Methods and compositions for targeting RNA polymerases and non-coding RNA biogenesis to specific loci
The present disclosure relates to the use of recombinant proteins for inducing epigenetic modifications at specific loci, as well as to methods of using these recombinant proteins for reducing the expression of genes in plants.
1. A method for reducing expression of a target nucleic acid in a plant, comprising:
(a) providing a plant comprising a recombinant nucleic acid, wherein the recombinant nucleic acid encodes a recombinant polypeptide comprising a first amino acid sequence comprising a DNA-binding domain capable of binding a target nucleic acid and a second amino acid sequence comprising a DNA methyltransferase polypeptide selected from the group consisting of an SssI polypeptide comprising an amino acid sequence having at least 90% amino acid identity to SEQ ID NO: 680 and a DRM2 polypeptide comprising an amino acid sequence having at least 90% amino acid identity to SEQ ID NO: 679; and
(b) growing the plant under conditions whereby the recombinant polypeptide encoded by the recombinant nucleic acid is expressed and binds to the target nucleic acid, thereby reducing expression of the target nucleic acid, wherein the target nucleic acid exhibits a specific increase in methylation over 5 to 3,000 nucleotides 5′ and/or 3′ to the target nucleotide sequence in the nucleic acid as compared to a corresponding control nucleic acid.
2. The method of claim 1 , wherein the DNA-binding domain comprises a zinc finger domain.
3. The method of claim 1 , wherein the DNA methyltransferase polypeptide is an SssI polypeptide, and wherein said polypeptide comprises an amino acid sequence having at least 95% amino acid identity to SEQ ID NO: 680.
4. The method of claim 1 , wherein the DNA methyltransferase polypeptide is a DRM2 polypeptide, and wherein said polypeptide comprises an amino acid sequence having at least 95% amino acid identity to SEQ ID NO: 679.
5. The method of claim 1 , wherein the target nucleic acid is an endogenous nucleic acid.
6. The method of claim 1 , wherein the target nucleic acid is a heterologous nucleic acid.
7. The method of claim 1 , wherein expression of the target nucleic acid is silenced.
8. A plant cell comprising a recombinant polypeptide comprising a first amino acid sequence comprising a DNA-binding domain and a second amino acid sequence comprising a DNA methyltransferase polypeptide selected from the group consisting of an SssI polypeptide comprising an amino acid sequence having at least 90% amino acid identity to SEQ ID NO: 680 and a DRM2 polypeptide comprising an amino acid sequence having at least 90% amino acid identity to SEQ ID NO: 679, wherein the plant cell comprises a nucleic acid with reduced expression and a specific increase in methylation over 5 to 3,000 nucleotides 5′ and/or 3′ to a target nucleotide sequence in the nucleic acid as compared to a corresponding control nucleic acid.
9. A recombinant plant comprising the plant cell of claim 8 .