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1.
Braz. j. biol ; 83: e246436, 2023. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1339391

RESUMO

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.


Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.


Assuntos
Animais , Proteínas Hemolisinas/genética , Mariposas , Fósforo , Proteínas de Bactérias/genética , Resistência a Inseticidas , Plantas Geneticamente Modificadas/genética , Endotoxinas/genética , Fertilizantes , Toxinas de Bacillus thuringiensis , Larva , Nitrogênio
2.
Int J Mol Sci ; 23(21)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36361800

RESUMO

Methionine aminopeptidases (MetAPs) catalyze the cleavage of the N-terminal initiator methionine (iMet) in new peptide chains and arylamides, which is essential for protein and peptide synthesis. MetAP is differentially expressed in two diamondback moth (DBM; Plutella xylostella) strains: the G88 susceptible strain and the Cry1S1000 strain, which are resistant to the Bt toxin Cry1Ac, implicating that MetAP expression might be associated with Bt resistance. In this study, we identified and cloned a MetAP gene from DBMs, named PxMetAP1, which has a CDS of 1140 bp and encodes a 379 amino acid protein. The relative expression of PxMetAP1 was found to be ~2.2-fold lower in the Cry1S1000 strain compared to that in the G88 strain. PxMetAP1 presents a stage- and tissue-specific expression pattern, with higher levels in the eggs, adults, integument, and fatbody of DBMs. The linkage between PxMetAP1 and Cry1Ac resistance is verified by genetic linkage analysis. The knockout of PxMetAP1 in G88 by CRISPR/Cas9 leads to a ~5.6-fold decrease in sensitivity to the Cry1Ac toxin, further supporting the association between the PxMetAP1 gene and Bt tolerance. Our research sheds light on the role of MetAP genes in the development of Bt tolerance in P. xylostella and enriches the knowledge for the management of such a cosmopolitan pest.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Resistência a Inseticidas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mariposas/metabolismo , Metionil Aminopeptidases/metabolismo , Metionina/metabolismo , Larva/metabolismo
3.
Toxins (Basel) ; 14(11)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36356018

RESUMO

Chemical products still represent the most common form of controlling crop pests and diseases. However, their extensive use has led to the selection of resistances. This makes the finding of new solutions paramount to countering the economic losses that pests and diseases represent in modern agriculture. Bacillus thuringiensis (Bt) is one of the most reliable alternatives to chemical-based solutions. In this study, we aimed to further expand the global applicability of Bt strains beyond their spores and crystals. To this end, we selected a new Bt strain (BST-122) with relevant toxicity factors and tested its activity against species belonging to different phyla. The spore and crystal mixture showed toxicity to coleopterans. Additionally, a novel Cry5-like protein proved active against the two-spotted spider mite. In vivo and plant assays revealed significant control of the parasitic nematode, Meloidogyne incognita. Surprisingly, our data indicated that the nematocidal determinants may be secreted. When evaluated against phytopathogenic fungi, the strain seemed to decelerate their growth. Overall, our research has highlighted the potential of Bt strains, expanding their use beyond the confinements of spores and crystals. However, further studies are required to pinpoint the factors responsible for the wide host range properties of the BST-122 strain.


Assuntos
Bacillus thuringiensis , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas/metabolismo , Endotoxinas/metabolismo , Proteínas de Bactérias/metabolismo
4.
Appl Environ Microbiol ; 88(20): e0119422, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36200769

