provided for application to a host plant to reduce, inhibit or impair one or more of growth and development of the host plant. A method of inhibiting growth plant growth and development is also provided as a means of controlling weedy species. The method comprises: selecting a suitable gene for growth suppression in a target plant; identifying an at least one target site accessible to base pairing in the suitable gene; identifying an at least one divergent site in the at least one target site; designing a construct complementary to the at least one divergent site; adding an at least one RNAi inducer to the construct; and delivering the construct to the target plant.

## Background
BACKGROUND OF THE INVENTION
The present technology is directed to a formulation and a method for controlling growth of plant species. More specifically, it is a formulation comprising a targeting construct and RNAi inducer to produce small interfering RNAs for use in non-stable expression in weedy plant species. Targeting constructs are designed to target endogenous genes in the weedy species while having no effect in off-target species.
DESCRIPTION OF THE RELATED ART
The impact of invasive and pest plant species has been called an “invisible tax” on our environment and economy. With ever increasing global transportation and travel has come an unprecedented spread of invasive and noxious plant species throughout the world. These weeds adapt quickly to new environments and go largely unchallenged by local flora and fauna. Many are unreachable by or have developed resistance to conventional control techniques. Invasive species cause direct economic losses in sectors such as forestry, ranching, and agriculture.
The current strategies for invasive species management consist of the application of different combinations of chemical herbicides and physical removal, coupled with bio-control techniques as available. The available chemicals are often toxic to a wide array of native plants, animals and insects and can have negative consequences for human health. Many cannot be used in riparian or aquatic environments as the compounds would quickly spread. In addition, they have a limited half-life and efficacy and must be reapplied year after year. Bio-control and physical removal are costly and labour intensive requiring large investments and again, often resulting in collateral damage to other organisms. Some invasive pest plants are now so well established that they are widely considered impossible to remove by any available technique, for example, Eurasian Milfoil. Others, having been subjected to years of treatment with chemicals, have developed resistance to them.
In an attempt to target the species of interest and reduce the damage done by spraying with broad spectrum herbicides, U.S. Pat. No. 7,805,884 discloses an injector system for injecting a dose of weed-killing fluid into the stem of a Japanese knotweed, including a fluid dispenser system with a fluid passage, a collared needle with a fluid delivery aperture in communication with the fluid dispenser system, and an actuator connected to the fluid dispenser system for actuating the transmission of fluid from the fluid dispenser system to the fluid delivery aperture. This employs chemical herbicides.
Control of insect pests is largely through the use of chemical insecticides. Some biological control methods also exist, for example, the use of pheromones in insect traps. These are relatively labour intensive as the traps have to be baited, set and removed.
Another example of biological control is the use of Bacillus thuringiensis toxin. It can be provided as a spray or produced in transgenic plants. In transgenic plants, the gene or genes are expressed in the plant, the plant produces the toxin, the foraging insect ingests the plant material and is killed. One could argue that these are quasi-chemical control methods, as toxic chemicals are still being produced and used to kill the insect pests.
Rather than using toxins, U.S. Pat. No. 7,943,819 provides methods for genetic control of insect infestations in plants and compositions thereof by inhibiting one or more biological functions by feeding one or more recombinant double stranded RNA molecules to the insect pest. This reportedly results in a reduction in pest infestation through suppression of gene expression.
U.S. Pat. No. 8,148,604 discloses methods and materials for conferring insect pest resistance to plants and controlling parasitic plant pests. Plants are stably transformed with a silencing construct homologous to a gene of a plant pest that is essential for the survival, development, or pathogenicity of the pest. This results in the plant producing RNA interference (RNAi), specifically short interfering RNA (siRNA) to the selected gene, which, when ingested by the insect pest results in silencing of the gene and a subsequent reduction of the pest's ability to harm the plant. In other embodiments, the pest's reduced ability to harm the plant is passed on to pest progeny. It is also suggested that parasitic plants pests, for example striga, dodder and mistletoe can also be controlled by stably transforming plants with a silencing construct homologous to a gene of the parasitic plant that is essential for survival or development.
Without being bound by theory, RNA interference (RNAi) is considered to be an ancient defense mechanism wherein the host organism recognizes as foreign a double-stranded RNA molecule and hydrolyzes it. The resulting hydrolysis products are small RNA fragments of 21-30 nucleotides in length, called siRNAs. The siRNAs then diffuse or are carried throughout the host, where they hybridize to the complementary Viral RNA or complementary endogenous polynucleotide sequences where they act as guides for RISC mediated hydrolysis and thus knock-down or dysregulation.
For example, the different Dicer-Like proteins (DCL) of Arabidopsis cleave dsRNA molecules into different sized (21-25 nt) small dsRNA products depending on which DCL is processing them. Arabidopsis encodes 10 Argonaute proteins (AGOI-10) which bind these small RNAs and, as a part of RISC, elicit different effects depending on which AGO the small RNA has been recruited into and the size of the recruited small RNA. AGOI is largely responsible for the miRNA pathway and also post transcriptional gene silencing. The pathway it is involved in has been shown to result in both targeted degradation of mRNAs and transitivity (RNA-dependent RNA polymerase (RdRP) dependent generation of 2° siRNA products and amplification of the initial signal). It has previously been found that AGOI prefers to recruit small dsRNAs that are 21 nt in length with a 2 nt, 3′ overhang on each end and will prefer sequences with a 5′ terminal U as the guide strand (the strand that is responsible for guiding complementary base pairing to a target mRNA sequence) (Mi et al. 2008. Sorting of small RNAs into Arabidopsis Argonaute Complexes Is Directed by the 5′ Terminal Nucleotide. DOI 10.1016/j.cell.2008.02.034.)
A species-specific herbicide that can be used to kill, weaken or impair growth of a weed species is needed. This is accomplished through miRNA, siRNA, DNA, or single- or double-stranded RNA designed to elicit an RNAi response that spreads systemically once inside a plant cell (RNAi Payload). The RNAi inducer elements cause the payload to be processed producing siRNAs. A region of the RNAi payload contains sequence complementary to endogenous target genes (Targeting construct). siRNAs produced from this region direct the knock-down of those genes leading to cell death. This knock-down is strengthened by RdRP mediated transitivity, phasing, and systemic spread. The result is a herbicide that can be tuned to affect any number of plant species.
SUMMARY OF THE INVENTION
The present technology provides a non-chemical herbicide that can be used to kill, weaken or impair growth of weedy species. In general, the formulation is for application to a host plant to reduce, inhibit or impair one or more of growth and development of the host plant. The formulation comprises an interfering Ribonucleic Acid (RNAi) payload, and at least one of a liquid carrier, a surfactant, a binder and tackifier, a thickener, a colourant, a spreader, an antifreezing agent, a sticker, an anticaking agent, a stabilizer, a disintegrator, an emulsifier, a synergistic compound, an abrasive, an emulsifier, a penetrating agent and a preservative.
In the formulation, the RNAi payload may comprise an at least one sequence specific to the host plant.
The RNAi payload comprises at least 20 contiguous nucleotides of at least one sequence selected from the group consisting of SEQ ID NOs 1 to 66.
The formulation may comprise an RNAi payload, the liquid carrier and the surfactant. It may further comprise the abrasive and still further comprise a synergistic compound.
The formulation is in an exemplary embodiment, for stem injection, and comprises the liquid carrier and the penetrating agent.
A method of inhibiting or impairing plant growth and development is also provided. The method comprises delivering a formulation to a host plant, by spraying, imbibing, irrigating, or injecting the formulation, the formulation comprising an interfering Ribonucleic Acid (RNAi) payload, an at least one of a liquid carrier, a surfactant, a binder and tackifier, a thickener, a colourant, a spreader, an antifreezing agent, a sticker, an anticaking agent, a stabilizer, a disintegrator, an emulsifier, a synergistic compound, an abrasive, an emulsifier, a penetrating agent and a preservative, thereby inhibiting or impairing growth and development. The method comprises delivering the formulation to at least one of a leaf, a root, a stem, a petiole, a seed and a cotyledon. The RNAi payload may comprise a sequence selected from the group consisting of SEQ ID NOs 1 to 66.
The method comprises injecting the stem or petiole or spraying the host plant.
The method further comprises inducing expression of any of SEQ ID NOs 7, 8, 9, 10, 11 and 12 thereby producing any of SEQ ID NOs 1, 2, 3, 4, 5, and 6.
A method of weed control is also provided, the method comprising:

    
    
        selecting a weed plant species to be controlled;
        synthesizing or obtaining at least one RNAi or RNAi encoding sequence;
        formulating a species-specific RNAi payload; and
        delivering the species-specific RNAi payload to the weed plant species while minimally impacting an at least one other plant species.
    
    


The RNAi payload comprises at least 20 contiguous nucleotides from or complementary to one or more of SEQ ID NOs 1 to 66.
The method may involve spraying the weed plant species or injecting the weed plant species
A method of designing a species-specific construct for RNAi suppression of growth of a target plant species is also provided, the method comprising the steps of:

    
    
        selecting a suitable gene for growth suppression;
        identifying an at least one target site accessible to base pairing in the suitable gene; identifying an at least one divergent site in the at least one target site;
        designing a construct complementary to the at least one divergent site; and
        adding an at least one RNAi inducer element to the construct, thereby designing a species-specific gene construct for siRNA suppression of growth of the target plant species.
        The method may further comprise adding an at least one helper sequence to the species specific gene construct.
        The method may further comprise sequencing an at least one gene from the target plant to select the suitable gene.
        In the method, the construct may include any one of SEQ. ID No. 1 to 66 or their complement.
        A method of inhibiting or impairing plant growth and development of a target plant is also provided, the method comprising:
        selecting a suitable gene for growth suppression;
        identifying an at least one target site accessible to base pairing in the suitable gene; identifying an at least one divergent site in the at least one target site;
        designing a construct complementary to the at least one divergent site;
        adding an at least one RNAi inducer element to the construct; and
        delivering the construct to the target plant.
        The method may further comprise adding an at least one helper sequence to the species specific gene construct.
        The method may further comprise sequencing an at least one gene from the target plant to select the suitable gene.
        In the method, the construct may include any one of SEQ ID No. 1 to 66 or their complement.
    
    





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a DPC targeting construct for photobleaching-based death in multiple species in accordance with an embodiment of the technology. Ath=Arabidopsis thaliana, Nto=Nicotiana tobacum, Bra=Brassica napus, Zma=Zea mays, Mtr=Medicago truncatula. 
FIG. 2 shows an apoptosis targeting construct for Brassica rapa in accordance with an embodiment of the technology. Inserted into vector for E. coli production or transcribed in vitro. Resultant dsRNA is applied to plants.
FIG. 3 shows an apoptosis targeting construct 2, for Nicotiana sylvestris in accordance with an embodiment of the technology. sgP=subgenomic promoter. Cloned into RNA2-MCS vectors or co-expressed with TRV replicase.
FIG. 4 shows an apoptosis targeting construct 3, for Nicotiana sylvestris inside TRV RNA2 in accordance with an embodiment of the technology. RNA applied to plants along with TRV RNA1.
FIG. 5 shows a T7-driven helper construct in accordance with an embodiment of the technology. RNA is added directly to plants, or cloned into RNA2-MCS or RNAl-MCS vectors.
FIG. 6 shows an empty VIGS-based vector to produce coated RNAl and 2 based RNAi inducers in E. coli in accordance with an embodiment of the technology. Targeting constructs such as FIG. 3 are cloned into the MCS contained in RNA2, usually with flanking subgenomic promoters.
FIG. 7 shows an empty VIGS-based vector to produce coated RNAl and 2 based RNAi inducers in accordance with an embodiment of the technology. Targeting constructs such as FIG. 3 are cloned into the MCS contained in RNA2, usually with flanking subgenomic promoters.
FIG. 8 shows an empty VIGS-based vector to produce coated RNAl and 2 based RNAi inducers in yeast in accordance with an embodiment of the technology. Targeting construct is cloned into the MCS contained in RNA2, usually with flanking subgenomic promoters.
FIG. 9 shows an empty VIGS-based vector to produce naked TRV RNAl and RNA2 based RNAi inducers in accordance with an embodiment of the technology. Functional in E. coli with T7 Polymerase and for in vitro production. Ribozymes cleave the RNA into separate strands.
FIG. 10 shows a generic model of a DNA construct for an RNAi herbicide. The core of the herbicide is the targeting construct, tuned to affect one or a few plant species. RNAi inducer elements are either inserted into the targeting sequence (introns to make hairpins, direct or inverted repeats with/without base pairing mismatches), or are inserted around the targeting construct (subgenomic, viral, or endogenous RdRP promoters). This is all driven by either single or flanking promoters for RNA production in the chosen production species, and a circular or linear backbone for maintaining the construct in the production species.
FIG. 11 shows a construct for producing an RNAi herbicide in E. coli, without a target construct. In bacteria the TRV coat protein is transcribed and translated. Targeting constructs are inserted into the MCS. The TRV RNAl fragments facilitate coating of the RNA. In target plants this RNA is transcribed to produce viral replicase, which produces dsRNA from the entire RNA. This induces the RNAi response.



