WO2019108619A1 - Procédés et compositions pour améliorer la résistance des plantes aux maladies - Google Patents

Procédés et compositions pour améliorer la résistance des plantes aux maladies Download PDF

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WO2019108619A1
WO2019108619A1 PCT/US2018/062800 US2018062800W WO2019108619A1 WO 2019108619 A1 WO2019108619 A1 WO 2019108619A1 US 2018062800 W US2018062800 W US 2018062800W WO 2019108619 A1 WO2019108619 A1 WO 2019108619A1
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plant
protein
amino acid
nrc
nlr
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PCT/US2018/062800
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English (en)
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Lida DEREVNINA
Chih-Hang Wu
Sophien KAMOUN
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Two Blades Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 070294-0l49SEQLST.TXT, created on November 28, 2018, and having a size of 198 kilobytes, and is filed concurrently with the specification.
  • sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • the present invention relates to the fields of plant disease resistance and crop plant improvement, particularly to enhancing the resistance of crop plants to plant disease using of engineered disease resistance genes.
  • genetic solutions instead of chemical solutions (e.g. synthetic pesticides) to protect crops against pathogens and pests (Jones et al. (2014) Philos. T. Roy. Soc. B 369:20130087).
  • genetic solutions include, for example, crop plants which have been bred to be resistant to pathogens through introgression of naturally-occurring resistance (R) genes which provide the plant with resistance against plant pathogens such as, for example, bacteria, oomycetes, viruses, fungi, and nematodes.
  • R naturally-occurring resistance
  • R genes have been successfully used to enhance the resistance of crop plants to plant pathogens, the resistance conferred to plants by most R genes has not been durable as pathogens evolve to overcome the resistance provided by the R genes.
  • the reliance on monocultures in modem agriculture promotes the rapid emergence of new virulent isolates of plant pathogens, because plant pathogens experience a strong selective pressure as cultivars with new genes are released (McDonald & Linde (2002) Euphytica 124: 163-180).
  • NLR leucine-rich repeat-containing
  • NRC NLR-required for cell death proteins
  • sensor NLR proteins Wu et al. 2017 PNAS 114(30): 8113-8118.
  • paired NLRs have been described across flowering plants, the degree to which plant NLRs have evolved to form higher order networks is poorly known (Wu et al, 2017 PNAS 114(30): 8113-8118). Further investigations designed to increase our understanding of the role of sensor NLR-NRC protein pairs in mediating plant immune responses are likely to provide the foundation for the development of new strategies for enhancing the resistance of plants to plant pathogens.
  • the present invention provides methods for making engineered nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins—particularly engineered NRC (NLR-required for cell death) proteins (also known as“helper NLR proteins”) and engineered sensor NLR proteins— that are capable of conferring to a plant enhanced resistance to at least one plant pathogen.
  • NLR leucine-rich repeat-containing
  • the present invention provides methods for making engineered NRC proteins that are capable of conferring to a plant enhanced resistance to a plant pathogen comprising a pathogen suppressor.
  • Such methods comprise modifying the amino acid sequence of an NRC protein, so as to produce an amino acid sequence of an engineered NRC protein that is not suppressible in the plant in the presence of the pathogen suppressor and is capable of causing a cell death response when paired with a sensor NLR protein that detects the plant pathogen.
  • the methods involve domain swapping using two or more NRC proteins to produce an engineered NRC protein that is a chimeric NRC protein comprising domains or parts of domains from two or more NRC proteins.
  • the present invention provides methods for making engineered sensor NLR proteins that are capable of evading suppression of cell death caused by a pathogen effector.
  • Such methods comprise modifying the amino acid sequence of a sensor NLR protein of interest that is capable of pairing with a first NRC protein to cause a cell death response in a plant in response to the pathogen contacting the plant only in the absence of the pathogen suppressor, whereby an engineered sensor NLR protein is produced that is capable of pairing with a second NRC protein to cause a cell death response in the plant in the presence of presence of the pathogen suppressor.
  • the methods involve selecting one or more amino acids for modification in the sensor NLR protein of interest by comparing the amino acid sequence of the sensor NLR protein of interest with the amino acid sequence of at least one other sensor NLR protein to identify one or more amino acid polymorphisms between the amino acid sequence of the sensor NLR protein of interest and the amino acid sequence of at least one other sensor NLR protein, wherein the at least one other sensor NLR protein is capable of pairing with the second NRC protein to cause a cell death response in the plant.
  • the methods involve modifying the amino acid sequence of the sensor NLR protein of interest by replacing one or more regions or blocks of multiple contiguous amino acids in one or more its domains with the
  • the present invention further provides methods for making nucleic acid molecules that encode the engineered NRC proteins and the engineered sensor NLR proteins described above.
  • such methods involve synthesizing in vitro nucleic acid molecules comprising the engineered NRC and sensor NLR proteins of the present invention.
  • the methods involve synthesizing the engineered NRC and sensor NLR proteins in plants cells through the use of genome editing with engineered nucleases that are capable of inducing double-strand breaks at specific locations in the genomes of cells.
  • the present invention further provides of methods for producing plants with enhanced resistance to a plant pathogen comprising modifying a plant cell to express an engineered NRC or sensor NLR protein of the present invention.
  • the methods comprise introducing into a plant cell a nucleic acid molecule comprising a nucleotide sequence that encodes an engineered NRC or sensor NLR protein, and optionally
  • the methods comprise modifying the genome of a plant or at least one cell thereof to comprise a polynucleotide comprising a nucleotide sequence that encodes an engineered NRC or sensor NLR protein using genome editing with engineered nucleases that are capable of inducing double-strand breaks in specific locations in the genomes of cells, whereby a plant with enhanced resistance to one or more plant pathogens is produced.
  • the present invention provides methods for enhancing the resistance of a plant to a plant pathogen, particularly a plant comprising partial resistance to the plant pathogen.
  • the methods comprise modifying a plant cell to be capable of increased expression of at least one NRC protein wherein the plant comprises a resistance gene against the pathogen but is only partially resistant to the pathogen.
  • the methods optionally further comprise regenerating the modified plant cell into a modified plant comprising enhanced resistance to the plant pathogen, relative to a control plant that is not modified for increased expression of the at least one NRC protein.
  • the present invention provides methods for enhancing the resistance of a plant to at least one plant pathogen when a resistance gene that is active for resistance to the pathogen in a first plant species is transferred to a second plant species, wherein the resistance gene is inactive or otherwise does not enhance the resistance of the second plant species to a plant pathogen.
  • the methods comprise modifying at least one cell of a plant of the second plant species to co-express a sensor NLR protein and an NRC protein, whereby the co-expressed sensor NLR and NRC proteins are together capable of causing a cell death response in the plant or part or cell thereof in response to the plant pathogen.
  • the methods optionally further comprise regenerating the modified plant cell into a modified plant comprising enhanced resistance to the plant pathogen, relative to a control plant that is not modified for expression of the sensor NLR protein and/or the NRC protein.
  • the methods comprise planting a seed, seedling, a tuber, or other plant part, wherein the seed, seedling, a tuber, or other plant part comprises a nucleic acid molecule comprising a nucleotide sequence encoding an engineered NRC or sensor NLR protein of the present invention.
  • the methods comprise planting a seed, seedling, a tuber, or other plant part, wherein the seed, seedling, a tuber, or other plant part from a plant that has been modified to be capable of increased expression of at least one NRC protein.
  • the methods comprise planting a seed, seedling, a tuber, or other plant part, wherein the seed, seedling, a tuber, or other plant part comprises either a nucleic acid molecule encoding both a sensor NLR protein and an NRC protein that is capable of causing a cell death response in a plant or part or cell thereof in response to a plant pathogen or two nucleic acid molecules, one nucleic acid molecule encoding the sensor NLR and the other nucleic acid molecule encoding the NRC.
  • the methods further comprise growing the plant under conditions favorable for the growth and development of the plant, and then optionally harvesting from the plant at least one seed, tuber, fruit, flower, or other plant part or parts.
  • engineered NRC and sensor NLR proteins and nucleic acid molecules encoding the engineered NRC and sensor NLR proteins are further provided. Further provided are transgenic and non-transgenic plants, plant parts, seeds, fruits, tubers, plant cells, other host cells, expression cassettes, and vectors comprising one or more of the nucleic acid molecules of the present invention.
  • FIG. 1 depicts an NRC-dependent NLR immune signalling network provides resistance to diverse pathogens.
  • NLRs that confer resistance to diverse pathogens, including virus, bacteria, oomycete, nematodes and insects converge on the three NRC proteins. These three NRC proteins are functionally redundant but also display specificity toward some of the sensor NLRs.
  • the downstream signalling remains largely unknown (adapted from Wu el al, 2017 PNAS 114(30): 8113—8118).
  • FIG. 2 is a photographic illustration that shows AVRcaplb and SPRYSEC15 can suppress cell death mediated by an autoactive NRC2 and NRC3 mutants, but not the autoactive NRC4 mutant.
  • Autoactive NRC2, NRC3 and NRC4 mutants were generated by mutating the MHD motif within the NB-ARC domain of the NLR proteins (van Ooijen el al. (2008) J Exp. Bot. 59: 1383-1397).
  • NRC2 H480R , NRC3 D480V and NRC4 D478V (autoactive mutants) were co-expressed with either an empty vector (EV), AVRcaplb * (upper panel) or SPRYSEC15 (lower panel) by agroinfiltration into N. benthamiana leaves.
  • EV empty vector
  • AVRcaplb * upper panel
  • SPRYSEC15 lower panel
  • SPRYSEC15 suppressed cell death mediated by NRC2 H480R (leftmost leaf in upper and lower panels) and NRC3 D480V (middle leaf in upper and lower panels), but not cell death mediated by NRC4 D478V (rightmost leaf in upper and lower panels).
  • FIG. 3 is a schematic illustration of NRC3 and NRC4 chimera design, and summary of suppression of immune responses by AVRcaplb and SPRYSEC15.
  • the various conserved domains of the resistance genes are indicated: CC, coiled coil domain; NB-ARC (nucleotide binding adaptor shared with APAF-l plant resistance proteins and CED-4 domain) and its three sub-domains (NB, ARC1 and ARC2); LRR1-13, leucine-rich repeat domain with 13 repeats.
