RESUMEN
Synthetic chemical elicitors, so called plant strengtheners, can protect plants from pests and pathogens. Most plant strengtheners act by modifying defense signaling pathways, and little is known about other mechanisms by which they may increase plant resistance. Moreover, whether plant strengtheners that enhance insect resistance actually enhance crop yields is often unclear. Here, we uncover how a mechanism by which 4-fluorophenoxyacetic acid (4-FPA) protects cereals from piercing-sucking insects and thereby increases rice yield in the field. Four-FPA does not stimulate hormonal signaling, but modulates the production of peroxidases, H2O2, and flavonoids and directly triggers the formation of flavonoid polymers. The increased deposition of phenolic polymers in rice parenchyma cells of 4-FPA-treated plants is associated with a decreased capacity of the white-backed planthopper (WBPH) Sogatella furcifera to reach the plant phloem. We demonstrate that application of 4-PFA in the field enhances rice yield by reducing the abundance of, and damage caused by, insect pests. We demonstrate that 4-FPA also increases the resistance of other major cereals such as wheat and barley to piercing-sucking insect pests. This study unravels a mode of action by which plant strengtheners can suppress herbivores and increase crop yield. We postulate that this represents a conserved defense mechanism of plants against piercing-sucking insect pests, at least in cereals.
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Acetatos/farmacología , Conducta Alimentaria/efectos de los fármacos , Flavonoides , Hemípteros , Inmunidad de la Planta/efectos de los fármacos , Animales , Bioensayo , Productos Agrícolas/efectos de los fármacos , Flavonoides/análisis , Flavonoides/metabolismo , Herbivoria , Hordeum/efectos de los fármacos , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/metabolismo , Oryza/efectos de los fármacos , Peroxidasas/análisis , Peroxidasas/metabolismo , Control de Plagas/métodos , Hojas de la Planta/química , Triticum/efectos de los fármacosRESUMEN
Suberin in roots acts as a physical barrier preventing water/mineral losses. In Arabidopsis, root suberization is regulated by abscisic acid (ABA) and ethylene in response to nutrient stresses. ABA also mediates coordination between microbiota and root endodermis in mineral nutrient homeostasis. However, it is not known whether this regulatory system is common to plants in general, and whether there are other key molecule(s) involved. We show that serotonin acts downstream of ABA in regulating suberization in rice and Arabidopsis and negatively regulates suberization in rice roots in response to salinity. We show that ABA represses transcription of the key gene (OsT5H) in serotonin biosynthesis, thus promoting root suberization in rice. Conversely, overexpression of OsT5H or supplementation with exogenous serotonin represses suberization and reduces tolerance to salt stress. These results identify an ABA-serotonin regulatory module controlling root suberization in rice and Arabidopsis, which is likely to represent a general mechanism as ABA and serotonin are ubiquitous in plants. These findings are of significant importance to breeding novel crop varieties that are resilient to abiotic stresses and developing strategies for production of suberin-rich roots to sequestrate more CO2 , helping to mitigate the effects of climate change.
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Arabidopsis , Oryza , Ácido Abscísico/farmacología , Arabidopsis/fisiología , Dióxido de Carbono/farmacología , Etilenos/farmacología , Regulación de la Expresión Génica de las Plantas , Oryza/fisiología , Fitomejoramiento , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente , Salinidad , Tolerancia a la Sal , Serotonina/farmacología , Estrés Fisiológico , Agua/farmacologíaRESUMEN
Plant viruses can manipulate their hosts to release odours that are attractive or repellent to their insect vectors. However, the volatile organic compounds (VOCs), either individually or as mixtures, which play a key role in the olfactory behaviour of insect vectors remains largely unknown. Our study focused on green rice leafhoppers (GRLHs) vectoring rice dwarf virus (RDV) revealed that RDV infection significantly induced the emission of (E)-ß-caryophyllene and 2-heptanol by rice plants, which influenced the olfactory behaviour of both non-viruliferous and viruliferous GRLHs. (E)-ß-caryophyllene attracted non-viruliferous GRLHs to settle on RDV-infected plants, but neither attracted nor repelled viruliferous GRLHs. In contrast, 2-heptanol repelled viruliferous GRLHs to settle on RDV-infected plants, but neither repelled nor attracted non-viruliferous GRLHs. Suppression of (E)-ß-caryophyllene synthase OsCAS via CRISPR-Cas9 to generate oscas-1 plants enabled us to confirm the important role played by (E)-ß-caryophyllene in modulating the virus-vector-host plant interaction. These novel results reveal the role of these virus-induced VOCs in modulating the behaviour of its GRLH insect vector and may facilitate the design of new strategies for disease control through manipulation of plant volatile emissions.
