RESUMO
Overexpression of carboxyl/cholinesterase (CCE) genes has been reported to be associated with many cases of pesticide resistance in arthropods. However, it has been rarely documented that CCE genes participate in spirodiclofen resistance in Panonychus citri. In previous research, we found that spirodiclofen resistance is related to increased P450 and CCE enzyme activities in P. citri. In this study, we identified two CCE genes, PcCCE3 and PcCCE5, which were significantly upregulated in spirodiclofen-resistant strain and after exposure to spirodiclofen. RNA interference of PcCCE3 and PcCCE5 increased the spirodiclofen susceptibility in P. citri. In vitro metabolism indicated that PcCCE3 and PcCCE5 could interact with spirodiclofen, but metabolites were detected only in the PcCCE3 treatment. Our results indicated that PcCCE3 participates in spirodiclofen resistance through direct metabolism, and PcCCE5 may be involved in the spirodiclofen resistance by passive binding and sequestration, which provides new insights into spirodiclofen resistance in P. citri.
Assuntos
Proteínas de Artrópodes , Compostos de Espiro , Animais , Compostos de Espiro/farmacologia , Compostos de Espiro/metabolismo , Compostos de Espiro/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/química , Resistência a Medicamentos/genética , Carboxilesterase/genética , Carboxilesterase/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , 4-Butirolactona/farmacologiaRESUMO
Selection of chemical-resistant predatory mites is a good alternative to balance the contradiction between chemical control and biological control. Previously, a resistant strain of Neoseiulus barkeri for chlorpyrifos was obtained. In the current study, two up-regulated (NbCYP3A6, NbCYP3A16) and one down-regulated (NbCYP3A24) P450s were screened through differential expression analysis and other detoxification-related genes such as CCEs, GST, etc. were not found. 3D modelling and molecular docking indicated that the chlorine at position 5 on the pyridine ring of chlorpyrifos, as well as a methyl group, were closest to the heme iron of the enzymes (less than 5 Å). Three active recombinant P450 proteins were heterologously expressed and metabolized with chlorpyrifos in vitro. HPLC assay showed that only NbCYP3A24 could metabolize chlorpyrifos, with a metabolism rate of 21.60 %. Analysis of the m/z of metabolites by LC-MS/MS showed that chlorine at the 5C position of chlorpyrifos was stripped and hydroxylated, whereas chlorpyrifos-oxon, a common product of oxidation by P450, was not found. Knockdown of the NbCYP3A24 gene in the susceptiblestrain did reduce the susceptibility of N. barkeri to chlorpyrifos, suggesting that the biological activity of the metabolite may be similar to chlorpyrifos-oxon, thus enhancing the inhibitory effect on the target.
Assuntos
Clorpirifos , Sistema Enzimático do Citocromo P-450 , Inseticidas , Ácaros , Simulação de Acoplamento Molecular , Clorpirifos/metabolismo , Clorpirifos/química , Clorpirifos/análogos & derivados , Animais , Ácaros/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Inseticidas/metabolismo , Inseticidas/química , Inseticidas/toxicidade , Regulação para Baixo , HidroxilaçãoRESUMO
Insecticide susceptibility is mainly determined by the insect host, but symbiotic bacteria are also an important affecting factor. In this study, we investigate the relationship between the structure of gut bacterial symbionts and insecticide susceptibility in Diaphorina citri, the important carrier of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). Our results indicated that antibiotic treatment significantly increased the susceptibility of D. citri to bifenthrin and thiamethoxam, and significantly decreased the relative abundance of Wolbachia and Profftella, enzyme activities of CarEs, and expression level of multiple CarE genes. The relative loads of Wolbachia and Profftella were positively correlated with DcitCCE13, DcitCCE14, DcitCCE15, and DcitCCE16. RNAi and prokaryotic expression revealed that DcitCCE15 is associated with bifenthrin metabolism. These results revealed that bacterial symbionts might regulate DcitCCE15 expression, which is involved in the susceptibility of D. citri to bifenthrin.