  • Both AVRcaplb and SPRYSEC15 are able to suppress cell death induced by NRC3, but the chimeric variants (NRC4-3 LRR and NRC4-3 LRR8 13 ) evade suppression.
  • FIGS. 4A-4D depict strategies for i? gene resurrection.
  • FIG. 4A depicts the NRC network in its natural state.
  • FIG. 4B depicts the NRC network with pathogen suppressors targeting NRC 2/3, thereby suppressing cell death mediated by sensor NLRs that signal through NRC2/3.
  • FIG. 4C depicts the strategy of engineering synthetic NRCs that evade suppression by pathogen effectors. These synthetic NRCs will signal through the NRC4 downstream immune pathway.
  • FIG. 4D depicts the strategy of engineering sensor NLRs that evade suppression by signalling through NRC4.
  • FIG. 5 is an alignment of amino acid sequences of the resistance proteins Gpa2 and Rx.
  • the black boxes represent the regions to be swapped in Gpa2 with that of Rx, and are labelled as polymorphic regions 1-5.
  • FIG. 6 is a photographic illustration showing different Nicotiana benthamiana lines infected with Phytophthora infestans strain P/NL07434.
  • Wild-type (WT) and NRC4 overexpression N. benthamiana transgenic lines, driven by the 35S promoter (35S :.NRC4) are susceptible to P/NL07434, while Rpiblb2 N. benthamiana transgenic lines are partially susceptible to P/NL07434.
  • Crosses between 35S : :NRC4 and Rpiblb2 resulted in F3 lines (Line 3.3, 7.4 and Line 7.3, right 3 panels) that display resistance to P/NL07434.
  • FIG. 7A is a schematic illustration of the genomic position of NRC6 and NLR protein Hero in Solanum lycopersicum.
  • FIG. 7B is photographic illustration showing that the autoactive Hero H855A mutant induces cell death only when co-expressed with NRC6 in Nicotiana benthamiana leaves.
  • the autoactive Hero H855A mutant was generated by mutating the MHD motif within the NB-ARC domain (van Ooijen et al. (2008) J. Exp. Bot. 59: 1383-1397).
  • 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.
  • 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.
  • SEQ ID NO: 1 sets forth a nucleotide sequence encoding the engineered NRC protein, NRC4-3 lrr .
  • a stop codon e.g. TAA, TAG, or TGA
  • TAA a stop codon
  • TAG a stop codon that can be operably linked to the 3' end of nucleic acid molecule comprising SEQ ID NO: 1. It is noted that in the stop codon used in the examples herein below with SEQ ID NO: 1 is TAA.
  • SEQ ID NO: 2 sets forth the amino acid sequence of the engineered NRC gene, NRC4-3 lrr .
  • SEQ ID NO: 3 sets forth a nucleotide sequence encoding the engineered NRC protein, NRC4-3 LRR8 13 . It is noted that in the stop codon used in the examples herein below with SEQ ID NO: 3 is TAA.
  • SEQ ID NO: 4 sets forth the amino acid sequence of the engineered NRC gene, NRC4-3 LRR8 13 .
  • SEQ ID NO: 5 sets forth a nucleotide sequence encoding the engineered sensor NLR protein, Gpa2/Rx-polymorphic region 1. It is noted that in the stop codon used in the examples herein below with SEQ ID NO: 5 is TAG.
  • SEQ ID NO: 6 sets forth the amino acid sequence of the engineered sensor NLR gene, Gpa2/Rx-polymorphic region 1.
  • SEQ ID NO: 7 sets forth a nucleotide sequence encoding the engineered sensor NLR protein, Gpa2/Rx-polymorphic region 2. It is noted that in the stop codon used in the examples herein below with SEQ ID NO: 7 is TAG.
  • SEQ ID NO: 8 sets forth the amino acid sequence of the engineered sensor NLR gene, Gpa2/Rx-polymorphic region 2.
  • SEQ ID NO: 9 sets forth a nucleotide sequence encoding the engineered sensor NLR protein, Gpa2/Rx-polymorphic region 3. It is noted that in the stop codon used in the examples herein below with SEQ ID NO: 9 is TAG.
  • SEQ ID NO: 10 sets forth the amino acid sequence of the engineered sensor NLR gene, Gpa2/Rx-polymorphic region 3.
  • SEQ ID NO: 11 sets forth a nucleotide sequence encoding the engineered sensor NLR protein, Gpa2/Rx-polymorphic region 4. It is noted that in the stop codon used in the examples herein below with SEQ ID NO: 11 is TAG.
  • SEQ ID NO: 12 sets forth the amino acid sequence of the engineered sensor NLR gene, Gpa2/Rx-polymorphic region 4.
  • SEQ ID NO: 13 sets forth a nucleotide sequence encoding the engineered sensor NLR protein, Gpa2/Rx-polymorphic region 5. It is noted that in the stop codon used in the examples herein below with SEQ ID NO: 13 is TAG.
  • SEQ ID NO: 14 sets forth the amino acid sequence of the engineered sensor NLR gene, Gpa2/Rx-polymorphic region 5.
  • SEQ ID NO: 15 sets forth the nucleotide sequence of the coding region of the cDNA of NRC2a from Nicotiana benthamiana.
  • a stop codon e.g. TAA, TAG, or TGA
  • the stop codon is TAA.
  • SEQ ID NO: 16 sets forth the amino acid sequence of NRC2a from Nicotiana benthamiana (Sol Genomics Network Accession NbS00026706g00l6. l).
  • SEQ ID NO: 17 sets forth the nucleotide sequence of the coding region of the cDNA of NRC2b from Nicotiana benthamiana.
  • a stop codon e.g. TAA, TAG, or TGA
  • the stop codon is TGA.
  • SEQ ID NO: 18 sets forth the amino acid sequence of NRC2b from Nicotiana benthamiana (Sol Genomics Network Accession NbS000l8282g00l9. l).
  • SEQ ID NO: 19 sets forth the nucleotide sequence of the coding region of the cDNA oiNRC3 from Nicotiana benthamiana.
  • a stop codon e.g. TAA, TAG, or TGA
  • the stop codon is TAA.
  • SEQ ID NO: 20 sets forth the amino acid sequence of NRC3 from Nicotiana benthamiana (Sol Genomics Network Accession NbSOOOl !087g0003.1).
  • SEQ ID NO: 21 sets forth the nucleotide sequence of the coding region of the cDNA of NRC4a from Nicotiana benthamiana.
  • a stop codon e.g. TAA, TAG, or TGA
  • the stop codon is TAA.
  • SEQ ID NO: 22 sets forth the amino acid sequence of NRC4a from Nicotiana benthamiana (Sol Genomics Network Accession NbS000l6l03g0004. l).
  • SEQ ID NO: 23 is an artificial or synthetic nucleotide sequence encoding NRC2a (SED IN: 15) which has been engineered to remove the recognition sites for the restriction enzyme Bpil and Bsal that occur in the wild-type NRC2a cDNA (SEQ ID NO: 14).
  • a stop codon e.g. TAA, TAG, or TGA
  • TAA the stop codon
  • SEQ ID NO: 24 is an artificial or synthetic nucleotide sequence encoding NRC2a (SED IN: 19) which has been engineered to remove the recognition sites for the restriction enzyme Bpil and Bsal that occur in the wild-type NRC3 cDNA (SEQ ID NO: 18).
  • a stop codon e.g. TAA, TAG, or TGA
  • TAA the stop codon
  • SEQ ID NO: 25 is an artificial or synthetic nucleotide sequence encoding NRC4a (SED IN: 21) which has been engineered to remove the recognition sites for the restriction enzyme Bpil and Bsal that occur in the wild-type NRC4a cDNA (SEQ ID NO: 20).
  • a stop codon e.g. TAA, TAG, or TGA
  • TAA the stop codon
  • SEQ ID NO: 26 sets forth the nucleotide sequence of the coding region of the cDNA of A V Reap lb from Phytophthora infestans (Genbank Accession XM_002896885. l).
  • a stop codon e.g. TAA, TAG, or TGA
  • the stop codon is TAA.
  • SEQ ID NO: 27 sets forth the amino acid sequence of AVRcaplb from Phytophthora infestans (Genbank Accession XP_00289693l. l).
  • SEQ ID NO: 28 sets forth the nucleotide sequence of the coding region of the cDNA of truncated SPRYSEC15 from Globodera rostochiensis (Genbank Accession KF963514.1). If desired, a stop codon (e.g. TAA, TAG, or TGA) can be operably linked to the 3' end of nucleic acid molecule comprising SEQ ID NO: 28. It is noted that in the cDNA derived from the truncated SPRYSEC15 transcript, the stop codon is TAG.
  • SEQ ID NO: 29 sets forth the amino acid sequence of truncated SPRYSEC15 from Globodera rostochiensis (Genbank Accession AHZ59334.1).
  • SEQ ID NO: 30 is an artificial or synthetic nucleotide sequence encoding a truncated AVRcaplb protein in which the signal peptide, RxLR and dEER domains have been omitted from the N-terminal end of AVRcaplb protein (corresponding to amino acids 1-61 of SEQ ID NO: 27).
  • a start codon was added to the 3' end of SEQ ID NO: 30 which results in an N- terminal methionine in the encoded protein.
  • a stop codon e.g. TAA, TAG, or TGA
  • the stop codon is TAA.
  • SEQ ID NO: 31 is an artificial or synthetic amino acid sequence of the truncated AVRcaplb protein encoded by SEQ ID NO: 30. Amino acids 2-618 of SEQ ID NO: 31 correspond to amino acids 62 to 678 of SEQ ID NO: 27.
  • SEQ ID NO: 32 is an artificial or synthetic nucleotide sequence encoding a fusion protein comprising 4HA-tag operably linked operably linked to the truncated SPRYSEC15 protein having the amino acid sequence set forth in SEQ ID NO: 29.
  • a stop codon e.g. TAA, TAG, or TGA
  • TAA AA, TAG, or TGA
  • SEQ ID NO: 33 is an artificial or synthetic amino acid sequence of the truncated SPRYSEC15 protein encoded by SEQ ID NO: 32. Amino acids 52-273 of SEQ ID NO: 33 correspond to amino acids 1 to 222 of SEQ ID NO: 29.
  • SEQ ID NO: 34 sets forth the nucleotide sequence of the coding region of the cDNA of Hero from Solarium lycopersicum.
