Patent Application
20180023092
The present invention relates generally to disease resistance in plants.
Animals and plants rely on pattern recognition receptors (“PRRs”) to detect conserved microbe-associated molecular patterns (“MAMPs”) in potential pathogens (Zhang et al., “Plant Immunity Triggered by Microbial Molecular Signatures,” Mol Plant 3:783-793 (2010)). Two well understood peptide MAMPs, flg22 and flgII-28, are derived from flagellin, which forms the ‘tail’ bacteria use for motility (see FIG. 1 of Gomez-Gomez et al., “Flagellin Perception: A Paradigm for Innate Immunity,” Trends Plant Sci 7:251-256 (2002)). In humans, the Toll-like receptor 5 (TLRS) binds a flagellin-derived MAMP that involves a peptide region known as flgII-28 thereby activating innate immunity. In plants, the best-characterized PRR is FLS2 that binds flg22. FLS2, which is conserved in monocots and dicots, is a leucine-rich repeat (“LRR”) receptor-like kinase (“RLK”) that activates pattern-triggered immunity (“PTI”) (Gomez-Gomez et al., “FLS2: An LRR Receptor-like Kinase Involved in the Perception of the Bacterial Elicitor Flagellin in Arabidopsis,” Mol Cell 5:1003-1011 (2000)). Tomato and other species in the family Solanaceae, but not Arabidopsis and other plants, recognizes flgII-28 (Cai et al., “The Plant Pathogen Pseudomonas syringae pv. tomato is Genetically Monomorphic and Under Strong Selection to Evade Tomato Immunity,” PLoS Pathogens 7:e1002130 (2011)). This finding is significant because some pathogens have a divergent flg22 region that allows them to evade detection by FLS2 (Sun et al., “Within-species Flagellin Polymorphism in Xanthomonas campestris pv. campestris and Its Impact on Elicitation of Arabidopsis FLAGELLIN SENSING2-dependent Defenses,” Plant Cell 18:764-779 (2006), which is hereby incorporated by reference in its entirety). The existence of a PRR that recognizes flgII-28 may therefore allow tomato and other plant species to recognize a broader range of bacterial pathogens.
Importantly, PRRs can be transferred from one plant species to another and remain functional. For example, the Arabidopsis receptor EFR, which recognizes the bacterial MAMP elongation factor (“EF”) Tu, was transformed into tomato and N. benthamiana, which are normally unable to detect EF-Tu. (Lacombe et al., “Interfamily Transfer of a Plant Pattern-Recognition Receptor Confers Broad-spectrum Bacterial Resistance,” Nat Biotechnol 28:365-369 (2010)). It was shown that the EFR-expressing plants were resistant to previously virulent pathogens (Lacombe et al., “Interfamily Transfer of a Plant Pattern-Recognition Receptor Confers Broad-spectrum Bacterial Resistance,” Nat Biotechnol 28:365-369 (2010)). Other recent examples include the transfer of the ReMAX PRR from Arabidopsis to tobacco and the expression of the tomato Ve1 gene in Arabidopsis (Jehle et al., “The Receptor-Like Protein ReMAX of Arabidopsis Detects the Microbe-Associated Molecular Pattern eMax from Xanthomonas” Plant Cell 25(6):2330-2340 (2013) and Fradin et al., “Interfamily Transfer of Tomato Ve1 Mediates Verticillium Resistance in Arabidopsis” Plant Physiol 156(4): 2255-2265 (2011)). In both cases, the genes provided new recognition specificity.
Significant agricultural benefits may arise from the identification of PRRs that occur in only some plant species. Such species-specific PRRs can be used to broaden and enhance disease resistance when transferred into economically important and taxonomically diverse plants that do not naturally express them (see Lacombe et al., “Interfamily Transfer of a Plant Pattern-Recognition Receptor Confers Broad-spectrum Bacterial Resistance,” Nat Biotechnol 28:365-369 (2010)). Despite this potential, there are few cloned PRR genes available from plants and the majority of these are widely conserved, so they do not offer the possibility of interspecies transfer. Further, despite progress in understanding the genetic control of plant resistance to pathogens, little progress has been reported in the identification and analysis of key regulators of pathogen resistance. Characterization of such genes would allow for the genetic engineering of plants with a variety of desirable traits.
The present invention is directed to overcoming these and other deficiencies in the art.
One aspect of the present invention relates to a nucleic acid construct that includes a nucleic acid molecule that encodes a FLAGELLIN-SENSING 3 (“FLS3”) protein; a 5′ heterologous DNA promoter sequence; and a 3′ terminator sequence, where the nucleic acid molecule, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleic acid molecule.
Another aspect of the present invention relates to a method of expressing a nucleic acid molecule in a plant. This method involves providing a transgenic plant or plant seed transformed with a nucleic acid construct comprising a nucleic acid molecule that encodes an FLS3 protein; a 5′ heterologous DNA promoter sequence; and a 3′ terminator sequence, where the nucleic acid molecule, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleic acid molecule. The method also involves growing the transgenic plant or a plant grown from the transgenic plant seed under conditions effective to express the nucleic acid molecule in said transgenic plant or said plant grown from the transgenic plant seed.
Yet another aspect of the present invention relates to a method of imparting disease resistance to a plant. This method involves transforming a plant or a plant seed with a nucleic acid molecule that increases expression of an FLS3 protein, where said transforming is effective in imparting disease resistance to the transformed plant or to a transgenic plant produced from the transformed plant seed.
A further aspect of the present invention relates to a method of imparting disease resistance to a plant. The method involves providing a plant having a gene encoding an FLS3 protein, and applying to the plant and/or area of cultivation of the plant a flgII-28 peptide or an FLS3-binding portion thereof, thereby imparting disease resistance to the plant.
A further aspect of the present invention relates to a method of identifying a candidate plant suitable for breeding that displays enhanced disease resistance. This method involves providing a candidate plant; analyzing the candidate plant for the presence, in its genome, of a gene encoding an FLS3 protein; identifying, based on said analyzing, a candidate plant suitable for breeding that includes in its genome, a gene encoding an FLS3 protein; and breeding the identified plant with at least one other plant.
Yet a further aspect of the present invention relates to a method for enhancing efficiency of transformation of a plant by Agrobacterium. This method involves transforming a plant or a plant seed with a nucleic acid construct effective to silence expression of a nucleic acid molecule that encodes an FLS3 protein, where said transforming is effective to reduce or eliminate expression of FLS3 protein in the plant and said nucleic acid construct. The nucleic acid construct includes a nucleic acid molecule configured to silence FLS3 protein expression; a 5′ DNA promoter sequence; and a 3′ terminator sequence, where the nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit expression of the nucleic acid molecule.
