Patent Application
20130318657
This application contains a sequence listing including a file named P34078US00_SequenceListing.txt, which is 73,728 bytes in size (measured in MS-DOS) and was created on May 23, 2013, which is herein incorporated by reference in its entirety.
The present invention, in some embodiments thereof, relates to methods of introducing dsRNA to plant seeds for modulating gene expression.
With a growing world population, increasing demand for food, fuel and fiber, and a changing climate, agriculture faces unprecedented challenges. Development of plants with improved traits is highly desirable, with some of the major traits that are of major interest to farmers and seed companies include improved abiotic stress tolerance, fertilizer use efficiency, disease resistance, yield and more.
Plant trait improvement is typically performed by either genetic engineering or classical breeding. New methods for trait improvement through specific gene alteration are highly desirable. These include methods for over-expression of genes or gene silencing. A powerful technique for sequence-specific gene silencing is through RNA interference (RNAi). First discovered in the nematode C. elegans (Fire et al 1998, Nature, 391:806-811), RNAi is a mechanism in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA (dsRNA) that is homologous to the selected gene, into cells. Inside the cell, dsRNA molecules are cut into shorter fragments of 21-27 nucleotides by an RNase III-related enzyme (Dicer). These fragments, called small interfering RNAs (siRNAs), get incorporated into the RNA-induced silencing complex (RISC). After additional processing, the siRNAs are transformed into single-stranded RNAs that act as guide sequences to eventually cleave target messenger RNAs. By using RNAi to specifically silence relevant target genes, one can alter basic traits of an organism. Specifically for plants, it holds incredible potential for modifications that may lead to increased stress resistance and better crop yield.
In plants, RNAi is typically performed by producing transgenic plants that over-express a DNA fragment that is transcribed to produce a dsRNA. This dsRNA is then processed into siRNAs that mediate the cleavage and silencing of target genes.
The major technical limitation for this technology is that many important plant crop species are difficult or impossible to transform, precluding the constitutive expression of constructs directing production of dsRNA. Moreover, questions concerning the potential ecological impact of virus-resistant transgenic plants have so far significantly limited their use [Tepfer, 2002, Annu. Rev. Phytopathol. 40, 467-491].
An additional hurdle for obtaining transgenic plants is attributed to the difficulty of having the transformation and regeneration events occur in the same cell types.
Therefore the development of a method for obtaining transformed seeds which is independent of the methods inherent to tissue culture procedures is at the cutting edge of plant molecular biology research.
Additional background art includes:
U.S. 20040055041 teach seed transformation by making use of the sonication system followed by infection by Agrobacterium.
Chee et al. 1989 Plant Physiol. 91:1212-1218 teach soybean transformation by inoculating the plumule, cotyledonary node and adjacent cotyledon tissues of germinating soybean using an Agrobacterium that contained a binary vector expressing a transgene.
Additional related background art: WO2011/001434, U.S. 20080022423, WO 2011112570, WO 2007/080127 WO 2007/080126, U.S. 20060272049, U.S. 2010068172, U.S. 20070250947, WO9926467, U.S. 20030154508, WO 02/14472, U.S. 20030150017, U.S. 201000154083.
According to an aspect of some embodiments of the present invention there is provided a method of introducing naked dsRNA into a seed, the method comprising contacting the seed with the naked dsRNA under conditions which allow penetration of the dsRNA into the seed, thereby introducing the dsRNA into the seed.
According to an aspect of some embodiments of the present invention there is provided an isolated seed comprising an exogenous naked dsRNA, wherein the seed is devoid of a heterologous promoter for driving expression of the dsRNA in a plant.
According to an aspect of some embodiments of the present invention there is provided an isolated seed comprising an exogenous naked dsRNA.
According to an aspect of some embodiments of the present invention there is provided an isolated seed comprising an exogenous dsRNA being present at a similar concentration in an embryo and an endosperm of the seed.
According to an aspect of some embodiments of the present invention there is provided an isolated seed comprising an exogenous dsRNA being spatially distributed in an embryo and an endosperm of the plant seed in a spatial distribution that differs from a spatial distribution of the exogenous dsRNA in a seed derived from a transgenic plant that recombinantly expresses the exogenous dsRNA.
According to an aspect of some embodiments of the present invention there is provided an isolated seed comprising an exogenous dsRNA, wherein a concentration ratio of the exogenous dsRNA to siRNA maturing therefrom is higher in the seed as compared to a transgenic seed recombinantly expressing the exogenous dsRNA.
According to an aspect of some embodiments of the present invention there is provided an isolated seed comprising an exogenous dsRNA, wherein the plant seed is devoid of a heterologous promoter for driving expression of the exogenous dsRNA, wherein a spatial distribution of the exogenous dsRNA and/or siRNA maturing therefrom is altered in the seed as compared to same in a transgenic seed recombinantly expressing the exogenous dsRNA.
According to an aspect of some embodiments of the present invention there is provided a plant or plant part comprising an exogenous naked dsRNA and being devoid of a heterologous promoter for driving expression of the dsRNA in the plant.
According to an aspect of some embodiments of the present invention there is provided a seed containing device comprising a plurality of the seeds.
According to an aspect of some embodiments of the present invention there is provided a sown field comprising a plurality of any of the seeds.
According to some embodiments of the invention, the method further comprises drying said seed following said contacting.
According to some embodiments of the invention, the method further comprises growing said plant under abiotic or biotic stress following said generating.
According to some embodiments of the invention, the naked dsRNA is designed for down regulating expression of a gene of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of producing a plant, the method comprising:
(a) providing any of the seeds; and
(b) germinating the seed so as to produce the plant.
According to an aspect of some embodiments of the present invention there is provided a method of modulating gene expression, the method comprising:
(a) contacting a seed of a plant with a naked dsRNA, under conditions which allow penetration of the dsRNA into the seed, thereby introducing the dsRNA into the seed; and optionally
(b) generating a plant of the seed.
According to some embodiments of the invention, the naked dsRNA is designed for down regulating expression of a gene of the plant.
According to some embodiments of the invention, the naked dsRNA is designed for down regulating expression of an edogenous gene of the plant.
According to some embodiments of the invention, the naked dsRNA is designed for down regulating expression of a gene of a viral pathogen.
According to some embodiments of the invention, the penetration is to an endosperm and alternatively or additionally an embryo of the seed.
According to some embodiments of the invention, the naked dsRNA does not integrate into the genome of the seeds.
According to some embodiments of the invention, the conditions result in presence of the dsRNA in the plant for at least 10 days following germination.
According to an aspect of some embodiments of the present invention there is provided a method of inhibiting expression of a target gene in a plant virus, the method comprising providing to the plant virus the plant or plant part, thereby inhibiting expression of a target gene in the plant virus.
According to some embodiments of the invention, the method further comprises observing reduced infectability or replicability of the viral pathogen following the providing.
According to an aspect of some embodiments of the present invention there is provided a kit for introducing naked dsRNA to seeds comprising;
(i) naked dsRNA; and
(ii) a priming solution.
According to some embodiments of the invention, the naked dsRNA and the priming solutions are comprised in separate containers.
According to some embodiments of the invention, the dsRNA comprises siRNA.
According to some embodiments of the invention, the dsRNA comprises siRNA and dsRNA.
According to some embodiments of the invention, the contacting is effected by inoculating the seed with the dsRNA.
According to some embodiments of the invention, the method further comprises priming the seed prior to the contacting.
According to some embodiments of the invention, the priming is effected by:
(i) washing the seed prior to the contacting; and
(ii) drying the seed following step (i).
According to some embodiments of the invention, the washing is effected in the presence of double deionized water.
According to some embodiments of the invention, the washing is effected for 2-6 hours.
According to some embodiments of the invention, the washing is effected at 4-28° C.
According to some embodiments of the invention, the drying is effected at 25-30° C. for 10-16 hours.
According to some embodiments of the invention, the contacting is effected in a presence of the naked dsRNA at a final concentration of 0.001-100 μg/μl.
According to some embodiments of the invention, the contacting is effected in a presence of the naked dsRNA at a final concentration of 0.001-0.5 μg/μl.
According to some embodiments of the invention, the method further comprises treating the seed with an agent selected from the group consisting of a pesticide, a fungicide, an insecticide, a fertilizer, a coating agent and a coloring agent following the contacting.
According to some embodiments of the invention, the treating comprises coating the seed with the agent.
According to some embodiments of the invention, the seed is free of an agent selected from the group consisting of a pesticide, a fungicide, an insecticide, a fertilizer, a coating agent and a coloring agent.
According to some embodiments of the invention, the dsRNA is for downregulating expression of a coding gene.
According to some embodiments of the invention, the dsRNA is for downregulating expression of a non-coding gene.
According to some embodiments of the invention, the seed is of the Viridiplantae super-family.
According to some embodiments of the invention, the conditions allow accumulation of the dsRNA in the endosperm and alternatively or additionally embryo of the seed.
According to some embodiments of the invention, a concentration of the naked dsRNA is adjusted according to a parameter selected from the group consisting of, seed size, seed weight, seed volume, seed surface area, seed density and seed permeability.
According to some embodiments of the invention, the contacting is effected prior to breaking of seed dormancy and embryo emergence.
According to some embodiments of the invention, the seed is a primed seed.
According to some embodiments of the invention, the seed or the plant comprises RNA dependent RNA polymerase activity for amplifying expression of the dsRNA.
According to some embodiments of the invention, the seed is a hybrid seed.
According to an aspect of some embodiments of the present invention there is provided a seed obtainable according to the methods described herein.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The present invention, in some embodiments thereof, relates to methods of introducing dsRNA to plant seeds for modulating gene expression.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
With the extensive growth of the world-population and the limited habitats for plant growth and cultivation, there is an urging need to improve plant yields under these changing conditions.
RNAi has emerged as a powerful tool for modulating gene expression which can be used for generating plants with improved stress tolerance.
In plants, RNAi is typically performed by producing transgenic plants that express a DNA fragment that is transcribed to produce a dsRNA. This dsRNA is then processed into siRNAs that mediate the silencing of target genes, typically by targeting cleavage of the target gene by an RNA Induced Silencing Complex (RISC) or by translational repression.
The major technical limitation for this technology is that many important plant crop species are difficult or impossible to transform, precluding the constitutive expression of constructs directing production of dsRNA. Moreover, questions concerning the potential ecological impact of virus-resistant transgenic plants have so far significantly limited their use [Tepfer, 2002, Annu. Rev. Phytopathol. 40, 467-491].
The present inventors have now devised a novel technology for introducing dsRNA molecules directly to the plant seed. These dsRNA molecules enter seeds and start a silencing process, which is continued during the life cycle of the plant, resulting in a plant with an improved trait of interest. The introduced dsRNA is naked and as such no exogenous transcription regulatory elements are introduced into the plant thus lowering the environmental concerns associated with transgenic plants. In addition, the modified seed can be germinated to generate a plant without the need of going through the laborious and cumbersome steps of tissue culture regeneration.
As is illustrated herein below and in the Examples section, which follows, embodiments of the present invention includes configuring the conditions necessary to introduce naked dsRNA into the seeds (see e.g., Example 1). The naked dsRNA doesn't integrate into the genome and is highly stable in the plant and in solution (Examples 2-4). The naked dsRNA penetrates through the seed coat (i.e., testa) of both monocot and dicot plants and distributes in the endosperm and seed embryo (Examples 5-6). In one aspect, embodiments of the present invention include altering expression of endogenous genes (Examples 7-14). In another aspect, embodiments of the present invention include introducing into seeds dsRNA directed to viral genes (Example 2). These results were reproduced over a number of plants of both monocot and dicot groups. In a further aspect, embodiments of the present invention include providing a wide range of doses and kinetics which resulted in a significant alteration of gene expression (see e.g., Examples 23-29). These results are further established in wheat and substantiated by a biological effect showing delayed germination ((Example 30). Further, as shown in Examples 31 and 32, gene expression is altered in Corn following introduction of dsRNA for various targets. Examples 33-37 show dsRNA treatment of other vegetables, including cucumber and lettuce ( ). In a further aspect, embodiments of the present invention include altering gene expression in another commercial crop i.e., soy (see, e.g., Example 42). Thus, the present results are sufficient to show that the present teachings provide a cost-effective treatment of plant seeds to achieve a desired agricultural and horticultural phenotype. Without being bound by theory, it is suggested that the newly suggested transformation modality and modulation of gene expression is dependent on and associated with: (i) Introduction of naked dsRNA into the interior of seeds (as opposed to mere seed coating). The introduction is effected by soaking the seeds in a solution which comprises the dsRNA such that the dsRNA penetrates through the seed coat or by dipping such that the dsRNA coats the seed and penetrates through the coat after sowing;
(ii) Amplification of the dsRNA signal; and
(iii) Spreading of the dsRNA signal throughout the plant.