  • a stop codon e.g. TAA, TAG, or TGA
  • TAA the native stop for Hero codon
  • SEQ ID NO: 35 sets forth the amino acid sequence of the Hero protein from Solarium lycopersicum.
  • SEQ ID NO: 36 is an artificial or synthetic nucleotide sequence encoding the Hero H855A protein.
  • a stop codon e.g. TAA, TAG, or TGA
  • TAA a stop codon
  • TAG a stop codon for a stop codon that is operably linked to the 3' end of nucleic acid molecule comprising SEQ ID NO: 36. It is noted that in certain embodiments of the invention disclosed herein below the stop codon is TAA.
  • SEQ ID NO: 37 is an artificial or synthetic amino acid sequence of the Hero H855A protein encoded by SEQ ID NO: 36.
  • SEQ ID NO: 38 sets forth the nucleotide sequence of the coding region of the cDNA of NRC6 from Solanum lycopersicum.
  • a stop codon e.g. TAA, TAG, or TGA
  • TAA, TAA, TAG, or TGA can be operably linked to the 3' end of nucleic acid molecule comprising SEQ ID NO: 38. It is noted that the native stop for NRC6 codon is TGA.
  • SEQ ID NO: 39 sets forth the amino acid sequence of the NRC6 protein from
  • the present invention relates methods for making and using synthetic or artificial NLR proteins and nucleic acid molecules encoding such NLR proteins.
  • the NLR proteins and nucleic acid molecules of the present invention are synthetic or artificial (i.e non-naturally occurring) proteins and nucleic acid molecules.
  • Such synthetic or artificial NLR proteins of the present invention are also referred to herein as“engineered NLR proteins”.
  • the NLR proteins and nucleic acid molecules encoding them find use in enhancing the resistance of plants, particularly crop plants, to plant pathogens. Such crop plants with enhanced resistance to plant pathogens find use in agriculture by limiting or reducing plant disease.
  • the present invention further relates to compositions comprising the NLR proteins of the present invention and/or the nucleic acid molecules of the present invention including, but not limited to, plants, plant cells, and other host cells comprising one or more of such NLR proteins and/or nucleic acid molecules and expression cassettes and vectors comprising one or more of such nucleic acid molecules.
  • the NLR proteins of the present invention include both sensor NLR proteins and helper NLR (or NRC) proteins. Plants rely on NLR proteins to respond to invading pathogens and to activate immune responses.
  • An emerging concept in NLR biology is that “sensor” NLR proteins are paired with“helper” NLR proteins to mediate immune signaling.
  • NRC NLR-required for cell death
  • NRC3 NRC-required for cell death protein family members
  • the NRC protein superclade comprises approximately one-third of all NLR proteins in the Solanaceae family.
  • the NRC protein family is functionally redundant but display specificities towards different sensor NLR proteins.
  • the sensor NLR protein, Prf signals via NRC2 or NRC3 but not NRC4, whereas the sensor NLR protein, Rpi-blb2, signals only via NRC4, while other sensor NLR proteins, such as R8, Rx, Bs2 and Sw5, signal via interchangeable NRC2, NRC 3 or NRC4.
  • AVRcaplb a Phytophthora infestans effector was recently identified that can suppress NRC2- and NRC3-dependent cell death but not NRC4- dependent cell death. This pathogen effector has been designated as AVRcaplb.
  • AVRcaplb When AVRcaplb was co-expressed with different sensor NLR/ AVR protein pairs in Nicotiana benthamiana leaves, suppression of cell death was observed only in NRC2/3 -dependent sensor NLR proteins (Prf, Gpa2, Rpiamrla) but not with other sensor NLR proteins that can signal interchangeable via NRC2, NRC3, or NRC4, or only with NRC4.
  • AVRcaplb and other pathogen effectors that function similarly to suppress a cell death response are referred to herein as“pathogen suppressors” to distinguish them from other pathogen effects that do not display such a suppression function.
  • the present inventors endeavored to produce modified or engineered NRC proteins and to test such engineered NRC proteins for the ability to overcome suppression caused by a pathogen suppressor such as, for example, AVRcaplb.
  • the present invention is based in part on the present inventors’ discovery that they can produce an engineered NRC protein that when expressed in plant cells can overcome the suppression of the cell death response in the presence of a pathogen suppressor.
  • the present invention provides not only methods for making and using engineered NRC proteins and nucleic acid molecules encoding engineered NRC proteins, but also compositions comprising one or more of the engineered NRC proteins and/or the nucleic acid molecules encoding engineered NRC proteins.
  • the present invention provides methods for making an engineered NRC protein that is capable of conferring to a plant enhanced resistance to a plant pathogen comprising a pathogen suppressor.
  • Such methods comprise modifying the amino acid sequence of an NRC protein to produce an engineered NRC protein that is not suppressible in the plant in the presence of the pathogen suppressor.
  • Such“modifying” comprises the substitution, addition, and/or deletion of one or more amino acids of amino acid sequence of the NRC protein or other protein (e.g. a sensor NLR).
  • modified amino acid sequence of an engineered NRC protein or any other engineered protein disclosed elsewhere herein comprising a modified amino acid sequence
  • an engineered sensor NLR e.g. an engineered sensor NLR
  • modified amino acid sequence can be produced by, for example, designing the modified amino acid sequence on paper and/or with the use of a computer using methods disclosed hereinbelow or otherwise known in the art.
  • the methods can further comprise producing an engineered NRC protein or other engineered protein comprising the modified amino acid sequence by, for example, chemically synthesizing a polypeptide comprising the modified amino acid sequence or by producing a nucleic acid molecule encoding a polypeptide comprising the modified amino acid sequence and expressing the nucleotide acid molecule in a cell or in vitro, whereby an engineered NRC or other engineered protein comprising the modified amino acid sequence is produced.
  • nucleic acid molecules can be produced, for example, by routine molecular biology methods disclosed elsewhere or otherwise known in the art or by chemical synthesis using a DNA synthesizer.
  • Such molecular biology methods include, but are not limited to, gene editing, PCR amplification, cloning, site-directed mutagenesis, restriction nuclease enzyme digestion, ligation, and the like. It is further recognized that a nucleic acid molecule encoding an engineered NRC or other engineered protein comprising a modified amino acid sequence can be produced within the genome of a plant cell or other host cell using genome-editing methods that are disclosed herein below or are otherwise known in the art.
  • modifying the amino acid sequence of an NRC protein comprises assembling an amino acid sequence encoding a chimeric NRC protein comprised of (a) the amino acid sequences of one or more domains or parts of domains of a first NRC protein operably linked to (b) the amino acid sequences of one or more domains or parts of domains of at least one additional NRC protein, whereby an amino acid sequence encoding the chimeric NRC is produced.
  • the NRC proteins are neither suppressible by the pathogen suppressor nor known to pair with a sensor NLR protein that senses the plant pathogen comprising the pathogen suppressor, and at least one NRC protein that is suppressible by the pathogen suppressor and known to pair with a sensor NLR protein that senses the plant pathogen comprising the pathogen suppressor.
  • the amino acid sequences for the various domains or parts of domains are operably linked and arranged in a manner to produce a primary protein structure that is analogous to a naturally-occurring NRC protein such as, for example, NRC3 and NRC4. See FIG. 3A.
  • the domains are the following order from N-terminal end of the amino acid sequence to C-terminal end: CC, NB, ARC1, ARC2, and LRR.
  • the methods for making an engineered NRC protein comprise modifying the amino acid sequence of an NRC protein to produce a chimeric NRC protein comprising the CC, NB, the ARC1, and the ARC2 domains of a first NRC protein and the LRR domain from a second NRC protein.
  • the first NRC protein is not suppressible by the pathogen suppressor produced by the pathogen of interest but is known to pair with a sensor NLR protein that senses the plant pathogen comprising the pathogen suppressor.
  • the second NRC protein is suppressible by the pathogen suppressor and known to pair with a sensor NLR protein that senses the plant pathogen comprising the suppressor.
  • the plant is a solanaceous plant, particularly a potato, tomato, or tobacco plant
  • the first NRC protein is NRC4.
  • the second NRC protein is NRC2 or NRC3. More preferably, the first NRC protein is NRC4 and the second NRC protein is NRC3.
  • the preferred pathogens are those that are known to infect solanaceous plants and comprise a pathogen suppressor that can suppress cell death in solanaceous plants.
  • Such preferred pathogens include, but are not limited to, strains of the oomycete pathogen Phytophthora infestans comprising the pathogen suppressor AVRcaplb and strains of the nematode pathogen Globodera rostochiensis comprising the pathogen suppressor SPRYSEC15.
  • Examples of such engineered NRC proteins that are producible by the method the present invention are NRC4-3 LRR and NRC4-3 LRR8 13 , which comprise the amino acid sequences set forth in SEQ ID NOS: 2 and 4, respectively.
  • the present invention further provides methods for making nucleic acid molecules encoding engineered NRC proteins of the present invention that are capable of conferring to a plant enhanced resistance to at least one plant pathogen.
  • the methods comprise synthesizing a nucleic acid molecule encoding the amino acid sequence of an engineered NRC of the present invention.
  • nucleic acid molecule encoding a protein of interest and that such a nucleic acid molecule can be synthesized using, for example, a DNA synthesizer and/or using standard molecular biology methods described hereinbelow or otherwise known the art such as, for example, restriction endonuclease digestion, ligation, polymerase chain reaction (PCR) amplification, site-directed mutagenesis, sequencing, and the like.
  • PCR polymerase chain reaction
  • nucleic acid molecules encoding such engineered NRC proteins that are producible by the methods of the present invention are nucleic molecules comprising at least one of the nucleotide sequences set forth in SEQ ID NOS: 1 and 3, which encode NRC4-3 LRR and NRC4-3 LRR8 13 , respectively.
  • the present invention further provides methods for making engineered sensor NLR proteins that evade suppression of cell death caused by a pathogen suppressor.
  • the methods comprise modifying the amino acid sequence of a sensor NLR protein of interest that is capable of pairing with a first NRC protein to cause a cell death response in a plant, or in at least one cell thereof, in response to a plant pathogen contacting the plant but is not capable of pairing with a second NRC protein to cause a cell death response in the plant, or in at least one cell thereof, whereby an engineered sensor NLR protein is produced that is capable of pairing with the second NRC protein to cause a cell death response in the plant.