RESUMO

The Cry proteins from Bacillus thuringiensis (Bt) are major insecticidal toxins in formulated Bt sprays and are expressed in genetically engineered Bt crops for insect pest control. However, the widespread application of Bt toxins in the field imposes strong selection pressure on target insects, leading to the evolution of insect resistance to the Bt toxins. Identification and understanding of mechanisms of insect resistance to Bt toxins are an important approach for dissecting the modes of action of Bt toxins and providing knowledge necessary for the development of resistance management technologies. In this study, cabbage looper (Trichoplusia ni) strains resistant to the transgenic dual-Bt toxin WideStrike cotton plants, which express Bt toxins Cry1Ac and Cry1F, were selected from T. ni strains resistant to the Bt formulation Bt-DiPel. The WideStrike-resistant T. ni larvae were confirmed to be resistant to both Bt toxins Cry1Ac and Cry1F. From the WideStrike-resistant T. ni, the Cry1F resistance trait was further isolated to establish a T. ni strain resistant to Cry1F only. The levels of Cry1F resistance in the WideStrike-resistant and the Cry1F-resistant strains were determined, and the inheritance of the Cry1F-resistant trait in the two strains was characterized. Genetic association analysis of the Cry1F resistance trait indicated that the Cry1F resistance in T. ni isolated in this study is not shared with the Cry1Ac resistance mechanism nor is it associated with a mutation in the ABCC2 gene, as has so far been reported in Cry1F-resistant insects. IMPORTANCE Insecticidal toxins from Bacillus thuringiensis (Bt) are highly effective for insect control in agriculture. However, the widespread application of Bt toxins exerts strong selection for Bt resistance in insect populations. The continuing success of Bt biotechnology for pest control requires the identification of resistance and understanding of the mechanisms of resistance to Bt toxins. Cry1F is an important Bt toxin used in transgenic cotton, maize, and soybean varieties adopted widely for insect control. To understand the mode of action of Cry1F and mechanisms of Cry1F resistance in insects, it is important to identify Cry1F-specific resistance and the resistance mechanisms. In this study, Trichoplusia ni strains resistant to commercial "WideStrike" cotton plants that express Bt toxins Cry1Ac and Cry1F were selected, and a Cry1F-specific resistant strain was isolated. The isolation of the novel Cry1F-specific resistance in the T. ni provided an invaluable biological system to discover a Cry1F-specific novel resistance mechanism.


Assuntos
Bacillus thuringiensis , Brassica , Mariposas , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/metabolismo , Endotoxinas/genética , Endotoxinas/farmacologia , Endotoxinas/metabolismo , Gossypium/genética , Gossypium/metabolismo , Brassica/metabolismo , Mariposas/genética , Mariposas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Larva/genética , Larva/metabolismo , Insetos , Resistência a Inseticidas/genética
5.
Toxins (Basel) ; 14(10)2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-36287921

RESUMO

The three-domain Cry4Aa toxin produced from Bacillus thuringiensis subsp. israelensis was previously shown to be much more toxic to Culex mosquito larvae than its closely related toxin-Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from Culex quinquefasciatus larvae, Cq-mALP1263and Cq-mALP1264, predicted to be GPI-linked was cloned and functionally expressed in Spodoptera frugiperda (Sf9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that both Cq-mALP isoforms share significant sequence similarity to Aedes aegypti-mALP-a Cry4Ba toxin receptor. In cytotoxicity assays, Sf9 cells expressing Cq-mALP1264, but not Cq-mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each Cq-mALP expressed on the cell membrane surface. Molecular docking of the Cq-mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to Cq-mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain-DII, including Thr512, Tyr513 and Lys514 in the ß10-ß11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain-DIII to interact with such a Culex counterpart receptor. Ala-substitutions of selected ß10-ß11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against C. quinquefasciatus larvae. Further substitution of Lys514 with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔGbind) in Cry4Aa-Cq-mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔGbind value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position. Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same Culex membrane-bound ALP isoform-Cq-mALP1264 for mediating differential toxicity against Culex target larvae.


Assuntos
Aedes , Bacillus thuringiensis , Culex , Animais , Toxinas de Bacillus thuringiensis , Culex/metabolismo , Proteínas Hemolisinas/genética , Endotoxinas/toxicidade , Endotoxinas/química , Larva/metabolismo , Fosfatase Alcalina/metabolismo , Simulação de Acoplamento Molecular , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Proteínas de Bactérias/química , Bacillus thuringiensis/genética , Aedes/genética , Isoformas de Proteínas
6.
Sci Rep ; 12(1): 16706, 2022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36202979

RESUMO

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.


