WO2017173318A1 - Gènes et marqueurs pour augmenter la résistance à la brûlure de l'épi causée par le fusarium et leurs utilisations - Google Patents

Gènes et marqueurs pour augmenter la résistance à la brûlure de l'épi causée par le fusarium et leurs utilisations Download PDF

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WO2017173318A1
WO2017173318A1 PCT/US2017/025454 US2017025454W WO2017173318A1 WO 2017173318 A1 WO2017173318 A1 WO 2017173318A1 US 2017025454 W US2017025454 W US 2017025454W WO 2017173318 A1 WO2017173318 A1 WO 2017173318A1
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seq
plant
tahrc
gene
susceptibility gene
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PCT/US2017/025454
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English (en)
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Zhenqi SU
Guihua BAI
Bin Tian
Harold TRICK
Amy BERNARDO
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Kansas State University Research Foundation
The United States Of America As Represented By The Secretary Of Agriculture
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Publication of WO2017173318A1 publication Critical patent/WO2017173318A1/fr

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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
    • C12N15/8282—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 for fungal resistance
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Definitions

  • the present invention is directed to plants having increased resistance to Fusarium Head Blight (FHB) disease through alteration of plant susceptibility genes and methods of producing the same.
  • FHB Fusarium Head Blight
  • Fusarium Head Blight mainly caused by Fusarium gramineamm
  • Fusarium Head Blight is one of the most devastating diseases of wheat.
  • FHB Fusarium Head Blight
  • DON deoxynivalenol
  • the present invention provides a new strategy for disease management based on knocking down the expression of susceptibility genes and transferring non-functional susceptible genes using diagnostic markers from the genes.
  • Described herein are resistant plants having increased resistance to Fusarium Head Blight relative to a control plant, where the control plant comprises a wild-type susceptibility gene TaHRC that is normally expressed in the presence of a Fusarium species.
  • the resistant plant comprises a corresponding susceptibility gene TaHRC in which the expression, activity, or function of the susceptibility gene is inhibited.
  • the resistant plants comprise altered or non-functional susceptibility genes TaHRC, reducing the susceptibility of the plant to Fusarium Head Blight infection.
  • these altered susceptibility genes can be transferred to progeny resulting in resistant progeny plants as well.
  • the methods generally comprise inhibiting the expression, activity, or function of a wild-type susceptibility gene TaHRC in the normally-susceptible plant cultivar to thereby produce the resistant plant. As such, there is a reduction or loss in protein levels in the presence of the Fusarium species in the resistant plant.
  • the wild-type plant susceptibility gene TaHRC targeted for silencing comprises: a coding sequence comprising SEQ ID NO: 8 or SEQ ID NO: 19; a nucleotide sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6; a nucleotide sequence encoding a TaHRC protein comprising SEQ ID NO:9; or a nucleotide sequence encoding a TaHRC protein having at least about 80% amino acid identity to SEQ ID NO:9 and retaining the functional characteristics thereof.
  • the methods generally comprise culturing immature plant embryos to form callus tissue; inhibiting expression or activity of the susceptibility gene TaHRC in said tissue to produce modified plant cells, and regenerating resistant plants from the modified plant cells. In the resistant plants, the expression, activity, or function of the susceptibility gene is inhibited, such that there is a reduction or loss in protein levels in the presence of the Fusarium species.
  • Seeds of resistant plants produced in accordance with embodiments of the invention are also disclosed.
  • Transgenic plants are also described herein, having decreased expression, activity, or function of wild-type plant susceptibility gene TaHRC, wherein the plant has stably incorporated into its genome a DNA construct, wherein the DNA construct comprises at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence comprising SEQ ID NO: 10 or SEQ ID NO: 11; a nucleotide sequence comprising an antisense sequence corresponding to SEQ ID NO: 10 or SEQ ID NO: 11; a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11; a nucleotide sequence encoding an TaHRC protein comprising SEQ ID NO:9; and a nucleotide sequence encoding an TaHRC protein having at least about 80%) amino acid identity to SEQ ID NO:9 and retaining the functional characteristics thereof, wherein the nucleotide sequence is operably linked to a promoter capable of regulating transcription of the sequence in the plant.
  • Embodiments of the invention are also concerned with isolated nucleotide sequences comprising: a nucleotide sequence comprising SEQ ID NO:8 or SEQ ID NO: 19; a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; a nucleotide sequence having at least about 50% sequence identity to SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6; a nucleotide sequence encoding a TaHRC protein comprising SEQ ID NO:9; or a nucleotide sequence encoding a TaHRC protein having at least about 30% amino acid identity to SEQ ID NO:9 and retaining the functional characteristics thereof.
  • Plant breeding methods are also described herein for increasing or enhancing resistance to Fusarium Head Blight in a plant. Such methods include providing a first parent plant having resistance to Fusarium Head Blight ("resistant parent"), in which the expression, activity, or function of the susceptibility gene TaHRC is inhibited; crossing the first parent plant with a second parent plant to produce progeny plants; and selecting for progeny plants having inhibited expression, activity, or function of the susceptibility gene, such that the progeny plants also have increased or enhanced resistance to Fusarium species.
  • resistant parent a first parent plant having resistance to Fusarium Head Blight
  • TaHRC susceptibility gene
  • the methods comprise screening genomic DNA from at least one plant for the presence of a genetic marker that is associated with decreased Fusarium Head Blight susceptibility activity; and selecting at least one plant comprising an allele of at least one of the genetic markers.
  • Genetic markers for use in marker-assisted plant breeding are also described.