  • the sensor NLR protein of interest is only capable of pairing with the first NRC protein and causing a cell death response in the absence of the pathogen suppressor. It is recognized that in a plant, or in at least one plant cell thereof, and in the absence of the plant suppressor, such a sensor NLR protein of interest is capable of binding to, or at least perceiving or recognizing, the presence of the pathogen effector produced by the plant pathogen and mediating the cell death response through pairing with the first NRC protein.
  • the cell death response is suppressed or otherwise does not occur in the plant, or in at least one plant cell thereof, when the plant is contacted by a different strain or race of the same pathogen that comprises both the pathogen effector and the pathogen suppressor.
  • the second NRC protein is from the same plant or plant species as the first NRC protein and is known to be capable of pairing with a different sensor NLR to cause a cell death response in the plant, or in at least one plant cell thereof, in the presence of the pathogen suppressor.
  • the second NRC protein is not known to be suppressed by any pathogen suppressor.
  • the methods for making an engineered sensor NLR protein are used to produce engineered sensor NLR proteins that are not only capable of pairing with the second NRC to cause a cell death response in the presence of the pathogen suppressor, but also are capable of pairing with both the first NRC protein and the second NRC to cause a cell death response in the absence of the pathogen suppressor. More preferably, the engineered sensor NLR proteins are not only capable of pairing with the second NRC to cause a cell death response in the presence of the pathogen suppressor, but also are capable of pairing with the first NRC protein, the second NRC, and one or more other NRC proteins to cause a cell death response in the absence of the pathogen suppressor.
  • the one or more other NRC proteins are NRC proteins, other than first NRC protein, for which the sensor NLR protein of interest is known to pair with and cause a cell death response in the absence of the pathogen suppressor.
  • the methods for making engineered sensor NLR proteins comprise modifying the amino acid sequence of a sensor NLR protein of interest.
  • Such“modifying” comprises the substitution, addition, and/or deletion of one or more amino acids of an amino acid sequence of the sensor NLR protein of interest.
  • the particular amino acids that are selected for modification in the amino acid sequence of the sensor NLR protein of interest can depend on a number of factors.
  • one or more amino acids of the amino acid sequence of the sensor NLR protein of interest are selected for modification by comparing the amino acid sequence of the sensor NLR protein of interest with the amino acid sequence of at least one other sensor NLR protein to identify one or more amino acid polymorphisms between the amino acid sequence of the sensor NLR protein of interest and the amino acid sequence of at least one other sensor NLR protein.
  • the one or more other NLR proteins are capable of pairing with the second NRC protein to cause a cell death response in the plant in the presence of the pathogen suppressor.
  • the amino acid sequence of the sensor NLR protein of interest is modified by replacing one or more regions or blocks of two or more contiguous amino acids in one or more its domains with the corresponding region(s) or block(s) of continuous amino acids in the same domain or domains of at least one other sensor NLR protein that is capable of pairing with the second NRC protein to cause a cell death response in the plant in the presence of the pathogen suppressor, so as to produce a chimeric, engineered sensor NLR protein.
  • Such domains of sensor NLR proteins include, for example the CC, NB, ARC1, ARC2, and LRR domains.
  • the regions or blocks of contiguous amino acids can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more contiguous amino acids or even an entire domain.
  • the methods of the present invention are not limited to modifying the amino acid sequence of a sensor NLR protein of interest by replacing a region of multiple contiguous amino acids within one domain.
  • the region of contiguous amino acids that is replaced is comprised of contiguous amino acids in two or more adjacent domains.
  • the region of contiguous amino acids that is replaced in the amino acid sequence of the sensor NLR protein of interest can be replaced with a corresponding region of amino acids that either has the same number of contiguous amino acids as the region of amino acids in the sensor NLR protein of interest or has a smaller or larger number of contiguous amino acids that the number of contiguous amino acids in the region of amino acids in the sensor NLR protein of interest.
  • a region of contiguous amino acids comprises multiple amino acid polymorphisms when the region in the sensor NLR protein of interest is compared to same region in at least one other sensor NLR protein that is capable of pairing with the second NRC protein to cause a cell death response in the plant in the presence of the pathogen suppressor. It is recognized that such polymorphisms between the amino acid sequences of two or more sensor NLR proteins can be identified through amino acid sequence alignments using methods described elsewhere herein or otherwise know in the art.
  • the amino acid sequence of the sensor NLR protein of interest is modified by replacing one or more regions of contiguous amino acids in its LRR domain with the corresponding region(s) of continuous amino acids in the LRR domain of at least one other sensor NLR protein that is capable of pairing with the second NRC protein to cause a cell death response in the plant in the presence of the pathogen suppressor, so as to produce a chimeric, engineered sensor NLR protein.
  • a region of contiguous amino acids comprises multiple amino acid polymorphisms when the region in the sensor NLR protein of interest is compared to same region in at least one other sensor NLR protein that is capable of pairing with the second NRC protein to cause a cell death response in the plant in the presence of the pathogen suppressor. It is recognized that such polymorphisms between the amino acid sequences of two or more sensor NLR proteins can be identified through amino acid sequence alignments using methods described elsewhere herein or otherwise know in the art.
  • the sensor NLR protein of interest is the nematode resistance protein Gpa2.
  • Gpa2 is capable of separately pairing with each of NRC2 and NRC3 to cause a cell death response in a plant but is not capable of pairing with NRC4 to cause a cell death response.
  • the nematode resistance conferred by Gpa2 is capable of being suppressed in the presence of pathogens comprising a pathogen suppressor such as, for example, SPRYSEC15 or
  • Avrcaplb each of which is capable of suppressing NRC2- and NRC3-dependent cell death but not NRC4-dependent cell death.
  • Engineered sensor NLR proteins can be produced by replacing the contiguous amino acids of one or more these regions of Gpa2 with the contiguous amino acids of the corresponding region of Rx.
  • Examples of such engineered sensor NLR proteins comprise the amino acid sequences set forth in SEQ ID NOS: 6, 8, 10, 12, and 14, and examples of nucleic acid molecules encoding such engineered sensor NLR proteins comprise the nucleotide sequences set forth in SEQ ID NOS: 5, 7, 9, 11, and 13, respectively.
  • the present invention further provides methods for making nucleic acid molecules encoding the engineered sensor NLR proteins of the present invention that evade suppression of cell death caused by a pathogen suppressor.
  • the methods comprise synthesizing a nucleic acid molecule encoding the amino acid sequence of an engineered sensor NLR protein of the present invention.
  • it is routine to synthesize a nucleic acid molecule of a protein of interest using standard molecular biology methods discussed elsewhere herein or otherwise known in the art.
  • Examples of nucleic acid molecules encoding such engineered sensor NLR proteins that a producible by the methods of the present invention are nucleic molecules comprising at least one of the nucleotide sequences set forth in SEQ ID NOS: 5, 7, 9, 11, and 13.
  • the present invention provides methods for enhancing the resistance of a plant to a plant pathogen, particularly a plant comprising partial resistance to the plant pathogen.
  • full or complete resistance is defined as the inability of the pathogen to spread within the host plant genotype. With full resistance, localized cell death is observed on the plant after being contacted by the pathogen but there are no spreading lesions. In contrast with partial resistance, the pathogen may still be able to infect the host plant and cause a spreading lesion, but the spread of the lesion is restricted or limited, when compared to a susceptible plant.
  • Such methods for enhancing the resistance of a plant comprise modifying a plant cell to be capable of increased expression (also referred to herein as overexpression) of at least one NRC protein, wherein the plant comprises a resistance gene against the pathogen but the plant is only partially resistant to the pathogen.
  • the methods optionally further comprise regenerating the modified plant cell into a modified plant comprising enhanced resistance to the plant pathogen.
  • the NRC protein is an NRC protein that is capable of pairing with a sensor NLR protein present in the plant and is capable of causing at least a weak or partial cell death response in the plant when the plant is contacted with the pathogen.
  • the NRC protein is endogenous or native to the genome of the plant.
  • the methods comprise introducing into at least one plant cell a polynucleotide construct comprising a promoter that drives expression in a plant and an operably linked nucleic acid molecule encoding the NRC protein using plant transformation methods described below or otherwise known in the art.
  • the methods comprising introducing into at least one plant cell a
  • polynucleotide construct comprising a promoter that drives expression in a plant and further comprise genome editing with a site-specific, engineered nuclease to replace the existing promoter of a gene encoding a HRC protein that is present in the genome of the cell with promoter that provides for stronger gene expression than the existing promoter.
  • Preferred promoters for enhancing the resistance of a plant to a plant pathogen are promoters known to drive high-level gene expression such as, for example, the CaMV 35S promoter. Additional promoters that are suitable for use in the methods of the present invention are described below.
  • the present invention provides methods for enhancing the resistance of a plant to at least one plant pathogen when a resistance gene that is active for resistance to a plant pathogen in a first plant species is transferred to a second plant species, wherein the resistance gene is inactive or otherwise does not enhance the resistance of the second plant species to a plant pathogen.
  • a sensor NLR gene is Hero from Solarium lycopersocum. Hero is known to confer resistance to potato cyst nematodes, Globodera pallida and G. rostochiensis in tomato (Ernst et al., 2002 Plant ./ 3 1 (2): 127-136) However, Nero does not confer resistance when transferred by itself into potato (Solarium tuberosum). As disclosed in Example 7 below, an autoactive mutant of Hero is not capable of causing a cell death response when expressed in leaves of the solanaceous plant, N.
  • NRC6 a cell death response is observed indicating that Hero requires the presence of a tomato NRC, particularly NRC6, to produce the cell death response associated with resistance activity. While the present invention does not depend on a particular biological mechanism, it is believed that in tomato, the Hero protein pairs with the NRC6 protein to cause a cell death repsonse in response to an attack by one or more potato cyst nematodes (e.g. Globodera pallida and G. rostochiensis) and that lack of resistance activity against these same pathogens when Hero is expressed in potato plants is due to the lack of an NRC protein that is capable of pairing with the Hero protein to initiate the cell death response that is known to be associated with resistance against plant pathogens.
  • potato cyst nematodes e.g. Globodera pallida and G. rostochiensis
  • the methods of the present invention can overcome the problem of the resistance inactivity of Hero and other sensor NLRs that are known to confer resistance to one or more plant pathogens in a first plant species but do not confer resistance against the one or more plant pathogen when the sensor NLR is expressed in a second plant species.
