Patent Application
20230227838
This invention was made with Government support under Federal Grant no. R35GM118036 awarded by the National Institutes of Health. The Federal Government has certain rights to this invention.
The Sequence Listing written in file 560198_SeqListing_ST25.txt is 257 kilobytes in size, was created Jun. 15, 2021, and is hereby incorporated by reference.
An essential feature of immunity is to ensure defense against pathogens without collateral damage to self. In plants, pathogen effector-triggered immunity (ETI) often results in programmed cell death, which is restricted through the function of NPR1, a positive regulator of systemic acquired resistance. Despite the importance of this protein and the extent to which it has been studied, the biochemical activities of NPR1 that promote both defense and cell survival remain unclear.
The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.
The present disclosure provides, is based, in part on the discovery by the inventors that NPR1 promotes cell survival by targeting substrates for ubiquitination and degradation through salicylic acid (SA)-driven phase separation into cytoplasmic condensates. Further, the inventors show that NPR1 condensates are enriched in cell death regulators including nucleotide-binding leucine-rich repeat immune receptors, redox metabolism proteins, DNA damage repair and protein quality control machineries. Phase separation of NPR1 is required for recruitment of the Cullin 3 RING E3 ligase complex to the condensates and NPR1 can promote cell survival by degrading EDS1 and specific WRKY transcription factors required for ETI. The discovery of distinct functional groups of proteins in the SA-induced NPR1 condensates, herein termed “SINCs”, suggests that NPR1 modulates survival by converging multiple stress-responsive processes in this quasi organelle.
Other aspects and embodiments of the present disclosure provide compositions and methods for promoting cell survival of a cell comprising, consisting of, or consisting essentially of modulating NPR1 in the cell. In some embodiments, modulating may comprise upregulating the expression of and/or enhancing the function of, NPR1 in the cell. In another embodiment, the cell comprises a plant cell.
In one aspect, provided are nucleic acids encoding a npr1 protein, wherein the npr1 protein forms salicylic acid-independent NPR1 condensates. In some such nucleic acids, the nucleic acid is operably linked to one or more expression control elements. In some such nucleic acids, the one or more expression control elements comprise: a promoter, one or more upstream open reading frames (uORFs), or the promoter and the one or more uORFs. In some such nucleic acids, the promoter is selected from the group consisting of: a constitutive promoter, an inducible promoter, a temporally-regulated promoter, a developmentally regulated promoter, a chemically regulated promoter, a tissue-preferred promoter, a tissue-specific promoter, a TBF1 promoter, a 35S promoter, a ubiquitin promoter, a tCUP cryptic constitutive promoter, a Rsyn7 promoter, a pathogen-inducible promoter, a maize In2-2 promoter, a tobacco PR-1a promoter, a glucocorticoid-inducible promoter, an estrogen-inducible promoter, a tetracycline-inducible promoter, a tetracycline-repressible promoter, a T3 promoter, a T7 promoter, and a SP6 promoter. In some such nucleic acids, the promoter is the TBF1 promoter. In some such nucleic acids, the uORF comprises a TBF1 gene uORF. In some such nucleic acids, the expression control elements comprise the TBF1 promoter and one or more TBF1 uORFs.
In some such nucleic acids, the npr1 protein is an A. thaliana npr1 protein comprising one or more mutations. In some such nucleic acids, the npr1 protein comprises one or more mutations in at least one redox-sensitive intrinsically disordered region (RDR). In some such nucleic acids, the one or more mutations comprises a substitution of one or more cysteines in at least one RDR, a deletion of one or more cysteines in at least one RDR, or a combination thereof. In some such nucleic acids, the one or more cysteines are located in a region corresponding to residues 140-160, 368-404, or 510-539 of SEQ ID NO: 1. In some such nucleic acids, the one or more cysteines are located in the region corresponding to residues 368-404 of SEQ ID NO: 1. In some such nucleic acids, the npr1 protein has increased interaction with CUL3 compared to wild-type NPR1 in the absence of salicylic acid. In some such nucleic acids, the one or more mutations reduces the redox-sensitivity of the RDR. In some such nucleic acids, the npr1 protein comprises a mutation of a cysteine corresponding to the cysteine at position 378, of SEQ ID NO: 1, position 385 of SEQ ID NO: 1, position 394 of SEQ ID NO: 1, positions 378 and 385 of SEQ ID NO: 1, positions 378 and 394 of SEQ ID NO: 1, positions 385 and 394 of SEQ ID NO: 1, or positions 378, 385, and 394 of SEQ ID NO: 1. In some such nucleic acids, the npr1 protein comprises the mutation of the cysteine corresponding to the cysteines at positions 378, 385, and 394 of SEQ ID NO: 1. In some such nucleic acids, the mutation of the cysteine comprises an alanine substitution. In some such nucleic acids, the npr1 protein comprises a mutation of one or more cysteines corresponding to the cysteines at positions 150, 155, 156, and 160 of SEQ ID NO: 1.
In some such nucleic acids, the nucleic acid encodes a ΔCTD npr1 ACTD protein, a BTB domain npr1 protein, or a sim3 npr1 protein.
In some such nucleic acids, the nucleic acid encodes: (a) a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160 or an ortholog thereof; or (b) a protein having at least 70% identity to a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160. In some such nucleic acids, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 134.
In another aspect, provided are plants or plant cells expressing any of the above nucleic acids. In some such plants or plant cells, the plant is a monocot or a dicot. In some such plants or plant cells, the plant is a food crop plant, a biofuel plant, a corn plant, a legume plant, a bean plant, a rice plant, a soybean plant, a cotton plant, a sugarcane plant, a tobacco plant, a palm oil plant, a date palm, a wheat, a vegetable plant, a squash plant, a Solanaceae plant, a tomato, a banana plant, a potato plant, a pepper plant, a moss plant, a parsley plant, a sunflower plant, a mustard plant, a sorghum plant, a millet plant, a citrus plant, an apple plant, a strawberry plant, a rapeseed plant, a cabbage plant, a cassava plant, a coffee plant, a sweet potato plant, a jatropha plant, or a switchgrass plant. In some such plants or plant cells, the plant expresses a wild-type NPR1 gene. In some such plants or plant cells, the plant does not express a wild-type NPR1 gene.
In another aspect, provided are methods of increasing stress tolerance in a plant, comprising expressing in the plant any of the above nucleic acids. In some such methods, the stress is biotic or abiotic stress. In some such methods, the biotic stress comprises pathogen infection. In some such methods, the pathogen is a bacteria or a virus. In some such methods, the abiotic stress comprises high temperature (heat shock) stress, low temperature (cold shock) stress, oxidative stress, or DNA damage. In some such methods, increasing stress tolerance comprises one or more of: decreasing programmed cell death, decreasing effector-triggered immunity (ETI)-induced cell death, increasing formation of NPR1 condensates, and degrading EDS1 and specific WRKY transcription factors required for pathogen ETI. In some such methods, the plant is a monocot or a dicot. In some such methods, the plant is a food crop plant, a biofuel plant, a corn plant, a legume plant, a bean plant, a rice plant, a soybean plant, a cotton plant, a sugarcane plant, a tobacco plant, a palm oil plant, a date palm, a wheat, a vegetable plant, a squash plant, a Solanaceae plant, a tomato, a banana plant, a potato plant, a pepper plant, a moss plant, a parsley plant, a sunflower plant, a mustard plant, a sorghum plant, a millet plant, a citrus plant, an apple plant, a strawberry plant, a rapeseed plant, a cabbage plant, a cassava plant, a coffee plant, a sweet potato plant, a jatropha plant, or a switchgrass plant. In some such methods, the plant expresses a wild-type NPR1 gene. In some such methods, the plant does not express a wild-type NPR1 gene.
Another aspect of the present disclosure provides all that is described and illustrated herein.
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:
(Avr). At 2 dpi, the adjacent halves were infiltrated with 50 μM estradiol, followed by cell death measurement using the conductivity assay. Data are presented as mean±SD. In
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.
“About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
The use herein of the terms “including,” “comprising,” or “having,” 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.”
Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
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.
A nucleic acid is “operably connected” or “operably linked” when it is placed into a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter is connected to the coding sequence or insert site such that it may affect transcription or expression of the coding sequence.
The nucleic acids described herein be operably linked to one or more expression control elements. “Expression control elements” are sequences that modulate expression of the gene, either through modulation of transcription of the gene, modulation of translation on an mRNA transcribed from the gene, or a combination of modulation of transcription and translation. Expression control elements include, but are not limited to, promoters, enhancers, 3′ untranslated sequence, and 5′ untranslated sequences.
The nucleic acids described herein may be operably linked to a promoter or a combination of a promoter and one or more uORFs. The terms “promoter,” “heterologous promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ end of the coding region, or within the coding region of the heterologous coding sequence, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. The typical 5′ promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be the endogenous promoter of an endogenous gene modified to include heterologous R-motif, uORF, and/or 5′ regulatory sequences (i.e., separately or in combination). The promoter may be natively associated with the 5′ UTR chosen, but be operably connected to a heterologous coding sequence.
Promoters include, but are not limited to, constitutive promoters, inducible promoters, temporally-regulated promoters, developmentally regulated promoters, chemically regulated promoters, tissue-preferred promoters, and tissue-specific promoters. A promoter suitable for expression in plants includes, but is not limited to, a TBF1 promoter (from any plant species including Arabidopsis), a 35S promoter (such as from a cauliflower mosaic virus), a ubiquitin promoter, a tCUP cryptic constitutive promoter, a Rsyn7 promoter, a pathogen-inducible promoter, a maize In2-2 promoter, a tobacco PR-1a promoter, a glucocorticoid-inducible promoter, an estrogen-inducible promoter, a tetracycline-inducible promoter, a tetracycline-repressible promoter, a T3 promoter, a T7 promoter, and a SP6 promoter. Those of skill in the art are familiar with a wide variety of additional promoters for use in various cell types. In some embodiments, the promoter includes a plant promoter. In some embodiments, the promoter includes a plant promoter inducible by a plant pathogen or chemical inducer. The promoter may be a seed-specific or fruit-specific promoter.
An upstream open reading frame (uORF) is an open reading frame (ORF) within the 5′ untranslated region (5′ UTR) of an mRNA. uORFs can regulate eukaryotic gene expression, such as through suppression of translation. Translation of the uORF may inhibit or increase downstream expression of the primary ORF (e.g., by translation suppression). As one example, a uORF can be a TBF1 uORF (e.g., Arabidopsis thaliana TBF1), such as uORF1 or uORF2. See, e.g., Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22 (2): 103-112; Xu et al. (2017) Nature 545(7655):491-494; US 2018-0273965; U.S. Pat. No. 10,584,346; US 2015-0113685; U.S. Pat. No. 10,017,773; WO 2013/096567; US 2019-0352664; and WO 2018/144831, each of which is herein incorporated by reference in its entirety for all purposes.