  • the genetic markers comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
  • Fig. 1A is a series of photographs showing near-isogenic lines (NIL) carrying Fhbl as compared to those in NILs without Fhbl;
  • NIL near-isogenic lines
  • Fig. IB is a schematic showing wheat chromosome arm map of 3BS, the map of the Fhbl candidate gene region, the predicted genes in the candidate region of Fhbl, and a diagram of gene structure of TaHRC-S from Clark;
  • Fig. 1C is a photograph showing PCR products amplified from c-DNA of 0 h, 24 h and 48 h F. graminearum-m ' oculated Fhbl near-isogenic lines, NIL22i (NIL-R) and NIL22h (NIL-S);
  • Fig. 2A is a table showing physical positions of representative polymorphic sequences in the ORF of TaHRC among the seven haplotypes (left) and mean percentage of symptomatic spikelets (PSS) for each haplotype (right) evaluated in the greenhouse experiments;
  • Fig. 2B is a graph showing candidate gene association analysis using six representative polymorphic sequence data from the TaHRC and other nine marker data from the 300k Fhbl candidate gene region;
  • Fig. 2C is a pair of photographs showing Tl transgenic plants of RNA interference of TaHRC-?> gene as compared to the non-transgenic Bobwhite control;
  • Fig. 2D is a graphs showing mean PSS for three Tl RNAi transgenic lines and Bobwhite control;
  • Fig. 3 A is a pair of photographs showing expression of 10 candidate genes in the resistant (bottom) and susceptible (top) near-isogenic lines (NILs);
  • Fig. 3B is a graph showing relative expression levels of TaHRC in the resistant and susceptible NILs at different inoculation time points;
  • Fig. 4A is a schematic diagram of cloned TaHRC fragment from Clark and its physical position in ctg0954;
  • Fig. 4B is a gel picture of the cloned TaHRC from Ning7840, Clark, and two sets of Fhbl
  • Fig. 5 is a sequence showing protein motifs of TaHRC
  • Fig. 6 is a graphic presentation of TaHRC gene structure showing the normal transcript from TaHRC-S and mis-spliced transcripts from TaHRC-R;
  • Fig. 7 is a cluster analysis of sequence data of TaHRC from 143 accessions of a worldwide wheat collection
  • Fig. 8 is a photograph showing segregation of the codominant Fhbl diagnostic marker in a subset of wheat accessions
  • Fig. 9 is a diagram showing two constructs (URC-R and HRC-F) used for transformation in the RNA interference experiment;
  • Fig. 10 is a graph showing relative expression levels of TaHRC in the RNAi transgenic plants and Bobwhite controls analyzed 48 h after point inoculation in a growth chamber;
  • Fig. 11 is a diagram showing a phylogenetic tree of grass species based on the putative protein sequences of TaHRC
  • Fig. 12 is a series of photographs showing Subcellular localization of TaHRC
  • Fig. 13 A is a photograph comparing FHB symptoms in a Bobwhite plant generated by genome editing to a non-edited Bobwhite control plant in the left.
  • Fig. 13B is a graph showing the mean FHB severity as reflected by percentage of symptomatic spikelets (PSS);
  • Fig. 14 is a graph showing polymorphism between resistant and genotypes.
  • Fig. 15 shows a sequence alignment of TaHRC among seven wheat cultivars. DETAILED DESCRIPTION OF PREFERRED EMB ODEVIENT S
  • the present invention is concerned with plants having inhibited expression, activity, or function of a target susceptibility gene, which thereby have increased resistance to FHB relative to corresponding control plants in which the wild-type (or endogenous) susceptibility gene is normally expressed in the presence of a Fusarium species pathogen (e.g., Fusarium graminearum). Such plants are referred to herein as "resistant" plants.
  • the invention is also concerned with various methods of increasing resistance to FHB by knocking down a target susceptibility gene in a plant, or a tissue, organ, part, or cell thereof.
  • the invention is further concerned with genetic markers that can be used in identifying resistant plants as part of marker- assisted plant breeding.
  • inventive methods can be applied to increase resistance in susceptible plants, and particularly cereal grains susceptible to FHB.
  • Exemplary plants for use in the invention include Triticum species (e.g., common wheat, spelt, durum), Hordeum species (e.g., barley), Secale species (e.g., rye), and Triticale.
  • the method of the invention can be used to increase plant resistance to various Fusarium species.
  • the method can increase plant resistance to a Fusarium species selected from the group consisting of F. avenaceum, F. bubigeum, F. culmorum, F. graminearum, F. langsethiae, F. oxysporum, F. poae, F. sporotrichioides, F. tricinctum, F. verticillioides, and F. virguliforme .
  • susceptibility is reduced by as much as 50% as compared to a control plant.
  • the target susceptibility gene is a histidine-rich calcium binding protein-like gene, named TaHRC.
  • the TaHRC gene is normally expressed at a low level in the tissue of uninfected wheat plants, but is induced to high levels of expression in susceptible wheat following infection of a Fusarium pathogen species. Therefore, manipulation of the TaHRC expression levels can improve FHB resistance in a target plant, and knocking down TaHRC expression reduces plant susceptibility to FHB.
  • the full TaHRC gene sequence will vary among species and between specific cultivars of a given species. For reference, the sequence alignment for the TaHRC gene in seven different wheat cultivars is shown in Fig.
  • the invention provides plants with increased resistance to FHB caused by Fusarium pathogen species, preferably due to inhibited expression, activity, or function of the endogenous TaHRC gene, a portion thereof, or a functional equivalent thereof.
  • “Functional equivalents,” as used herein, include sequences derived from or having at least about 90% sequence identity (preferably at least about 950% sequence identity, and more preferably at least about 99% sequence identity) to SEQ ID NOS: l-6 or the coding region of TaHRC (SEQ ID NO: 8), and which encode the wild-type protein (SEQ ID NO: 9), an amino acid sequence comprising at least about 70% amino acid identity (preferably at least about 80% amino acid identity, and more preferably at least about 90% amino acid identity) with SEQ ID NO:9, or which encode a protein having essentially the same functional characteristics thereof (i.e., conferring resistance when down regulated).
  • the invention provides a resistant plant which has reduced or no (lost) expression of the TaHRC wild-type protein (SEQ ID NO:9) or an amino acid sequence comprising at least about 30% amino acid identity (preferably at least about 50%) amino acid identity, and more preferably at least about 80% amino acid identity) with SEQ ID NO:9 in the presence of a Fusarium pathogen species.
  • resistance to a Fusarium pathogen species in a plant is increased by inhibiting the expression, function, or activity of a susceptibility gene which comprises a sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO:8; (b) a nucleotide sequence comprising an antisense sequence corresponding to SEQ ID NO: 8; (c) a nucleotide sequence having at least about 50% sequence identity (preferably at least about 80% sequence identity, and more preferably at least about 90% sequence identity) to SEQ ID NOS: l-6 or SEQ ID NO:8 (i.e., conservatively modified variants thereof); (d) a nucleotide sequence encoding an TaHRC protein comprising SEQ ID NO:9; and (e) a nucleotide sequence encoding an TaHRC protein having at least about 30% amino acid identity (preferably at least about 50% amino acid identity, and more preferably at least about 80% amino acid identity) to S
  • Manipulation of TaHRC expression levels can be caused by a deletion in the start codon region of the open reading frame (ORF) of TaHRC, or any other alteration that results in the loss- of-function of the TaHRC gene in the plant and thus a significant reduction in FHB susceptibility.
