Throwing Brazilian strains into the melting pot of P. xylostella resistance to Bacillus thuringiensis.

The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.

Impact of Bt corn expressing Bacillus thuringiensis Berliner insecticidal proteins on the growth and survival of Spodoptera frugiperda larvae in Colombia.

Bioassays were conducted under controlled conditions to determine the response of Spodoptera frugiperda (J. E. Smith) larvae fed with corn materials expressing Bacillus thuringiensis (Bt) insecticidal endotoxins: (1) VT Double Pro® (VT2P) expressing Cry1A.105-Cry2Ab2 proteins and (2) VT Triple Pro® (VT3P) expressing Cry1A.105-Cry2Ab2-Cry3Bb1 proteins. The parameters assessed were: (i) mortality rate, and (ii) growth inhibition (GI) with respect to the control. To conduct this study, larvae were collected from commercial non-Bt corn fields, in four agricultural sub-regions in Colombia, between 2018 and 2020. Fifty-two populations were assessed from the field and neonate larvae from each of the populations were used for the bioassays. The study found that mortality rates in the regions for larvae fed with VT2P corn ranged from 95.1 to 100.0%, with a growth inhibition (%GI) higher than 76.0%. Similarly, mortality rate for larvae fed with VT3P corn were between 91.4 and 100.0%, with a %GI above 74.0%. The population collected in Agua Blanca (Espinal, Tolima; Colombia) in 2020, showed the lowest mortality rate of 53.2% and a %GI of 73.5%, with respect to the control. The population that exhibited the lowest %GI was collected in 2018 in Agua Blanca (Espinal, Tolima, Colombia) with a 30.2%, growth inhibition, with respect to the control. In recent years, the use of plant tissue to monitor susceptibility to fall armyworm has proven to be useful in the resistance management program for corn in Colombia determining that the FAW populations are still susceptible to Bt proteins contained in VT2P and VT3P.

Residual Effects of Transgenic Cotton on the Intestinal Microbiota of Dysdercus concinnus.

The interaction among plants, insects, and microbes (PIM) is a determinant factor for the assembly and functioning of natural and anthropic ecosystems. In agroecosystems, the relationships among PIM are based on the interacting taxa, environmental conditions, and agricultural management, including genetically modified (GM) organisms. Although evidence for the unintended effects of GM plants on non-target insects is increasingly robust, our knowledge remains limited regarding their impact on gut microbes and their repercussions on the host's ecology, especially in the wild. In this study, we compared the gut microbial community of Dysdercus concinnus bugs collected on wild cotton (Gossypium hirsutum), with and without insecticidal transgenes (cry1ab/ac), in its center of origin and diversity. By sequencing the V4-V5 region of 16S rRNA, we show differences in the diversity, structure, and topology of D. concinnus gut microbial interactions between specimens foraging cotton plants with and without transgenes. Identifying unintended residual effects of genetic engineering in natural ecosystems will provide first-line knowledge for informed decision-making to manage genetic, ecological, and evolutionary resources. Thus, determining which organisms interact with GM plants and how is the first step toward conserving natural ecosystems with evidence of transgenic introgression.

A major conformational change of N-terminal helices of Bacillus thuringiensis Cry1Ab insecticidal protein is necessary for membrane insertion and toxicity.

