Patent Application
20240368617
The Sequence Listing written in file DU8135_SeqListing.xml is 80 kilobytes in size, was created Apr. 30, 2024, and is hereby incorporated by reference.
The Nonexpressor of Pathogenesis-Related Genes 1 (NPR1) gene is a positive regulator of the salicylic acid (SA)-mediated systemic acquired resistance (SAR) in plants. Overexpression of Arabidopsis NPR1 can enhance disease resistance against a variety of pathogens and stresses. However, this strategy often results in retardation in plant growth and reproduction dampening the potential for using NPR1 in agricultural applications. Thus, there is an ongoing need for improved systems and methods for engineered NPR1.
Described are genetically modified plants comprises a nucleic acid encoding a npr1(SAL) protein. The nucleic acid can be a heterologous nucleic acid encoding the npr1(SAL) protein or a modified endogenous NPR1 gene.
The heterologous nucleic acid encoding the npr1(SAL) protein can encode an Arabidopsis npr1(SAL) protein or an ortholog of an Arabidopsis npr1(SAL) protein. In some embodiments, the Arabidopsis npr1(SAL) protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the ortholog of the Arabidopsis npr1(SAL) comprises the amino acid sequence of any one of SEQ ID NOs: 5-22. In some embodiments, the heterologous nucleic acid comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the nucleic acid sequence of SEQ ID NO:2, or an ortholog thereof, and encodes a npr1(SAL) protein. In some embodiments, the described genetically modified plants comprise a heterologous nucleic acid sequence encoding an ortholog of the Arabidopsis npr1(SAL) protein, wherein the ortholog is derived from a plant of the same species or cultivar as the genetically modified plant.
In some embodiments, the modified endogenous NPR1 gene comprises cysteine substitutions at positions 400 and 506, wherein the positions are relative to the AtNPR1 amino acid sequence (SEQ ID NO:3). In some embodiments, the modified endogenous NPR1 further comprises an alanine or leucine substitution at position 401 and/or an alanine or leucine substitution at position 402, wherein the positions are relative to the AtNPR1 amino acid sequence (SEQ ID NO:3) (e.g., as provided in SEQ ID NOs. 5-22). An endogenous NPR1 gene in a plant can be modified using CRISPR or other gene modification tools.
In some embodiments, the described genetically modified plants have improved or increased growth under stress conditions. The stress can comprise biotic stress or abiotic stress. In some embodiments, the described genetically modified plants have increased resistance to pathogen infection. In some embodiments, the described genetically modified plants have increased growth under stress conditions relative to a similar plant overexpressing NPR1.
Also described are methods of increasing growth of a plant during stress, the method comprising: expressing in the plant a nucleic acid encoding a npr1(SAL) protein. The stress can comprise biotic stress or abiotic stress. In some embodiments, expressing in the plant a nucleic acid encoding a npr1(SAL) protein comprises introducing a heterologous nucleic acid encoding the npr1 (SAL) protein into a plant cell. In some embodiments, expressing in the plant a nucleic acid encoding a npr1(SAL) protein comprises modifying an endogenous NPR1 gene in a plant cell to encode cysteines at positions 400 and 506, wherein the positions are relative to the Arabidopsis NPR1 protein (SEQ ID NO:3). In some embodiments, modifying the endogenous NPR1 gene in a plant cell further comprises modifying the NPR1 gene to encode an alanine or leucine at position 401 and/or 402, wherein the positions are relative to the Arabidopsis NPR1 protein (SEQ ID NO:3).
In some embodiments, expressing the nucleic acid encoding the npr1(SAL) protein in the plant increases resistance to pathogen infection. In some embodiments, the resulting plants have increased growth under stress conditions relative to similar plants overexpressing wild-type NPR1.
In some embodiments, expressing in the plant the nucleic acid encoding the npr1(SAL) protein comprises introducing into the plant cell's genome a heterologous nucleic acid encoding the npr1(SAL) protein. In some embodiments, expressing in the plant the nucleic acid encoding the npr1(SAL) protein comprises introducing a CRISPR system into a plant cell, wherein the CRISPR system introduces into the plant cell's genome a heterologous nucleic acid encoding the npr1(SAL) protein. In some embodiments, the heterologous nucleic acid encodes an Arabidopsis npr1(SAL) protein. In some embodiments, the heterologous nucleic acid encodes an ortholog of an Arabidopsis npr1(SAL) protein. In some embodiments, the heterologous nucleic acid encodes: SEQ ID NO: 1 or an ortholog thereof or any one of SEQ ID NOs: 5-22. n some embodiments, the heterologous nucleic acid comprises a nucleic acid having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, identity to SEQ ID NO: 2, or an ortholog thereof, and encodes a npr1(SAL) protein. In some embodiments, the ortholog of the Arabidopsis npr1(SAL) protein is derived from a plant of the same species or cultivar as the plant.
In some embodiments, expressing in the plant the nucleic acid encoding the npr1(SAL) protein comprises modifying the endogenous NPR1 gene to encode cysteines at positions 400 and 506, wherein the positions are relative to the Arabidopsis NPR1 protein (SEQ ID NO:3). In some embodiments, modifying the endogenous NPR1 gene further comprises further comprises modifying the NPR1 gene to encode an alanine or leucine at position 401 and/or 402, wherein the positions are relative to the Arabidopsis NPR1 protein (SEQ ID NO:3)
In some embodiments, the methods comprise generating one or more regenerants following introducing the heterologous nucleic acid or the modifying the endogenous NPR1 gene into a plant or plant cell. In some embodiments, the methods further comprise genotyping one or more regenerants for the presence of the nucleic acid encoding the npr1 (SAL) protein and/or selecting one or more To plants containing the nucleic acid encoding the npr1 (SAL) protein.
Also described are nucleic acid encodings npr1(SAL) proteins. In some embodiments, the nucleic acid encoding the npr1(SAL) protein comprises a nucleic acid encoding an Arabidopsis npr1(SAL) protein or an ortholog of the Arabidopsis npr1(SAL) protein. In some embodiments, the nucleic acid encoding the npr1(SAL) protein comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 2 or an ortholog thereof. In some embodiments, the nucleic acid encoding the npr1 (SAL) protein comprises a sequence encoding any one of SEQ ID NOs: 5-22.
Also described are nucleic acid constructs for modifying a plant or plant cell, wherein the nucleic acid construct comprises a nucleic acid encoding a npr1 (SAL) protein; or a nucleic acid for modifying an endogenous NPR1 gene in the plant or plant cell to encode cysteines at positions 400 and 506, and optionally an alanine or leucine at position 401 and/or 402, wherein the positions are relative to the Arabidopsis NPR1 protein (SEQ ID NO:3).
The accompanying Figures and Examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying example figures (also “FIG.”) relating to one or more embodiments, in which:
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
The term “about” or “approximately” indicates within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of 0 to 20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” means within an acceptable error range for the particular value should be assumed.
The use herein of the terms “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).
As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
The term “effective amount” refers to an amount sufficient to effect beneficial or desirable biological results.
The term “locus” refers to a position in the genome that corresponds to a measurable characteristic (e.g., a trait) or gene. A locus can be a genomic region or section of DNA (the locus) which correlates with a variation in a phenotype. A locus can comprise a single or multiple genes or other genetic information within a contiguous genomic region or linkage group.
A “heterologous” sequence is a sequence which is not normally present in a cell, genome, or gene in the genetic context in which the sequence is currently found. A heterologous sequence can be a sequence derived from the same gene (e.g., a different allele) and/or cell type, but introduced into the cell or a similar cell in a different context, such as on an expression vector or in a different chromosomal location or with a different promoter. A heterologous sequence can be a sequence derived from a different gene or species than a reference gene or species. A heterologous sequence can be from a homologous gene from a different species, from a different gene in the same species, or from a different gene from a different species. For example, a regulatory sequence may be heterologous in that it is linked to a different coding sequence relative to the native regulatory sequence.
“Orthologs” are genes and products thereof in different species that evolved from a common ancestral gene by speciation and retain the same or similar function. An ortholog is a gene that is related by vertical descent and is responsible for substantially the same or identical functions in different organisms. For example, Arabidopsis thaliana NPR1 and maize NPR1 can be considered orthologs. Genes may share sequence similarity of sufficient amount to indicate they are orthologs. Protein may share three-dimensional structure of sufficient amount to indicate the proteins and the genes encoding them are orthologs. Methods of identifying orthologs are known in the art.
A “homeolog” or “homeologous” genes refer to genes found in the same species that originated by a speciation event. Homeologs can evolve in polyploidy species from allopolyploidization, a whole-genome duplication via hybridization followed by genome doubling. For example, an octaploid plant may contain four homeologous genes that evolved from allopolyploidization, which are homologous to a single gene in the diploid plant.
The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof (“polynucleotides”) in either single-or double-stranded form. Unless specifically limited, the term polynucleotide encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited, the term polynucleotide encompasses nucleic acids having one or more modified nucleotides. Modified nucleotides can modify binding properties or alter in vitro or in vivo stability. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985 J. Biol. Chem. 260:2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.
The term “expression vector” or “expression construct” or “expression cassette” refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. Eukaryotic cells are generally known to utilize promoters, enhancers, and termination and polyadenylation signals, although some elements may be deleted, and other elements added without sacrificing the necessary expression.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms, or by manual alignment and visual inspection.
