Unveiling gene expression regulation of the Bacillus thuringiensis Cry3Aa toxin receptor ADAM10 by the potato dietary miR171c in Colorado potato beetle.

BACKGROUND: The Colorado potato beetle (CPB) is a worldwide devastating pest of potato plants and other Solanaceae characterized by its remarkable ability to evolve resistance to insecticides. Bacillus thuringiensis (Bt) Cry3Aa toxin represents an environmentally safe alternative for CPB control but larvae susceptibility to this toxin has been reported to vary depending on the host plant on which larvae feed. To gain more insight into how nutrition mediates Bt tolerance through effects on gene expression, here we explored the post-transcriptional regulation by microRNAs (miRNAs) of the CPB-ADAM10 gene encoding the Cry3Aa toxin functional receptor ADAM10. RESULTS: The lower CPB-ADAM10 gene expression in CPB larvae fed on potato plants cv. Vivaldi than those fed on potato cv. Monalisa or tomato plants was inversely related to Cry3Aa toxicity. By high-throughput sequencing we identified seven CPB miRNAs and one potato miRNA predicted to base pair with the CPB-ADAM10 messenger RNA. No differential expression of the endogenous lde-miR1175-5p was found in larvae feeding on any of the two potato plant varieties. However, statistically significant increased amounts of potato stu-miR171c-5p were detected in CPB larvae fed on potato cv. Vivaldi compared to larvae fed on potato cv. Monalisa. CONCLUSION: Our results support a role for dietary miRNAs in Bt toxicity by regulating the CPB-ADAM10 gene encoding the Cry3Aa toxin receptor ADAM10 in CPB larvae and opening up the possibility of exploiting plant natural variation in miRNAs to provide more sustainable potato crop protection against CPB. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin.

When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to hexanoic acid but not to Cry3Aa toxin.

Prohibitin, an essential protein for Colorado potato beetle larval viability, is relevant to Bacillus thuringiensis Cry3Aa toxicity.

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.

Combining hexanoic acid plant priming with Bacillus thuringiensis insecticidal activity against Colorado potato beetle.

Interaction between insect herbivores and host plants can be modulated by endogenous and exogenous compounds present in the source of food and might be successfully exploited in Colorado potato beetle (CPB) pest management. Feeding tests with CPB larvae reared on three solanaceous plants (potato, eggplant and tomato) resulted in variable larval growth rates and differential susceptibility to Bacillus thuringiensis Cry3Aa toxin as a function of the host plant. An inverse correlation with toxicity was observed in Cry3Aa proteolytic patterns generated by CPB midgut brush-border membrane vesicles (BBMV) from Solanaceae-fed larvae, being the toxin most extensively proteolyzed on potato, followed by eggplant and tomato. We found that CPB cysteine proteases intestains may interact with Cry3Aa toxin and, in CPB BBMV from larvae fed all three Solanaceae, the toxin was able to compete for the hydrolysis of a papain substrate. In response to treatment with the JA-dependent plant inducer Hexanoic acid (Hx), we showed that eggplant reduced OPDA basal levels and both, potato and eggplant induced JA-Ile. CPB larvae feeding on Hx-induced plants exhibited enhanced Cry3Aa toxicity, which correlated with altered papain activity. Results indicated host-mediated effects on B. thuringiensis efficacy against CPB that can be enhanced in combination with Hx plant induction.