Assuntos
Hemípteros , Inseticidas , Simbiose , Animais , Inseticidas/farmacologia , Hemípteros/microbiologia , Hemípteros/genética , Hemípteros/efeitos dos fármacos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Wolbachia/efeitos dos fármacos , Wolbachia/genética , Piretrinas/farmacologia , Bactérias/genética , Bactérias/efeitos dos fármacos , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , Inativação Metabólica/genéticaRESUMO
The citrus red mite, Panonychus citri, is one of the most notorious and devastating citrus pests around the world that has developed resistance to multiple chemical acaricides. In previous research, we found that spirodiclofen-resistant is related to overexpression of P450, CCE, and ABC transporter genes in P. citri. However, the regulatory mechanisms of these detoxification genes are still elusive. This study identified all hormone receptor 96 genes of P. citri. 8 PcHR96 genes contained highly conserved domains. The expression profiles showed that PcHR96h was significantly upregulated in spirodiclofen resistant strain and after exposure to spirodiclofen. RNA interference of PcHR96h decreased expression of detoxification genes and increased spirodiclofen susceptibility in P. citri. Furthermore, molecular docking, heterologous expression, and drug affinity responsive target stability demonstrated that PcHR96h can interact with spirodiclofen in vitro. Our research results indicate that PcHR96h plays an important role in regulating spirodiclofen susceptibility and provides theoretical support for the resistance management of P. citri.
Assuntos
Compostos de Espiro , Animais , Compostos de Espiro/farmacologia , Compostos de Espiro/metabolismo , Acaricidas/farmacologia , Propionatos/farmacologia , Propionatos/metabolismo , Tetranychidae/efeitos dos fármacos , Tetranychidae/genética , Tetranychidae/metabolismo , Simulação de Acoplamento Molecular , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Resistência a Medicamentos/genética , 4-Butirolactona/análogos & derivadosRESUMO
Panonychus citri (McGregor) strains have developed a high level of resistance to abamectin, but the underlying molecular mechanism is unknown. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are critical for the removal of a variety of exogenous and endogenous substances. In this study, an enzyme activity assay revealed that UGTs potentially contribute to P. citri abamectin resistance. Spatiotemporal expression profiles showed that only PcUGT202A9 was significantly overexpressed in the abamectin-resistant strain (AbR) at all developmental stages. Moreover, UGT activity decreased significantly, whereas abamectin susceptibility increased significantly, in AbR after PcUGT202A9 was silenced. Three-dimensional modeling and molecular docking analyses revealed that PcUGT202A9 can bind stably to abamectin. Recombinant PcUGT202A9 activity was detected when α-naphthol was used, but the enzymatic activity was inhibited by abamectin (50â¯% inhibitory concentration: 803.3⯱â¯14.20⯵mol/L). High-performance liquid chromatography and mass spectrometry analyses indicated that recombinant PcUGT202A9 can effectively degrade abamectin and catalyze the conjugation of UDP-glucose to abamectin. These results imply PcUGT202A9 contributes to P. citri abamectin resistance.
Assuntos
Glicosiltransferases , Ivermectina , Simulação de Acoplamento Molecular , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Glicosiltransferases/química , Animais , Resistência a Medicamentos/genéticaRESUMO
The progression of liver fibrosis is determined by the interaction of damaged hepatocytes, active hepatic stellate cells, and macrophages, contributing to the development of oxidative stress and inflammatory environments within the liver. Unfortunately, the current pharmacological treatment for liver fibrosis is limited by its inability to regulate inflammation and oxidative stress concurrently. In this study, we developed a cell membrane biomaterial for the treatment of liver fibrosis, which we designated as PM. PM is a biomimetic nanomaterial constructed by encapsulating polydopamine (PDA) with a macrophage membrane (MM). It is hypothesized that PM nanoparticles (NPs) can successfully target the site of inflammation, simultaneously inhibit inflammation, and scavenge reactive oxygen species (ROS). In vitro experiments demonstrated that PM NPs exhibited strong antioxidant properties and the ability to neutralize pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß). Moreover, the capacity of PM NPs to safeguard cells from oxidative stress and their anti-inflammatory efficacy in an inflammatory model were validated in subsequent cellular experiments. Additionally, PM NPs exhibited a high biocompatibility. In a mouse model of hepatic fibrosis, PM NPs were observed to aggregate efficiently in the fibrotic liver, displaying excellent antioxidant and anti-inflammatory properties. Notably, PM NPs exhibited superior targeting, anti-inflammatory, and ROS scavenging abilities in inflamed tissues compared to MM, PDA, or erythrocyte membrane-encapsulated PDA. Under the synergistic effect of anti-inflammation and antioxidant, PM NPs produced significant therapeutic effects on liver fibrosis in mice. In conclusion, the synergistic alleviation of inflammation and ROS scavenging by this specially designed nanomaterial, PM NPs, provides valuable insights for the treatment of liver fibrosis and other inflammatory- or oxidative stress-related diseases.