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Hemípteros/efectos de los fármacos , Interacciones Huésped-Patógeno/fisiología , Oryza/virología , Reoviridae/patogenicidad , Compuestos Orgánicos Volátiles/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Enzimas/genética , Enzimas/metabolismo , Regulación de la Expresión Génica de las Plantas , Hemípteros/fisiología , Heptanol/metabolismo , Heptanol/farmacología , Repelentes de Insectos/metabolismo , Repelentes de Insectos/farmacología , Odorantes , Oryza/genética , Oryza/metabolismo , Enfermedades de las Plantas/virología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Virus de Plantas/patogenicidad , Plantas Modificadas Genéticamente , Sesquiterpenos Policíclicos/metabolismo , Compuestos Orgánicos Volátiles/farmacologíaRESUMEN
Cupriavidus necator H16 is gaining significant attention as a microbial chassis for range of biotechnological applications. While the bacterium is a major producer of bioplastics, its lithoautotrophic and versatile metabolic capabilities make the bacterium a promising microbial chassis for biofuels and chemicals using renewable resources. It remains necessary to develop appropriate experimental resources to permit controlled bioengineering and system optimization of this microbe. In this study, we employed statistical design of experiments to gain understanding of the impact of components of defined media on C. necator growth and built a model that can predict the bacterium's cell density based on medium components. This highlighted medium components, and interaction between components, having the most effect on growth: fructose, amino acids, trace elements, CaCl2, and Na2HPO4 contributed significantly to growth (t values of <-1.65 or >1.65); copper and histidine were found to interact and must be balanced for robust growth. Our model was experimentally validated and found to correlate well (r2 = 0.85). Model validation at large culture scales showed correlations between our model-predicted growth ranks and experimentally determined ranks at 100 ml in shake flasks (ρ = 0.87) and 1 liter in a bioreactor (ρ = 0.90). Our approach provides valuable and quantifiable insights on the impact of medium components on cell growth and can be applied to model other C. necator responses that are crucial for its deployment as a microbial chassis. This approach can be extended to other nonmodel microbes of medical and industrial biotechnological importance.IMPORTANCE Chemically defined media (CDM) for cultivation of C. necator vary in components and compositions. This lack of consensus makes it difficult to optimize new processes for the bacterium. This study employed statistical design of experiments (DOE) to understand how basic components of defined media affect C. necator growth. Our growth model predicts that C. necator can be cultivated to high cell density with components held at low concentrations, arguing that CDM for large-scale cultivation of the bacterium for industrial purposes will be economically competitive. Although existing CDM for the bacterium are without amino acids, addition of a few amino acids to growth medium shortened lag phase of growth. The interactions highlighted by our growth model show how factors can interact with each other during a process to positively or negatively affect process output. This approach is efficient, relying on few well-structured experimental runs to gain maximum information on a biological process, growth.
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Medios de Cultivo/metabolismo , Cupriavidus necator/crecimiento & desarrollo , Medios de Cultivo/química , Cupriavidus necator/metabolismo , Modelos EstadísticosRESUMEN
The glasshouse whitefly (Trialeurodes vaporariorum Westwood) is a polyphagous arthropod pest that is of particular detriment to glasshouse grown tomato (Solanum lycopersicum) across temperate regions of the world. Control of whiteflies with synthetic pesticides has resulted in the evolution of resistant genotypes and a reduction in natural enemies, thus highlighting the need for environmentally sound control strategies. Volatile organic compounds (VOCs) offer an environmentally benign alternative to synthetic chemical sprays and this study explored the use of VOCs as insect repellents and plant defence elicitors to control whiteflies on tomato in a commercial glasshouse setting. Limonene in the form of a volatile dispenser system was found to successfully repel whitefly from the target crop and increased fruit yield by 32% during a heavy whitefly infestation. Analysis of tomato herbivore induced plant volatiles (HIPVs) led us to select methyl salicylate (MeSA) as the plant elicitor and application of MeSA to un-infested tomato plants was found to successfully reduce whitefly population development and increase yield by 11%, although this difference was marginally statistically significant. Combination of these two methods was also effective but whitefly abundance in combined plots was similar to the standalone limonene treatment across the course of the experiment. All of the VOC based control methods we used had a negative impact on whitefly performance, with more pronounced effects during the first few weeks of infestation. In subsequent laboratory experiments, we found elevated peroxidase (POD) activity and a significant increase in TPX1 and PR1 transcripts in MeSA treated plants. This led us to deduce that MeSA immediately induced plant defences, rather than priming them. We did however see evidence for residual priming, as plants treated with MeSA and infested with whiteflies produced significantly higher levels of POD activity than whitefly infestation alone. Despite the fact that our treatments failed to synergise, our methods can be optimised further, and the effectiveness of the standalone treatments is promising for future studies. In particular, our repellent limonene dispensers were extremely effective at deterring whiteflies and offer a low economic cost and easy to implement whitefly control option. The methods we have used here could be incorporated into current integrated pest management (IPM) systems, a sustainable approach to pest control which will be central to our efforts to manage whitefly populations under glass in the future.