The first step occurs only once, during and shortly after the initial seed treatment, while the second and third steps occur in a repetitive loop for as long as the silencing signal remains active in the plant.
A suggested unbinding mode of action for the described invention is based on each step:
Introduction of dsRNA into Seeds
A typical mature seed consists of an embryo encapsulated within a maternal seed coat (testa) and an abundant layer of endosperm tissue between the embryo and seed coat. The endosperm serves as a nutrient source for the embryo during seed development, germination and seedling establishment.
Seed germination typically begins with exposure of the seeds to water, which is absorbed by the embryo and endosperm. The endosperm then expands in volume, with the endosperm of some plant species being able to grow several-fold from their original volume. The embryo, which was dormant until this stage, is now released from dormancy and cell division, expansion and differentiation begin. The endosperm feeds the developing embryo until it is developed enough to begin photosynthesis and autotrophic growth.
Based on these known mechanisms of seed germination, two possible modes of action for the initial step of “Introduction of dsRNA into seeds” are suggested:
The dsRNA molecules enter the embryo directly, carried by the water-based solution which is used for the seed treatment.
The dsRNA molecules enter the endosperm as part of the endosperm's water-absorption process. These molecules then feed the embryo as it develops as part of the nutrient flow from the endosperm during germination and seed development.
Based on the results described in
Amplification of the dsRNA Signal
Once the dsRNA molecules enter the embryo, they are recognized and processed by RNase III-like enzymes such as Dicer or Dicer-like (DCL) enzymes. DCL enzymes process the long dsRNA molecules into short, double strand RNAs (known as siRNAs or shRNAs), which are typically 21-24 nucleotides (nt) long. One of the siRNA strands is typically rapidly degraded and the second one can be incorporated in RISC(RNA Induced Silencing Complex) protein complexes, which contain an Argonaute (AGO) protein. AGO proteins contain a PIWI domain to bind siRNAs and a PAZ domain with RNase activity. Subsequently, the siRNA/AGO complex identifies an mRNA molecule, which is complementary to the siRNA and results in its silencing by cleavage or translational repression.
The siRNA is then released from the RISC complex and can now act as a primer for an RNA-Dependant RNA Polymerase (RDRP), this is an enzyme which is unique to the plant kingdom and can generate amplification of the silencing signal by generating new dsRNA molecules (secondary siRNA). These newly-synthesized dsRNAs can be processed again as described above, therefore maintaining and amplifying the silencing signal.
Spreading of the Silencing Signal
Silencing spreading is a known and well-understood phenomenon in plants. It is believed that short distance, cell-to-cell spreading occurs through plasmodesmata. This process is thought to be mediated by a 21 nt-long siRNA, which is the product of a DCL enzyme. Additionally, systemic spreading is achieved through the phloem across the entire plant from source to sink.
It is assumed that in the described methodology, spreading of the silencing signal occurs once the silencing signal begins and is amplified as described above. This may include both short-distance and systematic spreading by various siRNA signal molecules.
Thus according to an aspect of the invention, there is provided a method of introducing naked double-stranded RNA (dsRNA) into a seed, the method comprising contacting the seed with the naked dsRNA under conditions which allow penetration of the dsRNA into the seed, thereby introducing the dsRNA into the seed.
As used herein the phrase “naked dsRNA” refers to a dsRNA nucleic acid molecule which is non-transcribable in the plant cell. Thus, the naked dsRNA molecule is not comprised in a nucleic acid expression construct such as a viral vector. According to some embodiments of the invention, the naked dsRNA molecule is not derived from a viral vector. According to other embodiments of the invention, the naked dsRNA molecule is not produced from a natural viral infection. The naked dsRNA may comprise regulatory elements for in-vitro transcription, such as the T7 promoter. According to some embodiments of the invention, the naked dsRNA may be modified e.g., chemically modified, to confer higher bioavailability, penetration into the seeds and/or improved shelf-life.
As used herein the term “dsRNA” relates to two strands of anti-parallel polyribonucleic acids held together by base pairing. The two strands can be of identical length or of different lengths provided there is enough sequence homology between the two strands that a double stranded structure is formed with at least 80%, 90%, 95% or 100% complementarity over the entire length. According to an embodiment of the invention, there are no overhangs for the dsRNA molecule. According to another embodiment of the invention, the dsRNA molecule comprises overhangs. According to other embodiments, the strands are aligned such that there are at least 1, 2, or 3 bases at the end of the strands which do not align (i.e., for which no complementary bases occur in the opposing strand) such that an overhang of 1, 2 or 3 residues occurs at one or both ends of the duplex when strands are annealed.
As mentioned any dsRNA molecule can be used in accordance with the present teachings. In some embodiments, dsRNA used in the present application is subject to amplification by RNA-Dependant RNA Polymerase (RDRP). Without being limited, dsRNA can be siRNA, shRNA, pre-miRNA, or pri-miRNA.
The present teachings relate to various lengths of dsRNA, whereby the shorter version i.e., x is shorter or equals 50 bp (e.g., 17-50), is referred to as siRNA or miRNA. Longer dsRNA molecules of 51-600 are referred to herein as dsRNA, which can be further processed for siRNA molecules.
In one embodiment, the dsRNA in the present application is between 20 and 100 bp, between 25 and 90 bp, between 30 and 80 bp, between 30 and 70 bp, between 30 and 60 bp, or between 30 and 50 bp. In another embodiment, the dsRNA in the present application is about 50 bp. In a further embodiment, the 5′ and/or 3′ ends of the sense and/or antisense strands of the dsRNA comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide overhangs. In some embodiments, the dsRNA comprises 1, 2, or 3 nucleotide overhangs on the 5′ and/or 3′ ends of the sense and/or antisense strands. In another embodiment, the ends of the dsRNA are blunt.
In one embodiment, the dsRNA in the present application is between 15 and 500 bp, between 15 and 450 bp, between 15 and 400 bp, between 15 and 350 bp, between 15 and 300 bp, between 15 and 250 bp, between 15 and 200 bp, between 15 and 150 bp, between 15 and 100 bp, between 15 and 90 bp, between 15 and 80 bp, between 15 and 70 bp, between 15 and 60 bp, between 15 and 50 bp, between 15 and 40 bp, between 15 and 35 bp, between 15 and 30 bp, or between 15 and 25 bp. In another embodiment, the dsRNA in the present application is at least about 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600, 800, 900, 1000 nucleotides long. In a further embodiment, the dsRNA in the present application is between 100 and 1000 bp, between 200 and 1000 bp, between 300 and 1000 bp, between 400 and 1000 bp, between 500 and 1000 bp, between 600 and 1000 bp, between 700 and 1000 bp, between 800 and 1000 bp, or between 900 and 1000 bp. The term “siRNA” refers to small inhibitory RNA duplexes (generally between 17-30 basepairs, but also longer e.g., 31-50 bp) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3′-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21 mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27 mer) instead of a product (21 mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.
It has been found that position of the 3′-overhang influences potency of an siRNA and asymmetric duplexes having a 3′-overhang on the antisense strand are generally more potent than those with the 3′-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.
The strands of a double-stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). Thus, as mentioned the RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
The term “shRNA”, as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. et al. (2002) Science 296: 550, DEQ ID NO: 22) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al. (2002) RNA 8:1454, SEQ ID NO: 23). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.
As used herein, the phrase “microRNA (also referred to herein interchangeably as “miRNA” or “miR”) or a precursor thereof” refers to a microRNA (miRNA) molecule acting as a post-transcriptional regulator. Typically, the miRNA molecules are RNA molecules of about 20 to 22 nucleotides in length which can be loaded into a RISC complex and which direct the cleavage of another RNA molecule, wherein the other RNA molecule comprises a nucleotide sequence essentially complementary to the nucleotide sequence of the miRNA molecule.
Typically, a miRNA molecule is processed from a “pre-miRNA” or as used herein a precursor of a pre-miRNA molecule by proteins, such as DCL proteins, present in any plant cell and loaded onto a RISC complex where it can guide the cleavage of the target RNA molecules.
Pre-microRNA molecules are typically processed from pri-microRNA molecules (primary transcripts). The single stranded RNA segments flanking the pre-microRNA are important for processing of the pri-miRNA into the pre-miRNA. The cleavage site appears to be determined by the distance from the stem-ssRNA junction (Han et al. 2006, Cell 125, 887-901, 887-901).
As used herein, a “pre-miRNA” molecule is an RNA molecule of about 100 to about 200 nucleotides, preferably about 100 to about 130 nucleotides which can adopt a secondary structure comprising an imperfect double stranded RNA stem and a single stranded RNA loop (also referred to as “hairpin”) and further comprising the nucleotide sequence of the miRNA (and its complement sequence) in the double stranded RNA stem. According to a specific embodiment, the miRNA and its complement are located about 10 to about 20 nucleotides from the free ends of the miRNA double stranded RNA stem. The length and sequence of the single stranded loop region are not critical and may vary considerably, e.g. between 30 and 50 nt in length. The complementarity between the miRNA and its complement need not be perfect and about 1 to 3 bulges of unpaired nucleotides can be tolerated. The secondary structure adopted by an RNA molecule can be predicted by computer algorithms conventional in the art such as mFOLD. The particular strand of the double stranded RNA stem from the pre-miRNA which is released by DCL activity and loaded onto the RISC complex is determined by the degree of complementarity at the 5′ end, whereby the strand which at its 5′ end is the least involved in hydrogen bounding between the nucleotides of the different strands of the cleaved dsRNA stem is loaded onto the RISC complex and will determine the sequence specificity of the target RNA molecule degradation. However, if empirically the miRNA molecule from a particular synthetic pre-miRNA molecule is not functional (because the “wrong” strand is loaded on the RISC complex), it will be immediately evident that this problem can be solved by exchanging the position of the miRNA molecule and its complement on the respective strands of the dsRNA stem of the pre-miRNA molecule. As is known in the art, binding between A and U involving two hydrogen bounds, or G and U involving two hydrogen bounds is less strong that between G and C involving three hydrogen bounds. Exemplary hairpin sequences are provided in Tables 1-8, below.
Naturally occurring miRNA molecules may be comprised within their naturally occurring pre-miRNA molecules but they can also be introduced into existing pre-miRNA molecule scaffolds by exchanging the nucleotide sequence of the miRNA molecule normally processed from such existing pre-miRNA molecule for the nucleotide sequence of another miRNA of interest. The scaffold of the pre-miRNA can also be completely synthetic. Likewise, synthetic miRNA molecules may be comprised within, and processed from, existing pre-miRNA molecule scaffolds or synthetic pre-miRNA scaffolds. Some pre-miRNA scaffolds may be preferred over others for their efficiency to be correctly processed into the designed microRNAs, particularly when expressed as a chimeric gene wherein other DNA regions, such as untranslated leader sequences or transcription termination and polyadenylation regions are incorporated in the primary transcript in addition to the pre-microRNA.
According to the present teachings, the dsRNA molecules may be naturally occurring or synthetic.
The dsRNA can be a mixture of long and short dsRNA molecules such as, dsRNA, siRNA, siRNA+dsRNA, siRNA+miRNA, or a combination of same. According to a specific embodiment, the dsRNA is an siRNA (100%). According to a specific embodiment the dsRNA is an siRNA+dsRNA combination in various ratios. Any dsRNA to siRNA ratio can be used for the siRNA+dsRNA combination.
For example a ratio of 1 to 1: one dsRNA mixed with the same sequence after RNAse treatment. According to another embodiment, the dsRNA to siRNA ratio is 2:1, 1.5:1, 1.3:1, 1:0.01, 1:0.05 or 1:0.1. According to a further embodiment, the dsRNA to siRNA ratio is 2:1 to 1:0.1. According to a specific embodiment, the dsRNA is purified dsRNA (100%). According to another embodiment, the dsRNA to siRNA ratio is 1:2, 1:5, 1:10, 1:20, or 1:50. According to a further embodiment, the dsRNA is purified siRNA (100%).
