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.

The Histone Marks Signature in Exonic and Intronic Regions Is Relevant in Early Response of Tomato Genes to Botrytis cinerea and in miRNA Regulation.

Research into the relationship between epigenetic regulation and resistance to biotic stresses provides alternatives for plant protection and crop improvement. To unravel the mechanisms underlying tomato responses to Botrytis cinerea, we performed a chromatin immunoprecipitation (ChIP) analysis showing the increase in H3K9ac mark along the early induced genes SlyDES, SlyDOX1, and SlyLoxD encoding oxylipin-pathway enzymes, and SlyWRKY75 coding for a transcriptional regulator of hormonal signaling. This histone mark showed a more distinct distribution than the previously studied H3K4me3. The RNAPol-ChIP analysis reflected the actual gene transcription associated with increased histone modifications. A different pattern of marks in the oxylipin-related genes against P. syringae supported a pathogen-specific profile, while no significant differences occurred in SlyWRKY75. The epigenetic regulation of SlyWRKY75 by the intron-binding miR1127-3p was supported by the presence of SlyWRKY75 pre-mRNA in control plants. Interestingly, mRNA was found to be accumulated in response to B. cinerea and P. syringae, while reduction in miRNA only occurred against B. cinerea. The intronic region presented a similar pattern of marks than the rest of the gene in both pathosystems, except for H3K4me3 in the miRNA binding site upon B. cinerea. We located the gene encoding Sly-miR1127-3p, which presented reduced H3K4me3 on its promoter against B. cinerea.

Proteomic insights into the immune response of the Colorado potato beetle larvae challenged with Bacillus thuringiensis.

Bacillus thuringiensis (Bt) toxins constitute effective, environmentally safe biopesticides. Nevertheless, insects' tolerance to Bt is influenced by environmental factors affecting immunity. To understand larval immune response in the devastating coleopteran insect pest Colorado potato beetle (CPB), we undertook a proteomic analysis of hemolymph of non-treated control larvae and larvae consuming non-lethal doses of spore-crystal mixtures containing the coleopteran-active Cry3Aa toxin. Results revealed lower amount of proteins involved in insect growth and higher amount of immune response-related proteins in challenged insects, sustaining the larval weight loss observed. Additionally, we found a potential regulatory role of the evolutionary conserved miR-8 in the insect's immune response relying on antimicrobial peptides (AMPs) production. Upon toxin challenge, different patterns of hemolymph AMPs expression and phenoloxidase activity were observed in CPB larvae reared on different Solanaceae plants. This suggests that diet and diet-associated insect midgut microbiota might modulate this insects' tolerance to non-lethal doses of Bt.

Expression of miR159 Is Altered in Tomato Plants Undergoing Drought Stress.

In a scenario of global climate change, water scarcity is a major threat for agriculture, severely limiting crop yields. Therefore, alternatives are urgently needed for improving plant adaptation to drought stress. Among them, gene expression reprogramming by microRNAs (miRNAs) might offer a biotechnologically sound strategy. Drought-responsive miRNAs have been reported in many plant species, and some of them are known to participate in complex regulatory networks via their regulation of transcription factors involved in water stress signaling. We explored the role of miR159 in the response of Solanum lycopersicum Mill. plants to drought stress by analyzing the expression of sly-miR159 and its target SlMYB transcription factor genes in tomato plants of cv. Ailsa Craig grown in deprived water conditions or in response to mechanical damage caused by the Colorado potato beetle, a devastating insect pest of Solanaceae plants. Results showed that sly-miR159 regulatory function in the tomato plants response to distinct stresses might be mediated by differential stress-specific MYB transcription factor targeting. sly-miR159 targeting of SlMYB33 transcription factor transcript correlated with accumulation of the osmoprotective compounds proline and putrescine, which promote drought tolerance. This highlights the potential role of sly-miR159 in tomato plants' adaptation to water deficit conditions.

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.

Identification of Stress Associated microRNAs in Solanum lycopersicum by High-Throughput Sequencing.

