FIG. 1.

Plasmid constructs. (A) TMV-based 5′ modules, including pICH20111 and pICH22455, for the cytosolic localization of proteins of interest, without and with a His₆ tag and an enterokinase cleavage site, respectively; pICH20030, for chloroplast targeting, containing the consensus sequence of the transit peptide (CTP) for the small subunit of Rubisco (dicotyledonous plants); pICH20155 and pICH22488, for apoplast targeting, containing native and codon-optimized signal peptides (apoplast-targeting signal peptide [ATP]) from rice alpha-amylase, respectively; pICH20188, for apoplast targeting, containing a signal peptide from Nicotiana plumbaginifolia calreticulin; pICH20199, for apoplast targeting, containing a signal peptide of the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris; and pICH20388 and pICH20999, for apoplast targeting, containing signal peptides of apple pectinase and barley alpha-amylase, respectively. Plasmids pICH22464, pICH27757, pICH27784, and pICH27770 contain signal peptides from Nicotiana plumbaginifolia calreticulin, rice alpha-amylase, barley alpha-amylase, and apple pectinase, respectively, followed by a His₆ tag and an enterokinase cleavage site. (B) TMV-based 3′ provector modules, including cloning vector pICH28575 and 3′ modules for the expression of full-length hemagglutinin (rHA0) from NIBRG-14 influenza virus (pICH30392) and GFP (pICH7410). (C) Integrase module pICH14011 providing the site-specific integrase of phage C31 (PhiC31). LB and RB, left and right T-DNA borders, respectively; Pact, Arabidopsis actin 2 promoter; Phsp, promoter of the Arabidopsis gene encoding heat shock protein Hsp81.1; T, nos terminator; RdRp, RNA-dependent RNA polymerase; MP, movement protein; int, intron; AttP and AttB, recombination sites; his, His₆ tag; BsaI, cloning sites recognized by class IIs restriction enzyme BsaI; 3′NTR, 3′ untranslated region of TMV; ATP, apoplast-targeting signal peptide; CTP, chloroplast-targeting transit peptide; NLS, nuclear localization signal.