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EP-4737575-A2 - MODIFICATION OF TRANSCRIPTIONAL REPRESSOR BINDING SITE IN NF-YC4 PROMOTER FOR INCREASED PROTEIN CONTENT AND RESISTANCE TO STRESS

EP4737575A2EP 4737575 A2EP4737575 A2EP 4737575A2EP-4737575-A2

Abstract

Method of increasing protein content in a eukaryotic cell comprising an NF-YC4 gene comprising modifying the transcriptional repressor binding site; method of producing a plant with increased protein content comprising crossing and selecting for increased protein content; method of increasing resistance to a pathogen or a pest in a plant cell or plant comprising an NF-YC4 gene comprising modifying the transcriptional repressor binding site, alone or in further combination with expressing QQS in the plant cell or plant; method for producing a plant with increased resistance to a pathogen or a pest comprising crossing and selecting for increased resistance to the pathogen or the pest; a cell, collection of cells, tissue, organ, or organism, such as a plant, in which the NF-YC4 gene comprises a promoter comprising a transcriptional repressor binding site that has been modified so that the transcriptional repressor cannot prevent transcription of the NF-YC4; plants and hybrids thereof; and seeds.

Inventors

  • LI, LING
  • WURTELE, EVE SYRKIN

Assignees

  • Iowa State University Research Foundation, Inc.

Dates

Publication Date
20260506
Application Date
20160217

Claims (15)