“Salicylic acid-induced NPR1 condensates” (SINCs) are cytoplasmic organelle-like structures that contain NPR1 protein and Cullin 3 RING E3 ligase (CLR3) and are enriched in proteins that regulate death, redox metabolism, DNA damage response, and protein quality control machineries, including ubiquitination. SINC formation in wild-type plants is dependent on NPR1 and salicylic acid. In the absence of salicylic acid, wild-type NPR1 does not induce formation of SINCs. However, in the presence or salicylic acid, wild-type NPR1 initiates formation of condensates.
“Salicylic acid-independent NPR1 condensates” are NPR1 condensates that are functionally similar to SINCs and form in the absence of salicylic acid. Formation of salicylic acid-independent NPR1 condensates is induced by certain mutant npr1 proteins described herein in the absence of salicylic acid.
An “intrinsically disordered region” (IDR) is a region of a protein that does not have, or is not predicted to have, a fixed or ordered three-dimensional structure in the absence of interaction partners. IDRs can be determined or predicted using methods known in the art. IDRs can be predicted using algorithms such as, but not limited to, the IUPred2a algorithm. IUPred2A can be used to predict disordered protein regions using the IUPred2 algorithm and optionally disordered binding regions using ANCHOR2. IUPred2 returns a score between 0 and 1 for each residue in an input protein (amino acid) sequence corresponding to the probability that the given residue is part of a disordered region. IUPred2A is also capable of identifying protein regions that do or do not adopt a stable structure depending on the redox state of their environment.
A “redox-sensitive intrinsically disordered region” (RDR) is an IDR that is sensitive to oxidations. Redox-sensitivity can be determined or predicted using methods known in the art. Redox-sensitivity can be predicted using algorithms such as, but not limited to, IUPred2a algorithm. RDRs can be determined or predicted using methods known in the art. RDRs can be predicted using algorithms such as, but not limited to, the IUPred2a algorithm. In some embodiments, an RDR contains one or more cysteine residues. In some embodiments, an RDR region comprises a string of 5 or more contiguous amino acids wherein the differential IDR score ((Redox minus)−(Redox plus)) determined for each amino acid is greater than or equal to about 0.15. Redox minus and redox plus scores can be determined using the IUPred2a algorithm. In some embodiments, an RDR region comprises a string of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive amino acids wherein the differential IDR score ((Redox minus)−(Redox plus)) each amino acid is greater than or equal to about 0.15.
A “homolog” or “homologous” sequence (e.g., nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the known reference sequence. 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. Homologous sequences can include, for example, orthologs (orthologous sequences) and paralogs (paralogous sequences). Homologous genes, for example, typically descend from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). “Orthologous” genes include genes in different species that evolved from a common ancestral gene by speciation. Orthologs typically retain the same function in the course of evolution. “Paralogous” genes include genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution.
“NPR1” (non-expresser of pathogenesis related (PR) genes 1) is a plant gene that encodes the NPR1 protein. NPR1 is a positive regulator of systemic acquired resistance. NPR1 contains a Broad-Complex, Tramtrack and Bric a brac (BTB) domain, and three ankyrin-repeat domains.
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.
Treating plants with salicylic acid (SA) can protect them from cell death induced by both biotic and abiotic stresses, such as a broad spectrum of pathogens, high and low temperature, oxidative stress, and DNA damage through the formation of NPR1 condensates. These SA-induced NPR1 condensates (SINCs) contain many plant nucleotide-binding and leucine-rich repeat (NB-LRR) immune receptors and signaling components, such as EDS1 and PAD4, as well as components in the heat response, redox regulation, DNA damage repair and protein homeostasis (ubiquitination/degradation) pathways. SINC formation serves as a sink for maintaining protein homeostasis during a diverse array of stresses and make plants more resistant. Since SINC formation is an intrinsic property of NPR1, it can be used as a target for engineering broad spectrum stress resistance in different organisms because many of the SINC proteins and the processes that they represent are highly conserved in evolution.
The present disclosure provides, is based, in part on the discovery by the inventors that NPR1 promotes cell survival by targeting substrates for ubiquitination and degradation through salicylic acid (SA)-driven phase separation into cytoplasmic condensates. Further, the inventors show that NPR1 condensates are enriched in cell death regulators including nucleotide-binding leucine-rich repeat immune receptors, redox metabolism proteins, DNA damage repair and protein quality control machineries. Phase separation of NPR1 is required for recruitment of the Cullin 3 RING E3 ligase complex to the condensates and NPR1 can promote cell survival by degrading EDS1 and specific WRKY transcription factors required for ETI. The discovery of distinct functional groups of proteins in the SA-induced NPR1 condensates, herein termed “SINCs”, suggests that NPR1 modulates survival by converging multiple stress-responsive processes in this quasi organelle.
As described above and further described below in the examples, in response to SA, wild-type NPR1 can be found in cytoplasmic condensates (SINCs) that correlate with decreased plant cell death and increased plant tolerance to various stresses, including biotic and abiotic stress. Described herein are variant NPR1 proteins (npr1 proteins) that spontaneously or constitutively form cytoplasmic condensates (NPR1 condensates), i.e., in the absence of SA. The described npr1 proteins induce formation of NPR1 condensates at greater frequency compared to wild type NPR1 protein in the absence of SA. These salicylic acid (SA)-independent NPR1 condensates also correlate with decreased plant cell death and increased plant tolerance to various stresses, including biotic and abiotic stress. Expression of a npr1 protein in a plant cell or plant can be used to increase plant stress tolerance and reduce plant cell death in response to stress.
In some embodiments, a npr1 protein comprises a NPR1 protein having one or more mutations in at least one redox-sensitive intrinsically disordered region (RDR), wherein the one or more mutations result in the npr1 protein forming salicylic acid-independent NPR1 condensates. In some embodiments, the npr1 protein retains the cytoplasmic functions of NPR1. In some embodiments, the npr1 protein retains the nuclear functions of NPR1. In some embodiments, the npr1 protein retains both the cytoplasmic and nuclear functions of NPR1. Formation of salicylic acid-independent NPR1 condensates is readily determined using the methods described herein. NPR1 contains three RDRs (RDR1, RDR2, and RDR3). The three RDRs of the A. thaliana NPR1 are located at amino acids 140-160, 368-404, and 510-539 (shown in boxes) of
The RDR regions of homologs and/or orthologs of A. thaliana NPR1 can be identified using RDR predicting algorithms as is described for A. thaliana NPR1. Alternatively, RDR regions of homologs and/or orthologs of A. thaliana NPR1 can be identified by identifying the regions of the NPR1 homologs and/or orthologs corresponding to amino acids 140-160, 368-404, and 510-539 of SEQ ID NO: 1. Suitable orthologs of A. thaliana NPR1 include, but are not limited to, the NPR1 of tobacco, tomato, grape, barley, rice, soybean, melon, corn, rapeseed, cabbage, broccoli, radish, and mustard. Orthologs of SEQ ID NO: 1 include, but are not limited to: SEQ ID NOS: 2-12 and 30-34. The RDR2 region of A. thaliana NPR1 corresponds to amino acids 368-404 or SEQ ID NO: 1. The corresponding RDR2 regions of SEQ ID NOS. 2-12 and 30-34 are shown in Table 1. The corresponding RDR1 and RDR3 regions of SEQ ID NOS. 2-12 and 30-34 can be similarly determined by homology alignment with SEQ ID NO: 1.
thaliana and orthologs.
Arabidopsis thaliana
Nicotiana benthamiana
Solanum Lycopersicon
Vitis vinifera
Hordeum vulgare
Medicago truncatula
Nicotiana tabacum
Oryza sativa
Glycine max
Populus trichocarpa
Cucumis melo
Zea mays
Raphanus sativus
Brassica napus
Brassica oleracea
Brassica rapa
Brassica juncea
In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having one or more mutations in at least one redox-sensitive intrinsically disordered region (RDR), wherein the one or more mutations result in the npr1 protein forming salicylic acid-independent NPR1 condensates. In some embodiments, a npr1 protein comprises an ortholog of an A. thaliana NPR1 protein having one or more mutations in at least one redox-sensitive intrinsically disordered region (RDR), wherein the one or more mutations result in the npr1 protein forming salicylic acid-independent NPR1 condensates. Overexpression of A. thaliana NPR1 or its orthologs has been shown to enhance resistance in a number of horticultural crop plants, including grape, carrot, tomato, apple, citrus, tobacco, and strawberry and in high-acreage agronomic crops such as rice, wheat, soybean, peanut, and potato. See, e.g., Silva et al. (2018) Hortic. Res. 5:15, herein incorporated by reference in its entirety for all purposes. The ortholog can be, but is not limited to, a Nicotiana benthamiana NPR1, a Solanum lycopersicon NPR1, a Vitis vinifera NPR1, a Hordeum vulgare NPR1, a Medicago truncatula NPR1, a Nicotiana tabacum NPR1, a Oryza sativa NPR1, a Glycine max NPR1, a Populus trichocarpa NPR1, a Cucumis melo NPR1, a Zea mays NPR1, a Raphanus sativus NPR1, a Brassica napus NPR1, a Brassica oleracea NPR1, a Brassica rapa NPR1, a Brassica juncea NPR1. The ortholog can be, but is not limited to, a protein comprising the amino acid sequence of SEQ ID NO: 2-12 and 30-34.
In some embodiments, the one or more mutations in the at least one RDR reduce the redox-sensitivity of the RDR.
In some embodiments, a npr1 protein comprises a NPR1 protein having mutations of one or more cysteines in at least one RDR. The one or more cysteines can be located in a single RDR, 2 RDRs (e.g., RDR1 and RDR2, RDR2, and RDR3, or RDR1 and RDR3), 3 RDRs (e.g., RDR1, RDR1 and RDR3), or a combination thereof. In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having mutations of one or more cysteines in at least one RDR. The one or more cysteines can be located in a single RDR, 2 RDRs (e.g., RDR1 and RDR2, RDR2, and RDR3, or RDR1 and RDR3), 3 RDRs (e.g., RDR1, RDR1 and RDR3), or a combination thereof. In some embodiments, a npr1 protein comprises an ortholog of an A. thaliana NPR1 protein having mutations of one or more cysteines in at least one RDR. The ortholog can be, but is not limited to, a Nicotiana benthamiana NPR1, a Solanum lycopersicon NPR1, a Vitis vinifera NPR1, a Hordeum vulgare NPR1, a Medicago truncatula NPR1, a Nicotiana tabacum NPR1, a Oryza sativa NPR1, a Glycine max NPR1, a Populus trichocarpa NPR1, a Cucumis melo NPR1, a Zea mays NPR1, a Raphanus sativus NPR1, a Brassica napus NPR1, a Brassica oleracea NPR1, a Brassica rapa NPR1, a Brassica juncea NPR1. The ortholog can be, but is not limited to, a protein comprising the amino acid sequence of SEQ ID NO: 2-12 and 30-34. The npr1 protein can have a mutation of a single cysteine, 2 cysteines, 3 cysteines, 4 cysteines, 5 cysteines, 6 cysteines, or 7 or more cysteines. The mutation can be a substitution, a deletion, or a combination thereof. The mutation at each position can independently be an alanine substitution, a glycine substitution, a serine substitution, a threonine substitution, or a deletion. In some embodiments, the substitution is an alanine.