  • the methods comprise modifying at least one cell of a plant of the second plant species to co-express a sensor NLR protein and an NRC protein that is capable of causing a cell death response in the plant or part or cell thereof in response to the plant pathogen when co-expressed with the sensor NLR protein.
  • the methods optionally further comprise regenerating the modified plant cell into a modified plant comprising enhanced resistance to the plant pathogen, relative to a control plant that is not modified for expression of the sensor NLR protein and/or the NRC protein.
  • polynucleotides comprising a nucleotide sequences encoding the sensor NLR protein and NRC protein are introduced into a plant cell using plant transformation methods disclosed elsewhere herein or otherwise known in the art. It is recognized the polynucleotide comprising a nucleotide sequence encoding the sensor NLR protein can be introduced into the plant cell at the same time as the
  • polynucleotide comprising a nucleotide sequence encoding the NRC protein linked together in a single nucleic acid molecule or vector or as two, separate nucleic acid molecules.
  • the polynucleotide comprising a nucleotide sequence encoding the sensor NLR protein can be introduced into a first plant cell that is regenerated into a first plant comprising stably incorporated in its genome the sensor NLR-encoding polynucleotide, and the polynucleotide comprising a nucleotide sequence encoding the NRC protein can be introduced into a second plant cell that is regenerated into a second plant comprising stably incorporated in its genome the NRC-encoding polynucleotide.
  • a plant comprising both the sensor NLR-encoding polynucleotide and the NRC-encoding polynucleotide can be produced, for example using sexual reproduction methods involving cross pollination the are described elsewhere herein or otherwise known in the art. Additionally or alternatively, genome editing methods described elsewhere herein or otherwise known in the art can be used to modify a plant cell to express either one or both of the sensor NLR and the NRC proteins.
  • the methods of the present invention find use in producing plants with enhanced resistance to a plant disease caused by a plant pathogen.
  • the methods of the present invention will enhance or increase the resistance of the subject plant to one strains of a plant pathogen or to each of two or more strains of the plant pathogen by at least 25%, 50%, 75%, 100%, 150%, 200%, 250%, 500% or more when compared to the resistance of a control to same strain or strains of the plant pathogen.
  • a control plant for the present invention is a plant that does not comprise the polynucleotide construct of the present invention.
  • the control plant is essentially identical (e.g. same species, subspecies, and variety) to the plant comprising the
  • control plant will comprise a polynucleotide construct but not comprise one or more nucleotide sequences encoding an engineered sensor NLR protein, an engineered NRC protein, or other NRC protein that are in a polynucleotide construct of the present invention. In other embodiments, the control plant will not comprise an engineered NRC protein or an engineered sensor NLR protein of the present invention.
  • the plants of the present invention comprising an engineered sensor NLR protein, an engineered NRC protein, expressing an increased level of another NRC protein, or co expressing a sensor NLR protein and NRC protein disclosed herein find use in methods for limiting plant disease caused by at least one plant pathogen in agricultural crop production, particularly in regions where such a plant disease is prevalent and is known to negatively impact, or at least has the potential to negatively impact, agricultural yield.
  • the methods of the invention comprise planting a plant (e.g.
  • a seedling a seedling
  • tuber, or seed of the present invention wherein the plant, tuber, or seed comprises: at least one engineered sensor NLR protein or an engineered NRC protein of the present invention; at least one nucleotide molecule encoding engineered sensor NLR protein or an engineered NRC protein; is modified to express a high level of another NRC protein of the present invention; and/or co expresses a sensor NLR protein and NRC protein is necessary for resistance activity of the sensor NLR protein in the plant.
  • the methods further comprise growing the plant that is derived from the seedling, tuber, or seed under conditions favorable for the growth and development of the plant, and optionally harvesting at least one fruit, tuber, leaf, or seed from the plant.
  • the present invention provides plants, seeds, and plant cells produced by the methods of present invention and/or comprising a polynucleotide construct of the present invention. Also provided are progeny plants and seeds thereof comprising a polynucleotide construct of the present invention.
  • the present invention also provides seeds, vegetative parts, and other plant parts produced by the transformed plants and/or progeny plants of the invention as well as food products and other agricultural products produced from such plant parts that are intended to be consumed or used by humans and other animals including, but not limited to pets (e.g., dogs and cats) and livestock (e.g., pigs, cows, chickens, turkeys, and ducks).
  • the present invention encompasses isolated or substantially purified polynucleotide (also referred to herein as“nucleic acid molecule”,“nucleic acid” and the like) or protein (also referred to herein as“polypeptide”) compositions including, for example,
  • polynucleotides and proteins comprising the sequences set forth in the accompanying Sequence Listing as well as variants and fragments of such polynucleotides and proteins.
  • An “isolated” or“purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally-occurring environment.
  • an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an“isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
  • the isolated polynucleotide can contain less than about 5 kb, 4 kb,
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.
  • optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention.
  • fragment is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby.
  • Fragments of polynucleotides comprising coding sequences may encode protein fragments that retain biological activity of the full-length or native protein.
  • fragments of a polynucleotide that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity.
  • fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.
  • a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a“native” polynucleotide or polypeptide comprises a naturally-occurring nucleotide sequence or amino acid sequence, respectively.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the engineered NRC or sensor NLR proteins of the invention.
  • Variant polynucleotides include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an engineered NRC or sensor NLR protein of the invention.
  • variants of a polynucleotide of the invention will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
  • variants of a particular polynucleotide of the invention will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 23, 24, 25, 30, 32, 34, and 38.
  • Variants of a polynucleotide of the invention can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
  • a polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 31, 33, 35, or 39 is disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein.
  • the percent sequence identity between the two encoded polypeptides is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • variants of a particular polypeptide of the invention will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 31, 33, 35, and 39.
  • “Variant” protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C- terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • Biologically active variants of an engineered NRC or sensor NLR protein of the present invention will have at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein (e.g.
  • a biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • substitutions such as exchanging one amino acid with another having similar properties, may be optimal.
  • deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein.
  • a plant that is susceptible to a plant diseases caused by a pathogen or interest can be transformed polynucleotide construct encoding an engineered sensor NLR protein, an engineered NRC protein or other sensor NLR or NRC protein of the present invention, regenerated into a transformed or transgenic plant comprising the polynucleotide constructs, and tested for resistance using standard disease resistance assays known in the art or described elsewhere herein.
  • Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.
  • the engineered NLR and NRC proteins of the present invention and the polynucleotides encoding them confer, or are capable of conferring upon a plant comprising such a protein or polynucleotide, enhanced resistance to at least one plant pathogen, but preferably to two, three, four, five, or more plant pathogens.
  • PCR amplification can be used in certain embodiments of the methods of the present invention.
  • Methods for designing PCR primers and PCR amplificaiton are generally known in the art and are disclosed in Sambrook et al. ( 1 89) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., eds. (1990 ) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).
  • PCR amplification includes, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector- specific primers, partially-mismatched primers, and the like.
  • the engineered sensor NLR proteins, NRC proteins, and fusion proteins protein suppressor coding sequences of the present invention encompass nucleic acid molecules comprising a nucleotide sequence that is sufficiently identical to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 23, 24, 25, 30, 32, 34, and/or 38.
  • the term “sufficiently identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
  • amino acid or nucleotide sequences that contain a common structural domain having at least about 45%, 55%, or 65% identity, preferably 75% identity, more preferably 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently identical.
  • the sequences are aligned for optimal comparison purposes.
  • the two sequences are the same length.
  • the percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J Mol. Biol. 215:403.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
  • PSI- Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST; available on the world-wide web at ncbi.nlm.nih.gov).
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Alignment may also be performed manually by inspection.
  • sequence identity/similarity values provided herein refer to the value obtained using the full-length sequences of the invention and using multiple alignment by mean of the algorithm Clustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) using the program AlignX included in the software package Vector NTI Suite Version 7 (InforMax, Inc., Bethesda, MD, USA) using the default parameters; or any equivalent program thereof.
  • “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by CLUSTALW (Version 1.83) using default parameters (available at the European Bioinformatics Institute website on the world-wide web at:
  • polynucleotide is not intended to limit the present invention to polynucleotides comprising DNA.
  • polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides.
  • deoxyribonucleotides and ribonucleotides include both naturally-occurring molecules and synthetic analogues.
  • the polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
  • the polynucleotide constructs comprising engineered NLR or NRC protein coding regions can be provided in expression cassettes for expression in the plant or other organism or in a host cell of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to the protein coding region.
  • “Operably linked” is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a polynucleotide or gene of interest and a regulatory sequence i.e., a promoter
  • Operably linked elements may be contiguous or non-contiguous.
  • the cassete may additionally contain at least one additional gene to be cotransformed into the organism.
  • the additional gene(s) can be provided on multiple expression cassetes.
  • Such an expression cassete is provided with a plurality of restriction sites and/or recombination sites for insertion of the protein coding region to be under the transcriptional regulation of the regulatory regions.
  • the expression cassete may additionally contain selectable marker genes.
  • the expression cassete will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), an engineered NLR or NRC protein coding region of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants or other organism or non human host cell.
  • the regulatory regions i.e., promoters, transcriptional regulatory regions, and translational termination regions
  • the engineered NLR or NRC protein coding region or of the invention may be native/analogous to the host cell or to each other.
  • regulatory regions and/or the engineered NLR or NRC protein coding region of the invention may be heterologous to the host cell or to each other.
  • heterologous in reference to a nucleic acid molecule or nucleotide sequence is a nucleic acid molecule or nucleotide sequence that originates from a foreign species, or, if from the same species, is modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • the present invention provides host cells comprising at least of the nucleic acid molecules, expression cassetes, and vectors of the present invention.
  • a host cell is a plant cell.
  • a host cell is selected from the group consisting of a bacterium, a fungal cell, and an animal cell.
  • a host cell is non-human animal cell.
  • the host cell is an in-vitro cultured human cell. While it may be optimal to express the engineered NLR and NRC proteins using heterologous promoters, the native promoter of a corresponding NLR or NRC gene may be used.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked engineered NLR and NRC protein coding region of interest, may be native with the plant host, or may be derived from another source ( /. e..
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991 )Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671- 674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261- 1272; Munroe et al. (1990) Gene 91 : 151-158; Ballas et al. (1989) Nucleic Acids Res.
  • the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the expression cassettes may additionally contain 5' leader sequences.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); poty virus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene l65(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) ( Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al.