  • the manipulation of TaHRC expression levels is caused by an insertion or deletion of one or more base pairs within the ORF of TaHRC, thereby causing a frameshift mutation and malfunction of the gene (e.g., by forming a stop codon).
  • the insertion is a single base pair.
  • the insertion occurs within a fragment of the gene having SEQ ID NO: 18.
  • this insertion is in residues 197 to 216, and preferably between residues 213 and 214, of SEQ ID NO: 19.
  • the knockdown of TaHRC expression in accordance with embodiments described herein can result in the reduction of expression levels by at least about 50%, and preferably at least about 60%after inoculation with a species of Fusarium.
  • the method of increasing resistance of a plant to FHB comprises inhibiting the expression, activity, or function of the target susceptibility gene (i.e., TaHRC gene) in a plant cell to produce a modified cell.
  • the method can comprise culturing immature plant embryos to form callus tissue, followed by inhibiting the expression, activity, or function of the susceptibility gene in the tissue to produce modified plant cells (and in the case of transgenic techniques, transformed cells).
  • the modified cells are then used to regenerate resistant plants having inhibited expression, activity, or function of the susceptibility gene, as described herein.
  • the expression, activity, or function of the gene is reduced or diminished in the presence of & Fusarium species as compared to the normal expression, activity, or function of the corresponding susceptibility gene in a control (i.e., non-modified) plant or cell.
  • the invention also provides resistant and/or transgenic cells, tissue, and seeds of plants produced by the methods described herein, and the progeny thereof.
  • the expression, activity, or function of the susceptibility TaHRC gene in the plant can be inhibited by any suitable gene down-regulation technique, which can include modifying the target gene itself, as well as methods involving modification of adjacent sequences.
  • transgenic techniques can be used to alter expression of the target gene.
  • the resistant plant can comprise a nucleic acid construct, preferably stably incorporated into its genome, which inhibits expression, activity, or function of the susceptibility gene.
  • the resulting transgenic plant is prepared by introducing into a plant cell a nucleic acid construct that inhibits expression, activity, or function of the target susceptibility gene.
  • the nucleic acid encodes a double-stranded RNA that inhibits expression, activity, or function of the susceptibility gene.
  • the nucleic acid construct can comprise a nucleotide sequence specific for the susceptibility gene which is operably linked to a promoter that drives expression in the plant cell.
  • the transgenic plant is prepared by introducing into a plant cell a vector comprising the nucleic acid construct.
  • the construct or vector can be introduced by any suitable method, including, without limitation, a biolistic particle delivery system, microprojectile bombardment, viral infection, Agrobacterium-mediated transformation (Agrobacterium tumefaciens), electroporation, and liposomal delivery, to produce transformed cells.
  • the term "bombardment” with respect to transformation refers to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample.
  • a target biological sample e.g., cell, tissue, etc.
  • RNA interference is used to inhibit the expression, activity, or function of the target gene. More specifically, RNAi is used to reduce the expression of one or more transcripts normally induced during a compatible interaction between a Fusarium species and a corresponding host plant. RNAi relies on sequence-specific, post-transcriptional gene silencing, and is broadly defined herein to include all post-transcriptional and transcriptional mechanisms of RNA-mediated inhibition of gene expression. Generally, in RNAi, all or a portion of the target gene (typically greater than 200 bp) is duplicated in an expression vector in a sense/anti sense or an anti sense/sense orientation so that the resulting mRNA hybridizes to itself forming a large hairpin loop.
  • RNAi RNA interference
  • RNAi small interfering RNAs
  • the nucleic acid construct preferably comprises a sense and/or an antisense sequence for the target susceptibility gene and encodes double stranded RNA that inhibits the expression, activity, or function of the target susceptibility gene.
  • the nucleic acid construct will preferably comprise a sense sequence operably linked to its complementary antisense sequence and encoding double stranded RNA that inhibits expression, activity, or function of the target susceptibility gene.
  • the nucleic acid construct can further comprise a selection gene, such as those selected from the group consisting of herbicide resistant genes (e.g., bar gene), antibiotic resistant genes, and other positively selectable genes.
  • a selection gene such as those selected from the group consisting of herbicide resistant genes (e.g., bar gene), antibiotic resistant genes, and other positively selectable genes.
  • transformed cells are preferably grown on selection media corresponding to the desired selection gene to confirm transformation (i.e., incorporation of the construct). Transformation can also be confirmed by specific DNA detection (e.g., PCR) or other positive selection methods.
  • Transgenic plants having inhibited expression, activity, or function of the target susceptibility gene are then regenerated from the transformed cells. The reduction in transcript level from gene silencing results in lowered levels of the target protein, resulting in phenotypic changes in the modified plant, cell, or tissue.
  • the cells are transformed by delivering a selection gene and DNA coding for an antisense TaHRC sequence into the cells of the callus tissue. Plants with FHB resistance are then developed and selected by growing transformed cells on media and selecting for the selection gene, wherein the resulting transgenic plant transcribes the TaHRC sequence to form dsRNA, which inhibits the expression, activity, or function of the TaHRC gene.
  • the cells are transformed by delivering a selection gene and DNA coding for a sense TaHRC sequence and antisense TaHRC sequence into the cells of the callus tissue and selecting for FHB resistance as described above.
  • Virus-induced gene silencing can also be used to inhibit the expression, activity, or function of the target susceptibility gene.
  • This technology uses plant viruses to express a small fragment of a host gene in the form of dsRNA in inoculated plants.
  • the replication of the viral vector which includes the target gene fragment, induces a host response that knocks down or inhibits expression of the endogenous target gene.
  • the target sequence itself may be native or transgenic.
  • the plant is inoculated with a viral vector comprising a sequence (sense or antisense) of the targeted susceptibility gene, which encodes for RNA that inhibits the expression, activity, or function of the target gene to thereby produce the transgenic plant.
  • transgenic methods can also be utilized to silence the target gene, including microRNAs, artificial microRNAs, antisense RNA, or T-DNA insertional inactivation of the target gene or associated promoter.
  • DNA or RNA that is complementary to the mRNA of the target gene is introduced into a cell and inhibits translation of the gene product. This sequence can be all or a portion of the mRNA.
  • miRNAs are another class of small RNAs which can cause specific gene silencing.
  • More recent genome editing techniques can also be used to knock out or inhibit the function of the target gene, such as zinc finger proteins (ZNFs), transcription activator-like effector nucleases (TALENS), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- based technology.
