Susceptible and mCry3A resistant corn rootworm larvae killed by a non-hemolytic Bacillus thuringiensis Cyt1Aa mutant

Alejandra Bravo,Mario Soberón,Kathleen Harding,Mary-Carmen Torres-Quintero,Gusui Wu,Mark E Nelson,Takashi Yamamoto,Amit Sethi,Janette Onofre,Gretel Mendoza,Jian-Zhou Zhao,Jazmin A López-Diaz

doi:10.1038/s41598-018-36205-6

Alejandra Bravo, Mario Soberón + Show 10 more

Open Access

https://doi.org/10.1038/s41598-018-36205-6

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Abstract

The western corn rootworm (WCR) Diabrotica virgifera virgifera causes substantial damage in corn. Genetically modified (GM) plants expressing some Bacillus thuringiensis (Bt) insecticidal Cry proteins efficiently controlled this pest. However, changes in WCR susceptibility to these Bt traits have evolved and identification of insecticidal proteins with different modes of action against WCR is necessary. We show here for the first time that Cyt1Aa from Bt exhibits toxicity against WCR besides to the dipteran Aedes aegypti larvae. Cyt1Aa is a pore-forming toxin that shows no cross-resistance with mosquitocidal Cry toxins. We characterized different mutations in helix α-A from Cyt1Aa. Two mutants (A61C and A59C) exhibited reduced or absent hemolytic activity but retained toxicity to A. aegypti larvae, suggesting that insecticidal and hemolytic activities of Cyt1Aa are independent activities. These mutants were still able to form oligomers in synthetic lipid vesicles and to synergize Cry11Aa toxicity. Remarkably, mutant A61C showed a five-fold increase insecticidal activity against mosquito and almost 11-fold higher activity against WCR. Cyt1Aa A61C mutant was as potent in killing WCR that were selected for resistance to mCry3A as it was against unselected WCR indicating that this toxin could be a useful resistance management option in the control of WCR.

Highlights

The western corn rootworm (WCR) Diabrotica virgifera virgifera is an insect pest that attacks cornfields eating the roots of the plants causing extensive damage
Our results show that two mutations located in helix α-A of Cyt1A reduced hemolysis of red blood cells, but did not alter oligomerization, synergism with Cry11Aa, or toxicity against A. aegypti or WCR larvae
The evaluation of the mean growth inhibition concentration (IC50) value was performed with solubilized protoxin proteins (Fig. 1A) that were obtained from crystal inclusions purified by sucrose gradients as reported in Methods

Summary

Introduction

The western corn rootworm (WCR) Diabrotica virgifera virgifera is an insect pest that attacks cornfields eating the roots of the plants causing extensive damage It is principally found in North America and Europe resulting in grower-losses in excess of one billion dollars per year[1]. New proteins that kill WCR have been identified from non-Bt bacteria such as the small IPD072Aa protein from Pseudomonas chlororaphis or the PIP-47Aa protein from P. mosselii[10,11] These novel toxins do not share receptors with Cry3A or Cry34/35Ab1 and neither showed cross-resistance in Cry3A-selected insects[10,11], while IPD072Aa showed no cross-resistance to Cry34/35Ab1-selected insects[10]. It has been proposed that Cyt1Aa functions as a receptor of Cry11Aa since binding of this toxin to Cyt1Aa facilitates oligomer formation of Cry11Aa and its membrane insertion[29,32]

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