Plants are able to detect the presence of foreign invading organisms through the perception of conserved MAMPs by cell surface PRRs. Extensive research has characterized the perception of the flagellin-derived MAMP flg22 by the PRR FLS2, and the subsequent intracellular signaling events which lead to induction of immune responses. Several other MAMPs have been isolated in recent years, but few plant PRR proteins have been characterized. Recently, an additional epitope from flagellin, termed flgII-28, was found to be detected specifically by tomato and other related solanaceous plants in an FLS2-independent manner (Clarke et al., “Allelic Variation in Two Distinct Pseudomonas syringae Flagellin Epitopes Modulates the Strength of Plant Immune Responses But Not Bacterial Motility,” New Phytol 200:847-860 (2013), which is hereby incorporated by reference in its entirety). As described herein, to identify the gene encoding the receptor responsible for this recognition, termed FLS3 (i.e., FLAGELLIN-SENSING 3, previously referred to as FlgII-28 Sensitivity 3 in U.S. Provisional Application No. 62/021,995), natural variation in tomato heirloom varieties and a mapping-by-sequencing approach was used. Expression of the wild type FLS3 gene in non-responsive plants confers recognition of flgII-28 and enhances immune responses. Photo-affinity labeling demonstrated specific binding of the MAMP by FLS3, indicating it is the flgII-28 receptor. FLS3 represents an orthogonal means for flagellin perception and therefore expression of this solanaceous-specific PRR in crop plants that are unable to detect flgII-28 could be deployed as a tactic to combat pathogens that have evolved to evade flg22 detection and offers a strategy of controlling bacterial diseases without the use of pesticides.
The importance of PRRs has been demonstrated recently when it was shown that they can be introduced into plants lacking such proteins to bolster disease resistance (Lacombe et al., “Interfamily Transfer of a Plant Pattern-Recognition Receptor Confers Broad-spectrum Bacterial Resistance,” Nat Biotechnol 28:365-369 (2010), which is hereby incorporated by reference in its entirety); however, the identification of plant immune receptors has proven difficult, particularly in crop species. Thus, the findings described herein mark an important and significant step in understanding plant immunity and modulating (e.g., enhancing) disease resistance in plants. These findings add to the molecular and genetic toolbox available for research in crop plants, and highlight advantages of using natural variation to increase understanding about plant immunity. Perception of an additional element of flagellin likely allows tomato to recognize a broader range of bacterial pathogens. Therefore, significant agricultural benefits may be achieved by the introduction of FLS3 into other plants (e.g., crop plants).
One aspect of the present invention relates to a nucleic acid construct that includes a nucleic acid molecule that encodes an FLS3 protein; a 5′ heterologous DNA promoter sequence; and a 3′ terminator sequence, where the nucleic acid molecule, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleic acid molecule.
The nucleic acid molecule may (i) include the nucleotide sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 9 and/or (ii) encode a polypeptide or protein having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10, as set forth below.
The coding sequence for FLS3 (Solyc04g009640, which is hereby incorporated by reference in its entirety) from Solanum lycopersicum cv. ‘Heinz1706’ (SEQ ID NO:1) is as follows:
The amino acid sequence for FLS3 from S. lycopersicum cv. ‘Heinz1706’ (SEQ ID NO:2) is as follows:
The coding sequence for FLS3 from S. pimpinellifolium LA1589 (SEQ ID NO:3) is as follows:
The amino acid sequence for FLS3 from S. pimpinellifolium LA1589 (SEQ ID NO:4) is as follows:
A conserved amino acid sequence for FLS3 from S. pimpinellifolium and S. lycopersicum cv. ‘Heinz 1706’ (SEQ ID NO:5) is as follows:
The coding sequence for FLS3 (CA05g03880, which is hereby incorporated by reference in its entirety) from pepper (Capsicum annum) (SEQ ID NO:7) is as follows:
The amino acid sequence for FLS3 from pepper (Capsicum annum) (SEQ ID NO:8) is as follows:
The coding sequence for FLS3 (Sotub04g009590 or PGSC0003DMT400041350, each of which is hereby incorporated by reference in its entirety) from potato (Solanum tuberosum group phureja DM1-3) (SEQ ID NO:9) is as follows:
The amino acid sequence for FLS3 from potato (Solanum tuberosum group phureja DM1-3) (SEQ ID NO:10) is as follows:
A conserved amino acid sequence for FLS3 from S. lycopersicum cv. ‘Heinz 1706’, S. pimpinellifolium, pepper, and potato (SEQ ID NO:11) (see
In one embodiment, the nucleic acid molecule may include the nucleotide sequence of SEQ ID NO:1 and/or encode a polypeptide or protein having the amino acid sequence of SEQ ID NO:2. In one embodiment, the nucleic acid molecule may include the nucleotide sequence of SEQ ID NO:3 and/or encode a polypeptide or protein having the amino acid sequence of SEQ ID NO:4. In another embodiment, the nucleic acid molecule may encode the conserved amino acid sequence of SEQ ID NO:5. In one embodiment, the nucleic acid molecule may include the nucleotide sequence of SEQ ID NO:7 and/or encode a polypeptide or protein having the amino acid sequence of SEQ ID NO:8. In one embodiment, the nucleic acid molecule may include the nucleotide sequence of SEQ ID NO:9 and/or encode a polypeptide or protein having the amino acid sequence of SEQ ID NO:10. In another embodiment, the nucleic acid molecule may encode the conserved amino acid sequence of SEQ ID NO:11.
While activity in particular polypeptide or protein sequences has been identified, variants of those polypeptides or proteins are also contemplated and may also be used as described herein (e.g., to impart or enhance disease resistance). In some embodiments, the polypeptides or proteins of the invention comprise one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid insertions, deletions, or modifications (e.g., substitution of one amino acid for another) compared to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, and/or SEQ ID NO:11 or are otherwise substantially identical (e.g., having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical) with the entire sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, and/or SEQ ID NO:11. It is contemplated that such variants retain the function of, for example, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, and/or SEQ ID NO:11 (e.g., in imparting or enhancing disease resistance and/or in binding to flgII-28). For example, polypeptides or proteins comprising or consisting of an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, or more) conservative amino acid substitutions relative to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, and/or SEQ ID NO:11, but retaining the function of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, and/or SEQ ID NO:11 (e.g., in imparting or enhancing disease resistance and/or in binding to flgII-28) are encompassed. Further, nucleic acid molecules encoding such variants of the peptides of the present invention are also contemplated. Such nucleic acid molecules may have, for example, a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical) with the entire sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, and/or SEQ ID NO:9.
The FLS3 nucleic acid molecules of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire FLS3 sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. “Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode for an FLS3 protein and which hybridize under stringent conditions to at least one of the FLS3 nucleic acid molecules disclosed herein, or to variants or fragments thereof, are encompassed by the present invention. Accordingly, the nucleic acid molecule and encoded protein according to the present invention may be an ortholog of FLS3 from S. lycopersicum cv. ‘Heinz1706’, from S. pimpinellifolium LA1589, from potato (S. tuberosum), and/or from pepper (Capsicum annum) referred to herein. Plants that are members of the Solanaceae family include, but not limited to, tomato, potato, pepper, tobacco, eggplant, tomatillo, and petunia.
Components of nucleic acid constructs according to the present invention may be heterologous. A polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it is synthetic or originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence (or vice versa) refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g., a genetically engineered coding sequence or an allele from a different ecotype or variety).
Methods of producing recombinant nucleic acids for purposes of, e.g., making transgenic plants are well-known. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., Cold Spring Harbor Press (1989), and Ausubel et al., Current Protocols in Molecular Biology, New York, N.Y., John Wiley & Sons (1989), which are hereby incorporated by reference in their entirety.
