Toxicity and binding capacity of Cry1 proteins to Helicoverpa armigera (Lepidoptera: Noctuidae) intestine receptors

O objetivo deste trabalho foi avaliar a toxicidade e a capacidade de ligação das proteínas Cry1Aa, Cry1Ab, Cry1Ac e Cry1Ca, de Bacillus thuringiensis, a receptores intestinais de Helicoverpa armigera. Realizou-se análise de ligação das proteínas ativadas às vesículas de membrana da microvilosidade apical (VMMA) do intestino médio de H. armigera, além de ensaios de competição heteróloga para avaliar sua capacidade de ligação. Cry1Ac destacou-se como a proteína mais tóxica, seguida por Cry1Ab e Cry1Aa. A proteína Cry1Ca não foi tóxica às lagartas e, portanto, não foi possível determinar os seus parâmetros de toxicidade CL50 e CL90. As proteínas Cry1Aa, Cry1Ab e Cry1Ac são capazes de se ligar a um mesmo receptor nas membranas intestinais, o que aumenta o risco do desenvolvimento de resistência cruzada. Portanto, a utilização conjunta dessas proteínas deve ser evitada.The objective of this work was to evaluate the toxicity and the binding capacity of the Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ca proteins, from Bacillus thuringiensis, to Helicoverpa armigera intestine receptors. Binding analysis of the activated proteins to the brush‑border membrane vesicles (BBMV) in the midgut of H. armigera, besides heterologous competition assays to evaluate their binding capacity, was performed. Cry1Ac stood out as the most toxic protein, followed by Cry1Ab and Cry1Aa. The Cry1Ca protein had no toxicity to the caterpillars and, therefore, it was not possible to evaluate its LC50 and LC90 toxicity parameters. The Cry1Aa, Cry1Ab, and Cry1Ac proteins are able to bind themselves to the same receptor in the midgut membrane, which increases the risk of developing cross‑resistance. Therefore, the use of these proteins together should be avoided

Interação de proteínas Cry1 e Vip3A de Bacillus thuringiensis para controle de lepidópteros‑praga

The objective of this work was to evaluate the susceptibility of Anticarsia gemmatalis (Lepidoptera: Erebidae) and Chrysodeixis includens (Lepidoptera: Noctuidae) caterpillars to Cry1 and Vip3A proteins, as well as to determine if there is any interaction between these proteins on the control of the two species. Bioassays with both isolated and combined proteins were carried out, and lethal concentrations LC50 and LC90 were estimated for each condition. Cry1Aa, Cry1Ac, and Vip3Af were the more effective proteins for the control of A. gemmatalis, while Cry1Ac, Vip3Aa, and Vip3Af were more effective for the control of C. includens. Cry1Ac and Cry1Ca proteins caused the highest inhibition to the development of larvae that survived the LC50 dose in both species. Different combinations of Vip3A and Cry1 have synergistic effect in the control of both species, and the combination Vip3Aa + Cry1Ea showed an outstanding control of A. gemmatalis and C. includens. These proteins are promising for building pyramided plants for the simultaneous control of the pests.O objetivo deste trabalho foi avaliar a suscetibilidade das lagartas Anticarsia gemmatalis (Lepidoptera: Erebidae) e Chrysodeixis includens (Lepidoptera: Noctuidae) às proteínas Cry1 e Vip3A, bem como determinar se há a interação entre essas proteínas no controle das duas espécies. Bioensaios com as proteínas isoladas e em combinações foram realizados, e as concentrações letais CL50 e CL90 foram estimadas para cada condição. As proteínas Cry1Aa, Cry1Ac e Vip3Af foram as mais efetivas no controle de A. gemmatalis, enquanto Cry1Ac, Vip3Aa e Vip3Af foram mais efetivas no de C. includens. As proteínas Cry1Ac e Cry1Ca causaram maior inibição do desenvolvimento das larvas sobreviventes à CL50, em ambas as espécies. Combinações entre Vip3A e Cry1 apresentam efeito sinérgico no controle das espécies e a combinação Vip3Aa+Cry1Ea destaca-se no controle de A. gemmatalis e C. includens. Essas proteínas combinadas são promissoras na construção de plantas piramidadas, para o controle simultâneo das pragas

