In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium

Abstract

We have developed an efficient, versatile, and user-friendly viral engineering and expression system that is based on in planta assembly of functional viral vectors from separate pro-vector modules. With this new system, instead of supplying a plant cell with a complete viral vector as a mature viral particle, an RNA or a linear DNA molecule, we use agrobacteria to deliver various modules that are assembled inside the cell with the help of a site-specific recombinase. The resulting DNA is transcribed, and undesired elements such as recombination sites are spliced out, generating a fully functional RNA replicon. The proposed protocol allows us, by simply treating a plant with a mixture of two or more agrobacteria carrying specific prefabricated modules, to rapidly and inexpensively assemble and test multiple vector/gene combinations, without the need to perform the various engineering steps normally required with alternative protocols. The process described here is very fast (expression requires 3-4 days); it provides very high protein yield (up to 80% of total soluble protein); more than before, it is carried out using in vivo manipulations; it is based on prefabricated genetic modules that can be developed/upgraded independently; and it is inherently scalable.

Fig. 1.

Transient expression by agroinfiltration of N. benthamiana leaves using nonviral expression cassettes (A–C) and viral constructs (D–F). Protoplasts from a leaf coinfiltrated with constructs containing GFP or DsRed under control of the 35S promoter viewed under blue (A) or red (B) light 4 dpi. (C) GUS staining of a leaf coinfiltrated with pICH11330 and pICH7900 (infiltration 1) or pICH11330 alone (infiltration 2) 3 dpi. (D) GFP expression in a leaf of a plant stably transformed with pICH10881 and coinfiltrated with pICH4851 and pICH6891 6 dpi. (E) GFP expression (7 dpi) in leaf sectors coinfiltrated with the following constructs: pICH4371 and pICH4461 together with pICH1754 (infiltration 1) or without (infiltration 2), pICH4851 and pICH6891 together with pICH10881 (infiltration 3) or without (infiltration 4), and pICH4351 alone (infiltration 5). (F) DsRed expression in a leaf infiltrated with pICH-NOP and pICH-RED, viewed under normal light. (G) Schematic representation of the T-DNA regions of some of the constructs used for infiltration.

Fig. 2.

Schematic representation of intron-containing viral pro-vector modules. (A) T-DNA regions of 5′ modules. ubi, Arabidopsis ubiquitin sequence; cal, tobacco calreticulin apoplast targeting presequence; ctp, synthetic chloroplast targeting presequence; int, intron 5′ half; P, ACT2 promoter; T, nos terminator. (B) Product of recombination between 5′ modules and pICH-GFP (the sequence labeled “pectinase” is expected by recombination of the 5′ module pICH-PEC). Exon and intron sequences are displayed as uppercase and lowercase letters, respectively. Amino acids are shown in italics, and a stop codon is indicated by an asterisk. Protein cleavage sites are indicated with arrows. The end of the MP coding sequence is displayed in an open box.

Fig. 3.

N. benthamiana plant expressing GFP in systemic leaves 14 days after infiltration of one of the lower leaves (hidden from view) with pICH-NOP, pICH-GFPSYS, and pICH10881. Int, intron 3′ half; 3′PK, cr-TMV pseudoknot-like motifs; sgp, TMGMV U5 subgenomic promoter; 3′ NTR, TMGMV U5 3′ untranslated region; T, Nos terminator.

Fig. 4.

Targeted expression of GFP using different 5′ pro-vector modules. (A) GFP expression in N. benthamiana leaves 12 dpi. (a–e) Leaves viewed under UV illumination. (f–j) Mesophyll protoplasts viewed under blue light. (a and f) Uninfiltrated leaf. (b and g) Cytosolic localization of GFP provided by ubiquitin fusion (pICH-UBI). (c and h) Targeting to the secretory pathway with calreticulin module (pICH-CAL). (d and i) Targeting to the chloroplast (pICH-CHL). (e and j) No presequence for expression in the cytosol (pICH-NOP). (Scale bar, 10 μM.) (B) RT-PCR analysis of recombined pro-vector modules for ubiquitin-GFP, calreticulin-GFP, and CTP-GFP fusions. (C) Coomassie-stained gel (SDS/PAGE) containing total soluble protein extracts from leaves inoculated with the GFP 3′ module in combination with pICH-UBI (lane 2), pICH-CAL (lane 3), pICH-CHL (lane 4), pICH-CPF (lane 5), pICH-NOP (lane 6), and modules for His tag fusion without a cleavage site (lane 7) or with an enterokinase cleavage site (lane 8). Lane 1 shows noninfiltrated leaf tissue.

Fig. 5.

Coomassie-stained polyacrylamide gels showing protein expressed with the viral pro-vector system. (A) Total soluble proteins extracted from leaves infiltrated with pICH-NOP, pICH-GFP, and integrase (lane 1), from leaves infiltrated with pICH4351 (lane 2), from a systemic leaf containing WT cr-TMV virus (lane 3), and from uninfiltrated leaf tissue (lane 4). (B) Extracts from leaves infiltrated with pICH-NOP, pICH-GFP, and integrase 7 dpi (lane 1), 10 dpi (two different infiltrations 2 and 3), and 17 dpi (lane 4). (C) Extracts from uninfiltrated tissue (lane 1), from leaves infiltrated with a 35S-GFP cassette without suppressor of silencing (lane 2) or together with 35S-HcPro (lane 3) or 35S-p19 (lane 4), pICH-NOP infiltrated with integrase and pICH-GFP (lane 5) or pICH-GFPSYS (lane 6), WT cr-TMV virus (lane 7), and recombinant GFP (lane 8). Rbc, Rubisco large subunit.

Fig. 6.

Schematic overview of in planta assembly of viral pro-vector modules. SD, splice donor site; SA, splice acceptor site.