In preparing a nucleic acid vector for expression, the various nucleic acid sequences may normally be inserted or substituted into a bacterial plasmid. Any convenient plasmid may be employed, which will be characterized by having a bacterial replication system, a marker which allows for selection in a bacterium, and generally one or more unique, conveniently located restriction sites. Numerous plasmids, referred to as transformation vectors, are available for plant transformation. The selection of a vector will depend on the preferred transformation technique. A variety of vectors are available for stable transformation using Agrobacterium tumefaciens, a soilborne bacterium that causes crown gall. Crown gall are characterized by tumors or galls that develop on the lower stem and main roots of the infected plant. These tumors are due to the transfer and incorporation of part of the bacterium plasmid DNA into the plant chromosomal DNA. This transfer DNA (“T-DNA”) is expressed along with the normal genes of the plant cell. The plasmid DNA, pTi, or Ti-DNA, for “tumor inducing plasmid,” contains the vir genes necessary for movement of the T-DNA into the plant. The T-DNA carries genes that encode proteins involved in the biosynthesis of plant regulatory factors, and bacterial nutrients (opines). The T-DNA is delimited by two 25 bp imperfect direct repeat sequences called the “border sequences.” By removing the oncogene and opine genes, and replacing them with a gene of interest, it is possible to transfer foreign DNA into the plant without the formation of tumors or the multiplication of Agrobacterium tumefaciens (Fraley et al., “Expression of Bacterial Genes in Plant Cells,” Proc. Nat'l Acad. Sci. 80:4803-4807 (1983), which is hereby incorporated by reference in its entirety).
Further improvement of this technique led to the development of the binary vector system (Bevan, “Binary Agrobacterium Vectors for Plant Transformation,” Nucleic Acids Res. 12:8711-8721 (1984), which is hereby incorporated by reference in its entirety). In this system, all the T-DNA sequences (including the borders) are removed from the pTi, and a second vector containing T-DNA is introduced into Agrobacterium tumefaciens. This second vector has the advantage of being replicable in E. coli as well as A. tumefaciens, and contains a multiclonal site that facilitates the cloning of a transgene. An example of a commonly-used vector is pBin19 (Frisch et al., “Complete Sequence of the Binary Vector Bin19,” Plant Molec. Biol. 27:405-409 (1995), which is hereby incorporated by reference in its entirety). Any appropriate vectors now known or later described for genetic transformation are suitable for use with the present invention.
U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture.
Certain “control elements” or “regulatory sequences” are also incorporated into the vector-construct. These include non-translated regions of the vector, promoters, and 5′ and 3′ untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. Tissue-specific and organ-specific promoters can also be used.
A constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism. Examples of some constitutive promoters that are widely used for inducing expression of transgenes include the nopaline synthase (“NOS”) gene promoter, from Agrobacterium tumefaciens (U.S. Pat. No. 5,034,322 to Rogers et al., which is hereby incorporated by reference in its entirety), the cauliflower mosaic virus (CaMV) 35S and 19S promoters (U.S. Pat. No. 5,352,605 to Fraley et al., which is hereby incorporated by reference in its entirety), those derived from any of the several actin genes, which are known to be expressed in most cells types (U.S. Pat. No. 6,002,068 to Privalle et al., which is hereby incorporated by reference in its entirety), and the ubiquitin promoter, which is a gene product known to accumulate in many cell types.
An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed. The inducer can be a chemical agent, such as a metabolite, growth regulator, herbicide, or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, heat, salt, toxins, or through the action of a pathogen or disease agent such as a virus or fungus. A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating, or by exposure to the operative pathogen. An example of an appropriate inducible promoter is a glucocorticoid-inducible promoter (Schena et al., “A Steroid-Inducible Gene Expression System for Plant Cells,” Proc. Natl. Acad. Sci. 88:10421-5 (1991), which is hereby incorporated by reference in its entirety). Expression of the transgene-encoded protein is induced in the transformed plants when the transgenic plants are brought into contact with nanomolar concentrations of a glucocorticoid, or by contact with dexamethasone, a glucocorticoid analog (Schena et al., “A Steroid-Inducible Gene Expression System for Plant Cells,” Proc. Natl. Acad. Sci. USA 88:10421-5 (1991); Aoyama et al., “A Glucocorticoid-Mediated Transcriptional Induction System in Transgenic Plants,” Plant J. 11:605-612 (1997); and McNellis et al., “Glucocorticoid-Inducible Expression of a Bacterial Avirulence Gene in Transgenic Arabidopsis Induces Hypersensitive Cell Death,” Plant J. 14(2):247-57 (1998), which are hereby incorporated by reference in their entirety). In addition, inducible promoters include promoters that function in a tissue specific manner to regulate the gene of interest within selected tissues of the plant. Examples of such tissue specific or developmentally regulated promoters include seed, flower, fruit, or root specific promoters as are well known by those of ordinary skill in the art (U.S. Pat. No. 5,750,385 to Shewmaker et al., which is hereby incorporated by reference in its entirety).
A number of tissue- and organ-specific promoters have been developed for use in genetic engineering of plants (Potenza et al., “Targeting Transgene Expression in Research, Agricultural, and Environmental Applications: Promoters Used in Plant Transformation,” In Vitro Cell. Dev. Biol. Plant 40:1-22 (2004), which is hereby incorporated by reference in its entirety). Examples of such promoters include those that are floral-specific (Annadana et al., “Cloning of the Chrysanthemum UEP1 Promoter and Comparative Expression in Florets and Leaves of Dendranthema grandiflora,” Transgenic Res. 11:437-445 (2002), which is hereby incorporated by reference in its entirety), seed-specific (Kluth et al., “5′ Deletion of a gbss1 Promoter Region Leads to Changes in Tissue and Developmental Specificities,” Plant Mol. Biol. 49:669-682 (2002), which is hereby incorporated by reference in its entirety), root-specific (Yamamoto et al., “Characterization of cis-acting Sequences Regulating Root-Specific Gene Expression in Tobacco,” Plant Cell 3:371-382 (1991), which is hereby incorporated by reference in its entirety), fruit-specific (Fraser et al., “Evaluation of Transgenic Tomato Plants Expressing an Additional Phytoene Synthase in a Fruit-Specific Manner,” Proc. Natl. Acad. Sci. USA 99:1092-1097 (2002), which is hereby incorporated by reference in its entirety), and tuber/storage organ-specific (Visser et al., “Expression of a Chimeric Granule-Bound Starch Synthase-GUS Gene in Transgenic Potato Plants,” Plant Mol. Biol. 17:691-699 (1991), which is hereby incorporated by reference in its entirety). Targeted expression of an introduced gene (transgene) is necessary when expression of the transgene could have detrimental effects if expressed throughout the plant. On the other hand, silencing a gene throughout a plant could also have negative effects. However, this problem could be avoided by localizing the silencing to a region by a tissue-specific promoter.