  1. A plant cell or plant with increased resistance to a pathogen or a pest, wherein the plant cell or plant comprises an NF-YC4 gene, which comprises a promoter comprising a transcriptional repressor binding site comprising an ERF motif, a RAVI motif, or both an ERF motif and a RAVI motif that has been modified so that the transcriptional repressor cannot prevent transcription of the NF-YC4 gene, wherein the resistance to the pathogen or the pest is increased in the plant cell or plant.
  2. The plant cell or plant of claim 1, further comprising a polynucleotide comprising a nucleotide sequence encoding a Qua-Quine Starch (QQS) polypeptide having the amino acid sequence as set forth in SEQ ID NO: 16, wherein the nucleotide sequence is operably linked to a promoter.
  3. The plant cell or plant of claim 1, wherein the plant cell or plant is a rice cell or a rice plant and (i) two ERF motifs are deleted, (ii) a RAFI motif is deleted, or (iii) a RAVI motif and an ERF motif are deleted.
  4. The plant cell or plant of claim 1, wherein the plant cell or plant is a soybean cell or a soybean plant and either (i) two RAV1 motifs are deleted or (ii) a RAVI motif and an ERF motif are deleted.
  5. The plant cell or plant of claim 1, wherein the ERF motif is deleted, the RAV1 motif is deleted, or both the ERF motif and the RAV1 motif are deleted by using TALENS or CRISPR/Cas9.
  6. The plant cell or plant of claim 1 or 2, wherein the pathogen is a bacterium, a virus, a fungus or a seed plant, preferably wherein the pathogen is a bacterium or a virus.
  7. The plant cell or plant of claim 1 or 2, wherein the pest is an insect, a plasmodiophorid, a mite, or a nematode, preferably wherein the pest is an insect that is an aphid.
  8. The plant cell or plant of claim 1 or 2, wherein the plant is a crop plant, preferably wherein the crop plant is soybean, rice or corn.
  9. The plant cell or plant of claim 1 or 2, wherein the plant is a monocot or a dicot.
  10. The plant cell or plant of claim 1, wherein the wild-type of the plant does not comprise and express QQS and into which a polynucleotide comprising a nucleotide sequence encoding a QQS polypeptide having the amino acid sequence as set forth in SEQ ID NO: 16 has been introduced and expressed therein, wherein the nucleotide sequence is operably linked to a promoter.
  11. A genetically modified plant, comprising a NF-YC4 transgene with a manipulated or modified promotor, wherein the NF-YC4 transgene has been isolated and transferred from another plant, wherein the NF-YC4 transgene increases the protein production or the resistance to a pathogen or a pest.
  12. The plant of claim 11, wherein the NF-YC4 transgene has been derived from a plant of the same species, the same genus but a different species, or from a plant of a different family.
  13. The plant of claims 11 or 12, (I) wherein the plant is a monocot or a dicot, or (II) wherein the plant is a crop plant, root crop, or a horticultural crop, preferably, the plant is a crop plant selected from the group of soybean rice, corn, wheat, millet, barley, alfalfa, tomato, apple, pear, strawberry, orange, watermelon, pepper, carrot, potato, sugar beets, yam, lettuce, spinach, sunflower, and rape seed, a flowering plant, such as petunia, rose, and chrysanthemum, conifers and pine trees, a plant used in phytoremediation, and a plant used for experimental purposes such as Arabidopsis.
  14. The plant of any of claims 11 to 13, wherein the NF-YC4 transgene is from Brassica napus, Medicago truncatula, Arabidopsis thaliana, Glycine max, Oryza sativa, Zea mays, or Chlamydomonas reinhardtii.
  15. The plant of any of claims 11 to 14, wherein the NF-YC4 transgene is stably incorporated in the plant and can be introduced into other plants by sexual crossing.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT The work described herein was supported, at least in part, by grants from the National Science Foundation under MCB award nos. 0209789 and 0951170. Therefore, the Government of the United States of America has certain rights in the invention. TECHNICAL FIELD The present disclosure relates to increasing protein content in a eukaryotic cell, increasing a plant's resistance to stress, such as abiotic (e.g., salt, drought, and pollution) or biotic (e.g., pathogens and pests) stress, a promoter with a modified transcriptional repressor binding site, such that the transcriptional repressor cannot prevent transcription of the gene, NF-YC4, QQS, TALENS, CRISPR/Cas9, tissue culture, crossing and backcrossing plants, hybrid plants, regenerable cells, and seeds. BACKGROUND Protein deficiency is a major health problem throughout the developing world. Low protein intake contributes to mental retardation, stunting, susceptibility to disease, wasting diseases and/or death in hundreds of millions of children each year (Forrester et al., PloS one 7: e35907 (2012); Gomes et al., J. Neuroscience Res. 87: 3568-3575 (2009)). Plants provide over 60% of human dietary protein (Young et al., Am. J. Clin. Nutr. 59: 1203S-1212S (1994)). Increasing the protein content of staple crops could help alleviate protein deficiency, particularly when the use of animals requires about 100 times more water and 11 times more energy to produce an equivalent amount of protein (Pimentel et al., Am. J. Clin. Nutr. 78: 6605-6635 (2003)) and increasing the protein content of animals is often accompanied by a decrease in protein quality or yield (Bellaloui et al., Agricultural Sciences 1: 110-118 (2010); Wenefrida et al., J. Agricultural Food Chem. 61: 11702-11710 (2013)). The Arabidopsis thaliana QQS (At3g30720, Qua-Quine Starch) orphan gene modulates protein content in Arabidopsis. Arabidopsis thaliana starch synthase 3 (Atss3) mutants, which are morphologically similar to wild-type (WT) control lines but differ in starch levels (Zhang et al., Plant Physiol. 138: 663-674 (2005)), have more than five-fold the amount of QQS transcripts found in WT (Li et al., Plant J. 58: 485-498 (2009)). Over-expression of QQS in Arabidopsis increases total protein content and decreases total starch content in leaves, while down-regulation of QQS has the converse effect (Li et al., Plant Biotech. J. 13: 177-187 (2015); Li et al. (2009), supra). Expression of QQS as a transgene increases protein content in other plants, such as soybean (var. Williams 82; Li et al. (2014), supra; Li et al. (2009), supra). QQS expression has been observed to be tightly linked with a variety of developmental, environmental, and genetic perturbations (see, e.g., Arendsee et al., Trends in Plant Sci doi:10.1016/j.tplants.2014.07.003 (2014); Li et al. (2009), supra; and Li et al. (2015), supra). Its role, however, in such perturbations has not been elucidated. For example, PEN3 (Penetration Resistance 3 (At1g59870, PEN3, ABC binding cassette transporter gene) confers non-host resistance to fungal and oomycete pathogens. QQS has been reported to be the only gene that is up-regulated in pen3 knock-out (KO) mutants; however, QQS is up-regulated in infected and non-infected mutants (Stein et al., Plant Cell 18(3): 731-746 (2006)). As another example, two syntaxins, namely SYP121 (At3g11820, PEN1) and SYP122 (At3g52400) confer resistance to powdery mildews. Knock-outs of these genes result in increased sensitivity to these pathogens; QQS has been reported to be the only gene that is up-regulated in both (Zhang et al. (2008)). In contrast, while PEN3 and EXL1 are up-regulated following exposure to some pathogens, QQS is down-regulated in response to infection by some pathogens, such as Pseudomonas syringae (Kwon et al., Planta 236(3): 887-900 (2012); and Thilmony et al., Plant J. 46(1): 34-53 (2006)). When Arabidopsis plants were inoculated with Phytopthera infestans, QQS reportedly was first down-regulated at 6 hrs post-inoculation and then up-regulated at 12 and 24 hrs post-inoculation (Scheel et al., Experiment ID "E-GEOD-5616" in ArrayExpress). Thus, in view of the above, it is an object of the present disclosure to identify a gene with which QQS interacts. In Arabidopsis QQS interacts with nuclear factor Y, subunit C4 (NF-YC4, At5g63470). It is another object to provide materials and methods for manipulating a gene so identified. In an embodiment, the manipulation of such a gene results in increased protein content and/or decreased carbohydrate content. It is yet another object to provide materials and a method for increasing a plant's resistance to a pathogen or a pest. These and other objects, as well as inventive features, will be apparent from the detailed description provided herein. SUMMARY A method of increasing protein content in a eukaryotic cell comprising an NF-YC4 gene, which comprises a promoter comprising a transcrip