  • ZNFs zinc finger proteins
  • TALENS transcription activator-like effector nucleases
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • RNA-guided nucleases such as Cas9.
  • bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes can be used for gene editing.
  • CRISPR-Cas9 relies on sequence-specific double strand break (DSB) induction by the endonuclease Cas9 that is recruited to a single guiding (sg) RNA complex.
  • the complex directly binds to a 20-nucleotides recognition site on the target gene, and the sequence motif 'NGG' downstream is crucial for binding recognition.
  • all or a portion of the target gene is introduced to the double strand break and repaired either by homology-directed repair (HDR) or by nonhomologous end joining (NHEJ).
  • HDR homology-directed repair
  • NHEJ nonhomologous end joining
  • a knockout mutation (loss-of-function) of the target genes can be generated when the sequence changes cause a frameshift or a stop codon mutation in the target susceptibility gene.
  • the sense sequence used in the nucleic acid construct preferably comprises SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence having at least about 50% sequence identity (preferably at least about 80% sequence identity, and more preferably at least about 90% sequence identity) with SEQ ID NO: 10 or SEQ ID NO: 11.
  • the antisense sequence preferably corresponds to SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence having at least about 50% sequence identity (preferably at least about 80%) sequence identity, and more preferably at least about 90% sequence identity) with SEQ ID NO: 10 or SEQ ID NO: 11.
  • transgenic plant comprising a nucleotide sequence that encodes or is complementary to a sequence that encodes a TaHRC polynucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11, wherein the transgenic plant has increased resistance to FHB.
  • the nucleic acid construct comprises at least one nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 10 or SEQ ID NO: 11; (b) a nucleotide sequence comprising an antisense sequence corresponding to SEQ ID NO: 10 or SEQ ID NO: 11; (c) a nucleotide sequence having at least about 50% sequence identity (preferably at least about 80% sequence identity, and more preferably at least about 90% sequence identity) to SEQ ID NO: 10 or SEQ ID NO: 11; (d) a nucleotide sequence encoding a TaHRC protein comprising SEQ ID NO: 9; and (e) a nucleotide sequence encoding a TaHRC protein having at least about 30% amino acid identity (preferably at least about 50% amino acid identity, and more preferably at least about 80%) amino acid identity) to SEQ ID NO:3 and retaining the functional characteristics thereof.
  • nucleotide sequence is operably linked to a promoter capable of regulating transcription of the sequence in the transgenic plant.
  • nucleic acid construct could comprise any portion of SEQ ID NO: 1 or SEQ ID NO:2 (similar to embodiments using SEQ ID NO:4 above), which could be used to inhibit the expression, activity, or function of the target susceptibility gene.
  • the expression, activity, or function of the susceptibility gene in the resistant plant is inhibited by mutagenesis of the target susceptibility gene itself or of associated adjacent sequences affecting the expression, activity, or function of the target gene.
  • the resistant plant preferably comprises an insertion, deletion, or point mutation which directly or indirectly inhibits the expression, activity, or function of the susceptibility gene.
  • the mutation(s) can be induced by any suitable means, including chemically or with radiation according to known methods. Natural mutations which inhibit the expression, activity, or function of the target susceptibility gene or corresponding protein are also encompassed by the present invention.
  • TaHRC inhibition of the expression, activity, or function of TaHRC can be based on conditional expression of TaHRC driven by conditional promoters, according to known methods.
  • resistant cultivars can also be produced by interfering with other targets, such as gene products (e.g., proteins), in the TaHRC pathway.
  • resistant plants can be produced indirectly by breeding parent plants having inhibited expression, activity, or function of the target susceptibility gene or corresponding gene products (whether naturally occurring or induced) with other resistant plants, or with other cultivars having additional desired characteristics (e.g., drought tolerance, geographic adaptation, stalk strength, etc.).
  • the resulting progeny can then be screened to identify resistant progeny with inhibited expression, activity, or function of the corresponding target susceptibility gene or gene products.
  • resistant plants according to the invention preferably exhibit a decrease in the expression of the susceptibility gene in the presence of a Fusarium species as compared to a corresponding control plant. More preferably, the plant has a decreased level of TaHRC protein in the presence of the Fusarium species as compared to a corresponding control plant. Accordingly, the inventive plants have an increased or enhanced resistance to infection and spreading of infection by Fusarium pathogen species.
  • the methods described herein can be combined with other methods of increasing plant resistance to FHB.
  • the methods described herein can be combined with the transfer of exogenous resistance gene Fhbl into susceptible plant varieties by backcrossing or transgenic methods. Examples of increasing resistance through transfer of Fhbl can be found in WO 2015/184331, filed May 29, 2015, incorporated by reference in its entirety to the extent not inconsistent with the present disclosure. Together, these techniques yield plants having JaHRC-regulated FHB resistance.
  • the methods described herein can be further combined with additional methods for inducing or selecting other desirable wheat characteristics, including resistance to other pests, or environmental conditions (e.g., heat stress), and the like.
  • the present invention is directed to a method of identifying a plant comprising at least one allele associated with decreased FHB susceptibility activity in a plant that comprises: (a) screening genomic DNA from at least one plant for the presence of a nucleic acid marker that is associated with decreased FHB susceptibility activity; and (b) selecting at least one plant comprising an allele of at least one of said nucleic acid marker.
  • the nucleic acid marker is preferably developed from the nucleotide sequence of the TaHRC in a resistant plant.
  • the nucleic acid diagnostic marker can be used to identify a mutation (e.g., causal deletion) in the TaHRC gene of a plant for marker-assisted selection of the Fhbl in breeding (i.e., marker-assisted plant breeding).
  • diagnostic markers used in embodiments of the present invention comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
  • the term "susceptibility gene” refers to a gene that, when functional and expressed in the plant, renders the plant vulnerable to infection by a pathogen.
  • expression of the gene products (i.e., corresponding protein) in the plant results in the plant being receptive to infection.
  • the susceptibility gene is non-functional (and accordingly its gene products are reduced or not expressed), the plant displays a reduction in observable signs of infection and a resistance to spreading of the infection.
  • the susceptibility gene is TaHRC, which when expressed in a susceptible plant, renders the plant vulnerable to infection and spreading (e.g., within a spike) by a Fusarium species.
  • the susceptibility gene TaHRC is altered in accordance with the invention, such that the gene is non-functional, the Fusarium species is unable to spread within the host plant, and the resulting plants displays a reduction in observable signs of FHB infection and a resistance to spreading of the infection.