Assuntos
Bacillus thuringiensis , Mariposas , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Agrícolas/genética , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Humanos , Resistência a Inseticidas/genética , Larva/genética , Mariposas/genética , Mariposas/metabolismo , Plantas Geneticamente Modificadas/genética , RNA Guia/metabolismo , Zea mays/genética
7.
Nat Commun ; 13(1): 6024, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224245

RESUMO

Maintaining fitness during pathogen infection is vital for host survival as an excessive response can be as detrimental as the infection itself. Fitness costs are frequently associated with insect hosts countering the toxic effect of the entomopathogenic bacterium Bacillus thuringiensis (Bt), which delay the evolution of resistance to this pathogen. The insect pest Plutella xylostella has evolved a mechanism to resist Bt toxins without incurring significant fitness costs. Here, we reveal that non-phosphorylated and phosphorylated forms of a MAPK-modulated transcription factor fushi tarazu factor 1 (FTZ-F1) can respectively orchestrate down-regulation of Bt Cry1Ac toxin receptors and up-regulation of non-receptor paralogs via two distinct binding sites, thereby presenting Bt toxin resistance without growth penalty. Our findings reveal how host organisms can co-opt a master molecular switch to overcome pathogen invasion with low cost, and contribute to understanding the underlying mechanism of growth-defense tradeoffs during host-pathogen interactions in P. xylostella.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Medicamentos de Ervas Chinesas , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Insetos/metabolismo , Resistência a Inseticidas/genética , Larva/metabolismo , Fatores de Transcrição/metabolismo
8.
Pest Manag Sci ; 78(12): 5234-5242, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36053801

RESUMO

BACKGROUND: Crops genetically engineered to make insect-killing proteins from Bacillus thuringiensis (Bt) have revolutionized management of some pests. However, the benefits of such transgenic crops are reduced when pests evolve resistance to Bt toxins. We evaluated resistance to Bt toxins and Bt cotton plants using laboratory bioassays and complementary field trials focusing on Helicoverpa zea, one of the most economically important pests of cotton and other crops in the United States. RESULTS: The data from 235 laboratory bioassays demonstrate resistance to Cry1Ac, Cry1Fa, and Cry2Ab occurred in most of the 95 strains of H. zea derived from Arkansas, Louisiana, Mississippi, Tennessee, and Texas during 2016 to 2021. Complementary field data show efficacy decreased for Bt cotton producing Cry1Ac + Cry1Fa or Cry1Ac + Cry2Ab, but not Cry1Ac + Cry1Fa + Vip3Aa. Moreover, analysis of data paired by field site and year shows higher survival in bioassays was generally associated with lower efficacy of Bt cotton. CONCLUSIONS: The results confirm and extend previous evidence showing widespread practical resistance of H. zea in the United States to the Cry toxins produced by Bt cotton and corn, but not to Vip3Aa. Despite deployment in combination with Cry toxins in Bt crops, Vip3Aa effectively acts as a single toxin against H. zea larvae that are highly resistant to Cry toxins. Furthermore, Vip3Aa adoption is increasing and previous work provided an early warning of field-evolved resistance. Thus, rigorous resistance management measures are needed to preserve the efficacy of Vip3Aa against this highly adaptable pest. © 2022 Society of Chemical Industry.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Estados Unidos , Toxinas de Bacillus thuringiensis , Bacillus thuringiensis/genética , Zea mays/metabolismo , Gossypium/metabolismo , Resistência a Inseticidas , Proteínas de Bactérias/farmacologia , Proteínas Hemolisinas/farmacologia , Endotoxinas/farmacologia , Produtos Agrícolas/metabolismo , Plantas Geneticamente Modificadas/metabolismo
9.
Pest Manag Sci ; 78(12): 5150-5163, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36070208