Assuntos
Antioxidantes , Indóis , Inflamação , Cirrose Hepática , Macrófagos , Nanopartículas , Estresse Oxidativo , Polímeros , Espécies Reativas de Oxigênio , Animais , Polímeros/química , Espécies Reativas de Oxigênio/metabolismo , Indóis/química , Indóis/farmacologia , Indóis/administração & dosagem , Camundongos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Nanopartículas/química , Estresse Oxidativo/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Antioxidantes/farmacologia , Antioxidantes/química , Inflamação/tratamento farmacológico , Inflamação/patologia , Humanos , Masculino , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/administração & dosagem , Células RAW 264.7 , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Citocinas/metabolismoRESUMO
Cyetpyrafen is a recently developed acaricide. The citrus red mite, Panonychus citri (McGregor), has developed significant resistance to cyetpyrafen. However, the molecular mechanism underlying the cyetpyrafen resistance in P. citri remains unclear. Glutathione S-transferases (GSTs) play a critical role in arthropod pesticide resistance. This study showed that GSTs were potentially related to the resistance of P. citri to cyetpyrafen through synergistic experiments and enzyme activity analysis. An omega-family GST gene, PcGSTO1, was significantly up-regulated in the egg, nymph, and adult stages of the cyetpyrafen-resistant strain. Additionally, silencing of PcGSTO1 significantly increased the mortality of P. citri to cyetpyrafen and recombinant PcGSTO1 demonstrated the ability to metabolize cyetpyrafen. Our results indicated that the overexpression of PcGSTO1 is associated with cyetpyrafen resistance in P. citri, and they also provided valuable information for managing resistance in P. citri.
Assuntos
Acaricidas , Tetranychidae , Animais , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Tetranychidae/genética , Tetranychidae/metabolismo , Acaricidas/farmacologia , Acaricidas/metabolismoRESUMO
Diaphorina citri is a global citrus pest. As a vector insect, it can transmit the causative agents of citrus huanglongbing, causing irreversible losses to the citrus industry. The acquisition of genomic information can provide a molecular genetic basis for effective control of D. citri. Here, the DNBSEQ™ , Oxford Nanopore Technologies, and Hi-C technologies are applied to generate a high-quality chromosome-level genome of D. citri. The genome size of D. citri was 523.78 Mb with a scaffold N50 of 47.05 Mb distributed on 13 chromosomes. A total of 250.64 Mb (47.85%) repeat sequences and 24 048 protein-coding genes were predicted. Genome resequencing of female and male individuals indicated that the sex chromosome system of D. citri is XO. Phylogenetic analysis demonstrated that D. citri and Pachypsylla venusta, which separated from their most recent common ancestor about 336.62 million years ago, were the most closely related. Additionally, we identified genes potentially involved in detoxification metabolism, pathogen transmission, and honeydew secretion for further investigation. The high-quality genome provides an important reference for developing effective management strategies of D. citri.
Assuntos
Citrus , Hemípteros , Humanos , Animais , Feminino , Masculino , Hemípteros/genética , Filogenia , Análise de Sequência de DNA , Cromossomos , Citrus/genéticaRESUMO
A novel metal-organic framework (MOF) Zn-PBC (H2PBC = pyridine-3,5-bis(phenyl-4-carboxylic acid)) was designed and synthesized via a solvothermal reaction with the H2PBC ligand, and produced a strong fluorescence. The material exhibited good stability and an ideal luminescent property in water. In addition, it was found that Zn-PBC displayed a different fluorescent response to different types of amino acids, and the mechanism was investigated. This research might give insight to the interaction between MOFs and amino acids, which would provide a strategy to fabricate MOF-based sensors for biomolecules in future.
