Targeted gene demethylation in plants

Abstract

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 modulating the expression of genes in plants.

1 - 38 . (canceled) 
     
     
         39 . A method for reducing methylation of a target nucleic acid in a plant, comprising:
 (a) providing a plant comprising a recombinant methylcytosine dioxygenase polypeptide that comprises the amino acid sequence of SEQ ID NO: 189 and that is capable of being targeted to a target nucleic acid; and   (b) growing the plant under conditions whereby the recombinant polypeptide is targeted to the target nucleic acid, thereby reducing methylation of the target nucleic acid.   
     
     
         40 . The method of  claim 39 , wherein the recombinant methylcytosine dioxygenase polypeptide is targeted to the target nucleic acid via a SunTag targeting system. 
     
     
         41 - 45 : (canceled) 
     
     
         46 . The method of  claim 39 , wherein the recombinant methylcytosine dioxygenase polypeptide is targeted to the target nucleic acid via a DNA-binding domain. 
     
     
         47 . The method of  claim 39 , wherein the methylcytosine dioxygenase polypeptide is a TET polypeptide. 
     
     
         48 . The method of  claim 47 , wherein the TET polypeptide is a TET1 polypeptide 
     
     
         49 . The method of  claim 48 , wherein the TET1 polypeptide comprises the catalytic domain of TET1. 
     
     
         50 . The method of  claim 49 , wherein the TET1 polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 8. 
     
     
         51 - 52 . (canceled) 
     
     
         53 . The method of  claim 39 , wherein expression of the target nucleic acid is activated as compared to a corresponding control nucleic acid. 
     
     
         54 . A recombinant nucleic acid comprising a plant promoter and which encodes a recombinant polypeptide comprising a DNA-binding domain and a methylcytosine dioxygenase polypeptide that comprises the amino acid sequence of SEQ ID NO: 189. 
     
     
         55 . An expression vector comprising the recombinant nucleic acid of  claim 54 . 
     
     
         56 . A host cell comprising the expression vector of  claim 55 . 
     
     
         57 . A recombinant plant comprising the recombinant nucleic acid of  claim 54 . 
     
     
         58 . A plant having reduced methylation of a target nucleic acid as a consequence of the method of  claim 39 . 
     
     
         59 . A progeny plant of the plant of  claim 58 . 
     
     
         60 . The progeny plant of  claim 59 , wherein the progeny plant has reduced methylation of the target nucleic acid and does not comprise the recombinant methylcytosine dioxygenase polypeptide. 
     
     
         61 . (canceled) 
     
     
         62 . A recombinant vector comprising:
 a first nucleic acid sequence comprising a plant promoter and that encodes a recombinant polypeptide comprising a nuclease-deficient CAS9 polypeptide (dCAS9) or fragment thereof and a multimerized epitope;   a second nucleic acid sequence comprising a plant promoter and that encodes a recombinant polypeptide comprising a methylcytosine dioxygenase polypeptide that comprises the amino acid sequence of SEQ ID NO: 189, and an affinity polypeptide that specifically binds to the epitope; and   a third nucleic acid sequence comprising a promoter and that encodes a crRNA and a tracrRNA, or fusions thereof.   
     
     
         63 . A host cell comprising the vector of  claim 62 . 
     
     
         64 . A recombinant plant comprising the vector of  claim 63 . 
     
     
         65 - 67 . (canceled)