  • control when used with respect to control plants includes wild-type (native) plants, as well as cultivars and genetically altered plants (such as plants containing resistance genes) that otherwise contain a wild-type, non-modified, or native (endogenous) susceptibility gene targeted for gene silencing (inhibition) according to the invention.
  • a "control plant” refers not only to the same plant species, but also to the same cultivar as the comparative resistant plant without a modified susceptibility gene.
  • the "control plant” is susceptible for FHB infection and includes a functional susceptibility gene that is normally expressed in the presence of a Fusarium species.
  • a "wild-type” gene is one that has the characteristics of a gene isolated from a naturally occurring source.
  • a “wild-type” gene product is one that has the characteristics of a gene product isolated from a naturally occurring source, whereas “modified” genes or gene products are those having modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product.
  • modified cells, tissues, seeds, etc. are those that have been altered to change the expression, activity, or function of the target genes or gene products, as opposed to non- modified cells, tissues, etc.
  • the term “modified,” as used herein encompasses both transgenic and non-transgenic techniques (e.g., natural or induced mutagenesis).
  • references to "inhibition,” “silencing,” or “knock down” of the expression, activity, or function of a gene are used synonymously to refer to any suitable approach of reducing or even completely suppressing the normal activity or function of the gene, such that there is a reduction or even loss of gene function (i.e., a malfunction of some kind), and accordingly a reduction or loss of protein expression from the gene or coding sequence.
  • modified plants with inhibited expression, activity, or function of the gene cannot express normal (or any) levels of the associated protein.
  • Approaches for achieving such inhibition or silencing including methods of reducing the levels of protein produced as a result of gene transcription to mRNA and subsequent translation of the mRNA.
  • Gene inhibition may be effective against a wild-type plant gene associated with a trait, e.g., to provide the plant with a diminished level of a protein encoded by the wild-type gene or with reduced levels of an affected metabolite.
  • RNAi RNAi
  • VIGS RNAi
  • mutagenesis mutagenesis
  • CRISP gene editing and the like as described herein.
  • gene expression refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through transcription of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein, through translation of mRNA. Gene expression can be regulated (and inhibited) at many stages in the process. References to altered “levels” of expression refers to the production of gene product(s) in modified plants, such as transgenic plants, in amounts or proportions that differ from that of normal, control, or non-modified "wild-type" plants.
  • operably linked refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced
  • vector refers to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
  • the term includes recombinant DNA molecules containing a desired coding sequence(s) and appropriate nucleic acid sequences (e.g., promoters) necessary for the expression of the operably linked coding sequence in a particular host organism.
  • transformation is used herein to refer to the introduction of foreign DNA into cells. Transformation may be accomplished by a variety of means known to the art and described herein.
  • heterologous gene refers to a gene encoding a factor that is not in its natural environment.
  • a heterologous gene includes a gene from one species introduced into another species.
  • a heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.).
  • Heterologous genes are distinguished from endogenous plant genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
  • a “sense” strand of nucleic acid construct refers to a strand that is transcribed by a cell in its natural state into a “sense” mRNA.
  • the term “antisense” refers to a DNA sequence whose sequence of deoxyribonucleotide residues is complementary to all or part of the sequence of deoxyribonucleotide residues in a sense strand.
  • an “antisense” sequence is a sequence having the same sequence as the non-coding strand in a DNA duplex.
  • RNA transcript refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript or mRNA.
  • the complementarity of an antisense RNA or DNA may be with any part of the specific gene or transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • isolated when used in relation to a nucleic acid, refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural environment. That is, an isolated nucleic acid is one that is present in a form or setting that is different from that in which it is found in nature.
  • sequence identity or “amino acid identity” are used herein to describe the sequence relationships between two or more nucleic acid or amino acid sequences when aligned for maximum correspondence over a specified comparison window.
  • the percentage of “identity” is determined by comparing two optimally aligned sequences over the comparison window.
  • the portion of the sequence in the comparison window may include gaps (e.g., deletions or additions) as compared to the reference sequence, which does not contain additions or deletions.
  • the number of matched positions i.e., positions where the identical nucleic acid base or amino acid residue occurs in both sequences is determined and then divided by the total number of positions in the comparison window.
  • sequence having a certain % of sequence identity to a reference sequence does not necessarily have to have the same total number of nucleotides or amino acids (see e.g., microRNAs discussed above).
  • a sequence having a certain level of "identity" includes sequences that correspond to only a portion (i.e., 5' non-coding regions, 3' non-coding regions, coding regions, etc.) of the reference sequence.
  • the phrase "and/or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed.
  • the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
  • Ning7840 (a derivative line of Sumai3, "SM3") is a highly resistant Chinese line that carries Fhbl
  • Clark is a U.S. FFIB-susceptible cultivar from Purdue University. Both the parents and RIL population were evaluated for FFIB resistance in several greenhouse experiments.
  • hybrid plants from each backcross were genotyped using the flanking markers, Xgwm533 and Xgwm494, to select heterozygous Fhbl plants for further backcrossing.
  • selected BC7F1 plants with Fhbl were selfed to generate enough Bc7F 2 seeds for fine mapping.
  • NIL22 One plant, NIL22, was found to be heterozygous at XumnlO, and its BC7F4 seeds were individually planted and genotyped to select for homozygous plants with contrasting alleles at XumnlO. The selected plants were advanced and evaluated for FFIB resistance in the greenhouse using the single floret inoculation method. FFIB data showed that NIL22i and 22w were FHB -resistant, and 22h and 22v were FHB-susceptible in three consecutive greenhouse experiments with 30 plants per line tested. The four lines were selected as two sets of NILs for RNA-Seq. The resistant line NIL22i (NIL-R) and the susceptible line NIL22h (NIL-S) were used for candidate gene expression and sequencing.
  • a Kansas strain of F. graminearum (isolate GZ3639) was used as inocula to evaluate FHB resistance in the fine mapping population and the association panel.
  • a conidial spore suspension was prepared following Bai et al. (1999). At early anthesis, wheat spikes were inoculated by injecting 10 ⁇ of the conidial spore suspension (-1000 spores/spike) into a floret of a central spikelet in a spike using a syringe (Hamilton, Reno, NV). Five spikes per pot and three pots per line were inoculated in each experiment.
  • the inoculated spikes were either enclosed in a plastic moist chamber, or bagged with a thin plastic sandwich bag in each spike, to keep 100% relative humidity at 20-22 °C for 48 h to initiate fungal infection.
  • the infected plants were then moved to the greenhouse benches for further FHB development.