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Hemípteros/efectos de los fármacos , Control de Insectos/métodos , Repelentes de Insectos/química , Solanum lycopersicum/química , Compuestos Orgánicos Volátiles/química , Animales , Proteínas de Arabidopsis/metabolismo , Moléculas de Adhesión Celular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Herbivoria/efectos de los fármacos , Repelentes de Insectos/metabolismo , Limoneno/metabolismo , Solanum lycopersicum/metabolismo , Peroxidasa/metabolismo , Salicilatos/química , Salicilatos/metabolismo , Receptores Depuradores de Clase E , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
A major cause of yield loss in wheat worldwide is the fungal pathogen Zymoseptoria tritici, a hemibiotrophic fungus which causes Septoria leaf blotch, the most destructive wheat disease in Europe. Resistance in commercial wheat varieties is poor, however, a link between reduced nitrogen availability and increased Septoria tolerance has been observed. We have shown that Septoria load is not affected by nitrogen, whilst the fungus is in its first, symptomless stage of growth. This suggests that a link between nitrogen and Septoria is only present during the necrotrophic phase of Septoria infection. Quantitative real-time PCR data demonstrated that WRKYs, a superfamily of plant-specific transcription factors, are differentially expressed in response to both reduced nitrogen and Septoria. WRKY39 was downregulated over 30-fold in response to necrotrophic stage Septoria, whilst changes in the expression of WRKY68a during the late biotrophic phase were dependent on the concentration of nitrogen under which wheat is grown. WRKY68a may therefore mediate a link between nitrogen and Septoria. The potential remains to identify key regulators in the link between nitrogen and Septoria, and as such, elucidate molecular markers for wheat breeding, or targets for molecular-based breeding approaches.
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Ascomicetos/patogenicidad , Nitrógeno/metabolismo , Proteínas de Plantas/genética , Factores de Transcripción/genética , Triticum/microbiología , Ascomicetos/genética , ADN Espaciador Ribosómico/genética , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Triticum/genética , Triticum/metabolismoRESUMEN
A genetically modified (GM) commercial corn variety, MON810, resistant to European corn borer, has been shown to be non-toxic to mammals in a number of rodent feeding studies carried out in accordance with OECD Guidelines. Insect resistance results from expression of the Cry1Ab gene encoding an insecticidal Bt protein that causes lysis and cell death in susceptible insect larvae by binding to midgut epithelial cells, which is a key determinant of Cry toxin species specificity. Whilst whole animal studies are still recognised as the 'gold standard' for safety assessment, they only provide indirect evidence for changes at the cellular/organ/tissue level. In contrast, omics-based technologies enable mechanistic understanding of toxicological or nutritional events at the cellular/receptor level. To address this important knowledge-gap and to gain insights into the underlying molecular responses in rat to MON810, differential gene expression in the epithelial cells of the small intestine of rats fed formulated diets containing MON810, its near isogenic line, two conventional corn varieties, and a commercial (Purina™) corn-based control diet were investigated using comparative proteomic profiling. Pairwise and five-way comparisons showed that the majority of proteins that were differentially expressed in the small intestine epithelial cells in response to consumption of the different diets in both 7-day and 28-day studies were related to lipid and carbohydrate metabolism and protein biosynthesis. Irrespective of the diet, a limited number of stress-related proteins were shown to be differentially expressed. However these stress-related proteins differed between diets. No adverse clinical or behavioural effects, or biomarkers of adverse health, were observed in rats fed GM corn compared to the other corn diets. These findings suggest that MON810 has negligible effects on the small intestine of rats at the cellular level compared with the well-documented toxicity observed in susceptible insects.