The dsRNA molecule is designed for specifically targeting a target gene of interest. It will be appreciated that the dsRNA can be used to down-regulate one or more target genes. If a number of target genes are targeted, a heterogenic composition which comprises a plurality of dsRNA molecules for targeting a number of target genes is used. Alternatively said plurality of dsRNA molecules are separately applied to the seeds (but not as a single composition). According to a specific embodiment, a number of distinct dsRNA molecules for a single target are used, which may be separately or simultaneously (i.e., co-formulation) applied.
According to an embodiment of the invention, the target gene is endogenous to the plant. Downregulating such a gene is typically important for conferring the plant with an improved, agricultural, horticultural, nutritional trait (“improvement” or an “increase” is further defined hereinbelow). It will be appreciated that the treatment with the dsRNA may result in an up-regulation of the target gene (which follows a suggested mechanism that is provided hereinbelow) however such an up-regulation may be transient. The present inventors are able to confer resistance to biotic stress by modulating expression of endogenous genes in cucumber and tomato thereby conferring resistance to infections as demonstrated in Examples 35 and 41.
As used herein “endogenous” refers to a gene which expression (mRNA or protein) takes place in the plant. Typically, the endogenous gene is naturally expressed in the plant or originates from the plant. Thus, the plant may be a wild-type plant. However, the plant may also be a genetically modified plant (transgenic).
Downregulation of the target gene may be important for conferring improved one of-, or at least one of (e.g., two of- or more), biomass, vigor, yield, abiotic stress tolerance, biotic stress tolerance or improved nitrogen use efficiency.
Exemplary target genes include, but are not limited to, an enzyme, a structural protein, a plant regulatory protein, a miRNA target gene, or a non-coding RNA such as a miRNA of the plant. WO2011067745, WO 2009125401 and WO 2012056401 provide examples of miRNA sequences or targets of miRNAs (e.g., mRNA167, miRNA 156, miR164 and targets thereof NFY, SPL17 and NAC, respectively) which expression can be silenced to improve a plant trait. Other examples of target genes which may be subject to modulation according to the present teachings are described in the Examples section which follows.
The target gene may comprise a nucleic acid sequence which is transcribed to an mRNA which codes for a polypeptide.
Alternatively, the target gene can be a non-coding gene such as a miRNA or a siRNA.
For example, in order to silence the expression of an mRNA of interest, synthesis of the dsRNA suitable for use with some embodiments of the invention can be selected as follows. First, the mRNA sequence is scanned including the 3′ UTR and the 5′ UTR.
Second, the mRNA sequence is compared to an appropriate genomic database using any sequence alignment software, such as the BLAST software available from the NCBI server (wwwdotncbidotnlmdotnihdotgov/BLAST/). Putative regions in the mRNA sequence which exhibit significant homology to other coding sequences are filtered out.
Qualifying target sequences are selected as template for dsRNA synthesis. Preferred sequences are those that have as little homology to other genes in the genome to reduce an “off-target” effect.
In one embodiment, the dsRNA of the present application may comprise a target sequence in an intron, exon, 3′ UTR, 5′ UTR, or a regulatory element of an endogenous gene, or combinations thereof. In one embodiment, the dsRNA of the present application may comprise a target site residing in a promoter.
It will be appreciated that the RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
The dsRNA may be synthesized using any method known in the art, including either enzymatic syntheses or solid-phase syntheses. These are especially useful in the case of short polynucleotide sequences with or without modifications as explained above. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example: Sambrook, J. and Russell, D. W. (2001), “Molecular Cloning: A Laboratory Manual”; Ausubel, R. M. et al., eds. (1994, 1989), “Current Protocols in Molecular Biology,” Volumes I-III, John Wiley & Sons, Baltimore, Md.; Perbal, B. (1988), “A Practical Guide to Molecular Cloning,” John Wiley & Sons, New York; and Gait, M. J., ed. (1984), “Oligonucleotide Synthesis”; utilizing solid-phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting, and purification by, for example, an automated trityl-on method or HPLC.
As mentioned, the naked dsRNA molecule is directly contacted with the seed.
The seed may be of any plant, such as of the Viridiplantae super family including monocotyledon and dicotyledon plants. Other plants are listed hereinbelow. According to an embodiment of the invention, the cells of the plant comprise RNA dependent RNA polymerase activity and the target RNA molecule of the dsRNA to ensure amplification of the dsRNA.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and isolated plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. It will be appreciated, that the plant or seed thereof may be transgenic plants.
As used herein the phrase “plant cell” refers to plant cells which are derived and isolated from disintegrated plant cell tissue or plant cell cultures. Plant cells may be reproductive cells (i.e., cells from a tissue contributing directly to the sexual reproduction of a plant) or non-reproductive cells (i.e., cells from a tissue not involved in the sexual reproduction of a plant). Plant cells may be cells that are capable of regenerating into a whole plant or cells that cannot regenerate into a whole plant, for example, enucleated mature sieve tube cells.
As used herein the phrase “plant cell culture” refers to any type of native (naturally occurring) plant cells, plant cell lines and genetically modified plant cells, which are not assembled to form a complete plant, such that at least one biological structure of a plant is not present. Optionally, the plant cell culture of this aspect of the present invention may comprise a particular type of a plant cell or a plurality of different types of plant cells. It should be noted that optionally plant cultures featuring a particular type of plant cell may be originally derived from a plurality of different types of such plant cells.
Any commercially or scientifically valuable plant is envisaged in accordance with some embodiments of the invention. Plants that are particularly useful in the methods of the invention include all plants which belong to the super family Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barley, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively algae and other non-Viridiplantae can be used for the methods of the present invention.
According to some embodiments of the invention, the plant used by the method of the invention is a crop plant including, but not limited to, cotton, Brassica vegetables, oilseed rape, sesame, olive tree, palm oil, banana, wheat, corn or maize, barley, alfalfa, peanuts, sunflowers, rice, oats, sugarcane, soybean, turf grasses, barley, rye, sorghum, sugar cane, chicory, lettuce, tomato, zucchini, bell pepper, eggplant, cucumber, melon, watermelon, beans, hibiscus, okra, apple, rose, strawberry, chili, garlic, pea, lentil, canola, mums, Arabidopsis, broccoli, cabbage, beet, quinoa, spinach, squash, onion, leek, tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, and also plants used in horticulture, floriculture or forestry, such as, but not limited to, poplar, fir, eucalyptus, pine, an ornamental plant, a perennial grass and a forage crop, coniferous plants, moss, algae, as well as other plants listed in World Wide Web (dot) nationmaster (dot) com/encyclopedia/Plantae.
According to a specific embodiment, the plant is selected from the group consisting of corn, rice, wheat, tomato, cotton and sorghum.
According to a specific embodiment, the seed is an uncoated or fresh seed that hasn't been subjected to chemical/physical treatments.
Washing of the seeds is effected for 30 min to 4 hours. Other exemplary wash ranges are 1 minute to 10 minutes, 10 minutes-30 minutes. According to some embodiments, washing of the seeds can be as short as 5, 10, 20, 30, 45, or 60 seconds. The wash solution may include a weak detergent such as Tween-20. The concentration of the detergent may be 0.01-0.2% or 0.2-1%. According to another embodiment, the detergent concentration can be about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1% or higher.
The seed may be subjected to priming or washing prior to contacting with the dsRNA.
As used herein the term “priming” refers to controlling the hydration level within seeds so that the metabolic activity necessary for germination can occur but radicle emergence is prevented. Different physiological activities within the seed occur at different moisture levels (Leopold and Vertucci, 1989; Taylor, 1997). The last physiological activity in the germination process is radicle emergence. The initiation of radicle emergence requires a high seed water content. By limiting seed water content, all the metabolic steps necessary for germination can occur without the irreversible act of radicle emergence. Prior to radicle emergence, the seed is considered desiccation tolerant, thus the primed seed moisture content can be decreased by drying. After drying, primed seeds can be stored until time of sowing.
Several different priming methods are used commercially. Among them, liquid or osmotic priming and solid matrix priming appear to have the greatest following (Khan et al., 1991).
According to an embodiment of the invention, priming is effected in the presence of salt, a chelating agent, polyethylene glycol or a combination of same (e.g., chelating agent and salt).
Alternatively priming is effected in the presence of water such as deionized water or double deionized water. According to a specific embodiment, the priming is effected in the presence of 100% ddW.
Several types of seed priming are commonly used:
Osmopriming (osmoconditioning)—is the standard priming technique. Seeds are incubated in well aerated solutions with a low water potential, and afterwards washes and dried. The low water potential of the solutions can be achieved by adding osmotica like mannitol, polyethyleneglycol (PEG) or salts like KCl.
Hydropriming (drum priming)—is achieved by continuous or successive addition of a limited amount of water to the seeds. A drum is used for this purpose and the water can also be applied by humid air. ‘On-farm steeping’ is a cheap and useful technique that is practiced by incubating seeds (cereals, legumes) for a limited time in warm water.
Matrixpriming (matriconditioning)—is the incubation of seeds in a solid, insoluble matrix (vermiculite, diatomaceous earth, cross-linked highly water-absorbent polymers) with a limited amount of water. This method confers a slow imbibition.
Pregerminated seeds—is only possible with a few species. In contrast to normal priming, seeds are allowed to perform radicle protrusion. This is followed by sorting for specific stages, a treatment that reinduces desiccation tolerance, and drying. The use of pregerminated seeds causes rapid and uniform seedling development.
Thus, according to an exemplary embodiment, the seeds are primed seeds.
Of note, it may be possible that the seeds are treated with water (double-distilled water, ddW), prior to contacting with the dsRNA without effecting any priming on the seeds. For instance, treatment for a short while with water (e.g., 30 seconds to 1 hours, 30 seconds to 0.5 hour, 30 seconds to 10 min, 30 seconds to 5 min or 45 seconds to 5 min). According to some embodiments, treatment with water can be as short as 5, 10, 20, or 30 seconds.
It will be appreciated that the dsRNA can be comprised in water (e.g., tap water, distilled water or double distilled water) i.e., free of any of the above mentioned priming effective concentration of salts, a chelating agents, polyethylene glycol or combinations of same (e.g., chelating agent and salt).
According to an exemplary embodiment, the seeds are non-primed seeds.
A non-limiting exemplary method of introducing the dsRNA into the seed is provided in Example 1, which is considered as an integral part of the specification.
The temperature at the washing/priming and drying steps may be the same or differ.
According to an exemplary embodiment, the washing/priming is effected at 4-28° C.
According to an exemplary embodiment, the priming/washing solution or the dsRNA containing solution is devoid of a solid carrier.
According to an exemplary embodiment, the priming/washing solution or the dsRNA containing solution is devoid of a transferring agent such as a surfactant or a salt.
According to a further embodiment of the invention, the seeds subject to contacting with the dsRNA molecule are washed in order to remove agents, to which the seeds have been subjected, such as a pesticide, a fungicide, an insecticide, a fertilizer, a coating agent and a coloring agent.
Thus, according to an exemplary embodiment, the seeds (prior to treatment with dsRNA) are substantially free (i.e., do not comprise effective amounts) of pesticide, a fungicide, an insecticide, a fertilizer, a coating agent and a coloring agent.
The seeds are then subjected to drying. In some embodiments, drying is optional.
According to an exemplary embodiment, the drying is effected at 20-37° C., 20-30° C., 22-37° C., 15-22° C. or 20-25° C. for 10-20 hours, 10-16 hours or even 2-5 hours.
Various considerations are to be taken when calculating the concentration of the naked dsRNA in the contacting solution.
These are dependent on at least one of seed size, seed weight, seed volume, seed surface area, seed density and seed permeability.
For example, related to seed size, weight, volume and surface area, it is estimated that corn seeds will require longer treatment than Arabidopsis and tomato seeds. Regarding permeability and density, it is estimated that wheat seeds will require longer treatments at higher concentrations than tomato seeds.
Exemplary concentrations of dsRNA in the treating solution include, but are not limited to, 0.01-0.3 μg/μl, 0.01-0.15 μg/μl, 0.04-0.15 μg/μl, 0.1-100 μg/μl; 0.1-50 μg/μl, 0.1-10 μg/μl, 0.1-5 μg/μl, 0.1-1 μg/μl, 0.1-0.5 μg/μl, 0.15-0.5 μg/μl, 0.1-0.3 μg/μl, 0.01-0.1 μg/μl, 0.01-0.05 μg/μl, 0.02-0.04 μg/μl, 0.001-0.02 μg/μl. According to a specific embodiment, the concentration of the dsRNA in the treating solution is 0.01-0.15 or 0.04-0.15 μg/μl.