  • FHB symptom spread within a spike (type II resistance) was evaluated by counting the symptomatic spikelets and total spikelets in the inoculated spike 15 d after inoculation.
  • FHB severity for each line was calculated using percentage of symptomatic spikelets (PSS).
  • Leaf tissue was collected at two-leaf stage in 96-deepwell plates before vernalization, dried in a freeze dryer (ThermoSavant, Holbrook, NY) for 48 h, and ground using a Mixer Mill (MM 400, Retsch, Germany). Genomic DNA was isolated using a modified cetyltrimethyl ammonium bromide protocol (Bernardo et al. 2012). Polymerase chain reaction (PCR) was done in an MJ Research PTC-200 Thermal Cycler (Bio-Rad, Hercules, CA). For SSR and STS marker detection, an M13 tail (5'- ACGACGTTGTAAAACGAC) (SEQ ID NO: 17) was added to 5'-end of all forward primers.
  • PCR Polymerase chain reaction
  • a 10- ⁇ 1 PCR master mix contained IX ASB buffer, 2.5 mM of MgCk, 200 ⁇ of dNTP, 100 nM of fluorescent dye-labeled M13 primer, 100 nM of M13 tailed forward primer, 200 nM of a reverse primer, 0.6 U of Taq polymerase, and 40 ng of template genomic DNA.
  • PCR amplification was done using a touchdown program. The PCR mixture was incubated initially at 95°C for 5 min, followed by five cycles of 96°C for 1 min, annealing at 68°C for 3 min with a decrease of 2°C in each subsequent cycle, and extension at 72°C for 1 min.
  • annealing temperature started from 58°C for 2 min with a decrease of 2°C in each subsequent cycle, and then PCR went through an additional 25 cycles of 96°C for 1 min, 50°C for 1 min, and 72°C for 1 min, ending with a final extension at 72°C for 5 min.
  • Amplified PCR products labeled with different fl orescent dyes FAM, VIC, NED, and PET were pooled and analyzed in an ABI PRISM 3730 DNA Analyzer (Applied Biosystems, Foster City, CA). Data were scored using GeneMarker vl .75 (SoftGenetics LLC, State Collage, PA).
  • KASP assays were designed from the sequences harboring the SNPs in the Fhbl region between two contrasting NILs.
  • KASP assays consisted of three KASP primers: two allele-specific forward primers and one common reverse primer.
  • the KASP master mix for each reaction comprised of 3 ⁇ of 2x KASP reaction mix, 0.0825 ⁇ of KASP primer mix (100 ⁇ ) and 3 ⁇ of DNA (-40 ng). Reaction mixes were incubated at 94°C for 15 min, followed by 10 cycles of 94°C for 20 s and 65°C for 1 min with a decrease of 0.8°C in each subsequent cycle.
  • the PCR went through an additional 40 cycles of 94°C for 20 sec and 57°C for 1 min. After PCR, plates were read in an Applied Biosystems 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA).
  • the 15ul PCR mix contains IX ASB buffer, 2.5 mM MgC12, 200 ⁇ of each dNTP, 100 nM of each primer, 1 U Taq polymerase, and 60ng genomic DNA.
  • PCR amplification was conducted using a touchdown program that started at 95°C for 5 min, followed by 10 cycles of 95°C for 30 s min, 63°C for 30 s, with a decrease of 0.5°C in each subsequent cycle, and 72°C for 3 min. PCR then went through an additional 28 cycles of 95°C for 30 S, 58°C for 30 s, and 72°C for 3 min with a final extension at 72°C for 10 min.
  • the PCR product was analyzed in a 1.0% agarose gel.
  • Genomic DNA of TaHRC was amplified by PCR using the gene-specific primer pair of Cfb6058R and Cg8UTR-F.
  • the PCR fragments were purified using the Zymolean Gel DNA Recovery kit and then cloned into a pGEM-T Easy vector (Promega, Madison, WI) following the manufacturer's instruction.
  • PCR was performed using the primers Cfb6058R and Cg8UTR-F to select the positive clones for sequencing.
  • Partial genomic DNA of TaHRC-S and TaHRC -R was amplified using the primer combination of TaFIRC-S-3B-F/Cg8UTR-F and TaHRC -R-3B- F/Cg8UTR-F, respectively.
  • the PCR products were purified with shrimp alkaline phosphatase and exonuclease I and then sequenced in an AB 13730 DNA Sequencer (Thermo Fisher Scientific Inc.). Sequence analysis, gene function prediction,
  • DNA sequence was assembled and aligned using DNAstar Lasergene and Bioedit.
  • FGENESH was used to predict genes in the monocot (maize, rice, wheat, and barley) model.
  • a phylogenetic tree was constructed using the neighbor-jointing method, and the bootstrap test was carried out with 1000 replicates using Mega 5.0 (Tamura et al., 2011).
  • Prediction of biological function and conserved domain of the TaHRC protein was conducted using PROSITE.
  • Statistical analysis was conducted using SPSS16.0 for Windows (SPSS Inc., Chicago, USA).
  • the markers in the candidate gene region of Fhbl were used to conduct association analysis using the Tassel Pipeline and a threshold of -loglO > 3 to claim a significant association between a marker and FUB severity.
  • RNA-Seq cDNA libraries were constructed using the Ion Total RNA-Seq Kit V2 (Thermo Fisher Scientific Inc.), and the samples were barcoded with Ion Xpress RNA-seq barcode. Equimolar amounts of the same time-point libraries from the four sister lines were pooled and ran at least twice on an Ion Proton Sequencer with PI chip (Thermo Fisher Scientific Inc). NILs 22h and 22v are sister lines and were considered as biological replicates, and likewise with NILs 22i and 22w.
  • RNA-Seq data Two replicated sets of RNA-Seq data were combined to increase the sequencing depth.
  • the sequences of ERCC and F. graminearum were removed from the raw sequencing reads using an in-house Perl script.
  • the adapters were removed from the reads using Cutadapt (Martin, 2012).
  • the low-quality sequences of reads were trimmed using Fastx-toolkit (Pearson 1997).
  • GSNAP Thimas et al., 2005
  • TvVGSC Poly-N fragments
  • RNA was treated with RNase-free DNase I (Invitrogen, Carlsbad, CA).
  • the first- strand cDNA was synthesized using the Superscript II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA).