In some embodiments, a npr1 protein comprises a NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 140-160, 368-404, and/or 510-539 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 140-160, 368-404, and/or 510-539 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an ortholog of an A. thaliana NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 140-160, 368-404, and/or 510-539 of SEQ ID NO: 1. The ortholog can be, but is not limited to, a Nicotiana benthamiana NPR1, a Solanum lycopersicon NPR1, a Vitis vinifera NPR1, a Hordeum vulgare NPR1, a Medicago truncatula NPR1, a Nicotiana tabacum NPR1, a Oryza sativa NPR1, a Glycine max NPR1, a Populus trichocarpa NPR1, a Cucumis melo NPR1, a Zea mays NPR1, a Raphanus sativus NPR1, a Brassica napus NPR1, a Brassica oleracea NPR1, a Brassica rapa NPR1, a Brassica juncea NPR1. The ortholog can be, but is not limited to, a protein comprising the amino acid sequence of SEQ ID NO: 2-12 and 30-34. The npr1 protein can have a mutation of a single cysteine, 2 cysteines, 3 cysteines, 4 cysteines, 5 cysteines, 6 cysteines, or 7 or more cysteines. The mutation can be a substitution, a deletion, or a combination thereof. The mutation at each position can independently be an alanine substitution, a glycine substitution, a serine substitution, a threonine substitution, or a deletion. In some embodiments, the substitution is an alanine. The cysteine residues in an ortholog of A. thaliana NPR1 may not be in the exact same numerical positions as the cysteines in A. thaliana NPR1. For example, the cysteine residue at position 394 of SEQ ID NO: 1 occurs at position 396 of the Zea mays ortholog (SEQ ID NO: 12).
In some embodiments, a npr1 protein comprises a NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 368-404 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 368-404 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an ortholog of an A. thaliana NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 368-404 of SEQ ID NO: 1. The ortholog can be, but is not limited to, a Nicotiana benthamiana NPR1, a Solanum lycopersicon NPR1, a Vitis vinifera NPR1, a Hordeum vulgare NPR1, a Medicago truncatula NPR1, a Nicotiana tabacum NPR1, a Oryza sativa NPR1, a Glycine max NPR1, a Populus trichocarpa NPR1, a Cucumis melo NPR1, a Zea mays NPR1, a Raphanus sativus NPR1, a Brassica napus NPR1, a Brassica oleracea NPR1, a Brassica rapa NPR1, a Brassica juncea NPR1. The ortholog can be, but is not limited to, a protein comprising the amino acid sequence of SEQ ID NO: 2-12 and 30-34. The npr1 protein can have a mutation of a single cysteine, 2 cysteines, 3 cysteines, 4 cysteines, 5 cysteines, 6 cysteines, or 7 or more cysteines. The mutation can be a substitution, a deletion, or a combination thereof. The mutation at each position can independently be an alanine substitution, a glycine substitution, a serine substitution, a threonine substitution, or a deletion. In some embodiments, the substitution is an alanine.
In some embodiments, a npr1 protein comprises a NPR1 protein having a mutation of a cysteine located at a position corresponding to residue 394 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having a mutation of a cysteine at position 394 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an ortholog of an A. thaliana NPR1 protein having a mutation of a cysteine located at a position corresponding to residue 394 of SEQ ID NO: 1. The ortholog can be, but is not limited to, a Nicotiana benthamiana NPR1, a Solanum lycopersicon NPR1, a Vitis vinifera NPR1, a Hordeum vulgare NPR1, a Medicago truncatula NPR1, a Nicotiana tabacum NPR1, a Oryza sativa NPR1, a Glycine max NPR1, a Populus trichocarpa NPR1, a Cucumis melo NPR1, a Zea mays NPR1, a Raphanus sativus NPR1, a Brassica napus NPR1, a Brassica oleracea NPR1, a Brassica rapa NPR1, a Brassica juncea NPR1. The ortholog can be, but is not limited to, a protein comprising the amino acid sequence of SEQ ID NO: 2-12 and 30-34. The mutation can be a substitution or a deletion. The substitution can be an alanine substitution, a glycine substitution, a serine substitution, or a threonine substitution. In some embodiments, the substitution is an alanine. In some embodiments, the npr1 protein comprises the amino acid sequence of SEQ ID NO: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, or 153.
In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having a mutation of a cysteine at position 378 of SEQ ID NO: 1, position 385 of SEQ ID NO: 1, position 394 of SEQ ID NO: 1, positions 378 and 385 of SEQ ID NO: 1, positions 378 and 394 of SEQ ID NO: 1, positions 385 and 394 of SEQ ID NO: 1, or positions 378, 385, and 394 of SEQ ID NO: 1. The mutations can be substitutions, deletions, or a combination thereof. The mutation at each position can independently be an alanine substitution, a glycine substitution, a serine substitution, a threonine substitution, or a deletion. In some embodiments, the substitution is an alanine. In some embodiments, the npr1 protein comprises the amino acid sequence of SEQ ID NO: 134.
In some embodiments, a npr1 protein comprises a NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 140-160 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 140-160 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an ortholog of an A. thaliana NPR1 protein having mutations of one or more cysteines, wherein the cysteines are located in a region corresponding to residues 140-160 of SEQ ID NO: 1. The ortholog can be, but is not limited to, a Nicotiana benthamiana NPR1, a Solanum lycopersicon NPR1, a Vitis vinifera NPR1, a Hordeum vulgare NPR1, a Medicago truncatula NPR1, a Nicotiana tabacum NPR1, a Oryza sativa NPR1, a Glycine max NPR1, a Populus trichocarpa NPR1, a Cucumis melo NPR1, a Zea mays NPR1, a Raphanus sativus NPR1, a Brassica napus NPR1, a Brassica oleracea NPR1, a Brassica rapa NPR1, a Brassica juncea NPR1. The ortholog can be, but is not limited to, a protein comprising the amino acid sequence of SEQ ID NO: 2-12 and 30-34. The mutation can be a substitution, a deletion, or a combination thereof. The mutation at each position can independently be an alanine substitution, a glycine substitution, a serine substitution, a threonine substitution, or a deletion. In some embodiments, the substitution is an alanine.
In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having a mutation of a cysteine at one or more of positions 150, 155, 156, and 160 of SEQ ID NO: 1. In some embodiments, a npr1 protein comprises an A. thaliana NPR1 protein having a mutation of a cysteine at position 150 of SEQ ID NO: 1; position 155 of SEQ ID NO: 1; position 156 of SEQ ID NO: 1; position 160 of SEQ ID NO: 1; positions 150 and 155 of SEQ ID NO: 1; positions 150 and 156 of SEQ ID NO: 1; positions 150 and 160 of SEQ ID NO: 1; positions 155 and 156 of SEQ ID NO: 1; positions 155 and 160 of SEQ ID NO: 1; positions 156 and 160 of SEQ ID NO: 1; positions 150, 155, and 156 of SEQ ID NO: 1; positions 150, 156, and 160 of SEQ ID NO: 1; positions 150, 155, and 156 of SEQ ID NO: 1; positions 155, 156, and 160 of SEQ ID NO: 1; or positions 150, 155, 156, and 160 of SEQ ID NO: 1. The mutation can be a substitution, a deletion, or a combination thereof. The mutation at each position can independently be an alanine substitution, a glycine substitution, a serine substitution, a threonine substitution, or a deletion. In some embodiments, the substitution is an alanine. In some embodiments, the npr1 protein comprises the amino acid sequence of SEQ ID NO: 134.
In some embodiments, a npr1 protein comprises a npr1 ACTD protein, a BTB domain npr1 protein, or a npr1 sim3 protein. A ACTD npr1 comprises a NPR1 protein having a deletion of amino acids corresponding to amino acids 1-364 of SEQ ID NO: 1 or an ortholog thereof. A BTB npr1 protein consists the BTB domain of NPR1 corresponding to amino acids 65-144 of SEQ ID NO: 1 or an ortholog thereof. A npr1sim3 protein comprises a SUMOylation-deficient mutant of NPR1.
In some embodiments, the npr1 protein has increased interaction with CUL3 compared to wild-type NPR1 in the absence of salicylic acid when measured under the same conditions. In some embodiments, the npr1 protein has increased interaction with CUL3 compared to wild-type NPR1 at lower concentrations of salicylic acid when measured under the same conditions Interaction may be determined using methods known in the art, including, but not limited to, co-immunoprecipitation, yeast two-hybrid assay, and BiFC assay.
Any of the described npr1 proteins that form salicylic acid-independent NPR1 condensates can be expressed in a plant or plant cell by introducing into the plant or plant cell or a progenitor plant or plant cell, a nucleic acid encoding the npr1 protein. Nucleic acids encoding the described npr1 proteins (npr1 genes) are readily made using methods known in the art. Nucleic acid sequences encoding A. thaliana NPR1 (SEQ ID NO: 1) and its orthologs, such as SEQ ID NOS: 2-12 and 30-34, are known in the art. Modification (mutation) of a nucleic acid sequence encoding a NPR1 gene to form a nucleic acid encoding a described npr1 protein can be done using methods known in the art for site directed mutagenesis of a nucleic acid.
A npr1 gene encoding a npr1 protein that forms salicylic acid-independent NPR1 condensates can be a nucleic acid encoding: (a) a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160 or an ortholog thereof; or (b) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160, wherein the protein retains salicylic acid-independent NPR1 condensate formation function.
A npr1 gene encoding any of the described npr1 proteins can be operably linked to one or more expression control elements (e.g., one or more heterologous expression control elements). The expression control elements can comprise a promoter (e.g., a heterologous promoter), one or more upstream open reading frames (uORFs) (e.g., one or more heterologous uORFs), or a promoter and one or more uORFs. In a specific example, a npr1 gene can be operably linked to a TBF1 promoter (e.g., Arabidopsis TBF1 promoter) and one or more TBF1 uORFs (e.g., Arabidopsis TBF1 uORF1 and/or uORF2).
The promoter can be, but is not limited to, a constitutive promoter, an inducible promoter, a temporally-regulated promoter, a developmentally regulated promoter, a chemically regulated promoter, a tissue-preferred promoter, a tissue-specific promoter, a TBF1 promoter, a 35S promoter, a ubiquitin promoter, a tCUP cryptic constitutive promoter, a Rsyn7 promoter, a pathogen-inducible promoter, a maize In2-2 promoter, a tobacco PR-1a promoter, a glucocorticoid-inducible promoter, an estrogen-inducible promoter, a tetracycline-inducible promoter, a tetracycline-repressible promoter, a T3 promoter, a T7 promoter, or a SP6 promoter. In a specific example, the promoter is a TBF1 promoter, such as an Arabidopsis TBF1 promoter. See, e.g., Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22(2):103-112; Xu et al. (2017) Nature 545(7655):491-494; US 2018-0273965; U.S. Pat. No. 10,584,346; US 2015-0113685; U.S. Pat. No. 10,017,773; WO 2013/096567; US 2019-0352664; and WO 2018/144831, each of which is herein incorporated by reference in its entirety for all purposes. TBF1 is an important transcription factor for the growth-to-defense switch upon immune induction. In one example, the promoter can comprise the sequence set forth in SEQ ID NO: 167. In another example, the promoter can comprise the sequence set forth in SEQ ID NO: 168.
