TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector

Abstract

Transient expression is a rapid, useful approach for producing proteins of interest in plants. Tobacco mosaic virus (TMV)-based transient expression vectors can express very high levels of foreign proteins in plants. However, TMV vectors are, in general, not efficiently delivered to plant cells by agroinfection. It was determined that agroinfection was very efficient with a 35S promoter-driven TMV replicon that lacked the TMV coat protein gene sequence. This coat protein deletion vector had several useful features as a transient expression system, including improved ease of use, higher protein expression rates, and improved biocontainment. Using this TMV expression vector, some foreign proteins were expressed at levels of 3 to 5 mg/g fresh weight of plant tissue. It is proposed that this new transient expression vector will be a useful tool for expressing recombinant proteins in plants for either research or production purposes.

Figure 1.

Maps of plasmids used in this project. The T-DNA regions of binary plasmids used in this project are represented. Block arrow, CaMV duplicated 35S promoter. Black box, CaMV polyA signal sequence/terminator. Dark gray box, Tobacco etch virus 5′-nontranslated leader sequence. Light gray box, Ribozyme. Bent arrows, Subgenomic promoters. ORFs are represented by white boxes. Identities of ORFs are labeled in white boxes. Replicase, TMV 126K/183K ORF; MP, movement protein; P19, 19-kD RNA-silencing suppressor gene from Tomato bushy stunt virus.

Figure 2.

Comparison of agroinfection efficiency of pJL24 and pJL-TRBO vectors. T-DNAs of TMV-based expression vectors were introduced into N. benthamiana by agroinfection. Sections of an N. benthamiana leaf were infiltrated with A. tumefaciens (A.t.) cell suspensions transformed with plasmids as follows. A, A.t./pJL24 (OD₆₀₀ 1.0). B, Mixture of A.t./pJL24 + A.t./pJL3:P19 (each at final OD₆₀₀ of 0.5). C, A.t./pJL-TRBO-G (OD₆₀₀ 1.0). D, Mixture of A.t./pJL-TRBO-G + A.t./pJL3:P19 (each at final OD₆₀₀ of 0.5). Photo taken under UV illumination 4 dpi. In grayscale, GFP fluorescence appears as a light color. [See online article for color version of this figure.]

Figure 3.

Effect of A. tumefaciens (A.t.) cell density on agroinfection of plants with pJL-TRBO-G expression vector. Leaves of N. benthamiana plants were infiltrated with A.t. cell suspensions transformed with various binary (modified Ti) plasmids. A.t. cell suspensions were diluted, as noted in the figure, from an initial OD₆₀₀ of 1.0. A, Individual leaves infiltrated with A.t./pJL-TRBO-G cell suspensions were photographed under UV illumination at 3 and 4 dpi as noted. B, Left half of leaf infiltrated with 1:100 dilution of A.t./pJL24. Right half of leaf infiltrated with a mixture of 1:100 dilution of A.t./pJL24 and 1:10 dilution of A.t./pJL3:P19. Photo taken under UV illumination 3 dpi. [See online article for color version of this figure.]

Figure 4.

TRBO-G replicon does not move systemically in plants. One leaf of an N. benthamiana plant was infiltrated with A. tumefaciens carrying pJL24 or pJL-TRBO-G plasmids. Plants were photographed under UV light to visualize the GFP expressed by either expression vector. [See online article for color version of this figure.]

Figure 5.

Quantitative analysis of GFP expression levels from TMV vectors JL24 and TRBO-G. Leaves of N. benthamiana were infiltrated with A. tumefaciens cells transformed with plasmids identified in the figure. Bottom, Images of individual infiltrated leaves photographed under UV illumination at 4 dpi. Top, Quantitation of GFP fluorescence activity levels in extracts prepared from infiltrated leaves 6 dpi. Extracts were analyzed by a plate-based GFP fluorescence assay. Purified recombinant His-6-tagged GFP was used to generate a standard curve. Results are presented in micrograms GFP produced per gram of infiltrated tissue. [See online article for color version of this figure.]

Figure 6.

GFP expression from TRBO-G vector. N. benthamiana leaves were infiltrated with A. tumefaciens cultures transformed with pJL-TRBO-G. Total protein extracts were prepared from infiltrated leaf tissue from 3 to 7 dpi. Equal volumes of extracts were analyzed by SDS-PAGE, followed by staining with Coomassie Blue. Location of TRBO-expressed 27-kD GFP is noted by black arrowhead. Amount of Rubisco large subunit protein (white arrowhead) is greatly reduced in 3- to 6-dpi samples because they were subjected to a freeze-thaw cycle. Molecular mass of protein standards (kD) is noted at left of image. Lanes: M, molecular mass marker; H, healthy plant extract; 3 to 7, extracts from pJL-TRBO-G-agroinfiltrated leaves, 3 to 7 dpi, respectively.

Figure 7.

Expression of various proteins from the TRBO vector. N. benthamiana leaves were agroinoculated with pJL-TRBO vectors expressing various genes. Total soluble protein extracts were prepared approximately 5 dpi. Equal volumes of extract were loaded per lane. In some cases, proteins were expressed as fusions to a peptide tag of His-6-hemaglutinin peptide (duplicated), H₆HA₂. A, Coomassie Blue-stained SDS-PAGE gel of extracts. B, Western-blot (immunoblot) analysis of extracts using anti-HA peptide primary antibody. Lanes: M, molecular mass marker; M2, SeeBlue (Invitrogen) molecular mass marker; H, healthy plant extract. Extracts from tissue infected with JL-TRBO vector expressing the following genes: 1, Phytopthora infestans Avr3a (15 kD); 2, Aequorea victoria GFP (27 kD); 3, GFP:H₆HA₂ fusion (31 kD); 4, Arabidopsis adenosine kinase (38 kD); 5, 10th type III (FN10) domain from human fibronectin:H₆HA₂ (13 kD); 6, tomato RCR-3 proteinase:H₆HA₂ (42 kD); 7, tomato P69b proteinase:H₆HA₂ (73 kD). White and gray circles denote location of FN10 and RCR-3 proteins on Coomassie Blue-stained gel, respectively. [See online article for color version of this figure.]