  • the primers used for amplifying the first-strand cDNA of 10 Fhbl candidate genes were designed based on the Ctg0954 sequences of Chinese Spring. Quantitative real-time PCR was performed on ABI7900 using SYBR green (ThermoFisher Scientific Inc.). Actin gene was used as an internal control. Gene expression level was analyzed using ⁇ cycle threshold method using RQ study software (ThermoFisher Scientific Inc.).
  • Primers were designed from the TaHRC sequence of wheat cultivar Bobwhite, a highly FUB susceptible cultivar with a high transformation efficiency using Primer Premier 6.0 (Premier Biosoft, Interpairs, Palo Alto, CA).
  • Primer Premier 6.0 Premier Biosoft, Interpairs, Palo Alto, CA.
  • nucleotides CACC were added to the 5'- end of both forward primers for directional cloning of the PCR fragments into the entry vector pENTER-D/TOPO (Invitrogen).
  • RNAi-F (SEQ ID NO: 10) was made from a 428- bp fragment that corresponds to the nucleotides 33-460 in the ORF of HRC, and the construct RNAi-R (SEQ ID NO: 11) contains a 406-bp fragment that corresponds to the nucleotides 212-618 in the ORF (Fig. 9). Both fragments were independently cloned into the pANDA-mini vector (Miki and Shimamoto, 2004) by homologous recombination using LR Clonase (Invitrogen). In each final RNAi construct, two identical TaHRC fragments were inserted into both sides of the 920-bp GUS linker in antisense and sense orientations (Fig.
  • the plasmids containing the constructs were transformed into Top 10 competent Escherichia coli cells (Invitrogen) and purified using an E.Z.N.A. Plasmid Mini Kit (Omega Bio-Tek, Norcross, GA). Sanger sequencing was used to determine the sequences and orientation of the insertions.
  • the constructs were co-bombarded with the pAHC20 construct into the cultured immature embryo of the wheat cultivar Bobwhite as described by Cruz et al. (2014).
  • the vector pAHC20 (Christiansen et al., 1996) contained the bar gene under control of the ubiquitin promoter for selection.
  • the wheat transformation was performed through biolistic particle delivery. Tissue culture media and methods of selection and culture followed Anand et al. (2003).
  • Each construct had six bombardments using 160 embryos in each bombardment. Briefly, embryos were excised from immature seeds at 10 to 14 d post anthesis and placed on a callus introduction medium CM4 for 4 to 7 d in the dark at room temperature.
  • Somatic embryos selected for bombardment were placed in the fresh medium and co-bombarded with pAHC20 and RNAi constructs at 1 : 1 ratio through the particle inflow gun.
  • Wheat calli were placed on a CM4 medium containing glufosinate for selection after 5 d. After three transfers on to a new CM4 medium containing glufosinate, the growing calli were transferred to a shoot production medium (MSP), and then to an elongation and rooting medium (MSE) with selection by glufosinate all the time.
  • Putative TO transgenic plants were transferred to soil and tested for glufosinate resistance by brushing a 0.2% v/v Liberty (glufosinate) solution (AgroEvo USA, Wilmington, DE).
  • Transgenic plants were identified by PCR for the presence of the TaHRC inserts using the gus-linker primer, gus-Fl, gus-F2 and the TaHRC specific reverse primers, HRCF-Rv440-460 and HRCR-Rv598-618, respectively.
  • the transgenic plants (Tl and T2) with both sense and antisense fragments of TaHRC were selected for FUB phenotyping.
  • 35S:7aHRC-GFP expression vector was constructed by fusing the coding sequence of TaHRC-S from Bobwhite to GFP coding sequence.
  • the resultant construct was introduced into wheat protoplasts of leaf and onion epidermal cells, respectively.
  • the nuclei in transformed cells were stained with 4',6-diamidino-2- phenylindole (DAPI, 5 mg ml-1 in PBS), then examined using a LSM 510 META confocal microscope (Zeiss Inc, Jena, Germany). Wheat leaf protoplast isolation and transfection was performed as described by Zhai et al. (2009).
  • Fhbl was originally mapped to the short arm of wheat chromosome 3B in Sumai3 and
  • Ning7840 (Fig. IB).
  • a fine mapping population of (Clark* 8/Ning7840)F2 was developed using marker-assisted backcross, and two flanking markers, Xsts3B-163 and Xsts3B-142, for Fhbl (Fig. IB) were identified after screening 1750 (Clark* 8/Ning7840)F 2 plants.
  • An additional 13,000 (Clark* 8/Ning7840)F 2 plants were screened to fine map Fhbl using the flanking markers, and 22 recombinants were identified (Fig. IB).
  • NIL22i a histidine-rich calcium binding protein-like gene, designated as TaHRC
  • RNA-Seq experiment was also conducted using the 48 h inoculated spike samples collected from the same NILs, and 21 differentially expressed genes were identified on the chromosome 3B.
  • TaHRC was selected as the candidate gene for Fhbl based on the fine mapping and gene expression result.
  • TaHRC was cloned from Ning7840, Clark, and the Fhbl NILs by PCR using the common primers, and it was sequenced to obtain a 2650 bp DNA fragment from Clark and NIL-S, designated TaHRC-S, and a 2041 bp DNA fragment from Ning7840 and NIL-R, designated TaHRC- (Figs. 4A and 4B).
  • a sequence comparison between TaHRC- in Ning7840 and TaHRC-S in Clark revealed a low similarity (72.5%) compared with that (98.5%) between two susceptible genotypes Clark and Chinese Spring.
  • Sequencing of the cDNA confirmed that the TaHRC-S contains three exons and two introns. In the resistant NIL22i and Ning7840, however, a functional gene could not be predicted from the TaHRC-R sequence due to missing start codon in the ORF. Sequencing the full-length cDNA from TaHRC-K identified at least three types of cDNA sequences with different lengths. All three cDNA sequences were different from the one transcribed from the TaHRC-S (Fig. 1C). Two of the transcripts have three small exons in the 5 'UTR, and one retains the intron 3 to form a large exon 3 (Fig. 6), indicating alternative-splicing occurred in the TaHRC- K.
  • TaHRC was sequenced from a worldwide collection of 143 wheat accessions with various levels of FHB resistance.
  • Cluster analysis using the sequence data identified seven clusters (Fig. 7). Haplotype analysis showed that the seven clusters could be separated by six representative sequence polymorphisms in the ORF (Fig. 2A). Based on the presence and absence of the start codon, the seven clusters could be merged into two haplotype groups: the TaHRC-K group containing only the Ning7840 (Ning) cluster (60 accessions) and the TaHRC-S group containing the rest of six clusters (83 accessions).