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Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Plantas Modificadas Genéticamente/genética , Proteómica , Zea mays/genética , Alimentación Animal , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/farmacología , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Endotoxinas/farmacología , Alimentos Modificados Genéticamente , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Hemolisinas/farmacología , Humanos , Insectos/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Plantas Modificadas Genéticamente/microbiología , Ratas , Ratas Wistar , Zea mays/químicaRESUMEN
Plants have co-evolved with a diverse array of pathogens and insect herbivores and so have evolved an extensive repertoire of constitutive and induced defence mechanisms activated through complex signalling pathways. OXI1 kinase is required for activation of mitogen-activated protein kinases (MAPKs) and is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses. Furthermore, changes in the levels of OXI1 appear to be crucial for appropriate signalling. Callose deposition also plays a key role in defence. Here we demonstrate, for the first time, that OXI1 plays an important role in defence against aphids. The Arabidopsis mutant, oxi1-2, showed significant resistance both in terms of population build-up (p < 0.001) and the rate of build-up (p < 0.001). Arabidopsis mutants for ß-1,3-glucanase, gns2 and gns3, showed partial aphid resistance, significantly delaying developmental rate, taking two-fold longer to reach adulthood. Whilst ß-1,3-glucanase genes GNS1, GNS2, GNS3 and GNS5 were not induced in oxi1-2 in response to aphid feeding, GNS2 was expressed to high levels in the corresponding WT (Col-0) in response to aphid feeding. Callose synthase GSL5 was up-regulated in oxi1-2 in response to aphids. The results suggest that resistance in oxi1-2 mutants is through induction of callose deposition via MAPKs resulting in ROS induction as an early response. Furthermore, the results suggest that the ß-1,3-glucanase genes, especially GNS2, play an important role in host plant susceptibility to aphids. Better understanding of signalling cascades underpinning tolerance to biotic stress will help inform future breeding programmes for enhancing crop resilience.
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Proteínas de Arabidopsis/genética , Arabidopsis/parasitología , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Áfidos/genética , Áfidos/patogenicidad , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Tolerancia a Medicamentos , Regulación de la Expresión Génica de las Plantas/genética , Fitomejoramiento , Enfermedades de las Plantas/parasitología , Transducción de Señal , Activación TranscripcionalRESUMEN
The glasshouse whitefly, Trialeurodes vaporariorum, is an important pest of many crop plants including tomato, Solanum lycopersicum. Many wild tomato species exhibit a higher resistance to whiteflies. Therefore, locating the source of this enhanced resistance and breeding it into commercial tomato species is an important strategy to reduce the impact of pests on crops. Here, we assessed the pest resistance of Lycopersicon pimpinellifolium by comparing oviposition and feeding data from T. vaporariorum on this wild tomato species with data collected from a susceptible commercial tomato, S. lycopersicum var. 'Elegance'. The location of resistance factors was examined by use of electrical penetration graph (EPG) studies on these tomato species. Results show that whiteflies preferentially settled on the commercial tomato more often in 80 % of the replicates when given free choice between the two tomato species and laid significantly fewer eggs on L. pimpinellifolium. Whiteflies exhibited a shorter duration of the second feeding bout, reduced pathway phase probing, longer salivation in the phloem and more non-probing activities in the early stages of the EPG on the wild tomato species compared to the commercial tomato. These findings evidence that a dual mode of resistance is present in this wild tomato against T. vaporariorum: a post-penetration, pre-phloem resistance mechanism and a phloem-located factor, which to the best of our knowledge is the first time that evidence for this has been presented. These findings can be used to inform future breeding strategies to increase the resistance of commercial tomato varieties against this important pest.
RESUMEN
Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a 'carrier' protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a 'carrier', is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD50>100 µg bee(-1)) or injection of fusion protein. Bees fed acute (100 µg bee(-1)) or chronic (0.35 mg ml(-1)) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.