Nucleic acid constructs of the present invention include an operable 3′ regulatory region, selected from among those which are capable of providing correct transcription termination and polyadenylation of mRNA for expression in the host cell of choice, operably linked to a nucleic acid molecule configured to silence BBTV. A number of 3′ regulatory regions are known to be operable in plants. Exemplary 3′ regulatory regions include, without limitation, the nopaline synthase (“nos”) 3′ regulatory region (Fraley et al., “Expression of Bacterial Genes in Plant Cells,” Proc. Nat'l Acad. Sci. USA 80:4803-4807 (1983), which is hereby incorporated by reference in its entirety) and the cauliflower mosaic virus (“CaMV”) 3′ regulatory region (Odell et al., “Identification of DNA Sequences Required for Activity of the Cauliflower Mosaic Virus 35S Promoter,” Nature 313(6005):810-812 (1985), which is hereby incorporated by reference in its entirety). Virtually any 3′ regulatory region known to be operable in plants would be suitable for use in conjunction with the present invention.
The different components described supra can be ligated together to produce the expression systems which contain the nucleic acid constructs of the present invention, using well known molecular cloning techniques as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition Cold Spring Harbor, N.Y., Cold Spring Harbor Press (1989), and Ausubel et al. Current Protocols in Molecular Biology, New York, N.Y., John Wiley & Sons (1989), which are hereby incorporated by reference in their entirety.
Once the nucleic acid construct of the present invention has been prepared, it is ready to be incorporated into a host cell. Accordingly, another aspect of the present invention relates to a recombinant host cell containing one or more of the nucleic acid constructs of the present invention. Basically, this method is carried out by transforming a host cell with a nucleic acid construct of the present invention under conditions effective to achieve transcription of the nucleic acid molecule in the host cell. This is achieved with standard cloning procedures known in the art, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. Preferably the host cells are either a bacterial cell or a plant cell. Methods of transformation may result in transient or stable expression of the nucleic acid under control of the promoter. Preferably, a nucleic acid construct of the present invention is stably inserted into the genome of the recombinant plant cell as a result of the transformation, although transient expression can serve an important purpose.
Plant tissue suitable for transformation includes leaf tissue, root tissue, meristems, zygotic and somatic embryos, callus, protoplasts, tassels, pollen, embryos, anthers, and the like. The means of transformation chosen is that most suited to the tissue to be transformed.
Transient expression in plant tissue can be achieved by particle bombardment (Klein et al., “High-Velocity Microprojectiles for Delivering Nucleic Acids Into Living Cells,” Nature 327:70-73 (1987), which is hereby incorporated by reference in its entirety), also known as biolistic transformation of the host cell, as disclosed in U.S. Pat. Nos. 4,945,050; 5,036,006; and 5,100,792, all to Sanford et al., and in Emerschad et al., “Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera),” Plant Cell Reports 14:6-12 (1995), which are hereby incorporated by reference in their entirety.
In particle bombardment, tungsten or gold microparticles (1 to 2 μm in diameter) are coated with the DNA of interest and then bombarded at the tissue using high pressure gas. In this way, it is possible to deliver foreign DNA into the nucleus and obtain a temporal expression of the gene under the current conditions of the tissue. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells. Other variations of particle bombardment, now known or hereafter developed, can also be used.
An appropriate method of stably introducing the nucleic acid construct into plant cells is to infect a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with the nucleic acid construct of the present invention. As described supra, the Ti (or RI) plasmid of Agrobacterium enables the highly successful transfer of a foreign nucleic acid molecule into plant cells. A variation of Agrobacterium transformation uses vacuum infiltration in which whole plants are used (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety).
Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies (Fraley et al., “Liposome-mediated Delivery of Tobacco Mosaic Virus RNA Into Tobacco Protoplasts: A Sensitive Assay for Monitoring Liposome-protoplast Interactions,” Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), which is hereby incorporated by reference in its entirety). The nucleic acid molecule may also be introduced into the plant cells by electroporation (Fromm et al., “Expression of Genes Transferred into Monocot and Dicot Plant Cells by Electroporation,” Proc. Natl. Acad. Sci. USA 82:5824 (1985), which is hereby incorporated by reference in its entirety). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate. Other methods of transformation include polyethylene-mediated plant transformation, micro-injection, physical abrasives, and laser beams (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety). The precise method of transformation is not critical to the practice of the present invention. Any method that results in efficient transformation of the host cell of choice is appropriate for practicing the present invention.
Yet a further method for introduction is by use of known techniques for genome editing or alteration. Such techniques for targeted genomic insertion involve, for example, inducing a double stranded DNA break precisely at one or more targeted genetic loci followed by integration of a chosen transgene or nucleic acid molecule (or construct) during repair. Such techniques or systems include, for example, zinc finger nucleases (“ZFNs”) (Urnov et al., “Genome Editing with Engineered Zinc Finger Nucleases,” Nat Rev Genet. 11: 636-646 (2010), which is hereby incorporated by reference in its entirety), transcription activator-like effector nucleases (“TALENs”) (Joung & Sander, “TALENs: A Widely Applicable Technology for Targeted Genome Editing,” Nat Rev Mol Cell Biol. 14: 49-55 (2013), which is hereby incorporated by reference in its entirety), clustered regularly interspaced short palindromic repeat (“CRISPR”)-associated endonucleases (e.g., CRISPR/CRISPR-associated (“Cas”) 9 systems) (Wiedenheft et al., “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nat 482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems,” Science 339(6121): 819-23 (2013); and Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based Methods for Genome Engineering,” Cell 31(7):397-405 (2013), each of which is hereby incorporated by reference in its entirety).
After transformation, the transformed plant cells must be regenerated. Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1, New York, N.Y., MacMillan Publishing Co. (1983); Vasil, ed., Cell Culture and Somatic Cell Genetics of Plants, Vol. I (1984) and Vol. III (1986), Orlando, Acad. Press; and Fitch et al., “Somatic Embryogenesis and Plant Regeneration from Immature Zygotic Embryos of Papaya (Carica papaya L.),” Plant Cell Rep. 9:320 (1990), which are hereby incorporated by reference in their entirety.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
Preferably, transformed cells are first identified using a selection marker simultaneously introduced into the host cells along with the nucleic acid construct of the present invention. Suitable selection markers include, without limitation, markers encoding for antibiotic resistance, such as the neomycin phosphotransferae II (“nptII”) gene which confers kanamycin resistance (Fraley et al., “Expression of Bacterial Genes in Plant Cells,” Proc. Natl. Acad. Sci. USA 80:4803-4807 (1983), which is hereby incorporated by reference in its entirety), and the genes which confer resistance to gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like. Cells or tissues are grown on a selection medium containing the appropriate antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow. Other types of markers are also suitable for inclusion in the expression cassette of the present invention. For example, a gene encoding for herbicide tolerance, such as tolerance to sulfonylurea is useful, or the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104 (1983), which is hereby incorporated by reference in its entirety). Similarly, “reporter genes,” which encode for enzymes providing for production of an identifiable compound are suitable. The most widely used reporter gene for gene fusion experiments has been uidA, a gene from Escherichia coli that encodes the β-glucuronidase protein, also known as GUS (Jefferson et al., “GUS Fusions: β Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plants,” EMBO J. 6:3901-3907 (1987), which is hereby incorporated by reference in its entirety). Similarly, enzymes providing for production of a compound identifiable by luminescence, such as luciferase, are useful. The selection marker employed will depend on the target species; for certain target species, different antibiotics, herbicide, or biosynthesis selection markers are preferred.
Plant cells and tissues selected by means of an inhibitory agent or other selection marker are then tested for the acquisition of the transgene (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., Cold Spring Harbor Press (1989), which is hereby incorporated by reference in its entirety).
