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

Abstract

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.

Keywords: Nicotiana benthamiana; RNA‐dependent RNA polymerase 6; TAS3; auxin response factor; miR390; transient expression; trans‐acting short interference RNA.

Figure 1

Schematic view of the miR390‐TAS3‐ARF pathway in the wild‐type, rdr6, and TAS3i plants. In the rdr6 plants, the function of RDR6 is lost, impairing the negative regulation of the ARF genes. In the TAS3i plants, the function of RDR6 is lost; the double‐stranded RNA of the NbTAS3 sequence is supplied from the NbTAS3 inverted repeat expression cassette. Thus, the negative regulation of ARF genes could be recovered.

Figure 2

Alignment of the NtTAS3 and putative NbTAS3 sequences. Nucleotide sequences of ta‐siARF loci from Nicotiana benthamiana (Niben101Scf07184Ctg012:211877‐212 317). The cloned NbTAS3 and previously reported TAS3 sequences for N. tabacum (NCBI accession number FJ804751) were aligned and compared. The putative miR390 and ta‐siARF sequences, ta‐siARF (U, upstream) and ta‐siARF (D, downstream), are indicated by the dotted and black lines, respectively.

Figure 3

The phenotypes of the TAS3i and rdr6 plants at 58 days after seeding. Leaves (a) and flowers (b and c) were collected from the respective plants. The whole plant bodies were photographed from the side (d) and above (e).

Figure 4

Transient expression of GFP and human FGF1 in WT, TAS3i, and rdr6 plants. GFP and human FGF1 were transiently expressed in WT, TAS3i, and rdr6 plants, and the infiltrated leaves were harvested at 5 dpi. (a) The relative expression level of GFP mRNA was estimated by real‐time RT‐PCR using NAD(P)H dehydrogenase (quinone) (NQO) as the housekeeping gene. (b) The amount of GFP protein was estimated using SDS‐PAGE followed by Coomassie blue staining. (c) Relative GFP fluorescence was measured by 20‐fold diluting the centrifuged supernatant of the crude extract (prepared in PBS at five times the volume of the leaves) from GFP‐expressing leaves. (d) The relative expression level of FGF1 mRNA was estimated by real‐time RT‐PCR using NQO as the housekeeping gene. (e) Relative band intensities of FGF1 were estimated from Western blot analysis. Data are shown as mean ± standard deviation (n = 3). t‐test; *P < 0.05; n.s., not significant.