In one embodiment, the dsRNA concentration in the treating solution is 0.01-0.3 μg/ml, 0.01-0.15 μg/ml, 0.04-0.15 μg/ml, 0.1-100 μg/ml; 0.1-50 μg/ml, 0.1-10 μg/ml, 0.1-5 μg/ml, 0.1-1 μg/ml, 0.1-0.5 μg/ml, 0.15-0.5 μg/ml, 0.1-0.3 μg/ml, 0.01-0.1 μg/ml, 0.01-0.05 μg/ml, 0.02-0.04 μg/ml, or 0.001-0.02 μg/ml.
In another embodiment, the dsRNA concentration in the treating solution is 0.0001-3 μg/μl, 0.0001-2.5 μg/μl, 0.0001-2 μg/μl, 0.0001-1.5 μg/μl, 0.0001-1 μg/μl, 0.0001-0.9 μg/μl, 0.0001-0.8 μg/μl, 0.0001-0.7 μg/μl, 0.0001-0.6 μg/μl, 0.0001-0.5 μg/μl, 0.0001-0.4 μg/μl, 0.0001-0.3 μg/μl, 0.0001-0.2 μg/μl, 0.0001-0.1 μg/μl, 0.0001-0.05 μg/μl, 0.0001-0.02 μg/μl, 0.0001-0.01 μg/μl, 0.0001-0.005 μg/μl, 0.0001-0.001 μg/μl, or 0.0001-0.0005 μg/μl.
In another embodiment, the dsRNA concentration in the treating solution is 0.0001-3 μg/μl, 0.0005-3 μg/μl, 0.001-3 μg/μl, 0.005-3 μg/μl, 0.01-3 μg/μl, 0.05-3 μg/μl, 0.1-3 μg/μl, 0.2-3 μg/μl, 0.3-3 μg/μl, 0.4-3 μg/μl, 0.5-3 μg/μl, 0.6-3 μg/μl, 0.7-3 μg/μl, 0.8-3 μg/μl, 0.9-3 μg/μ1,1-3 μg/μl, or 2-3 μg/μl.
In another embodiment, the dsRNA concentration in the treating solution is 0.0001-3 μg/μl, 0.0005-2.5 μg/μl, 0.001-2 μg/μl, 0.005-1.5 μg/μl, 0.01-1 μg/μl, 0.05-0.5 μg/μl, 0.1-0.4 μg/μl, or 0.2-0.3 μg/μl.
According to a specific embodiment, the contacting with the dsRNA is effected in the presence of a chelating agent such as EDTA or another chelating agent such as DTPA (0.01-0.1 mM).
The contacting solution may comprise a transferring agent such as a surfactant or a salt.
Examples of such transferring agents include but are not limited salts such as sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids lipofectamine or lipofectin (1-20 nM, or 0.1-1 nM)) and organosilicone surfactants. Other useful surfactants include organosilicone surfactants including nonionic organosilicone surfactants, e.g., trisiloxane ethoxylate surfactants or a silicone polyether copolymer such as a copolymer of polyalkylene oxide modified heptamethyl trisiloxane and allyloxypolypropylene glycol methylether (commercially available as Silwet™ L-77 surfactant having CAS Number 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, N.Y.).
Useful physical agents can include (a) abrasives such as carborundum, corundum, sand, calcite, pumice, garnet, and the like, (b) nanoparticles such as carbon nanotubes or (c) a physical force. Carbon nanotubes are disclosed by Kam et al. (2004) J. Am. Chem. Soc., 126 (22):6850-6851, Liu et al. (2009) Nano Lett., 9(3):1007-1010, and Khodakovskaya et al. (2009) ACS Nano, 3(10):3221-3227. Physical force agents can include heating, chilling, the application of positive pressure, or ultrasound treatment. Agents for laboratory conditioning of a plant to permeation by polynucleotides include, e.g., application of a chemical agent, enzymatic treatment, heating or chilling, treatment with positive or negative pressure, or ultrasound treatment. Agents for conditioning plants in a field include chemical agents such as surfactants and salts.
Contacting of the seeds with the dsRNA can be effected using any method known in the art as long as a suppressive amount of the dsRNA enters the seeds. These examples include, but are not limited to, soaking, spraying or coating with powder, emulsion, suspension, or solution; similarly, the polynucleotide molecules are applied to the plant by any convenient method, e.g., spraying or wiping a solution, emulsion, or suspension.
As used herein “a suppressive amount” refers to an amount of dsRNA which is sufficient to down regulate the target gene by at least 20%, 30%, 40%, 50%, or more, say 60%, 70%, 80%, 90% or more even 100%. The suppressive amount can be a result of the formation of amplification in the plant.
According to a specific embodiment contacting may be effected by soaking (i.e., inoculation) so that shaking the seeds with the treating solution may improve penetration and soaking and therefore reduce treatment time. Shaking is typically performed at 50-150 RPM and depends on the volume of the treating solution. Shaking may be effected for 4-24 hours (1-4 hours, 10 minutes to 1 hour or 30 seconds to 10 minutes). The present teachings further envisage short incubation time such as up to 10 minutes. Examples include but are not limited to 30 seconds to 7 min, to 30 seconds to 5 min, to 30 seconds to 3 min, to 30 seconds to 2 min, to 30 seconds to 1 min, 1 min to 10 min or to 1 min to 5 min.
In one embodiment, the incubation time may be between 1 and 60, between 2 and 60, between 5 and 60, between 10 and 60, between 20 and 60, between 30 and 60, between 40 and 60, between 50 and 60, between 1 and 50, between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 10, between 1 and 5, between 5 and 50, between 10 and 40, and between 20 and 30 seconds.
In another embodiment, the incubation time may be between 1 and 60, between 2 and 60, between 5 and 60, between 10 and 60, between 20 and 60, between 30 and 60, between 40 and 60, between 50 and 60, between 1 and 50, between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 10, between 1 and 5, between 5 and 50, between 10 and 40, and between 20 and 30 minutes.
Dipping is also considered under the scope of the present invention. Thus, the seeds are dipped into the dsRNA solution for seconds e.g., 1-10 seconds, 1-5 seconds, 1-3 seconds or 1-2 seconds. During this period, the dsRNA may adsorb on the seed surface. The adsorbed dsRNA which coats the seed may penetrate the seed or the seedling during germination. The incubation takes place in the dark at 4-28° C. or 15-22° C. (e.g., 8-15° C., 4-8° C., 22-28° C.).
In one embodiment, the dipping time may be between 1 and 60, between 2 and 60, between 5 and 60, between 10 and 60, between 20 and 60, between 30 and 60, between 40 and 60, between 50 and 60, between 1 and 50, between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 10, between 1 and 5, between 5 and 50, between 10 and 40, and between 20 and 30 minutes.
In one embodiment, the dipping time may be between 1 and 60, between 2 and 60, between 5 and 60, between 10 and 60, between 20 and 60, between 30 and 60, between 40 and 60, between 50 and 60, between 1 and 50, between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 10, between 1 and 5, between 5 and 50, between 10 and 40, and between 20 and 30 seconds.
According to a specific embodiment, contacting occurs prior to breaking of seed dormancy and embryo emergence.
Following contacting, preferably prior to breaking of seed dormancy and embryo emergence, the seeds may be subjected to treatments (e.g., coating) with the above agents (e.g., pesticide, fungicide etc.).
Contacting is effected such that the dsRNA enters the embryo, endosperm, the coat, or a combination of the three.
After contacting with the treatment solution, the seeds may be subjected to drying for up to 30 hours at 25-37° C. For example, the seeds may be dried for 16 hours at 30° C.
According to a specific embodiment, the seed (e.g., isolated seed) comprises the exogenous naked dsRNA and wherein at least 10 or 20 molecules of the dsRNA are in the endosperm of the isolated seed.
As used herein the term “isolated” refers to separation from the natural physiological environment. In the case of seed, the isolated seed is separated from other parts of the plant. In the case of a nucleic acid molecule (e.g., dsRNA) separated from the cytoplasm.
According to a specific embodiment, the dsRNA is not expressed from the plant genome, thereby not being an integral part of the genome.
According to a specific embodiment there is provided an isolated seed comprising an exogenous dsRNA being present at a similar concentration (e.g., about 1:1, 2:1 or 1:2) in an embryo and an endosperm of the seed. It is suggested that the direct introduction of the naked dsRNA to the seed results in higher concentration of the dsRNA in the endosperm than that observed when the dsRNA is expressed from a nucleic acid expression construct.
According to a specific embodiment there is provided an isolated seed comprising an exogenous dsRNA being spatially distributed in an embryo and an endosperm of the plant seed in a spatial distribution that differs from a spatial distribution of the exogenous dsRNA in a seed derived from a transgenic plant that recombinantly expresses said exogenous dsRNA.
Methods of measuring the localization of RNA molecules in the seed are well known in the art. The use of siGlo as described in the Examples section is an example for such.
According to an alternative or an additional embodiment, there is provided an isolated seed comprising an exogenous dsRNA, wherein a concentration ratio of said exogenous dsRNA to siRNA maturing therefrom is higher in the seed as compared to a transgenic seed recombinantly expressing said exogenous dsRNA.
As used herein the term “higher” refers to at least about 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90% or even a few folds higher.
According to an alternative or an additional embodiment, there is provided an isolated seed comprising an exogenous dsRNA, wherein the plant seed is devoid of a heterologous promoter for driving expression of said exogenous dsRNA, wherein a spatial distribution of said exogenous dsRNA and/or siRNA maturing therefrom is altered in the seed as compared to same in a transgenic seed recombinantly expressing said exogenous dsRNA.
The term “recombinantly expressing” refers to an expression from a nucleic acid construct.
According to a further embodiment there is provided a plant seed obtainable (or obtained) by any of the methods described herein.
Methods of qualifying successful introduction of the dsRNA include but are not limited to, RT-PCR (e.g., quantifying the level of the target gene or the naked dsRNA), phenotypic analysis such as biomass, vigor, yield and stress tolerance, root architecture, leaf dimensions, grain size and weight, oil content, cellulose, as well as cell biology techniques.
According to embodiments of the invention, up regulation of the gene targeted by the Seed treatment, as described herein, is sometimes observed. This has been mostly noted in genes that function as master regulators, such as targets of microRNAs (e.g. SPL and NAC) and other genes involved in regulating key processes (e.g. HY5). See for instance Examples 23 and 32 of the Examples section which follows.
Without being bound by theory, it is suggested that this phenomenon could be related to a potential feedback loop in the regulation of the expression of these genes. These genes are probably tightly regulated and therefore plants could react to changes in their expression in one direction by over compensating a strong change in the genes' expression in the opposite direction. Accordingly, it is possible for example that a gene will initially present down-regulation in the first hours or days following treatment, which could be altered into up-regulation later on in the plant's life cycle. See for instance Example 32 of the Examples section which follows. Thus, the present inventors observed up regulation of the NAC gene in corn five days following treatment and down regulation 10 and 12 days following treatment. This is further substantiated in lettuce for the Hy 5.5 or 5.6 gene.
Seeds may be stored for 1 day to several months prior to planting (e.g., at 4-10° C.).
The resultant seed can be germinated in the dark so as to produce a plant.
Thus there is provided a plant or plant part comprising an exogenous naked dsRNA and devoid of a heterologous promoter for driving expression of the dsRNA in the plant.
As used herein “devoid of a heterologous promoter for driving expression of the dsRNA” means that the plant or plant cell doesn't include a cis-acting regulatory sequence (e.g., heterologous) transcribing the dsRNA in the plant. As used herein the term “heterologous” refers to exogenous, not-naturally occurring within the native plant cell (such as by position of integration, or being non-naturally found within the plant cell). Thus the isolated seed in the absence of a heterologous promoter sequence for driving expression of the dsRNA in the plant, comprises a homogenic (prior to amplification) or heterogenic (secondary siRNAs, following amplification) population of plant non-transcribable dsRNA.
The present methodology can be used for modulating gene expression such as in a plant, the method comprising:
(a) contacting a seed of the plant with a naked dsRNA, under conditions which allow penetration of the dsRNA into the seed, thereby introducing the dsRNA into the seed; and optionally
(b) generating a plant of the seed.
When used for down-regulating a plant gene, the naked dsRNA is designed of the desired specificity using bioinformatic tools which are well known in the art (e.g., BLAST).