In one embodiment, the transgenic plant is transformed with a bacterial artificial chromosome (“BAC”). A BAC is a cloning vector derived from the naturally occurring F factor of Escherichia coli. BACs can accept large inserts of a DNA sequence. In maize, a number of BACs, each containing a large insert of maize genomic DNA, have been assembled into contigs (overlapping contiguous genetic fragments, or “contiguous DNA”). BACs have a propensity for coming together to form contiguous stretches of DNA. A BAC “assembles” to a contig based on sequence alignment, if the BAC is sequenced, or via the alignment of its BAC fingerprint to the fingerprints of other BACs. The assemblies can be found using the Maize Genome Browser, which is publicly available on the internet.
Accordingly, one aspect of the present invention relates to a plant or plant seed transformed with one or more nucleic acid constructs described herein. The present invention also encompasses the whole plant, or a component part of a plant, including shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same.
Suitable plants for use in accordance with the present invention include both monocots and dicots. Suitable plants for use in accordance with the present invention also include both crop plants and ornamentals. For example, suitable plants include rice, corn, soybean, canola, potato, wheat, mung bean, alfalfa, barley, rye, cotton, sunflower, peanut, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, tobacco, tomato, sorghum, sugarcane, banana, Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, crocus, marigold, daffodil, pine, Medicago truncatula, Sandersonia aurantiaca, and zinnia.
Another aspect of the present invention relates to a method of expressing a nucleic acid molecule in a plant. This method involves providing a transgenic plant or plant seed transformed with a nucleic acid construct comprising a nucleic acid molecule that encodes an FLS3 protein; a 5′ heterologous DNA promoter sequence; and a 3′ terminator sequence, where the nucleic acid molecule, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleic acid molecule. The method also involves growing the transgenic plant or a plant grown from the transgenic plant seed under conditions effective to express the nucleic acid molecule in said transgenic plant or said plant grown from the transgenic plant seed.
Suitable nucleic acid molecules are described above.
In one embodiment a transgenic plant is provided. In another embodiment, a transgenic plant seed is provided.
In one embodiment, providing a transgenic plant or plant seed involves transforming a non-transgenic plant or a non-transgenic plant seed with the nucleic acid construct to yield the transgenic plant or plant seed. Transformation is described above and may include Agrobacterium-mediated transformation, whisker method transformation, vacuum infiltration, biolistic transformation, electroporation, micro-injection, polyethylene-mediated transformation, or laser-beam transformation.
Yet another aspect of the present invention relates to a method of imparting disease resistance to a plant. This method involves transforming a plant or a plant seed with a nucleic acid molecule that increases expression of an FLS3 protein, where said transforming is effective in imparting disease resistance to the transformed plant or to a transgenic plant produced from the transformed plant seed.
In one embodiment, a plant is transformed. In another embodiment, a plant seed is transformed and the method also involves planting the transformed plant seed under conditions effective for a plant to grow from the planted plant seed. Suitable nucleic acid molecules and are described above.
Imparting disease resistance or enhancing disease resistance refers to an increase in the ability of a plant to prevent pathogen infection or pathogen-induced symptoms. Disease resistance may be increased compared to a control plant (for example, an unmodified or non-transgenic plant). In one embodiment, the level of resistance in a non-naturally occurring transgenic plant of the invention is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% greater than the resistance exhibited by a control plant. The level of resistance is measured using conventional methods. For example, the level of resistance to a pathogen may be determined by comparing physical features and characteristics (for example, plant height and weight) or by comparing disease symptoms (for example, delayed lesion development, reduced lesion size, leaf wilting and curling, water-soaked spots, amount of pathogen growth, and discoloration of cells) of the non-naturally occurring plant (e.g., a transgenic plant).
Disease resistance can be increased resistance relative to a particular pathogen species or genus or can be increased resistance to a broad range of pathogens (e.g., pattern-triggered immunity, systemic acquired resistance). In one embodiment, the pathogen is a bacterial plant pathogen. By way of example, bacterial pathogens may belong to Acidovorax, Agrobacterium, Burkholderia, Candidatus Liberibacter, Clavibacter, Curtobacterium, Dickeya, Erwinia, Pantoea, Pectobacterium, Phytoplasma, Pseudomonas, Ralstonia, Spiroplasma, Streptomyces, Xanthomonas, and Xylella. See P
A further aspect of the present invention relates to a method of imparting disease resistance to a plant. The method involves providing a plant having a gene encoding an FLS3 protein, and applying to the plant and/or area of cultivation of the plant a flgII-28 peptide or an FLS3-binding portion thereof, thereby imparting disease resistance to the plant.
In one embodiment, the gene is a transgene. In one embodiment, providing the plant includes transforming a plant or a plant seed with a nucleic acid molecule that increases expression of an FLS3 protein. Suitable nucleic acid molecules are described above.
In one embodiment, the flgII-28 peptide comprises the amino acid sequence of SEQ ID NO:12, as follows: ESTNILQRMREL[A/V]VQ[S/F]RNDSNSATDR[E/D]A (Cai et al., “The Plant Pathogen Pseudomonas syringae pv. tomato is Genetically Monomorphic and Under Strong Selection to Evade Tomato Immunity,” PLoS Pathog. 7(8):e1002130 (2011) and Clarke et al., “Allelic Variation in Two Distinct Pseudomonas syringae Flagellin Epitopes Modulates the Strength of Plant Immune Responses But Not Bacterial Motility,” New Phytol 200:847-860 (2013), each of which is hereby incorporated by reference in its entirety). In another embodiment, the flgII-28 peptide comprises the amino acid sequence of SEQ ID NO:13, as follows: ES[T/V][N/S]ILQRMRELAVQSRNDSNS[A/S][T/E][D/G]R[E/D]A (Clarke et al., “Allelic Variation in Two Distinct Pseudomonas syringae Flagellin Epitopes Modulates the Strength of Plant Immune Responses But Not Bacterial Motility,” New Phytol 200:847-860 (2013), which is hereby incorporated by reference in its entirety). In another embodiment, the flgII-28 peptide comprises the amino acid sequence of SEQ ID NO:14, as follows: EIGSNLQRIRELSVQSSNATNSASDRDA. In another embodiment, the flgII-28 peptide comprises the amino acid sequence of SEQ ID NO:15, as follows: EINNNLQRVRELAVQSANSTNSQSDLDS (Meng et al., “Salmonella colonization activates the plant immune system and benefits from association with plant pathogenic bacteria,” Environ Microbiol 15(9):2418-2430 (2013), which is hereby incorporated by reference in its entirety). Also contemplated are variants of SEQ ID NOs:12-15, which retain the function of binding FLS3 and/or causing pattern-triggered immunity in a plant. In some embodiments, such peptides comprise one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid insertions, deletions, or modifications (e.g., substitution of one amino acid for another) compared to SEQ ID NOs:12-15 or are otherwise substantially identical (e.g., having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical) with the entire sequence of SEQ ID NOs:12-15. Further, nucleic acid molecules encoding such variants of the peptides of SEQ ID NOs:12-15 are also contemplated. Such nucleic acid molecules may have, for example, a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical) with the entire sequence of SEQ ID NOs:12-15. The flgII-28 peptide or variant as described herein may include a heterologous portion (e.g., fusion protein or peptide). For example, a fusion protein may include two or more flgII-28 peptides or variants optionally joined by a linker. A fusion protein may include one or more flgII-28 peptides or variants joined (optionally with a linker) to a further peptide capable of, e.g., eliciting PTI and/or hypersensitive response in a plant.