RESUMO

BACKGROUND: Brazil is the largest grower of the world's 26 million ha of sugarcane, Saccharum officinarum. Pest damage mainly by the sugarcane borer, Diatraea saccharalis (F.), is a great challenge to the sugarcane industry. To control D. saccharalis, Brazil launched the world's first commercial use of Bt sugarcane in 2017. As part of the resistance management programs for Bt sugarcane planting, 535 F2 isoline families of D. saccharalis collected from three major sugarcane planting states (Goiás, Minas Gerais and São Paulo) in Brazil during 2019-2020 were screened for resistance to two Bt sugarcane varieties: CTC20BT expressing Cry1Ab and CTC9001BT expressing Cry1Ac. Here we report the results of the first study related to Bt resistance in a sugarcane cropping system. RESULTS: Larval survivorships of these families in an F2 screen on CTC20BT were highly correlated with their survival on CTC9001BT, whereas the Cry1Ac tissues exhibited greater insecticidal activities than Cry1Ab. Resistance allele frequencies (RAFs) for populations from Goiás and Minas Gerais were relatively low at 0.0034 for Cry1Ab and 0.0045 to Cry1Ac. By contrast, RAFs for São Paulo populations were considerably greater (0.0393 to Cry1Ab, 0.0245 to Cry1Ac). CONCLUSIONS: RAFs to Cry1Ab and Cry1Ac varied among Brazilian D. saccharalis populations. Prior selection resulting from an intensive use of single-gene Bt maize under low compliance of refuge planting could be a main factor contributing to the high RAF in São Paulo. The results suggest that mitigation measures including sufficient non-Bt maize refuge planting, effective resistance monitoring, and use of pyramided Bt sugarcane traits should be implemented promptly to prevent further increase in the RAF to ensure the sustainable use of Bt sugarcane in Brazil. MINI ABSTRACT: To control Diatraea saccharalis, Brazil launched the world's first commercial use of Bt sugarcane in 2017. As part of the resistance management programs for Bt sugarcane planting in Brazil, 535 F2 isoline families of D. saccharalis collected from three major sugarcane planting states (Goiás, Minas Gerais and São Paulo) in Brazil during 2019-2020 were screened for resistance to Cry1Ab and Cry1Ac sugarcane plants Resistance allele frequencies (RAFs) for the populations from Goiás and Minas Gerais were relatively low at 0.0034 for Cry1Ab and 0.0045 to Cry1Ac. By contrast, RAFs for the São Paulo populations were considerably greater (0.0393 to Cry1Ab, 0.0245 to Cry1Ac). Prior selection resulting from an intensive use of single-gene Bt maize under low compliance of non-Bt maize refuge planting could be a main factor contributing to the high RAF in São Paulo. The results suggest that effective mitigation measures including sufficient non-Bt maize refuge planting, effective resistance monitoring and use of pyramided Bt sugarcane traits should be implemented promptly to prevent further increase in the RAF to ensure the sustainable use of Bt sugarcane in Brazil. © 2022 Society of Chemical Industry.


Assuntos
Mariposas , Saccharum , Animais , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas/farmacologia , Endotoxinas/farmacologia , Brasil , Alelos , Proteínas de Bactérias/farmacologia , Zea mays/genética , Grão Comestível , Plantas Geneticamente Modificadas
10.
Mol Biol Rep ; 49(11): 10557-10564, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36169899

RESUMO

BACKGROUND: Cotton is continuously exposed to sucking and chewing insect pest pressure since emergence to harvesting. Pink bollworm (Pectinophora gossypiella) has become major chewing insect pest to reduce the cotton yield and results in bad lint quality even in transgenic crops. The efficiency of insecticidal genes has been compromised due to extensive utilization of transgenic crops. METHODS AND RESULTS: The present study was conducted to evaluate the efficacy of an alternate cry1Ia12 insecticidal gene against pink bollworm (PBW) in cotton. Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA2300 expression vector containing cry1Ia12 gene under the control of 35S CaMV was used to transform a local cotton cultivar GS-01. The various molecular analyses revealed the transgene integration and expression in primary transformants. Among five selected transgenic plants, tcL-08 showed maximum (16.06-fold) mRNA expression of cry1Ia12 gene whereas tcL-03 showed minimum (2.33-fold) expression. Feeding bioassays of 2nd and 3rd instar pink bollworm (PBW) larvae on immature cotton bolls, flowers and cotton squares revealed up to 33.33% mortality on tcL-08 while lowest mortality (13.33%) was observed in tcL-03 and tcL-15. Furthermore, the average weight and size of survived larvae fed on transgenic plants was significantly lesser than the average weight of larvae survived on non-transgenic plants. CONCLUSIONS: The present study suggests the cry1Ia12 gene as an alternate insecticidal gene for the resistance management of cotton bollworms, especially PBW.


