Use of RNAi as a preliminary tool for screening putative receptors of nematicidal toxins from Bacillus thuringiensis.

Bacillus thuringiensis is a potential control agent for plant-parasitic nematodes. Nematode intestinal receptors for Cry21-type toxins are poorly known. Therefore, a strategy was tested as a primary screening tool to find possible Cry toxin receptors, using a nematicidal Bt strain and the RNAi technique on Caenorhabditis elegans. Six genes encoding intestinal membrane proteins were selected (abt-4, bre-1, bre-2, bre-3, asps-1, abl-1) as possible targets for Cry proteins. Fractions of each selected gene were amplified by PCR. Amplicons were cloned into the L4440 vector to transform the E. coli HT155 (DE3) strain. Transformed bacteria were used to silence the selected genes using the RNAi feeding method. Nematodes with silenced genes were tested with the Bt strain LBIT-107, which harbors the nematicidal protein Cry21Aa3, among others. Results indicated that nematodes with the silenced abt-4 gene were 69.5% more resistant to the LBIT-107 strain, in general, and 79% to the Cry21Aa3 toxin, specifically.

Insecticidal Activity of a Cry1Ca toxin of Bacillus thuringiensis Berliner (Firmicutes: Bacillaceae) and Its Synergism with the Cyt1Aa Toxin Against Aedes aegypti (Diptera: Culicidae).

The Cry1C protein family of Bacillus thuringiensis form bipyramidal crystals, which are commonly associated with toxic activity against lepidopteran species; however, some members of this family may also be toxic to dipterans. In the present work, the Cry1Ca16 protein, synthesized by the B. thuringiensis LBIT-1217 strain, was analyzed. The gene coding for this protein was amplified, sequenced, and cloned into the pSTAB vector, which was electro-transferred into the acrystalliferous B. thuringiensis 4Q7 strain. The recombinant strain showed the expected bipyramidal crystal morphology, identical to the original LBIT-1217 strain and exhibited toxicity against larvae of Aedes aegypti (Diptera). Pure crystals from the recombinant strain were used in bioassays against Ae. aegypti larvae, estimating an LC50 of 4.61 µg/ml. Further studies on Cry1Ca16 mosquitocidal potential included joint-action tests with the Cyt1Aa protein crystals from B. thuringiensis israelensis. An LC50 using pure Cyt1Aa crystals was estimated at 0.73 µg/ml, whereas an LC50 of 0.61 µg/ml was estimated when both toxins were tested together. Data from these bioassays was analyzed using joint-action tests such as the Tammes-Bakuniak graphical method and the formula proposed by Tabashnik (1992). Both tests clearly showed a synergistic effect between these two toxins.