FlgII-28 peptides or fragments or variants thereof may be formulated (either together or separately) in a manner common for agrochemical formulations. Agricultural formulations of active substances are well known. Non-limiting examples include solutions, emulsions, suspensions, dusts, powders, pastes, and granules. The particular formulation chosen may vary depending on the particular intended application. In each case, it is typically advantageous to ensure a fine and even distribution of the active ingredient(s) in a liquid or solid carrier. Formulation methods are taught, e.g., in U.S. Pat. No. 3,060,084 to Littler and European Patent No. 0707445 to BASF AG (for liquid concentrates); Browning, “Agglomeration,” Chemical Engineering pp. 147-48 (1967); Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963; PCT Publication No. WO 91/13546 to E.I. Du Pont De Nemours and Co.; U.S. Pat. No. 4,172,714 to Albert; U.S. Pat. No. 4,144,050 to Frensch et al; U.S. Pat. No. 3,920,442 to Albert; U.S. Pat. No. 5,180,587 to Moore; U.S. Pat. No. 5,232,701 to Ogawa et al; U.S. Pat. No. 5,208,030 to Hoy et al, Great Britain Patent No. 2,095,558; U.S. Pat. No. 3,299,566 to Macmullen; Klingman, Weed Control as a Science, J. Wiley & Sons, New York, 1961; Hance et al, Weed Control Handbook, 8th Ed., Blackwell Scientific, Oxford, 1989; and Mollet and Grubemann, Formulation Technology, Wiley VCH Verlag, Weinheim, 2001, each of which is hereby incorporated by reference in its entirety.
The area of cultivation is any type of environment, soil, area, or material where the plant is growing or intended to grow. The composition comprising the flgII-28 peptide or fragment or variant thereof may be applied in the form of directly sprayable solutions, powders, suspensions, dispersions, emulsions, oil dispersions, pastes, dustable products, materials for spreading, or granules, by means of spraying, atomizing, dusting, spreading, brushing, immersing, or pouring. The application form depends on the intended purpose to ensure in each case the finest possible distribution of the active compound(s).
A further aspect of the present invention relates to a method of identifying a candidate plant suitable for breeding that displays enhanced disease resistance. This method involves providing a candidate plant; analyzing the candidate plant for the presence, in its genome, of a gene encoding an FLS3 protein; identifying, based on said analyzing, a candidate plant suitable for breeding that includes in its genome, a gene encoding an FLS3 protein; and breeding the identified plant with at least one other plant.
In one embodiment, analyzing the candidate plant for the presence, in its genome, of a gene encoding an FLS3 protein involves isolating genomic DNA from the plant, germplasm, pollen, or seed of the plant; analyzing genomic DNA from the plant, germplasm, pollen, or seed of the plant for the presence of the gene encoding the FLS3 protein; and detecting the gene encoding the FLS3 protein.
In one embodiment, the breeding involves crossing, making hybrids, backcrossing, self-crossing, double haploid breeding, and/or combinations thereof.
In one embodiment, a transgenic plant transformed with a nucleic acid molecule that encodes an FLS3 protein is provided as the candidate plant. In one embodiment, providing the transgenic plant involves transforming a plant or plant seed with a nucleic acid construct according to the present invention and growing the transgenic plant or a plant grown from the transgenic plant seed under conditions effective to express the nucleic acid molecule in the transgenic plant or said plant grown from the transgenic plant seed.
Yet a further aspect of the present invention relates to a method for enhancing efficiency of transformation of a plant by Agrobacterium. This method involves transforming a plant or a plant seed with a nucleic acid construct effective to silence expression of a nucleic acid molecule that encodes an FLS3 protein, where said transforming is effective to reduce or eliminate expression of FLS3 protein in the plant and said nucleic acid construct. The nucleic acid construct includes a nucleic acid molecule configured to silence FLS3 protein expression; a 5′ DNA promoter sequence; and a 3′ terminator sequence, where the nucleic acid molecule, the promoter, and the terminator are operatively coupled to permit expression of the nucleic acid molecule.
In one embodiment, this method involves simultaneous or sequential exposure of the plant cell to an Agrobacterium strain that transforms a plant variety with a nucleic acid molecule of interest.
In one embodiment, the bacterium is A. tumefaciens having a functional type IV secretion system.
The FLS3 protein may comprise the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO:10, or SEQ ID NO:11 and/or may be encoded by the nucleotide sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO:7, or SEQ ID NO: 9.
In one embodiment, a plant is transformed. In another embodiment according to this aspect of the claimed invention, a plant seed is transformed and the method also involves planting the transformed plant seed under conditions effective for a plant to grow from the planted plant seed.
In another embodiment, the nucleic acid molecule is positioned in the nucleic acid construct to result in suppression or interference of endogenous mRNA encoding the FLS3 protein.
In one aspect of the present invention, the nucleic acid construct results in suppression or interference of FLS3 protein expression by the nucleic acid molecule of the construct containing a dominant negative mutation and encoding a non-functional FLS3 protein. Examples of such mutations are described with respect to
In another aspect of the present invention, the nucleic acid construct results in interference of FLS3 protein expression by sense or co-suppression in which the nucleic acid molecule of the construct (e.g., that encoding FLS3 or a fragment thereof) is in a sense (5′→3′) orientation. Co-suppression has been observed and reported in many plant species and may be subject to a transgene dosage effect or, in another model, an interaction of endogenous and transgene transcripts that results in aberrant mRNAs (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998); Waterhouse et al., “Exploring Plant Genomes by RNA-Induced Gene Silencing,” Nature Review: Genetics 4: 29-38 (2003), which are hereby incorporated by reference in their entirety). A construct with the nucleic acid molecule (or fragment thereof) in the sense orientation may also give sequence specificity to RNA silencing when inserted into a vector along with a construct of both sense and antisense nucleic acid orientations as described infra (Wesley et al., “Construct Design for Efficient, Effective and High-Throughput Gene Silencing in Plants,” Plant Journal 27(6) 581-590 (2001), which is hereby incorporated by reference in its entirety).
In another embodiment of the present invention, the nucleic acid construct results in interference of FLS3 expression by the use of antisense suppression in which the nucleic acid molecule of the construct (e.g., that encoding FLS3 or a fragment thereof) is an antisense (3′→5′) orientation. The use of antisense RNA to down-regulate the expression of specific plant genes is well known (van der Krol et al., Nature, 333:866-869 (1988) and Smith et al., Nature, 334:724-726 (1988), which are hereby incorporated by reference in their entirety). Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, “Antisense RNA and DNA,” Scientific American 262:40 (1990), which is hereby incorporated by reference in its entirety). In the target cell, the antisense nucleic acids hybridize to a target nucleic acid and interfere with transcription, and/or RNA processing, transport, translation, and/or stability. The overall effect of such interference with the target nucleic acid function is the disruption of protein expression (Baulcombe, “Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,” Plant Cell 8:1833-44 (1996); Dougherty, et al., “Transgenes and Gene Suppression: Telling us Something New?,” Current Opinion in Cell Biology 7:399-05 (1995); Lomonossoff, “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Phytopathol. 33:323-43 (1995), which are hereby incorporated by reference in their entirety). Accordingly, one aspect of the present invention involves a nucleic acid construct which contains an antisense nucleic acid molecule to a nucleic acid molecule encoding an FLS3 protein (or fragment thereof).