Assuntos
Inseticidas , Lepidópteros , Mariposas , Animais , Lepidópteros/genética , Toxinas de Bacillus thuringiensis , Inseticidas/farmacologia , Proteínas Hemolisinas/genética , Endotoxinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mariposas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Larva/genética , Larva/metabolismo , Gossypium/genética , Gossypium/metabolismo , Controle de Pragas , Resistência a Inseticidas/genética
11.
Sci Rep ; 12(1): 13580, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35945334

RESUMO

The fall armyworm (Spodoptera frugiperda) is a highly polyphagous lepidopteran pest of relevant food and fiber staple crops. In the Americas, transgenic corn and cotton producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have controlled and reduced the damage caused by S. frugiperda. However, cases of field-evolved S. frugiperda resistance to Bt corn producing the Cry1F insecticidal protein have been documented in North and South America. When characterized, field resistance to Cry1F is linked to insertions and mutations resulting in a modified or truncated ABC transporter subfamily C2 (SfABCC2) protein that serves as Cry1F receptor in susceptible S. frugiperda. In this work, we present detection of a large genomic deletion (~ 8 kb) affecting the SfABCC2 and an ABC transporter gene subfamily 3 -like gene (SfABCC3) as linked to resistance to Cry1F corn in a S. frugiperda strain from Florida (FL39). Monitoring for this genomic deletion using a discriminatory PCR reaction in field-collected S. frugiperda moths detected individuals carrying this allele in Florida, but not in surrounding states. This is the first report of a large genomic deletion being involved in resistance to a Bt insecticidal protein.


Assuntos
Bacillus thuringiensis , Inseticidas , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/genética , Florida , Genômica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Plantas Geneticamente Modificadas/genética , Spodoptera/metabolismo , Zea mays/genética , Zea mays/metabolismo
12.
Biol Open ; 11(9)2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36017723

RESUMO

The δ-endotoxin Cry4Aa from Bacillus thuringiensis israelensis (Bti) has insecticidal characteristics specific to insects of the order Diptera. Although Cry4Aa has shown potential as an effective proteinaceous pesticide against mosquitoes, it has an ultraviolet (UV)-intolerant property that limits its outdoor use. Our previous research showed that protein microcrystal polyhedra from Bombyx mori cypovirus can encapsulate diverse foreign proteins and maintain long-term protein activity under hostile environmental conditions, including UV irradiation. In this study, we report the development of polyhedra encapsulating the Cry4Aa insecticidal activity domain by using a modified baculovirus expression system. We confirmed the oral intake of recombinant polyhedra introduced into the experimental environment by the larvae of a mosquito, Aedes albopictus, and delivery of encapsulated proteins into the digestive tract. The polyhedra encapsulating partial Cry4Aa showed mosquito larvicidal activity during incubation of larvae with 50% lethal-dose value of 23.717×104 cubes for 10 Aedes albopictus larvae in 1 ml water. In addition, polyhedra showed a specific property to reduce the impact of UV-C irradiation on the activity of encapsulated partial Cry4Aa, thus demonstrating the effectiveness of encapsulating Bti δ-endotoxins inside polyhedra to increase the availability of proteinaceous pesticides for outdoor use for mosquito control.


Assuntos
Aedes , Bacillus thuringiensis , Praguicidas , Reoviridae , Aedes/metabolismo , Animais , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Endotoxinas/química , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Larva/metabolismo , Praguicidas/metabolismo , Praguicidas/farmacologia , Reoviridae/metabolismo , Água/metabolismo
13.
J Agric Food Chem ; 70(36): 11419-11428, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36040024

RESUMO

Rapid evolution of resistance in crop pests to Bacillus thuringiensis (Bt) products threatens their widespread use, especially as pests appear to develop resistance through a range of different physiological adaptations. With such a diverse range of mechanisms reported, researchers have resorted to multi-omic approaches to understand the molecular basis of resistance. Such approaches generate a lot of data making it difficult to establish where causal links between physiological changes and resistance exist. In this study, a combination of RNA-Seq and iTRAQ was used with a strain of diamondback moth, Plutella xylostella (L.), whose resistance mechanism is well understood. While some of the causal molecular changes in the resistant strain were detected, other previously verified changes were not detected. We suggest that while multi-omic studies have use in validating a proposed resistance mechanism, they are of limited value in identifying such a mechanism in the first place.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Larva/genética , Mariposas/genética
14.
Int J Biol Macromol ; 219: 587-596, 2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-35952810