  • Ning Ning7840
  • the TaHRC-K group mainly consisted of the Chinese landraces (38 accessions) and
  • Chokwang is an accession in DHP cluster and shares identical sequence with DHP.
  • a previous QTL mapping study indicated that Chokwang does not carry Fhbl, thus DHP may not carry Fhbl either.
  • the Ning cluster showed significantly lower mean PSS (19.8%) than that in the DHP cluster (55%), which further supports that the start codon deletion in TaHRC-K underlines the FHB resistance of Fhbl.
  • Two diagnostic markers based on the sequence of TaHRC -R were developed: one can be analyzed using Agarose gel and another is a SNP marker that is analyzed using KASP chemistry.
  • the primer sequences of the KASP (competitive allele-specific PCR) diagnostic marker for selecting Fhbl in breeding are provided as SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.
  • SEQ ID NO: 12 is Fhbl-R (Resistance allele).
  • SEQ ID NO: 13 is Fhbl-S (Susceptible allele).
  • SEQ ID NO: 14 is Kasp-Fhbl-rev.
  • the primers for the Agarose gel-based Fhbl diagnostic marker for breeding are SEQ ID NO: 15 (TaHRC-STS-F) and SEQ ID NO: 16 TaHRC-STS-R).
  • the KASP marker for TaHRC clearly separates Fhb 1 resistance allele (upper left cluster) and Fhbl susceptible allele (bottom right cluster).
  • the HRC-STS marker was used to capture the causal deletion in TaHRC-K for marker-assisted selection of the Fhbl in breeding. Specifically, Fig.
  • RNA interference technique was used to knock down the expression of TaHRC- in a highly susceptible cultivar, Bobwhite.
  • three 4798R, 483 OR, and 5428F were confirmed to carry both the sense and the antisense strands of the TaHRC constructs (Fig. 9) in the Tl plants.
  • the Tl plants were inoculated with F. graminearum using point inoculation, and the blight symptoms were spread much slower than the Bobwhite control after inoculation (Fig. 2C).
  • the disease symptoms were not spread to nearby spikelets, even at 20 d after inoculation in the two Tl plants (Fig.
  • TaHRC is a conserved gene in grass species (Fig. 11). Function prediction of TaHRC-S could not find any known conserved domain for plant disease resistance genes. Its biological functions in plants remain to be characterized. Motif scan of the putative protein of URC-S identified with a serine-rich region at the aa positions 54-213, an arginine-rich region at the positions 70-191, a histidine-rich region at the positions 232-247, and bipartite nuclear localization signal at the positions 82-96 (Fig. 5). Transient expression of the green fluorescence protein (GFP) labeled TaHRC-S gene in wheat leaf protoplast and onion epidermal cells localized the protein in the nucleus (Fig. 12), thus it is believed that TaHRC is a novel nuclear gene regulating FHB resistance in wheat.
  • GFP green fluorescence protein
  • TaHRC is an FHB susceptibility gene, and knocking down TaHRC-S expression in Bobwhite wheat using transformation and transferring Fhbl into highly susceptible varieties by backcrossing significantly reduced FHB spread rate in wheat spikes.
  • manipulation of the TaHRC expression levels can greatly improve FHB resistance.
  • Fhbl remaining effective to all isolates tested worldwide for a half-century demonstrates that the TaHRC-K regulated FHB resistance is durable.
  • the gene can be used to quickly relieve the FHB damage by manipulating TaHRC-S expression in locally adapted susceptible cultivars. This can be achieved by changing the functional TaHRC-S in susceptible cultivars to TaHRC- using the diagnostic marker, RNA interference, and gene editing techniques, using transformation to provide a quick solution to minimizing the FHB damage in wheat. To reach adequate protection, minor genes from locally adapted cultivars are preferred to combine with Fhbl.
  • TaHRC was knocked down in FHB susceptible Bobwhite using CRISP/Cas9 genome editing technology to show that using gene editing to change susceptible TaHRC sequence can slow down FHB spread in wheat therefore increase wheat resistance to FHB.
  • the plant codon optimized Streptococcus pyogenes Cas9 and sgRNA were synthesized by Genscript.
  • the plasmid pAHC17 with the maize ubiquitin 1 promoter was used to construct the Cas9 expression vector by BamHI restriction site to generate expression vector, pAHC17- tawoCas9.
  • the wheat U6 promoters with wheat sgRNA were cloned into pUC57 vectors (Genscript) to generate the AtU6sgRNA plasmid.
  • PAM 5'NGG protospacer-adjacent motif
  • a target sequence (SEQ ID NO: 18) was identified for gene editing within the TaHRC-S coding sequence (SEQ ID NO: 19) of Bobwhite.
  • the target region was in residues 197 to 216 of SEQ ID NO: 19.
  • the target insertion was an adenine base pair insertion between residues 213 and 214 of SEQ ID NO: 19.
  • 6 positive transgenic lines harboring guard RNA and CAS9 were obtained, but only one of them (9427) has 1 base pair insertion in the target region causing a reading frame shift resulting in a stop codon at position 264. This one base insertion caused loss-of-function in TaHRC and reduced FHB severity in the transgenic plants.
  • Fig. 13 A the plant on the right generated by genome editing shows significantly slower FHB development in the inoculated spike after genome editing to knockdown TaHRC than the non-edited Bobwhite control plant in the left.
  • Fig. 13B shows the mean FHB severity as reflected by percentage of symptomatic spikelets (PSS) for Bobwhite control (Bob-CK), two negative transgenic controls (9427-A-Neg and 9427-B-Neg), two positive TaHRC gene edited Tl lines (9427-A-Edi, 9427-B-Edi).
  • PSS symptomatic spikelets

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Abstract

La présente invention concerne des plantes ayant une résistance accrue à la brûlure de l'épi causée par le Fusarium. Les plantes ont été modifiées de telle sorte qu'un gène de susceptibilité de type sauvage TaHRC dans la plante qui est normalement exprimé en présence d'une espèce Fusarium a été modifié, de sorte que l'expression, l'activité ou la fonction du gène de susceptibilité est inhibée ou perdue pour produire une plante résistante. L'invention concerne en outre des procédés de modification de plantes normalement susceptibles pour une résistance accrue, ainsi que des marqueurs génétiques pour la sélection de plantes assistée par marqueur pour sélectionner des plantes résistantes.
PCT/US2017/025454 2016-03-31 2017-03-31 Gènes et marqueurs pour augmenter la résistance à la brûlure de l'épi causée par le fusarium et leurs utilisations WO2017173318A1 (fr)

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