Pore forming toxins rely on oligomerization for membrane insertion to kill their targets. Bacillus thuringiensis produces insecticidal Cry-proteins composed of three domains that form pores that kill the insect larvae. Domain I is involved in oligomerization and membrane insertion, whereas Domains II and III participate in receptor binding and specificity. However, the structural changes involved in membrane insertion of these proteins remain unsolved. The most widely accepted model for membrane insertion, the 'umbrella model', proposed that the α-4/α-5 hairpin of Domain I swings away and is inserted into the membrane. To determine the topology of Cry1Ab in the membrane, disulfide bonds linking α-helices of Domain I were introduced to restrict their movement. Disulfide bonds between helices α-2/α-3 or α-3/α-4 lost oligomerization and toxicity, indicating that movement of these helices is needed for insecticidal activity. By contrast, disulfide bonds linking helices α-5/α-6 did not affect toxicity, which contradicts the 'umbrella model'. Additionally, Föster resonance energy transfer closest approach analyses measuring distances of different points in the toxin to the membrane plane and collisional quenching assays analysing the protection of specific fluorescent-labeled residues to the soluble potassium iodide quencher in the membrane inserted state were performed. Overall, the data show that Domain I from Cry1Ab may undergo a major conformational change during its membrane insertion, where the N-terminal region (helices α-1 to α-4) participates in oligomerization and toxicity, probably forming an extended helix. These data break a paradigm, showing a new 'folding white-cane model', which better explains the structural changes of Cry toxins during insertion into the membrane.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

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.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

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.

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.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton

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.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

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.(AU)

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.(AU)

Varied frequencies of resistance alleles to Cry1Ab and Cry1Ac among Brazilian populations of the sugarcane borer, Diatraea saccharalis (F.).

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.

MON 95379 Bt maize as a new tool to manage sugarcane borer (Diatraea saccharalis) in South America.

BACKGROUND: The sugarcane borer (SCB), Diatraea saccharalis (Lepidoptera: Crambidae), is a key pest of maize in Argentina, and genetically modified maize, producing Bacillus thuringiensis (Bt) proteins, has revolutionized the management of this insect in South America. However, field-evolved resistance to some Bt technologies has been observed in SCB in Argentina. Here we assessed a new Bt technology, MON 95379, in the laboratory, greenhouse and field for efficacy against SCB. RESULTS: In a laboratory leaf disc bioassay, both MON 95379 (producing Cry1B.868 and Cry1Da_7) and Cry1B.868_single maize (producing only Cry1B.868) resulted in 100% mortality of SCB. The level of Cry1B.868 in the Cry1B.868_single maize is comparable to that in MON 95379 maize. However, the Cry1Da_7 protein does not have high efficacy against SCB, as evidenced by < 20% mortality on Cry1Da_7_single leaf tissue. Total (100%) mortality of SCB in a Cry1B.868_single tissue dilution bioassay indicated that Cry1B.868_single maize meets the criteria to be classified as a high dose. Similar median lethal concentration (LC50 ) values were observed for MON 89034-R and susceptible SCB strains exposed to Cry1B.868 protein. MON 95379 also controlled SCB strains resistant to MON 89034 (Cry1A.105/Cry2Ab2) and Cry1Ab. Under field conditions in Brazil and Argentina, MON 95379 maize plants were consistently protected from SCB damage. CONCLUSION: MON 95379 maize will bring value to maize growers in South America by effectively managing SCB even in locations where resistance to other Bt-containing maize technologies has been reported. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Recombinant Expression of ABCC2 Variants Confirms the Importance of Mutations in Extracellular Loop 4 for Cry1F Resistance in Fall Armyworm.

Fall armyworm (FAW), Spodoptera frugiperda, is a highly destructive and invasive global noctuid pest. Its control is based on insecticide applications and Bacillus thuringiensis (Bt) insecticidal Cry toxins expressed in transgenic crops, such as Cry1F in Bt corn. Continuous selection pressure has resulted in populations that are resistant to Bt corn, particularly in Brazil. FAW resistance to Cry1F was recently shown to be conferred by mutations of ATP-binding cassette transporter C2 (ABCC2), but several mutations, particularly indels in extracellular loop 4 (ECL4), are not yet functionally validated. We addressed this knowledge gap by baculovirus-free insect cell expression of ABCC2 variants (and ABCC3) by electroporation technology and tested their response to Cry1F, Cry1A.105 and Cry1Ab. We employed a SYTOXTM orange cell viability test measuring ABCC2-mediated Bt toxin pore formation. In total, we tested seven different FAW ABCC2 variants mutated in ECL4, two mutants modified in nucleotide binding domain (NBD) 2, including a deletion mutant lacking NBD2, and S. frugiperda ABCC3. All tested ECL4 mutations conferred high resistance to Cry1F, but much less to Cry1A.105 and Cry1Ab, whereas mutations in NBD2 hardly affected Bt toxin activity. Our study confirms the importance of indels in ECL4 for Cry1F resistance in S. frugiperda ABCC2.