Interações moleculares das proteínas CRY1 e VIP3 no controle de lepidópteros em cana-de-açúcar

O potencial biotecnológico das proteínas Cry e Vip provenientes da bactéria Bacillus thuringiensis é amplamente conhecido. Porém, a evolução da resistência de pragas é a principal ameaça a longo prazo do controle de insetos-praga por plantas transgênicas com toxinas desta bactéria. Estudos relatam a necessidade de se retardar a evolução da resistência e dentre as possibilidades, a utilização de mais de um gene na construção de plantas transgênicas mostra-se eficiente. Dessa forma, é importante buscar novos genes, com diferentes modos de ação, e selecionar os que apresentam atividade entomotóxica diferenciada para Diatraea flavipennella e Elasmopalpus lignosellus, que são pragas secundárias e potenciais da cana-de-açúcar. Para tanto, proteínas Cry1 e Vip3 foram expressas em Escherichia coli e a toxicidade verificada por meio de bioensaios com lagartas neonatas de ambas as espécies de insetos-praga. As proteínas foram purificadas, solubilizadas, ativadas com tripsina e biotiniladas. As BBMVs (Brush Border Membrane Vesicles) foram preparadas a partir dos intestinos de lagartas das duas espécies para realização de ensaios de competição homóloga e heteróloga. Considerando a CL50, as proteínas Cry1Ac e Vip3Aa foram as mais efetivas no controle dos insetos-praga em estudo. O ensaio de ligação mostrou que ocorreu interação entre todas as proteínas e os receptores das duas espécies de lagartas. Os ensaios de competição heteróloga demonstraram não haver competição entre as proteínas Cry1 e Vip3 pelos mesmos sítios de ligação para ambas espécies de insetos estudadas. Os resultados obtidos sugerem a combinação de Cry1Aa ou Cry1Ac com Vip3Aa para futuros eventos de geração de cana-de-açúcar geneticamente modificada o que poderia contribuir para o manejo da resistência por estes insetos.The biotechnological potential of Cry and Vip proteins from the bacterium Bacillus thuringiensis is widely known. However, the evolution of pest resistance is a major threat to long-term control of insect pests by transgenic plants with toxins of this bacterium. Studies have reported the need to slow down the evolution of resistance and, among the possibilities, the use of more than one gene in the construction of transgenic plants is shown to be efficient. Thus, it is important to look for new genes with different modes of action, and select those with different entomotoxic activity to Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary and potential sugarcane pests. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli and the toxicity was verified by bioassays using neonate larvae of both species of the insect pests. The proteins were purified, solubilized, and activated with trypsin and biotinylated. The BBMVs (Brush Border Membrane Vesicles) were prepared using the intestines of the two species to perform the homologous and heterologous competition assays. Considering the LC50, the Cry1Ac and Vip3Aa proteins were the most effective in controlling the insect pests in this study. The binding assays showed that there was interaction between the proteins and the receptors of the two species of larvae. The heterologous competition assays showed no competition between Cry1 and Vip3 proteins for the same binding sites for both species studied. The results suggest that the combination of Cry1Aa or Cry1Ac with Vip3Aa for future events of sugarcane generation genetically modified could contribute to resistance management for these insects.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Proteínas Cry1 e Vip3A de Bacillus thuringiensis: sinergismo e efeito sub-letal no controle de Heliothis virescens