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Abejas/efectos de los fármacos , Bloqueadores de los Canales de Calcio/toxicidad , Insecticidas/toxicidad , Lectinas de Unión a Manosa/toxicidad , Lectinas de Plantas/toxicidad , Venenos de Araña/toxicidad , Animales , Abejas/crecimiento & desarrollo , Bloqueadores de los Canales de Calcio/metabolismo , Galanthus/química , Insecticidas/metabolismo , Larva/efectos de los fármacos , Aprendizaje/efectos de los fármacos , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad , Venenos de Araña/genética , Venenos de Araña/metabolismoRESUMEN
The full-length cDNA of four Ofapn aminopeptidases were cloned and sequenced from susceptible and resistant Ostrinia furnacalis strains. Four sequences were identified as APN because they shared the common structural features with APN from Lepidoptera, including the signal peptide, GPI anchor signal, the zinc binding/gluzincin motif HEX2HX18E and the gluzincin aminopeptidase motif GAMEN. Compared with APN sequences from the susceptible strain, there were 9, 5, 10 and 12 amino acid variations in the deduced protein sequences from the resistant strain. There were also differences in mRNA expression of the four Ofapn genes between resistant and susceptible O. furnacalis strains.
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Antígenos CD13/genética , Resistencia a los Insecticidas/genética , Lepidópteros/enzimología , Lepidópteros/genética , Secuencia de Aminoácidos , Animales , Bacillus thuringiensis , ADN Complementario/análisis , Proteínas de Insectos/genética , Isoenzimas/genética , Datos de Secuencia Molecular , Control Biológico de Vectores/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: RNA interference (RNAi) is an endogenous eukaryote viral defence mechanism representing a unique form of post-transcriptional gene silencing. Owing to its high specificity, this technology is being developed for use in dsRNA-based biopesticides for control of pest insects. Whilst many lepidopteran species are recalcitrant to RNAi, Tuta absoluta, a polyphagous insect responsible for extensive crop damage, is sensitive. Ryanodine receptors (RyRs) are intracellular calcium channels regulating calcium ion (Ca2+) release. The chemical pesticide class of diamides functions agonistically against lepidopteran RyR, resulting in uncontrolled Ca2+ release, feeding cessation and death. Resistance to diamides has emerged in T. absoluta, derived from RyR point mutations. RESULTS: RNAi was used to target RyR transcripts of T. absoluta. Data presented here demonstrate the systemic use of exogenous T. absoluta RyR-specific (TaRy) dsRNA in tomato plants (Solanum lycopersicum) to significantly downregulate expression of the target gene, resulting in significant insect mortality and reduced leaf damage. Using a leaflet delivery system, daily dosing of 3 µg TaRy dsRNA for 72 h resulted in 50% downregulation of the target gene and 50% reduction in tomato leaf damage. Corrected larval mortality and adult emergence were reduced by 38% and 33%, respectively. TaRy dsRNA demonstrated stability in tomato leaves ≤72 h after dosing. CONCLUSIONS: This work identifies TaRy as a promising target for RNAi control of this widespread crop pest. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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The endosymbiotic bacterium Buchnera aphidicola allows its host Acyrthosiphon pisum to utilise a nutritionally limited phloem sap diet without significant mortality by providing essential amino acids (EAAs), which it biosynthesises de novo via complex pathways consisting of multiple enzymes. Previous studies have reported how non-essential amino acids (NEAAs) provided by the host are utilised by B. aphidicola, along with how genes within the biosynthetic pathways respond to amino acid deficiency. Although the effect on B. aphidicola gene expression upon the removal of a single EAA and multiple NEAAs from the A. pisum diet has been reported, little is known about the effects of the complete simultaneous removal of multiple EAAs, especially branched-chain amino acids (BCAAs). To investigate this, A. pisum was provided with amino acid deficient diets ilv- (lacking isoleucine, leucine, valine) or thra- (lacking threonine, methionine, lysine). Due to their involvement in the production of several amino acids, the expression of genes ilvC, ilvD (both involved in isoleucine, leucine and valine biosynthesis) and thrA (involved in threonine, methionine and lysine biosynthesis) was analysed and the expression of trpC (involved in tryptophan biosynthesis) was used as a control. Survival was reduced significantly when A. pisum was reared on ilv- or thra- (P < 0.001 and P = 0.000 respectively) compared to optimal artificial diet and was significantly lower on ilv- (P < 0.001) than thra-. This is likely attributed to the EAAs absent from ilv- being required at higher concentrations for aphid growth, than those EAAs absent from thra-. Expression of ilvC and ilvD were upregulated 2.49- and 2.08-fold (respectively) and thrA expression increased 2.35- and 2.12-fold when A. pisum was reared on ilv- and thra- (respectively). The surprisingly large upregulation of thrA when reared on ilv- is likely due to threonine being an intermediate in isoleucine biosynthesis. Expression of trpC was not affected by rearing on either of the two amino acid deficient diets. To our knowledge this study has shown, for the first time, how genes within the biosynthetic pathways of an endosymbiont respond to the simultaneous complete omission of multiple EAAs as well as all three BCAAs (leucine, isoleucine, valine), from the host diet.