Sequence identity can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated the window of comparison between two sequences is defined by the entire length of the shorter of the two sequences.
A residue (e.g., amino acid) in an ortholog of AtNPR1 can be determined to correspond with a residue in AtNPR1 by optimally aligning the two sequences for maximum correspondence over the full sequence or over a specified comparison window. In aligning the sequences, either sequence (AtNPR1 or the ortholog of ANPR1) may contain amino acid additions (insertions) or deletions (gaps) relative to the other sequence. An amino acid residue of an ortholog of AtNPR1 corresponds to an amino acid residue at a particular position of AtNPR1 if amino acid residues are located at the same position when the AtNPR1 sequence and the AfNPR1 ortholog sequence are optimally aligned.
The term “plant” includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, reproductive organs, embryos, and parts thereof, etc.), seedlings, seeds and plant cells, and progeny thereof. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, (e.g., octoploid, diploid, haploid, and hemizygous).
An “RNA-guided DNA endonuclease” is an enzyme (endonuclease) that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. An RNA-guided DNA endonuclease may be, but is not limited to, a zCas9 nuclease, a Cas9 nuclease, type II Cas nuclease, an nCas9 nuclease, a type V Cas nuclease, a Cas12a nuclease, a Cas12b nuclease, a Cas12c nuclease, a CasY nuclease, a CasX nuclease, a Cas12i nuclease, or an engineered RNA-guided DNA endonuclease.
A “guide RNA” (gRNA) comprises an RNA sequence (tracrRNA) bound by Cas and a spacer sequence (crRNA) that hybridizes to a target sequence and defines the genomic target to be modified. The tracrRNA and crRNA may be linked to form a “single chimeric guide RNA” (sgRNA).
The term “CRISPR RNA (crRNA)” has been described in the art (e.g., in Makarova et al. (2011) Nat Rev Microbiol 9:467-477; Makarova et al. (2011) Biol Direct 6:38; Bhaya et al. (2011) Annu Rev Genet 45:273-297; Barrangou et al. (2012) Annu Rev Food Sci Technol 3:143-162; Jinek et al. (2012) Science 337:816-821; Cong et al. (2013) Science 339:819-823; Mali et al. (2013) Science 339:823-826; and Hwang et al. (2013) Nature Biotechnol 31:227-229). A crRNA contains a sequence (spacer sequence or guide sequence) that hybridizes to a target sequence in the genome. A target sequence can be any sequence that is unique compared to the rest of the genome and is adjacent to a protospacer-adjacent motif (PAM).
A “protospacer-adjacent motif” (PAM) is a short sequence recognized by the CRISPR complex. The precise sequence and length requirements for the PAM differ depending on the CRISPR system used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (i.e., target sequence). Non-limiting examples of PAMs include NGG, NNGRRT, NN [A/C/T] RRT, NGAN, NGCG, NGAG, NGNG, NGC, and NGA.
A “trans-activating CRISPR RNA” (tracrRNA) is an RNA species facilitates binding of the RNA-guided DNA endonuclease (e.g., Cas) to the guide RNA.
A “CRISPR system” comprises a guide RNA, either as a crRNA and a tracrRNA (dual guide RNA) or an sgRNA, and RNA-guided DNA endonuclease. The guide RNA directs sequence-specific binding of the RNA-guided DNA endonuclease to a target sequence. In some embodiments, the RNA-guided DNA endonuclease contains a nuclear localization sequence. In some embodiments, the CRISPR system further comprises one or more fluorescent proteins and/or one or more endosomal escape agents. In some embodiments, the gRNA and RNA-guided DNA endonuclease are provided in a complex. In some embodiments, the gRNA and RNA-guided DNA endonuclease are provided in one or more expression constructs (CRISPR constructs) encoding the gRNA and the RNA-guided DNA endonuclease. Delivery of the CRISPR construct(s) to a cell results in expression of the gRNA and RNA-guided DNA endonuclease in the cell. The CRISPR system can be, but is not limited to, a CRISPR class 1 system, a CRISPR class 2 system, a CRISPR/Cas system, a CRISPR/Cas9 system, a CRISPR/zCas9 system and a CRISPR/Cas3 system.
A “DNA donor template” is a nucleic acid, such as a single stranded DNA, linear double strand DNA, plasmid DNA or AAV sequence that provides the homology necessary for precise repair of a double-strand break. A DNA donor template contains two regions of homology, one region of homology to either side of the double strand break. A DNA donor template can also contain a heterologous sequence between the two regions of homology. Each region of homology can be about 30 to about 100 nucleotides in length.
A “regenerant” is a plant produced from a plant tissue cell, such as a genetically modified plant tissue cell.
“Introgression” or “introgressing” of a locus means introduction of a locus from a donor plant comprising the locus of interest into a recipient plant by standard breeding techniques, wherein selection can be done phenotypically by means of observation of a plant characteristic including, but not limited to, characteristics such as the internodal length or plant height, or selection can be done with the use of markers through marker-assisted breeding, or combinations of these. The process of introgressing is often referred to as “backcrossing” when the process is repeated two or more times. In introgressing or backcrossing, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. Selection is started in the F1 or any further generation from a cross between the recipient plant and the donor plant, suitably by using markers as identified herein. The skilled person is, however, familiar with creating and using new molecular markers that can identify or are linked to a locus of interest.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The Nonexpressor of Pathogenesis-Related Genes 1 (NPR1) gene is a positive regulator of the salicylic acid (SA)-mediated systemic acquired resistance (SAR) in plants. NPR1 is evolutionary conserved across a wide range of species, and overexpression of A/NPR1 protein enhances broad-spectrum disease resistance in numerous plants. NPR1 is recognized as a key regulator of defense across diverse plant species. NPR1 orthologs have been identified in numerous plant species, including, but not limited to, Brassica rapa (includes turnip Komatsuna, napa cabbage, bomdong, bok choy, and rapini, mustard seed/rapeseed), Brassica juncea (includes mustard, oilseed mustard), Brassica napus (rapeseed/canola), Oryza sativa (rice), Raphanus sativus (radish), Nicotiana tabacum (tobacco), Glycine max (soybean), Populus trichocarpa (black cottonwood), Cucumis melo (melon), Zea mays (corn/maize), Solanum lycopersicum (tomato), Vitis vinifera (grape), Hordeum vulgare (barley), Medicago truncatula (barrelclover), Sorghum bicolor (Sorghum), Saccharum species (sugar cane), Beta vulgaris (sugar beet), Panicum virgatum (switchgrass), Fragaria species (strawberry), and Citrus species (citrus; e.g., Citrus×sinensis (orange)).
The inventors have modeled a molecular mechanism of NPR1 activity in plant protection in which NPR1 promotes cell survival by targeting over-accumulated stress proteins for ubiquitination and degradation through formation of SA-induced NPR1 condensates (SINCs). SINCs are enriched with defense proteins, including a number of immune receptors, oxidative and DNA damage response proteins, and protein quality control machineries. Transition of NPR 1 into condensates is required for formation of the NPR1-Cullin 3 E3 ligase complex to ubiquitinate SINC-localized proteins and promote cell survival. NPR1 not only sequesters stress proteome in SINCs, but also induces transcription of a large number of defense genes, including SINC components, through its SA-dependent transcriptional coactivation function.
Structural analysis of NPR1 revealed a bird-shaped homodimer consisting of a central BTB domain, a BTB-and carboxy terminal Kelch helix bundle (BHB), four ankyrin repeats (ANK), and a disordered SA-binding domain (SBD). In the BTB domain, NPR1 harbors a unique zinc-finger motif important for interacting with ankyrin repeats and mediating NPR1 oligomerization. Cryo-EM analysis of NPR1 in complex with SA revealed that SA induces not only folding of the otherwise unstructured SBD domain, but also promotes its docking onto the ankyrin repeats.
A NPR1 protein includes AtNPR1 and orthologs of AtNPR1 from other plants (i.e., NPR1 proteins encoded by NPR1 genes). In some embodiments, AtNPR1 comprises SEQ ID NO: 3. NPR1 orthologs include, but are not limited to, Brassica rapa BrNPR1, Brassica juncea BjNPR1, Brassica napus BnNPR1, Raphanus sativus RsNPR1, Oryza sativa OsNPR1, Nicotiana tabacum NINPR1, Glycine max GmNPR1, Populus trichocarpa PINPR1, Cucumis melo CmNPR1, Zea mays ZmNPR1, Solanum lycopersicum SINPR1, Vitis vinifera WyNPR1, Hordeum vulgare HvNPR1, Medicago truncatula MINPR1, Sorghum bicolor SbNPR1, Musa acuminata MaNPR 1 and Musa balbisiana MbNPR1.
A NPR1 gene comprises a nucleotide sequence that encodes a NPR1 protein. A NPR1 gene includes AtNPR1 and orthologs of AtNPR1 form other plants. In some embodiments, ANPR1 comprises SEQ ID NO:4. NPR1 orthologs include, but are not limited to, Brassica rapa BrNPR1, Brassica juncea BjNPR1, Brassica napus BnNPR1, Raphanus sativus RsNPR1, Oryza sativa OsNPR1, Nicotiana tabacum NINPR1, Glycine max GmNPR1, Populus trichocarpa PINPR1, Cucumis melo CmNPR1, Zea mays ZmNPR1, Solanum lycopersicum SINPR1, Vitis vinifera VvNPR1, Hordeum vulgare HvNPR1, Medicago truncatula MINPR1, Sorghum bicolor SbNPR1, Avena sativa (oats), Triticum species (wheat), Secale cereale (rye), Rubus species (raspberries), Vaccinium species (blueberries, cranberries, etc.), Malus domestica (apples), Prunus species (peaches, plums, cherries, almonds, etc.), Gossypium species (cotton), Coffee species (coffee), Arachis hypogaea (peanut), Cannabisspecies (hemp), and palms (e.g., Cocos nucifera, Elaeis species, Nypa fruticans, Phoenix species, and Arenga pinnata).
