Patent ID: 8729339

Claim:
A method of inhibiting expression of a target sequence in a plant cell comprising: (a) introducing into the plant cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified SEQ ID NO:1, wherein the polynucleotide comprises in the following order: (i) a first heterologous sequence, (ii) the modified SEQ ID NO:1, wherein SEQ ID NO:1 has been modified only 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 (iii) a second heterologous sequence, wherein at least one of the first and the second oligonucleotide is heterologous to SEQ ID NO:1, and further wherein the polynucleotide encodes an RNA precursor capable of forming a 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.