Modification of transcriptional repressor binding site in NF-YC4 promoter for increased protein content and resistance to stress

Patent metadata

Field	Value
Publication number	US-12559767-B2
Application number	US-202418679743-A
Country	US
Kind code	B2
Filing date	May 31, 2024
Priority date	Feb 18, 2015
Publication date	Feb 24, 2026
Grant date	Feb 24, 2026

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.

First claim

What is claimed is:

1 . A genetically modified plant, or a part from the genetically modified plant, comprising a genetically modified endogenous nuclear factor Y subunit C4 (NF-YC4) promoter operably linked to an endogenous NF-YC4 protein coding sequence, wherein an agent for modifying a nucleotide sequence of said NF-YC4 promoter, modifies at least one transcriptional repressor binding site in the nucleotide sequence of said NF-YC4 promoter, and whereupon the transcriptional repressor cannot prevent transcription of the NF-YC4 coding sequence and thus the modified endogenous NF-YC4 promoter increases the transcription of the endogenous NF-YC4 protein coding sequence in the genetically modified plant or the part from the genetically modified plant, and wherein said genetically modified plant or said part from the genetically modified plant that was modified to increase NF-YC4 expression was selected for increased total protein content due to overexpression of said endogenous NF-YC4 protein as compared to the total protein content in the corresponding wild-type (WT) plant or the corresponding WT part thereof of the same plant species lacking said modified NF-YC4 promoter, and wherein said genetically modified plant, or said part from the genetically modified plant, is non-naturally occurring.

2 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the agent for modifying the NF-YC4 promoter is a zinc-finger nuclease (ZFN) protein, a transcription activator-like effector nuclease (TALEN) protein, an RNA-guided, engineered nuclease (RGENs) complex, a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas complex, a chemical mutagen, or a nucleic acid construct comprising a ZFN protein coding region, a TALEN protein coding region, an RGEN protein coding region and RNA guide sequence, or a Cas protein coding region and a guide sequence.

3 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the transcriptional repressor binding site is modified by a deletion.

4 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the transcriptional repressor binding site is modified by one or more nucleotide substitutions.

5 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the transcriptional repressor binding site comprises an ERF motif, a RAV1 motif, or both an ERF motif and a RAV1 motif.

6 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the genetically modified plant or the part from the genetically modified plant is rice and wherein said transcriptional repressor binding site has (i) two ERF motifs are deleted, (ii) RAV1 motif is deleted, or (iii) a RAV1 motif and an ERF motif are deleted.

7 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the genetically modified plant or the part from the genetically modified plant is soybean and wherein said transcriptional repressor binding site has either (i) two RAV1 motifs are deleted or (ii) a RAV1 motif and an ERF motif are deleted.

8 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the total protein content is increased in the leaves or the seeds of the genetically modified plant or the part from the genetically modified plant.

9 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the genetically modified plant or the part from the genetically modified plant is a crop plant.

10 . The genetically modified plant, or the part from the genetically modified plant, of claim 9 , wherein the crop plant is soybean, rice, or corn.

11 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the genetically modified plant or the part from the genetically modified plant is a monocot.

12 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the genetically modified plant or the part from the genetically modified plant is a dicot.

13 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the plant is Glycine max , and wherein the NF-YC4 promoter of the endogenous NF-YC4 protein coding sequence comprises SEQ ID NO: 8.

14 . The genetically modified plant, or the part from the genetically modified plant, of claim 9 , wherein the crop plant is selected from the group consisting of corn, soybean, alfalfa, canola, wheat, rice, potato and lettuce.

15 . The genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein the NF-YC4 protein is selected from the group consisting of Brassica napus NF-YC4 protein, Medicago truncatula NF-Y C4 protein, Arabidopsis thaliana NF-YC4 protein, Glycine max NF-YC4 protein, Oryza sativa NF-YC4 protein, and Zea mays NF-YC4 protein.

16 . A genetically modified plant progeny obtained from the genetically modified plant, or the part from the genetically modified plant, of claim 1 , wherein said genetically modified plant progeny comprises said genetically modified endogenous NF-YC4 promoter and exhibits increasing protein content as compared to a corresponding wild-type control plant of the same plant species lacking said modified NF-YC4 promoter.

Assignees

• Univ Iowa State Res Found Inc

Inventors

• Li Ling
• Wurtele Eve Syrkin

Classifications

• C12N15/8279

  for biotic stress resistance, pathogen resistance, disease resistance · CPC title

• C12N15/8216

  Methods for controlling, regulating or enhancing expression of transgenes in plant cells · CPC title

• C12N15/8205

  Agrobacterium mediated transformation · CPC title

• C12N15/8241

  Phenotypically and genetically modified plants via recombinant DNA technology · CPC title

• C12N15/8247

  involving modified lipid metabolism, e.g. seed oil composition · CPC title