Described are npr1 variants comprising cysteine substitutions at positions 400 and 506 relative to the Arabidopsis thaliana NPR1 protein (AtNPR1, SEQ ID NO:3), Q400C and R506C, respectively. The cysteine at position 400 is in an ANK domain, and the cysteine at position 506 is in the SBD domain. In some embodiments, the cysteines substituted at positions 400 and 506 (relative to SEQ ID NO:3) form a disulfide bond that crosslinks the SBD-ANK docking conformation. In some embodiments, the npr1 variants further comprise an Alanine or Leucine substitution at position 401 relative to ANPR1) protein (A1NPR1, SEQ ID NO:3), e.g., E401L. In some embodiments, the npr1 variants further comprise an Alanine or Leucine substitution at position 402 relative to AfNPR1), e.g., or 402A or 402L. The npr1(SAL) 401 (A or L) substitution or the 402 (A or L) substitution may compensate for the loss of the E401-R506 hydrogen bond and avoid burial of a charged residue at the protein interface. The resulting npr1 variants (400C/506C; 400C/401 (A or L/506C; 400C/402 (A or L)/506C), which can be referred to as npr1 (SAL) (for SBD-ANK Locked), significantly elevate induction of PRI gene expression in a transient reporter assay without changing the interaction with the TGA3 transcription factor. Activity of npr1 (SAL) indicates that the SBD-ANK-docking conformation is important for NPR1 activity in defense gene transcription.
In some embodiments, a Arnprl (Sal) variant comprises AtNPR1 having an Q400C substitution and a R506C substation. In some embodiments, the Atnprl (Sal) variant further comprises an E401A (SEQ ID NO:1) or E401L substitution.
Similar substitutions can be made in orthologs of AtNPR1 from other plant species. NPR1 orthologs (and genes encoding the NPR1 orthologs) can be readily identified using methods and analyses available in the art for identifying orthologs in plants. Thus, a npr1 (SAL) variant includes Atnpr1(Sal) and orthologs of Amnpr1(SAL) from other plant species. In some embodiments, an Atnpr1(SAL) ortholog comprises an ortholog of ANPR 1 comprising cysteine substitutions at positions corresponding to positions 400 and 506 of AtNPR1 (SEQ ID NO:3). In some embodiments, an Atnpr1(SAL) ortholog further comprises an alanine or leucine substitution at a position corresponding to position 401 or 402 of AtNPR1 (SEQ ID NO:3)). In some embodiments, npr1(SAL) variants include, but are not limited to: Brassica rapa Brnpr1(SAL) (SEQ ID NO:5), Brassica juncea Bjnpr1(SAL) (SEQ ID NO:6), Brassica napus Bnnpr1(SAL) (SEQ ID NO:7), Raphanus sativus Rsnpr1(SAL) (SEQ ID NO:8), Oryza sativa Osnpr1(SAL) (SEQ ID NO:9), Nicotiana tabacum Ntnpr1(SAL) (SEQ ID NO:10), Glycine max Gmnpr1(SAL) (SEQ ID NO:11), Populus trichocarpa Pinpr1(SAL) (SEQ ID NO:12), Cucumis melo (mnpr1(SAL) (SEQ ID NO:13), Zea mays Zmnpr1(SAL) (SEQ ID NO:14), Solanum lycopersicum S/npr1(SAL) (SEQ ID NO:15), Vitis vinifera Vvnpr1(SAL) (SEQ ID NO: 16), Hordeum vulgare Hynpr1(SAL) (SEQ ID NO:17), Medicago truncatula Minpr1(SAL) (SEQ ID NO:18), Sorghum bicolor Sbnpr1(SAL) (SEQ ID NO:19), Triticum species Trnpr1(SAL) (SEQ ID NO:20), Citrus×sinensis Csnpr1(SAL) (SEQ ID NO:21), Fragaria species Fynpr1(SAL) (SEQ ID NO: 22). Alignment of the BTB domains and zinc finger regions of various NPR 1 orthologs is shown in
In some embodiments, a npr1(SAL) variant comprises a protein having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of Amnpr1(SAL) (SEQ ID NO:1) or a protein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an ortholog of Atnprl (SAL), wherein the npr1 (SAL) protein comprises a cysteine at positions corresponding to positions 400 and 506 of AtNPR1 (SEQ ID NO:3) and optionally an alanine or leucine substitution at a position corresponding to position 401 and/or 402 of AtNPR1 (SEQ ID NO:3), and wherein the npr1 (SAL) variant induces expression of PRI gene expression in a transient in response to salicylic acid (SA). In some embodiments, the npr1(SAL) variant interacts with the TGA3 transcription factor.
In some embodiments, a npr1(SAL) variant comprises a protein having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NO:5-22, wherein the npr1(SAL) protein comprises a cysteine at positions corresponding to positions 400 and 506 of AtNPR1 (SEQ ID NO: 3) and optionally an alanine or leucine substitution at a position corresponding to position 401 and/or 402 of AtNPR1 (SEQ ID NO:3), and wherein the npr1(SAL) variant induces expression of PRI gene expression. In some embodiments, the npr1(SAL) variant interacts with the TGA3 transcription factor.
Described are nucleic acid sequences, expression vectors, or CRISPR constructs encoding a npr1(SAL) variant or a portion thereof. A npr1(SAL) gene comprises a nucleic acid encoding a npr1(SAL) protein. In some embodiments, a npr1(SAL) gene comprises SEQ ID NO: 2 (Atnpr1(SAL)). In some embodiments, a npr1(SAL) gene comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2, wherein the nucleic acid sequence encodes an Atnpr1(SAL protein). In some embodiments, a npr1(SAL) gene encodes an ortholog of Atnpr1(SAL). In some embodiments, a npr1(SAL) gene comprises a nucleotide sequence encoding any one of SEQ ID NO:5-22 or a protein having at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NO:5-22, wherein the npr1(SAL) protein comprises a cysteine at positions corresponding to positions 400 and 506 of AtNPR1(SEQ ID NO:3) and optionally an alanine or leucine substitution at a position corresponding to position 401 or 402 of AtNPR1(SEQ ID NO: 3), and wherein the npr1(SAL) variant induces expression of PRI gene expression in response to SA.
Described are compositions for genetically modifying a plant to express a npr1(SAL) variant. Genetically modifying a plant to express a npr1(SAL) variant comprises expressing a heterologous nucleic acid encoding a npr1 (SAL) gene in the plant or modifying an endogenous NPR1 gene in the plant to encode a npr1 (SAL) variant. Described are compositions for modifying a NPR1 locus to encode a npr1(SAL) variant.
Also described are methods of using the compositions to produce plants having increased resistance to stress. In some embodiments, plants expressing a npr1 (SAL) variant have improved or increased growth or biomass production under stress conditions relative to genetically similar plants that do not express the npr1 (SAL) variant, when grown under the same conditions. In some embodiments, plants expressing a npr1(SAL) variant have increased stress resistance relative to genetically similar plants that do not express the npr1(SAL) variant, when grown under the same conditions. In some embodiments, plants expressing a npr1(SAL) variant have improved or increased growth or biomass production and increased stress resistance relative to genetically similar plants that do not express the npr l (SAL) variant, when grown under similar stress conditions. In some embodiments, plants expressing a npr1(SAL) variant have improved or increased growth or biomass production under stress conditions relative to genetically similar plant that do not express the npr1(SAL) variant, when grown under the same conditions. In some embodiments, expression of a npr1(SAL) variant in a plant results in improved growth under conditions of immunity activation compared to a genetically similar plant that does not express npr1(SAL), when grown under similar conditions. The stress resistance can be biotic stress and/or abiotic stress. Biotic stress includes, but is not limited to, bacterial or viral infection. Abiotic stress includes, but is not limited to, heat and drought.
Described are methods of improving plant growth under stress conditions comprising expressing in the plant a npr1 (SAL) gene. In some embodiments, the methods comprise expressing a heterologous npr l (SAL) gene in the plant. In some embodiments, the methods comprise modifying an endogenous NPR1 gene to generate a npr 1 (SAL) gene.
In some embodiments, plants expressing a npr1(SAL) variant have increased biomass production under stress conditions relative to genetically similar plants overexpressing wild-type NPR1, when grown under stress conditions. The stress resistance can be biotic stress and/or abiotic stress. Biotic stress includes, but is not limited to, bacterial or viral infection. Abiotic stress includes, but is not limited to, heat and drought.
In some embodiments, plants expressing a npr1(SAL) variant have increased survival under pathogen effector-triggered (ETI) cell death relative to genetically similar plants that do not express the npr1(SAL) variant, when grown under the same conditions. In some embodiments, plants expressing a npr1(SAL) variant have increase cell survival against pathogen-induced cell death relative to genetically similar plants that do not express the npr1(SAL) variant, when grown under the same conditions.
