Toxicidade de proteínas bacterianas e análise in silico de Vip3A em população Cry-resistente de Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae)
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2023-02-28
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Universidade Estadual Paulista (Unesp)
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RESUMO - A lagarta-do-cartucho (Spodoptera frugiperda) é uma praga de alto impacto econômico em grandes culturas, como o milho. Para reduzir os prejuízos causados pelos ataques, são utilizadas cultivares transgênicos expressando proteínas de Bacillus thuringiensis (Bt). Entretanto, a alta pressão de seleção em campo favoreceu a seleção de populações Bt-resistentes dessa praga. Até o momento, não foram relatadas populações de S. frugiperda de campo resistentes a Vip3Aa20, proteína Bt expressa em milhos transgênicos para o controle de lepidópteros-praga. Portanto, é necessário explorar o potencial das proteínas de Bt e de outras espécies de bactérias no manejo da resistência. O objetivo desse trabalho foi avaliar a atividade inseticida de diferentes proteínas bacterianas de Bt, Xenorhabdus nematophila e Photorhabdus akhurstii em duas populações da lagarta-do-cartucho (suscetível e resistente às proteínas Cry1A.105, Cry2Ab2 e Cry1F de Bt). As proteínas Txp40 de P. akhurstii, XnGroel de X. nematophila e diferentes Bt (Vip3Aa20, Vip3Ae e Vip3Af) foram obtidas por expressão heteróloga em Escherichia coli BL21 (DE3) “One Shot”. Posteriormente, ensaios de toxicidade in vitro foram conduzidos para estimar a concentração letal média (CL50) para cada proteína. A CL50 foi estimada a partir de uma curva de nove doses, com quatro repetições de 16 lagartas/dose, previamente definidas para cada população. A comparação da cinética proteolítica também foi realizada para as populações suscetível e resistente de S. frugiperda. As proteínas testadas foram incubadas com o conteúdo intestinal de lagartas de 3º instar, nas condições de 30ºC a 100 rpm e amostras foram coletadas ao longo de 120 min. O produto da proteólise foi quantificado por densitometria para avaliar a intensidade das bandas referentes às proteínas após o processamento no intestino do inseto. As proteínas Txp40 e XnGroel não apresentaram toxicidade em ambas as populações. As CL50 estimadas de Vip3Aa20 e Vip3Af foram, respectivamente, 4x e 14x superior na população resistente de S. frugiperda em relação à população suscetível, e a de Vip3Ae foi semelhante entre as populações. Na cinética proteolítica, diferenças entre as populações avaliadas não foram identificadas. A atividade inseticida diferencial detectada em Vip3Ae foi correlacionada à estrutura molecular da proteína. A localização de motifs diferenciais na região C–terminal, onde a ausência de um sítio foi detectada para Vip3Ae e as alterações nos resíduos Glu(546)->Gly(546) e Asn(568)->Lys(568) do domínio IV também podem estar associadas a susceptibilidade das populações em relação a esta proteína. Os resultados obtidos neste trabalho são importantes para auxiliar no desenvolvimento de estratégias para manejo de resistência e prolongar a durabilidade das tecnologias Bt.
