Toward mosquito control with Chlamydomonas : expression of Cry genes from Bacillus thuringiensis israelensis in the chloroplast of Chlamydomonas reinhardti

Although crop plants that express Cry genes have been developed and used in the field to help control insect pests, genes that are toxic to mosquito larvae have not been exploited successfully for mosquito control. Chlamydomonas reinhardtii is a feasible platform for Bacillus thuringiensis israelensis (Bti) toxin expression and deployment as a mosquito larvicide. C. reinhardtii chloroplast genome engineering is well established and ideal for this application. The Bti Cry proteins Cry4Ba (128 kDa), Cry11Aa (72 kDa), and a truncated Cry4Aa (Cry4A700; 74 kDa) were recoded and expressed from the chloroplast genome of C. reinhardtii. Since these proteins can be toxic to heterologous hosts, the inducible Cyc6:Nac2-psbD expression system was used. The codon-optimized Cry genes were outfitted with a modified psbD 5’ region, and integrated into the chloroplast genome of the Ind41_18 strain; homoplasmicity of the transformants was verified by PCR. Western blots of total cell protein showed the accumulation of all three proteins under induction (i.e., minus Cu2+) conditions, with relative protein expression in the order of Cry4Aa700 >> Cry11Aa > Cry4Ba. A live-cell bioassay demonstrated the toxicity of the Cry4Aa700 and Cry11Aa transformants against larvae of Aedes aegypti and Culex quinquefasciatus. Surprisingly, the growth rates of the transformants under inducing and noninducing conditions were very similar, suggesting that these Cry gene constructs were not very toxic to these cells (Ind41_18 host strain under minus-Cu2+ conditions). Finally, RT-PCR analysis of the mRNAs suggested that Cry11Aa expression may be limited at the post-translational level. Constitutive expression of Cry toxins is essential for field application of Bti-Chlamydomonas. The same synthetic genes that were inducibly expressed in the Ind41_18 strain (above) were transformed into a wild-type strain of C. reinhardtii, but only Cry4Ba and Cry11Aa transformants reached homoplasmicity. Western blotting results confirmed the accumulation of Cry11Aa in those transformants, and indicated that it was at least as high as in the inducible system. The Cry11Aa wild-type strain was also toxic to A. aegypti larvae in a live cell bioassay. These results lay the foundation for obtaining Bti-Chlamydomonas strains that are highly toxic to mosquito larvae, and safe for non-target organisms. Taken together with the environmental and genetic controls on this organism and its chloroplast genes should make it an attractive biocontrol agent.