Increasing resistance to stress often has an indirect relationship with plant growth. Increasing stress resistance often results in decreased plant growth. For example, overexpression of wild-type NPR1 in plants increases disease resistance, but results in reduced growth under stress conditions. The described nucleic acids and methods can be used to improve plant crop yield while maintaining or increasing resistance to stress. In some embodiments, plants expressing a npr I (SAL) gene have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 100% increased biomass production under stress conditions relative to genetically similar plants that do not express a npr1(SAL) gene and overexpress wild-type NPR1. In some embodiments, stress-induced loss of crop yield (e.g., plant biomass) in plants expressing a npr1(SAL) gene may be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or up to 100% compared to genetically similar plants that do not express a npr (SAL) gene and overexpress wild-type NPR1.
In some embodiments, genetically modified plants expressing a npr1(SAL) gene are described. The plant can, but is not limited to, a crop plant. Using the described nucleic acids encoding a npr1(SAL) variants or portions thereof, genetically modified plants that express a npr1(SAL) variant can be made. The genetically modified plants can be made using methods available in the art for generating genetically modified plants.
In some embodiments, the genetically modified plants contain a nucleic acid sequence encoding a heterologous npr1(SAL) gene. In some embodiments, the heterologous npr1(SAL) gene is introduced into a plant to improve or increase plant growth under conditions of immunity activation. In some embodiments, the heterologous npr1(SAL) gene is introduced into a plant to improve or increase plant growth under conditions of biotic or abiotic stress. In some embodiments, the heterologous npr1(SAL) gene is introduced into a plant to increase stress resistance without inhibiting growth.
In some embodiments, an endogenous NPR1 gene or locus in a plant is modified to encode a npr1(SAL) protein (i.e., an endogenous NPR1 gene is modified to contain a Q400C substitution and a R400C substitution, and optionally a 410 or 402 alanine or leucine substitution, wherein the positions correspond to the positions in the AtNPR1 (SEQ ID NO:3)). In some embodiments, the endogenous NPR1 gene or locus in a plant is modified to encode a npr1(SAL) protein to improve or increase plant growth under conditions of immunity activation. In some embodiments, the endogenous NPR1 gene or locus in a plant is modified to encode a npr1(SAL) protein improve or increase plant growth under conditions of biotic or abiotic stress. In some embodiments, the endogenous NPR1 gene or locus in a plant is modified to encode a npr1(SAL) protein increase stress resistance without inhibiting growth.
Various methods for introducing a transgene expression vector constructs of the invention into a plant or plant cell are well known to those skilled in the art, and any method capable of transforming the target plant or plant cell may be utilized.
Nucleic acids may be introduced into a plant, plant cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, via the use of various DNA-based vectors such as Agrobacterium tumefaciens and Agrobacterium rhizogenes vectors, and CRISPR or CRISPR/Cas9. Once a plant cell has been successfully transformed, it may be cultivated to regenerate a transgenic plant (regenerant). T0 transgenic plants may be used to generate subsequent generations (e.g., T1, T2, etc.) by selfing of primary or secondary transformants, or by sexual crossing of primary or secondary transformants with other plants (transformed or untransformed).
In some embodiments, Agrobacterium tumefaciens is used to deliver an expression vector or CRISPR system nucleic acids to a plant. Agrobacterium-mediated transformation of a large number of plants are extensively described in the literature (see, for example, Agrobacterium Protocols, Wan, ed., Humana Press, 2nd edition, 2006). Various methods for introducing DNA into Agrobacteria are known, including electroporation, freeze/thaw methods, and triparental mating. In some embodiments, a pMON316-based vector is used in the leaf disc transformation system of Horsch et al. Other commonly used transformation methods include, but are not limited to, microprojectile bombardment, biolistic transformation, and protoplast transformation of naked DNA by calcium, polyethylene glycol (PEG) or electroporation (Paszkowski et al., 1984, EMBO J. 3:2727-2722; Potrykus et al., 1985, Mol. Gen. Genet. 199:169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828; Shimamoto et al., 1989, Nature, 338:274-276.)
In some embodiments, unaided homologous recombination, recombinase-based insertion, and DNA repair-based insertion targeted genetic modification (see, e.g., Dong et al., 2021, PNAS. 118 (22) e2004834117) can be used to insert (knock in) a heterologous npr 1 (SAL) gene into a plant, replace an endogenous NPR1 allele with a npr1(SAL) gene, or modify an endogenous NPR1 gene to express a npr1(SAL) protein. Recombinase-based insertion can comprise systems and constructs involving site-specific recombinases. Such systems include, but not limited to, Cre: loxP systems, Flp: FRT systems, Dre: rox systems, VCre: loxV systems, Gin: gix systems, Bxb1: attP attB systems, phiC31: attP attB systems. DNA repair-based insertion methods rely upon the activity of a nuclease agent. The nuclease agent can be, but is not limited to, a Transcription Activator-Like Effector Nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, a restriction endonuclease (Type I, Type II, Type III, or Type IV endonucleases), or a CRISPR/Cas system.
A CRISPR system comprises an RNA-guided DNA endonuclease enzyme and a CRISPR RNA. In some embodiments, a CRISPR RNA is part of a guide RNA. In some embodiments, the RNA-guided DNA endonuclease enzyme is a Cas9 protein. In some embodiments, a CRISPR system comprises one or more nucleic acids encoding an RNA-guided DNA endonuclease enzyme (such as, but not limited to a Cas9 protein) and a guide RNA. A guide RNA can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), either as separate molecules or a single chimeric guide RNA (sgRNA). The guide RNA contains a guide sequence having complementarity to a sequence in the target gene genomic region. Suitable crRNAs or gRNAs and nucleic acids encoding crRNAs or gRNAs can be made using methods and tools available in the art to design and manufacture crRNAs or gRNAs. The Cas protein can be introduced into the plant in the form of a protein or a nucleic acid (DNA or RNA) encoding the Cas protein (e.g., operably linked to a promoter expressible in the plant). The guide RNA can be introduced into the plant in the form of RNA or a DNA encoding the guide RNA (e.g., operably linked to a promoter expressible in the plant). In some embodiments, the CRISPR system further includes a DNA donor template. In some embodiments, the CRISPR system can be delivered to a plant or plant cell via a bacterium. The bacterium can be, but is not limited to, Agrobacterium tumefaciens.
In some embodiments, the CRISPR system is designed to knock in a heterologous npr1(SAL) gene into a target locus in the plant suitable for expression of a heterologous npr 1 (SAL) gene. The target locus can be an endogenous NPR1 locus or a safe harbor locus. In some embodiments, the CRISPR system is designed to modify an endogenous NPR1 locus.
The CRISPR/Cas system can be, but is not limited to, a CRISPR class 1 system, a CRISPR class 2 system, a CRISPR/Cas system, a CRISPR/Cas9 system, a CRISPR/zCas9 system or a CRISPR/Cas3 system.
Suitable guide sequences for use in a CRISPR system include 17-20 nucleotide sequences along the target locus that are unique compared to the rest of the genome and immediately adjacent (5′) to a protospacer-adjacent motif (PAM) site. For the RNA-guided DNA endonuclease enzyme zCas9, a PAM site is NGG. Thus, any unique 17-20 nucleotide sequence immediately 5′ of a 5′-NGG-3′ in the target locus can be used in forming a gRNA. In some embodiments, the guide sequence is 100% complementary to a target sequence present in the target locus. In some embodiments, the guide sequence is at least 90% or at least 95% complementary to the target sequence. In some embodiments, the guide sequence contains 0, 1, or 2 mismatches when hybridized to the target sequence. In some embodiments, a mismatch, if present, is located distal to the PAM, in the 5′ end of the guide sequence.
The DNA donor template contains sequence to be inserted into the genome of the plant. In some embodiments, the DNA donor template to be used with a CRISPR system comprises sequences for a heterologous npr 1 (SAP) gene, coding sequence, or fragment thereof. The donor template can further comprise one or more regulator sequence operatively linked to the coding sequence that drive expression of the coding sequence in the plant cell.
In some embodiments, the DNA donor template to be used with a CRISPR system comprises sequence for modifying an endogenous NPR1 gene. CRISPR constructs (nucleic acids) for modifying an endogenous NPR1 gene can be designed and manufactured using methods available in the art for genetically modifying a genomic locus in plants.
DNA donor templates further comprise 5′ and 3′ homology regions located 5′ and 3′ to the sequence to be inserted into the genome. The homology regions comprise about 30 to about 100 nucleotides that are complementary to a corresponding number of nucleotides in the genome on either side of the double strand break created by the CRISPR nuclease.
The DNA donor template can be provided a single strand DNA, double strand DNA, plasmid DNA, or adeno-associated vector DNA.
It is understood that RNA equivalents of any listed DNA sequences, substituting uracils (U) for thymines (T), may be used. An “RNA equivalent” is an RNA molecule having essentially the same complementary base pair hybridization properties as the listed DNA sequence.
CRISPR modification of a target locus is not limited to the CRISPR/zCas9 system. Other CRISPR systems using different nucleases and having different PAM sequence requirements are known in the art. PAM sequences vary by the species of RNA-guided DNA endonuclease. For example, Class 2 CRISPR-Cas type II endonuclease derived from S. pyogenes utilizes an NGG PAM sequence located on the immediate 3′ end of the guide sequence. Other PAM sequences include, but are not limited to, NNNNGATT (Neisseria meningitidis), NNAGAA (Streptococcus thermophilus), and NAAAAC (Treponema denticola). Guide sequences for CRISPR systems having nucleases with different PAM sequence requirements are identified as described above for zCas9, substituting the different PAM sequences.
