**1. Introduction**

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164 Biotechnology

The combination, 'pyramiding' or 'stacking' of multiple genes in plants is a fundamental aspect of modern plant research and biotechnology. The most widely adopted stacked traits (herbi‐ cide tolerance and insect protection) provide growers with benefits of increased crop yield, simplified management of weed control and reduced insecticide use. The global acreage of stacked traits or more precisely genetically modified organisms bearing stacked traits is expected to increase rapidly in the near future, with the introduction of nutritional and/or industrial traits to satisfy the needs of consumers and producers [1]. Several approaches have been used to stack multiple genes into plant genomes and then to coordinate expression [2-4]. Stacking approaches include sexual crossing between plants carrying distinct transgenes [5,6], sequential re-transformation [7], and single-plasmid [8] or multiple-plasmid co-transforma‐ tion [9]. These strategies, however, suffer from the inherent weakness that co-expression of the heterologous proteins is unreliable.

Due to limited genomic coding space, many viruses encode more than one protein from a single mRNA transcript. Internal ribosome entry site (IRES) sequences serve as a launching pad for internal initiation of translation, allowing expression of two or more genes from a single transcript [reviewed in 10]. A number of IRES motifs from plant [11] and animal [12] viruses have been used to direct the expression of multiple recombinant proteins in plants and plant cells [13,14]. However, widespread use of IRES motifs in plant biotechnology is limited: they are not small (~600 base pairs), adding to the size of the transgene; translation efficiency of a gene placed after the IRES is much lower than that of a gene located before the IRES [14]. One promising gene/protein strategy adopted by some viruses to ensure a balance of proteins *in vivo* is to express polyprotein precursors with cleavable linkers between the proteins of interest [15]. Several groups demonstrated the potential of this approach by co-expressing two proteins

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separated by the tobacco etch virus (TEV) NIa protease recognition sequence (heptapeptide cleavage recognition sequence ENLYFQS) together with the NIa proteinase [16-18]. The utility of the NIa protease is limited due to the presence of a nuclear-localizing signal (NLS) within the protease and the amount of energy necessary to express the 49 kDa protease. It is also possible to use linker sequences that are putative substrates of known endogenous plant proteases [19].

To bypass the need for an endogenous or recombinant accessory protease acting on the translated polypeptide product a different approach involves the use of self-processing viral 2A peptide bridges [reviewed in 20, 21]. The designation "2A" derives from the systematic nomenclature of protein domains within the polyproteins of picornaviruses. In foot-andmouth disease virus (FMDV) and some other picornaviruses the oligopeptide 2A region of the polyprotein manipulates the ribosome to "skip" the synthesis of the glycyl-prolyl peptide bond at its own carboxyl terminus leading to the release of the nascent protein and translation of the downstream sequence [22]. Under the monikers of "Skipping", "Stop-Carry On" and "StopGo" translation, it allows the stoichiometric production of multiple, discrete, protein products from a single transgene [23,24]. Several recent review articles have amply covered the role of 2A biotechnology in animal systems [20, 25]. This summary-review will provide an up-to-date overview of 2A and cover the wider application of 2A-polyproteins to the expres‐ sion of multiple proteins in plants.
