Abstract
Due to their great adaptability to management strategies, insect pests are a constant threat to crops. Maize is the third most important staple crop in the world after wheat and rice. The western corn rootworm (WCR) Diabrotica virgifera virgifera (LeConte, 1868) and the fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith, 1797) are among the most serious pests of maize and are responsible for massive yield losses every year. Bt maize expressing insecticidal proteins from Bacillus thuringiensis has long been a good alternative to chemicals. However, their overuse has led to the development of resistant populations. Hence, the need to find new compounds and strategies for sustainable control of these insects. In this PhD project, we aimed to develop biotech and biocontrol solutions for both WCR and FAW. In the first part, I was particularly interested in the study of binary insecticidal proteins named GDI0005A/GDI0006A identified by screening bacterial genomes and test against WCR larvae. These proteins were identified in Chryseobacterium arthrosphaerae genome. Heterologous expression of the two components was performed in E. coli system. One of the two components (GDI0006A) was a membrane lipoprotein, and resulted in low amounts successfully purified. Nonetheless, the combination of the two proteins was active against WCR including WCR resistant to the currently marketed Cry3Bb1 and Gpp34Ab1/Tpp35Ab1 proteins. These results suggest that the new binary toxin may have different binding sites and thus a distinct mode of action compared to commercial toxins. The binary proteins were then expressed successfully in plant expression systems. However, the expression at low level of GDI0006A lead to no significant activity in planta. Finally, we tested the bacteria C. arthrospharae and show that it had activity in vitro against WCR, making of it a potential BCA against WCR.The second part was devoted to studying the possible synergy between Junonia coenia densovirus (JcDV) and a Bt insecticidal protein Cry2Ab2 for biocontrol applications. Both are known to infect FAW and disturb the midgut permeability. However, when tested together either with ex vivo or in vivo approaches, we have noticed an antagonism suggesting a competition for the same midgut receptors. We also checked the feasibility of using VP4, the major capsid protein of JcDV in a biotech approach to enhance the activity of other gut binding insecticidal proteins. The preliminary results have shown a good expression of VP4 in Nicotiana benthamiana leaves without any sign of phytotoxicity. We demonstrated the ability of VP4 to self-assemble into virus-like particles (VLPs) in plant. When tested ex vivo, VLP-VP4 were able to penetrate the FAW intestinal cells. This proof of concept will be useful to explore the possible synergies with gut-binding insecticidal proteins to create new generations of transgenic maize resistant to FAW.