Differential capability of Bacillus thuringiensis Cry1Ac protoxin and toxin to induce in vivo activation of dendritic cells and B lymphocytes.

The insecticidal Bacillus thuringiensis protein Cry1Ac is produced as a protoxin and becomes activated to a toxin when ingested by larvae. Both proteins are immunogenic and able to activate macrophages. The proposed mechanism of immunostimulation by Cry1Ac protoxin has been related to its capacity to activate antigen-presenting cells (APC), but its ability to activate dendritic cells (DC) has not been explored. Here we evaluated, in the popliteal lymph nodes (PLN), spleen and peritoneum, the activation of DC CD11c+ MHC-II+ following injection with single doses (50 µg) of Cry1Ac toxin or protoxin via the intradermal (i.d.) and intraperitoneal (i.p.) routes in C57BL/6 mice. In vivo stimulation with both Cry1Ac proteins induced activation of DC via upregulation of CD86, primarily in PLN 24 h after i. d. injection. Moreover, this activation was detected in DC, displaying CD103+, a typical marker of migratory DC, while upregulation of CD80 was uniquely induced by toxin. Tracking experiments showed that Cy5-labeled Cry1Ac proteins could rapidly reach the PLN and localize near DC, but some label remained in the footpad. When the capacity of Cry1Ac-activated DC to induce antigen presentation was examined, significant proliferation of naïve T lymphocytes was induced exclusively by the protoxin. The protoxin elicited a Th17-biased cytokine profile. Moreover, only the Cry1Ac toxin induced a pronounced proliferation of B cells from both untreated and Cry1Ac-injected mice, suggesting that it acts as a polyclonal activator. In conclusion, Cry1Ac protoxin and toxin show a distinctive capacity to activate APCs.

Cryptochrome 1a depends on blue light fluence rate to mediate osmotic stress responses in tomato.

The participation of plant cryptochromes in water deficit response mechanisms has been highlighted in several reports. However, the role of tomato (Solanum lycopersicum L.) cryptochrome 1a (cry1a) in the blue light fluence-dependent modulation of the water deficit response remains largely elusive. The tomato cry1a mutant and its wild-type counterpart were grown in water (no stress) or PEG6000 (osmotic stress) treatments under white light (60 µmol m-2 s-1) or from low to high blue light fluence (1, 5, 10, 15 and 25 µmol m-2 s-1). We first demonstrate that under nonstress conditions cry1a regulates seedling growth by mechanisms that involve pigmentation, lipid peroxidation and osmoprotectant accumulation in a blue light-dependent manner. In addition, we further highlighted under osmotic stress conditions that cry1a increased tomato growth by reduced malondialdehyde (MDA) and proline accumulation. Although blue light is an environmental signal that influences osmotic stress responses mediated by tomato cry1a, specific blue light fluence rates are required during these responses. Here, we show that CRY1a manipulation may be a potential biotechnological target to develop a drought-tolerant tomato variety. Nevertheless, the complete understanding of this phenomenon requires further investigation.

Cryptochrome 1a of tomato mediates long-distance signaling of soil water deficit.

