Direct protein delivery into intact Arabidopsis cells for genome engineering.

Intracellular delivery of biomolecules is a prerequisite for genetic techniques such as recombinant engineering and genome editing. Realizing the full potential of this technology requires the development of safe and effective methods for delivering protein tools into cells. In this study, we demonstrated the spontaneous internalization of exogenous proteins into intact cells and root tissue of whole plants of Arabidopsis thaliana. We termed this internalization phenomenon as protein Delivery Independent of Vehicles or Equipment (DIVE), which efficiently delivered genome engineering proteins including Cre recombinase and zinc-finger nucleases (ZFN) into plant cells. Using protein DIVE, we achieved less toxic protein delivery than electroporation with up to 94% efficiency in Arabidopsis cell culture and 19% genome modification in Arabidopsis plants that was maintained in regenerated tissue. This work illustrates the potential of protein DIVE for a wide range of applications, including genome engineering in plants.

A Method for Electroporation of Cre Recombinase Protein into Intact Nicotiana tabacum Cells.

The Cre/lox recombination system has become a powerful technology for gene function analysis in a broad spectrum of cell types and organisms. In our previous report, Cre protein had been successfully delivered into intact Arabidopsis thaliana cells using electroporation. To expand the feasibility of the method of protein electroporation to other plant cells, here we attempt the protein electroporation into tobacco-derived BY-2 cells, one of the most frequently used plant cell lines for industrial production. In this study, we successfully deliver Cre protein into BY-2 cells with intact cell walls by electroporation with low toxicity. Targeted loxP sequences in the BY-2 genome are recombined significantly. These results provide useful information for genome engineering in diverse plant cells possessing various types of cell walls.

Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system.

Bioluminescent detection has become a powerful method that is used extensively in numerous areas in life science research. Given that fluorescence detection in plant cells is difficult owing to the autofluorescence of chlorophyll, the use of a luciferin-luciferase system should be effective in plant biology. However, the suitable optical window for a luminescence system in plants remains unexplored. In this study, we sought to determine the optical window and optimal luciferase reporter system for terrestrial plant analyses using Arabidopsis thaliana as a model organism. We compared six different luciferase systems and found the green enhanced Nano-lantern (GeNL)-furimazine combination to be the optimal luciferase reporter. Spectral measurements of GeNL-furimazine showed that its luminescence peak falls within the range of optical transparency for chlorophyll and, therefore, enables greater penetration through a layer of cultured A. thaliana cells. Moreover, A. thaliana plants expressing GeNL with furimazine emitted strong luminescence, which could be detected even with the naked eye. Thus, the GeNL-furimazine combination should facilitate biological analyses of genes and cellular functions in A. thaliana and all other terrestrial plants.

A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall.

Genome engineering in plants is highly dependent on the availability of effective molecular techniques. Despite vast quantities of research, genome engineering in plants is still limited in terms of gene delivery, which requires the use of infectious bacteria or harsh conditions owing to the difficulty delivering biomaterial into plant cells through the cell wall. Here, we describe a method that uses electroporation-mediated protein delivery into cultured Arabidopsis thaliana cells possessing an intact cell wall, and demonstrate Cre-mediated site-specific recombination. By optimizing conditions for the electric pulse, protein concentration, and electroporation buffer, we were able to achieve efficient and less-toxic protein delivery into Arabidopsis thaliana cells with 83% efficiency despite the cell wall. To the best of our knowledge, this is the first report demonstrating the electroporation-mediated protein delivery of Cre recombinase to achieve nucleic acid-free genome engineering in plant cells possessing an intact cell wall.