DESCRIPTIONS OF THE PREFERRED EMBODIMENTS
Brief Description of the Sequences
SEQ ID NO: 1 is the short interfering sequence Actin 2 siRNA-A used according to the present technology.
SEQ ID NO: 2 is the short interfering sequence Actin 2 siRNA-B used according to the present technology.
SEQ ID NO: 3 is the short interfering sequence CHLI siRNA-A used according to the present technology.
SEQ ID NO: 4 is the short interfering sequence CHLI siRNA-B used according to the present technology.
SEQ ID NO: 5 is the short interfering sequence 18S siRNA-A used according to the present technology.
SEQ ID NO: 6 is the short interfering sequence 18S siRNA-B used according to the present technology.
SEQ ID NO: 7 is the DNA sequence encoding the short interfering sequence Actin 2 siRNA-A used according to the present technology.
SEQ ID NO: 8 is the DNA sequence encoding the short interfering sequence Actin 2 si RNA-B used according to the present technology.
SEQ ID NO: 9 is the DNA sequence encoding the short interfering sequence CHLI siRNA-A used according to the present technology.
SEQ ID NO: 10 is the DNA sequence encoding the short interfering sequence CHLI siRNA-B used according to the present technology.
SEQ ID NO: 11 is the DNA sequence encoding the short interfering sequence 18S siRNA-A used according to the present technology.
SEQ ID NO: 12 is the DNA sequence encoding the short interfering sequence 18S siRNA-B used according to the present technology.
SEQ ID No. 13: Synthetic construct targeting CHLI1 in A. thaliana, B. rapa, M. truncatula, Z. mays, and N. Tobacum. 
SEQ ID No. 14: Synthetic construct targeting mGFP5er, Acdll, Acd2, Catl, Cat2, and Lsdl in B. rapa pekinensis. 
SEQ ID No. 15: Synthetic construct targeting Atg5, Catl, Jazh, MC2, and Beclinl in Nicotiana sylvestris. 
SEQ ID No. 16: Synthetic construct targeting Acd2, Bl-1, Llsl, NtTCTP, and Beclinl in Nicotiana sylvestris. 
SEQ ID No. 17: Synthetic construct consisting of CaMV35s promoter, TRV Ppk20 RNA1, ribozyme sequence and NOS terminator.
SEQ ID No. 18: TRV RNA2-MCS for transcription in plant cells.
SEQ ID No. 19: Truncated T7 driven Tobacco Rattle Virus RNA1 (T7-RNA1 inducer).
SEQ ID No. 20: Synthetic sequence consisting of optimized TRV coat protein driven by T7 promoter and a strong Ribosome binding site (RBS), and Tobacco rattle virus (TRV) isolate Ppk20 RNA1 and ribozyme sequence driven by T7 promoter. All elements are in the pUC57 vector.
SEQ ID No. 21: Synthetic T7-RNA2-MCS inducer sequence.
SEQ ID No. 22: Synthetic T7 driven RNA2 with sample construct (C3) in MCS, ribozyme, NOS.
SEQ ID No. 23: Synthetic T7-RNA2-sgP-C3 sequence.
SEQ ID No. 24: Synthetic sequence consisting of pUC57 MCS flanked by Pea Early Browning virus (PEBV) subgenomic promoters, all of which are flanked by T7 promoters.
SEQ ID No. 25: Synthetic RNA1 of TRV Ppk20 sequence.
SEQ ID No. 26: Synthetic RNA2 of pTRV2 with C3 insert sequence.
SEQ ID NO 27: Synthetic sequence consisting of SEQ ID NO 15 flanked by PEBV subgenomic promoters
SEQ ID NO 28: Synthetic pRNAi-GG sequence.
SEQ ID No. 29: Synthetic pRNAi-GG with SEQID 14 inserts.
SEQ ID No. 30: Human cytomegalovirus immediate early enhancer and promoter sequence.
SEQ ID No. 31: Synthetic TRV coat protein CDS DNA from pTRV2 sequence.
SEQ ID No. 32: Synthetic Tobacco Rattle Virus Codon-optimized Coat Protein mRNA sequence.
SEQ ID No. 33: Tomato Bushy Stunt Virus P19 suppressor protein CDS from Tomato Bushy Stunt Virus M21958.1 sequence.SEQ ID No. 34: Papaya Ringspot Virus strain P isolate pFT3-NP HCpro peptide CDS sequence.
SEQ ID NO 35: Tobacco Mosaic Virus TMV 30 kDa movement protein CDS sequence.
SEQ ID No. 36: Arabidopsis thaliana TOR gene CDS (TAIR accession AT1G50030).
SEQ ID No. 37: Arabidopsis thaliana ATG5 sequence.
SEQ ID NO 38: Arabidopsis thaliana Beclin 1 sequence.
SEQ ID No. 39: Nicotiana attenuata ZIM domain protein h mRNA sequence.
SEQ ID NO. 40: Nicotiana benthamiana Bax inhibitor 1 mRNA sequence.
SEQ ID No. 41: Nicotiana sylvestris Acd2 partial transcript sequence derived from N. sylvestris transcriptome.
SEQ ID No. 42: Lycopersicon esculentum lethal leaf spot 1-like protein mRNA sequence.
SEQ ID NO 43: Nicotiana tobacum mRNA for catalase 1 (catl gene), cultivar NC89 sequence.
SEQ ID NO 44: Arabidopsis thaliana MC2 sequence.
SEQ ID No. 45: Nicotiana benthamiana NbTCTP mRNA for translationally controlled tumor protein sequence.
SEQ ID No. 46: Arabidopsis thaliana Lsdl sequence.
SEQ ID No. 47: Arabidopsis thaliana Acdll sequence.
SEQ ID No. 48: Nicotiana sylvestris PDS gene target construct.
SEQ ID No. 49: T7 driven RNA2 with NSYL PDS target construct in MCS.
SEQ ID No. 50: T7 driven truncated PPK20 RNA1 consisting of 5′ sequence, replicase CDS, PUC57 MCS, 3′ sequence, ribozyme and NOS terminator.
SEQ ID No. 51: TRV PPK20 RNAl replicase CDS.
SEQ ID No. 52: TRV PPK20 RNA2 5′ replication element containing sequence
SEQ ID No. 53: TRV Ppk20 RNA2 3′ replication element containing sequence
SEQ ID No. 54: Arabidopsis thaliana ESR gene CDS.
SEQ ID No. 55: Arabidopsis thaliana SAG12 (senescence associated gene 12) CDS.
SEQ ID No. 56: Arabidopsis thaliana PAD4 (phytoalexin deficient 4) gene CDS.
SEQ ID No. 57: Arabidopsis thaliana CPR5 (constitutive expression of PR genes 5) gene CDS.
SEQ ID No. 58: Arabidopsis thaliana ACD1 (accelerated cell death 1) gene CDS.
SEQ ID No. 59: Arabidopsis thaliana ATG18 (homolog of yeast autophagy gene 18 G) gene CDS.
Additional sequences included in this application are from Arabidopsis. Each line provides the gene symbol, genes name and Arabidopsis accession number.
Starvation:
SEQ ID No. 60: HDH (HISTIDINOL DEHYDROGENASE) AT5G63890
SEQ ID No. 61: ATHMEE2 (MATERNAL EFFECT EMBRYO ARREST 2/=SHI KIMATE DEHYDROGENASE) AT3G06350
SEQ ID No. 62: ICDH (ISOCITRATE DEHYDROGENASE) AT1G54340
Early Senescence:
SEQ ID No. 63: APG 9 (AUTOPHAGY 9) AT2G31260
SEQ ID No. 64: ATG 2 (AUTOPHAGY 2) AT3G19190
SEQ ID No. 65: SRI (SIGNAL RESPONSIVE 1) AT2G22300
SEQ ID No. 66: APG7 (AUTOPHAGY 7) AT5G45900
Definitions
RNAi Payload means a payload consisting of at least one specific nucleic acid sequence or analogue sequence that, when introduced into the body of a plant, will trigger or initiate an RNAi cascade.
Cell (or host plant cell) means a cell or protoplast of a plant cell and includes isolated cells and cells in a whole plant, plant organ, or fragment of a plant. It also includes non-isolated cells.
Double stranded region means a region of a polynucleotide wherein the nucleotides or analogues are capable of hydrogen bonding to each other. Such hydrogen bonding can be intramolecular or intermolecular (e.g. single transcription unit forming a double stranded region with the so-called hairpin or two transcription units that align appropriately for complementary sequences to hydrogen bond). To be a double stranded region, according to the present invention, it is not necessary for 100% of the nucleotides to be complementary and hydrogen bonded within a region. It is merely necessary for sufficient base pairing to occur to give the RNA a substantial double stranded character (e.g. an indicative melting point).
RNAi Inducer means at least one specific nucleic acid sequence or analogue sequence that, when introduced into the body of a plant, will trigger or initiate an RNAi cascade. This can be, for example, but is not limited to DNA, dsRNA, ssRNA, siRNA, and miRNA sequences. RNAi inducers are usually capable of activating RNAi in a number of species.
Targeting constructs are added to the RNAi inducer sequence to direct the RNAi response against specific endogenous polynucleotides.
Targeting construct means a region of nucleic acid sequence that is complementary to one or more endogenous or exogenous polynucleotides. siRNAs released from the processing of a targeting construct direct RNAi machinery to knock-down endogenous polynucleotides.
RdRP means a RNA-dependent RNA polymerase. An RdRP creates a complementary strand of RNA using RNA as a template. Endogenous RdRPs include components of RISC machinery, and DNA-dependent RNA polymerases when recruited by special RNA sequences/structures. Exogenous RdRPs come from virus, retrotransposons, or are harvested from another organism.
Exogenous gene means a gene that is not normally present in a given host genome in the present form. In this respect, the gene itself may be native to the host genome, however the exogenous gene will comprise the native gene altered by the addition or deletion of one or more different regulatory elements or additional genes.
Gene or genes means nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any functional portion of such whole RNA or whole protein sufficient to possess a desired characteristic.
Marker gene means a gene that, when its activity is altered, imparts a distinct phenotype.
Essential gene means a gene that, when inhibited, results in a negative effect on at least one of plant growth and development. They are required for normal plant growth and reproduction.
Heterologous polynucleotide means any polynucleotide that is introduced (transiently or stably) into a non-transformed host plant. A polynucleotide is not excluded from being a heterologous polynucleotide by the presence of matching endogenous polynucleotide sequences.
Homologous means having sequence similarity sufficient to allow hybridization in vivo, in vitro, and/or ex vivo under low stringency conditions between the antisense sequence and the sense gene mRNA.
Inhibition of gene expression means a decrease in the level of protein and/or RNA product from a target gene. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, polymerase chain reaction (PCR), reverse transcription (RT) reverse transcription PCR(RT/PCR), gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence assisted cell sorting (FACS).
Substantially complementary, with respect to the sense and antisense sequences means sufficiently complementary to allow for formation of a double stranded molecule.
Transcript means RNA encoded by DNA. In the context of sense and antisense transcripts of the present invention, such sense and antisense transcripts can be part of the same polynucleotide or they can be 2 separate polynucleotides (i.e., each having its own 5′ and 3′ end).
Treating a weed plant means a method to cause a deleterious effect on the weed, for example, but not limited to, interfering with development, reducing growth, triggering programmed cell death such as apoptosis, senescence, or autophagy, reducing vigour, interfering with reproductive viability, or result in death.
hpRNA is hairpin RNA, produced through inverted repeats with or without a single stranded loop region.
RISC is an RNA-induced silencing complex.
dsRNA is double stranded RNA. siRNA is short interfering RNA.
miRNA is microRNA and is a small non-coding RNA molecule (ca. 22 nucleotides) found in plants and animals. They function in transcriptional and post-transcriptional regulation of gene expression.
pTRVl and pTRV2 are well proven RNAi inducers. One skilled in the art can use other virus based sequences to create an inducer by placing the virus sequence between a suitable promoter and terminator and incorporating an MCS into it.
Weeds mean members of the Amaranthaceae family, such as green pigweed and redroot pigweed, members of the Anacardiaceae family, such as western poison-oak, central poison-ivy, eastern poison-ivy, rydberg's poison-ivy, and poison sumac, members of the
Asclepiadaceae family, such as common milkweed, black dog-strangling vine, and dog-strangling vine, members of the Balsaminaceae family such as spotted jewelweed, members of the Berberidaceae family such as common barberry, members of the Boraginaceae family such as blueweed, and stickseed, members of the Caryophyllaceae family such as purple cockle, mouse-eared chickweed, bouncingbet, night-flowering catchfly, white cockle, bladder campion, corn spurry, chickweed, grass-leaved stichwort, and cow cockle, members of the Chenopodiaceae family such as Russian pigweed, lamb's quarters, Kochia, and Russian thistle, members of the Compositae family (Asteraceae) such as common yarrow, Russian knapweed, common ragweed, perennial ragweed, giant ragweed, stinking mayweed, common burdock, woolly burdock, absinth, biennial wormwood, mugwort, New England aster, nodding beggarticks, tall beggarticks, plumeless thistle, nodding thistle, diffuse knapweed, brown knapweed, spotted knapweed, black knapweed, chicory, Canada thistle, bull thistle, Canada fleabane, smooth hawk's-beard, narrow-leaved hawk's-beard, Philadelphia fleabane, rough fleabane, spotted Joe-Pye weed, hairy galinsoga, orange hawkweed, mouse-eared hawkweed, king devil hawkweed, spotted cat's-ear, elecampane, poverty weed, false ragweed, prickly lettuce, blue lettuce, nipplewort, fall hawkbit, ox-eye daisy, pineapple weed, scentless chamomile, black-eyed Susan, tansy ragwort, Canada goldenrod, perennial sow-thistle, spiny annual sow-thistle, annual sow-thistle, tansy, dandelion, goat's-beard, meadow goat's-beard, colt's-foot, and cocklebur, members of the Convolvulaceae family such as field bindweed, and field dodder, members of the Crassulaceae family such as mossy stonecrop, members of the Cruciferae family (Brassicaceae) such as garlic mustard, yellow rocket, hoary alyssum, Indian mustard, bird rape, small-seeded false flax, shepherd's purse, lens-podded hoary cress, hare's-ear mustard, flixweed, wood whitlow-grass, dog mustard, wormseed mustard, tall wormseed mustard, dame's-rocket, field pepper-grass, common pepper-grass, poor-man's pepper-grass, ball mustard, wild radish, creeping yellow cress, wild mustard, tumble mustard, tall hedge mustard, and stinkweed, members of the Cucurbitaceae family such as wild cucumber, members of the Cyperaceae family such as yellow nut sedge, members of the Equisetaceae family such as field horsetail, members of the Euphorbiaceae family such as three-seeded mercury, cypress spurge, leafy spurge, and hairy-stemmed spurge, members of Gramineae family (Poaceae) such as wild oats, smooth brome, downy brome, smooth crab grass, large crab grass, barnyard grass, quack grass, foxtail barley, Persian darnel, witch grass, common reed, annual blue grass, Kentucky blue grass, green foxtail, and yellow foxtail, members of the Guttiferae family such as St. John's-wort, member of the Haloragaceae family such as Eurasian water-milfoil, members of the Hydrocharitaceae family such as European frogbit, members of the Labiatae family such as ajuga, American dragonhead, hemp-nettle, ground-ivy, motherwort, catnip, heal-all, andnnarsh hedge-nettle, members of the Leguminosae family (Fabaceae) such as hog-peanut, bird's-foot trefoil, black medick, white sweet-clover, yellow sweet-clover, crown vetch, white clover, and tufted vetch, members of the Liliaceae family such as false hellebore, showy false hellebore, smooth camas, and meadow camas, members of the Lythraceae family such as purple loosestrife, members of the Malvaceae family such as velvetleaf, round-leaved mallow, and common mallow, members of the Onagraceae family such as fireweed, and yellow evening-primrose, members of the Oxalidaceae family such as European wood-sorrel, members of the Plantaginaceae family including narrow-leaved plantain, broad-leaved plantain, hoary plantain, and Rugel's plantain, members of the Polygonaceae family such as Tartary buckwheat, striate knotweed, prostrate knotweed, wild buckwheat, pale smartweed, lady's-thumb, green smartweed, sheep sorrel, curled dock, long-leaved dock, field dock, serrate-valved dock, and broad-leaved dock, members of the Pteridaceae family such as bracken, members of the Portulacaceae family such as purslane, members of the Ranunculaceae family such as tall buttercup, and creeping buttercup, members of the Rhamnaceae family such as European buckthorn, members of the Rosaceae such as silvery cinquefoil, rough cinquefoil, sulfur cinquefoil, narrow-leaved meadowsweet, and hardhack, members of the Rubiaceae family such as smooth bedstraw, members of the Scrophulariaceae family such as dwarf snapdragon, yellow toadflax, Dalmation toadflax, moth mullein, common mullein, and thyme-leaved speedwell, members of the Solanaceae family such as climbing nightshade, and eastern black nightshade, members of the Typhaceae family such as narrow-leaved cattail, and cattail, members of the Umbelliferae (Apiaceae) family such as goutweed, caraway, western water-hemlock, spotted water-hemlock, poison-hemlock, wild carrot, giant hogweed, wild parsnip, and water-parsnip, and members of the Urticaceae family such as stinging nettle.