RESUMO

The diamondback moth (DBM), Plutella xylostella (L.), has evolved resistance to multiple insecticides including Bacillus thuringiensis (Bt). ATP-binding cassette (ABC) transporters are a class of transmembrane protein families, involved in multiple physiological processes and pesticide resistances in insects. However, the role and regulatory mechanism of ABC transporter in mediating the response to Bt Cry1Ac toxin remain unclear. Here, we characterized a MAPK signaling pathway-enriched ABCG subfamily gene PxABCG20 from DBM, and found it was differentially expressed in the Cry1Ac-resistant and Cry1Ac-susceptible strains. RNAi knockdown of PxABCG20 increased the tolerance of DBM to Cry1Ac protoxin. To explore the regulatory mechanism of PxABCG20 expression, we predicted the potential miRNAs targeting PxABCG20 using two target prediction algorithms. Luciferase reporter assay confirmed that novel-miR-310 was able to down-regulate PxABCG20 expression in HEK293T cells. Furthermore, injection of novel-miR-310 agomir markedly inhibited PxABCG20 expression, resulting in increased tolerance to Cry1Ac protoxin in susceptible strain, while injection of novel-miR-310 antagomir markedly induced the expression of PxABCG20, leading to decreased tolerance to Cry1Ac protoxin. Our work provides theoretical basis for exploring novel targets for the DBM response to Cry1Ac toxin and expands the understanding of miRNA role in mediating the susceptibility of insect pest to Cry1Ac toxin.


Assuntos
Bacillus thuringiensis , Inseticidas , MicroRNAs , Mariposas , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antagomirs/metabolismo , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Células HEK293 , Humanos , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/metabolismo
15.
Sci Total Environ ; 851(Pt 2): 158120, 2022 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-35987246

RESUMO

The widespread adoption of Bt crops expressing insecticidal proteins derived from Bacillus thuringiensis has created a need to assess the potential effects of these toxins on non-target organisms, especially species such as Arma custos, a generalist predator that provides important biological control services in many field crops in Asia. Direct dietary exposure of A. custos to Cry1Ah and Vip3Aa proteins produced no adverse effects on life history traits, despite continuous exposure throughout development and early adult life to concentrations significantly higher than the Bt protein concentration likely encountered by A.custos in the field, even when feeding directly on Bt plants. Enzyme-linked immunosorbent assay confirmed the presence of Bt proteins in A. custos midguts, but quantitative real-time PCR analysis of 12 genes associated with detoxification, antioxidative responses, immune responses, and metabolism revealed no significant changes in expression in adult bugs. Indirect exposure to these toxins via consumption of intoxicated prey, larvae of Helicoverpa armigera (Hübner), likewise produced no negative impacts on survival, development, adult weight, or female fecundity in either the F0 (exposed) or F1 (unexposed) generation, but female fresh weight was reduced in the F0 generation by the Cry1Ah (50 µg/g) treatment. Finally, a competitive binding assay with labelled protein and a ligand blotting assay both demonstrated that the Cry1Ah protein could not bind to receptors on the midgut brush border membrane vesicles (BBMVs) of A. custos adults. Therefore, we conclude that Cry1Ah and Vip3Aa proteins are unlikely to have significant negative effects on A. custos populations if employed as plant-incorporated protectants in field crops.


