Expression of a Synthetic Gene for the Major Cytotoxin (Cyt1Aa) of Bacillus thuringiensis subsp. israelensis in the Chloroplast of Wild-Type Chlamydomonas.

Seongjoon Kang,David Herrin,Candice Malone,Saravanan Thangamani,Obed Odom

doi:10.3390/biology7020029

Seongjoon Kang, David Herrin + Show 3 more

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https://doi.org/10.3390/biology7020029

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Abstract

Chlamydomonas reinhardtii (Chlamydomonas) strains that are toxic to mosquito larvae because they express chloroplast transgenes that are based on the mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis (Bti) could be very useful in mosquito control. Chlamydomonas has several advantages for this approach, including genetic controls not generally available with industrial algae. The Bti toxin is produced by sporulating bacteria and has been used for mosquito control for >30 years without creating highly resistant mosquito populations. The suite of toxins is four main proteins: three Cry proteins and the cytotoxic Cyt1Aa (27 kDa). Cyt1Aa is not very toxic to mosquitoes by itself, but it prevents the development of resistance. The production of Cyt1Aa in other microbes, however, has been challenging due to its affinity for certain membrane phospholipids. Here we report on the production of recombinant Cyt1Aa (rCyt1A) in the chloroplast of photosynthetic Chlamydomonas at levels of at least 0.3% total protein. Live cell bioassays demonstrated toxicity of the rCyt1Aa Chlamydomonas to larvae of Aedes aegypti. We also expressed the chloroplast cyt1Aa gene in a wild-type Chlamydomonas strain (21 gr) that can grow on nitrate. These results have implications for developing a Chlamydomonas strain that will be toxic to mosquito larvae but will not induce strongly resistant populations.

Highlights

Mosquitoes are vectors for some important parasites and viruses that cause diseases in humans, which include the malarial parasite, plasmodium, and the emerging viruses, West Nile, chikungunya and Zika [1]
Chlamydomonas protein atpB gene, we examined the rCyt1Aa protein on western blots of total Chlamydomonas protein (Figure 2)
We have demonstrated that the cytotoxic Cyt1Aa protein from the mosquitocidal bacterium, Bacillus thuringiensis subsp. israelensis (Bti), can be produced in the chloroplast at reasonably high levels in wild-type photosynthetic cells

Summary

Introduction

Mosquitoes are vectors for some important parasites and viruses that cause diseases in humans, which include the malarial parasite, plasmodium, and the emerging viruses, West Nile, chikungunya and Zika [1]. Since treatments for the aforementioned viruses are generally lacking, and notwithstanding the sexual transmission of Zika, vector control is essential for limiting the prevalence of these diseases. Mosquito control has proven to be an effective approach, that is both preventative and powerful because it blocks the transmission of multiple diseases simultaneously. The preferred strategy is integrated pest management (IPM), which targets all stages of the insect life cycle. The pesticides used in IPM are increasingly problematic, especially the chemicals that kill adult insects, which are toxic to non-target organisms, such as honey bees. The development of Biology 2018, 7, 29; doi:10.3390/biology7020029 www.mdpi.com/journal/biology

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