Interference of FLS3 expression is also achieved in the present invention by the generation of double-stranded RNA (“dsRNA”) through the use of inverted-repeats, segments of gene-specific sequences oriented in both sense and antisense orientations. In one embodiment of this aspect of the present invention, sequences in the sense and antisense orientations are linked by a third segment, and inserted into a suitable expression vector having the appropriate 5′ and 3′ regulatory nucleotide sequences operably linked for transcription. The expression vector having the modified nucleic acid molecule is then inserted into a suitable host cell or subject. In the present invention, the third segment linking the two segments of sense and antisense orientation may be any nucleotide sequence such as a fragment of the β-glucuronidase (“GUS”) gene. In another embodiment of this aspect of the present invention, a functional (splicing) intron of the FLS3 gene may be used for the third (linking) segment, or, in yet another aspect of the present invention, other nucleotide sequences without complementary components in the FLS3 gene may be used to link the two segments of sense and antisense orientation (Chuang et al., “Specific and Heritable Genetic Interference by Double-Stranded RNA in Arabidopsis thaliana,” Proc. Nat'l Academy of Sciences USA 97(9):4985-4990 (2000); Smith et al., “Total Silencing by Intron-Spliced Hairpin RNAs,” Nature 407:319-320 (2000); Waterhouse et al., “Exploring Plant Genomes by RNA-Induced Gene Silencing,” Nature Review: Genetics 4:29-38 (2003); Wesley et al., “Construct Design for Efficient, Effective and High-Throughput Gene Silencing in Plants,” Plant Journal 27(6):581-590 (2001), which are hereby incorporated by reference in their entirety). In any of the embodiments with inverted repeats of FLS3, the sense and antisense segments may be oriented either head-to-head or tail-to-tail in the construct.
Another aspect of the present invention involves using hairpin RNA (“hpRNA”) which may also be characterized as dsRNA. This involves RNA hybridizing with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem. Though a linker may be used between the inverted repeat segments of sense and antisense sequences to generate hairpin or double-stranded RNA, the use of intron-free hpRNA can also be used to achieve silencing of FLS3 expression.
Alternatively, in another aspect of the present invention, a plant may be transformed with constructs encoding both sense and antisense orientation molecules having separate promoters and no third segment linking the sense and antisense sequences (Chuang et al., “Specific and Heritable Genetic Interference by Double-Stranded RNA in Arabidopsis thaliana,” Proc. Nat'l Academy of Sciences USA 97(9):4985-4990 (2000); Waterhouse et al., “Exploring Plant Genomes by RNA-Induced Gene Silencing,” Nat Rev Genet. 4:29-38 (2003); Wesley et al., “Construct Design for Efficient, Effective and High-Throughput Gene Silencing in Plants,” Plant Journal 27(6):581-590 (2001), which are hereby incorporated by reference in their entirety).
Altering expression (e.g., inhibition of, or interference with, endogenous expression) of FLS3 can also be accomplished using known techniques for targeted alteration of genes, such as zinc finger nucleases (“ZFNs”) (Urnov et al., “Genome Editing with Engineered Zinc Finger Nucleases,” Nat Rev Genet. 11: 636-646 (2010), which is hereby incorporated by reference in its entirety), transcription activator-like effector nucleases (“TALENs”) (Joung & Sander, “TALENs: A Widely Applicable Technology for Targeted Genome Editing,” Nat Rev Mol Cell Biol. 14: 49-55 (2013), which is hereby incorporated by reference in its entirety), clustered regularly interspaced short palindromic repeat (“CRISPR”)-associated endonucleases (e.g., CRISPR/CRISPR-associated (“Cas”) 9 systems) (Wiedenheft et al., “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nat 482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems,” Science 339(6121): 819-23 (2013); and Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based Methods for Genome Engineering,” Cell 31(7):397-405 (2013), each of which is hereby incorporated by reference in its entirety).
The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
Materials:
Induction media (0.05 M MES, 0.5% D-glucose, 0.025% NaH2PO4, 0.1% NH4Cl, 0.03% MgSO4 7H2O, 0.015% KCl, 0.00025% FeSO4 7H2O, 0.01% CaCl2 2H2O, pH 5.6); infiltration media (0.01 M MES, 0.01 M MgCl2, pH 5.6); 200 mM acetosyringone (19.6 mg in 0.5 mL DMSO).
Procedure:
The Agrobacterium strains GV2260 or GV3101 containing constructs expressing genes of interest were streaked on Lysogeny Broth (LB) solid media with antibiotics and grown for 2 days at 30° C. Bacteria were suspended in induction media supplemented with antibiotics and 200 μM acetosyringone and incubated at room temperature with shaking for 5-6 hours. Bacterial cultures were washed twice with infiltration media and the bacterial pellet was resuspended in infiltration media. The OD600 of the culture at 1:100 dilution was measured and a cell suspension made of the appropriate (i.e., 0.1-0.2) OD600 in infiltration media supplemented with 200 μM acetosyringone. All constructs were mixed at equal concentrations with Agrobacterium containing a construct expressing the viral suppressor of silencing p19. Fully expanded leaves of Nicotiana benthamiana or tomato were used for infiltration. First, a hole was made in the leaf using a needle, and then the leaf was infiltrated with the solution containing the Agrobacterium using a needle-less syringe. Plants were kept for 24 hours before collecting samples for ROS analysis (see infra Example 3), or for 48 hours before collecting samples for immunoblot analysis (see infra Example 4).
Materials:
LB solid media supplemented with antibiotics; infiltration buffer (10 mM MES, 10 mM MgCl2, pH 5.5; 200 mM acetosyringone (0.039 g dissolved in 1 mL DMSO).
Procedure:
The Agrobacterium strains GV2260 or GV3101 containing constructs expressing genes of interest were streaked on Lysogeny Broth (LB) solid media with antibiotics and grown for 2 days at 30° C. Bacteria were suspended in LB liquid media supplemented with antibiotics and 200 μM acetosyringone and incubated at 30° C. overnight with shaking. Bacterial cultures were washed twice with infiltration media and the bacterial pellet was suspended in infiltration media. The OD600 of the culture at 1:100 dilution was measured and a cell suspension made of the appropriate (i.e., 0.1-0.2) OD600 in infiltration media supplemented with 200 μM acetosyringone. The two constructs (i.e., TRV1 and TRV2 with the gene of interest) were mixed at equal concentrations. Leaves of 3-week-old Nicotiana benthamiana plants were used for infiltration. First, a hole was made in the leaf using a needle, then the leaf was infiltrated with the solution containing the Agrobacterium using a needle-less syringe. Plants were kept for 4-5 weeks before use.