This methodology can be used in various applications starting from basic research such as in order to asses gene function and lasting in generating plants with altered traits which have valuable commercial use.
Such plants can exhibit agricultural beneficial traits including altered morphology, altered flowering, altered tolerance to stress (i.e., viral biotic stress and/or abiotic), altered biomass vigor and/or yield and the like.
The phrase “abiotic stress” as used herein refers to any adverse effect on metabolism, growth, viability and/or reproduction of a plant. Abiotic stress can be induced by any of suboptimal environmental growth conditions such as, for example, water deficit or drought, flooding, freezing, low or high temperature, strong winds, heavy metal toxicity, anaerobiosis, high or low nutrient levels (e.g. nutrient deficiency), high or low salt levels (e.g. salinity), atmospheric pollution, high or low light intensities (e.g. insufficient light) or UV irradiation. Abiotic stress may be a short term effect (e.g. acute effect, e.g. lasting for about a week) or alternatively may be persistent (e.g. chronic effect, e.g. lasting for example 10 days or more). The present invention contemplates situations in which there is a single abiotic stress condition or alternatively situations in which two or more abiotic stresses occur.
According to an exemplary embodiment the abiotic stress refers to salinity.
According to another exemplary embodiment the abiotic stress refers to drought.
According to another exemplary embodiment the abiotic stress refers to a temperature stress.
As used herein the phrase “abiotic stress tolerance” refers to the ability of a plant to endure an abiotic stress without exhibiting substantial physiological or physical damage (e.g. alteration in metabolism, growth, viability and/or reproducibility of the plant).
As used herein the phrase “nitrogen use efficiency (NUE)” refers to a measure of crop production per unit of nitrogen fertilizer input. Fertilizer use efficiency (FUE) is a measure of NUE. Crop production can be measured by biomass, vigor or yield. The plant's nitrogen use efficiency is typically a result of an alteration in at least one of the uptake, spread, absorbance, accumulation, relocation (within the plant) and use of nitrogen absorbed by the plant. Improved NUE is with respect to that of a non-transgenic plant (i.e., lacking the transgene of the transgenic plant) of the same species and of the same developmental stage and grown under the same conditions.
As used herein the phrase “nitrogen-limiting conditions” refers to growth conditions which include a level (e.g., concentration) of nitrogen (e.g., ammonium or nitrate) applied which is below the level needed for optimal plant metabolism, growth, reproduction and/or viability.
As used herein the term/phrase “biomass”, “biomass of a plant” or “plant biomass” refers to the amount (e.g., measured in grams of air-dry tissue) of a tissue produced from the plant in a growing season. An increase in plant biomass can be in the whole plant or in parts thereof such as aboveground (e.g. harvestable) parts, vegetative biomass, roots and/or seeds or contents thereof (e.g., oil, starch etc.).
As used herein the term/phrase “vigor”, “vigor of a plant” or “plant vigor” refers to the amount (e.g., measured by weight) of tissue produced by the plant in a given time. Increased vigor could determine or affect the plant yield or the yield per growing time or growing area. In addition, early vigor (e.g. seed and/or seedling) results in improved field stand.
As used herein the term/phrase “yield”, “yield of a plant” or “plant yield” refers to the amount (e.g., as determined by weight or size) or quantity (e.g., numbers) of tissues or organs produced per plant or per growing season. Increased yield of a plant can affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time.
According to an exemplary embodiment the yield is measured by cellulose content, oil content, starch content and the like.
According to another exemplary embodiment the yield is measured by oil content.
According to another exemplary embodiment the yield is measured by protein content.
According to another exemplary embodiment, the yield is measured by seed number, seed weight, fruit number or fruit weight per plant or part thereof (e.g., kernel, bean).
A plant yield can be affected by various parameters including, but not limited to, plant biomass; plant vigor; plant growth rate; seed yield; seed or grain quantity; seed or grain quality; oil yield; content of oil, starch and/or protein in harvested organs (e.g., seeds or vegetative parts of the plant); number of flowers (e.g. florets) per panicle (e.g. expressed as a ratio of number of filled seeds over number of primary panicles); harvest index; number of plants grown per area; number and size of harvested organs per plant and per area; number of plants per growing area (e.g. density); number of harvested organs in field; total leaf area; carbon assimilation and carbon partitioning (e.g. the distribution/allocation of carbon within the plant); resistance to shade; number of harvestable organs (e.g. seeds), seeds per pod, weight per seed; and modified architecture [such as increase stalk diameter, thickness or improvement of physical properties (e.g. elasticity)].
Improved plant NUE is translated in the field into either harvesting similar quantities of yield, while implementing less fertilizers, or increased yields gained by implementing the same levels of fertilizers. Thus, improved NUE or FUE has a direct effect on plant yield in the field.
As used herein the term “improving” or “increasing” refers to at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or greater increase in NUE, in tolerance to stress, in yield, in biomass or in vigor of a plant, as compared to a native or wild-type plants [i.e., isogenic plants (not modified to comprise the dsRNA) of the invention].
As mentioned, the target gene of the dsRNA may not be an endogenous plant gene but rather a gene exogenous to the plant, such as that of a plant virus or bacteria which feeds on the plant or depends thereon for growth, replication and/or survival.
Thus, according to an aspect of the invention there is provided a method of inhibiting expression of a target gene in a plant virus, the method comprising providing (contacting under infecting conditions) to the plant virus the plant as described herein (at least part thereof includes the naked dsRNA), thereby inhibiting expression of a target gene in the plant virus.
A number of virus genera are transmitted, both persistently and non-persistently, by soil borne zoosporic protozoa. These protozoa are not viral pathogenic themselves, but parasitic. Transmission of the virus takes place when they become associated with the plant roots. Examples include Polymyxa graminis, which has been shown to transmit plant viral diseases in cereal crops and Polymyxa betae which transmits Beet necrotic yellow vein virus. Plasmodiophorids also create wounds in the plant's root through which other viruses can enter.
Specific examples of viruses which can be targeted according to the present teachings include, but are not limited to:
(1) Tobacco mosaic virus (TMV, RNA virus) which infects plants, especially tobacco and other members of the family Solanaceae).
(2) Tomato spotted wilt virus (TSWV, RNA virus) which causes serious diseases of many economically important plants representing 35 plant families, including dicots and monocots. This wide host range of ornamentals, vegetables, and field crops is unique among plant-infecting viruses. Belongs to tospoviruses in the Mediterranean area, affect vegetable crops, especially tomato, pepper and lettuce (Turina et al., 2012, Adv Virus Res 84; 403-437).
(3) Tomato yellow leaf curl virus (TYLCV) which is transmitted by whitefly, mostly affects tomato plants. Geminiviruses (DNA viruses) in the genus Begomovirus (including sweepoviruses and legumoviruses)—most devastating pathogens affecting a variety of cultivated crops, including cassaya, sweet potato, beans, tomato, cotton and grain legumes (Rey et al 2012, Viruses 4; 1753-1791). Members include TYLCV above and tomato leaf curl virus (ToLCV).
(4) Cucumber mosaic virus (CMV)—CMV has a wide range of hosts and attacks a great variety of vegetables, ornamentals, and other plants (as many as 191 host species in 40 families). Among the most important vegetables affected by cucumber mosaic are peppers (Capsicum annuum L.), cucurbits, tomatoes (Lycopersicon esculentum Mill.), and bananas (Musa L. spp.).
Other vegetable hosts include: cucumber, muskmelon, squash, tomato, spinach, celery, peppers, water cress, beet, sweet potato, turnip, chayote, gherkin, watermelon, pumpkin, citron, gourd, lima bean, broad bean, onion, ground-cherry, eggplant, potato, rhubarb, carrot, dill, fennel, parsnip, parsley, loofah, and artichoke (Chabbouh and Chemf, 1990, FAO Plant Prot. Bull. 38:52-53.).
Ornamental hosts include: China aster, chrysanthemum, delphinium, salvia, geranium, gilia, gladiolus, heliotrope, hyacinth, larkspur, lily, marigold, morning glory, nasturtium, periwinkle, petunia, phlox, snapdragon, tulip, and zinnia (Chupp and Sherf, 1960; Agrios, 1978).
(5) Potato virus Y (PVY)— one of the most important plant viruses affecting potato production.
(6) Cauliflower mosaic virus (CaMV, DNA virus (Rothnie et al., 1994)).
(7) African cassaya mosaic virus (ACMV).
(8) Plum pox virus (PPV) is the most devastating viral disease of stone fruit from the genus Prunus.
(9) Brome mosaic virus (BMV)—commonly infects Bromus inermis and other grasses, can be found almost anywhere wheat is grown.
(10) Potato virus X (PVX)— There are no insect or fungal vectors for this virus. This virus causes mild or no symptoms in most potato varieties, but when Potato virus Y is present, synergy between these two viruses causes severe symptoms in potatoes.
Additional Viruses:
Citrus tristeza virus (CTV)—causes the most economically damaging disease to Citrus, including sour orange (Citrus aurantium), and any Citrus species grafted onto sour orange root stock, sweet orange (C. sinensis), grapefruit (C. paradisi), lime and Seville orange (C. aurantifolia), and mandarin (C. reticulata). CTV is also known to infect Aeglopsis chevalieri, Afraegle paniculata, Pamburus missionis, and Passiflora gracilis. CTV is distributed worldwide and can be found wherever citrus trees grow.
Barley yellow dwarf virus (BYDV)—most widely distributed viral disease of cereals. It affects the economically important crop species barley, oats, wheat, maize, triticale and rice.
Potato leafroll virus (PLRV) infects potatoes and other members of the family Solanaceae.
Tomato bushy stunt virus (TBSV), RNA virus, a member of the genus Tombusvirus and mostly affects tomatoes and eggplant.
Additional Reviews:
Hamilton et al., 1981, J Gen Virol 54; 223-241—mentions TMV, PVX, PVY, CMV, CaMV.
Additional Scientific Papers:
Makkouk et al., 2012, Adv Virus Res 84; 367-402—Viruses affecting peas and beans with narrow (Faba bean necrotic yellow virus (FBNYN)) and wide (alfalfa mosaic virus (AMV) and CMV) host range.
The target gene of the plant virus encodes a product essential to the viability and/or infectivity of the viral pathogen, therefore its down-regulation (by the naked dsRNA) results in a reduced capability of the pathogen to survive and infect host cells. Hence, such down-regulation results in a “deleterious effect” on the maintenance viability and/or infectivity of the viral pathogen, in that it prevents or reduces the pathogen's ability to feed off and survive on nutrients derived from host cells. By virtue of this reduction in the viral pathogen's viability and/or infectivity, resistance and/or enhanced tolerance to infection by a pathogen is facilitated in the cells of the plant. Genes in the pathogen may be targeted at the mature (adult), immature (juvenile) or embryo stages.
As used herein, a “plant virus resistance” trait is a characteristic of a plant that causes the plant host to be resistant to attack from a viral pathogen that typically is capable of inflicting damage or loss to the plant. Once the viral pathogen is provided with the plant material comprising the naked dsRNA, expression of the gene within the target virus is suppressed by the dsRNA, and the suppression of expression of the gene in the target virus results in the plant being resistant to the virus.
To substantiate the anti-viral activity, the present teachings also contemplate observing reduced infectability or replicability and the degree of host symptomatology following said providing.
To improve the anti-viral activity, embodiments of the present invention further provide a composition that contains two or more different agents each toxic to the same plant virus, at least one of which comprises a dsRNA described herein. In certain embodiments, the second agent can be an agent selected from the group consisting of inhibitors of metabolic enzymes involved in amino acid or carbohydrate synthesis; inhibitors of cell division; cell wall synthesis inhibitors; inhibitors of DNA or RNA synthesis, gyrase inhibitors, tubulin assembly inhibitors, inhibitors of ATP synthesis; oxidative phosphorylation uncouplers; inhibitors of protein synthesis; MAP kinase inhibitors; lipid synthesis or oxidation inhibitors; sterol synthesis inhibitors; and melanin synthesis inhibitors.
In addition, plants generated according to the teachings of the present invention or parts thereof can exhibit altered nutritional or therapeutic efficacy and as such can be employed in the food or feed and drug industries. Likewise, the plants generated according to the teachings of the present invention or parts thereof can exhibit altered oil or cellulose content and as such can be implemented in the construction or oil industry.
The seeds of the present invention can be packed in a seed containing device which comprises a plurality of seeds at least some of which (e.g., 5%, 10% or more) containing an exogenous naked dsRNA, wherein the seed is devoid of a heterologous promoter for driving expression of the dsRNA.
