Simultaneous gene silencing and suppressing gene silencing in ihe same cell

A process for selecting a plant cell with a desired property resulting from an increased level of an RNA of interest and/or amount of protein encoded by the RNA of interest, and a reduced level of target RNA encoded by a first polynucleotide of interest and/or amount of the protein encoded by the target RNA, the process comprising:

• i) obtaining one or more plant cells comprising,
• (a) a polynucleotide of interest which encodes a target RNA,
• b) a first exogenous polynucleotide encoding a double stranded RNA (dsRNA) molecule which comprises a stem-loop and a first nucleotide sequence which is complementary to a region of the target RNA encoded by the polynucleotide of interest,
• c) a second exogenous polynucleotide encoding a candidate silencing suppressor polypeptide,
• d) a third exogenous polynucleotide, different to the first and second exogenous polynucleotides and the polynucleotide of interest, which encodes an RNA of interest, and
• e) a reduced level of the target RNA encoded by the polynucleotide of interest and/or the amount of a protein encoded by the target RNA when compared to a corresponding cell lacking the first exogenous polynucleotide,
• wherein each exogenous polynucleotide is operably linked to one or more promoters that are capable of directing expression of the polynucleotide in the cell, wherein the first and second exogenous polynucleotides form part of one DNA construct, and wherein the candidate silencing suppressor polypeptide comprises amino acids having the sequence set forth in any one of SEQ ID NOs 39 to 51, or a sequence which is at least 95% identical to the sequence set forth in any one of SEQ ID NOs 39 to 51,
• ii) analysing the cell(s) for the desired property,
• iii) if the cell(s) does not have the desired property, substituting the second exogenous polynucleotide encoding the candidate silencing suppressor polypeptide with a second exogenous polynucleotide encoding a different candidate silencing suppressor polypeptide comprising amino acids having the sequence set forth in any one of SEQ ID NOs 1 or 39 to 51, or having a sequence which is at least 95% identical to the sequence set forth in any one of SEQ ID NOs 1 or 39 to 51, and analysing the resultant cell(s) for the desired property,
• iv) if necessary, repeating step iii) until the desired property is obtained, and
• v) selecting a plant cell with the desired property.