Multimodal protein constructs for herbivore insect control

Abstract

Transgenic plants expressing combinations of microbial or plant pesticidal proteins represent a promising tool for the efficient, durable control of herbivorous insects. In this review we describe current strategies devised for the heterologous co-expression of pesticidal proteins in planta, some of which have already shown usefulness in plant protection. Emphasis is placed on protein engineering strategies involving the insertion of single DNA constructs within the host plant genome. Multimodal fusion proteins integrating complementary pesticidal functions along a unique polypeptide are first considered, taking into account the structural constraints associated with protein or protein domain grafting to biologically active proteins. Strategies that allow for the co- or post-translational release of two or more pesticidal proteins are then considered, including polyprotein precursors releasing free proteins upon proteolytic cleavage, and multicistronic transcripts for the parallel translation of single protein-encoding mRNA sequences.

Keywords: Bt plants; Cry toxins; defense proteins; fusion proteins; gene stacking; insect-resistant transgenic plants; polyproteins; protein pyramiding.

Figure 1

Cleavable polyprotein precursor constructs for the heterologous co-expression of two hypothetical pesticidal proteins, Protein 1 and Protein 2, in transgenic plants. The polyprotein precursor includes a cleavable linker peptide (clp) (in green) between the two protein moieties, which is post-translationally processed by exogenous or endogenous proteases (Pro) to release the two mature proteins. (a) Exogenous protease-mediated cleavage. The polyprotein precursor may be cleaved off by a recombinant protease expressed as part of a processing functional unit (cleavage in cis) or after the integration of an independent, co-expressed protease-encoding transgene (cleavage in trans). (b) Endogenous protease-mediated cleavage. Alternatively, the mature proteins may be released by cleavage of a clp recognized by the host plant endogenous proteases. Black arrows on panels (a) and (b) indicate the direction of ribosome-mediated mRNA translation. Red arrows point to protease-susceptible sites on cleavable linker peptides.

Figure 2

Polycistronic constructs for the heterologous co-expression of two hypothetical pesticidal proteins, Protein 1 and Protein 2, in transgenic plants. (a) IRES-mediated translation. An internal ribosome entry site (IRES) sequence is included between the codingsequences of Protein 1 and Protein 2 to drive a cap-independent, internal initiation of Protein 2 translation, in parallel to Protein 1 cap-dependent translation initiated at the polycistron transcript 5' end. (b) 2A peptide-mediated translation. A viral 2A peptide sequence is included between the coding sequences of Protein 1 and Protein 2 to induce ‘ribosomal skipping’ during translation leading to the co-translational release of the two proteins.