The upstream uORF can comprise one or more TBF1 gene uORFs. See, e.g., Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22(2):103-112; Xu et al. (2017) Nature 545(7655):491-494; US 2018-0273965; U.S. Pat. No. 10,584,346; US 2015-0113685; U.S. Pat. No. 10,017,773; WO 2013/096567; US 2019-0352664; and WO 2018/144831, each of which is herein incorporated by reference in its entirety for all purposes. The TBF1 uORFs can comprise, for example, Arabidopsis TBF1 uORFs, such as uORF1 (SEQ ID NO: 162, or encoding SEQ ID NO: 164) and uORF2 (SEQ ID NO: 163, or encoding SEQ ID NO: 165). In one example, the uORFs can comprise uORF1 (SEQ ID NO: 162 or encoding SEQ ID NO: 164), uORF2 (SEQ ID NO: 163 or encoding SEQ ID NO: 165), or both uORF1 and uORF2. In a specific example, the npr1 gene can be operably linked to a regulatory sequence (e.g., 5′ regulatory sequence) comprising SEQ ID NO: 166, which includes both uORF1 and uORF2. In another specific example, the npr1 gene can be operably linked to a regulatory sequence (e.g., 5′ regulatory sequence) comprising SEQ ID NO: 168, which includes a TBF1 promoter, uORF1, and uORF2.
A nucleic acid encoding a npr1 protein may be introduced into a plant or plant cell using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, lipofection, nucleofection, 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. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).
In some embodiments, Agrobacterium tumefaciens is used to generate a transgenic plant. There are numerous vectors designed for Agrobacterium transformation. For stable transformation, Agrobacterium systems can utilize “binary” vectors that permit plasmid manipulation in both E. coli and Agrobacterium, and typically contain one or more selectable markers to recover transformed plants. Binary vectors for use in Agrobacterium transformation systems typically comprise the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, and selectable marker and reporter genes. 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.
Methods of producing a plant that expresses a npr1 protein as described herein or genetically modifying a plant to express a npr1 protein as described herein using a CRISPR/Cas system are described. Stress resistant plants created using a CRISPR/Cas system and nucleic acids for producing a stress resistant plant using a CRISPR/Cas system are also described.
A CRISPR system can comprise an RNA-guided DNA endonuclease enzyme and a guide RNA. The RNA-guided DNA endonuclease enzyme can be, but is not limited to, a Cas9 protein. A CRISPR system can comprise 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. 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 can be delivered to a plant or plant cell via a bacterium. The bacterium can be, but is not limited to, Agrobacterium tumefaciens.
The CRISPR system is designed to target insertion of a nucleic acid encoding a npr1 protein into the plant genome. The CRIPSR system can be designed to target insertion of the nucleic acid encoding a npr1 protein into the NPR1 locus. The CRISPR/Cas system can be, but is not limited to, a CRISPR class 1 system, CRISPR class 2 system, CRISPR/Cas system, a CRISPR/Cas9 system, a CRISPR/zCas9 system or CRISPR/Cas3 system.
To 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).
Plant cells including or expressing any of the npr1 proteins described herein are provided. The plants cells have increased stress tolerance, increased cell survival (decreased cell death) against biotic and/or abiotic stress, and/or increased cell survival against plant immune response, relative to a similar plant cell not expressing the nrp1 protein. The plant cell can be a monocot plant cell or a dicot plant cell. The plant cell can be, but is not limited to, a food crop plant cell, a biofuel plant cell, a corn plant cell, a legume plant cell, a bean plant cell, a rice plant cell, a soybean plant cell, a cotton plant cell, a sugarcane plant cell, a tobacco plant cell, a palm oil plant cell, a date palm cell, a wheat cell, a vegetable plant cell, a squash plant cell, a Solanaceae plant cell, a tomato cell, a banana plant cell, a potato plant cell, a pepper plant cell, a moss plant cell, a parsley plant cell, a sunflower plant cell, a mustard plant cell, a sorghum plant cell, a millet plant cell, a citrus plant cell, an apple plant cell, a strawberry plant cell, a rapeseed plant cell, a cabbage plant cell, a cassava plant cell, a coffee plant cell, a sweet potato plant cell, a jatropha plant cell, or a switchgrass plant cell. The npr1 gene can be integrated into the genome of the plant cell. The npr1 gene can be integrated into the genome of the plant cell at the NPR1 locus or a heterologous locus.
A plant cell can contain a npr1 gene encoding: (a) a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160 or an ortholog thereof; or (b) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160, wherein the protein retains salicylic acid-independent NPR1 condensate formation function.
In some embodiments, the plant cells expresses a wild-type NPR1 gene. In some embodiments, the plant cell does not express a wild-type NPR1 gene. For example, in some embodiments, the plant cell is npr1-null.
Plants including or expressing any of the npr1 proteins described herein are provided. The plant can be a monocot plant or a dicot plant. The plants have increased stress tolerance, increased cell survival (decreased cell death) against biotic and/or abiotic stress, and/or increased cell survival against plant immune response, relative to a similar plant not expressing the nrp1 protein. The plant can be, but is not limited to, a food crop plant, a biofuel plant, a corn plant, a legume plant, a bean plant, a rice plant, a soybean plant, a cotton plant, a sugarcane plant, a tobacco plant, a palm oil plant, a date palm, a wheat, a vegetable plant, a squash plant, a Solanaceae plant, a tomato, a banana plant, a potato plant, a pepper plant, a moss plant, a parsley plant, a sunflower plant, a mustard plant, a sorghum plant, a millet plant, a citrus plant, an apple plant, a strawberry plant, a rapeseed plant, a cabbage plant, a cassava plant, a coffee plant, a sweet potato plant, a jatropha plant, or a switchgrass plant. The npr1 gene can be integrated into the genome of the plant. The npr1 gene can be integrated into the genome of the plant at the NPR1 locus or a heterologous locus.
A plant can contain a npr1 gene encoding: (a) a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160 or an ortholog thereof; or (b) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160, wherein the protein retains salicylic acid-independent NPR1 condensate formation function.
In some embodiments, the plant expresses a wild-type NPR1 gene. In some embodiments, the plant does not express a wild-type NPR1 gene. For example, in some embodiments, the plant is npr1-null.
Described are methods of increasing stress tolerance, increasing cell survival (decreasing cell death) against biotic and/or abiotic stress, and/or increasing cell survival against plant immune response in a plant or plant cell comprising expressing in the plant or plant cell a npr1 protein that forms salicylic acid-independent NPR1 condensates. The npr1 protein can be any of the npr1 proteins described herein. The methods comprise introducing into the plant, the plant cell, or a progenitor of the plant or plant cell, a nucleic acid encoding any of the described npr1 proteins such that the nucleic acid is expressed in the plant or plant cell. In some embodiments, the nucleic acid is operatively linked to one or more expression control elements that are functional in the plant or plant cell. In some embodiments, the nucleic acid is operatively linked to a promoter, or a promoter and one or more uORFs. In some embodiments, the nucleic acid comprises a npr1 gene encoding: (a) a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160 or an ortholog thereof; (b) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a protein comprising the amino acid sequence of any of SEQ ID NOS: 134-160, wherein the protein retains salicylic acid-independent NPR1 condensate formation function; (c) a npr1 ΔCTD protein; (d) a BTB domain npr1 protein, or (e) a npr1sim3 protein. Expression of the npr1 protein in the plant or plant cell results in the plant or plant cell having increased stress tolerance, increased cell survival (decreased cell death) against biotic and/or abiotic stress, and/or increased cell survival against plant immune response relative to a similar plant or plant cell that does not express the npr1 protein. A biotic stress can be, but is not limited to, a viral or bacterial infection. An abiotic stress can be, but is not limited to, high temperature (heat shock) stress, low temperature (cold shock) stress, oxidative stress, or DNA damage.
In some embodiments, increasing stress tolerance comprises one or more of: decreasing programmed cell death, decreasing effector-triggered immunity (ETI)-induced cell death, increasing formation of NPR1 condensates, and degrading EDS1 and specific WRKY transcription factors required for pathogen ETI.
The plant, plant cell, or progenitor of the plant or plant cell can be, but is not limited to, a monocot or a dicot. The plant, plant cell, or progenitor of the plant or plant cell can be, but is not limited to, a food crop plant, a biofuel plant, a corn plant, a legume plant, a bean plant, a rice plant, a soybean plant, a cotton plant, a sugarcane plant, a tobacco plant, a palm oil plant, a date palm, a wheat, a vegetable plant, a squash plant, a Solanaceae plant, a tomato, a banana plant, a potato plant, a pepper plant, a moss plant, a parsley plant, a sunflower plant, a mustard plant, a sorghum plant, a millet plant, a citrus plant, an apple plant, a strawberry plant, a rapeseed plant, a cabbage plant, a cassava plant, a coffee plant, a sweet potato plant, a jatropha plant, or a switchgrass plant, or a cell derived from any of these plants. The npr1 gene can be integrated into the genome of the plant or plant cell. The npr1 gene can be integrated into the genome of the plant or plant cell at the NPR1 locus or a heterologous locus.
The nucleic acid encoding the npr1 protein can be introduced into a plant, plant cell, or progenitor of the plant or plant cell that expresses a wild-type NPR1 gene. Alternatively, the nucleic acid encoding the npr1 protein can be introduced into a plant, plant cell, or progenitor of the plant or plant cell that does not express a wild-type NPR1 gene (e.g., a npr1-null plant, plant cell, or progenitor of the plant or plant cell). The nucleic acid encoding the npr1 protein can be introduced into a plant, or progenitor of the plant having one genotype and introgressed into a plant having a different genotype. “Introgression” of a gene or locus means introduction of the gene or locus from a donor plant comprising the gene or locus into a recipient plant by standard breeding techniques. Selection of can be done phenotypically or selection can be done with the use of genetic 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 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.
In some embodiments, producing a plant having increased stress tolerance, increased cell survival (decreased cell death) against biotic and/or abiotic stress, and/or increased cell survival against plant immune response comprises crossing a first plant expressing any of the described npr1 proteins with a second plant to produce at least a first progeny plant, and selecting one or more progeny plants that express the npr1 protein any or have increased stress tolerance, increased cell survival (decreased cell death) against biotic and/or abiotic stress, and/or increased cell survival against plant immune response compared to a control plant that doesn't express the npr1 protein.
Also described are methods of improving plant growth under conditions of stress, the methods comprising introducing into one or more plants a nucleic acid encoding any of the described npr1 proteins such that the npr1 protein is expressed in the plant, subjecting the one or more plants to stress; and selecting a plant having improved plant growth under the stress when compared to a plant that lacks the nucleic acid encoding the npr1 protein.
Another aspect of the present disclosure provides all that is described and illustrated herein.
The following Examples and attached Appendices are provided by way of illustration and not by way of limitation.