The seed containing device can be a bag, a plastic bag, a paper bag, a soft shell container or a hard shell container.
Reagents of the present invention can be packed in a kit including the naked dsRNA, instructions for introducing the dsRNA into the seeds and optionally a priming solution.
Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, which may contain one or more dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for introduction to the seed.
According to an exemplary embodiment, the naked dsRNA and priming solution are comprised in separate containers.
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Generating the dsRNA/siRNA Sequences
The dsRNA sequences were custom-created for each gene using in vitro transcription of PCR products. Part of the mRNA, including either the ORF, 3′ UTR or 5′ UTR for which dsRNA to be produced was PCR-amplified using gene-specific primers, which contain the sequence of the T7 promoter on either side. This product was used as a template for dsRNA production using commercial kits such as the MaxiScript dsRNA kit (Life Technologies) or T7 High Yield RNA Synthesis kit (NEB). Next, the sample is treated with DNase Turbo at 37° C. for 15-30 min followed by phenol treatment and nucleic acid precipitation. Next, one of two different reactions is carried out: (1) dsRNA is ready to use, (2) processing of the dsRNA with Dicer (Shortcut RNase III (NEB)) to create small interfering RNAs (siRNA).
Either dsRNA or a combination of dsRNA and siRNA were used for seed treatments as described below. All dsRNA sequences provided herein are listed as DNA (simple transformation is done by converting T>U).
General Seed Treatment Protocol for Gene Silencing using a dsRNA/siRNA Mixture
Uncoated organic corn seeds were from variety “popcorn”, uncoated organic whole grain rice seeds and organic soybean were purchased from Nitsat Haduvdevan (Israel). Wheat seeds were from A.B. Seeds (Israel). Lettuce seeds were from the variety Sun Valley. Fresh tomato seeds were retrieved from M82 tomato fruits, which are propagated in-house. Uncoated or fresh plant seeds were washed with double distilled water (DDW) prior to treatment for four hours. Next, seeds were dried at 20-30° C. for up to 24 hours. Following the drying step, seeds were treated with a solution containing the dsRNA formulation, which is made of dsRNA at a final concentration of 1-261 μg/ml in 0.1 mM EDTA. Treatment was performed by gently shaking the seeds in the solution for up to 60 hours in a dark growth chamber at 15-25° C. Finally, seeds were washed up to three times briefly and planted on soil or germinated at 25° C. in a dark growth chamber and planted in soil or dried for 0-30 hours and germinated at 25° C. in a dark growth chamber and planted in soil or planted directly in soil. Control seeds were treated in a similar way, with a formulation that lacked the dsRNA or with non-specific dsRNA.
As an example for an exogenous gene that is not present/expressed in plants, the ORFs encoding the replicase and coat protein of CGMMV (Cucumber green mottle mosaic virus, accession number AF417242) were used as targets for dsRNA treatment of plant seeds using the protocol described in Example 1. Rice, tomato and sorghum seeds were washed for 4 hours at 20° C., tomato and sorghum were dried at 30° C. and rice at 20° C. for overnight. Seeds were immediately treated at 15° C. with 132.7 μg/ml dsRNA (final concentration) for 39 hours for rice, as shown in
The exogenous dsRNA was found to be stable for at least three weeks in rice seedlings as can be seen in
Rice seeds were treated with an exogenous dsRNA as in Example 2. Plants were germinated and grown for five weeks, DNA was extracted and PCR reactions were performed to demonstrate that the dsRNA did not integrate into the Rice's genome (
Corn seeds were treated using the protocol described in Example 1, seeds were washed for 4 h at 20° C., dried at 30° C. overnight and immediately treated with 40 μg/ml dsRNA (final concentration) directed against the β-glucuronidase (GUS) reporter gene for 60 hours at 15° C., dried and were germinated. Leaves and roots were harvested from control and dsGUS-treated plants 7 and 15 days following germination. RNA was extracted from the harvested tissues and RT-PCR with specific GUS primers was run (Table 3). In addition, a corn endogenous housekeeping gene (ubiquitin) was used as a positive control for the PCR reaction. The GUS dsRNA molecules were found to be extremely stable in the treated seeds, and can be detected in corn plants 7 and 15 days post germination of the seeds (
Plant seeds were treated as per the protocol described in Example 1. Seeds were washed for 4 h at 20° C., dried at 25° C. and were immediately treated with a fluorescent siRNA (siGLO, 2 nM final concentration, Thermo Scientific) at 15° C. for 24 h.
The quality of the siGLO was verified by gel electrophoresis analysis as can be seen on
Fluorescent pictures of the seeds were taken 24-48 hours post treatment using an Olympus microscope at the lowest objective magnification (5× for bigger seeds such as rice and tomato seeds, and 10× for smaller seeds such as arabidopsis seeds). To eliminate the possibility of non-specific auto-fluorescence, each dsRNA-treated seed is shown alongside a control seed that was left untreated (
In order to evaluate the distribution efficiency of the fluorescent siRNA inside the seeds, different plant seeds were cut into slices and imaged with a fluorescent binocular 48 hours after treatment. Each treated seed was imaged alongside a control untreated seed. Light and fluorescent images were taken where applicable for rice, tomato, cucumber, bean, sorghum and wheat seed samples (
The dsRNA molecules enter the embryo directly, carried by the water-based solution which is used for the seed treatment.
The dsRNA molecules enter the endosperm as part of the endosperm's water-absorption process. These molecules then feed the embryo as it develops as part of the nutrient flow from the endosperm during germination and seed development.
These present findings suggest the RNA molecules used to treat the seeds both penetrate the embryo and function in the embryo as it develops and also penetrate the endosperm and feed the embryo following germination.
A time course experiment was performed on rice seeds to monitor the kinetics of siGLO penetration into the seeds following the seed treatment (
Rice seeds were washed in wash solution for 4 h at 20° C., dried at 25° C. and immediately treated with a mixture of dsRNA/siRNA at a total concentration of 60 μg/ml at 15° C. for 40 hours. Seeds were germinated at room temperature for several days and seed development was monitored. Seeds treated with the PDS and dsRNA/siRNA mixture exhibited stunted and delayed development, as seen by smaller seedlings and reduced rooting. In these experiments, two products of the PDS-1 gene are combined (see Table 4).
Zea mays
The experiment was performed in three biological repeats and the results are presented in
Rice seeds were treated as described in Example 7 and their subsequent development and seedling growth were monitored. Thirty days post PDS-1 silencing treatment the overall phenotype of the two plant groups, control and PDS-silenced, was recorded. PDS silencing has been reported to cause chlorophyll bleaching and growth inhibition (Peretz et al., 2007, Plant Physiol 145: 1251-1263), which correlates with the phenotype of the PDS-silenced plants of the invention, as they appeared smaller in size and paler in color, respectively, compared to control plants (see
Following treatment with the dsRNA/siRNA mixture (ratio 1:1) as described in Example 7, expression levels of PDS-1 gene products are determined by real-time PCR using specifically designed primers (Forward: GATTGCTGGAGCAGGATTAG SEQ ID NO: 46, Reverse: CCCTTGCCTCAAGCAATATG, SEQ ID NO: 47). For normalization purposes, UBQS expression was also determined using primers (forward—ACCACTTCGACCGCCACTACT, SEQ ID NO: 48, reverse—ACGCCTAAGCCTGCTGGTT, SEQ ID NO: 49). The results are shown in
Rice seeds were treated using the protocol described in Example 1, Seeds were washed for 4 h at room temperature, dried overnight at 25° C. and immediately treated with a Hap2e dsRNA concentration of 152 μg/ml, for 41 hours at 15° C. (for Hap2e dsRNA sequences see Table 11, below). Control and Hap2e dsRNA-treated rice seeds that were germinated 5 days post treatment did not exhibit any differences in their root development (
Other rice seeds were treated under the same conditions as in
Corn seeds were treated using the protocol described in Example 1, Seeds were washed for 4 h at room temperature, dried overnight at 30° C. and immediately treated with a NFY dsRNA concentration of 56 μg/ml, for 40 hours at 15° C. (for NFY dsRNA sequence see table 11). RT-PCR on RNA extracted from shoots of control and NFY dsRNA-treated corn seeds 10 days after germination was performed to determine the expression level of NFY target gene (see Table 6). Down-regulation of the gene was successfully achieved as exhibited in
Tomato seeds were treated using the protocol described in Example 1. Un washed seeds were treated with a NFY dsRNA concentration of 200 μg/ml, for 24 hours at 15° C., seeds were washed twice briefly and immediately planted in soil without drying. RT-PCR on RNA extracted from shoots of control and NFY dsRNA-treated tomato seeds 3 weeks after germination was performed to determine the expression level of NFY target gene (see Table 7). Down-regulation of the gene was successfully achieved as exhibited in
Tomato plants 55 days post treatment with NFY dsRNA molecules were compared to same age control plants. Major phenotypic differences were evident upon comparison, most notably was a shift in height, where treated plants appeared significantly shorter than untreated control plants (
Corn seeds were treated using the protocol described in Example 1, seeds were washed for 4 h at room temperature, dried overnight at 30° C. and immediately treated with a NAC dsRNA concentration of 90 μg/ml, for 40 hours at 15° C. and immediately germinated (for NAC dsRNA sequence see Table 11). RT-PCR on RNA extracted from shoots of control and NAC dsRNA-treated corn seeds 10 days after germination was performed to determine the expression level of NAC target gene (see Table 8). Down-regulation of the gene was successfully achieved as exhibited in
Rice seeds were treated using the protocol described in Example 1, seeds were washed for 4 h, dried overnight at 20° C. and immediately treated with an ARF-8 dsRNA concentration of 66.2 μg/ml, for 42 hours at 15° C. RT-PCR on RNA extracted from control and ARF-8 dsRNA-treated rice seeds 18 days after germination was performed to determine the expression level of ARF-8 target gene (see Table 9). Down-regulation of the gene was successfully achieved as exhibited in
Rice seeds were treated using the protocol described in Example 1, seeds were washed for 4 h, dried overnight at 20° C. and immediately treated with a SPL17 dsRNA concentration of 200 μg/ml, for 41 hours at 15° C. (for SPL17 dsRNA sequence see table 11). Control and SPL17 dsRNA-treated rice seeds that were germinated 5 days post treatment did not exhibit any visual differences (
The high specificity and efficiency of posttranscriptional gene silencing by target gene-specific dsRNA has become a preferred method to generate preferred phenotype eukaryotic organisms, wherein expression of one or more genes is reduced or inactivated. Specific dsRNA sequences designed to silence corn (Zea mays) and rice (Oryza sative) microRNA target genes. Specifically, microRNAs shown to associate with improved abiotic stress tolerance will be used. Table 11 below provides several examples for target gene sequences that are produced using PCR amplification to test the gene silencing capabilities of their respective dsRNA/siRNA mixture. These dsRNA molecules will then be used to knock down the endogenous level of the selected target genes.
Zea mays
Oryza
sativa
Oryza
sativa
Zea mays
Oryza
sativa
Zea mays
Tomato seeds were treated using the protocol described in Example 1, unwashed seeds were treated with an ARF-8 dsRNA concentration of 200 μg/ml, for 24 hours at 15° C. and immediately planted in soil. Expression levels of the gene were examined using RT-PCR, 3 and 8 weeks after treatment (see Table 12). Changes in expression were observed in dsRNA-treated plants 3 weeks after treatment (
Plants that were treated with dsRNA molecules specific for the ARF8 gene showed a phenotypic difference compared to control plants. This phenotypic difference was observed at different time points (55, 62 and 72 days) and was demonstrated by a decrease in height (
Tomato seeds were treated using the protocol described in Example 1, unwashed seeds were treated with a FW2.2 dsRNA concentration of 100 μg/ml, for 24 hours at 15° C. and immediately planted in soil Expression levels of the gene were examined using RT-PCR, 9 weeks following germination (primers are listed in Table 13). An approximate 2-fold reduction in the expression level of FW2.2 in dsRNA treated plants compared to control plants was detected (
Even so, plants that were treated with dsRNA molecules specific for the FW2.2 gene showed no phenotypic differences compared to control plants, ruling out a toxic effect as an alternative explanation for the phenotypic effects seen in the previous example. The plants presented similar height and appearance 72 days after treatment (
Rice seeds were treated using the protocol described in Example 1, seeds were washed for 4 h, dried for 24 h at room temperature and immediately treated with a DELLA dsRNA concentration of 66 μg/ml, for 36 hours at 15° C. Rice seeds were treated with dsRNA directed against the Della gene (see Table 15 below), which is a known plant growth repressor. Arabidopsis seedlings with mutant Della gene are larger with a longer root system (Josse, E. M., Gan, Y., Bou-Torrent, J., Stewart, K. L., Gilday, A. D., Jeffree, C. E., Vaistij, F. E., Martinez-Garcia, J. F., Nagy, F., Graham, I. A., and Halliday, K. J. (2011). A DELLA in disguise: SPATULA restrains the growth of the developing Arabidopsis seedling. Plant Cell 23: 1337-1351.).