In addition, the following weeds will be controlled, if not already listed above:
Abutilon theophrasti (Velvetleaf), Acroptilon repens (Russian Knapweed), Aegilops cylindrica (Jointed Goatgrass), Agropyron repens (Quackgrass), Alyssum, Hoary (Berteroa incana), Amaranthus retroflexus (Redroot Pigweed), Anchusa officinalis (Common Bugloss), Annual Bluegrass (Poa annua), Annual Sow-thistle (Sonchus oleraceus), Annual Sow-thistle, Spiny (Sonchus asper), Anthriscus sylvestris (Wild Chervil), Arctium spp. (Burdock), Asclepias speciosa (Showy Milkweed), Avena fatua (Wild Oats), Baby's-Breath (Gypsophila paniculata), Barley, Foxtail (Hordeum jubatum), Barnyardgrass (Echinochloa crusgalli), Beggar-Ticks, Nodding (Bidens cernua), Berteroa incana (Hoary Alyssum), Bidens cernua (Nodding Beggar-Ticks), Bindweed, Field (Convolvulus arvensis), Bladder Campion (Silene cucubalus), Bluegrass, Annual (Poa annua), Blueweed (Echium vulgare), Bog Rush (Juncus effusus), Broad-Leaved Plantain (Plantago major), Buckwheat, Tartary (Fagopyrum tataricum), Buckwheat, Wild (Polygonum convolvulus), Bugloss, Common (Anchusa officinalis), Bull Thistle (Cirsium vulgare), Burdock (Arctium spp.), Buttercup, Creeping (Ranunculus repens), Canada Thistle (Cirsium arvense), Capsella bursa-pastoris (Shepherd's-Purse), Cardaria spp. (Hoary Cress), Carduus nutans (Nodding Thistle, a.k.a. Musk Thistle), Carduus acanthoides (Plumeless Thistle), Centaurea diffusa (Diffuse Knapweed), Centaurea pratensis (Meadow Knapweed), Centaurea solstitialis (Yellow Starthistle), Centaurea maculosa (Spotted Knapweed), Chamomile, Scentless (Matricaria maritima), Chenopodium album (Lamb's-Quarters), Cichorium intybus (Chicory), Cirsium palustre (Marsh Plume Thistle), Chervil, Wild (Anthriscus sylvestris), Chicory (Cichorium intybus), Chondrilla juncea (Rush Skeletonweed), Chrysanthemum leucanthemum (Oxeye Daisy), Cicuta douglasii (Water Hemlock), Cinquefoil, Sulphur (Potentilla recta), Cirsium arvense (Canada Thistle), Cirsium vulgare (Bull Thistle), Cleavers (Galium aparine), Cluster Tarweed (Madia glomerata), Common Bugloss (Anchusa officinalis), Common Tansy (Tanacetum vulgare), Common Mallow (Malva neglecta), Common Chickweed (Stellaria media), Convolvulus arvensis (Field Bindweed), Corn Spurry (Spergula arvensis), Creeping Buttercup (Ranunculus repens), Crupina vulgaris (Crupina), Cudweed (Gnaphalium uliginosunn), Curled Dock (Rumex crispus), Cytisus scoparius (Scotch Broom), Dalmatian Toadflax (Linaria dalmatica), Diffuse Knapweed (Centaurea diffusa), Dodder, (Cuscuta spp.), Field Bindweed (Convolvulus arvensis), Field Scabious (Knautia arvensis), Foxtail Barley (Hordeum jubatum), Giant Hogweed (Heracleum mantegazzianum), Gorse (Tragopogon dubius), Green Foxtail (Setaria viridis), Groundsel (Senecio vulgaris), Gypsophila paniculata (Baby's-Breath), Hemp-Nettle (Galeopsis tetrahit), Henbit (Lamium amplexicaule), Heracleum mantegazzianum (Giant Hogweed), Himalayan Balsam (Impatiens glandulifera), Hoary Alyssum (Berteroa incana), Hoary Cress (Cardaria spp.), Hordeum jubatum (Foxtail Barley), Horsetail, Field (Equisetum arvense), Hound's-tongue (Cynoglossum officinale), Hypericum perforatum (St. John's-Wort), Impatiens glandulifera (Himalayan Balsam), Japanese Knotweed (Polygonum cuspidatum), Jointed Goatgrass (Aegilops cylindrica), Juncus effusus (Bog Rush), Knapweed, Meadow (Centaurea pratensis), Knapweed, Spotted (Centaurea maculosa), Knapweed, Russian (Acroptilon repens), Knapweed, Diffuse (Centaurea diffusa), Knautia arvensis (Field Scabious), Kochia scoparia (Kochia), Lady's-Thumb (Polygonum persicaria), Lamb's-Quarters (Chenopodium album), Lamium amplexicaule (Henbit), Leafy Spurge (Euphorbia esula), Lepidium latifolium (Perennial Pepperweed), Linaria dalmatica (Dalmatian Toadflax), Linaria vulgaris (Yellow Toadflax), Lychnis alba (White Cockle), Lythrum salicaria (Purple Loosestrife), Madia glomerata (Cluster Tarweed) Malva neglecta (Common Mallow), Marsh Plume Thistle (Cirsium palustre), Matricaria maritima (Scentless Chamomile), Matricaria matricariodes (Pineappleweed), Meadow Knapweed (Centaurea pratensis), Meadow Hawkweed (Hieracium pilosella), Milkweed, Showy (Asclepias speciosa), Mullein (Verbascum thapsus), Mustard, Wild (Sinapsis arvensis), Narrow-Leaved Plantain (Plantago lanceolata), Night-Flowering Catchfly (Silene noctiflora), Nightshade (Solanum spp.), Nodding Thistle, a.k.a. Musk Thistle (Carduus nutans), Nodding Beggar-Ticks (Bidens cernua), Nutsedge, Purple (Cyperus rotundus), Nutsedge, Yellow (Cyperus esculentus), Onopordum acanthium (Scotch Thistle), Orange Hawkweed (Hieracium aurantiacum), Oxeye Daisy (Chrysanthemum leucanthemum), Panicum capillare (Witchgrass), Perennial Pepperweed (Lepidium latifolium), Perennial Sowthistle (Sonchus arvensis), Pigweed, Red root (Amaranthus retroflexus), Pinea ppleweed (Matricaria matricariodes), Plantago lanceolata (Narrow-Leaved Plantain), Plantago major (Broad-Leaved Plantain), Plumeless Thistle (Carduus acanthoides), Poa annua (Annual Bluegrass), Polygonum convolvulus (Wild Buckwheat), Polygonum cuspidatum (Japanese Knotweed), Polygonum persicaria (Lady's-Thumb), Potentilla recta (Sulphur Cinquefoil), Puncture vine (Tribulus terrestris), Purple Nutsedge (Cyperus rotundus), Purple Loosestrife (Lythrum salicaria), Quackgrass (Agropyron repens), Ranunculus repens (Creeping Buttercup), Rumex acetosella (Sheep Sorrel), Rumex crispus (Curled Dock), Rush Skeletonweed (Chondrilla juncea), Russian Knapweed (Acroptilon repens), Russian Thistle (Salsola kali), Scentless Chamomile (Matricaria maritima), Scotch Broom (Cytisus scoparius), Scotch Thistle (Onopordum acanthium), Senecio jacobaea (Tansy Ragwort), Sheep Sorrel (Rumex acetosella), Shepherd's-Purse (Capsella bursa-pastoris), Sulphur Cinquefoil (Potentilla recta), Spotted Knapweed (Centaurea maculosa), St. John's-Wort (Hypericum perforatum), Stinkweed (Thlapsi arvense), Tansy Ragwort (Senecio jacobaea), Tartary Buckwheat (Fagopyrum tataricum), Tarweed, Cluster (Madia glomerata), Thistle, Bull (Cirsium vulgare), Thistle, Canada (Cirsium arvense), Nodding Thistle a.k.a. Musk Thistle (Carduus nutans), Plumeless Thistle (Carduus acanthoides), Russian Thistle (Salsola kali), Scotch Thistle (Onopordum acanthium), Thlapsi arvense (Stinkweed), Dalmatian Toadflax (Linaria dalmatica), Yellow Toadflax (Linaria vulgaris), Tragopogon dubius (Western Goat's-Beard), Tribulus terrestris (Puncture vine), Ulex europaeus (Gorse), Velvetleaf (Abutilon theophrasti), Verbascum thapsus (Mullein), Water Hemlock (Cicuta douglasii), Western Goat's-Beard (Tragopogon dubius), White Cockle (Lychnis alba), Wild Chervil (Anthriscus sylvestris), Wild Mustard (Sinapsis arvensis), Wild Buckwheat (Polygonum convolvulus), Wild Oats (Avena fatua), Witchgrass (Panicum capillare), Yellow Hawkweed (Hieracium pratense), Yellow Starthistle (Centaurea solstitialis), Kudzu (Pueraria lobata), Japanese dodder (Cuscuta japonica), water hyacinth (Eichhornia spp.) and Yellow Nutsedge (Cyperus esculentus).
Underlying the various embodiments of the present invention is treating a weed by introducing a heterologous polynucleotide or analogue into the weed plant, the heterologous polynucleotide comprising: 1) an RNAi inducer capable of recruiting RISC machinery to the sequence and 2) a targeting construct comprising (a) an antisense sequence having homology to an essential gene, or a marker gene, or (b) a sense sequence substantially complementary to said antisense sequence; wherein said sense and antisense sequences are capable of hybridizing to each other to form a double-stranded region.
Description:
Except as otherwise expressly provided, the following rules of interpretation apply to this specification (written description, claims and drawings): (a) all words used herein shall be construed to be of such gender or number (singular or plural) as the circumstances require; (b) the singular terms “a”, “an”, and “the”, as used in the specification and the appended claims include plural references unless the context clearly dictates otherwise; (c) the antecedent term “about” applied to a recited range or value denotes an approximation within the deviation in the range or value known or expected in the art from the measurements method; (d) the words “herein”, “hereby”, “hereof”, “hereto”, “hereinbefore”, and “hereinafter”, and words of similar import, refer to this specification in its entirety and not to any particular paragraph, claim or other subdivision, unless otherwise specified; (e) descriptive headings are for convenience only and shall not control or affect the meaning or construction of any part of the specification; and (f) “or” and “any” are not exclusive and “include” and “including” are not limiting. Further, The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.
To the extent necessary to provide descriptive support, the subject matter and/or text of the appended claims is incorporated herein by reference in their entirety.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Where a specific range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. All smaller sub ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Although any methods and materials similar or equivalent to those described herein can also be used, the acceptable methods and materials are now described.
Overview: An RNAi payload is introduced into a host plant, for example, a weed by application of a formulation comprising the payload. Application methods include spraying, irrigating, injecting (extracellular as opposed to microinjection), abrading or otherwise causing entry of the formulation into, for example, but not limited to, a seed, a seedling, a sapling, a mature plant, a reproducing plant or a senescing plant. Application methods do not include stable transformation methods. The RNAi payload comprises one or more RNAi inducer elements encouraging its processing by dicer. The RNAi payload also contains a targeting region complementary to corresponding essential genes, or marker genes or both. When the RNAi payload is processed it releases siRNAs against those genes. The siRNAs direct RISC machinery to knock down those genes.
A list of genes used to build targeting constructs is provided. For each gene, one or more of double stranded RNA fragments and the DNA coding sequences or analogues that generate them are provided. These fragments have sequences that allow them to initiate the RNAi cascade, hence the DNA sequences will have, in addition, suitable promoters, for example,
but not limited to, constitutive promoters that result in a high level of expression, and a suitable transcriptional stop element. The DNA sequences may be provided as crude viral or bacterial extracts, plasmid or viral DNA with the sequence and regulatory regions inserted therein, or may be synthesized. Each target in the targeting construct comprises at least about 19 nucleotides or at least about 50 nucleotides, or at least about 100 nucleotides, or at least about 150 nucleotides, and all sub ranges therebetween. During the knock-down process RdRPs ‘transcribe’ the target mRNAs. These transcripts are processed into more siRNAs targeting the whole mRNA. These are transported through the plant where they spread the cascade.
In the context of the present invention, there are three important steps to an effective RNAi herbicide. Firstly, the RNAi payload (DNA, RNA, or synthetic oligos) is delivered to the plant or part of the plant. Application methods affect delivery, with stem injection, spray, and vector-aided delivery (without stable transformation) being common techniques. Once applied, the inducer is introduced to the cytoplasm of the target cells. This may be mediated by, for example, but not limited to, additives, chemical modification of the inducer, or vectors such as viral coat protein, or nano-cages.
Secondly, a build-up of RNA occurs that can spread from cell to cell. This can happen prior to the RNAi response if exogenous RNA polymerases (such as viral RdRPs) are included as RNAi inducer elements, or if endogenous RNA polymerases (including DNA dependant RNA polymerases) are recruited to replicate the payload. It can also happen during the RNAi response if the inducer triggers RNAi-associated RdRPs. The entire inducer can be replicated, or only specific regions (using internal RNA promoters such as viral subgenomic promoters). Inducers can use one or both of these pathways for replication. Viral RNAi suppressor proteins can be included to increase the amount of RNA present before RNAi is triggered. Cell-to-cell spread can be accelerated using viral movement proteins and by targeting key plant genes.
Finally, the RNAi inducer elements elicit an RNAi response that targets RISC machinery to degrade critical endogenous RNAs. This is accomplished by complementarity between regions of the inducer and the target RNAs. Once the inducer is processed into siRNAs they are used by RISC to target further RNA. siRNAs produced from the targeting construct are complementary to endogenous target genes. These are knocked down as “Collateral damage” while the plant clears the payload.
In addition to the sequences for essential genes, the payload will include RNA fragments that will silence genes that modulate the RNAi cascade. These will be synthetic or virally derived RNA fragments targeting components of the RNAi pathway. Without being bound by theory, it is believed that the RNA payload used in the present technology will target and silence, knock-down, or dysregulate genes that are necessary for the proper growth and development and optimally, the survival of the weed.
Elements
Target genes: Apoptosis; Autophagy; Senescence; Starvation; Accessory (RISC components)
RNAi inducers: Replicase/promoter pairs; (Viral replicase and promoter/subgenomic promoter pairs; Recruitment and co-option of endogenous RNA polymerases; and Action of endogenous rdRPs [siRNA asymmetries, single base mismatches]); Recruitment of DNA ligases for RNA ligation; and that recruit dicer for RNAi processing (dsRNA regions [Inverted repeats; Hairpins; and Direct repeats])
Functional elements: Promoters; Terminators; Ribosome binding sites; Internal ribosome entry sites; Hammerhead ribozymes; Recruitment and co-option of endogenous DNA ligase to ligate RNA; and Cap stealing or RNA capping sequences.
Exogenous helper genes: Coat proteins; Movement Proteins; and RNAi suppressor proteins.
Without being bound to theory, there are three primary ways to kill plants using an RNAi cascade. The first way knocks down production of essential cellular components. This causes cells to starve, or to structurally degrade. Target genes include EPSP synthese, chalcone synthase, starch synthase, cellulose synthase, acetyl-COA reductase, transaminase, 18S rRNA, eEF-IB gamma, SAP130b, TRPT, PAI1, PDS, DGL
The second way is to induce apoptotic programmed cell death by knocking out key repressors in the pathway. This results in Hypersensitive response like (HR) and necrotic lesions. It is quicker than starvation but may in some situations be too quick, killing cells before the RNAi cascade can spread. Runaway hypersensitive response can also be elicited in this manner. Target genes include BECLIN1 PI3K/VPS30 ATG3 ATG7 CAT1 ACD2, NbTCTP, LLS1.
The final way is by inducing senescence, again by knocking out key repressors of that pathway. Once senescence is triggered cells undergo a slower, more regulated cell death. Target genes to induce senescence include: APG 9 (Autophagy 9), ATG 2 (Autophagy 2), SRI (Signal responsive 1, APG7 (Autophagy 7)
Autophagy can be triggered along with any of these responses. During autophagy plant cells engulf and digest their organelles.
Helper genes include the following:

    
    
        Protein coding sequences for:
        
            Viral movement proteins that interact with coat protein (VIGS based inducer)
            Viral movement proteins that do not require coat protein
            Viral coat proteins
        
        
    
    


Production of RNA is achieved through transcription of a DNA template, either in a cell such as E. coli or in vitro. DNA is produced in cells, or through PCR. Promoter-polymerase combinations such at the T7 system can be used for tight control of transcription and high yield. Eukaryotic expression systems such as yeast are also viable production factories. Viral coat proteins or other protective structures may be produced in the same cells or added later to purified RNA.
The simplest RNAi inducers are siRNAs. They are recognised by RISC machinery and used directly to guide knockdown of endogenous polynucleotides. If properly formatted they also encourage endogenous RdRPs to amplify the silencing signal and cause transitivity. The siRNA sequence is also the simplest targeting construct. Longer targeting constructs can be grouped together on an RNA. Using secondary structure such as hairpins, or by transcribing both DNA strands into RNA, large dsRNA regions are created. These are recognised and processed by RISC machinery. The dsRNA replication intermediate of many virus is a trigger for RNAi, allowing many virus to act as RNAi inducers. Incorporating a targeting construct into such a virus results in a functional RNAi herbicide. Finally, single stranded RNA or DNA transcribed in plant cells can trigger RNAi if the RNA is replicated. This is achieved by including coding sequences for exogenous RdRPs, or through RNA sequences that recruit endogenous RdRPs.
Examples of the RNAi payloads used in the present technology follow and were designed to target the sequences as shown:







SEQ ID NO: 1 


Actin 2 siRNA-A (target sequence: 5′-GCATCACACTTTC


GTACAA-3′);


 


SEQ ID NO: 2 


Actin 2 siRNA-B (target sequence: 5′-CGAGAAGAACTAT


GAATTA-3′);


 


SEQ ID NO: 3 


CHLI siRNA-A (target sequence: 5′-GGAGATAGAGGAACT


GGAA-3′);


 


SEQ ID NO: 4 


CHLI siRNA-B (target sequence: 5′-GGAACATCTTCTTCT


GCAA-3′);


 


SEQ ID NO: 5 


18S siRNA-A (target sequence: 5′-GGGAGGTAGTGACAATA


AA-3′);


and


 


SEQ ID NO: 6 


18S siRNA-B (target sequence: 5′-GGACGCATTTATTAGAT


AA-3′).






