15 Multifunctional Hybrid Proteins Useful in Plant Protection Meriem Benchabane • Dominique Michaud * CRH/INAF, Pavillon des Services/INAF, Université Laval, Québec QC, Canada G1K 7P4 Corresponding author: * Dominique.Michaud@crh.ulaval.ca Keywords: Bt toxins, fusion proteins, gene stacking, pest-resistant transgenic plants, plant defense proteins, polyproteins, protein pyramiding ABSTRACT The potential of transgenic plants expressing combinations of microbial or plant-derived pesticidal proteins for the efficient, long-term control of herbivorous pests has been addressed by several authors over the last few years. In this short review we describe current protein engineering strategies devised for the co-expression of recombinant proteins useful in plant protection. Strategies based on the expression of polyprotein precursors comprising distinct pesticidal proteins linked by intrinsic ‘cleavage’ sites for processing are first described. Multifunctional fusion proteins bearing complementary pesticidal functions are then considered, along with some challenges to address for the successful use of such hybrid proteins in plants. 1. INTRODUCTION The large-scale use of transgenic plant lines expressing insecticidal -endotoxins from the soil bacterium Bacillus thuringiensis clearly illustrates the potential of insect-resistant transgenic plants in current agricultural systems (James 2005). The development of Bt toxin-expressing lines (or ‘Bt lines’) highly resistant to insect pests of major agronomic importance, along with the adoption of deployment strategies aimed at preventing genetic resistance among target insect populations, have largely contributed to the success of these lines worldwide (Shelton et al. 2002, Glaser and Matten 2003, Tabashnik et al. 2003). After several years of commercial use, genetic resistance to Bt lines has not been documented in the field despite the pesticidal [toxic] nature of Bt toxins and the rapidly growing importance of culture areas devoted to these lines in several countries (Fox 2003, Tabashnik et al. 2003 2005). Contrasting with this positive account on Bt lines, experimental data – notably the isolation of Bt toxin-resistant insects from laboratory colonies (e.g., Tang et al. 1999a, Zhao et al. 2002) or field plots sprayed with Bt formulations (e.g., Tabashnik et al. 1990, Janmaat and Myers 2003) – suggest the intrinsic ability of herbivorous insects to overcome the pesticidal effects of Bt toxins. Current strategies to prevent insect resistance, such as the use of Bt lines with high levels of toxin and the concomitant use of non-Bt plant refuges for susceptible insects, have proved effective until now, but resistance problems in the future cannot be excluded (Sayyed et al. 2003, Tabashnik et al. 2003). A reliable strategy to promote the long-term effectiveness of Bt lines would be to consider these plants as components of much broader, integrated pest management systems involving several complementary means for pest control (Bates et al. 2005). The use of transgenic lines expressing combinations of Bt toxins has also been proposed to delay the development of resistance, compared with the use of single-toxin plants used alone, sequentially or in mosaics (Roush 1998, Gould 2003, Zhao et al. 2003 2005). In practice, such ‘pyramiding’ of Bt toxins could also provide improved protection against insects moderately susceptible to single toxins (Jackson et al. 2004), or broaden the pesticidal spectrum of Bt toxins against insect pests (Bohorova et al. 2001, Naimov et al. 2003). In a larger perspective, the pesticidal effect of Bt toxins could be broadened by the co-expression of complementary resistance factors with different modes of action (Bates et al. 2005), including for instance alternative bacterial toxins (Lee et al. 2003, Liu et al. 2003, Moellenbeck et al. 2004) or plant- derived defense genes (Tang et al. 1999b, Maqbool et al. 2001). The potential of protein [or gene] pyramiding has also been considered for the expression of plant defense proteins with complementary or synergistic effects against herbivorous pests and pathogens (Boulter et al. 1990, Michaud 1997, Campbell et al. 2002). Several approaches have been proposed over the last fifteen years to co-express multiple transgenes and recombinant proteins in plants (Halpin 2005). Here we briefly describe these approaches, with particular emphasis on protein engineering strategies enabling the coordinated expression of distinct pesticidal proteins under the control of single promoters.