The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5′ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3′ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. When a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon degenerate variants thereof that encode the same amino acid sequence are also provided. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.
Arabidopsis thaliana npr1 protein
Nicotiana benthamiana npr1 protein
Solanum lycopersicon npr1 protein
Vitis vinifera npr1 protein
Hordeum vulgare npr1 protein
Medicago truncatula npr1 protein
Nicotiana tabacum npr1 protein
Oryza sativa npr1 protein
Glycine max npr1 protein
Populus trichocarpa npr1 protein
Cucumis melo npr1 protein
Zea mays npr1 protein
Arabidopsis thaliana RDR2 sequence alignment
Nicotiana benthamiana RDR2 sequence alignment
Solanum lycopersicon RDR2 sequence alignment
Vitis vinifera RDR2 sequence alignment
Hordeum vulgare RDR2 sequence alignment
Medicago truncatula RDR2 sequence alignment
Nicotiana tabacum RDR2 sequence alignment
Oryza sativa RDR2 sequence alignment
Glycine max RDR2 sequence alignment
Populus trichocarpa RDR2 sequence alignment
Cucumis melo RDR2 sequence alignment
Zea mays RDR2 sequence alignment
Raphanus sativus RDR2 sequence alignment
Brassica napus RDR2 sequence alignment
Brassica oleracea RDR2 sequence alignment
Brassica rapa RDR2 sequence alignment
Brassica juncea RDR2 sequence alignment
Brassica napus npr1 protein
Brassica rapa npr1 protein
Brassica oleracea npr1 protein
Raphanus sativus npr1 protein
Brassica juncea npr1 protein
A. thaliana npr1 rdr2 protein
A. thaliana npr1 rdrl protein
A. thaliana npr1 rdr3 protein
Nicotiana benthamiana npr1 rdr2 protein
Solanum lycopersicon npr1 rdr2 protein
Vitis vinifera npr1 rdr2 protein
Hordeum vulgare npr1 rdr2 protein
Medicago truncatula npr1 rdr2 protein
Nicotiana tabacum npr1 rdr2 protein
Oryza sativa npr1 rdr2 protein
Glycine max npr1 rdr2 protein
Populus trichocarpa npr1 rdr2 protein
Cucumis melo npr1 rdr2 protein
Zea mays npr1 rdr2 protein
Brassica napus npr1 rdr2 protein
Brassica rapa npr1 rdr2 protein
Brassica oleracea npr1 rdr2 protein
Raphanus sativus npr1 rdr2 protein
Brassica juncea npr1 rdr2 protein
A. thaliana npr1 rdr2 protein v2
Glycine max npr1 rdr2 protein v2
Cucumis melo npr1 rdr2 protein v2
Brassica napus npr1 rdr2 protein v2
Brassica rapa npr1 rdr2 protein v2
Brassica oleracea npr1 rdr2 protein v2
Raphanus sativus npr1 rdr2 protein v2
Brassica juncea npr1 rdr2 protein v2
Arabidopsis thaliana TBF1 uORF1
Arabidopsis thaliana TBF1 uORF2
Arabidopsis thaliana TBF1 5′ regulatory sequence with uORF1 and uORF2
Arabidopsis thaliana TBF1 promoter
Arabidopsis thaliana TBF1 promoter region with uORF1 and uORF2
In plants, pathogen effector-triggered immunity (ETI) often leads to programmed cell death, which is restricted by NPR1, an activator of systemic acquired resistance. However, the biochemical activities of NPR1 enabling it to both promote defense and restrict cell death remain unclear. Here we show that NPR1 promotes cell survival by targeting substrates for ubiquitination and degradation through formation of salicylic acid-induced NPR1 condensates (SINCs). SINCs are enriched in stress response proteins, including nucleotide-binding leucine-rich repeat immune receptors, oxidative and DNA damage response proteins, and protein quality control machineries. Transition of NPR1 into condensates is required for the formation of the NPR1-Cullin 3 E3 ligase complex to ubiquitinate SINC-localized substrates, such as EDS1 and specific WRKY transcription factors, and promote cell survival during ETI. Our analysis of SINCs suggests that NPR1 is centrally integrated into the cell death/survival decisions in plant immunity by modulating multiple stress-responsive processes in this quasi organelle.
Eukaryotes have evolved mechanisms to effectively restrict infection while avoiding significant damage to self. Such a regulation is evident during effector-triggered immunity (ETI) in plants, which is activated upon recognition of pathogen effectors by the nucleotide-binding and leucine-rich repeat immune receptors (NB-LRRs). ETI often culminates in rapid programmed cell death (PCD) at the site of infection to restrict pathogen growth. However, without a counteractive mechanism, ETI can severely compromise host survival due to the spread of cell death to uninfected tissues. One of the counteractive mechanisms is the systemic acquired resistance (SAR) mediated by salicylic acid (SA) through the function of NPR1 [non-expresser of pathogenesis related (PR) genes 1]. NPR1 is a master regulator of genes in multiple cellular machineries, including antimicrobial PR genes and endoplasmic reticulum-resident genes, that protect plants against a broad spectrum of diseases and stresses. In addition to NPR1-mediated transcription, the ubiquitin-proteasome system (UPS) and autophagy are sequentially recruited to maintain defense protein homeostasis and promote host survival. Mutants in the autophagy pathway are defective in the containment of age- and immunity-associated PCD due to proteotoxic accumulation of ubiquitinated proteins, which is partly dependent on SA and NPR1. Though early studies have implicated SA and NPR1 in suppressing ETI-induced cell death, the underlying mechanism is unknown.
In NPR1, the presence of a Broad-Complex, Tramtrack and Bric a brac (BTB) domain, in combination with a putative substrate-binding ankyrin-repeat domain, suggests that it may function as a Cullin 3 RING E3 ligase (CRL3) adaptor. However, an early yeast two-hybrid study failed to detect NPR1-CUL3 interaction. Instead, two NPR1 paralogs, NPR3 and NPR4, were later found to function as CRL3 adaptors in mediating NPR1 degradation in the nucleus.
Different activities of NPR1 are likely controlled by posttranslational modifications (PTMs), such as SUMOylation which is preceded by dephosphorylation and phosphorylation at two distinct IKB-like degrons, S55/59 and S11/15, respectively. SUMOylation not only regulates NPR1 nuclear-cytoplasmic partitioning and affects its association with different transcription factors (TFs), but also promotes its degradation by NPR3/4. Another important PTM triggered by SA is the oligomer-to-monomer transition of NPR1 through its conserved cysteines in response to cellular redox changes. Recent studies indicate that PTMs and changes in the cellular redox environment can lead to phase transition in many essential hub proteins enabling them to carry out diverse cellular functions.
In this study, we report that NPR1 contains intrinsically disordered regions (IDRs). In response to SA, NPR1 undergoes transition into cytoplasmic condensate-like structures enriched in proteins regulating ETI cell death, DNA damage response, redox metabolism, and ubiquitination. This SA-triggered NPR1 condensation is mediated through conserved cysteine clusters present within its IDRs and is required for the formation of a functional NPR1-CRL3 adaptor complex in the cytoplasm. Using cell biology, molecular and genetic analyses, we demonstrate that recruitment of the CRL3 ubiquitination machinery into SA-induced NPR1 condensates is an essential function of NPR1 in mediating protein homeostasis and cell survival. We provide evidence that the master immune regulator, EDS1, and TFs WRKY54 and WRKY70 are among the substrates of the NPR1-CRL3 complex for SA-mediated cell survival during ETI.
SA Promotes Cell Survival and Ubiquitination through the Function of NPR1. To elucidate the role of NPR1 in the regulation of cell survival, we first infected half leaves of Arabidopsis plants with Pseudomonas syringae pv. maculicola ES4326 carrying the AvrRpt2 effector (Psm ES4326/AvrRpt2) to systemically activate NPR1 and induce SAR. Then the other halves of the leaves were challenged with the same pathogen to determine how the ETI-associated cell death was affected in the neighboring tissue by measuring tissue collapse and conductivity increases due to electrolyte leakage (
We found that in wild type (WT) plants, the first inoculation significantly suppressed PCD triggered by the second inoculation in the neighboring tissue. This suppression was SA- and NPR1-dependent because it was not observed in the SA biosynthesis mutant, sid2-2, or in the npr1-2 mutant. Consistent with our hypothesis that systemic activation of NPR1 inhibits ETI-induced cell death in the neighboring tissue, significant growth of the otherwise avirulent Psm ES4326/AvrRpt2, close to the level of the receptor mutant rps2, was observed in the second leaf halves in the WT plants, but not in npr1-2 or sid2-2 mutants (
To distinguish whether this SA- and NPR1-dependent cell survival occurs at the host cell level or is due to differential pathogen growth, we made use of an Arabidopsis line carrying the dexamethasone (dex)-inducible AvrRpt2 effector gene (dex:AvrRpt2) in either WT or the npr1-2 background. We found that in WT, induction by Psm ES4326/AvrRpt2 could protect plants against cell death triggered by subsequent in planta expression of AvrRpt2 in the neighboring tissue. This effect was abolished both in npr1-2 (
These results indicate that in pathogen-induced SAR, both SA and NPR1 are required at the host cell level for inhibiting ETI-induced PCD.
To determine whether SA is sufficient to inhibit ETI-triggered cell death, we pretreated plants with SA and found that at 0.5 and 1 mM, SA could fully suppress Psm ES4326/AvrRpt2-mediated cell death (
To determine how SA-induced and NPR1-dependent cell survival correlates with protein homeostasis, we examined the accumulation of ubiquitinated proteins after treating WT and npr1-2 plants with SA. We found SA concentration-dependent accumulation of ubiquitinated proteins in WT, which is absent in npr1-2 (
NPR1 Accumulates in the Cytoplasm and Undergoes SA-Triggered Condensate Formation. To determine the likely cellular compartment where NPR1-mediated ubiquitination occurs, we analyzed the subcellular accumulation of the endogenous NPR1 after SA treatment. As expected, NPR1 had predominant cytoplasmic accumulation in the mock-treated sample (0 mM SA), and an increased nuclear accumulation at 0.1 and 0.5 mM SA with corresponding decreases in the cytoplasmic fraction (
Surprisingly, at 1 mM SA, more NPR1 accumulated in the cytoplasm, accompanied by its relative reduction in the nuclear fraction. With a longer exposure of the western blot, we could observe ubiquitinated NPR1 (higher MW smear) in the nuclear fractions of the SA-treated samples (
To validate the fractionation result, we monitored the nuclear-cytoplasmic partitioning of NPR1-GFP in Arabidopsis using transient expression assay. We observed significant levels of NPR1-GFP in the cytoplasm of untreated plants (
Moreover, development-associated NPRS and NPR6 showed constitutive bodies as previously reported. Bimolecular fluorescence complementation (BiFC) showed that NPR1 interactions with other NPRs are unlikely to trigger its cytoplasmic body formation (
Because formation of NPR1 bodies is an induced process, and the morphology of the bodies is similar to that of phase separated proteins (Banani et al., 2017), we hypothesized that NPR1 undergoes conditional transition from soluble to condensed state, possibly through phase separation, to form the observed structures. To test our hypothesis, we performed single-cell time-lapse imaging of NPR1-GFP body formation (
To determine whether NPR1 harbors IDRs, we used all available prediction algorithms and found cysteine-dependent or redox-sensitive IDRs (RDRs) to have the highest probability (
In contrast, mutating RDR3 had no effect on protein state in untreated cells (
SINCs are Enriched with Stress Proteins and Ubiquitination Components. To uncover the function of cytoplasmic NPR1 condensates, we performed quantitative proteomic analysis using the cytoplasmic sim3 mutant. To ensure that SA-mediated transcription by the nuclear NPR1 is intact, the sim3-GFP was expressed in the WT NPR1 background (sim3-GFP/Col-0). After confirming that the line had proper SINC formation (
From these samples with good reproducibility (
We then sought to determine the role of SA/NPR1 in the specific cellular pathways identified in SINCs, such as heat shock, oxidative and DNA damage responses, based on the hypothesis that over-accumulation of these stress-responsive proteins would result in cell death. Indeed, we found that SA significantly suppressed cell death induced by these stresses in WT, but not in the npr1-2 mutant plants (
NPR1 recruits CUL3 to Cytoplasmic Condensates. The presence of ubiquitin, ubiquitin ligases and E3 ligase complex components in SINCs suggests that NPR1 may regulate protein homeostasis by recruiting ubiquitination machinery as a CRL3 adaptor. Previous studies have shown that members of the NPR family can associate with CUL3 and serve as adaptors for ubiquitination of cognate substrates.