The RNAi payloads used in the present technology were designed to target the sequences as shown:







SEQ ID NO: 7


Actin 2 siRNA-A (target sequence: 5′-GGCATCACACTTT


CTACAA-3′);


 


SEQ ID NO: 8


Actin 2 siRNA-B (target sequence: 5′-CGAGAAGAACTAT


GAATTA-3′);


 


SEQ ID NO: 9


CHLI siRNA-A (target sequence: 5′-GGAGATAGAGGAACTG


GAA-3′);


 


SEQ ID NO: 10


CHLI siRNA-B (target sequence: 5′-GGAACATCTTCTTCTG


CAA-3′);


 


SEQ ID NO: 11


18S siRNA-A (target sequence: 5′-GGGAGGTAGTGACAATA


AA-3′);


and


SEQ ID NO: 12


18S siRNA-B (target sequence: 5′-GGACGCATTTATTAGAT


AA-3′).






Design: For the target genes, siRNAs were designed with reference to the literature, the target site accessibility web tool, RNAxs (http://rna.tbi,unjvie.ac.at/cgi-bin/RNAxs?hakim-1) and BLAST searches.
The genes chosen were essential genes or marker genes, which, when knocked down would be expected to provide an easily identifiable phenotype.
The first siRNAs were designed to be incorporated into Argonaute protein (AGOI). They were 21 nt in length with a UU, 3′ overhang on each end and a 5′ terminal U. A 5′ phosphate was added to the guide strand of the siRNA.
Other designs include one or more of targeting different AGO complexes, using a different 5′ nucleotide, using chemically modified siRNA to increase stability, using different 3′ overhang nucleotides, and including a 5′ phosphate on both strands.
As the mRNA target site should be accessible, the RNAxs webserver was used to search through a given mRNA sequence and identify those sites based on the 2° structure of the mRNA and thermodynamic asymmetry and folding energies associated with the siRNAs themselves.
siRNA means “small interfering RNA” which are also commonly referred to as “short interfering RNA” or alternatively as “silencing RNA”.
Details of the process used to produce targeting constructs in Nicotiana sylvestris were as follows (this process applies to designing targeting constructs for any plant): Target gene mRNAs were run through the RNAxs program using standard settings.

    
    
        The top 20-25 hits (lowest “worst rank”) were mapped to the original mRNA sequence
        
            When available, homologous mRNA sequences from other N. sylvetris (or Solanacea spp. or Arabidopsis thaliana) were also run through RNAxs and have their highest 20-25 hits mapped.
            The homologues were then aligned to compare the regions of highest effective siRNA target concentration.
            For the present technology, regions with numerous “good” targets that also have perfect (or at most 2 mismatches in a stretch of 21 nt) sequence identity to the N. sylvetris sequence were sought.
            N. sylvetris was used as the reference sequence for all targets, therefore the whole construct had perfect sequence identity to N. sylvestris. 
        
        
        Regions of effective targets were cut from the original mRNA sequence to make smaller target regions of various lengths (21-120 nt). The 18-24 nt regions can be used directly as siRNA constructs (which have both RNAi inducer and targeting construct activity). Otherwise, the process to build longer, multi-gene targeting constructs is as follows:
        
            Sequences complementary to the most accessible mRNA regions were pulled out were trimmed to remove intervening sequences where no effective siRNAs are predicted.
            Multiple trimmed segments were joined together to make an approx. 120 nt targeting cluster that consist of 10s of predicted high-effectiveness siRNAs targeting a gene of interest.
        
        
    
    