Tomato (Solanum lycopersicum) is one of the most important crops around the world and also a model plant to study response to stress. High-throughput sequencing was used to analyse the microRNA (miRNA) profile of tomato plants undergoing five biotic and abiotic stress conditions (drought, heat, P. syringae infection, B. cinerea infection, and herbivore insect attack with Leptinotarsa decemlineata larvae) and one chemical treatment with a plant defence inducer, hexanoic acid. We identified 104 conserved miRNAs belonging to 37 families and we predicted 61 novel tomato miRNAs. Among those 165 miRNAs, 41 were stress-responsive. Reverse transcription quantitative PCR (RT-qPCR) was used to validate high-throughput expression analysis data, confirming the expression profiles of 10 out of 11 randomly selected miRNAs. Most of the differentially expressed miRNAs were stress-specific, except for sly-miR167c-3p upregulated in B. cinerea and P. syringae infection, sly-newmiR26-3p upregulated in drought and Hx treatment samples, and sly-newmiR33-3p, sly-newmiR6-3p and sly-newmiR8-3p differentially expressed both in biotic and abiotic stresses. From mature miRNAs sequences of the 41 stress-responsive miRNAs 279 targets were predicted. An inverse correlation between the expression profiles of 4 selected miRNAs (sly-miR171a, sly-miR172c, sly-newmiR22-3p and sly-miR167c-3p) and their target genes (Kinesin, PPR, GRAS40, ABC transporter, GDP and RLP1) was confirmed by RT-qPCR. Altogether, our analysis of miRNAs in different biotic and abiotic stress conditions highlight the interest to understand the functional role of miRNAs in tomato stress response as well as their putative targets which could help to elucidate plants molecular and physiological adaptation to stress.

Epigenetic regulation of the expression of WRKY75 transcription factor in response to biotic and abiotic stresses in Solanaceae plants.

KEY MESSAGE: SlyWRKY75: gene expression was induced in response to biotic stresses, especially in Botrytis cinerea-infected tomato plants, in which Sly-miR1127-3p is a putative SlyWRKY75 regulator and epigenetic marks were detected. WRKY75 transcription factor involved in Pi homeostasis was recently found also induced in defense against necrotrophic pathogens. In this study, we analyzed by RT-qPCR the expression of SlyWRKY75 gene in tomato plants in response to abiotic stresses (drought or heat) and biotic stresses (Colorado potato beetle larvae infestation, Pseudomonas syringae or Botrytis cinerea infection) being only differentially expressed following biotic stresses, especially upon B. cinerea infection (55-fold induction). JA and JA-Ile levels were significantly increased in tomato plants under biotic stresses compared with control plants, indicating that SlyWRKY75 might be a transcriptional regulator of the JA pathway. The contribution of miRNAs and epigenetic molecular mechanisms to the regulation of this gene in B. cinerea-infected tomato plants was explored. We identified a putative Sly-miR1127-3p miRNA predicted to bind the intronic region of the SlyWRKY75 genomic sequence. Sly-miR1127-3p miRNA was repressed in infected plants (0.4-fold) supporting that it might act as an epigenetic regulation factor of SlyWRKY75 gene expression rather than via the post-transcriptional mechanisms of canonical miRNAs. It has been proposed that certain miRNAs can mediate DNA methylation in the plant nucleus broadening miRNA functions with transcriptional gene silencing by targeting intron-containing pre-mRNAs. Histone modifications analysis by chromatin immunoprecipitation (ChIP) demonstrated the presence of the activator histone modification H3K4me3 on SlyWRKY75 transcription start site and gene body. The induction of this gene in response to B. cinerea correlates with the presence of an activator mark. Thus, miRNAs and chromatin modifications might cooperate as epigenetic factors to modulate SlyWRKY75 gene expression.

Oxylipin mediated stress response of a miraculin-like protease inhibitor in Hexanoic acid primed eggplant plants infested by Colorado potato beetle.

Insect-plant interactions are governed by a complex equilibrium between the mechanisms through which plant recognize insect attack and orchestrate downstream signaling events that trigger plant defense responses, and the mechanisms by which insects overcome plant defenses. Due to this tight and dynamic interplay, insight into the nature of the plant defense response can be gained by analyzing changes in the insect herbivores digestive system upon plant feeding. In this work we have identified a Solanum melongena miraculin-like protease inhibitor in the midgut juice of Colorado potato larvae feeding on eggplant plants treated with the natural inducer of plant defenses hexanoic acid. We analyzed the corresponding gene expression by qRT-PCR and our results showed that this eggplant miraculin-like gene enhanced induction contributes to the hexanoic acid priming effect in this Solanaceae species. Moreover, our data evidencing that OPDA might be involved in this gene regulation highlights its potential as biomarker in eggplant plant responses to stress mediated this oxylipin signaling pathway.

Tribolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity.

In Tribolium castaneum larvae we have demonstrated by RNA interference knockdown that the Bacillus thuringiensis Cry3Ba toxin receptors Cadherin-like and Sodium solute symporter proteins are also functional receptors of the less active Cry3Aa toxin. Differences in susceptibility to B. thuringiensis infection might not only rely on toxin-receptor interaction but also on host defense mechanisms. We compared the expression of the immune related genes encoding Apolipophorin-III and two antimicrobial peptides, Defensin3 and Defensin2 after B. thuringiensis challenge. All three genes were up-regulated following Cry3Ba spore-crystal intoxication whereas only Defensins gene expression was induced upon Cry3Aa spore-crystal treatment, evidencing a possible association between host immune response and larval susceptibility to B. thuringiensis. We assessed the antimicrobial activity spectra of T. castaneum defensins peptide fragments and found that a peptide fragment of Defensin3 was effective against the human microbial pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans, being S. aureus the most susceptible one.

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.

Sodium solute symporter and cadherin proteins act as Bacillus thuringiensis Cry3Ba toxin functional receptors in Tribolium castaneum.

Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that this protein is not relevant in the Cry3Ba toxin mode of action in Tc. Remarkable features of TcSSS protein were the presence of cadherin repeats in its amino acid sequence and that a TcSSS peptide fragment containing a sequence homologous to a binding epitope found in Manduca sexta and Tenebrio molitor Bt cadherin functional receptors enhanced Cry3Ba toxicity. This is the first time that the involvement of a sodium solute symporter protein as a Bt functional receptor has been demonstrated. The role of this novel receptor in Bt toxicity against coleopteran insects together with the lack of receptor functionality of aminopeptidase N proteins might account for some of the differences in toxin specificity between Lepidoptera and Coleoptera insect orders.

Tribolium castaneum Apolipophorin-III acts as an immune response protein against Bacillus thuringiensis Cry3Ba toxic activity.

In this study, a 2.1-fold Apolipophorin-III mRNA up-regulation was found in Tribolium castaneum larvae challenged with Bacillus thuringiensis Cry3Ba spore-crystal mixture. Knockdown of Apolipophorin-III by RNAi resulted in increased T. castaneum larvae susceptibility following Cry3Ba spore-crystal treatment, demonstrating Apolipophorin-III involvement in insect defense against B. thuringiensis. We showed that Apolipophorin-III participates in T. castaneum immune response to B. thuringiensis activating the prophenoloxidase cascade since: (i) phenoloxidase activity significantly increased after Cry3Ba spore-crystal treatment compared to untreated or Cry1Ac spore-crystal treated larvae and (ii) phenoloxidase activity in Cry3Ba spore-crystal treated Apolipophorin-III silenced larvae was 71±14% lower than that of non-silenced intoxicated larvae.

Proteome response of Tribolium castaneum larvae to Bacillus thuringiensis toxin producing strains.

Susceptibility of Tribolium castaneum (Tc) larvae was determined against spore-crystal mixtures of five coleopteran specific and one lepidopteran specific Bacillus thuringiensis Cry toxin producing strains and those containing the structurally unrelated Cry3Ba and Cry23Aa/Cry37Aa proteins were found toxic (LC(50) values 13.53 and 6.30 µg spore-crystal mixture/µL flour disc, respectively). Using iTRAQ combined with LC-MS/MS allowed the discovery of seven novel differentially expressed proteins in early response of Tc larvae to the two active spore-crystal mixtures. Proteins showing a statistically significant change in treated larvae compared to non-intoxicated larvae fell into two major categories; up-regulated proteins were involved in host defense (odorant binding protein C12, apolipophorin-III and chemosensory protein 18) and down-regulated proteins were linked to metabolic pathways affecting larval metabolism and development (pyruvate dehydrogenase Eα subunit, cuticular protein, ribosomal protein L13a and apolipoprotein LI-II). Among increased proteins, Odorant binding protein C12 showed the highest change, 4-fold increase in both toxin treatments. The protein displayed amino acid sequence and structural homology to Tenebrio molitor 12 kDa hemolymph protein b precursor, a non-olfactory odorant binding protein. Analysis of mRNA expression and mortality assays in Odorant binding protein C12 silenced larvae were consistent with a general immune defense function of non-olfactory odorant binding proteins. Regarding down-regulated proteins, at the transcriptional level, pyruvate dehydrogenase and cuticular genes were decreased in Tc larvae exposed to the Cry3Ba producing strain compared to the Cry23Aa/Cry37Aa producing strain, which may contribute to the developmental arrest that we observed with larvae fed the Cry3Ba producing strain. Results demonstrated a distinct host transcriptional regulation depending upon the Cry toxin treatment. Knowledge on how insects respond to Bt intoxication will allow designing more effective management strategies for pest control.