To test our hypothesis, NPR1 and CUL3 were co-expressed in Nicotiana benthamiana followed by 1 mM SA treatment and co-immunoprecipitation (co-IP). We found that the WT NPR1 had a weak pull-down signal of CUL3 in the SA-treated sample (
A similar interaction was also detected with the endogenous CUL3 in Arabidopsis (
To identify regions and residues that could influence CUL3-NPR1 interaction, we included different truncations and point mutants of NPR1 in our co-IP assay (
At the lower concentration of 1 mM SA used in this screen, the WT NPR1 is predominantly nuclear localized (
To test this hypothesis, we used the BiFC assay to examine the subcellular localization of NPR1-CUL3 interaction. Both NPR1 and sim3 showed clear interactions with CUL3 in discrete cytoplasmic condensates, with the signal more intense for sim3 (
In further support of our hypothesis that the NPR1-CUL3 interaction occurs in cytoplasmic condensates, rdr1 and rdr2, which constitutively form condensates, showed strong interactions with CUL3, whereas rdr3, which is deficient in condensate formation, displayed little interaction (
To demonstrate that it is NPR1 that recruits CUL3 to the condensate instead of CUL3 engaging NPR1 to a pre-existing complex, we analyzed GFP-CUL3 localization in the NbNPR1 knockdown plants (
NPR1-CUL3 Condensates Are Active Ubiquitination Complexes. To determine whether recruitment of CUL3 into SINCs is associated with increased ubiquitination activity, we first examined the co-localization of NPR1-CUL3 complex with known markers of protein bodies involved in protein homeostasis: ubiquitin, ATG8, NBR1 (a polyubiquitin receptor) and HSC70. Because the sim3 mutant showed a stronger interaction with CUL3, we used the sim3/CUL3 BiFC pair for this analysis. We found that sim3/CUL3 condensates co-localized strongly with all four markers of protein bodies, but not with markers of the morphologically similar membrane-bound bodies such as Golgi, TGN and MVB (
Importantly, co-localization of sim3-GFP with mCherry-NBR1, was significantly enhanced by the presence of CUL3 (
To further establish the correlation between the SA-dependent recruitment of CUL3 into condensates and protein ubiquitination, we performed NPR1-CUL3 and sim3-CUL3 pull down under dose response to SA. We found an increase in the interaction between 0-1 mM SA for both NPR1 and sim3 (
NPR1 Targets SINC-localized Proteins for Ubiquitination and Degradation. Many SINC proteins identified in our proteomic analysis, such as NB-LRRs and EDS1/PAD4 involved in ETI signaling (
Next, we observed complete co-localization of EDS1 with NPR1/CUL3 and sim3/CUL3 bodies (
In npr1-2, while the accumulation of EDS1 was low, the protein levels remained unchanged after SA treatment. Similar results were obtained with another SINC-localized and NPR1-dependent and interacting protein, NIM1-interacting 1 (NIMIN1) (
WRKY TFs Required for ETI Are Targets of the NPR1-CRL3 Complex. Because EDS1 is a major upstream immune regulator involved in not only ETI, but also SA synthesis, it would be difficult to use genetic epistasis to test the specific effect of NPR1 in recruiting EDS1 to SINCs for ubiquitination and degradation. To remedy this problem, we sought other potential candidates. Among the known NPR1-interacting proteins, WRKY70 TF has been shown to play opposing roles as a repressor of SA synthesis and SA-responsive genes and a positive regulator of various ETI. Like EDS1, WRKY70 interacts more strongly with the cytoplasmic sim3 mutant than the WT NPR1, suggesting that this WRKY TF and perhaps its close homolog WRKY54 may be recruited to SINCs. In support of this hypothesis, we found that these WRKYs could indeed co-localize with sim3/CUL3 bodies (
Moreover, analysis of the constitutively expressed WRKY70-GFP in WT and npr1-2 plants showed that while NPR1 was required for stabilization of WRKY70-GFP in the absence of SA, the protein level was reduced in an NPR1- and proteasome-dependent manner in the presence of SA (
We next sought to put NPR1-mediated degradation of these WRKY TFs in the context of NPR1's function in promoting cell survival by testing ETI induced by Psm ES4326/AvrRpt2 in wrky54 wrky70 double and npr1 wrky54 wrky70 triple mutants. Mutating WRKY54 and WRKY70 diminished the ETI-triggered cell death (
Moreover, these wrky54 wrky70 phenotypes were epistatic to those of npr1-2 in the npr1 wrky54 wrky70 triple mutant, once again rendering strong support for our hypothesis that NPR1 inhibits cell death by degrading positive regulators of ETI such as WRKY54 and WRKY70. Based on the molecular, cellular, and genetic data, we present the following model to describe the molecular function of NPR1 in mediating cell survival (
In this study, we found that NPR1 has a major role in controlling protein homeostasis through formation of previously unknown subcellular structures, SINCs, to sequester distinct stress-responsive components in the cytoplasm. Importantly, this cytoplasmic function of NPR1 likely occurs subsequent to its nuclear function in SA/NPR1-mediated transcriptional reprogramming, explaining why such a function was obscured in previous studies. Indeed, many of the proteins found in SINCs are SA-inducible (
The dynamic distribution of NPR1 between nucleus and cytoplasm induced by SA allows coordination of NPR1′s function between the two compartments to achieve proper control of plant immune responses. This process is regulated at multiple steps: Pathogen-induced SA increase is known to change the cellular redox state, leading to the release of NPR1 monomer from the homo-oligomer to translocate into the nucleus. In the absence of SA, the NPR1 homo-oligomer does not form cytoplasmic condensates, nor interact with CUL3, probably due to phosphorylation at S55/59, because the phosphomimic mutant, S55/59D, is defective in both of these processes. SA-induced dephosphorylation at these two residues is required for activation of NPR1 to either enter the nucleus or to form SINCs in the cytoplasm as the phospho-deficient mutant S55/59A has autoimmunity and is severely retarded in growth. Dephosphorylation at S55/59 is also a pre-requisite for SUMOylation in the nucleus, because S55/59D is incompetent for this PTM (Saleh et al., 2015). SUMOylation and subsequent ubiquitination and degradation of nuclear NPR1 mediated by NPR3/4-CRL3 also interplay with SINC formation in the cytoplasm, as shown by the increased SINC formation in sim3 (
Importantly, at all SA concentrations tested, the endogenous NPR1 was never completely depleted from the cytoplasm. In fact, we observed a clear increase in the levels of NPR1 protein in the cytoplasm at higher SA concentrations which correlated with protein condensation. However, we cannot rule out the presence of NPR1 condensates at lower SA concentrations because of the limitation in detection. Though high protein levels are required for visualizing condensates through microscopy, their formation is determined by the biological properties of the proteins. The phosphomimic S55/59D mutant and paralogs, NPR2, NPR3 and NPR4, all fail to form cytoplasmic condensates (
Condensate formation is a feature of proteins that occupy essential hub positions in chromatin organization, transcription, translation, maintenance of cell architecture and protein quality control. Recent development in NPR1 research expanded the list of its interactors and cellular processes in histone modification, cold acclimation, unfolded protein response and SAR. Carrying out these functions would require formation of multi-protein complexes with diverse signaling and metabolic activities. The intrinsic ability of NPR1 to transition from one conformational state to another by forming condensates is a remarkable adaptation enabling it to regulate complex cellular processes, such as signal transduction and protein homeostasis under stress, to promote host survival. This may explain why overexpressing Arabidopsis NPR1 in heterologous plant backgrounds proved to be effective in engineering broad-spectrum disease resistance. Besides serving as a hub for regulating protein homeostasis during cell stress, SINCs may also play a signaling role in plant immunity. The presence of 10 NB-LRR immune receptors, their downstream components EDS1/PAD4, cysteine proteases, cellular redox regulators, ubiquitination, and DNA damage response proteins opens new areas of inquiry for possible interplay between these processes inside SINCs to uncover novel signaling mechanisms.
Arabidopsis thaliana (At) wild type (WT), mutants, and transgenic plants used in this study were all in the Col-0 ecotype background, with the exception of the Ws-2 ecotype which was used for Pseudomonas fluorescens (Pf) Pf0-1 AvrRps4 infection. Unless otherwise indicated, transgenic Arabidopsis over-expressing GFP-fused NPR1 or its mutant/truncation variants, are all in the npr1-2 mutant background. Arabidopsis mutants npr1-2, sid2-2, rps2, wrky54 wrky70 double and eds1-2; and transgenic lines over-expressing NPR1-GFP and sim3-GFP in the npr1-2 background, the dex:AvrRpt2/rps2 line, and the estradiol-inducible AvrRpt2 (est:AvrRpt2) lines in the Col-0, sid2-2 and rps2 backgrounds were described previously. Transgenic Arabidopsis over-expressing sim3-GFP in the Col-0 background, ABTB-GFP and rdr1/2/3-GFP in the npr1-2 background, and WRKY70-GFP in the Col-0 background, were generated and plants homozygous for the transgenes were used. Transgenic Arabidopsis over-expressing WRKY70-GFP in the npr1-2 background was generated by crossing WRKY70-GFP/Col-0 with the npr1-2 mutant. The dexamethasone-inducible AvrRpt2 line in the npr1-2 mutant background (dex:AvrRpt2/npr1-2) was generated by crossing dex:AvrRpt2/Col-0 (McNellis et al., 1998) with the npr1-2 mutant. The npr1 wrky54 wrky70 triple mutant was generated by crossing wrky54 wrky70 double mutant with npr1-2. Seeds were stratified at 4° C. for three days and plants were grown under a 12 hr light and 12 hr dark cycle at 22° C. Nicotiana benthamiana WT plants were grown under the same conditions. Unless otherwise indicated, in all experiments, soil-grown Arabidopsis and N. benthamiana plants were used at three-week-old and four-week-old age, respectively.