Although the blue light photoreceptors cryptochromes mediate the expression of genes related to reactive oxygen species, whether cryptochrome 1a (cry1a) regulates local and long-distance signaling of water deficit in tomato (Solanum lycopersicum L.) is unknown. Thus the cry1a tomato mutant and its wild-type (WT) were reciprocally grafted (WT/WT; cry1a/cry1a; WT/cry1a; cry1a/WT; as scion/rootstock) or grown on their own roots (WT and cry1a) under irrigated and water deficit conditions. Plant growth, pigmentation, oxidative stress, water relations, stomatal characteristics and leaf gas exchange were measured. WT and cry1a plants grew similarly under irrigated conditions, whereas cry1a plants had less root biomass and length and higher tissue malondialdehyde concentrations under water deficit. Despite greater oxidative stress, cry1a maintained chlorophyll and carotenoid concentrations in drying soil. Lower stomatal density of cry1a likely increased its leaf relative water content (RWC). In grafted plants, scion genotype largely determined shoot and root biomass accumulation irrespective of water deficit. In chimeric plants grown in drying soil, cry1a rootstocks increased RWC while WT rootstocks maintained photosynthesis of cry1a scions. Manipulating tomato CRY1a may enhance plant drought tolerance by altering leaf pigmentation and gas exchange during soil drying via local and long-distance effects.

Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

Molecular characterization of Cry1F resistance in fall armyworm, Spodoptera frugiperda from Brazil.

Fall armyworm, Spodoptera frugiperda (J.E. Smith) is a major lepidopteran pest of maize in Brazil and its control particularly relies on the use of genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins such as Cry1F. However, control failures compromising the efficacy of this technology have been reported in many regions in Brazil, but the mechanism of Cry1F resistance in Brazilian fall armyworm populations remained elusive. Here we investigated the molecular mechanism of Cry1F resistance in two field-collected strains of S. frugiperda from Brazil exhibiting high levels of Cry1F resistance. We first rigorously evaluated several candidate reference genes for normalization of gene expression data across strains, larval instars and gut tissues, and identified ribosomal proteins L10, L17 and RPS3A to be most suitable. We then investigated the expression pattern of ten potential Bt toxin receptors/enzymes in both neonates and 2nd instar gut tissue of Cry1F resistant fall armyworm strains compared to a susceptible strain. Next we sequenced the ATP-dependent Binding Cassette subfamily C2 gene (ABCC2) and identified three mutated sites present in ABCC2 of both Cry1F resistant strains: two of them, a GY deletion (positions 788-789) and a P799 K/R amino acid substitution, located in a conserved region of ABCC2 extracellular loop 4 (EC4) and another amino acid substitution, G1088D, but in a less conserved region. We further characterized the role of the novel mutations present in EC4 by functionally expressing both wild type and mutated ABCC2 transporters in insect cell lines, and confirmed a critical role of both sites for Cry1F binding by cell viability assays. Finally, we assessed the frequency of the mutant alleles by pooled population sequencing and pyrosequencing in 40 fall armyworm populations collected from maize fields in different regions in Brazil. We found that the GY deletion being present at high frequency. However we also observed many rare alleles which disrupt residues between sites 783-799, and their diversity and abundance in field collected populations lends further support to the importance of the EC4 domain for Cry1F toxicity.

Performance of Field-Collected Spodoptera frugiperda (Lepidoptera: Noctuidae) Strains Exposed to Different Transgenic and Refuge Maize Hybrids in Argentina.

Spodoptera frugiperda (J. E. Smith) is one of the major pests of maize in Argentina. The main tool for its control is the use of genetically modified maize hybrids expressing Bacillus thuringiensis (Bt) insecticidal proteins. Maize growers in Argentina initially controlled this pest with Bt maize that expressed a single Bt protein (Cry1Ab or Cry1F). Currently it is necessary to plant maize cultivars that produce two Bt proteins to achieve the satisfactory control. Recently, Cry1F field-evolved resistant populations of this species were confirmed in Argentina. The objective of this study was to evaluate the performance of S. frugiperda field-collected strains on different Bt and non-Bt maize hybrids. Strains were collected from non-Bt maize (T1), Agrisure TDMax (T2), Agrisure Viptera (T3), Agrisure Viptera 3110 (T4), Genuity VT Triple Pro (T5), and Power Core (T6). Three experiments were performed to 1) determine the survivorship and reproduction of field-collected larvae (F0) from Bt maize hybrids, 2) evaluate Cry1F resistance using an F1 screen, and 3) assess the performance of F1 strains on different maize hybrids. In the F0, the survivorship from larva to adult ranged from 0 to 63%. We obtained adults from only the T1, T2, T5, and T6 strains with no significant differences in the reproductive parameters. Continuously rearing F1 larvae on their collected hosts affected larval duration, which was significantly shorter for a known-laboratory Bt-susceptible strain than the field-collected strains. Our results support the existence of Cry1F-resistance alleles in S. frugiperda field populations in Argentina.