  • EMCV leader Engelphalomyocarditis 5' noncoding region
  • poty virus leaders for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene l65(2):233-238
  • AMV RNA 4 alfalfa mosaic virus
  • TMV tobacco mosaic virus leader
  • MCMV maize chlorotic mottle virus leader
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • a number of promoters can be used in the practice of the invention.
  • the promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.
  • constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al. ( 1 85) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al.
  • Tissue-preferred promoters can be utilized to target enhanced expression of the engineered NLR and NRC protein coding sequences within a particular plant tissue.
  • tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root- preferred promoters, seed-preferred promoters, and stem-preferred promoters.
  • Tissue- preferred promoters include Yamamoto et al. (1997) Plant J. l2(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. ( ⁇ 991)Mol. Gen Genet. 254(3):337- 343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol.
  • an inducible promoter particularly from a pathogen-inducible promoter.
  • promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-l,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819, herein incorporated by reference.
  • promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton el al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch el al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988 )Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad Sci. USA 93: 14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al.
  • a wound-inducible promoter may be used in the constructions of the invention.
  • wound- inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498); wunl and wun2, U.S. Patent No. 5,428,148; winl and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225: 1570-1573); WIP1 (Rohmeier el al.
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical -inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid.
  • Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the
  • the expression cassette can also comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • Additional selectable markers include phenotypic markers such as b-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al.
  • selectable marker genes are not intended to be limiting. Any selectable marker gene can be used in the present invention.
  • the methods of the invention involve introducing a polynucleotide construct into a plant.
  • introducing is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant.
  • the methods of the invention do not depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant.
  • Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
  • stable transformation is intended that the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof.
  • transient transformation is intended that a polynucleotide construct introduced into a plant does not integrate into the genome of the plant.
  • nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell.
  • the selection of the vector depends on the preferred transformation technique and the target plant species to be transformed.
  • nucleotide sequences into plant cells and subsequent insertion into the plant genome
  • microinjection as Crossway et al. (1986) Biotechniques 4:320-334
  • electroporation as described by Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606.
  • Agrobaclerium-mediaied transformation as described by Townsend et al, U.S. Patent No. 5,563,055, Zhao et al, U.S. Patent No. 5,981,840
  • direct gene transfer as described by Paszkowski et al. (1984) EMBO J. 3:2717-2722
  • ballistic particle acceleration as described in, for example, Sanford et al, U.S.
  • the polynucleotides of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.
  • the modified viruses or modified viral nucleic acids can be prepared in formulations.
  • formulations are prepared in a known manner (see e.g. for review US 3,060,084, EP-A 707 445 (for liquid concentrates), Browning,“Agglomeration”, Chemical Engineering, Dec. 4, 1967, 147-48, Perry’s Chemical Engineer’s Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and et seq.
  • auxiliaries suitable for the formulation of agrochemicals such as solvents and/or carriers, if desired emulsifiers, surfactants and dispersants, preservatives, antifoaming agents, anti-freezing agents, for seed treatment formulation also optionally colorants and/or binders and/or gelling agents.
  • polynucleotide constructs and expression cassettes of the invention can be provided to a plant using a variety of transient transformation methods known in the art. Such methods include, for example, microinjection or particle
  • the polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and
  • Agrobacterium tumefaciens- mediated transient expression as described elsewhere herein.
  • the cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a polynucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
  • Such methods known in the art for modifying DNA in the genome of a plant include, for example, genome editing techniques, such as, for example, methods involving targeted mutagenesis, homologous recombination, and mutation breeding.
  • genome editing techniques such as, for example, methods involving targeted mutagenesis, homologous recombination, and mutation breeding.
  • Targeted mutagenesis or similar techniques are disclosed in U.S. Patent Nos. 5,565,350; 5,731,181; 5,756,325;
  • Methods for gene modification or gene replacement comprising homologous recombination can involve inducing double-strand breaks in DNA using zinc- finger nucleases (ZFN), TAL (transcription activator-like) effector nucleases (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas nuclease), or homing endonucleases that have been engineered endonucleases to make double-strand breaks at specific recognition sequences in the genome of a plant, other organism, or host cell. See, for example, Durai et al, (2005) Nucleic Acids Res.
  • TAL effector nucleases can be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination.
  • TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism.
  • TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, Fold.
  • TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity.
  • the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS l0. l073/pnas. l0l3l33l07; Scholze and Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi: 10. l093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology 29: 143-148; all of which are herein incorporated by reference.
  • the CRISPR/Cas nuclease system can also be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination.
  • the CRISPR/Cas nuclease is an RNA- guided (simple guide RNA, sgRNA in short) DNA endonuclease system performing sequence-specific double-stranded breaks in a DNA segment homologous to the designed RNA. It is possible to design the specificity of the sequence (Cho S.W. et al, Nat.
  • a ZFN can be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination.
  • the Zinc Finger Nuclease is a fusion protein comprising the part of the Fold restriction endonuclease protein responsible for DNA cleavage and a zinc finger protein which recognizes specific, designed genomic sequences and cleaves the double-stranded DNA at those sequences, thereby producing free DNA ends (Umov F.D. et al, Nat Rev Genet. 11 :636-46, 2010; Carroll D., Genetics. 188:773-82, 2011).
  • Breaking DNA using site specific nucleases can increase the rate of homologous recombination in the region of the breakage.
  • site specific nucleases such as, for example, those described herein above
  • coupling of such effectors as described above with nucleases enables the generation of targeted changes in genomes which include additions, deletions and other modifications.
  • the methods and compositions of the present invention can be used with any plant species including, for example, monocotyledonous plants, dicotyledonous plants, and conifers.
  • plant species of interest include, but are not limited to, com (Zea mays), Brassica sp. (e.g., B. napus,
  • B. rapa, B. junced particularly those Brassica species useful as sources of seed oil, alfalfa ( Medicago sativa ), rice ( Oryza sativa ), rye ( Secale cereale ), triticale (x Triticosecale or Triticum x Secale) sorghum ( Sorghum bicolor, Sorghum vulgar e), tefif ( Eragrostis tef), millet (e.g., pearl millet Pennisetum glaucum), proso millet ( Panicum miliaceum), foxtail millet ( Setaria italica), finger millet ( Eleusine coracana)), switchgrass ( Panicum virgatum), sunflower ( Helianthus annuus), safflower ( Carthamus tinctorius), wheat ( Triticum aestivum), soybean ( Glycine max), tobacco ( Nicotiana tabacum), potato (Solatium tuberosum), peanuts ( Ar
  • plants of the present invention are crop plants (e.g. maize, sorghum, wheat, millet, rice, barley, oats, sugarcane, alfalfa, soybean, peanut, sunflower, cotton, safflower, Bras sica spp., lettuce, strawberry, apple, citrus, etc.).
  • crop plants e.g. maize, sorghum, wheat, millet, rice, barley, oats, sugarcane, alfalfa, soybean, peanut, sunflower, cotton, safflower, Bras sica spp., lettuce, strawberry, apple, citrus, etc.
  • Vegetables include tomatoes (Lycopersicon esculentum), eggplant (also known as “aubergine” or“brinjal”) (Solanum melongena), pepper (Capsicum annuum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), chickpeas (Cicer arietinum), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
  • Fruit trees and related plants include, for example, apples, pears, peaches, plums, oranges, grapefruits, limes, pomelos, palms, and bananas.
  • Nut trees and related plants include, for example, almonds, cashews, walnuts, pistachios, macadamia nuts, filberts, hazelnuts, and pecans.
  • the plants of the present invention are crop plants such as, for example, maize (com), soybean, wheat, rice, cotton, alfalfa, sunflower, canola (Brassica spp., particularly Brassica napus, Brassica rapa, Brassica juncea), rapeseed (Brassica napus), sorghum, millet, barley, triticale, safflower, peanut, sugarcane, tobacco, potato, tomato, and pepper.
  • maize com
  • soybean wheat
  • rice cotton
  • alfalfa sunflower
  • canola Brassica napus
  • Brassica rapa Brassica rapa
  • Brassica juncea rapeseed
  • sorghum millet
  • barley triticale
  • safflower peanut, sugarcane, tobacco, potato, tomato, and pepper.
  • the plants of the present invention are solanaceous plants.
  • Solanaceous plants of the present invention include, but are not limited to, potato ( Solanum tuberosum), eggplant ( Solanum melongena), petunia ⁇ Petunia spp., e.g., Petunia x hybrida or Petunia hybrida ), tomatillo (Physalis philadelphica), Cape gooseberry (Physalis peruviana ), Physalis sp., woody nightshade ⁇ Solanum dulcamara), garden huckleberry ⁇ Solanum scabrum), gboma eggplant ⁇ Solanum macrocarpon), pepper ⁇ Capsicum spp.; e.g., Capsicum annuum, C.
  • solanaceous plants are solanaceous plants grown in agriculture including, but not limited to, potato, tomato, tomatillo, eggplant, pepper, tobacco, Cape gooseberry, and petunia.
  • plant is intended to encompass plants at any stage of maturity or development, as well as any cells, tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context.
  • Plant parts include, but are not limited to, fruits, stems, tubers, roots, flowers, ovules, stamens, petals, leaves, hypocotyls, epicotyls, cotyledons, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, seeds, and the like. It is recognized that the plant protoplasts of the present invention can be prepared from any one or more of the aforementioned plant parts and at any stage of development and/or maturity.
  • plant cell is intended to encompass plant cells obtained from or in plants at any stage of maturity or development unless otherwise clearly indicated by context.
  • Plant cells can be from or in plant parts including, but are not limited to, fruits, stems, tubers, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, in vitro-cultured tissues, organs or cells and the like.
  • the plant protoplasts of the present invention can be prepared from any one or more of the aforementioned plant cells and at any stage of development and/or maturity.
  • the term“plant cell” is intended to encompass a plant protoplast.
  • a plant cell is transformed with a polynucleotide construct encoding an engineered sensor NLR protein, an engineered NRC protein or other NRC protein of the present invention.
  • the term“expression” as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product.
  • The“expression” or“production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide
  • the“expression” or“production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide. Examples of polynucleotide constructs and nucleic acid molecules that encode engineered NLR and NRC proteins are described elsewhere herein.
