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

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.

Keywords: duckweed; expression vector; plant expression system; recombinant proteins; transient expression.

FIGURE 1

Duckweed species and vector constructs used in research: (A) Spirodela polyrhiza fronds and turions (white arrows show turions), bar = 1 cm. (B) Landoltia punctata fronds, bar = 1 cm. (C) Schematic representation of the T-DNA of vectors: Ubi prom–the promoter of the N. tabacum Ubiquitin4 U4 gene, mGFP5—coding sequence of modified GFP, sGFP–coding sequence of synthetic GFP, HSP term–the transcription terminator of the A. thaliana HEAT SHOCK PROTEIN 18.2 gene, 35S prom–the сauliflower mosaic virus 35S promoter, AtADH 5′-UTR–translational enhancer from the 5′-UTR of the A. thaliana ALCOHOL DEHYDROGENASE gene, AC1, AC2, AC3 and AC4–coding regions of SPLCV replication-associated proteins, IR - SPLCV intergenic region, p19—the tomato bushy stunt virus suppressor of gene silencing, Ω–the tobacco mosaic virus translational enhancer of 5′-leader sequence, NOS term–the transcription terminator of the A. tumefacience NOPALINE SYNTHASE gene, OCS term–the transcription terminator of the A. tumefacience OCTOPINE SYNTHASE gene, 8, 12, 25 K–PVX triple gene block movement proteins, PVX term–the PVX transcription terminator, CP–coding sequence of the PVX coat protein, PVX-pol–PVX RNA-dependent RNA polymerase gene, LB, RB–left and right borders of the T-DNA.

FIGURE 2

Duckweed transiently expressing GFP from the non-replicating vectors 35S-GFP-p19 and 35S-GFP and the replicating vector SPLCV-GFP. (A,D,G) The appearance of duckweed populations infiltrated with Agrobacterium bearing the 35S-GFP-p19 (A), 35S-GFP (D), and SPLCV-GFP vectors (G). (B,C) Selected fronds expressing GFP from S. polyrhiza population infiltrated with Agrobacterium bearing the 35S-GFP-p19 vector, 10 dpi. (E,F) Selected fronds expressing GFP from L. punctata population infiltrated with Agrobacterium bearing the 35S-GFP vector, 10 dpi. (H,I) Selected fronds expressing GFP from L. punctata population infiltrated with Agrobacterium bearing the SPLCV-GFP vector, 30 dpi. (C,F,I) Images were taken under artificial white light. (A,B) Images were taken under monochrome excitation light (400 nm) through a long wave pass light filter (450 nm). (D,E,G,H) Images were taken under monochrome excitation light (488 nm) through a long wave pass light filter (520 nm). Bar = 1 cm.

FIGURE 3

S. polyrhiza fronds and turions transiently expressing GFP from the PVX-GFP vector. (A) The appearance of fronds population maintained in liquid medium after agro-infiltration, 30 dpi. (B)–(G) The dynamics of GFP fluorescence in agro-infiltrated fronds maintained on solid medium at 8 dpi (B,C), 11 dpi (D,E), and 20 dpi (F,G). (H,I) Air-facing side of the turion. (J,K) Waterfacing side of the turion. (A,B,D,F,H,J) Images were taken under monochrome excitation light (400 nm) through a long wave pass light filter (450 nm). (C,E,G,I,K) Images were taken under artificial white light. (A–G) Bar = 1 cm, (H–K) Bar = 1 mm.

FIGURE 4

Analysis of GFP accumulation in duckweed biomass. (A,B) SDS-PAGE analysis of protein extracts from S. polyrhiza infiltrated with Agrobacterium bearing the PVX-GFP vector. (A) Representative image of the gel before staining; image was taken using artificial white light combined with light from a 365 nm LED without any long wave pass light filters. (B) Image of the gel shown in (A) after staining with Coomassie dye. (C) GFP accumulation in duckweed population infiltrated with Agrobacterium bearing different vectors (duckweed/vector): 1– L. punctata/Ubi-GFP vector (no accumulation), 10–12 dpi; 2 - L. punctata/35S-GFP vector, 10–12 dpi; 3– L. punctata/SPLCV-GFP vector, 25–30 dpi; 4– S. polyrhiza/35S-GFP-p19 vector, 10–12 dpi; 5– S. polyrhiza/PVX-GFP vector, 20–25 dpi. White arrows show gel notches marking the positions of the GFP fluorescence bands; black arrows show the positions of the 29 kDa bands of the protein molecular weight marker; black triangles indicate the position of large subunit RuBisCO. M—protein molecular weight marker. WT—extracts from S. polyrhiza not infiltrated by Agrobacterium. Error bars indicate doubled standard deviations (n = 3). Values with different letters are significantly different (Tukey Post Hoc multiple comparison, p < 0.05).