A bactéria Bacillus thuringiensis (Bt) possui a capacidade de produzir inclusões protéicas (proteína Cry) e proteínas vegetativas (Vip). Estas proteínas podem ser tóxicas para insetos e por meio de transgenia, a expressão em plantas, podem também proporcionar controle de importantes pragas agrícolas. Nesse sentido, esta pesquisa teve por objetivo avaliar o potencial de controle das proteínas Cry1Aa, Cry1Ac, Cry1Ca, Vip3A(1), Vip3A(2) e Vip3A(3) em uma população brasileira da lagarta-da-maçã, Heliothis virescens (Lepidoptera: Noctuidae), bem como o efeito subletal e efeito sinérgico entre estas proteínas e a proteólise pelo suco do intestino do inseto praga em estudo. Para tanto, clones de Escherichia coli recombinantes portadores de genes únicos foram cultivados em meio para a indução das proteínas e os lisados obtidos foram utilizados para as análises de toxicidade por meio de bioensaios. Diferentes concentrações protéicas foram utilizadas para conduzir os bioensaios. A mortalidade foi avaliada e obteve-se a CL50. Desta forma, observou-se que, dentre as proteínas testadas, Cry1Ac (CL50 39,89 ng.cm-2), Vip3A(2) (CL50 945,77 ng.cm-2) e Vip3A(3) (CL50 874,45 ng.cm-2 ) foram as mais tóxicas e houve correlação negativa entre a concentração de proteínas e o peso das lagartas. Nos ensaios referentes ao efeito sinérgico das proteínas, ativadas e não ativadas com tripsina comercial, foram encontradas possíveis combinações eficientes no controle da praga em estudo destacando-se Vip3A(2)/Cry1Aa, Vip3A(1)/Cry1Aa, Vip3A(1)/Cry1Ac, e Vip3A(2)/Cry1Ac. Os resultados referentes à interação das enzimas digestivas do intestino de H. virescens com as toxinas Cry1 e Vip3A permitiram constatar que as proteínas são ativadasBacillus thuringiensis (Bt) produces protein inclusions (Cry proteins) and vegetative proteins (Vip). Such proteins may act as toxic to some insects and through transgenesis plants they may be able to control important agricultural pests. Thus this work aimed to evaluate the control potential of Cry1Aa, Cry1Ac, Cry1Ca, Vip3A(1), Vip3A(2) and Vip3A(3) proteins in a Brazilian population of Heliothis virescens (tobacco budworm) (Lepidoptera: Noctuidae), as well as to analyze the sublethal and the synergic effects among these proteins and the proteolysis on the intestinal juice of this pest. In order to do this, Escherichia coli clones expressing each one of the above mentioned proteins were induced to produce them and the obtained lysates were used to determine the level of toxicity through bioassays with neonatal larvae of H. virescens. Different protein concentrations were used to carry out the bioassays. The mortality was evaluated and it was possible to detect the CL50. It was possible to observe that among the tested proteins, Cry1Ac (CL50 39.89 ng.cm-2), Vip3A(2) (CL50 945,77 ng.cm-2) and Vip3A (3) (CL50 874,45 ng.cm-2) were the most toxic and showed a negative correlation between the protein concentration and larvae weight. During the bioassays concerning the synergistic effect of these proteins, which were either previously activated or not using commercial trypsin, there we found efficient combinations for the control of the pest under study in this work, being that the combinations Vip3A(2)/Cry1Aa, Vip3A(1)/Cry1Aa, Vip3A(1)/Cry1Ac and Vip3A(2)/Cry1Ac were considered the best ones. The results with reference to the interaction of the digestive enzymes from the intestine of H. virescens larvae when using the toxins Cry1 and Vip3A allowed us to detect that these proteins are activatedCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Toxicidade e capacidade de ligação de proteínas Cry1 a receptores intestinais de Helicoverpa armigera (Lepidoptera: Noctuidae)

Resumo: O objetivo deste trabalho foi avaliar a toxicidade e a capacidade de ligação das proteínas Cry1Aa, Cry1Ab, Cry1Ac e Cry1Ca, de Bacillus thuringiensis, a receptores intestinais de Helicoverpa armigera. Realizou-se análise de ligação das proteínas ativadas às vesículas de membrana da microvilosidade apical (VMMA) do intestino médio deH. armigera, além de ensaios de competição heteróloga para avaliar sua capacidade de ligação. Cry1Ac destacou-se como a proteína mais tóxica, seguida por Cry1Ab e Cry1Aa. A proteína Cry1Ca não foi tóxica às lagartas e, portanto, não foi possível determinar os seus parâmetros de toxicidade CL50 e CL90. As proteínas Cry1Aa, Cry1Ab e Cry1Ac são capazes de se ligar a um mesmo receptor nas membranas intestinais, o que aumenta o risco do desenvolvimento de resistência cruzada. Portanto, a utilização conjunta dessas proteínas deve ser evitada