  • deoxyribonucleotides and ribonucleotides include both naturally-occurring molecules and synthetic analogues including, but not limited to, nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • polynucleotide molecules of the invention also encompass all forms of polynucleotide molecules including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. Furthermore, it is understood by those of ordinary skill in the art that the nucleotide sequences disclosed herein also encompasses the complement of that exemplified nucleotide sequence.
  • the invention is drawn to compositions and methods for producing a plant with enhanced resistance to a plant disease caused by one, two, three, four or more plant pathogens.
  • resistance to a plant disease or“disease resistance” is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. That is, one or more pathogens are prevented from causing a plant disease or plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the one or more pathogens is minimized or lessened.
  • the present invention encompasses the polynucleotide constructs disclosed herein or in the accompanying sequence listing and/or drawings including, but not limited to, a polynucleotide construct comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 23, 24, 25, 30, 32, 34, and 38.
  • the present invention further encompasses plants, plant cells, host cells, and vectors comprising at least one of such polynucleotide constructs, as well as food products produced from such plants. Additionally encompassed by the present invention are uses of plants comprising at least one of such polynucleotide constructs in the methods disclosed elsewhere herein such as, for example, methods of limiting plant diseases in agricultural crop production.
  • Plant pathogens include, for example, bacteria, fungi, oomycetes, viruses, nematodes, and the like. Specific pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var.
  • phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splend
  • Phytophthora megasperma Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii,
  • Leptosphaerulina briosiana Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium
  • Phomopsis helianthi Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Com: Colletotrichum graminicola, Fusarium moniliforme var.
  • nebraskense Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv.
  • zea Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis , Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum, C.
  • Corynebacterium michiganense pv. michiganense Pseudomonas syringae pv. tomato, Ralstonia solanacearum, Xanthomonas vesicatoria, Xanthomonas perforans, Alternaria solani, Alternaria porri, Collectotrichum spp., Fulvia fulva Syn. Cladosporium fulvum, Fusarium oxysporum f. lycopersici, Leveillula taurica/Oidiopsis taurica, Phytophthora infestans, other Phytophthora spp., Pseudocercospora fuligena Syn.
  • Cercospora fuligena Sclerotium rolfsii, Septoria lycopersici, Meloidogyne spp.
  • Potato Ralstonia solanacearum, Pseudomonas solanacearum, Erwinia carotovora subsp. Atroseptica Erwinia carotovora subsp. Carotovora, Pectobacterium carotovorum subsp. Atrosepticum, Pseudomonas fluorescens, Clavibacter michiganensis subsp.
  • Sepedonicus Corynebacterium sepedonicum, Streptomyces scabiei, Colletotrichum coccodes, Alternaria alternate, Mycovellosiella concors, Cercospora solani, Macrophomina phaseolina, Sclerotium bataticola, Choanephora cucurbitarum, Puccinia pittieriana, Aecidium cantensis, Alternaria solani, Fusarium spp., Phoma solanicola f. foveata, Botrytis cinerea, Botryotinia fuckeliana, Phytophthora infestans, Pythium spp., Phoma andigena var.
  • Pleospora herbarum Stemphylium herbarum, Erysiphe cichoracearum, Spongospora subterranean Rhizoctonia solani, Thanatephorus cucumeris, Rosellinia sp. Dematophora sp., Septoria lycopersici,
  • Helminthosporium solani Polyscytalum pustulans, Sclerotium rolfsii, Athelia rolfsii, Angiosorus solani, Ulocladium atrum, Verticillium albo-atrum, V. dahlia, Synchytrium endobioticum, Sclerotinia sclerotiorum, Candidatus Liberibacter solanacearum; Banana: Fusarium oxysporum f. sp.
  • Ceratocystis paradoxa Haplobasidion musae, Marasmiellus inoderma, Pseudomonas solanacearum, Radopholus similis, Lasiodiplodia theobromae, Fusarium pallidoroseum, Verticillium theobromae, Pestalotiopsis palmarum, Phaeoseptoria musae, Pyricularia grisea, Fusarium moniliforme, Gibberella fujikuroi, Erwinia carotovora, Erwinia chrysanthemi, Cylindrocarpon musae, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, Pratylenchus coffeae, Pratylenchus goodeyi, Pratylenchus brachyurus,
  • Pratylenchus reniformia Sclerotinia sclerotiorum, Nectria foliicola, Mycosphaerella musicola, Pseudocercospora musae, Limacinula tenuis, Mycosphaerella musae,
  • Helicotylenchus multicinctus Helicotylenchus dihystera, Nigrospora sphaerica,
  • Nematodes of interest include, but are not limited to, parasitic nematodes such as root-knot, cyst, and lesion nematodes, including, for example, Globodera spp., Meloidogyne spp., and Heterodera spp.
  • Globodera spp. include, but are not limited to, G. rostochiensis and G. pailida (potato cyst nematodes).
  • Meloidogyne spp. include, but are not limited to, M. javanica, M. arenaria, M. graminicola, M. incognita, M. hapla, andM chitwood.
  • Heterodera spp. include, but are not limited to, H. glycines (soybean cyst nematode), H. schachtii (beet cyst nematode), Heterodera goettingiana (pea cyst nematode), Heterodera zeae (com cyst nematode), and H. avenae (cereal cyst nematode).
  • Lesion nematodes include, for example, Pratylenchus spp.
  • Other nematodes of interest include, for example,
  • Radopholus similis (banana-root nematode) and Belonolaimus longicaudatus (sting nematode).
  • compositions and methods of the present invention are as follows:
  • a method for making an engineered helper nucleotide-binding domain and leucine-rich repeat-containing-required-for-cell-death (NRC) protein that is capable of conferring to a plant enhanced resistance to a plant pathogen comprising a pathogen suppressor comprising modifying the amino acid sequence of an NRC protein, so as to produce an engineered NRC protein that is not suppressible in the plant in the presence of the pathogen suppressor.
  • modifying the amino acid sequence of the NRC protein comprises the substitution, addition, and/or deletion of one or more amino acids of amino acid sequence of the NRC protein.
  • modifying the amino acid sequence of an NRC protein comprises substituting at least one domain or part thereof of the NRC protein with at least one corresponding domain or part thereof of at least one additional NRC protein that is not suppressible by the pathogen effector.
  • the at least one domain is selected from the group consisting of the coiled-coiled (CC) domain, the nucleotide-binding (NB) domain, the ARC1 domain, the ARC2 domain, and the leucine-rich repeat domain (LRR).
  • modifying the amino acid sequence of a NRC protein comprises substituting the CC, NB, the ARC1, and the ARC2 domains of the NRC protein with the CC, NB, the ARC1, and the ARC2 domains of an NRC protein that is not suppressible by the pathogen effector.
  • NRC protein and/or the at least one additional NRC protein is selected from the group consisting of NRC2, NRC3, and NRC4.
  • modifying the amino acid sequence of an NRC protein comprises producing a nucleic acid molecule encoding the engineered NRC protein.
  • a plant or plant cell comprising the engineered NRC protein of embodiment 14 or the nucleic acid molecule of embodiment 15.
  • a method for making a nucleic acid molecule encoding an engineered NRC protein that is capable of conferring to a plant enhanced resistance to a plant pathogen comprising a pathogen suppressor comprising synthesizing a nucleic acid molecule encoding the amino acid sequence of an engineered NRC that is designed by considering the amino acid sequence of an NRC protein that is suppressible by a pathogen effector and modifying the amino acid sequence of the NRC protein so as to produce the amino acid sequence of the, wherein the engineered NRC protein is not suppressible by the pathogen effector.
  • modifying the amino acid sequence of an NRC protein comprises the substitution, addition, and/or deletion of one or more amino acids of amino acid sequence of the NRC protein.
  • modifying the amino acid sequence of an NRC protein comprises substituting at least one domain or part thereof of the NRC protein with at least one corresponding domain or part thereof of at least one additional NRC protein that is not suppressible by the pathogen effector.
  • the at least one domain is selected from the group consisting of the coiled-coiled (CC) domain, the nucleotide-binding (NB) domain, the ARC1 domain, the ARC2 domain, and the leucine-rich repeat domain (LRR). 22.
  • modifying the amino acid sequence of an NRC protein comprises substituting the CC, NB, the ARC1, and the ARC2 domains of the NRC protein with the CC, NB, the ARC1, and the ARC2 domains of an NRC protein that is not suppressible by the pathogen effector.
  • NRC protein and/or the at least one additional NRC protein is selected from the group consisting of NRC2, NRC3, and NRC4.
  • a plant or plant cell comprising the nucleic acid molecule of embodiment 29 or the engineered NRC protein of embodiment 30.
  • a method for making an engineered sensor nucleotide-binding domain and leucine-rich repeat-containing (NLR) protein that evades suppression of cell death caused by a pathogen suppressor comprising modifying the amino acid sequence of a sensor NLR protein of interest that is capable of pairing with a first NRC protein to cause a cell death response in a plant in response to the pathogen contacting the plant in the absence of the pathogen suppressor, whereby an engineered sensor NLR protein is produced that is capable of pairing with a second NRC protein to cause a cell death response in the plant, wherein:
  • modifying the amino acid sequence of a sensor NLR protein of interest comprises the substitution, addition, and/or deletion of one or more amino acids of amino acid sequence of the sensor NLR protein of interest.
  • solanaceous plant is selected from the group consisting of potato, tomato, pepper, eggplant, and tobacco.
  • modifying the amino acid sequence of a sensor NLR protein of interest comprises producing a nucleic acid molecule encoding the engineered sensor NLR protein.
  • a plant or plant cell comprising the engineered sensor NLR protein of embodiment 45 or the nucleic acid molecule of embodiment 46.
  • a method for making a nucleic acid molecule encoding an engineered sensor NLR protein that evades suppression of cell death caused by a pathogen suppressor comprising synthesizing a nucleic acid molecule encoding the amino acid sequence of an engineered sensor NLR protein that is designed by modifying the amino acid sequence of a sensor NLR protein of interest that is capable of pairing with a first NRC protein to cause a cell death response in a plant in response to the pathogen contacting the plant in the absence of the pathogen suppressor, whereby an engineered sensor NLR protein is produced that is capable of pairing with a second NRC protein to cause a cell death response in the plant, wherein:
  • modifying the amino acid sequence of a sensor NLR protein of interest comprises the substitution, addition, and/or deletion of one or more amino acids of amino acid sequence of the sensor NLR protein.