The insect-protected CTC91087-6 sugarcane event expresses Cry1Ac protein preferentially in leaves and presents compositional equivalence to conventional sugarcane.

A Cry1Ac-expressing sugarcane cultivar, CTC91087-6, has been developed by Centro de Tecnologia Canavieira (CTC) to be resistant to the sugarcane borer (Diatraea saccharalis). This genetically modified event was developed using Agrobacterium-mediated transformation and the help of the selectable marker phosphinothricin N-acetyltransferase (PAT) expressed from bar gene. We describe here a detailed characterization of CTC91087-6 event with respect to protein expression, nutritional composition, and assessment of its derived DNA and proteins in raw sugar. Expression of the Cry1Ac and PAT (bar) proteins produced by CTC91087-6 was evaluated in different tissues and at different times during the growing season. The new proteins are preferentially expressed in leaves, are produced at low levels in stalks, and are near the limits of detection in root tissues. The levels of Cry1Ac were much higher than PAT in all evaluated tissues. Furthermore, Cry1Ac levels in CTC91087-6 leaves are stable at various times during sugarcane cultivation cycle, assuring borer control throughout the complete crop cycle. Assessment of CTC91087-6 tissues for key food and feed nutrients as recommended by OECD to assess the safety of new varieties of sugarcane showed compositional equivalence to the conventional counterpart CTC9001 and to other commercial sugarcane varieties used as references. Raw sugar samples produced from CTC91087-6 did not contain DNA corresponding to cry1Ac and bar genes nor DNA specifically derived from CTC91087-6. In a similar way, there is no detection of Cry1Ac and PAT proteins in raw sugar produced from CTC91087-6. Taken together these results show that CTC91087-6 stably expresses Cry1Ac and PAT proteins and is substantially equivalent to the conventional counterpart CTC9001.

Bt-induced hormesis in Bt-resistant insects: Theoretical possibility or factual concern?

The biphasic dose-response of a stressor where low amounts of a toxicant may stimulate some biological processes is a recent focus of attention in insecticide ecotoxicology. Nonetheless, the importance and management consequences of this phenomenon of pesticide-induced hormesis remain largely unrecognized. Curiously, the potential induction of hormesis by insecticidal proteins such as Bacillus thuringiensis toxins (i.e., Bt toxins), a major agriculture pest management tool of widespread use, has been wholly neglected. Thus, we aimed to circumvent this shortcoming while assessing the potential occurrence of hormesis induced by the Bt toxin Cry1Fa in its main target pest species - the fall armyworm Spodoptera frugiperda. Concentration-response bioassays were carried out in a Bt-susceptible and a Bt-resistant population providing the purified Cry1Fa toxin in artificial diet and recording the insect demographic parameters. As significant hormetic effect was detected in both populations with a significant increase in the net reproductive rate and the intrinsic rate of population growth, the potential occurrence of Bt-induced hormesis was subsequently tested providing the insects with leaves from transgenic Bt maize expressing the toxic protein. The performance of the Bt-resistant insects was not different in both maize genotypes, indicating that the leaf expression of the Bt protein did not promote hormesis in the resistant insects. Thus, despite the Bt-induced hormesis detected in the purified protein bioassays, the phenomenon was not detected with current levels of Bt expression in maize minimizing the risk of this additional efficacy constraint besides that of field occurrence of Bt resistance.

Biochemical characterization of digestive membrane-associated alkaline phosphatase from the velvet bean caterpillar Anticarsia gemmatalis.

In Brazil, the use of transgenic plants expressing the insect-toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane-associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in-gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme-linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.