Assuntos
Bacillus thuringiensis , Heterópteros , Animais , Bacillus thuringiensis/genética , Ligantes , Proteínas de Bactérias/genética , Toxinas de Bacillus thuringiensis , Endotoxinas/toxicidade , Endotoxinas/genética , Larva , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/genética
16.
J Agric Food Chem ; 70(37): 11510-11519, 2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-35944165

RESUMO

New insecticidal genes and approaches for pest control are a hot research area. In the present study, we explored a novel strategy for the generation of insecticidal proteins. The midgut cadherin of Helicoverpa armigera (H. armigera) was used as a target to screen materials that have insecticidal activity. After three rounds of panning, the phage-displayed human domain antibody B1F6, which not only binds to the H. armigera cadherin CR9-CR11 but also significantly inhibits Cry1Ac toxins from binding to CR9-CR11, was obtained from a phage-displayed human domain antibody (DAb) library. To better analyze the relevant activity of B1F6, soluble B1F6 protein was expressed by Escherichia coli BL21 (DE3). The cytotoxicity assays demonstrated that soluble B1F6 induced Sf9 cell death when expressing H. armigera cadherin on the cell membrane. The insect bioassay results showed that soluble B1F6 protein (90 µg/cm2) caused 49.5 ± 3.3% H. armigera larvae mortality. The midgut histological results showed that soluble B1F6 caused damage to the midgut epithelium of H. armigera larvae. The present study explored a new strategy and provided a basic material for the generation of new insecticidal materials.


Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Caderinas/genética , Caderinas/metabolismo , Endotoxinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Fragmentos de Imunoglobulinas/metabolismo , Inseticidas/química , Larva/genética , Larva/metabolismo , Mariposas/metabolismo
17.
PLoS One ; 17(8): e0272311, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35921368

RESUMO

Western corn rootworm (WCR), Diabrotica virgifera virgifera, LeConte, is an insect pest that poses a significant threat to the productivity of modern agriculture, causing significant economic and crop losses. The development of genetically modified (GM) crops expressing one or more proteins that confer tolerance to specific insect pests, such as WCR, was a historic breakthrough in agricultural biotechnology and continues to serve as an invaluable tool in pest management. Despite this, evolving resistance to existing insect control proteins expressed in current generation GM crops requires continued identification of new proteins with distinct modes of action while retaining targeted insecticidal efficacy. GM crops expressing insecticidal proteins must undergo extensive safety assessments prior to commercialization to ensure that they pose no increased risk to the health of humans or other animals relative to their non-GM conventional counterparts. As part of these safety evaluations, a weight of evidence approach is utilized to assess the safety of the expressed insecticidal proteins to evaluate any potential risk in the context of dietary exposure. This study describes the food and feed safety assessment of Vpb4Da2, a new Bacillus thuringiensis insecticidal protein that confers in planta tolerance to WCR. Vpb4Da2 exhibits structural and functional similarities to other insect control proteins expressed in commercialized GM crops. In addition, the lack of homology to known toxins or allergens, a lack of acute toxicity in mice, inactivation by conditions commonly experienced in the human gut or during cooking/food processing, and the extremely low expected dietary exposure to Vpb4Da2 provide a substantial weight of evidence to demonstrate that the Vpb4Da2 protein poses no indication of a risk to the health of humans or other animals.


Assuntos
Bacillus thuringiensis , Besouros , Inseticidas , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Produtos Agrícolas/metabolismo , Endotoxinas/metabolismo , Humanos , Resistência a Inseticidas , Inseticidas/farmacologia , Larva , Camundongos , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Zea mays/genética , Zea mays/metabolismo
18.
Sci Rep ; 12(1): 11639, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35804088

RESUMO

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious pest of maize (Zea mays L.) in the U.S. Corn Belt and parts of Europe. Transgenic maize hybrids expressing at least one of the four currently available insecticidal toxins from Bacillus thuringiensis (Bt) Berliner, currently the most widely adopted control method in continuous maize, have faltered due to the emergence of resistance. The resistance mechanisms of WCR to Bt toxins are not fully understood. We identified metabolic profiles of susceptible and resistant WCR larvae fed on maize hybrids expressing each of three available Cry3 proteins (eCry3Ab1, mCry3A, and Cry3Bb1) targeting corn rootworms and a control non-Bt maize via an untargeted metabolomics approach. Over 580 unique metabolites found in WCR larvae were classified into different pathways (amino acids, carbohydrates, cofactors and vitamins, energy, lipid, nucleotide, peptide, and xenobiotics). By exploring shifts in WCR larval metabolome exclusively by Bt toxins, several candidate metabolites and metabolic pathways were identified in susceptible and resistant larvae that may be involved in defense against or recovery from Bt ingestion by these larvae. These findings would provide mechanistic insights into altered metabolic pathways associated with the resistance mechanisms of WCR to Bt toxins.