Functional significance of membrane associated proteolysis in the toxicity of Bacillus thuringiensis Cry3Aa toxin against Colorado potato beetle.

Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when compared to the wild type toxin and impaired ability to compete CPB brush border membrane associated cleavage of an ADAM fluorogenic substrate. Although the proteolytic profile of Cry3Aa toxin mutants generated by brush border membrane associated proteases was similar to that of Cry3Aa toxin, the metalloprotease inhibitor 1,10-phenanthroline was less efficient on the proteolysis of mutants than on that of the wild type toxin. The relevance of the Cry3Aa-ADAM interaction through the predicted recognition sequence was further confirmed by analyzing the effect of membrane integrity disturbance on Cry3Aa toxin membrane associated proteolysis and CPB larvae toxicity. Data support that Cry3Aa proteolysis, as a result of the interaction with ADAM through the Cry3Aa recognition motif, is essential for Cry3Aa toxic action in CPB. Detailed knowledge of Cry3Aa interaction with CPB midgut membrane should facilitate the development of more effective Bt based products against this devastating pest and other Coleoptera.

Identification of a calmodulin-binding site within the domain I of Bacillus thuringiensis Cry3Aa toxin.

Bacillus thuringiensis Cry3Aa toxin is a coleopteran specific toxin highly active against Colorado Potato Beetle (CPB).We have recently shown that Cry3Aa toxin is proteolytically cleaved by CPB midgut membrane associated metalloproteases and that this cleavage is inhibited by ADAM metalloprotease inhibitors. In the present study, we investigated whether the Cry3Aa toxin is a calmodulin (CaM) binding protein, as it is the case of several different ADAM shedding substrates. In pull-down assays using agarose beads conjugated with CaM, we demonstrated that Cry3Aa toxin specifically binds to CaM in a calcium-independent manner. Furthermore, we used gel shift assays and (1)H NMR spectra to demonstrate that CaM binds to a 16-amino acid synthetic peptide corresponding to residues N256-V271 within the domain I of Cry3Aa toxin. Finally, to investigate whether CaM has any effect on Cry3Aa toxin CPB midgut membrane associated proteolysis, cleavage assays were performed in the presence of the CaM-specific inhibitor trifluoperazine. We showed that trifluoperazine significantly increased Cry3Aa toxin proteolysis and also decreased Cry3Aa larval toxicity.

An ADAM metalloprotease is a Cry3Aa Bacillus thuringiensis toxin receptor.

Bacillus thuringiensis insecticidal proteins toxic action relies on the interaction with receptor molecules on insect midgut target cells. Here, we describe an ADAM metalloprotease as a novel type of B. thuringiensis toxin receptor on the basis of the following data: (i) by ligand blot and N-terminal analysis, we detected a Colorado potato beetle Cry3Aa toxin binding molecule that shares homology with an ADAM10 metalloprotease; (ii) Colorado potato beetle brush border membrane vesicles display ADAM activity since it cleaves an ADAM fluorogenic substrate; (iii) Cry3Aa acts as a competitor of the cleavage of the ADAM fluorogenic substrate; (iv) Cry3Aa sequence contains the recognition motif R(345)FQPGYYGND(354) present in ADAM10 substrates. Accordingly, a peptide representative of the recognition motif localized within loop 1 of Cry3Aa domain II (Ac-F(341)HTRFQPGYYGNDSFN(358)-NH(2)) effectively prevented Cry3Aa proteolytic processing and nearly abolished pore formation, evidencing the functional significance of the Cry3Aa-ADAM interaction in relation to this toxin mode of action.

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins.

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.