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Background  Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.    Results  We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in  Nicotiana benthamiana  leaf tissue. TriTag-1 and TriTag-2 use alternative splicing to generate differentially localized GFP isoforms, localizing it to the chloroplasts, peroxisomes and cytosol. TriTag-1 shows a bias for targeting the chloroplast envelope while TriTag-2 preferentially targets the peroxisomes. TriTag-3 embeds a conserved peroxisomal targeting signal within a chloroplast transit peptide, directing GFP to the chloroplasts and peroxisomes.    Conclusions  Our novel signal sequences can reduce the number of cloning steps and the amount of genetic material required to target a heterologous protein to multiple locations in plant cells. This work harnesses alternative splicing and signal embedding for engineering plants to express multi-functional proteins from single genetic constructs.

Targeting a heterologous protein to multiple plant organelles via rationally designed 5′ mRNA tags