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A Cry1Ab-resistant population of Asian corn borer (ACB-AbR) exhibiting approximately 100 times greater resistance to activated Cry1Ab than a susceptible population (Ostrinia furnacalis; ACB-BtS), was previously shown to exhibit high levels of cross-resistance to Cry1Ah (131-fold), but no cross-resistance to Cry1Ie. It was suggested that the proposed mechanism of resistance was due to the alteration of specific receptors for Cry toxins in the midgut brush border membrane. In the present study a proteomic-based approach was used to identify proteins from brush border membrane vesicles (isolated from both resistant and susceptible Ostrinia furnacalis larvae) interacting with biotinylated Cry1Ab, Cry1Ah, and Cry1Ie. 2D-Electrophoresis in combination with ligand blots were employed and putative protein identities obtained using MALDI-ToF/ToF mass spectrometry. The V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins were identified as interacting with the Cry toxins tested in the ACB-AbR and ACB-BtS larvae. The biotinylated Cry toxins showed markedly stronger interactions with proteins in the resistant compared to the susceptible larvae, suggesting an up-regulation of the V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins in the resistant (ACB-AbR) larvae. Interestingly, Cry1Ie interactions with the V-type proton ATPase catalytic subunit A in the ACB-BtS larvae appeared to be absent.
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Proteínas Bacterianas/toxicidad , Tolerancia a Medicamentos/genética , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Lepidópteros/genética , Proteómica , Animales , Bacillus thuringiensis/patogenicidad , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Endotoxinas/genética , Proteínas Hemolisinas/genética , Larva/efectos de los fármacos , Larva/genética , Lepidópteros/efectos de los fármacosRESUMEN
Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of wheat to infestation by cereal aphid (Sitobion avenae) were investigated in a proteomic analysis. Approximately, 500 protein spots were reproducibly detected in the extracts from leaves of wheat seedlings after extraction and 2-DE. Sixty-seven spots differed significantly between control and infested plants following 24 h of aphid feeding, with 27 and 11 up-regulated, and 8 and 21 down-regulated, in local or systemic tissues, respectively. After 8 days, 80 protein spots differed significantly between control and aphid treatments with 13 and 18 up-regulated and 27 and 22 down-regulated in local or systemic tissues, respectively. As positive controls, plants were treated with salicylic acid or methyl jasmonate; 81 and 37 differentially expressed protein spots, respectively, were identified for these treatments. Approximately, 50% of differentially expressed protein spots were identified by PMF, revealing that the majority of proteins altered by aphid infestation were involved in metabolic processes and photosynthesis. Other proteins identified were involved in signal transduction, stress and defence, antioxidant activity, regulatory processes, and hormone responses. Responses to aphid attack at the proteome level were broadly similar to basal non-specific defence and stress responses in wheat, with evidence of down-regulation of insect-specific defence mechanisms, in agreement with the observed lack of aphid resistance in commercial wheat lines.