Two or more guide RNAs can used with the same RNA-guided DNA endonuclease (e.g., Cas nuclease) or different RNA-guided DNA endonucleases.
Any of the above-described guide RNAs can be provided as an RNA or a DNA encoding the RNA.
In some embodiments, a CRISPR system comprises one or more guide RNAs and a nucleic acid encoding an RNA-guided DNA endonuclease. In some embodiments, a CRISPR system comprises one or more guide RNAs, a nucleic acid encoding an RNA-guided DNA endonuclease, and a DNA donor template.
In some embodiments, a CRISPR system comprises one or more guide RNAs and a one or more nucleic acids encoding two or more different RNA-guided DNA endonucleases. In some embodiments, a CRISPR system comprises one or more guide RNAs, one or more nucleic acids encoding two or more different RNA-guided DNA endonucleases, and at least one DNA donor template.
In some embodiments, a CRISPR system comprises a guide RNA and an RNA-guided DNA endonuclease in a complex. In some embodiments, a CRISPR system comprises a guide two or more RNAs each in a complex with an RNA-guided DNA endonuclease. In some embodiments, a CRISPR system comprises a guide RNA and an RNA-guided DNA endonuclease in a complex and a DNA donor template.
CRISPR constructs and systems comprising Cas proteins and guide RNAs for directed modification at the NPR1 gene are described. In some embodiments, the CRISPR constructs and systems can be used to modify an endogenous NPR1 gene to encode cysteine substitutions at positions corresponding to positions 400 and 506 of AtNPR1 (SEQ ID NO:3). In the embodiments, the CRISPR constructs and systems can be used to modify an endogenous NPR1 gene to contain encode cysteine substitutions at positions corresponding to positions 400 and 506 of AtNPR1 (SEQ ID NO:3) and an alanine or leucine substitution at a position corresponding to positions 401 or 402 of AINPR1. In some embodiments, CRISPR constructs and systems can be used to insert (knock in) a heterologous sequence encoding a npr1(SAL) gene into the genome of a plant. The heterologous sequence encoding a npr1(SAL) gene can be inserted into an endogenous NPR1 locus or a different locus, such as a safe harbor site, suitable for expression of the npr1(SAL) gene.
In some embodiments, the CRISPR constructs and systems are used to generate genetically modified plant cells carrying a npr1(SAL) gene. The npr1(SAL) gene can be a modified endogenous NPR 1 gene or a heterologous npr1(SAL) gene. These plant cells can then be used to produce regenerant progeny transgenic plants that that express the npr1(SAL) gene.
The described methods can be used to modify a plant having one or more traits of interest. Such traits can be, but are not limited to, genes for herbicide tolerance, increased yield, insect control, other fungal disease resistance, virus resistance, bacterial disease resistance, germination and/or seedling growth control, enhanced animal and/or human nutrition, improved processing traits, or improved flavor, among others.
One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.
All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.
An important aspect of successful immunity is the ability to correctly manage defense-to-growth trade-offs. Repeated application of SA or its analog BTH has been traditionally used to induce growth retardation in Arabidopsis as a result of activation of NPR1-dependent immunity. To test the role of the npr1(SAL) variant in the SA-induced growth retardation, the npr1(SAL)-GFP fusion was stably expressed in transgenic plants and plant biomass after BTH treatment was measured (
It was also tested if npr1(SAL) expression could increase survival under pathogen effector-triggered (ETI) cell death (
The results show that expression of npr1(SAL) in crop plants can enhance their survival against not only pathogen-induced cell death. The results further indicate that expression of npr1(SAL) in crop plants can also enhance survival against environmental (abiotic) stress-associated cell death. Unlike overexpression of wild-type NPR1, expression of Nprl (SA) provides immune gene expression and resistance while maintaining improved or normal plant growth.
NPR1 is a master regulator of the defense transcriptome induced by the plant immune signal salicylic acid. Despite the important role of NPR1 in plant immunity, understanding of its regulatory mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding domain. Crystal structure analysis reveals a unique zinc-finger motif in BTB for interacting with ankyrin repeats and mediating NPR1 oligomerization. We found that, after stimulation, salicylic-acid-induced folding and docking of the salicylic-acid-binding domain onto ankyrin repeats is required for the transcriptional cofactor activity of NPR1, providing a structural explanation for a direct role of salicylic acid in regulating NPR1-dependent gene expression. Moreover, our structure of the TGA32-NPR12-TGA32 complex, DNA-binding assay and genetic data show that dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form an enhanceosome. The stepwise assembly of the NPR1-TGA complex suggests possible hetero-oligomeric complex formation with other transcription factors, revealing how NPR1 reprograms the defense transcriptome.
As a positive regulator that is required for systemic acquired resistance mediated by salicylic acid (SA) in plants, the NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) gene was identified more than two decades ago in Arabidopsis. Since then, the central role of NPR1 in controlling plant responses against a broad spectrum of pathogens has been firmly established. Notably, overexpression of Arabidopsis NPR1 in a wide range of plant species, including many crops, has been shown to enhance disease resistance against a variety of pathogens and stresses, indicating that the protein has intrinsic properties underlying its broad protective functions. Despite its importance in plant immunity and potential agricultural application, the molecular architecture of NPR1 has remained unclear, hindering mechanistic understanding of its regulation and function.
A. The cryo-EM structure of apo NPR1. As the first step towards structural analysis of NPR1, we expressed and purified the full-length Arabidopsis NPR1 using insect cells. Interestingly, this ˜66 kDa protein eluted between standards of 440 kDa and 158 kDa in size-exclusion chromatography (
The structure of the NPR1 dimer was determined using single-particle cryo-electron microscopy (cryo-EM) at a resolution of 3.8 Å (
After the BHB is an extended linker loop connecting the BHB to ankyrin repeats (ANKs) (
Beyond the ANKs, no density can be observed for the C-terminal putative SA-binding domain (SBD), consistent with it being disordered as revealed by small-angle X-ray scattering (SAXS) measurements (
B. NPR1 contains a zinc finger. As EM densities in some functionally important regions of NPR1 did not provide sufficient resolution for confident model building, we crystallized NPR1 (ASBD) (Thr39-Asp410), which diffracted to 3.06 Å (Extended Data Table 2). X-ray fluorescence scanning revealed the presence of Zn2+ ions in the protein crystal, but not in the buffer (
As part of its role in maintaining the structural integrity, the zinc finger bridges the BTB domain to ANK4 through a network of polar interactions (
Moreover, the formation of the zinc finger, with side chains of Cys155 and His157 pointing inwards to coordinate Zn2+, pivots Cys156 directly into solvent (
C. SA induces docking of the SBD onto ANK3 4. Even though NPR1 contains all of the SA-binding residues in the SA receptor NPR4, and binds to SA with a sub-micromolar dissociation constant (Kd) value, its binding capacity is low, suggesting that only a small percentage of the recombinant SBD has the ability to bind to SA in vitro, possibly due to the absence of a chaperone. To mitigate this problem, we refolded full-length NPR 1 in the presence of 0.2 mM SA and subjected it to extensive purification before preparing cryo-EM samples.
Analysis of cryo-EM 2D class averages of the NPR1-SA complex revealed similar features of the BTB-BHB-ANKs architecture as those in apo NPR1, confirming that NPR1 was properly refolded (
The reconstructed 3D density map revealed that the SBD largely consists of helices (
To test whether perturbation of the SBD-ANK interaction can affect NPR1 function, we mutated hydrophobic ANK residues at the interface (L346D, L393D and 1397D;
D. NPR1 bridges two TGA3 dimers through ANK1. To elucidate how SA-induced NPR1 activates TGA transcriptional activity, we determined the cryo-EM structure of NPR1 in complex with TGA3 in the presence of DNA carrying the TGA cis-element as-1 (LS7). Supporting the complex formation, extra densities are readily visible near ANK1 of NPR1 at one or both sides of the NPR1 dimer (
Surprisingly, EM analysis revealed that each extra density represents a dimer of the TGA3 C-terminal domain, which, to the best of our knowledge, has not been reported to form a stable dimer. Owing to its role of engaging NPR1, we named it the NPR1-interacting domain (NID) (
NPR1-TGA3 binding is mediated by elaborate interactions (
In the NPR1 complex with TGA3, weak density of the SBD can also be visualized (
E. The NPR1 dimer is required in plant immunity. The structure of the TGA32-NPR12-TGA32 complex, with the dimeric NPR1 engaging two TGA3 dimers, suggests that NPR1 may serve as a transcription cofactor by bridging two transcription factor complexes in an enhanceosome. Consistent with this hypothesis, the as-/element is significantly enriched in the promoters of top 100 SA-induced genes, and 77 contain at least two as-/elements that are frequently separated by <50 bp (
To test our hypothesis genetically, we designed a dimerization-deficient npr1(dim) mutant by introducing L49D/F53D/V56D/V83K mutations into the α0 and A1 helices of the NPR1 BTB dimer interface (
Before testing the transcriptional activity of npr1 (dim), we evaluated its ability to form a quiescent homo-oligomer, which has been shown to prevent NPR1 nuclear translocation. We found that npr1(dim) was deficient in oligomer formation and had increased nuclear accumulation (
This study establishes a structural framework for explaining data from over two decades of NPR1 studies (
A. Cryo-EM Studies of apo NPR1, NPR1-SA and the npr1(dim) Mutant
1. Cloning, expression and purification using insect cells. Full-length Arabidopsis thaliana NPR1 and the corresponding npr1 (dim) mutant (L49D/F53D/V56D/V83K) were cloned into the pFASTBac1 vector as a His10-GST-NPR1-Strep fusion protein containing a PreScission pro-tease site after GST. Baculovirus was generated and amplified according to the manufacturer's protocol (Thermo Fisher Scientific) using Sf9 cells (Expression Systems). For protein expression, Trichoplusiani (High Five) cells were cultured in the ESF 921 insect cell culture medium (Ex-pression Systems) at 27° C.; the suspension cell culture was infected with a high-titer baculovirus stock and collected after 50 h by centrifugation at 200 g for 5 min. Cell pellets were resuspended and lysed by sonication in the purification buffer (25 mM HEPES pH 7.5 and 150 mM NaCl) and supplemented with protease inhibitors (15 μM leupeptin, 1 μM pepstatin A, 2 μM E-64, 0.1 μM aprotinin, 1 mM phenylmethylsulphonyl fluoride) and DNase I. After centrifugation at 15,000 rpm and 4° C. for 15 min, the fusion protein was purified using the Strep-Tactin Superflow Plus resin (Qiagen). After PreScission protease cleavage to remove the His10-GST tag, the WT protein or the npr l (dim) mutant were further purified by passing it through the Talon resin (Takara Bio) and eluted from the column in the presence of 10-15 mM imidazole in the lysis buffer, whereas the His10-GST tag was retained on the cobalt column.