Assuntos
Bacillus thuringiensis , Besouros , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Besouros/genética , Endotoxinas/genética , Endotoxinas/toxicidade , Resistência a Inseticidas/genética , Larva , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Plântula , Zea mays/genética
19.
Sci Rep ; 12(1): 12529, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35869123

RESUMO

The insecticidal crystalline proteins (Crys) are a family of insect endotoxin functioning in crop protection. As insects keep evolving into tolerance to the existing Crys, it is necessary to discover new Cry proteins to overcome potential threatens. Crys possess three functional domains at their N-termini, and the most active region throughout evolution was found at the domain-III. We swapped domain-IIIs from various Cry proteins and generated seven chimeric proteins. All recombinants were expressed in Escherichia coli and their toxicity was assessed by dietary exposure assays. Three of the seven Crys exhibited a high toxicity to Asian corn borer over the controls. One of them, Cry1Ab-Gc, a chimeric Cry1Ab being replaced with the domain-III of Cry1Gc, showed the highest toxicity to rice stem borer when it was over-expressed in Oryza sativa. Furthermore, it was also transformed into maize, backcrossed into commercial maize inbred lines and then produced hybrid to evaluate their commercial value. Transgenic maize performed significant resistance to the Asian corn borer without affecting the yield. We further showed that this new protein did not have adverse effects on the environment. Our results indicated that domain III swapped of Crys could be used as an efficient method for developing new engineered insecticidal protein.


Assuntos
Bacillus thuringiensis , Inseticidas , Oryza , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Insetos/metabolismo , Inseticidas/metabolismo , Inseticidas/farmacologia , Oryza/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/metabolismo , Zea mays/genética , Zea mays/metabolismo
20.
Toxins (Basel) ; 14(8)2022 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-35893749

RESUMO

To control the fall armyworm (FAW), Spodoptera frugiperda (Smith), a serious threat to maize production in China, the Chinese government has issued biosafety certificates for transgenic insect-resistant maize expressing Bt (Bacillus thuringiensis) toxins including Bt-Cry1Ab maize (crop event DBN9936), Bt-Vip3Aa maize (event DBN9501), Bt-(Cry1Ab+Vip3Aa) maize with superimposed traits (event DBN9936 × DBN9501) and Bt-(Cry1Ab+Vip3Aa) maize with superimposed traits (event Bt11 × MIR162), but the susceptibility baselines of geographically distinct FAW populations to these events, which form the basis for managing resistance development in the pest to these events, are not clear. We used the diet-incorporated bioassays method to detect the susceptibilities of the seven FAW populations collected from Yunnan, Henan and Hubei provinces in China in 2021 to the insecticidal proteins of the four Bt maize events. The result showed that the susceptibilities of different geographical populations to Bt insecticidal proteins were significantly different. In the seven populations, the range in median lethal concentrations (LC50) of Cry1Ab expressed in DBN9936 was 0.87-2.63 µg/g, 0.14-0.30 µg/g for Vip3Aa expressed in DBN9501, 0.78-1.86 µg/g for Cry1Ab+Vip3Aa expressed in DBN9936 × DBN9501, and 0.36-1.42 µg/g for CryAb+Vip3Aa expressed in Bt11 × MIR162. The growth inhibition responses also showed that the susceptibilities varied with the different median growth inhibitory concentration (GIC50) ranges (0.38-1.22, 0.08-0.28, 0.28-0.87, and 0.24-0.78 µg/g, respectively). The variations in the ranges of the susceptibility baselines of the geographical populations of fall armyworm in China to the insecticidal proteins expressed in the four events provide a scientific basis for monitoring FAW population resistance to Bt maize and managing the populations using different Bt maize events.


Assuntos
Inseticidas , Zea mays , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/toxicidade , China , Endotoxinas/genética , Endotoxinas/metabolismo , Endotoxinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Spodoptera/metabolismo , Zea mays/metabolismo
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