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Áfidos/metabolismo , Interacciones Huésped-Parásitos/fisiología , Enfermedades de las Plantas/parasitología , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Triticum/metabolismo , Acetatos/farmacología , Animales , Ciclopentanos/farmacología , Electroforesis en Gel Bidimensional , Oxilipinas/farmacología , Mapeo Peptídico , Hojas de la Planta/química , Proteínas de Plantas/análisis , Proteínas de Plantas/clasificación , Proteoma/química , Ácido Salicílico/farmacología , Plantones/metabolismo , Plantones/parasitología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estrés FisiológicoRESUMEN
This paper provides recommendations on experimental design for early-tier laboratory studies used in risk assessments to evaluate potential adverse impacts of arthropod-resistant genetically engineered (GE) plants on non-target arthropods (NTAs). While we rely heavily on the currently used proteins from Bacillus thuringiensis (Bt) in this discussion, the concepts apply to other arthropod-active proteins. A risk may exist if the newly acquired trait of the GE plant has adverse effects on NTAs when they are exposed to the arthropod-active protein. Typically, the risk assessment follows a tiered approach that starts with laboratory studies under worst-case exposure conditions; such studies have a high ability to detect adverse effects on non-target species. Clear guidance on how such data are produced in laboratory studies assists the product developers and risk assessors. The studies should be reproducible and test clearly defined risk hypotheses. These properties contribute to the robustness of, and confidence in, environmental risk assessments for GE plants. Data from NTA studies, collected during the analysis phase of an environmental risk assessment, are critical to the outcome of the assessment and ultimately the decision taken by regulatory authorities on the release of a GE plant. Confidence in the results of early-tier laboratory studies is a precondition for the acceptance of data across regulatory jurisdictions and should encourage agencies to share useful information and thus avoid redundant testing.
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Artrópodos/efectos de los fármacos , Plantas Modificadas Genéticamente/toxicidad , Proyectos de Investigación/normas , Animales , Bacillus thuringiensis , Productos Agrícolas/genética , Guías como Asunto , Laboratorios , Plantas Modificadas Genéticamente/parasitología , Medición de Riesgo/métodos , Medición de Riesgo/normasRESUMEN
Cupriavidus necator H16 is a chemolithoautotroph with a range of industrial biotechnological applications. Advanced metabolic engineering in the bacterium, however, is impeded by low transformation efficiency, making it difficult to introduce and screen new genetic functions rapidly. This study systematically characterized the broad host range plasmids pBHR1, pBBR1MCS-2 and pKT230 used frequently for C. necator engineering. Kanamycin resistance cassette (KanR) and a truncated sequence of the replication origin (Rep) are contributing factors to C. necator low electroporation transformation efficiency. Consequently, a series of modular minimal plasmids, named pCAT, were constructed. pCAT vectors transform C. necator H16 with a > 3000-fold higher efficiency (up to 107 CFU/µg DNA) compared to control plasmids. Further, pCAT vectors are highly stable, expressing reporter proteins over several days of serial cultivation in the absence of selection pressure. Finally, they can be assembled rapidly from PCR or synthesized DNA fragments, and restriction-ligation reactions can be efficiently electroporated directly into C. necator, circumventing the requirement to use Escherichia coli for plasmid maintenance or propagation. This study demonstrates that an understanding of the behaviour of the constituent parts of plasmids in a host is key to efficient propagation of genetic information, while offering new methods for engineering a bacterium with desirable industrial biotechnological features.
Asunto(s)
Cupriavidus necator , Electroporación , Vectores Genéticos , Ingeniería Metabólica , Cupriavidus necator/genética , Escherichia coli/genética , Plásmidos/genéticaRESUMEN
Bt crops have been grown commercially for more than two decades. They have proven remarkably effective in the control of target insect pests. However, Bt crops can become less effective under various forms of environmental stress. Most studies in this area have considered the effect of environmental stress on Bt insecticidal protein levels or target pest mortality, but not both, resulting in a lack of mechanistic analysis. In this review, we critically examine previous research addressing the impact of environmental stress on the effectiveness of Bt crops. We find that the body of research data is not sufficiently robust to allow the reliable prediction of the performance of Bt crops under extreme climatic conditions.
Asunto(s)
Bacillus thuringiensis , Animales , Proteínas Bacterianas , Productos Agrícolas , Control Biológico de Vectores , Plantas Modificadas GenéticamenteRESUMEN
Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized pest control, but the benefits of this approach have been reduced by the evolution of resistance in pests. The widely adopted 'pyramid strategy' for delaying resistance entails transgenic crops producing two or more distinct toxins that kill the same pest. The limited experimental evidence supporting this strategy comes primarily from a model system under ideal conditions. Here we tested the pyramid strategy under nearly worst-case conditions, including some cross-resistance between the toxins in the pyramid. In a laboratory selection experiment with an artificial diet, we used Bt toxins Cry1Ab, Cry1F, and Cry1Ie singly or in pairs against Ostrinia furnacalis, one of the most destructive pests of corn in Asia. Under the conditions evaluated, pairs of toxins did not consistently delay the evolution of resistance relative to single toxins.