Other considerations included that the target sites should not cover splice junctions or start or stop codons and should avoid sites of single nucleotide polymorphisms between sequenced transcript variants.
Longer RNAi payloads require RNAi inducer elements to induce the processing of the payload into siRNAs. These mostly involve the production of a dsRNA region in the RNAi payload.
Synthesis: For using siRNAs directly as RNAi payloads the selected siRNAs were synthesized chemically. A 5′ phosphate was added to the guide strand of the siRNA.
Longer RNAi payloads are transcribed from DNA, either in vitro, in plantae, or in another organism such as Escherichia coli. The DNA sequences encoding longer RNAi payloads may also be synthesized or produced using standard cloning techniques and PCR, or a combination of both.
DNA production: The selected DNA encoding the RNAi payloads were cloned using standard cloning techniques, in, for example, but not limited to a replication system in E. coli, using vectors that comprise, for example, but are not limited to, pBR322, pUC series, M13 mp series, pACYC184, etc., and pCAMBIA 1201. The DNA sequence was inserted into the vector at a suitable restriction site. The resulting plasmid was used for transformation into E. coli. The E. coli cells were cultivated in a suitable nutrient medium, then harvested, lysed and optionally lyophilize and used directly, or the plasmid was recovered and used as such, or the specific sequence and the promoter and the transcription stop were recovered and used.
There are a wide number of promoters that can be employed, including constitutive inducible, and tissue or temporally specific promoters. Plant promoters include but are not limited to ribulose-l,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat-shock promoters, the enhanced Ca MV 35S promoter and tissue specific promoters.
Transcription stops include but are not limited to nopaline synthase (NOS) gene transcription stop, the Cauliflower mosaic virus (CaMV) 35S gene transcription stop, and the Rubisco small subunit (SSU) gene transcription stop.
Those skilled in the art will be aware of additional promoter sequences and terminator sequences suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.
Embodiments of the present invention are taught herein where it is desirable to have more than one terminator. Examples of such are embodiments are where the sense and antisense sequences are to be contained on separate transcripts (i.e. each having its own 3′ and 5′ end).
Delivery: The RNAi payloads are delivered to the weed as a formulation by spraying, irrigating, injecting, or abrading a seedling, a sapling, a mature plant, a reproducing plant or a senescing plant. Both the stem and the petiole will be injected. Leaves will be specifically targeted in addition to delivering the formulation to the entire plant. Seeds will also treated by dipping or imbibition. Roots will be treated by irrigation.
In addition to the RNAi payload, accessory targeting constructs, and helper genes, the formulations include any or all of a liquid carrier, a surfactant, a binder and tackifier, a thickener, a colourant, a spreader, an antifreezing agent, a sticker, an anticaking agent, a stabilizer, a disintegrator, an emulsifier, a synergistic compound, an abrasive, an emulsifier, a penetrating agent and a preservative.
The liquid carrier includes, for example, alcohols including monohydric alcohols such as methanol, ethanol, propanol, isopropanol, butanol and the like and polyhydric alcohols such as ethylene glycol, diethylene glycol, propylene glycol, hexylene glycol, poly(ethylene glycol), poly(propylene glycol), glycerol and the like; polyhydric alcohol-based compounds such as propylene glycol ether and the like; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, diisobutyl ketone, cyclohexanone and the like; ethers such as ethyl ether, dioxane, ethyleneglycol monoethyl ether, dipropyl ether, tetrahydrofuran and the like; aliphatic hydrocarbons such as normal paraffins, naphthenes, isoparaffins, kerosenes, minerals oil and the like; aromatic hydrocarbons such as benzene, toluene, xylene, solvent naphtha, alkylnaphthalenes and the like; halogenated hydrocarbons such as dichloroethane, chloroform, carbon tetrachloride and the like; esters such as ethyl acetate, diisopropyl phthalate, dibutyl phthalate, dioctyl phthalate, dimethyl adipate and the like; lactones such as gamma-butyrolactone and the like; amides such as dimethylformamide, diethylformamide, dimethylacetamide, N-alkylpyrrolidinone and the like; nitriles such as acetonitrile and the like; sulfur compounds such as dimethyl sulfoxide and the like; vegetable oils such as soybean oil, rapeseed oil, cottonseed oil, castor oil and the like; water; and so on. These can be used singly or can be used as a combination of two kinds or more.
The penetrating agents include dimethyl sulphoxide (DMSO), Azone (1-dodecylazacycloheptan-2-one or laurocapran), N-methyl-2-pyrolidone, glycols (diethylene glycol and tetraethyleneglycol), fatty acids (lauric acid, myristic acid, oleic acid and capric acid), terpenes such as the essential oils of eucalyptus, chenopodium and ylang-ylang, sesquiterpenes, polyethylene glycol (PEG) and L-menthol.
The surfactant includes, for example, nonionic surfactants such as sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene resinate esters, polyoxyethylene fatty acid diesters, polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene dialkyl phenyl ethers, polyoxyethylene alkyl phenyl ether-formalin condensate products, polyoxyethylene-polyoxypropylene block copolymers, alkyl polyoxyethylene-polypropylene block polymer ethers, polyoxyethylenealkylamines, polyoxyethylene fatty acid amides, polyoxyethylene fatty acid bisphenyl ethers, polyalkylene benzyl phenyl ethers, polyoxyalkylene styrylphenyl ethers, acetylene diols, polyoxyalkylene-added acetylene diols, polyoxyethylene ether-type silicones, ester-type silicones, fluorine surfactants, polyoxyethylene castor oils, hydrogenated polyoxyethylene castor oils and the like; anionic surfactants such as alkyl sulfate salts, polyoxyethylene alkyl ether sulfate salts, polyoxyethylene alkyl phenyl ether sulfate salts, polyoxyethylene styryl phenyl ether sulfate salts, alkylbenzenesulfonate salts, lignin sulfonate salts, alkylsulfosuccinate salts, naphthalenesulfonate salts, alkylnaphthalene sulfonate salts, salts of formalin condensate products of naphthalene sulfonic acid, salts of formalin condensate products of alkylnaphthalene sulfonic acid, fatty acid salts, polycarboxylate salts, N-methyl-fatty acid sarcosinate, resinates, polyoxyethylene alkyl ether phosphate salts, polyoxyethylene alkyl phenyl ether phosphate salts and the like; cationic surfactants such as laurylamine hydrochloride salts, stearylamine hydrochloride salts, oleylamine hydrochloride salts, stearylamine acetate salts, stearylaminopropylamine acetate salts, alkylamine salts including alkyltrimethylammonium chloride and alkyldimethylbenzalkonium chloride and the like; ampholytic surfactants such as amino acid type- or betaine type-surfactants and the like; and so on. These surfactants can be used singly or can be used as a combination of two kinds or more.
The binder and tackifier include, for example, carboxymethylcellulose and a salt thereof, dextrin, water-soluble starch, xanthan gum, guar gum, sucrose, poly(vinylpyrrolidone), gum arabic, polyvinyl alcohol), polyvinyl acetate), sodium polyacrylate, poly(ethylene glycol) with an average molecular weight of 6000 to 20000, polyethylene oxide with an average molecular weight of 100000 to 5000000, phospholipid (for example, cephalin, lecithin and the like) and so on.
The thickener includes, for example, water-soluble polymers such as xanthan gum, guar gum, carboxymethylcellulose, poly(vinylpyrrolidone), carboxyvinyl polymers, acrylic polymers, starch-based compounds and polysaccharides; inorganic fine powders such as high-purity bentonite and fumed silica (white carbon); and the like.
The colourant includes, for example, inorganic pigments such as iron oxide, titanium oxide, and Prussian blue; organic dyes such as an alizarin dye, azo dye, and metal phthalocyanine dye; and the like.
The spreader includes, for example, silicone-based surfactants, cellulose powders, dextrin, modified starch, a polyaminocarboxylic acid chelate compound, crosslinked poly(vinylpyrrolidone), a copolymer of maleic acid with a styrene compound, a (meth)acrylic acid copolymer, a half ester of a polymer composed of polyhydric alcohol with dicarboxylic anhydride, a water-soluble salt of polystyrenesulfonic acid and the like.
The sticker includes, for example, paraffin, terpene, a polyamide resin, polyacrylate, polyoxyethylene, wax, polyvinyl alkyl ether, an alkylphenol-formalin condensate product, a synthetic resin emulsion and the like.
The antifreezing agent includes, for example, polyhydric alcohols such as ethylene glycol, diethylene glycol, propylene glycol, glycerol and the like, and so on.
The anticaking agent includes, for example, polysaccharides such as starch, alginic acid, mannose, galactose and the like; poly(vinylpyrrolidone), fumed silica (white carbon), ester gum, a petroleum resin and the like.
The disintegrator includes, for example, sodium tripolyphosphate, sodium hexametaphosphate, metal stearates, a cellulose powder, dextrin, a methacrylate copolymer, poly(vinylpyrrolidone), a polyaminocarboxylic acid chelate compound, a sulfonated styrene-isobutylene-maleic anhydride copolymer, a starch-polyacrylonitrile graft copolymer and the like.
The stabilizer includes, for example, desiccants such as zeolite, calcined lime, magnesium oxide and the like; antioxidants such as phenol compounds, amine compounds, sulfur compounds, phosphoric acid compounds and the like; ultraviolet absorbers such as salicylic acid compounds, benzophenone compounds and the like; and so on.
The preservative includes, for example, potassium sorbate, 1,2-benzthiazolin-3-one and the like.
The abrasives include carborundum, silica, calcium oxalate, microbeads, nanobeads, nanoparticles and the like.
In a number of cases it is advantageous to add emulsifiers to the formulation. A first preferred group of emulsifiers encompasses non-ionic surfactants such as, for example: products of the addition of 2 to 30 mol ethylene oxide and/or 0 to 5 mol propylene oxide onto linear or branched, saturated or unsaturated C. sub.8-22 fatty alcohols, onto C. sub.12-22 fatty acids and onto alkyl phenols containing 8 to 15 carbon atoms in the alkyl group; C. sub.12/18 fatty acid monoesters and diesters of addition products of 1 to 30 mol ethylene oxide onto glycerol; glycerol mono- and diesters and sorbitan mono- and diesters of saturated and unsaturated fatty acids containing 6 to 22 carbon atoms and ethylene oxide addition products thereof; addition products of 15 to 60 mol ethylene oxide onto castor oil and/or hydrogenated castor oil; polyol esters and, in particular, polyglycerol esters such as, for example, polyglycerol polyricinoleate, polyglycerol poly-12-hydroxystearate or polyglycerol dimerate isostearate. Mixtures of compounds from several of these classes are also suitable; addition products of 2 to 15 mol ethylene oxide onto castor oil and/or hydrogenated castor oil and/or other vegetable oils; partial esters based on linear, branched, unsaturated or saturated C. sub.6/22 fatty acids, ricinoleic acid and 12-hydroxystearic acid and glycerol, polyglycerol, pentaerythritol, dipentaerythritol, sugar alcohols (for example sorbitol), alkyl glucosides (for example methyl glucoside, butyl glucoside, lauryl glucoside) and polyglucosides (for example cellulose); mono-, di and trialkyl phosphates and mono-, di- and/or tri-PEG-alkyl phosphates and salts thereof; wool wax alcohols; polysiloxane/polyalkyl polyether copolymers and corresponding derivatives; mixed esters of pentaerythritol, fatty acids, citric acid and fatty alcohol and/or mixed esters of C. sub.6-22 fatty acids, methyl glucose and polyols, preferably glycerol or polyglycerol, polyalkylene glycols and alkyl and glycerol carbonates.
The formulation may be prepared as a mixture with components other than those listed above, such as, for example, another herbicide, a plant growth regulator, a fertilizer and the like. It is proposed that these adjuvants would increase the efficacy of the treatment or would have a synergistic effect.
When the aforementioned additional ingredient is contained in the formulation, a content thereof is selected in the range of, on a mass basis, usually 5 to 95% or, preferably, 20 to 90% as a carrier, usually 0.1 to 30% or, preferably, 0.5 to 10% as a surfactant, and 0.1 to 30% or, preferably, 0.5 to 10% as other additional ingredients.
The formulation can be employed as prepared in any desired formulations including liquid formulations, emulsifiable concentrates, wettable powders, dust formulations, oil solutions, water dispersible granules, flowable, emulsion waters, granules, jumbo formulations, suspended-emulsions, microcapsules and others.
FIG. 1 shows a DPC targeting construct for photobleaching-based death in multiple species in accordance with an embodiment of the technology. Ath=Arabidopsis thaliana, Nto=Nicotiana tobacum, Bra=Brassica napus, Zma=Zea mays, Mtr=Medicago truncatula. 
FIG. 2 shows an apoptosis targeting construct for Brassica rapa in accordance with an embodiment of the technology. Inserted into vector for E. coli production or transcribed in vitro. Resultant dsRNA is applied to plants.
FIG. 3 shows an apoptosis targeting construct 2, for N. sylvetris in accordance with an embodiment of the technology. sgP=subgenomic promoter. Cloned into RNA2-MCS vectors or co-expressed with TRV replicase.
FIG. 4 shows an apoptosis targeting construct 3, for N. sylvetris inside TRV RNA2 in accordance with an embodiment of the technology. RNA applied to plants along with TRV RNA1.
FIG. 5 shows a T7-driven helper construct in accordance with an embodiment of the technology. RNA added directly to plants, or cloned into RNA2-MCS or RNA1-MCS vectors.
FIG. 6 shows an empty VIGS-based vector to produce coated RNAl and 2 based RNAi inducers in E. coli in accordance with an embodiment of the technology. Targeting constructs such as FIG. 3 are cloned into the MCS contained in RNA2, usually with flanking subgenomic promoters.
FIG. 7 shows an empty VIGS-based vector to produce coated RNAl and 2 based RNAi inducers in accordance with an embodiment of the technology. Targeting constructs such as FIG. 3 are cloned into the MCS contained in RNA2, usually with flanking subgenomic promoters.
FIG. 8 shows an empty VIGS-based vector to produce coated RNAl and 2 based RNAi inducers in yeast in accordance with an embodiment of the technology. A targeting construct is cloned into the MCS contained in RNA2, usually with flanking subgenomic promoters.
FIG. 9 shows an empty VIGS-based vector to produce naked TRV RNAl and RNA2 based RNAi inducers in accordance with an embodiment of the technology. Functional in E. coli with T7 Polymerase and for in vitro production. Ribozymes cleave the RNA into separate strands.
FIG. 10 shows a generic model of a DNA construct for an RNAi herbicide. The core of the herbicide is the targeting construct, tuned to affect one or a few plant species. RNAi inducer elements are either inserted into the targeting sequence (introns to make hairpins, direct or inverted repeats with/without base pairing mismatches), or are inserted around the targeting construct (subgenomic, viral, or endogenous RdRP promoters). This is all driven by either single or flanking promoters for RNA production in the chosen production species, and a circular or linear backbone for maintaining the construct in the production species.
FIG. 11 shows a construct for producing an RNAi herbicide in E. coli, without a target construct. In bacteria the TRV coat protein is transcribed and translated. Targeting constructs are inserted into the MCS. The TRV RNAl fragments facilitate coating of the RNA. In target plants this RNA is transcribed to produce viral replicase, which produces dsRNA from the entire RNA. This induces the RNAi response.
Treatment: By way of example, suitable exemplary treatments are outlined as follows:
Example 1
SEQ ID NO: 3 will be used to treat Medicago truncatula seeds by imbibition. This sequence targets the gene for the CHLI subunit of magnesium chelatase (SULFUR gene). Seeds will be imbibed in a solution containing SEQ ID NO: 3 and siRNA targeting AG06. The results will show that the seedlings, and more specifically, the cotyledons will be chlorotic in comparison to the controls.
Example 2
SEQ ID NO: 1 will be used to treat Arabidopsis thaliana plants, by abrading the leaves and delivering a solution of SEQ ID NO: 1, a surfactant, and siRNA targeting AG06. This sequence targets the Actin 2 gene. The results will show that the leaves senesce more rapidly than those of the controls.
Example 3
SEQ ID NO: 1 will be used to treat Arabidopsis thaliana plants, by abrading the leaves and applying a solution of SEQ ID NO: 1 and a surfactant. This sequence targets the Actin 2 gene. The results will show that the leaves senesce more rapidly than those of the controls.
Example 4
SEQ ID NO: 2 will be used to treat Arabidopsis thaliana plants, by spraying the leaves with a solution of SEQ ID NO: 2, an abrasive and a surfactant. This sequence targets the Actin 2 gene. The results will show that the leaves senesce more rapidly than those of the controls.
Example 5
SEQ ID NO: 4 will be used to treat Medicago truncatula plants, by injecting the stem or petiole with a solution of SEQ ID NO: 2, 2,4-Dichlorophenoxyacetic acid (2,4-D) DMSO and siRNA targeting AG06. SEQ ID NO: 4 targets the gene for the CHLI subunit of magnesium chelatase. The results will show that the leaves become chlorotic more rapidly and to a greater extent than those of the controls. Similarly, the leaves will become chlorotic more rapidly and to a greater extent than those treated with the formulation of Example 1. Without being bound to theory, it is expected that there is a synergistic effect caused by the combination of the siRNA and the 2,4-D.
Example 6
SEQ ID NO: 2 will be used to treat Physcomitrella patens, by spraying the moss with a solution of SEQ ID NO: 2 and a surfactant. SEQ ID NO: 4 targets the Actin 2 gene. The results will show that the moss senesces more rapidly than those of the controls.
Example 7
SEQ ID NO: 5 will be used to treat Medicago truncatula plants by irrigating the roots with a solution of SEQ ID NO: 5. SEQ ID NO: 5 targets the 18S ribosomal RNA gene. The results will show that the plants senesce more rapidly than the controls.
Example 8
SEQ ID NO: 8 will be used to treat Arabidopsis thaliana seeds by coating the seeds prior to imbibition. The formulation will include a sticker such as a terpene or wax or a tackifier such as xanthan gum. SEQ ID NO: 8 targets the 18S ribosomal RNA gene. The results will show that the seedlings senesce, whereas the controls do not.
Example 9
Other sequences will be synthesized and tested. The following functions and genes will be targeted:
Regulating water content (Lock stomata open or closed)
Targets: Effectors that open stomata, Regulators of effectors that open/close stomata.
Gene: ABI1 (AT4G26080) (component of negative feedback loop in abscisic acid (ABA) signalling).
Result: Without being bound to theory, stomata stay closed contributing to the overall damage to the plant and forcing it to retain water.
Example 10
Deregulate Starch Breakdown