Rice seeds were treated using the protocol described in Example 1, seeds were washed for 4 h, dried for 24 h at room temperature and immediately treated with a NRR dsRNA concentration of approximately 13 μg/ml, for 36 hours at 15° C. Rice seeds were treated with dsRNA directed against the NRR gene, which was found to regulate root growth in response to macronutrients in rice (Zhang et al., 2012, Mol Plant 5(1):63-72). Transgenic rice seedlings, with reduced NRR levels using RNAi were shown to have longer roots when grown under nitrogen limiting conditions.
In the present example, the effect of silencing three genes simultaneously is tested. Rice seeds were treated using the protocol described in Example 1, seeds were washed for 4 h, dried overnight at room temperature and immediately treated with a solution containing a mixture (152.3 μg/ml final concentration) of dsRNA against three genes: Hap2e (59.9 μg/ml, see Table 11), Della (44 μg/ml see table 15 below) and SQS (48.4 μg/ml see table 16 below) for 42 h at 15° C. RNA was extracted from shoots of germinated seeds, 18 days post germination, and RT-PCR for each of the three genes was run (see Table 15 below). As can be seen in
Tomato seeds were treated with a fluorescent siRNA (siGLO, 1nM final concentration, Thermo Scientific) at 15° C. for 24 h.
The seeds were cut into slices and fluorescent pictures were taken 24 hours post treatment using Leica confocal microscope. As shown in
Tomato seeds were treated with a dsRNA (SEQ ID NO: 126) derived from the SPL gene and with GUS dsRNA as a control according to the protocol described in Example 1. Treatment was performed by gently shaking the seeds in the solution for up to 24 hours in a dark growth chamber at 15-25° C. followed by washing with water three times for one minute. After treatment, seeds were planted in soil and grown at about 25° C. with 16 hours photoperiod. The plants were watered with tap water as necessary. The primers used for in-vitro transcription of SPL dsRNA and the sequence of the dsRNA are listed in Table 16. The sequence of GUS dsRNA appears in Table 3.
Prior to treatment, the dsRNA concentration and purity were assessed by absorbance (NanoDrop) and by HPLC. The concentration of the dsRNA according to the NanoDrop measurement was 1864 μg/ml for SPL and 1964 μg/ml for GUS. HPLC showed that the sample contains mostly dsRNA but other molecules (including nucleotides and ssRNA) were present in the solution as well. According to the HPLC analysis, the SPL solution contained 1219 μg/ml of dsRNA and 838 μg/ml of ssRNA. The GUS solution contained 633 μg/ml of dsRNA and 167 μg/ml of ssRNA (see
Seeds were treated with dsRNA at a concentration of 50 ng/ml (determined by the HPLC measurement) for 10 minutes, 2, 6 and 24 hours. An additional HPLC analysis of the SPL and GUS dsRNA solutions following seed incubation revealed that the ssRNA peak has disappeared (see
Total RNA was extracted from leaves of germinated seeds, 17 days (in plants treated for 24 hours) and 18 days (in plants treated for 10 minutes, 2 and 6 hours) post treatment. cDNA was prepared using oligo-dT primers and the expression level of SPL mRNA was determined in treated and control plants by real-time PCR with SYBR Green (Quanta BioSciences). Primers used for real-time PCR of SPL were located outside of the dsRNA region, at the 3′ UTR. Primers sequences are listed in Table 17 together with the house-keeping genes (Expressed and CAC) that were used as normalizers (in the case of SPL Expressed was used as the normalizer).
This analysis showed a significant (Wilcoxon rank-sum test, p-value<0.05) up-regulation of SPL mRNA at all incubation times. The median expression level of SPL in plants treated with SPL dsRNA for 10 minutes, 2 hours, 6 hours and 24 hours was 2.54, 2.85, 2.69 and 2.73-fold higher than in control plants treated with GUS dsRNA, respectively (
To verify that the effect on SPL mRNA results specifically from treatment with SPL dsRNA, the level of SPL mRNA was measured after treatment with both GUS dsRNA (a control sequence having no silencing relevant homology in the tomato genome) and FW2.2 dsRNA (which is a tomato endogenous sequence, see Table 13). Plants treated with 50 μg/ml dsRNA for 24 hours showed up-regulation of SPL when treated with SPL sequence, but not when treated with GUS or FW2.2 sequences (
In another experiment, tomato seeds of the Oregon Spring variety were treated with an independently made dsRNA for the SPL gene at a concentration of 50 μg/ml for 24 hours. The seeds were then washed 3 times with water, and transferred to seed germination boxes (9 seeds per box) containing 12 ml water. Seeds in boxes were germinated in a growth chamber in the light at 25° C. for 7 days, and shoot tissue was then harvested for RNA analysis. RNA analysis was conducted by Taqman, using a tomato CAC assay for normalization of the values to correct for differences in sample concentration (Table 17). Table 18 shows that in seedlings germinated from seeds treated with SPL dsRNA, mRNA levels of the SPL gene was elevated about 7-fold compared to seedlings germinated from seeds treated with GUS dsRNA. Treatment with AFR8 dsRNA and 0.1 mM EDTA (buffer) had no significant effect on SPL mRNA levels compared to treatment with GUS dsRNA.
Tomato seeds were treated with a dsRNA derived from the SPL gene as described in Examples 1 and 23.
Treatment with dsRNA at a concentration of 50 ng/ml was done by dipping the seeds in the dsRNA solution at room temperature and immediately washing with double distilled water (DDW). Total RNA was extracted from leaves of germinated seeds, 13 days post treatment. cDNA was prepared using oligo-dT primers and the expression level of SPL mRNA was determined in treated and control plants by real-time PCR, as described in Example 23.
This analysis showed a significant (p-value<0.05) up-regulation of SPL mRNA (
Tomato seeds were treated with a dsRNA derived from the SPL gene as described in Examples 1 and 23.
Seeds were treated with dsRNA at a concentration of 25 μg/ml for 10 minutes, 2 and 24 hours. Total RNA was extracted from leaves of germinated seeds, 17 days (in plants treated for 24 hours) and 18 days (in plants treated for 10 minutes and 2 hours) post treatment. cDNA was prepared using oligo-dT primers and the expression level of SPL mRNA was determined in treated and control plants by real-time PCR, as described in Example 23.
This analysis showed a significant (p-value<0.05) up-regulation of SPL mRNA for incubation times of 2 and 24 hours, and an up-regulation trend for 10 minutes incubation time (
Tomato seeds were treated with a dsRNA derived from the SPL gene as described in Examples 1 and 23.
Seeds were treated with dsRNA at a concentration of 1 or 5 ng/ml for 10 minutes, 2 and 24 hours. Total RNA was extracted from leaves of germinated seeds, 17 days (in plants treated for 24 hours) and 18 days (in plants treated for 10 minutes and 2 hours) post treatment. cDNA was prepared using oligo-dT primers and the expression level of SPL mRNA was determined in treated and control plants by real-time PCR, as described in Example 23.
An up-regulation trend in SPL mRNA expression was detected for all incubation times treated with 1 ng/ml dsRNA (
dsRNA derived from the SPL gene (Table 16) was processed with ShortCut® RNase III (NEB) as described in Example 1.
Tomato seeds were treated with the resulting siRNA at a concentration of 50 μg/ml for 2 hours at 25° C. Total RNA was extracted from leaves of germinated seeds 13 days post treatment. cDNA was prepared using oligo-dT primers and the expression level of SPL mRNA was determined in treated and control plants by real-time PCR, as described in Example 23.
An up-regulation trend in SPL mRNA expression was detected. The median expression level of SPL in plants treated with SPL siRNA was 1.89 higher than in control plants treated with GUS siRNA (
Tomato seeds were treated with a dsRNA derived from the FW2.2 gene as described in Examples 1 and 23. As a control, seeds were treated with GUS dsRNA at the same concentration. The FW2.2 dsRNA sequence appears in Table 13.
Before treatment, the dsRNA concentration and purity were assessed by absorbance (NanoDrop) and by HPLC. The concentration of the dsRNA according to the NanoDrop measurement was 1791 μg/ml. HPLC showed that the sample contains mostly dsRNA but other species (nucleotides and ssRNA) were present in the solution as well. According to the HPLC analysis, the FW2.2 solution contained 1410 μg/ml of dsRNA and 473 μg/ml of ssRNA (see
Seeds were treated with dsRNA at a concentration of 50 μg/ml (determined by the HPLC measurement) for 2, 6 and 24 hours. Total RNA was extracted from leaves of germinated seeds, 17 days (in plants treated for 24 hours) and 18 days (in plants treated for 2 and 6 hours) post treatment. cDNA was prepared using oligo-dT primers and the expression level of FW2.2 mRNA was determined in treated and control plants by real-time PCR. Primers used for real-time PCR of FW2.2 were TCTCTGGGCTTGTATCATCC (Forward primer, SEQ ID NO: 137) and GCTGCTCAAGGTGTTTGTG (Reverse primer, SEQ ID NO: 138). These primers are located outside of the dsRNA region, at the 3′UTR.
Down-regulation of FW2.2 mRNA following seed treatment was achieved at all incubation times (p-value≦0.1). The median expression level of FW2.2 in plants treated with FW2.2 dsRNA for 2, 6 and 24 hours was 1.26, 1.09 and 1.41-fold lower than in control plants treated with GUS dsRNA, respectively (
Rice seeds were treated using the protocol described in Example 1. Seeds were washed for 4 h, dried over night at room temperature and treated with DELLA dsRNA (see Table 15) at a concentration of 142 μg/ml, for 24 hours at 15° C. Total RNA was extracted from leaves of germinated seeds 13 days post treatment. cDNA was prepared using oligo-dT primers and the expression level of DELLA mRNA was determined in treated and control plants by real-time PCR (see Table 15).
This analysis showed a significant (p-value<0.05) down-regulation of DELLA mRNA. The median expression level of DELLA in plants treated with DELLA dsRNA was 3.73-fold lower than in control plants treated with GUS dsRNA (
Wheat seeds were treated using the protocol described in Example 1. Seeds were treated with a mixture of two PDS dsRNA fragments (see Table 19) for 4 hours at 15° C. and then germinated on a wet paper towel. The concentration of dsRNAs in the mixture was 136 ng/ml for fragment #1 (SEQ ID NO: 141) and 125 ng/ml for fragment #2 (SEQ ID NO: 144). Three days post treatment, seeds treated with the PDS dsRNA mixture exhibited stunted and delayed development, as seen by smaller seedlings and reduced rooting compared with control seeds treated with either 200 μg/ml GUS dsRNA or 0.1 mM EDTA (
Corn seeds were treated using the protocol described in Example 1. Seeds were washed for 4 h, dried over night at 30° C. and treated with TB1 dsRNA (see Table 20) at a concentration of 25 μg/ml, for 24 hours at 15° C. As a control, seeds were treated with CGMMV dsRNA (Table 1, product 1, SEQ ID NO: 8) at the same concentration. Total RNA was extracted from leaves of germinated seeds 7.5 weeks post germination. cDNA was prepared using oligo-dT primers and the expression level of TB1 mRNA was determined in treated and control plants by real-time PCR, using GPM120 as a normalizer (see Table 20).
This analysis showed down-regulation of TB1 mRNA following dsRNA treatment. The median expression level of TB1 in plants treated with TB1 dsRNA was 9.88-fold lower than in control plants treated with CGMMV dsRNA (
The two indicated NAC dsRNA sequences (Table 11) were synthesized in vitro using a convergent T7 RNA polymerase method, and diluted in 0.1 mM EDTA to a concentration of 50 μg/ml total nucleic acid. Fifteen inbred (LH244) maize seeds were incubated in 7.5 ml of this dsRNA solution in a 15 ml tube in the dark at 15° C., with gentle oscillation, for 24 hours. One set of seeds was washed 3 times with water after treatment with dsRNAs, and then dried overnight at 30° C. A second set of seeds was planted directly after imbibition in the dsRNA solution.