Interaction of Cry1 and Vip3A proteins of Bacillus thuringiensis for the control of lepidopteran insect pests

O objetivo deste trabalho foi avaliar a suscetibilidade das lagartas Anticarsia gemmatalis (Lepidoptera: Erebidae) e Chrysodeixis includens (Lepidoptera: Noctuidae) às proteínas Cry1 e Vip3A, bem como determinar se há a interação entre essas proteínas no controle das duas espécies. Bioensaios com as proteínas isoladas e em combinações foram realizados, e as concentrações letais CL50 e CL90 foram estimadas para cada condição. As proteínas Cry1Aa, Cry1Ac e Vip3Af foram as mais efetivas no controle de A. gemmatalis, enquanto Cry1Ac, Vip3Aa e Vip3Af foram mais efetivas no de C. includens. As proteínas Cry1Ac e Cry1Ca causaram maior inibição do desenvolvimento das larvas sobreviventes à CL50, em ambas as espécies. Combinações entre Vip3A e Cry1 apresentam efeito sinérgico no controle das espécies e a combinação Vip3Aa+Cry1Ea destaca-se no controle de A. gemmatalis e C. includens. Essas proteínas combinadas são promissoras na construção de plantas piramidadas, para o controle simultâneo das pragas.The objective of this work was to evaluate the susceptibility of Anticarsia gemmatalis (Lepidoptera: Erebidae) and Chrysodeixis includens (Lepidoptera: Noctuidae) caterpillars to Cry1 and Vip3A proteins, as well as to determine if there is any interaction between these proteins on the control of the two species. Bioassays with both isolated and combined proteins were carried out, and lethal concentrations LC50 and LC90 were estimated for each condition. Cry1Aa, Cry1Ac, and Vip3Af were the more effective proteins for the control of A. gemmatalis, while Cry1Ac, Vip3Aa, and Vip3Af were more effective for the control of C. includens. Cry1Ac and Cry1Ca proteins caused the highest inhibition to the development of larvae that survived the LC50 dose in both species. Different combinations of Vip3A and Cry1 have synergistic effect in the control of both species, and the combination Vip3Aa + Cry1Ea showed an outstanding control of A. gemmatalis and C. includens. These proteins are promising for building pyramided plants for the simultaneous control of the pests

Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane

The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests

Evaluation of antagonism, at the LC₅₀ and LC₉₀ level, of combinations of Vip3A and Cry1 protoxins to <i>H. virescens</i> neonate larvae.

a<p>Each data point was obtained from three replicates of 16 larvae per replicate (n = 48).</p>b<p>Proportions of proteins were chosen approximately to match those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107196#pone-0107196-t002" target="_blank">Table 2</a>.</p>c<p>Slope ± standard error.</p>d<p>FL₉₅: 95% fiducial limits.</p>e<p>Expected mortality considering simple similar action.</p>f<p>AF: Antagonism factor, calculated as the ratio of the observed LC₅₀ over the expected LC₅₀. NS = not significant.</p><p>Evaluation of antagonism, at the LC₅₀ and LC₉₀ level, of combinations of Vip3A and Cry1 protoxins to <i>H. virescens</i> neonate larvae.</p

Susceptibility of <i>H. virescens</i> neonate larvae to Cry1 and Vip3A protoxins.^a

a<p>Values represent the mean from three replicates of 16 larvae per replicate (n = 48).</p>b<p>Slope ± standard error.</p>c<p>Values are expressed as µg/cm² with 95% fiducial limits (at 5 days).</p><p>Susceptibility of <i>H. virescens</i> neonate larvae to Cry1 and Vip3A protoxins.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107196#nt101" target="_blank">a</a>}</p