The National Institute of Advanced Industrial Science and Technology (AIST) announced on October 1 that Senior Researcher Kouki Matsuo at the Biomanufacturing Process Research Center has successfully developed plants capable of producing high levels of functional proteins while maintaining normal growth without dwarfing by suppressing the plant RNA silencing mechanism. Seeds that inherited these traits and germinated normally were also obtained from the developed plants. This breakthrough is expected to enable mass production of functional proteins using plants. The results were published in The Plant Journal on July 18.
Methods for efficiently producing functional proteins such as vaccine antigens and enzymes through genetic introduction into microorganisms are widely used in pharmaceuticals and other fields, but there are potential risks of pathogen or viral infiltration. In contrast, technology for producing functional proteins in plants is expected to be commercialized from the perspective of safety and cost.
However, plants possess an "RNA silencing mechanism" that degrades messenger RNA derived from foreign genes to protect themselves from external threats such as viruses, which has posed challenges for expression efficiency. Higher expression levels and larger plant sizes result in greater production of functional proteins.
Previously, AIST had successfully developed rdr6 plants using tobacco, which significantly improved both gene introduction efficiency and production of functional proteins by disrupting the RDR6 gene. The RDR6 gene plays a central role in synthesizing double-stranded RNA in the RNA silencing mechanism. However, these plants were dwarfed compared with wild-type plants and did not form seeds.
In this study, Matsuo focused on "TAS3," a DNA sequence closely related to the RDR6 gene that is involved in plant morphogenesis.
The RDR6 gene plays an important role in the "miR390-TAS3-ARF pathway," which regulates the expression of ARF genes (auxin response factor genes) essential for controlling organ formation in plant leaves and flowers. This pathway regulates ARF gene expression through multiple steps, but because RDR6 is an essential enzyme gene in the middle of this pathway, rdr6 plants cannot properly regulate ARF gene expression.
Therefore, Matsuo introduced a new mechanism that generates double-stranded RNA derived from the TAS3 sequence. He verified the restoration of pathway function by generating double-stranded RNA in rdr6 plants.
Specifically, he created TAS3i plants by introducing into rdr6 plants a vector in which the TAS3 DNA sequence was arranged in opposite directions with a spacer sequence in between (TAS3 sequence - spacer sequence - inverted TAS3 sequence). He anticipated that even without RDR6 function, double-stranded RNA of TAS3 would be generated through the spacer region forming a hairpin-like loop structure.
As a result, expression of multiple ARF genes increased in TAS3i plants. Growth of the entire plant including leaf size recovered to nearly wild-type levels, no morphological abnormalities were observed, and seeds were obtained.
When transient expression of green fluorescent protein (GFP) was performed in wild-type, rdr6, and TAS3i plants, stronger GFP fluorescence was observed in rdr6 and TAS3i plants compared with wild-type, confirming mass production of GFP.
Furthermore, more than 90% of the seeds obtained germinated normally, confirming that the functions and properties were inherited. Similar manipulations using non-tabacco plants are expected to be applicable.
Anticipating its use for the production of functional proteins such as cytokines and considering that genetic introduction has been performed and variations in cultivation conditions affect the quality of the functional proteins produced, Matsuo envisions utilization in closed-system facilities with stable cultivation conditions.
Acceleration of social implementation and dissemination needed
Matsuo commented: "While commercial use of recombinant protein production using plants is advancing overseas, its application has been slow in Japan. Since the use of plant-produced recombinant proteins is expected to reduce costs in fields such as regenerative medicine and cultured meat, we believe it is necessary to advance technological development while accelerating social implementation and dissemination."
Journal Information
Publication: The Plant Journal
Title: 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
DOI: 10.1111/tpj.70350
This article has been translated by JST with permission from The Science News Ltd. (https://sci-news.co.jp/). Unauthorized reproduction of the article and photographs is prohibited.