RESUMO
Bacillus thuringiensis (Bt) is known for its Cry and Vip3A pesticidal proteins with high selectivity to target pests. Here, we assessed the potential of a novel neotropical Bt strain (UFT038) against six lepidopteran pests, including two Cry-resistant populations of fall armyworm, Spodoptera frugiperda. We also sequenced and analyzed the genome of Bt UFT038 to identify genes involved in insecticidal activities or encoding other virulence factors. In toxicological bioassays, Bt UFT038 killed and inhibited the neonate growth in a concentration-dependent manner. Bt UFT038 and HD-1 were equally toxic against S. cosmioides, S. frugiperda (S_Bt and R_Cry1 + 2Ab populations), Helicoverpa zea, and H. armigera. However, larval growth inhibition results indicated that Bt UFT038 was more toxic than HD-1 to S. cosmioides, while HD-1 was more active against Chrysodeixis includens. The draft genome of Bt UFT038 showed the cry1Aa8, cry1Ac11, cry1Ia44, cry2Aa9, cry2Ab35, and vip3Af5 genes. Besides this, genes encoding the virulence factors (inhA, plcA, piplC, sph, and chi1-2) and toxins (alo, cytK, hlyIII, hblA-D, and nheA-C) were also identified. Collectively, our findings reveal the potential of the Bt UFT038 strain as a source of insecticidal genes against lepidopteran pests, including S. cosmioides and S. frugiperda.
Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Animais , Humanos , Recém-Nascido , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Glycine max , Endotoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Inseticidas/farmacologia , Inseticidas/metabolismo , Spodoptera/metabolismo , Larva , Fatores de Virulência/metabolismo , Controle Biológico de VetoresRESUMO
BACKGROUND: Brea gum (BG) is an exudate from the Cercidium praecox tree that grows in semi-arid regions of Argentina. Some previous studies on BG have shown physicochemical characteristics and functional features similar to those of gum arabic. However, there is a need to elucidate the molecular structure of BG to understand the functionality. In this sense, BG was fractionated using hydrophobic interaction chromatography and the obtained fractions were analyzed by size exclusion chromatography. RESULTS: Analysis of the fractions showed that the bulk of the gum (approx. 84% of the polysaccharides) was a polysaccharide of 2.79 × 10(3) kDa. The second major fraction (approx. 16% of the polysaccharides) was a polysaccharide-protein complex with a molecular mass of 1.92 × 10(5) kDa. A third fraction consisted of protein species with a wide range of molecular weights. The molecular weight distribution of the protein fraction was analyzed by size exclusion chromatography. Comparison of the elution profiles of the exudates in native and reducing conditions revealed that some of the proteins were forming aggregates through disulfide bridges in native conditions. Further analysis of the protein fraction by SDS-PAGE showed proteins with molecular weight ranging from 6.5 to 66 kDa. CONCLUSIONS: The findings showed that BG consists of several fractions with heterogeneous chemical composition and polydisperse molecular weight distributions. © 2016 Society of Chemical Industry.
Assuntos
Fabaceae/química , Gomas Vegetais/química , Proteínas de Plantas/análise , Polissacarídeos/análise , Argentina , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Clima Desértico , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Fabaceae/crescimento & desenvolvimento , Aditivos Alimentares/análise , Aditivos Alimentares/química , Goma Arábica/química , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , Oxirredução , Fenóis/análise , Fenóis/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Proteínas de Plantas/química , Polissacarídeos/química , Agregados Proteicos/efeitos dos fármacos , Substâncias Redutoras/farmacologia , Reagentes de Sulfidrila/farmacologiaRESUMO
Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae.
Assuntos
Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Resistência a Inseticidas , Plantas Geneticamente Modificadas/parasitologia , Spodoptera/efeitos dos fármacos , Zea mays/parasitologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/enzimologia , Proteínas Hemolisinas/genética , Ligação Proteica , Porto Rico , Spodoptera/fisiologia , Estados UnidosRESUMO
Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.