Gene silencing

A method of inhibiting expression of a target sequence in a plant or plant cell comprising:

• (a) introducing into the plant or plant cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified full-length plant miRNA precursor, wherein the full-length plant miRNA precursor has been modified in a backside region or a miRNA template region, or both, as follows:
• (1) the backside region consists of a first oligonucleotide of 21 contiguous nucleotides, wherein the first oligonucleotide is either fully complementary to a second oligonucleotide of 21 contiguous nucleotides or has 1, 2, 3, 4, or 5 mismatches, and
• (2) the miRNA template region consists of the second oligonucleotide of 21 contiguous nucleotides, wherein the second oligonucleotide encodes a miRNA, and wherein the second oligonucleotide is either fully complementary to the target sequence or has 1, 2, 3, 4, or 5 mismatches, and
• wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the precursor is capable of forming a hairpin, wherein the precursor has only been modified in the backside region, the miRNA template region, or both, and optionally by addition of a restriction site recognition sequence or a recombination sequence flanking the hairpin;
• and
• (b) expressing the nucleic acid construct for a time sufficient to produce the miRNA, wherein the miRNA inhibits expression of the target sequence.