WT NPR1 was concentrated and further purified by size-exclusion chromatography using the Superose 6 increase 10/300 GL column (Cytiva) pre-equilibrated with the purification buffer containing 2 mM DTT. Peak fractions were concentrated and cross-linked using 2 mM BS3 (Thermo Fisher Scientific) at room temperature for 30 min, and the cross-linked sample was further purified by size-exclusion chromatography (Superose 6 increase 10/300 GL; Cytiva) for cryo-EM grid preparation.
The npr1(dim) mutant used for cryo-EM grid preparation was purified using identical procedures as the WT NPR1.
The SA-bound NPR1 was obtained by refolding NPR1 in the presence of SA. In brief, purified NPR1-Strep was concentrated and dialyzed against 8 m urea followed by dialysis in the refolding buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT and 0.2 mM SA. The refolded sample was purified by size-exclusion chromatography using the Superose 6 increase 10/300 GL column (Cytiva) pre-equilibrated with the refolding buffer. The peak fraction was then concentrated and cross-linked with 2 mM BS3 as described above; the cross-linked sample was repurified by size-exclusion chromatography (Superose 6 increase 10/300 GL; Cytiva) for cryo-EM grid preparation.
2. Cryo-EM sample preparation and data collection. The cryo-EM grids containing NPR1 were prepared using the Leica EM GP Automatic Plunge Freezer at 10° C. and 95% humidity. The UltrAuFoil R1.2/1.3 300-mesh grids were glow-discharged using the Tergeo-EM plasma cleaner (Pie Scientific). A 3 μl sample of BS3 cross-linked NPR1 (˜0.6 mg ml-1) was applied to the grids and blotted for 2-3 s with Whatman No. 1 filter paper (Whatman International) to remove excess sample and plunge-frozen into liquid ethane cooled by liquid nitrogen. A total of 1,638 micrograph stacks were collected with SerialEM35 on a Talos Arctica microscope at 200 kV equipped with a K2 Summit direct electron detector (Gatan), at a nominal magnification of 45,000× and defocus values from −2.5 μm to −0.8 μm, yielding a pixel resolution of 0.9317 Å px-1. Each stack was exposed for 8.4 s with an exposing time of 0.14 s per frame, resulting in 60 frames per stack. The total dose was approximately 60 e-Å-2 for each stack.
The cryo-EM grids containing NPR1-SA were prepared similarly to that of NPR1. A total of 9,486 micrograph stacks were collected with SerialEM on a Titan Krios microscope (Thermo Fisher Scientific) at 300 kV equipped with a K3 direct electron detector (Gatan), at a nominal magnification of 64,000× and defocus values from −2.5 μm to −0.8 μm, yielding a pixel resolution of 0.5325 Å px-1. Each stack was exposed for 8.069 s with an exposing time of 0.134 s per frame, resulting in 60 frames per stack. The total dose was approximately 60 e-Å-2 for each stack.
The cryo-EM grids containing the npr1 (dim) mutant were prepared similarly except that C-flat R1.2/1.3 300-mesh grids (Protochips) coated with gold were used. A total of 3,500 movies were recorded with a defocus range from −2.0 μm to −0.8 μm on the Talos Arctica electron microscope (Thermo Fisher Scientific) at 200 kV equipped with a K3 Summit direct electron detector (Gatan) at a nominal magnification of 45,000× (0.88 Å px-1) using SerialEM. Each stack was exposed for 2.716 s with an exposing time of 0.045 s per frame. The total dose was approximately 55 e-Å-2 distributed over 60 frames.
3. Cryo-EM data processing and model building. For cryo-EM data analysis of apo NPR1, movie alignment was performed in RELION36 and the parameters of the contrast transfer function (CTF) were determined on the motion corrected sum of frames using CTFFIND4.1 (ref. 37). Micrographs (1,391) were selected on the basis of the CTF fit resolution using a cut-off of 4.5 Å. A total of 615,647 NPR1 particles were boxed out using template-free particle picking. Two consecutive rounds of 2D classification were performed to clean the extracted particles. A total of 125,604 clean particles were used for further processing. The processing flow chart is shown in
The NPR1-SA dataset was processed in a similar manner to that described above. A total of 5,424 micrographs were selected on the basis of the CTF fit resolution with a cut-off value of 4.5 Å. Around 5.6 million particles were used for 2D classification after curating the template-free blob picking results. After three rounds of cleaning with 2D classification, 120,893 particles were selected and imported back to RELION for refinement. The apo NPR1 map and mask were used as the 3D reference for the consensus refinement with C2 symmetry focusing on the NPR1 dimer core domain, which resulted in a 4.0 Å map. 3D classification without alignment was conducted (no mask or symmetry applied). One good class containing 117,529 particles was selected and processed for local refinement with a shape mask yielding an overall resolution of 3.9 Å. A summary of the processing flow chart is shown in
For the npr1 (dim) dataset, movie alignment and the CTF estimation were conducted using cryoSPARC. A total of 2,377 micrographs were selected based on the CTF fit resolution cut-off of 4 Å. Manually picked particles were used as input for Topaz particle picking41. Rounds of 2D classification were performed to isolate good classes. Two ab initio models were generated and processed a subset of 18,795 particles showing better structural features was selected for homogeneous refinement.
4. Cloning, overexpression and purication of the NPR1 SBD for biological SAXS. The Arabidopsis NPR1 SBD (Pro412-Arg593) was cloned into the pFASTBacl vector as a His10-GST-NPR1-Strep fusion protein containing a PreScission protease site following GST. The baculovirus generation, protein expression and purification followed similar procedures as described for NPR1. The protein was concentrated to 1 mg ml-1 in a buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl and 2 mM DTT for biological SAXS measurements at beamline 12-ID-B of the Advanced Photon Source (APS).
1. Expression and purification of Arabidopsis NPR1 and TGA3 using Escherichia coli. Full-length Arabidopsis NPR1 was cloned into the pTrcHis vector (Thermo Fisher Scientific) as a His10-MBP-fusion protein containing a PreScission protease site following MBP and a C-terminal Strep tag (Strep3) with the last Lys residue of the Strep tag mutated to Arg. Transformed BL21 (DE3) E. coli cells were grown in Luria-Bertani (LB) medium at 37° C. to an optical density at 600 nm (OD600) of 0.5, induced with 0.1 mM IPTG, 0.1 mM ZnSO4 and 0.2 mM SA at 18° C. overnight. The purification of the E. coli-expressed NPR1 was performed as described above for the insect-cell-expressed NPR1, except that the purification buffer contained 0.2 mM SA.
Arabidopsis TGA3 (Asn87-Thr384) was cloned into the pTrcHis vector (Thermo Fisher Scientific) as a His10-MBP fusion protein containing a PreScission protease site following MBP and a C-terminal tandem Flag-HA tag. Transformed BL21 (DE3) E. coli cells were grown in LB medium at 37° C. to an OD600 of 0.5 and induced with 0.1 mM IPTG at 18° C. overnight. The purification was performed as described above for NPR1.
2. NPR1 TGA3 LS7-DNA cryo-EM sample preparation. The purified NPR1-Strep3 (20 μM), TGA3-Flag-HA (48 μM) and the LS7 DNA containing a palindromic TGA-binding site (underlined in 5′-CACTATTTTACTGACGTCATAGATGTGGCG-3′, 57.6 μM) were mixed and incubated at 4° C. for 30 min. The protein-DNA complex was cross-linked with 1.5 mM BS3 (Thermo Fisher Scientific) at room temperature for 30 min and quenched with 50 mM Tris (pH 8.0). The protein-DNA mixture was further purified using a Strep-Tactin column (Qiagen) and size-exclusion chromatography (Superose 6 in-crease 10/300 GL, Cytiva) in a buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT and 0.2 mM SA.
