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

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.

The Cyt1Aa toxin from Bacillus thuringiensis inserts into target membranes via different mechanisms in insects, red blood cells, and lipid liposomes

“…Bti Cyt1Aa toxin is a versatile protein that has proven to be toxic to different insect orders and also to erythrocytes and some mammalian cell lines (7,(14)(15)(16)(17). For more than 20 years, different groups have studied the mechanism of action of Cyt toxins.…”

“…2, left panel). Cyt1Aa-A59C and Cyt1Aa-A61C were not analyzed because their effect in hemolytic activity was previously reported, showing that Cyt1Aa-A59C was severely affected, and Cyt1Aa-A61C showed reduced hemolytic activity (17). The double mutant Cyt1Aa-C7S-C190S showed a similar hemolytic activity as the Cyt1Aa toxin.…”

“…The mechanism of action of Cyt1Aa toxin is not dependent on receptor proteins, because this toxin interacts with unsaturated membrane lipids present in brush border membranes from the mosquito larvae (13). Cyt1Aa toxin is toxic to dipteran insects, but toxicity against coleopteran larvae and pea aphid has also been reported, as well as cytolytic effect against some mammalian cell lines and erythrocytes (7,(14)(15)(16)(17).…”

“…strands b-6, b-7, and b-8; Fig. 1) is involved in membrane insertion of the toxin, whereas helices a-1 and a-3 are important for membrane recognition and oligomerization (12,17,20,22,23) to finally form high-molecular-weight oligomers, which insert into the membrane and form the lytic pores composed of 16 monomers (19,24,25). It has been shown that Cyt1Aa oligomerization is needed for membrane insertion and pore formation into insect brush border membrane vesicles (BBMV) because Cyt1Aa helix a-3 mutants were unable to oligomerize and lost toxicity against mosquitoes and hemolytic activity, suggesting that oligomerization is needed for membrane insertion (23).…”

“…In the case of Cyt2Aa, mutagenesis of helix a-1 showed that this helix could also be involved in toxin oligomerization and toxicity (26). However, mutations in helix a-1 in the Cyt1Aa toxin were not affected in oligomer formation, insecticidal activity, and synergism with Cry11Aa but were severely affected in their hemolytic activity, suggesting that insecticidal and hemolytic activities of Cyt1Aa could involve different mechanisms (17).…”