Robust Agrobacterium-Mediated Transient Expression in Two Duckweed Species (Lemnaceae) Directed by Non-replicating, Replicating, and Cell-to-Cell Spreading Vectors.

Anatoli Giritch,Olena Kishchenko,Yuzhen Zhou,Anton Peterson,Maksym Vasylenko,Nikolai Borisjuk,Jian Sun,Mykola Kuchuk

doi:10.3389/fbioe.2021.761073

Abstract

Plant-based transient expression systems have recognized potential for use as rapid and cost-effective alternatives to expression systems based on bacteria, yeast, insect, or mammalian cells. The free-floating aquatic plants of the Lemnaceae family (duckweed) have compact architecture and can be vegetatively propagated on low-cost nutrient solutions in aseptic conditions. These features provide an economically feasible opportunity for duckweed-based production of high-value products via transient expression of recombinant products in fully contained, controlled, aseptic and bio-safe conditions in accordance with the requirements for pharmaceutical manufacturing and environmental biosafety. Here, we demonstrated Agrobacterium-mediated high-yield transient expression of a reporter green fluorescent protein using deconstructed vectors based on potato virus X and sweet potato leaf curl virus, as well as conventional binary vectors, in two representatives of the Lemnaceae (Spirodela polyrhiza and Landoltia punctata). Aseptically cultivated duckweed populations yielded reporter protein accumulation of >1 mg/g fresh biomass, when the protein was expressed from a deconstructed potato virus X-based vector, which is capable of replication and cell-to-cell movement of the replicons in duckweed. The expression efficiency demonstrated here places duckweed among the most efficient host organisms for plant-based transient expression systems, with the additional benefits of easy scale-up and full containment.

Highlights

Plant-based transient expression systems are widely used for studying gene and protein functions, metabolic engineering, and biosynthesis of valuable recombinant products (Reed et al, 2017; Tusé et al, 2020)
To test the possibility of high-efficiency transient expression in duckweed (S. polyrhiza and L. punctata), we used a range of expression vectors carrying a reporter gene encoding green fluorescent protein (GFP)
N. benthamiana, a well-documented host plant for transient expression, was used as a positive control for 35S-GFP-p19 and potato virus X (PVX)-GFP vectors, and L. sativa was used as a positive control for the Ubi-GFP, 35SGFP, and sweet potato leaf curl virus (SPLCV)-GFP vectors

Summary

Introduction

Plant-based transient expression systems are widely used for studying gene and protein functions, metabolic engineering, and biosynthesis of valuable recombinant products (Reed et al, 2017; Tusé et al, 2020). RNA and DNA viruses have been used to generate plant expression vectors (Marillonnet et al, 2005; Lindbo, 2007; Chen et al, 2011; Peyret and Lomonossoff, 2013; Yu et al, 2020; Torti et al, 2021). The PVX-based deconstructed vectors that encode MPs and modified CPs are replicating vectors that can move from cell to cell and systematically These vectors are used for Agrobacterium-mediated transient expression in a number of dicotyledonous plant species (Giritch et al, 2006; Larsen and Curtis, 2012; Schulz et al, 2015; Mardanova et al, 2017; Torti et al, 2021). Geminivirus-based deconstructed vectors are replicating vectors but do not undergo cell-to-cell movement

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Conclusion