Plasmid construction and E. coli-based ubiquitination: The coding sequences for all Arabidopsis genes were amplified from cDNA. Point mutations of AtNPR1 (AT1G64280) were generated using the QuikChange II site-directed mutagenesis kit (Agilent). Overlap PCR was used to generate the deletion/truncation mutations of AtCUL3A (AT1G26830) and AtNPR1. The position of NPR1 truncations and point mutations are indicated in
To generate the RNAi silencing vectors for NbNPR1 and NbCUL3, protein sequences for all six AtNPRs [AtNPR1, AtNPR2 (AT4G26120), AtNPR3 (AT5G45110), AtNPR4 (AT4G19660), AtNPR5 (AT2G41370) and AtNPR6 (AT3G57130)] and all six AtCULLINs [AtCUL1 (AT4G02570), AtCUL2 (AT1G02980), AtCUL3A (AT1G26830), AtCUL3B (AT1G69670), AtCUL4 (AT5G46210) and AtCUL5 (AT4g12100)] were used to retrieve orthologs in the N. benthamiana genome assembly (Fernandez-Pozo et al., 2015a). The coding sequences of two genes found in the NPR1 clade [Niben101Scf14780g01001.1 (NbNPR1a) and Niben101Scf11512g01004.1 (NbNPR1b)], and four genes found in the CUL3 clade [Niben101Scf01326g05017.1 (NbCUL3Aa), Niben101Scf05060g00001.1 (NbCUL3Ab), Niben101Scf06545g03015.1 (NbCUL3Ba) and Niben101Scf00272g10015.1 (NbCUL3Bb)] were further submitted to the virus-induced gene silencing (VIGS) tool to predict conserved gene fragments for silencing each clade (Fernandez-Pozo et al., 2015b). A 300-bp fragment was designed to silence the two NbNPR1 genes, and a 600-bp fusion fragment was designed to silence the four NbCUL3 genes. The fragments were amplified from N. benthamiana genomic DNA using gene-specific primers (Table 6) and cloned into the pTRV2-LIC plasmid to generate pTRV2-NbNPR1 and pTRV2-NbCUL3 plant binary vectors. The control vector carrying the N. benthamiana phytoene desaturase gene (pTRV2-NbPDS) was described previously. The ubiquitination reaction was carried out according to the previously described principle of reconstituting basic ubiquitination cascade in E. coli. To reconstitute the complex, CUL3-mediated ubiquitination cascade, the reaction components consisting total of seven proteins (Substrate, NPR1, CUL3, RBX1, E1, E2 and Ubiquitin) were co-expressed in E. coli using a modified Duet vector system (Novagen). Three expression vectors were constructed: (1) pETDuet-1:FLAG-WRKY70+GST-NPR1 or pETDuet-1:FLAG-WRKY70+GST-sim3 or pETDuet-1:FLAG-WRKY70+GST (pET-AdS); (2) pACYCDuet-1:RBX1+Myc-CUL3 (pACYC-RC3); and (3) pCDFDuet-1:HA-Ub+UBC8+UBA1 (pCDF-Ub;
Plant transformation: 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 overnight at 28° C. in Luria-Bertani (LB) broth medium supplemented with appropriate antibiotics: spectinomycin (100 μg/ml), kanamycin (50 μg/ml), gentamycin (50 μg/ml), and rifampicin (25 μg/ml). The obtained culture was re-inoculated at 1:10 into fresh growth media with antibiotics and grown for another 4 hr. Cells were then spun down at 1,600 g for 10 min, and inoculum was prepared by resuspending cells to OD600 nm=1 in double distilled water (DDW) containing 200 μM acetosyringone (Sigma). Unless otherwise stated, for co-IP, co-localization, and BiFC assays in N. benthamiana, the proteins were co-expressed at 1:1 mixture of the relevant Agrobacteria inoculums. In all BiFC assays, the pair of YN/YC fusions was co-expressed together with free mCherry to mark the cytoplasm and nucleus, mCherry-NLS to mark the nucleus only, or mCherry-fused test proteins for co-localization analysis. The inoculum was pressure infiltrated into N. benthamiana leaves at the abaxial side using 1 ml syringe without the needle. Due to low overall levels of NPR1-GFP in transgenic plants, a transient expression assay in Arabidopsis seedlings was used to monitor NPR1-GFP subcellular localization after SA treatment (
Chemical treatment, pathogen infection and cell death assays. For soil-grown Arabidopsis plants, SA treatment was carried out with spray at indicated concentrations. For seedlings grown vertically on solid 1/2 MS medium for 10-12 days, SA 925 treatment (1 mM), with or without the proteasome inhibitor MG132 (50 μM), and protein synthesis 926 inhibitor cycloheximide (100 μM) was done by submerging them in solutions for indicated periods. Unless otherwise stated, in N. benthamiana, SA treatment was done by infiltrating leaves 24 hr after agroinfiltration. SA pre-treatment of mature Arabidopsis was done by spray 24 hr before bacterial infection or cell death induction. For bacterial infections, Pseudomonas syringae pv. maculicola ES4326 carrying AvrRpt2 or AvrRpm1 effectors, and Pf Pf0-1 carrying functional AvrRps4 or non-functional AvrRps4KRVY-AAAA effectors were grown for 2 days on solid King's B medium supplemented with appropriate antibiotics. An inoculum was prepared by resuspending cells in 10 mM MgSO4 to obtain the desired optical density: Psm ES4326/AvrRpt2 (OD600 nm=0.02); Psm ES4326/AvrRpm1 (OD600 nm=0.1); Pf Pf0-1/AvrRps4 and Pf Pf0-1/AvrRps4KRVY-AAAA (OD600 nm=0.2). Bacteria were pressure infiltrated into mature leaves of three-week-old Arabidopsis plants and cell death or bacterial growth were assessed at indicated times post inoculation (hpi, hours post inoculation; dpi, days post inoculation). Induction of cell death in dex:AvrRpt2 transgenic plants was performed by spraying plants or infiltrating individual leaves with 25 μM dexamethasone (Sigma). Induction of cell death in est:AvrRpt2 transgenic plants was performed by infiltrating individual leaves with 941 50 μM β-estradiol (Sigma). Heat stress was applied by incubating mature leaf disks from three-942 week-old Arabidopsis plants in 45° C. water bath for 45 min. Oxidative stress was induced by spraying three-week-old Arabidopsis plants with 0.25 mM water solution of MV (Methyl viologen dichloride hydrate; Sigma). UV-C irradiation was performed on leaf disks from three-week-old Arabidopsis plants using UV crosslinker with total dose of 20 kJ/m2. Cell death was monitored using electrolyte leakage assay 1 hr after pathogen infection, or induction of dex:AvrRpt2 and est:AvrRpt2, or application of stresses. For electrolyte leakage, 12 leaf disks were sampled from four plants for each treatment/genotype in three replicates. After sampling, the disks were washed with DDW and conductivity was measured every 3 hr using Orion Star™ A222 Portable Conductivity Meter (ThermoFisher). Monitoring the RPP1/ATR1-induced cell death in the presence of SA was done by addition of SA into the leaf disks incubation solution with subsequent subtraction of the conductivity reads contributed by SA from the total reads. For bacterial growth assay, leaf disks were sampled at 1 dpi from eight infected plants per treatment/genotype, ground in 10 mM MgSO4, and plated with sequential dilutions on King's B medium plates supplemented with appropriate antibiotics. At 2 days, the bacterial colonies were scored.
Trypan blue staining. Leaves of three-week-old Arabidopsis infected with Psm ES4326/AvrRpt2 were sampled at 1 dpi, boiled in 95% ethanol until completely bleached, followed by boiling for 3 min in a 1:1 mixture of 95% ethanol and staining solution (10 ml lactic acid, 10 ml glycerol, 10 g phenol and 10 mg trypan blue, dissolved in 10 ml DDW). The leaves were then de-stained in 2.5 g/ml chloral hydrate solution and transferred to 70% glycerol for subsequent imaging.
VIGS assay. The silencing of NbNPR1 (NbNPR1-RNAi) and NbCUL3 (NbCUL3-RNAi) was done using VIGS assay performed as previously described. Ten-day-old WT N. benthamiana plants were inoculated with Agrobacteria (GV3101) carrying the helper plasmid pTRV1-LIC mixed at 1:1 with a strain carrying either pTRV2-LIC (empty vector control, E.V.), pTRV2-NbPDS (positive control), pTRV2-NbNPR1 or pTRV2-NbCUL3 vectors. Ten days later, gene silencing in the mock and SA-treated plants was verified in the systemic leaves by qPCR using gene-specific and reference gene (eIF4a) primers (Table 6). NbNPR1 silencing was additionally confirmed by analysis of NbPR1 expression. The silenced leaves were used in the standard transient expression assay by agroinfiltration using strains carrying the indicated test genes; and gene expression was assayed at 2 dpi.
Quantitative PCR. Total RNA was extracted from fresh leaf tissue with Trizol reagent (Sigma). cDNA was synthetized using the SuperScript III cDNA Synthesis (Thermofisher). QPCR was performed with FastStart Universal SYBR Green Master Kit (Roche) using Mastercycler ep realplex (Eppendorf). Gene specific primers used for qPCR are listed in Table 6.
PR1 promoter activity and dual luciferase assay. For promoter transactivation assays, the PR1 promoter reporter (pPR1:DUAL-LUC) was transiently co-expressed in N. benthamiana together with free HA, or HA-fused WT NPR1 or npr1 mutants (effectors) followed by treatment with SA at 1 dpi. At 2 dpi (24 hr after SA treatment), leaf discs were collected, ground in liquid nitrogen, and lysed with the PLB buffer of the Dual-Luciferase Reporter Assay System (Promega, E1910). Lysate was spun down at 12,000 g for 1 min, and 10 μl was taken for measuring FLUC and RLUC activities according to manufacturer's instructions using a Victor3 plate reader (PerkinElmer). At 25° C., substrates for FLUC and RLUC 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 PR1 promoter activity, the ratio of F-LUC and R-LUC activities was calculated for each effector and plotted relative to that of free HA.
Confocal laser scanning microscopy. Unless otherwise indicated, all imaging data and micrographs for single protein imaging, co-localizations, BiFC and time-lapse were obtained from the transient expression in N. benthamiana using the relevant Agrobacteria strains. All imaging was done with confocal laser scanning fluorescence microscopy using Zeiss 880 airyscan inverted confocal microscope with a 40×/1.2 water correction objective. GFP (eGFP) was excited with a 488 nm argon laser and emission was collected with a 505-530 nm band pass filter. YFP was excited with an argon laser using a 514 nm beam splitter, and emission was detected with a 520-555 nm band pass filter. mCherry was excited with a 561 nm diode laser, and emission was detected with a 575-615 nm band pass filter. When eGFP/YFP were imaged together with mCherry, a spectral GASP detector was used to collect emission from eGFP/YFP. Propidium iodide (PI) was excited with 488 nm argon laser and emission was detected with a 590-620 nm band pass filter. Time-lapse imaging was carried out on live leaf tissue samples from N. benthamiana plants transiently expressing the protein of interest. Image acquisition was done in 5 min intervals for the duration of 2 hr by scanning 30 consecutive focal planes along the Z-axis covering the entire thickness of an epidermal cell. Z-projection was done to each time frame stack before composing a final time-lapse file.
