Recovery of RNA-dependent RNA polymerase 6 gene-knockout phenotypes in Nicotiana benthamiana via in vivo generation of inverted repeat construct of the trans-acting short interference RNA3 sequence

Pharmaceutical recombinant proteins are widely produced using plants; however, several challenges such as low productivity limit their application. To overcome these problems, RNA-dependent RNA polymerase 6-knockout Nicotiana benthamiana plants (rdr6 plants) were previously produced for the mass production of recombinant proteins. These rdr6 plants produced higher amounts of recombinant proteins than wild-type plants, but their practical use for recombinant protein production is limited by their sterility and small leaf size. As an essential component of the microRNA 390-trans-acting short interference RNA3-auxin response factor (miR390-TAS3-ARF) pathway, rdr6 modulates leaf morphology, flower architecture, and embryo development. In fact, the expression levels of ARF3 and 4 genes differed in leaves, flowers, and buds of wild-type and rdr6 plants. Therefore, a dysfunctional miR390-TAS3-ARF pathway might be responsible for the sterility and small leaf size of the rdr6 plants. Herein, to overcome the disadvantages of rdr6 plants while maintaining the high productivity of recombinant proteins, the miR390-TAS3-ARF pathway was reconstructed. Transgenic rdr6 plants expressing the inverted repeat constructs of the TAS3 sequence of N. benthamiana were successfully produced (TAS3i plants). These plants produced seeds and large leaves, like the wild-type plants, while maintaining a high productivity of recombinant proteins, like the rdr6 plants. Thus, the TAS3i plants can be considered useful for producing recombinant proteins. This is the first report that the miR390-TAS3-ARF pathway can function in plants in the absence of RDR6.