3. Cryo-EM sample preparation and data collection. Cryo-EM grids containing the NPR1-TGA3-LS7-DNA complex were prepared using the Leica EM GP2 Automatic Plunge Freezer at 4° C. and 85% humidity. The UltrAuFoil R1.2/1.3 300-mesh grids (Quantifoil) were glow-discharged using the PELCO easiGlow Glow Discharge Cleaning System (Ted Pella). A 3 μl sample of NPR1-TGA3-LS7-DNA (˜1.3 mg/ml) was applied to the grid, incubated for 60 s in the chamber and blotted for 2.4 s with Whatman No. 1 filter paper (Whatman International) to re-move excess sample, and then plunge-frozen into liquid ethane cooled by liquid nitrogen. A total of 9,442 micrograph stacks were collected with SerialEM on a Titan Krios microscope (Thermo Fisher Scientific) at 300 kV equipped with a K3 direct electron detector (Gatan), at a nominal magnification of 81,000× and defocus values from −2.0 μm to −1.0 μm, yielding a resolution of 1.066 Åfipx-1. Each stack was exposed for 8.3 s with an exposing time of 0.138 s per frame, resulting in 60 frames per stack. The total dose was approximately 60 e−Å−2 per stack.
4. Cryo-EM data processing and model building. For cryo-EM data analysis of TGA32-NPR12-TGA32, movie alignment and the CTF estimation were conducted using cryoSPARC38. A total of 8,988 micrographs were selected based on the CTF fit resolution using a cut-off value of 4.0 Å. A total of around 14 million particles were boxed out using the NPR1 as the template. Multiple rounds of 2D classification and selection were performed to clean the extracted particles. A total of around 881,000 clean particles were used for further processing. The processing flow chart is shown in
Similarly, 536,200 particles were selected for reconstruction of the NPR12-TGA32 complex. After ab initio reconstruction and 3D classification/heterogeneous refinement, 287,150 particles were selected for homogenous and non-uniform refinement, yielding an overall resolution of 3.6 Å. Focused refinement of NPR12 (excluding the weak ANK region), TGA32 and the weak ANK region extended local resolutions to 3.4 Å, 3.6 Å and 4.0 Å, respectively.
The de novo model building was conducted using COOT. Structure refinement was performed using PHENIX in real space with secondary structure and geometry restraints.
5. X-ray crystallography studies of NPR1 (ΔSBD)) and the TGA3 NID. Arabidopsis NPR1 (2SBD) (Thr39-Asp410) was cloned into the pTrcHis vector (Thermo Fisher Scientific) as a His10-MBP fusion protein containing a PreScission protease site following MBP and a C-terminal Strep tag. Expression and purification of NPR1 (2SBD) was performed using similar procedures to those described above for E. coli-expressed NPR1, except that the size-exclusion chromatography buffer contained 20 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM TCEP. Protein crystals were grown using the sitting-drop vapor diffusion method at 20° C. Each drop was prepared by mixing 1 μl of the protein solution at 6 mg ml-1 with 1 μl of the reservoir solution containing 0.1 M HEPES (pH 7.5), 19% PEG 3350, 0.25 m tri-sodium citrate. Diffraction-quality protein crystals were collected after 1 week and cryoprotected with the reservoir solution containing 25% glycerol.
The Arabidopsis TGA3 NID (Ala160-Thr384) was cloned into the pTrcHis vector (Thermo Fisher Scientific) as a His10-MBP fusion protein containing a PreScission protease site following MBP and a C-terminal tandem Flag-HA tag. The purification procedure was per-formed similarly to that of Arabidopsis TGA3 (Asn87-Thr384) except that the size-exclusion chromatography buffer contained 20 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM TCEP. Protein crystals were grown using the sitting-drop vapor diffusion method at 20° C. Each drop contained 1 μl of the protein solution mixed with 1 μl of the reservoir solution (0.1 m sodium acetate at pH 5.0, 20% (v/v) MPD). Diffraction quality protein crystals were collected after 1 week and cryoprotected with the reservoir solution also containing 25% glycerol.
X-ray fluorescence and diffraction datasets of NPR1 (2SBD) were collected at the Northeastern Collaborative Access Team (NECAT) 24-ID-C beamline at the Advanced Photon Source at the Argonne National Laboratory. The X-ray diffraction data were processed using XDS42. The phase information was obtained by molecular replacement with the PHASER module in the PHENIX suite using the cryo-EM coordinates of apo NPR1 and the TGA3 subunit in the NPR12-TGA32 complex as the search model for NPR1 (2SBD) and the TGA3 NID, respectively. Iterative model building and refinement were performed using COOT and PHENIX. For NPR1 (2SBD), the final model has 95.13% residues in the favored region, 4.87% in the allowed region and 0.0% in the outlier region of the Ramachandran plot. For the TGA3 NID, the final model has 99.01% residues in the favored region, 0.99% residues in the allowed region and 0.0% in the outlier region of the Ramachandran plot.
C. Lipid Extraction from the TGA3 NID and LC ESI MS Analysis
The fatty acid was extracted from the purified TGA3 NID protein using the two-phase Bligh-Dyer method. The extract was analyzed using normal-phase liquid chromatography-electrospray ionization/MS (LC-ESI/MS) in negative mode using an Agilent 1200 Quaternary LC system coupled to a high-resolution TripleTOF5600 mass spectrometer (Sciex) as previously described. Data acquisition and analysis were performed using the Analyst TF1.5 software (Sciex).
D. In vitro NPR1 Oxidation
WT NPR1 BTB/BHB (Thr39-Lys262) and the npr1(C156A), npr1 (C82A) and npr1(C212V/C216A/C223L) mutants were cloned into the pTrcHis vector (Thermo Fisher Scientific) as His10-MBP fusion proteins with a PreScission protease site following MBP and a C-terminal Strep tag. The proteins were expressed and purified similarly to the NPR1 (2SBD) as described above. The NPR1 BTB/BHB WT and mutant proteins at a concentration of 25 μM were incubated with 1 mM hydrogen peroxide in a buffer containing 125 mM HEPES and 150 mM NaCl at pH 8.0 at room temperature for 45 min. The oxidized samples were incubated with 50 mM iodoacetamide for 5 min, mixed with 4× SDS loading dye and analyzed using SDS-PAGE in the absence of any reducing reagent.
E. Promoter Analysis for as-1 Elements
For the promoter analysis, the scanMotifGenomeWide.pl function in HOMER (v.4.11) was initially used to generate a bed file containing all as-1 elements in the Arabidopsis genome. Moreover, using R (v.4.1.1) and the Araport 11 GTF, bed files for the 3,000 bp upstream regions of the top 100 induced and of all the uninduced genes in response to 8 h of treatment by the SA analogue benzothiadiazole were generated. The window function in bedtools (v.2.30.0) with a window of 0 bp was used to intersect both promoter and as-/element bed files to generate bed files containing all as-1 elements within the 3,000 bp upstream regions of the top 100 SA-induced genes and uninduced genes. The number of as-1 elements in each promoter was counted and the as-1-element count distribution of the top 100 SA-induced genes was compared to the distribution of uninduced genes using a Æ2 test. The promoters of the top 100 SA-induced genes were further analyzed for multiple as-1 elements and subset into groups containing 0, 1, 2 or more as-1 elements. The distances between as-1 elements in the same promoter were computed and plotted as a histogram binned every 50 bp using R (v.4.1.1).
The npr1(dim) mutant (L49D/F53D/V56D/V83K) was cloned into the pTrcHis vector (Thermo Fisher Scientific) as a His10-MBP fusion protein containing a PreScission protease site following MBP and a C-terminal Strep tag, and was expressed and purified similarly to the WT NPR 1 protein. Purified TGA3 (Asn87-Thr384) at a concentration of 0.6 μM and WT NPR1 or the npr1(dim) mutant at a concentration of 8 μM were used in the EMSA with the 6-fluorescein (6-FAM)-labelled oligonucleotide probes (200 nM) containing the LS5 and LS7 as-I cis-elements of the SA-responsive PRI gene promoter (LS5/LS7, 6-FAM-C-gggCTATGACGTAAGTAAAATAGTGACGTAGAGAggg) or DNA sequences with the two as-1 regions replaced with adenosines (LS5/LS7, 6-FAM-C-gggCTAAAAAAAAAGTAAAATAGAAAAAAAAAGAggg), with the LS7 as-1 element replaced with adenosines (LS5/LS7, 6-FAM-C-gggCTATGACGTAAGTAAAATAGAAAAAAAAA-GAggg) or with the LS5 as-1 element replaced with adenosines (LS5/LS7, 6-FAM-C-gggCTAAAAAAAAAGTAAAATAGTGACGTAGAGAggg) as well as their complementary oligonucleotides (Integrated DNA Technologies). The uppercase bases indicate the LS5/LS7 sequence; the lowercase bases indicate overhangs outside the LS5/LS7 sequence; and bases replaced with adenosines are indicated with A's in the small capital font. Pairs of complementary DNA strands were annealed before the binding reaction. For each binding reaction, the desired protein combination was mixed with the target DNA probe together with 30 ng/μL of poly (dI-dC) (non-specific competitor, Thermo Fisher Scientific) in a binding buffer containing 20 mM HEPES (pH 8.0), 300 mM NaCl, 10% glycerol, 1 mM DTT and 0.2 mM SA. The mixture was incubated at 4° C. for 30 min and then run on a 3.8% native polyacrylamide gel in the TB buffer (100 mM Tris-borate pH 8.3, 10% glycerol). After electrophoresis, the gel was scanned with the Typhoon FLA 7000 imager (GE Healthcare).