Protein analysis and IP. Recombinant protein analysis was performed using transient expression in N. benthamiana, or stable over-expression in transgenic Arabidopsis, or expression in E. coli. Total protein extraction from plants was performed by homogenizing leaf tissue in the plant extraction buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, 0.2% Nonidet P-40] supplemented with 1 mM PMSF, 100 μM MG132, and EDTA-free protease inhibitor cocktail (Roche). For ubiquitinated protein analysis in plants, 100 μM of DUB (deubiquitinase) inhibitor (LifeSensors) and 10 mM NEM were added. Next, the lysates were cleared by centrifugation at 14,000 rpm for 15 min followed by filtering through a 0.2 μm filter. Unless otherwise indicated, total protein extraction from E. coli was performed by mechanical disruption of cells with lysis buffer containing 125 mM Tris HCl (pH 7.5), 150 mM NaCl, cocktail of protease inhibitors, 1 mM PMSF, 7.15 mM BME, 1 mM EDTA. Pull-downs or IP experiments were done on total protein extracts by incubating protein samples in the lysis buffer overnight with indicated affinity tag or antibody conjugated to agarose or magnetic beads: GFP-trap agarose beads (Chromotek) for GFP fusions; RFP-trap agarose beads (Chromotek) for mCherry fusions; anti-HA magnetic beads (ThermoFisher) for HA tag fusions; anti-DYKDDDDK coupled magnetic agarose (ThermoFisher) for FLAG tag fusions; and glutathione magnetic agarose (ThermoFisher) for GST fusions. Unless otherwise indicated, after pull-down/IP, beads were washed 3 times and proteins were eluted by boiling in the 2×SDS sample buffer. For Western blotting, the SDS sample buffer was added to the protein extracts from a 4×stock solution supplemented with 50 mM DTT (dithiothreitol) and 715 mM BME. Protein samples were heated to 95° C. for 10 min, separated on SDS-PAGE gels, and transferred to nitrocellulose membranes. Western blots were probed with α-AtNPR1, α-AtEDS1 (Agrisera), α-AtNIMIN1 (ABclonal), α-HA (Biolegend), α-Myc (Santa 1036 Cruz), α-FLAG-HRP (Biolegend), α-V5 (Santa Cruz), α-GST HRP (GE Healthcare), α-GFP (Clonetech), α-RFP (Chromotek), α-AtCUL3A, α-Ubiquitin (Ub; P4D1; Santa Cruz), α-TUB (β-Tubulin F1; Santa Cruz), α-Histone H3 (H3; Agrisera), or α-Actin (ACT; Agrisera) primary antibodies. In Western blots, asterisks indicate non-specific bands from the α-NPR1 (
Cell fractionation. Cytoplasmic and nuclear proteins were sequentially isolated from three-week-old Arabidopsis (Col-0) plants using 1 g of leaf tissue and a sucrose gradient protocol described previously with modifications. Tissue was homogenized in lysis buffer (20 mM Tris-HCl, pH 7.5, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl2, 25% glycerol, 0.25 M Sucrose) supplemented with 100 μM MG132, EDTA-free protease inhibitor cocktail, 1 mM PMSF and 5 mM DTT. The lysate was filtered through a 70 μm filter and centrifuged at 20,000 g for 15 min at 4° C., and the supernatant was collected (cytoplasmic fraction). The pellet was washed four times with 5 ml of NRBT buffer (20 mM Tris-HCl, pH 7.4, 25% glycerol, 2.5 mM MgCl2, and 0.2% Triton X-100). After the last wash, the pellet was resuspended with 500 μl of NRB2 buffer (20 mM Tris-HCl, pH 7.5, 0.25 M Sucrose, 10 mM MgCl2, 0.5% Triton X-100) supplemented with protease inhibitor cocktail and 5 mM BME. The obtained suspension was layered at 1:1 on top of the NRB3 buffer (20 mM Tris-HCl, pH 7.5, 1.7 M Sucrose, 10 mM MgCl2, 0.5% Triton X-100) supplemented with protease inhibitor cocktail and 5 mM BME, centrifuged at 16,000 g for 45 min at 4° C. The top layer was removed and the pellet was resuspended with 200 μl of plant extraction buffer containing 1% Triton X-100, protease inhibitor cocktail and 5 mM BME (nuclear fraction). Samples were run on a reducing SDS-PAGE. Cell fractionation was confirmed by immunoblotting with antibodies against cytoplasmic marker actin (α-ACT) and nuclear marker histone H3 (α-H3).
Prediction of redox-sensitive disorder regions. Protein sequences of AtNPRs were submitted to the IUPred2a tool (iupred2a.elte.hu/; (Meszaros et al., 2018)) for prediction of intrinsically disordered regions (IDRs) with experimental redox-state option turned on. The differential IDR score per residue (
Mass spectrometry. For proteomic analysis of NPR1 condensates, total proteins from mock- and SA-treated transgenic Arabidopsis plants expressing sim3-GFP in the WT NPR1 background (sim3-GFP/Col-0) were isolated, followed by immunoprecipitation of sim3-GFP and a quantitative LC-MS analysis of the co-purified proteins. Lysates were prepared from 6 g of tissue from three-week-old plants treated with water (mock) or 1 mM SA for 24 hr using IP buffer (plant extraction buffer containing 1% Triton X-100) supplemented with 1 mM PMSF, 100 μM MG132, 100 μM DUB inhibitor, 10 mM NEM, 1.43 mM BME, EDTA-free protease inhibitor cocktail (Roche), and 100 μM SA for SA-treated sample. For each treatment the obtained lysate was filtered through a 0.2 μm filter, split into three replicates, mixed with GFP-trap agarose beads (Chromotek) under saturating conditions [25 μl beads (50% slurry)/3 ml of lysate] and subjected to three independent IP reactions per each sample by overnight incubation at 4° C. After incubation, the beads were washed five times with the IP buffer and three times with 50 mM ammonium bicarbonate (NH4HCO3). For silver stain, 5% of the beads were mixed with ×2 SDS sample buffer, boiled at 95° C. for 10 min and the supernatant was run on a 4-12% polyacrylamide gel. The gel was stained with Pierce™ Silver Stain Kit (ThermoFisher) according to the manufacturer's instructions. The remaining beads were submitted to the Duke Proteomics Core Facility for an on-bead trypsin digestion, peptide lyophilization and LC-MS/MS analysis. Quantitative one-dimensional liquid chromatography, tandem mass spectrometry (1D-LC-MS/MS) was performed on equal volumes of peptide digests of the three replicates from each treatment. Following the LC-MS/MS runs, data were imported into Rosetta Elucidator v. 4 (Rosetta Biosoftware, Inc.), and analyses were aligned based on the accurate mass and retention time of detected ions (“features”) using PeakTeller algorithm in Elucidator. Relative peptide abundance (expression intensity) was calculated based on area-under-the curve (AUC) of the selected ion chromatograms of the aligned features across all runs. The MS/MS data were searched against a custom Araport11 database with an additional entry for the sim3-GFP sequence and an equal number of reversed-sequence “decoys” for false discovery rate determination (96,720 total entries). Mascot Distiller and Mascot Server (v 2.5, Matrix Sciences) were utilized to produce fragment ion spectra and to perform the database searches. Database search parameters included precursor mass tolerance of 5 ppm, product ion mass tolerance of 0.8 Da, trypsin specificity with up to 2 missed cleavages, fixed modification on Cys (carbamidomethyl) and variable modification of deamidation (Asn/Gln), oxidation (Met) and N-terminal protein acetylation. After individual peptide scoring using the PeptideProphet algorithm in Elucidator, the data were annotated at a 1% peptide and 0.8% protein false discovery rates, respectively. The data were filtered to remove low quality peptides with poor chromatographic peak shape, and those quantified by less than two peptides. Only those proteins quantified from at least two replicates in each sample were accepted. Due to differences in the intensity values of sim3-GFP peptides between mock and SA treated samples, the data were normalized to the mean sim3-GFP expression across all 6 samples. In the normalized data the intensities of sim3-GFP were equalized, while other identified protein intensities varied based on their abundance. The variability between the samples and replicates were visualized from the normalized data by principal component analysis (PCA) and 2-dimentional hierarchical clustering (
Quantification And Statistical Analysis. For all image quantifications, 8-16 randomly sampled unsaturated confocal images (512×512 pixels, 225×225 μm) were used with an automated image analysis algorithm implemented in the ImageJ software as previously described. Briefly, for each experiment, images from control and test samples were grouped into a dataset. Next, a random image was selected from the dataset and parameters such as local threshold, background noise, object size and shape were determined. The obtained parameters were fed into an algorithm for automated analysis of the entire dataset. The output values, such as body number, size, and intensity/body, were used for statistical analysis between test and control groups using Student's t tests or one-way ANOVA tests. For quantification of co-localization, Pearson's correlation coefficients were calculated between GFP/BiFC and mCherry signals using the JACoP plugin in the ImageJ software with the default settings.
All experiments involving measurements/quantifications, imaging and quantifications from images were repeated at least two times with similar results. Data plotting and statistical tests were performed in GraphPad Prism 8. Statistical parameters such as mean±SD (standard deviation), SE (standard error), 95% confidence intervals are indicated in figure legends. In graphs showing quantification of co-localization and nuclear-cytoplasmic partitioning, all data points (number of images used, n) are plotted using box & whiskers with min/max range indication. In all graphs, asterisks indicate statistical significance (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001, ns, not significant) tested by Student's t test (two groups) or one/two-way ANOVA (multiple groups). Lowercase letters indicate statistical significance tested between multiple groups by one-way ANOVA at p<0.05. In conductivity assays n=3; in bacterial growth assays n=8; in PR1 promoter transactivation assay n=3.
syringae pv. tomato infection.
parasitica) which exhibits a pronounced upregulation
H. parasitica. LURP1 is required for full basal defense to
H. parasitica and resistance to this pathogen mediated
Pseudomonas syringae pv. maculicola ES4326/AvrRpt2
Pseudomonas syringae pv. maculicola ES4326/AvrRpm1
Pseudomonas fluorescens Pf0-1/AvrRps4
Pseudomonas fluorescens Pf0-1/AvrRps4KRVY-AAAA
Agrobacterium tumefaciens, strain GV3101
Escherichia coli (E. coli), strain BL21(DE3)
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.
No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. 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.
This application claims the benefit of U.S. Provisional Application No. 63/042,237, filed Jun. 22, 2020, which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/038430 | 6/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63042237 | Jun 2020 | US |