  • a plant or plant cell comprising the nucleic acid molecule of embodiment 61 or the engineered sensor NLR protein of embodiment 60.
  • a method for enhancing the resistance of a plant to at least one plant pathogen comprising modifying a plant cell to be capable of expressing at least one engineered protein selected from the group consisting of:
  • nucleic acid molecule is selected from the group consisting of:
  • modifying comprises or further comprises genome editing with one or more engineered nucleases.
  • a method for enhancing the resistance of a plant to a plant pathogen comprising modifying a plant cell to be capable of increased expression of at least one NRC protein wherein the plant comprises a resistance gene against the pathogen but is only partially resistant to the pathogen.
  • modifying comprises introducing into the plant cell a polynucleotide construct comprising (i) a promoter that drives expression in a plant or (ii) a promoter that drives expression in a plant and operably linked nucleic acid molecule encoding the NRC protein.
  • modifying comprises or further comprises genome editing with one or more engineered nucleases.
  • a nucleic acid molecule comprising at least one nucleotide sequence selected from the group consisting of:
  • nucleotide sequence having at least 80% nucleotide sequence identity to at least one nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 23, 24, 25, 30, and 32; and
  • nucleotide sequence encoding an amino acid sequence having at least 80% amino acid sequence identity to at least one amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 31, and 33.
  • a plant, a plant cell, or other host cell comprising the nucleic acid molecule of embodiment 78.
  • nucleotide sequence having at least 80% nucleotide sequence identity to at least one nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 23, 24, 25, 30, and 32.
  • a plant, a plant cell, or other host cell comprising the polypeptide of embodiment
  • a method of enhancing the resistance of a plant to at least one plant pathogen comprising modifying a plant or at least one plant cell to co-express a sensor NLR protein and an NRC protein, wherein the sensor NLR protein is capable of enhancing the resistance of a first plant species comprising the sensor NLR protein to at least one plant pathogen, wherein the sensor NLR protein is not capable of enhancing the resistance of a second plant species comprising the sensor NLR protein to at least one plant pathogen in the absence of the NRC protein, and wherein the modified plant or plant cell is from the second plant species and the modified plant or a plant regenerated from the modified plant cell comprises enhanced resistance to at least one plant pathogen, relative to the resistance of an unmodified control plant of the second plant species.
  • solanaceous plant is selected from the group consisting of potato, tomato, pepper, eggplant, and tobacco.
  • nucleotide sequence encoding an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of (b).
  • ⁇ Hi a nucleotide sequence having at least 80% nucleotide sequence identity to the sequence of (i), and
  • EXAMPLE 1 Identification of Pathogen Effectors that Suppress NRC -Dependent Cell
  • AVRcaplb which suppressed NRC2- and NRC3 -dependent cell death, but not NRC4- dependent cell death.
  • AVRcaplb was co-expressed with different R/AVR pairs in Nicotiana benthamiana, suppression of cell death was observed only in NRC2/3 dependent sensor NLRs (Prf, Gpa2, Rpiamrla), but not in sensor NLRs that signaled through NRC4 (Rl, R8, Rpiblb2, Rx, BS2, Mil .2) (data not shown).
  • SPRYSEC15 a candidate NRC2/3 suppressor (FIG. 2).
  • SPRYSEC15 suppressed cell death mediated by all sensor NLRs known to signal through NRC2/3, but not NRC4 (data not shown). Similar to AVRcaplb, SPRYSEC15 suppressed cell death in N. benthamiana mediated by an autoactive NRC2 H480R , NRC3 D480V variant, but not cell death mediated by an NRC4 D478V variant (FIG. 2, lower panel).
  • EXAMPLE 2 Creation of an Engineered NRC Protein that is Not Suppressed by a
  • EXAMPLE 3 Transformed Potatoes Expressing Engineered NRC Protein and Testing for Enhanced Pathogen Resistance
  • Susceptible cultivated potato and wild Solanum spp. are transformed to express the two NRC variants described above (NRC4-3 LRR and NRC4-3 LRR8 13 ).
  • the transformed potato lines are challenged with strains of Phytophthora infestans or Globodera rostochiensis and assayed for enhanced resistance to these pathogens.
  • Enhanced resistance suggests the presence of cryptic R genes, which are R genes that signal through NRC2/3 but that are no longer functional due to suppression of the helper NLR by the pathogen.
  • Cryptic R genes providing resistance in an NRC4-3 LRR or NRC4-3 LRR8 13 background are then isolated using an accelerated cloning technique such as RenSeq (Jupe et al. (2013) Plant J. 76:530-544).
  • the nematode resistance gene Gpa2 is NRC2/3 dependent whereas the viral resistance gene Rx is NRC2/3/4 dependent.
  • the two proteins are 88% identical.
  • Gpa2 variants that gain dependency on NRC4 are created by swapping five of the most polymorphic regions in the LRR domain of Gpa2 with that of Rx (FIG. 5) and testing the resulting Gpa2 variants for the ability to recognize the corresponding effector RBP1 in a transient assay such as that depicted in FIG. 2. If recognition is observed, NRC dependency is assessed using silencing and complementation assays (Wu et al. , 2017 PNAS 114(30): 8113-8118). A Gpa2 variant that is NRC4 dependent is then tested for evasion of suppression by AVRcaplb and SPRYSEC15. Chimeric variants that provide better resistance than the original Gpa2 are selected.
  • Cryptic R genes isolated as described in Example 3 are transformed with the engineered NRC variants described in Example 2 into susceptible potato lines, and the resulting transgenic plants are screened for enhanced resistance to P. infestans.
  • these cryptic R genes are engineered to signal through NRC4 as described in Example 4 and transformed into susceptible potato lines, and the resulting plants are screened for enhanced resistance to P. infestans.
  • P. infestans strains for example P/NL07434, have partially overcome disease resistance mediated by Rpi-blb2 (FIG. 6), an NRC4-dependent sensor NLR (unpublished). This partial resistance phenotype can also be observed on Rpi-blb2 transgenic N.
  • NRCs are genetically downstream of sensor NLRs
  • overexpression of NRCs may help intensify NLR-triggered immune responses, thus making a partially resistant plant display full resistance.
  • Rpi-blb2 N. benthamiana plants overexpressing NRC4 under the 35 S promoter, were generated by crossing two transgenic lines (Rpi-blb2xNRC4) and assayed for resistance to P/NL07434.
  • Three of the selected F3 lines displayed full resistance to P/NL07434 (FIG. 6, three rightmost leaves) indicating that increasing the expression of NRC4 can make a plant fully resistant to a strain of P. infestans that had partially overcome disease resistance mediated by Rpi-blb2 in control plant that does not overexpress NRC4 (FIG. 6, second leaf from the left).
  • potato lines containing Rpi-blb2 are transformed with NRC4 that are driven by the 35S promoter, potato plants from the transformed lines overexpressing NRC4 resistance are tested for resistance to P/NL07434. and lines that display full resistance to P/NL07434 are selected.
  • NLR proteins between closely related plant species such as between members of the Solanaceae plant clade, sometimes results in inactivity.
  • An example is the potato cyst nematode resistance gene, Hero, in Solanum lycopersocum. Hero is known to confer resistance to potato cyst nematodes, Globodera pallida and G. rostochiensis in tomato (Ernst et al., 2002 Plant J 31(2): 127-136) but not when transferred into potato ( Solanum tuberosum).
  • Hero is in a gene cluster with NRC6 (FIG. 7A). An ortholog of NRC6 exists in N. benthamiana but was shown not to be expressed.
  • Hero H855A A mutation in the highly conserved MHD motif from Histidine to Alanine at position 855 was introduced into Hero (Hero H855A ) (van Ooijen et al. (2008) J. Exp. Bot. 59: 1383-1397). This mutation is associated with autoimmunity of NLR proteins, however, Hero H855A did not trigger cell death when expressed in N. benthamiana. When Hero H855A was co-expressed with NRC6
  • FIG. 7B it induced cell death. This result indicates that the resistance gene Hero requires NRC6 to initiate immune responses. Moreover, the results disclosed in this example reveal that to transfer successfully resistance mediated by some NLR proteins from one species to another species can require both co-expression of the sensor NLR and its cognate helper NLR.
  • potato lines are transformed with a gene stack of Hero and NRC6.
  • Potato lines co-expresssing Hero and NRC6 are tested for resistance to Globodera spp., and lines that display full resistance are selected.
  • the article“a” and“an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article.
  • “an element” means one or more element.

Abstract

L'invention concerne des compositions et des procédés permettant d'améliorer la résistance des plantes aux maladies. Des procédés de fabrication de protéines contenant des répétitions riches en leucine et à domaine de liaison aux nucléotides (NLR) modifiées et des procédés d'utilisation de ces protéines NLR modifiées pour améliorer la résistance des plantes aux maladies provoquées par des phytopathogènes sont décrits. Les compositions selon l'invention comprennent des protéines NLR modifiées qui peuvent être utilisées pour améliorer la résistance des plantes aux maladies, des molécules d'acide nucléique codant pour lesdites protéines NLR modifiées, et des plantes comprenant les protéines NLR modifiées, des molécules d'acide nucléique codant pour les protéines NLR modifiées, des protéines détectrices NLR et NRC appariées, et/ou des molécules d'acide nucléique codant pour lesdites protéines détectrices NLR et NRC. Des procédés d'utilisation de ces plantes en agriculture pour limiter les maladies végétales provoquées par des phytopathogènes sont en outre décrits.
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WO2021255272A1 (fr) * 2020-06-18 2021-12-23 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Gène conférant une résistance au nématode de la nodosité des racines
WO2022129579A1 (fr) * 2020-12-18 2022-06-23 John Innes Centre Modulation des réponses d'une plante aux activités de facteurs de virulence d'agents pathogènes
EP4170039A1 (fr) 2021-10-22 2023-04-26 The Sainsbury Laboratory Modification de la réponse immunitaire chez les plantes
WO2023067197A1 (fr) 2021-10-22 2023-04-27 The Sainsbury Laboratory Modification de la réponse immunitaire dans des plantes
CN116171857A (zh) * 2023-02-27 2023-05-30 广西壮族自治区农业科学院 一种五指毛桃瓶外高效生根的方法
CN116171857B (zh) * 2023-02-27 2023-12-05 广西壮族自治区农业科学院 一种五指毛桃瓶外高效生根的方法

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