A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein

Abstract

Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering.

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

“…Engineering of complex regulatory circuits and traits might require the use of multiple components. Viral vectors permit coexpression of various proteins that can be targeted to diverse subcellular compartments (Majer et al, 2015). Multiple proteins have been expressed simultaneously using single viral vectors.…”

Efficient production of saffron crocins and picrocrocin inNicotiana benthamianausing a virus-driven system

“…Plasmids pGTEVΔNIb-CsCCD2L, -BdCCD4.1, -BdCCD4.3, -CsCCD2L/PaCrtB, - CsCCD2L/CsBCH2 and -CsCCD2L/CsLPT1 were built on the basis of pGTEVa (Bedoya et al, 2012) that contains a wild-type TEV cDNA (GenBank accession number DQ986288 with the two silent and neutral mutations G273A and A1119G) flanked by the CaMV 35S promoter and terminator in a binary vector that also derives from pCLEAN-G181. These plasmids were built, as explained above, by PCR amplification of CsCCD2L (Ahrazem et al, 2016c), BdCCD4.1 and BdCCD4.3 (Ahrazem et al, 2017), PaCrtB (Majer et al, 2017), CsBCH2 (Castillo et al, 2005) and CsLPT1 (Gómez-Gómez et al, 2010) cDNAs and assembly into a pGTEVa version in which the NIb cistron was deleted (pGTEVaΔNIb) (Majer et al, 2015). The exact sequences of the TEV recombinant clones (TEVΔNIb-CsCCD2L, -BdCCD4.1, -BdCCD4.3, -CsCCD2L/PaCrtB, - CsCCD2L/CsBCH2 and -CsCCD2L/CsLPT1) contained in the resulting plasmids are in Supplementary Fig.…”

Tobacco Etch Virus protease: A shortcut across biotechnologies

“…TEVp has been often used in vivo to facilitate stoichiometric expression of multiples genes in bacteria (Chen et al, 2010), yeast (Ghiaci et al, 2014), plants (Majer et al, 2015) and mammalian cells (Cesaratto et al, 2015). Using this strategy it is possible to achieve expression of protein subunits at stoichiometric equal levels and at the same physical location.…”

“…An alternative system was described to work in N. tabacum plants. A TEV-derived viral vector bearing a cassette with cDNAs of multiple proteins separated by the TS allowed the translation of equimolar amounts of up to three proteins (Majer et al, 2015).…”