A. thaliana mutants, and transgenic plants are all in the Col-0 ecotype background. Arabidopsis mutant npr1-2 (ref. 6), and transgenic lines expressing NPR1-GFP in the npr1-2 background and npr1(C82A)-GFP in the npr1-1 background were described previously. Transgenic Arabidopsis expressing npr1 (dim)-GFP in the npr1-2 background was generated and two independent lines homozygous for the transgene were used. Seeds were stratified at 4° C. for 3 days before sowing and plants were grown under 12 h-12 h light-dark cycles at 22° C. N. benthamiana plants were grown under the same conditions.
The coding sequences of WT AtNPR1 (AT1G64280) and AtTGA3 (AT1G22070), as well AtNPR1 mutants generated with the QuikChange II site-directed mutagenesis kit (Agilent), were subcloned into the pDONR207 gateway donor vector and confirmed by sequencing. The npr1 mutants were designed based on the structural information. The SBD-ANK cross-linked mutant npr1(Q400C/E401L/R506C) was designed based on the proximity and geometry of Gln400 and Arg506 side chains in the docked conformation of the NPR1 SBD onto ANKs. The E401L mutation was introduced to compensate for the loss of the Glu401-Arg506 hydrogen bond and avoid burial of a charged residue at the protein interface. The obtained entry vectors carrying WT or mutated genes were recombined into the plant binary vectors pK7FWG2, pLN462 and pSITE-4NB to generate C-terminal eGFP, HA and mRFP fusion proteins, respectively. The pPR1: DUAL-LUC reporter was described previously.
For plant transformations, the Agrobacterium tumefaciens strain GV3101 was transformed with plant binary vectors carrying the indicated transgenes. For stable expression in Arabidopsis, a floral dipping method was used. For transient expression in N. benthamiana, the Agrobacterium carrying the indicated construct was cultured over-night at 28° C. in LB broth medium supplemented with appropriate antibiotics: spectinomycin (100 μg ml-1), kanamycin (50 μg ml-1), gentamycin (50 μg ml-1) and rifampicin (25 μg ml-1). The obtained culture was re-inoculated at 1:10 into fresh growth medium with antibiotics and grown for another 4 h. Cells were then centrifuged at 1,600g for 10 min, and inoculum was prepared by resuspending cells to an OD600 of 1 in double-distilled water containing 200 μM acetosyringone (Sigma-Aldrich). The co-expression assays were per-formed with a 1:1 mixture of Agrobacteria inoculums. The inoculum was pressure-infiltrated into N. benthamiana leaves at the abaxial side using 1 ml syringe without the needle.
Expression of the defense genes PRI, WRKY18, WRKY38 and WRKY62 in transgenic Arabidopsis was performed by quantitative PCR (qPCR). Total RNA was extracted from fresh leaf tissue with Trizol reagent (Sigma-Aldrich). cDNA was synthetized using the SuperScript III cDNA Synthesis kit (Thermo Fisher Scientific). qPCR was performed using the FastStart Universal SYBR Green Master Kit (Roche) using the Mastercycler ep realplex (Eppendorf) system. A list of the gene-specific primers used for qPCR is provided in Supplementary Table 1.
The PRI promoter activity was tested using dual-luciferase (DUAL-LUC) reporter assay as described previously. The pPRI: DUAL-LUC reporter was transiently co-expressed in N. benthamiana together with HA-fused NPR1 or its mutants as effectors. The free HA effector was used as a negative control. Plants were treated with 2 mM SA for 24-30 h at 1 day after inoculation. At 2 days after inoculation, leaf discs were collected, ground in liquid nitrogen, and lysed with the passive lysis buffer (PLB) of the Dual-Luciferase Reporter Assay System (Promega, E1910). Lysate was centrifuged at 12,000g for 1 min, and 10 μl was taken for measuring firefly LUC (F-LUC) and Renilla LUC (R-LUC) activities according to the manufacturer's instructions using the Victor3 plate reader (Perkin Elmer). At 25° C., substrates for F-LUC and R-LUC were added using the automatic injector and, after 3 s shaking and 3 s delay, the signals were captured for 3 s and recorded as counts per second. To obtain the PRI promoter activity, the ratio of F-LUC over the R-LUC activities was calculated for each effector.
Recombinant protein analysis, co-immunoprecipitation and cell-free oligomerization were performed using total protein extracts from the transient expression assay in N. benthamiana as described previously. Interaction analysis of NPR1-HA with TGA3-mRFP or NPR1-GFP (self-interaction), was performed in vivo by transiently co-expressing the two proteins in N. benthamiana followed by treatment with water (mock) or 1 mM SA for 5 h. Total protein was extracted and used for co-immunoprecipitation with anti-RFP or anti-HA beads, respectively. In the NPR1-HA/TGA3-mRFP interaction experiments the npr1-1-HA was used as a negative control. Interaction between GST-NPR1 and Myc-CUL3A was performed by co-expression in E. coli as described. For western blotting under reducing conditions, the SDS sample buffer was added to the protein extracts from a 4× stock solution supplemented with 50 mM DTT and 715 mM B-mercaptoethanol. For western blotting under non-reducing conditions, a DDT-and B-mercaptoethanol-free SDS sample buffer was used. The blots were probed with anti-GFP (Clontech, 632381, 1:5,000), anti-HA (BioLegend, 901513, 1:1,000), anti-RFP (ChromoTek, 6G6, 1:5,000), anti-cMyc (Santa Cruz, SC-40, 9E10, 1:1,000) or anti-GST-HRP (GE Healthcare, RPN1236V, 1:10,000) primary antibodies. Secondary anti-mouse-HRP antibodies (Abcam, Ab97040) were used at 1:20,000.
For the cell-free oligomerization assay, total proteins were extracted with lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% Triton X-100, 0.2% Nonidet P-40, 5% glycerol) supplemented with 1 mM PMSF, 100 μM MG132, EDTA-free protease inhibitor cocktail and 14.3 mM B-mercaptoethanol. The obtained lysate was diluted 10-fold with the lysis buffer supplemented with 1 mM PMSF, 100 μM MG132 and EDTA-free protease inhibitor cocktail. The samples were incubated in the dark at 4° C. for 0, 1, 2 and 3 h time periods before SDS-PAGE analysis under reducing or non-reducing conditions.
For soil-grown Arabidopsis plants, SA treatment was carried out with spray at 1 mM concentration 24 h before gene expression analysis or bacterial infection. For bacterial infection, P. syringae pv. maculicola ES4326 (Psm ES4326) was grown for 2 days on solid King's B medium supplemented with 100 μg/mL streptomycin. An inoculum was prepared by resuspending cells in 10 mM MgSO4 to obtain an OD600 of 0.001. Bacteria were pressure-infiltrated into mature leaves of three-week old Arabidopsis plants and bacterial growth was assessed at 3 days after inoculation by collecting leaf disks from eight infected plants per treatment/genotype, ground in 10 mM MgSO4, and plated at sequential dilutions on King's B medium plates supplemented with 100 μg ml-1 streptomycin. At 2 days after inoculation, the bacterial colonies were scored.
Confocal fluorescence microscopy was performed using the Zeiss 880 airyscan inverted confocal microscope with a ×40/1.2 NA water correction objective. eGFP was excited with a 488 nm argon laser and emission was collected with a 505-530 nm bandpass filter. NPR1-GFP or npr1(dim)-GFP localization was tested in leaf epidermal cells of N. benthamiana after transient expression. To determine nucleocytoplasmic partitioning, the fluorescence intensities from the entire cell (total) and from nuclei were quantified from 15-20 randomly sampled unsaturated confocal images (512×512 px, 225×225 μm) using an automated image analysis algorithm implemented in the ImageJ software as previously described. The obtained intensity values were used to calculate the ratio of nuclear signal over total signal in Microsoft Excel.
Data plotting and statistical tests were performed in GraphPad Prism 8. Statistical parameters such as mean±s.d. and 95% confidence intervals are indicated in the figure legends. In the graphs, asterisks and lowercase letters indicate statistical significance tested either by Stu-dent's t-tests (two groups) or ANOVA (multiple groups) at a significance threshold of P<0.05. The number of biological replicates is indicated for all the data in figure captions. In the figures, the individual datasets (
The cryo-EM structures of the apo NPR1, NPR12-TGA32 and TGA32-NPR12-TGA32, and X-ray crystal structures of the NPR1 (2SBD) and TGA3 NID have been deposited at the PDB (www.pdb.org) under accession codes 7MK2, 7TAD, 7TAC, 7MK3 and 7TAE, respectively. The cryo-EM density maps of apo NPR1, NPR1-SA, NPR12-TGA32 and TGA32-NPR12-TGA32 have been deposited to the Electron Microscopy Data Bank under accession codes EMD-23884, EMD-23885, EMD-25771 and EMD-25769, respectively. There are no restrictions on data availability. Source data are provided with this paper.
This application claims the benefit of U.S. Provisional Application No. 63/463,338, filed May 2, 2023, which is incorporated herein by reference.
This invention was made with Government support under Federal Grant nos. R35 GM118036, GM145026, and GM115355 awarded by the National Institutes of Health National Institute of General Medical Sciences (NIH/NIGMS). The Federal Government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63463338 | May 2023 | US |