Materials:
Luminol (17 mg/mL in DMSO); horseradish peroxidase type VI (10 mg/mL in H2O; cork borer #1 or 2; 96-well microplates
Procedure:
Leaf disks were obtained using the cork borer, and disks were floated on water in 96-well microplates, adaxial (upper) side facing up. After overnight incubation, the water was removed and replaced with 100 μL per well ROS assay solution (34 μg/mL luminol, 20 μg/mL horseradish peroxidase, with an appropriate concentration of the MAMP of interest). The resulting luminescence was measured using a plate reader (i.e., Biotek Synergy 2 with Gen5 1.10 software). The luminescence was measured over 45 minutes with a 27 millisecond interval for a total of 101 reads.
Procedure: Approximately 25 grams of Nicotiana benthamiana tissue overexpressing FLS3 with a c-terminal GFP tag fusion was ground in a mortar with a pestle in liquid nitrogen. 200 mL of extraction buffer (50 mM MOPS-KOH, pH7.5, 0.5 M sorbitol, 5 mM DTT, 5 mM EDTA, pH8.0, 1% PVPP, 1 mM PMSF) was then added to the ground tissue and the extract was filtered through Miracloth. The extract was centrifuged in 40 mL round-bottom tubes at 10,500 rpm for 25 minutes at 4° C. 40 mL of supernatant was transferred to each of 6 open-top tubes and centrifuged for 75 minutes at 23,600 rpm (100,000×g) at 4° C. Microsomal membranes were suspended in 10 mL of suspension buffer (25 mM Tris-HCl, pH7.5, 0.25 M sucrose, 10 mM potassium phosphate, pH7.5, 28.8 mM NaCl). Samples were then sonicated for 2×30 seconds at half power with samples kept in an ice bath. 9 mL of suspended microsomal membranes was then added in 27 g phase mixture (6% w/w dextran T-500, 6% w/w PEG 3350, 0.25 M sucrose, 10 mM potassium phosphate, pH7.5, 28.8 mM NaCl) and mixed by inverting 20 times. The mixture was then centrifuged at 1,000 rpm for 5 minutes at 4° C. and then the upper layer was transferred to a fresh tube containing 10 mL of dextran phase solution while adding 10 mL of PEG phase solution to lower layer (phase solutions were generated using 6% w/w dextran T-500, 6% w/w PEG 3350, 0.25 M sucrose, 10 mM potassium phosphate, pH7.5, 28.8 mM NaCl). This was mixed by inversion and centrifuged. Then the upper layer (from original upper layer) was transferred to a fresh tube with 10 mL of dextran phase solution, mixed by inversion, and centrifuged. The upper layers of mixtures from 2 tubes were then combined into 40 mL open-top tube and filled with suspension buffer. This was then centrifuged for 120 minutes at 23,600 rpm (100,000×g) at 4° C. The supernatant was removed and the plasma membranes suspended in 1 mL binding buffer (25 mM MES, pH6.0, 3 mM MgCl2, 10 mM NaCl). This suspension was then sonicated for 5 sec using a sonicating water bath. 5-10 μM MAMP peptide was added and the plasma membrane-enriched microsomes/peptide mixture incubated on ice in the dark cold room for 15 min with mixing every 5 min. Suspended membranes were transferred to a cold watch glass (65 mm OD×50 mm ID×10 mm depth). Membranes were irradiated with UV lamp for 15 min; the watch glass/lamp were moved every 5 min to keep cold. The suspended membranes were them transferred to 1.5 mL tube and 10 μL Triton X-100 (final conc. 1%) and 5 μL of 20% SDS (final conc. 0.1%) were added. Next 20 μL of anti-GFP-nanotrap slurry was used per sample. Resin was washed with 1 mL cold binding buffer supplemented with detergents (1% Triton X-100 and 0.1% SDS). Washed resin was transferred into 1.5 mL tube containing the MAMP treated and UV photo-crosslinked membranes, incubated at 4° C. (cold room) with rotating for 2 hr. Resin was washed twice with 1 mL of chilled PBS buffer. The resin was washed three times with 1 mL of chilled RIPA buffer (PBS pH 7.4, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS). After the third wash, the beads were suspended in 465 μL of RIPA buffer and 35 μL of click reaction mix (500 μM LTTP Ligand, 250 μM CuSO4-5H2O, 2 mM Na ascorbate, 100 μM N3-Biotin) was added. Click reactions were incubated with rotating at 4° C. overnight. Resin was washed 5 times with 1 mL of RIPA buffer. Upon removal of the final wash, the 20 μL pellet was suspended in 30 μL of freshly prepared 2× sample buffer+10% β-mercaptoethanol. Material was eluted by boiling for 6 min. Precast BioRad TGX 4-20% gradient gels, 1 mm thick with 10 wells, were used. Samples were loaded on to the gel, and the gel was run at 200 V for 90 min and then transferred to PVDF membrane at 100 V for 90 min. Blocking was carried out in 5% milk TBS for 1 hr at room temperature. The primary antibody anti-GFP was used at 1:4000, 4° C. overnight, in 1% milk TBS-Tween20. The secondary antibody anti-mouse-HRP was used at 1:20,000, 1 hr at room temp, in 1% milk TBS-Tween20. Alternatively, the conjugated primary antibody streptavidin-HRP was used at 1:5,000, 4° C. overnight, in 1% milk TBS-Tween20. Detection was carried out using an HRP substrate reagent.
Using an F2 population, mapping-by-sequencing was performed followed by fine mapping to identify a gene encoding a RLK that is linked to flgII-28 responsiveness in tomato. In order to identify the responsible gene by bulked segregant analysis, segregating populations were generated by crossing flgII-28 sensitive (LA1589) and insensitive (Yellow Pear) accessions (
The FLS3 protein structure, its similarity to FLS2, and FLS3 orthologs from other Solanaceae species were elucidated. The wild type FLS3 allele encodes a class XII LRR RLK with 27 LRRs and an intracellular non-RD kinase domain (
After identification of FLS3 using a genetics approach, the identity of FLS3 was confirmed using independent gain-of-function approaches. First, the activity of FLS3 in tomato protoplasts was tested. Leaf protoplasts were generated from the tomato cultivar ‘Yellow Pear’ that does not respond to flgII-28 treatment. Protoplasts expressing FLS3 and treated with flgII-28 showed an increase in MAPK activation (
Next, FLS3 was expressed in N. benthamiana plants that lacked BAK1, a protein that functions as a co-receptor with many other PRRs. A decreased ROS burst was observed in bald-silenced plants compared to control plants (
In order to confirm that FLS3 contributes to plant immunity, F2 plants from the LA1589×Yellow Pear cross were infected with a bacterial pathogen. Decreased bacterial growth was observed in LA1589 (FLS3/FLS3) and in F2 plants that have a functional copy of FLS3 (FLS3/FLS3 or FLS3/fls3) (
Finally, using a novel photo-crosslinking and click chemistry approach (
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
This application claims benefit of U.S. Provisional Application No. 62/021,995 filed Jul. 8, 2014, which is hereby incorporated by reference in its entirety.
This invention was made with government support under the following grant numbers: IOS-1025642 awarded by the National Science Foundation; R01-GM078021 awarded by the National Institutes of Health; and 2010-65108-20503 awarded by the United States Department of Agriculture/National Institute of Food and Agriculture. The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/039520 | 7/8/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62021995 | Jul 2014 | US |