Following treatment, seeds were either transferred to seed germination boxes (15 seed per box) containing moistened filter paper and germinated in a growth chamber set at 25° C. in the dark, or were planted in soil and germinated in a greenhouse.
Shoot tissues, including mesocotyl and coleoptile base, were harvested from the germinating seeds in the germination boxes 5 days after treatment for RNA analysis. Seeds that had been dried had only 4 days to germinate, while seeds that were germinated without the drying step had 5 days for germination. However, drying resulted in improved synchronicity in germination so that the 2 sets of plants spanned similar developmental stages. RNA analysis was conducted by Taqman, using the Zm.GPM120 gene for normalization of the values to correct for differences in sample concentration (Table 21). RQ values were transformed to log 10 for analysis. Outlier data points are defined as being 3 standard deviations from the mean, and were removed from the dataset before analysis.
Treatment of seeds with NAC dsRNAs resulted in increased NAC expression so that NAC mRNA levels were 1.7 to 2.2 times those of GUS control values (Table 22 and
In another experiment, seeds were treated as described above and directly planted in soil. 12 days after planting, there was a decreased NAC mRNA expression in the V1 leaf in seeds that were not washed and dried before planting (
GUS_dry and NAC_dry, seeds were washed and dried after treatment with dsRNAs. The other set was put directly into seed germination boxes after treatment with dsRNAs. Comparisons with a control using Dunnett's Method, Control Group=GUS_dry or GUS, as appropriate.
The LONG HYPOCOTYL 5 (HY5) gene encodes a key positive regulator of plant response to light (Oyama, Shimura et al. 1997). Lettuce contains 2 genes related to Arabidopsis HY5 on chromosomes 5 and 6 which have 79% DNA sequence identity to each other. A ˜500 bp region was selected from the cDNA sequence of each gene as a trigger (Table 25), and dsRNA was made in vitro using the convergent T7 RNA polymerase method. The dsRNAs were solubilized in 0.1 mM EDTA at a concentration of 50 μg/ml. Thirty lettuce seeds of the variety Sun Valley were incubated in 1.5 ml of this dsRNA solution in a 2 ml eppendorf tube in the dark at 15° C., with gentle shaking, for 24 hours. The seeds were then washed 3 times with water, and then either transferred to seed germination boxes (9 seeds per box) containing 12 ml water, or to soil. Seeds in boxes were germinated in the light at 25° C. for 7 days, and shoot tissue was then harvested for RNA analysis. RNA analysis was conducted by Taqman, using a lettuce ubiquitin assay for normalization of the values to correct for differences in sample concentration (Table 25).
Table 26 and
In another experiment, plants were treated as described above and were grown for 2 weeks in the greenhouse. Then, a leaf punch was harvested and analyzed for mRNA concentration. In these older plants, a decrease in HY5 expression was observed. Table 28 and
(DHFR) mRNA in Lettuce Plants after Treatment of Seeds with DHFR dsRNA
Dihydroflavonol 4-Reductase (DHFR) is an enzyme in the anthocyanin biosynthetic pathway. One DHFR gene was identified in lettuce, and a 524 bp region was selected from the cDNA sequence as a trigger (Table 30). dsRNA was made in vitro using the convergent T7 RNA polymerase method for DFR and for the GUS control sequence.
The dsRNAs were solubilized in 0.1 mM EDTA at a concentration of 50 μg/ml. Lettuce variety 8N LLF 65-2713141 Batavia is a highly pigmented red variant used for these studies. Thirty lettuce seeds were incubated in 1.5 ml of the dsRNA solution in a 2 ml eppendorf tube in the dark at 15° C., with gentle shaking, for 24 hours. The seeds were then washed 3 times with water, and then either transferred to seed germination boxes (10 seeds per box) containing 12 ml water, or to soil. Seeds in boxes were germinated in the light at 20° C. for 7 days, and shoot tissue was then harvested for RNA analysis. RNA analysis was conducted by Taqman, using a lettuce ubiquitin assay for normalization of the values to correct for differences in sample concentration (Tables 25 and 30).
mRNA levels for DHFR were significantly reduced when seeds were treated with dsRNA for DHFR compared to seeds treated with GUS dsRNA or the buffer control (Table 31 and
DND1, defense no death, is a negative regulator of plant defenses. Mutations in DND1 lead to constitutive systemic resistance and elevated levels of salicylic acid (Clough et al. 2000). Seeds of Straight 8 cucumber variety were placed in a monolayer in a plastic box and covered with 5-12 volumes of water at room temperature (˜20 C). Seeds were washed for 4 hours in water with gentle agitation. Following the washing, seed were air dried on filter paper at ˜30 C for 12-24 hr. Cucumber DND1 dsRNAs (Table 32) were resuspended in 0.1 mM EDTA (diluted from a 0.5M pH8.0 stock solution) to 100 μg/ml. A 1:5(w/v) ratio of dsRNA solution to seed (e.g. for 1 gr of seed a 5 mL solution of dsRNA in EDTA) was used. Seeds were placed in a 50 mL conical tube and incubated in the dark at 15 C with gentle agitation for 24 hr. Following incubation, seeds were washed three times with gentle agitation for 1 minute each in a volume of water to completely fill the plastic container, and dried on filter paper before planting.
RNA analysis was conducted by Taqman real time PCR using a cucumber ELF1a assay for normalization of the values to correct for differences in sample concentration (see Table 32 for primers and Taqman probes sequences). Analysis on samples from fifteen days old cucumber leaves germinated from seeds treated with DND1 dsRNAs have demonstrated altered expression of the DND1 gene compared to control seeds treated with GFP dsRNA (see Table 32 for GFP dsRNA sequence and Table 33 and
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In another experiment, One hundred cucumber seeds were treated with dsRNA containing the cucumber DND1 transcribed sequence (Table 32, dsRNA #2 T33788) as described above. Treated seeds were plated on 1/4MS plates for 3 days. Ten ml dry sand was added to each glass vial and seedlings were planted by tilting the vial and laying the seedling in the correct orientation so that the cotyledons are just above the sand and then tilting back to cover the radicles with sand. 3.3 ml water was added to each vial and the vials placed in racks under fluorescent light banks. 250 vermiform eggs or 300 J2 RKN were inoculated in each tube in 50 ul of deionized or spring water. Plants were watered as needed. Harvest of the cucumber plants was performed at 11 days after inoculation by washing sand off the roots. A percent gall rating of the roots was taken for each sample, demonstrating a 26% and 21% reduction in RKN galling when compared to control treatments with 0.1 mM EDTA and GFP dsRNA, respectively (
PMR5 belongs to a family of plant-specific genes of unknown function.
Mutations in the Arabidopsis gene PMR5, powdery mildew resistance, have pectin enriched cell walls and confer powdery mildew resistance (Vogel et al. 2004).
Cucumber seeds were treated with PMR5 dsRNAs (Table 34) as described in Example 35. Leaves germinated from treated seeds were analyzed as described in Example 35. An altered expression of the PMR5 gene was demonstrated in PMR5 treated seeds compared to GFP treated seeds (Table 35 and
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TubG encodes a γ-tubulin protein (Snustad, D. P., et al. 1992). Cucumber seeds were treated with TubG dsRNAs (Table 36) as described in Example 35. Leaves germinated from treated seeds were analyzed as described in Example 35. An altered expression of the TubG gene was demonstrated in leaves of treated seeds compared to GFP treated seeds (Table 37 and
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Microtom variety tomato seeds were treated with DND1 dsRNAs (Table 38) as described in Example 35 (except for using 2 ml Eppendorf tube instead of a 50 ml Conical tube in the dsRNA incubation step). RNA analysis was conducted by Taqman real time PCR using a tomato TIP41 assay for normalization of the values to correct for differences in sample concentration (see Table 38 for primers and Taqman probes sequences). An altered expression of the DND1 gene was demonstrated in leaves of treated seeds compared to GFP treated seeds (Table 39 and
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Tomato seeds were treated with PMR5 dsRNAs (Table 40) as described in Example 35 and 39. Leaves germinated from treated seeds were analyzed as described in Example 38. An altered expression of the PMR5 gene was demonstrated in leaves of treated seeds compared to GFP treated seeds (Table 41 and
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MLO, mildew resistance locus O protein encodes a plant-specific membrane protein and been demonstrated to play a role in powdery mildew resistance in Arabidopsis, barley and tomato (Bai et. Al 2007). Tomato seeds were treated with MLO dsRNAs (Table 42) as described in Examples 35 and 38. Leaves germinated from treated seeds were analyzed as described in Example 38. An altered expression of the MLO gene was demonstrated in leaves of treated seeds compared to GFP treated seeds (Table 43 and
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Bi1 is a negative regulator of programmed cell death. Microtom tomato seeds were treated with dsRNAs containing the tomato Bi1 (Table 44, dsRNA #2) and PMR5 (Table 40, dsRNA #1) dsNAs as described Examples 35 and 38.
Seeds treated with dsRNA were planted in M200 soil with fertilizer (Hummerts) and placed in a growth chamber. One tomato seed is planted per pot, approximately ¼″ into the soil and the top of the soil was drenched with water. The growth chamber settings were at 26/24 C, 16 hr light cycle, 50% humidity, light intensity 3 and watered 4×/week by automated subirrigation. Fourteen days after planting pots were organized by similar size. The number of reps was 5 plants/treatment. On day 15 plants were randomized and shifted to the following growth chamber conditions: 22/20 C, 16 hr light cycle, 70% humidity, light intensity 3 and watered M,W,F. Infection with Tomato Powdery Mildew (Oidium neolycopersici) was as follows:
Eight days post-infection samples were taken for qPCR. PMR5 (Table 40, dsRNA #1) treated plants showed a significant up-regulation of PMRS expression compare to plants treated with GFP and 0.1 mM EDTA (Formulation) as controls (Table 45).
Fourteen days post-infection disease development was scored for the percentage of leaf area covered with powdery mildew. Ratings were set at 0,10,25 and 50% infection. Data was analyzed using Anova Single Factor Analysis (α=0.1). The ½ LSD was calculated and custom error bars were created for the graphs. Treatment averages are used to graph a percent disease reduction compared to the GFP control. This analysis demonstrated a 23% reduction in disease compared with GFP treated tomato seeds (p-value<0.1,
Soybean Phytochrome A (PHYA) genes E3 and E4 were targeted by treating seeds with dsRNAs containing sequences from those genes. PHYAE3 and PHYAE4 dsRNA sequences (Table 46) were synthesized in vitro using a convergent T7 RNA polymerase method. dsRNAs for the two genes were combined and brought to a final concentration of 50 μg/ml total nucleic acid in 0.1 mM EDTA. Fifteen soy (Williams 82) seeds were incubated in 7.5 ml of this dsRNA solution or in dsRNA from a GUS control in a 15 ml tube in the dark at 15 C, with gentle oscillation, for 24 hours. After treatment, seeds were transferred to seed germination boxes (15 seed per box) containing moistened filter paper and germinated in a growth chamber set at 25 C in the dark.
Shoot tissues, excluding cotyledons, were harvested from the germinating seeds in the germination boxes 5 days after treatment for RNA analysis. RNA analysis was conducted by Taqman, using the Gm.ref16 gene for normalization of the values to correct for differences in sample concentration (Table 46). RQ values were transformed to log 10 for analysis after outlier removal.
Treatment of seeds with PHYAE3 and PHYAE4 dsRNAs resulted in increased PHYAE3 expression in seedling shoots so that PHYAE3 mRNA levels were 1.7 those of GUS control values (Table 47 and
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations are apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application is specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
This application claims the benefit under 35 U.S.C. §119 of U.S. Provisional Application Nos. 61/651,131 filed May 24, 2012 (pending), 61/814,888 filed Apr. 23, 2013 (pending), 61/814,890 filed Apr. 23, 2013 (pending), 61/814,892 filed Apr. 23, 2013 (pending), 61/814,899 filed Apr. 23, 2013 (pending). Each of the foregoing applications is herein incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61651131 | May 2012 | US | |
| 61814888 | Apr 2013 | US | |
| 61814890 | Apr 2013 | US | |
| 61814892 | Apr 2013 | US | |
| 61814899 | Apr 2013 | US |