Targets: Starch synthesis genes and Repressors of starch breakdown genes (repressors of amylases).
Gene: ADP-glucose pyrophosphorylase (At5g48300).
Result: Without being bound to theory, increased concentration of simple sugars which will affect the energy state of the plant, alter the direction and volume of phloem transport, and make it more accessible to saprotrophs and other organisms.
Example 11
Gene targets that will limit the effectiveness of the RNAi cascade.
Targets: DCLs, AGOs, other RISC components etc.
Gene: AGO6 (AT2G32940)
Result: Without being bound to theory, a subset of sRNA processing is ablated altering regulation across the plant and increasing the relative number of available intracellular RISC components.
Example 12
Limit ability of plasmodesmata to close (lock them open via an effector or an element from a component of the blue light response pathway).
Targets: Effectors that close plasmodesmata, or repressors of effectors that open plasmodesmata
Gene: Cadmium-ion induced glycine rich protein cdiG RP homologues, an effector that closes plasmodesmata.
Result: Without being bound to theory, plasmodesmata will tend to be open thus maximizing the ability of the RNAi cascade to spread through the plant.
Example 13
Deregulate Cell Wall Modifying Enzymes
Targets: Repressors of cell wall modifying enzymes—specifically enzymes that break down main, load bearing and protective components of the cell wall (expansins, cellulases, proteases, glycoside hydrolases etc.). Gene: DREB binding factor homologues: an effector that negatively regulates leaf elongation.
Result: Without being bound to theory, in addition to contributing to the overall damage (lethality) to the plant, this will also make the plant matter easier to degrade by saprotrophs and other organisms.
Example 15
SEQ ID No. 13 is cloned into SEQ ID No. 28. DNA is grown in E. coli and then purified. Arabidopsis thaliana, Brassica rapa, Medicago truncatula, Zea mays, and Nicotiana leaves are treated with the resultant DNA. In all plants the result will be death through loss of chlorophyll production and chlorophyll degradation (photobleaching).
Example 16
SEQ ID No. 14 is cloned in between T7 promoters in a standard vector. DNA is grown in E. coli expressing T7 polymerase or transcribed in vitro. The resulting dsRNA is purified and applied directly to Brassica rapa subsp. pekinensis. Treated plants will undergo systemically spreading hypersensitive-response like cell death.
Example 17
SEQ ID No. 15 DNA is cloned into the MCS of the T7-RNA2-MCS inducer as shown in sequence ID No 26. The resultant DNA along with the T7-RNA1 inducer are transcribed in vitro. The resulting naked RNAs are applied directly to Nicotiana sylvestris. Viral replicase increases the amount of RNAi inducer and targeting construct present. Viral coat proteins and movement proteins initiate the systemic spread of the targeting construct before the RNAi response begins. Treated plants die from necrotic/HR-like lesions.
Example 18
T7 promoter driven RNA2 containing Seq ID NO 15 flanked by sgPs and T7 promoter driven RNAl are prepared and used on Nicotiana sylvestris as above. The subgenomic promoter sequences flanking the targeting construct cause it to be replicated by itself, further increasing the amount of targeting construct present before the RNAi response.
Example 19
A targeting construct flanked with sgPs is cloned into the MCS in FIG. 7. The resulting DNA is then cloned into a standard vector and grown in E. coli. Transcription results in production of coat protein mRNA and RNAl containing a targeting construct. Translation of the coat protein in E. coli results in monomers that recognise elements of the TRV RNAl sequences, coating the entire RNA. This adds stability and increases ease of transmission to target plants. Once inside the target plant, replicase is produced from the TRV RNAl fragment, replicating the entire fragment as well as producing dsRNA of the targeting construct.
Example 20
SEQ ID No. 13 is flanked by subgenomic promoters and cloned into the TRV1 RNAl fragment MCS of an RNAi inducer construct. Treatment of the above plants with the RNA produced will result in photobleaching of chloroplasts and resultant plant death through energy starvation.
Example 21
SEQ ID No. 14 is cloned in original and inverted orientation into an RNAi GG vector. This results in a hairpin with the two sequences separated by an intron. Treatment of plants with this DNA vector produces the hairpin RNA which is processed into siRNAs targeting the 5 endogenous genes. Treated plants die from spreading necrotic lesions as runaway apoptosis is initiated.
Example 22
SEQ ID No. 15 was cloned into the MCS of SEQ ID 18. The resulting DNA is delivered directly to plants along with a helper construct encoding a replicase (SEQ ID No. 17) Endogenous promoters transcribe the initial RNA, replicase is translated, and the construct RNA is replicated. Treated Nicotiana sylvestris die from runaway HR-associated apoptotic cell death.
Example 23
SEQ ID No. 16 was cloned into the MCS of SEQ ID 18. The resulting DNA is delivered directly to plants along with a helper construct encoding a replicase (SEQ ID No. 17) Endogenous promoters transcribe the initial RNA, replicase is translated, and the construct RNA is replicated. Treated Nicotiana sylvestris die from runaway HR-associated apoptotic cell death.
Example 24
SEQ ID No. 17 is a helper construct. Plants are treated with this helper construct DNA (linear or in a vector) in addition to a RNA2-MCS RNAi inducer containing a targeting construct (SEQ ID No. 13). Treated plants undergo photobleaching and death through energy starvation. SEQ id 17 contains elements such as replicase to produce dsRNA in plant cells.
Example 25
SEQ ID No. 18 is used to clone targeting construct (SEQ ID No. 16) directly. The resulting DNA is delivered directly to plants along with a helper construct encoding a replicase (SEQ ID No. 17) Endogenous promoters transcribe the initial RNA, replicase is translated, and the construct RNA is replicated. Treated Nicotiana sylvestris die from runaway HR-associated apoptotic cell death.
Example 26
SEQ ID No. 19 and SEQ ID No. 23 are transcribed in vitro (this DNA is used to produce TRV RNAl RNA in E. coli or in vitro). The resulting RNAs are delivered directly to plants with carborundum abrasive. Treated plants die from runaway HR like apoptotic cell death.
Example 27
SEQ ID No. 20 is co-transformed into E. coli containing inducible T7 polymerase along with a plasmid containing a targeting construct flanked with RNAl or 2 3′ and 5′ sequences and driven by T7 promoter (SEQ ID No. 21 containing SEQ ID No. 16 in the MCS). Upon induction the coat protein is translated and coats the two RNAs when they are transcribed. The resulting coated RNAs are delivered directly to plants. Treated plants exhibit a runaway HR-like apoptotic cell death phenotype.
Example 28
SEQ ID 16 is cloned into the MCS of SEQ ID No. 21. The product thereof is co-transformed into E. coli containing inducible T7 polymerase along with SEQ ID No. 20. Upon induction the coat protein is translated and coats the two RNAs when they are transcribed. The resulting coated RNAs are delivered directly to plants. Treated plants exhibit a runaway HR-like apoptotic cell death phenotype.
Example 29
SEQ ID No. 22 and SEQ ID 20 are transcribed in vitro using the T7 polymerase system. Coat protein from SEQ ID No. 20 isn't produced. The two RNAs that are produced are applied directly to N. sylvestris. Treated plants die from runaway HR like necrotic cell death.
Example 30
SEQ ID No. 23 and SEQ ID No. 20 are transcribed in vitro. Coat protein is not produced in vitro. Plants treated with the two RNAs die from runaway HR like necrotic cell death. The addition of the subgenomic promoter increases the amount of RNA produced in plant cells. This strengthens the RNAi signal.
Example 31
SEQ ID No. 15 is cloned into SEQ ID No. 24. The resultant sequence and SEQ ID 20 are transcribed in vitro. Coat protein is not produced in vitro. Plants treated with the two RNAs die from runaway HR like necrotic cell death. The addition of flanking subgenomic promoters results in the production of dsRNA of just the targeting construct region in addition to replication of the entire RNA. This strengthens the RNAi signal.
Example 32
SEQ ID No. 26 along with RNA produced from SEQ ID No. 23 is delivered directly to N. sylvetris with carborundum abrasive. Treated plants die from runaway HR like apoptotic cell death.
Example 33
SEQ ID No. 27 is cloned into the MCS of SEQ ID No. 24. The resultant sequence and SEQ ID No. 20 are transcribed in vitro. Coat protein is not produced in vitro. Plants treated with the two RNAs die from runaway HR like necrotic cell death. The addition of flanking subgenomic promoters results in the production of dsRNA of just the targeting construct region in addition to replication of the entire RNA. This strengthens the RNAi signal.
Example 34
SEQ ID No. 15 is cloned into SEQ ID No. 28 using Bsal sites. Plants treated with the resultant DNA transcribe a large hairpin RNA from it. This is processed into siRNAs that induce runaway HR like necrotic cell death.
Example 35
SEQ ID No. 29 is used to treat plants. Plants treated with this DNA transcribe a large hairpin RNA from it. This is processed into siRNAs that induce runaway HR like necrotic cell death.
Example 36
SEQ ID No. 30 will be used to drive transcription of RNAi herbicide components in eukaryotic platforms.
Example 37
SEQ ID No. 31 will be used in conjunction with full length TRV derived RNAs or RNA flanked with TRV RNA 3′ and 5′ ends. The protein produced coats such RNAs, protecting them and increasing the likelihood of them reaching plant tissues.
Example 38
SEQ ID No. 32 will be used in conjunction with full length TRV derived RNAs or RNA flanked with TRV RNA 3′ and 5′ ends. The protein produced coats such RNAs, protecting them and increasing the likelihood of them reaching plant tissues. Codon optimization has been used to create a sequence for optimal translation in E. coli. 
Example 39
SEQ ID No. 33 will be used in helper constructs. The protein produced suppresses RNAi temporarily in planta. This allows initial RNA elements time to replicate and reach a higher concentration before being processed by RNAi machinery.
Example 40
SEQ ID No. 34 will be incorporated in a plant expressible helper construct and DNA or RNA delivered directly to plants. It will lessen and slow the spread of the RNAi cascade.
Example 41
SEQ ID No. 35 will be incorporated into a plant-expressible helper construct and DNA or RNA applied directly to plant. It will aid in the spread of RNAs before RNAi is activated.
Example 42
SEQ ID No. 36 will be used to find homologues in other species. It is also a target gene used to build target constructs. Knockdown of this gene will result in autophagy.
Example 43
SEQ ID No. 37 will be used to find homologues in other species. It is also a target gene used to build target constructs. Knockdown of this gene will cause apoptosis.
Example 44
SEQ ID No. 38 will cause apoptosis. It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 45
SEQ ID No. 39 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 46
SEQ ID No. 40 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 47
SEQ ID No. 41 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 48
SEQ ID No. 42 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 49
SEQ ID No. 43 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 50
SEQ ID No. 44 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 51
SEQ ID No. 45 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 52
SEQ ID No. 46 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 53
SEQ ID No. 47 It is a target gene used to build target constructs. It will also be used to find homologues in other species.
Example 54
Nicotiana sylvestris was chosen as the target weed. Medicago truncatula was chosen as the non-target plant. The sequences for both species are publically available. Genes from the target list that act as negative regulators of apoptosis were selected and the Nicotiana sylvestris homologues were run through RNAxs. The most accessible sequence regions were compared to homologous stretches of Medicago truncatula genes to confirm divergence. Suitable sequences were incorporated into SEQ ID No. 15. This construct was then cloned into the MCS of SEQ ID No. 21 in an E. coli backbone (pUC57). SEQ ID No. 20 was also cloned into the E. coli backbone. E. coli containing inducible T7 polymerase was transformed with this construct. Transformed E. coli were grown up, spun down, lysed, and the lysate rubbed onto plants with carborundum. The lysate contains RNA1 and 2 from TRV, with the targeting construct inside the MCS of RNA2. These RNAs are coated with viral coat protein. Treated plants undergo spreading apoptotic cell death similar to a runaway hypersensitive response. Without being bound to theory, this is because replicase is produced from the TRV RNA1, which replicates both RNAs. This increases RNA concentration. Viral movement proteins aid in the spread of intact RNAs. Eventually the dsRNA replication intermediaries are recognized by RISC machinery and processed into siRNAs. The siRNAs produced from the targeting construct induce the knock-down of Atg5, Catl, Jazh, MC2, and Beclinl. This tips the plant cell's regulatory machinery toward hypersensitive response. siRNAS produced from the targeting construct, as well as phased siRNAs produced from RdRP replication of target mRNAs by RISC machinery, are transported throughout the plant, spreading the phenotype. Treating Medicago truncatula did not affect the plant because processing of the targeting construct does not result in siRNAs targeting endogenous genes.
Example 55
SEQ ID No. 13 is a target construct generated as follows: The CHLI1 gene in Arabidopsis was used to find homologues in Brassica rapa, Medicago truncatula, Zea mays, and Nicotiana tobacum. These sequences were searched for regions accessible by RISC machinery using RNAxs. The best regions from each homologue were incorporated into the target construct. This construct was then cloned into the MCS of SEQ ID No. 21 in an E. coli backbone (pUC57). SEQ ID No. 20 was also cloned into the E. coli backbone. E. coli containing inducible T7 polymerase was transformed with this construct. Transformed E. coli were grown up, spun down, lysed, and the lysate rubbed onto plants with carborundum. The lysate contains RNA1 and 2 from TRV, with the target construct inside the MCS of RNA2. These RNAs are coated with viral coat protein. Replicase is produced from the TRV RNA1, which replicates both RNAs. Without being bound to theory, this increases RNA concentration. Viral movement proteins aid in the spread of intact RNAs. Eventually the dsRNA replication intermediaries are recognizes by RISC machinery and processed into siRNAs. The siRNAs produced from the targeting construct induce the knock-down of CHLI1. Plant growth is retarded, sometimes fatally, as damaged photosystems are not repaired. Off target plants are unaffected.
Example 56
A helper construct is constructed from SEQ IDs 33, 34 and 35. Sequences are driven by the T7 promoter producing polycistronic RNA. IRES elements are used to ensure translation in plant tissues. This is inserted into the MCS of SEQ ID No. 18. This along with SEQ ID No. 22 is transcribed in vitro. The resulting RNAs are applied directly to plants. Treated plants exhibit runaway HR-like apoptotic cell death. The 30 kda movement protein and the HCpro proteins aid in the movement of the unprocessed RNAs. The P19 protein suppresses the RNAi response until the RNAs have moved further from the application site.
Example 57
A targeting construct was designed to induce senescence in Nicotiana sylvestris. Genes from the senescence gene list were used to identify homologues of APG-9, ATG 2, SRI, and APG7 in N. sylvestris. These were run through RNAxs to identify RISC accessible regions. Sequences complementary to the most accessible regions were strung together to make the targeting construct RNA. DNA encoding this RNA is cloned into the MCS of SEQ ID No. 18. This and SEQ ID No. 17 are cloned into a binary vector maintained in plants and E. coli. The resulting construct is replicated and purified from E. coli, and the DNA applied directly to plants. In the plant the DNA is transcribed. The resultant RNAs are directly replicated after replicase is translated from RNAl. Treated plants undergo spreading senescence which eventually overwhelms them. As senescence takes a while to develop after induction, the signal has time to spread through the plant.
Example 58
A targeting construct was designed to starve cells of amino acids. Genes from the starvation list were used to identify homologues of HDH, AthMee2, and ICDH in N. sylvestris. These were run through RNAxs to identify RISC accessible regions. Sequences complementary to the most accessible regions were strung together to make the targeting construct RNA. DNA encoding this RNA is cloned into the MCS of SEQ ID No. 18. This and SEQ ID No. 17 are cloned into a binary vector maintained in plants and E. coli. The resulting construct is replicated and purified from E. coli, and the DNA delivered directly to plants. In the plant the DNA is transcribed. The resultant RNAs are directly replicated after replicase is translated from RNA1. Processing of these RNAs by RISC machinery leads to loss of production of a number of amino acids. Plants die due to being unable to replace degraded or damaged proteins.
Example 59
SEQ ID No. 48 is a target construct generated as follows: The Arabidopsis PDS gene was used to find the homologue in Nicotiana sylvestris. These sequences were searched for regions accessible by RISC machinery using RNAxs. The best regions containing no perfect matches to Medicago truncatula were incorporated into the target construct. DNA encoding this RNA is cloned into the MCS of SEQ ID 18. This and SEQ ID No. 17 are cloned into a binary vector maintained in plants and E. coli. The resulting construct is replicated and purified from E. coli and the DNA applied directly to plants. In the plant the DNA is transcribed. The resultant RNAs are directly replicated after replicase is translated from RNA1. Processing of the targeting construct results in siRNAs that knock down Phytoene desaturase. Plants turn white and plant growth is retarded, sometimes fatally, as damaged photosystems are not repaired. Off target plants such as Medicago truncatula, Arabidopsis thaliana and Beta vulgaris are unaffected.
Example 60
SEQ ID No. 49 is DNA containing TRV RNA2 loaded with the Nicotiana sylvestris anti-PDS targeting construct. This, along with SEQ ID No. 19 are used to produce RNAs in Vitro with T7 polymerase. The resulting RNAs are applied directly to plants. The RNAs are directly replicated after replicase is translated from RNA1. Processing of the targeting construct results in siRNAs that knock down Phytoene desaturase. Plants turn white and plant growth is retarded, sometimes fatally, as damaged photosystems are not repaired. Off target plants such as Medicago truncatula, Arabidopsis thaliana and Beta vulgaris are unaffected.
Example 61
SEQ ID No. 32 is used in conjunction with full length TRV derived RNAs or RNA flanked with TRV RNA 3′ and 5′ ends. The protein produced coats such RNAs, protecting them and increasing the likelihood of them reaching plant tissues. Codon optimization has been used to create a sequence for optimal translation in E. coli. 
Example 62


