Production and stably maintenance of strigolactone by transient expression of biosynthetic enzymes in Nicotiana benthamiana.

Strigolactones (SLs) are phytohormones that play an essential role in plant-microbe interactions. The instability of SLs makes it challenging to use them for application to agriculture. In this study, we successfully produced a large amount of the 4-deoxyorobanchol (4DO), one of SLs, in the leaves of Nicotiana benthamiana, using a transient expression system to express SL biosynthetic enzymes. Using this system, the yield of 4DO was 2.1 ± 0.3 µg/gFM (fresh mass). Treatment of leaves at 80°C for 16 h killed Agrobacterium and approximately half amount of 4DO was left in the leaves (1.0 µg/gFM (calculated based on the original FM) ± 0.3). Interestingly, incubation of dried leaves at room temperature for 1 month maintained an almost equal amount of 4DO (0.9 ± 0.2 µg/gFM) in the leaves. These results suggest that high accumulation of 4DO with stability for long periods can be achieved in plant leaves.

High-Yield Production of the Major Birch Pollen Allergen Bet v 1 With Allergen Immunogenicity in Nicotiana benthamiana.

Type I allergy is an immunological disorder triggered by allergens and causes significant health problems. The major allergen of birch pollen is Bet v 1, which belongs to the pathogen-related protein 10 (PR-10) family. Here, we established a rapid and robust method for the production of Bet v 1 in Nicotiana benthamiana leaves, with binding activity to allergic patients' IgE. The Bet v 1 allergen was expressed in N. benthamiana using a strong agroinfiltration-based transient protein expression system, which consists of a deconstructed geminiviral vector system with a double terminator. Five days post-infiltration, the allergen concentration in N. benthamiana leaves was 1.2 mg/g of fresh mass, being this the maximum yield of Bet v 1 in plants reported up to now. A part of plant-derived Bet v 1 was glycosylated. Bet v 1 purified from N. benthamiana or Brevibacillus brevis was used to carry out enzyme-linked immunoassays; both recombinant allergens were found to have comparable binding properties to the IgE of allergic patients. These results suggest that our plant expression system allows rapid and robust production of the allergen, which keeps the immunogenicity.