ABSTRACT - The fall armyworm (Spodoptera frugiperda) is a pest of high economic impact on large crops such as maize. To reduce damages caused by their attacks, transgenic cultivars expressing Bacillus thuringiensis (Bt) proteins have been adopted. However, the high selection pressure in the field has favored this pest Bt-resistant populations selection. Until now, no field populations of S. frugiperda resistant to Vip3Aa20, a Bt protein expressed in commercial transgenic maize used to control Lepidoptera pests. Therefore, explore the Bt proteins potential and other bacterial species is needed on resistance management. The aim of this work was to evaluate the insecticidal activity of different bacterial proteins from Bt, Xenorhabdus nematophila and Photorhabdus akhurstii in two fall armyworm populations (susceptible and resistant to Bt proteins Cry1A.105, Cry2Ab2 and Cry1F). Txp40 proteins from P. akhurstii, XnGroel from X. nematophila and different Bt (Vip3Aa20, Vip3Ae and Vip3Af) were obtained by heterologous expression in Escherichia coli BL21 (DE3) “One Shot”. Subsequently, in vitro toxicity assays were conducted to estimate the lethal concentration mean (LC50) for each protein. The LC50 was estimated by a nine doses curve, with four replications of 16 caterpillars/dose, previously defined for each population. The proteolytic kinetics comparison was also performed on susceptible and resistant S. frugiperda populations. The tested proteins were incubated with the intestinal contents of 3rd larvae instar, under 30ºC at 100 rpm conditions and the samples were collected during 120 min. The proteolysis product was quantified by densitometry to evaluate bands intensity referring the proteins after processing in the insect's intestine. The Txp40 and XnGroel proteins did not show toxicity in both populations. The estimated LC50 of Vip3Aa20 and Vip3Af were 4x and 14x higher, respectively, in S. frugiperda resistant population compared to the susceptible population, and the Vip3Ae CL50 was similar among populations. In proteolytic kinetics, differences among the evaluated populations were not identified. The differential insecticidal activity detected in Vip3Ae was correlated to the protein molecular structure. The differential motifs location on C–terminal region, where a site absence was detected for Vip3Ae and changes in Glu(546)->Gly(546) and Asn(568)->Lys(568) residues of domain IV may also be associated with populations performance compared to this protein. The results of this work are important to held the strategies development for resistance management and to extend Bt technologies durability.
ABSTRACT - The fall armyworm (Spodoptera frugiperda) is a pest of high economic impact on large crops such as maize. To reduce damages caused by their attacks, transgenic cultivars expressing Bacillus thuringiensis (Bt) proteins have been adopted. However, the high selection pressure in the field has favored this pest Bt-resistant populations selection. Until now, no field populations of S. frugiperda resistant to Vip3Aa20, a Bt protein expressed in commercial transgenic maize used to control Lepidoptera pests. Therefore, explore the Bt proteins potential and other bacterial species is needed on resistance management. The aim of this work was to evaluate the insecticidal activity of different bacterial proteins from Bt, Xenorhabdus nematophila and Photorhabdus akhurstii in two fall armyworm populations (susceptible and resistant to Bt proteins Cry1A.105, Cry2Ab2 and Cry1F). Txp40 proteins from P. akhurstii, XnGroel from X. nematophila and different Bt (Vip3Aa20, Vip3Ae and Vip3Af) were obtained by heterologous expression in Escherichia coli BL21 (DE3) “One Shot”. Subsequently, in vitro toxicity assays were conducted to estimate the lethal concentration mean (LC50) for each protein. The LC50 was estimated by a nine doses curve, with four replications of 16 caterpillars/dose, previously defined for each population. The proteolytic kinetics comparison was also performed on susceptible and resistant S. frugiperda populations. The tested proteins were incubated with the intestinal contents of 3rd larvae instar, under 30ºC at 100 rpm conditions and the samples were collected during 120 min. The proteolysis product was quantified by densitometry to evaluate bands intensity referring the proteins after processing in the insect's intestine. The Txp40 and XnGroel proteins did not show toxicity in both populations. The estimated LC50 of Vip3Aa20 and Vip3Af were 4x and 14x higher, respectively, in S. frugiperda resistant population compared to the susceptible population, and the Vip3Ae CL50 was similar among populations. In proteolytic kinetics, differences among the evaluated populations were not identified. The differential insecticidal activity detected in Vip3Ae was correlated to the protein molecular structure. The differential motifs location on C–terminal region, where a site absence was detected for Vip3Ae and changes in Glu(546)->Gly(546) and Asn(568)->Lys(568) residues of domain IV may also be associated with populations performance compared to this protein. The results of this work are important to held the strategies development for resistance management and to extend Bt technologies durability.
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Plantas transgênicas, Plantas Resistência aos insetos, Toxinas bacterianas, Pragas agrícolas Controle integrado, Estrutura molecular