    
    
        The generalized steps for controlling a weed species are as follows:
        Selecting a weed plant species to be controlled;
        Sequencing genes from the target list in the weed and in non-target neighbor plants;
        Designing a targeting construct complementary to accessible regions of target genes that are divergent in off-target plants;
        Incorporating the target construct into an effective RNAi inducer;
        Adding helper components as required to increase silencing efficiency and spread;
        Producing the DNA or RNA to apply to plants;
        Formulating the DNA or RNA for optimum penetration into plant tissues; and
    
    


Delivering the formulated DNA or RNA to plants.
Example 63
SEQ ID No. 48 was examined and found to have 0 off-target perfect matches in Medicago truncatula, Brassica napus, Arabidopsis thaliana, Beta vulgaris, Gossypium spp. and Oryza sativa. 
Example 64
An accessory targeting construct was built targeting AB1, ADP-glucose pyrophosphorylase, AG06, cdiGRP, and DREB binding factor homologues in Nicotiana sylvestris. These were run through RNAxs to identify RISC accessible regions. Sequences complementary to the most accessible regions were strung together to make the targeting construct RNA. DNA encoding this RNA is cloned into the MCS of SEQ ID No. 19 (T7 RNA1) and applied in conjunction with SEQ ID No. 49: (T7 driven RNA2 with Nsyl PDS targeting construct in MCS). RNA from these sequences was produced in vitro using the T7 system. Treated plants turn white and plant growth is retarded, sometimes fatally, as damaged photosystems are not repaired. ABI1 knock-down will cause stomata to stay closed contributing to the overall damage to the plant and forcing it to retain water. ADP-glucose pyrophosphorylase knock-down will cause increased concentration of simple sugars which will affect the energy state of the plant, alter the direction and volume of phloem transport, and make it more accessible to saprotrophs and other organisms. AGO6 knockdown will ablate a subset of small RNA processing, freeing RISC machinery to process endogenous gene targets. cdiGRP knock-down will keep plasmodesmata open even during conditions of stress, increasing the rate of spread of the RNAi cascade. Knock-down of homologues of DREB binding factor caused unregulated leaf elongation, including increasing production of cell-wall degrading enzymes such as expansins. This contributes to the overall damage of the plant.
Example 65
The process for creating a RNAi herbicide against a specific species is as follows.

    
    
        1. Obtain sequence data for target species
        
            a. Publically available,
            b. If no sequence data is available use PCR to amplify regions corresponding to genes in target list. Have those PCR fragments sequenced.
        
        
        2. Select target regions based on RNA accessibility,
        3. Obtain sequence data for other plants in area where herbicide will be applied
    
    


a. Use public data where available, otherwise PCR and sequence as in 1.

    
    
        4. Search for target region sequences with no more than 18 nt sequential complementarity to non-target plants.
        
            a. And preferably less than 18 nt complementarity or none.
        
        
        5. Assemble 2 or more (usually at least 5) target regions into a targeting construct
        6. Add an RNAi inducer to the targeting construct
        
            a. Replicase/promoter pairs
            b. Secondary structure elements (bulges, single base mismatches outside of core region, hairpins and inverted repeats)
        
        
        7. Chose the most appropriate RNA production method
        
            a. In planta
            
                i. Construct flanked with plant expression promoters (CaMV35 for example) DNA added to plants.
            
            
            b. In prokaryotes
            
                i. Add prokaryotic promoters (one or flanking promoters) to produce single or double stranded RNA.
                ii. Add coating elements from virus (such as TRV) and co-express construct with bacterially translated coat protein.
            
            
            c. In other eukaryotes
            
                i. Same as i for prokaryotes
                ii. Express VIGS based RNAs along with viral replicase
                
                    1. Results in production of coated RNAs containing construct
                
                
            
            
            d. In vitro
        
        
    
    


Certain elements enhance the systemic spread of RNAi. Mismatched base pairs in the stem region of a dsRNA are a powerful way to ensure the derived siRNAs trigger RdRP activity at their targets. Mismatches at the 5′′ end of the siRNA are not tolerated. Moving from the 5′ to the 3′ end, mismatches become better tolerated. Using endogenous miRNA generating sequences such as tasi-RNA as backbones also aids in systemic spread.
Without being bound to theory, there are two reasons why the process described in this patent is generalizable to all known plant species. First, due to the degenerate nature of the genetic code there are many sequence level differences between species in genes that code for identical proteins. Because RNAi requires at least 18 nt of sequence complementarity (usually 21 nt) it is relatively easy to find stretches of RNA that are different in the target species and its neighbors. A single mismatch is usually sufficient to prevent knock-down in off-target plants. Sequences with 2 or more mismatches to off-target plants are sought to ensure knock-down does not occur. Plants can develop resistance to a specific targeting construct through mutation. The likelihood of developing enough spontaneous mutations in all the target genes simultaneously however is low. Even if resistance emerges those individuals can be sequenced and used to produce a new targeting construct.
Secondly, the core RISC machinery is highly conserved and RNAi is critical for defense against virus and pathogenic sequence elements. In order to develop resistance to this process a plant would have to shut down this response. Those plants would be highly susceptible to disease preventing them from gaining a foothold in the population.
List of potential genes of interest for use in this technology:
Essential Gene Targets

    
    
        3-phosphoshikimate 1-carboxyvinyltransferase (EPSP synthase) AT2G45300
        Chalcone synthase (CHS) AT5G13930
        Starch synthase (SSI) AT5G24300
        Starch synthase 3 (SS3) AT1G11720
        Cellulose synthase 1 (CESA1) AT4G32410
        Cellulose synthase 8 (CESA8) AT4G18780
        Histidinol dehydrogenase (HDH) AT5G63890
        Maternal effect embryo arrest 2/Shikimate dehydrogenase (AthMEE2) AT3G06350
        Isocitrate dehydrogenase (ICDH) AT1G54340
        Hydroxyl methylglutaryl coA reductase 1 (HMG1) AT1G76490
        Pyruvate dehydrogenase El alpha subunit (PDH-E1 ALPHA) AT1G01090
        Branched chain amino acid transaminase 1 (BCAT1) AT1G10060
        Branched chain amino acid transaminase 2 (BCAT2) AT1G10070
        18s ribosomal RNA (18S rRNA) 1005246134
        Eukaryotic elongation factor (eEF-IB beta) AT1G30230
        Spliceosome associated protein 130b (SAP130b) AT3G55220
        2′ tRNA phosphotransferase (TRPT) AT2G45330
        Phosphoribosylanthranilate isomerase 1 (PAI1) AT1G07780
        Phytoene desaturase (PDS) AT4G14210
        Dongle (DGL) AT1G05800
    
    


Apoptosis Gene Targets

    
    
        Beclin 1 (BECLIN1) AT3G61710
        Bax inhibitor 1 (Bl-l) JX481914
        Phosphatidylinositol 3-kinase (PI3K/VPS30) AT1G60490
        Lesion simulating disease 1 (LSD1) AT4G20380
        Accelerated cell death 1 (ACD1) AT3G44880
        Autophagy related protein 3 (ATG3) AT5G61500
        Autophagy related protein 7 (ATG7) AT5G45900
        Accelerated cell death 11 (ACD11) AT2G34690
        Catalase 1 (CAT1) HF564631.1
        Accelerated cell death 2/Putative red chlorophyll catabolite reductase (ACD2) EU294213.1
        Translationally controlled tumor protein (NbTCTP) AB780363.1
        Jasmonate ZIM domain protein h (JazH) JQ172766.1
        Lethal leaf spot 1-like (LLS1) AF321984.1
        Metacaspase 2 (MCS) AT4G25110
    
    


Senescence and Autophagy

    
    
        Target of rapamycin (TOR) AT1G50030
        Autophagy 9 (APG9) AT2G31260
        Autophagy 2 (ATG2) AT3G19190
        Autophagy 5 (ATG5) AT5G17290
        Signal responsive 1 (SRI) AT2G22300
        Autophagy 7 (APG7) AT5G45900
        Senescence associated gene (SAG12) AT5G45890
        Phytoalexin deficient (PAD4) AT3G52430
        Constitutive expression of PR genes 5 (CPR5) AT5G64930
        Homologue of yeast autophagy gene 18 (ATG18) AT1G03380
    
    


Helper Elements

    
    
        Tobacco mosaic virus 30 kDa movement protein, V01408.1
        Papaya Ringspot virus HCpro peptide, JX448373.1
        Tomato bushy stunt virus P19 suppressor, AJ288943.1
    
    


Inducers

    
    
        pTRV2, AF406991.1
        pRNAi-GG, JQ085427.1
        TRV Ppk20 RNA1, AF314165.1
    
    


All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.
Advantages of the exemplary embodiments described herein may be realized and attained by means of the instrumentalities and combinations particularly pointed out in this written description. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims below. While example embodiments have been described in detail, the foregoing description is in all aspects illustrative and not restrictive. It is understood that numerous other modifications and variations can be devised without departing from the scope of the example embodiment. For example, other genes may be targeted, such as chloroplast and mitochondrial nuclear encoded genes, trafficking and translocation signal sequences, energy metabolism genes, high level regulatory sequences, regulators of cellulases and cell-wall remodelling enzymes and regulators of apoptosis.

## Claims

1. A RNAi payload construct comprising the nucleotide sequences set forth in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:27 and SEQ ID NO:29.