RESUMO
Asian citrus psyllid (Diaphorina citri, D. citri) is the important vector of "Candidatus Liberibacter asiaticus" (CLas), associated with Huanglongbing, the most devastating citrus disease worldwide. CLas can affect endosymbiont abundance of D. citri. Here, we generated the high-quality gut endosymbiont metagenomes of Diaphorina citri on the condition of CLas infected and uninfected. The dataset comprised 6616.74 M and 6586.04 M raw reads, on overage, from CLas uninfected and infected psyllid strains, respectively. Taxonomic analysis revealed that a total of 1046 species were annotated with 10 Archaea, 733 Bacteria, 234 Eukaryota, and 69 Viruses. 80 unique genera in CLas infected D. citri were identified. DIAMOND software was used for complement function research against various functional databases, including Nr, KEGG, eggNOG, and CAZy, which annotated 84543 protein-coding genes. These datasets provided an avenue for further study of the interaction mechanism between CLas and D. citri.
Assuntos
Hemípteros , Rhizobiaceae , Animais , Hemípteros/genética , Hemípteros/microbiologia , Metagenoma , Rhizobiaceae/genéticaRESUMO
Huanglongbing (HLB), the most prevalent citrus disease worldwide, is responsible for substantial yield and economic losses. Phytobiomes, which have critical effects on plant health, are associated with HLB outcomes. The development of a refined model for predicting HLB outbreaks based on phytobiome markers may facilitate early disease detection, thus enabling growers to minimize damages. Although some investigations have focused on differences in the phytobiomes of HLB-infected citrus plants and healthy ones, individual studies are inappropriate for generating common biomarkers useful for detecting HLB on a global scale. In this study, we therefore obtained bacterial information from several independent datasets representing hundreds of citrus samples from six continents and used these data to construct HLB prediction models based on 10 machine learning algorithms. We detected clear differences in the phyllosphere and rhizosphere microbiomes of HLB-infected and healthy citrus samples. Moreover, phytobiome alpha diversity indices were consistently higher for healthy samples. Furthermore, the contribution of stochastic processes to citrus rhizosphere and phyllosphere microbiome assemblies decreased in response to HLB. Comparison of all constructed models indicated that a random forest model based on 28 bacterial genera in the rhizosphere and a bagging model based on 17 bacterial species in the phyllosphere predicted the health status of citrus plants with almost 100% accuracy. Our results thus demonstrate that machine learning models and phytobiome biomarkers may be applied to evaluate the health status of citrus plants.
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pH detection is related to human beings' life closely. Herein, a porous metal hydrogen-bonded organic framework (MHOF) Co-CDI-CO32- was designed and synthesized, which could be utilized to detect the pH values of strong acids. The sensitivity of pH detection was in the range of pH 2.0-2.4 with an accuracy of 0.1, which mainly depended on the different degree of crystal surface damage. The MHOF Co-CDI-CO32- had potential applications with the advantages of low cost, easy storage, and great sensitivity for pH detection as a pH sensor.
Assuntos
Estruturas Metalorgânicas , Humanos , Ligação de Hidrogênio , Hidrogênio , Porosidade , Concentração de Íons de HidrogênioRESUMO
Spirodiclofen is one of the most widely used acaricides in China. The citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), is one of the most destructive citrus pests worldwide and has developed a high resistance to spirodiclofen. However, the molecular mechanism of spirodiclofen resistance in P. citri is still unknown. In this study, we identified a field spirodiclofen-resistant strain (DL-SC) that showed 712-fold resistance to spirodiclofen by egg bioassay compared to the susceptible strain. Target-site resistance was not detected as non-synonymous mutations were not found by amplification and sequencing of the ACCase gene of resistant and susceptible strains; in addition, the mRNA expression levels of ACCase were similar in both resistant and susceptible strains. The activity of detoxifying enzymes P450s and CCEs in the resistant strain was significantly higher than in the susceptible strain. The transcriptome expression data showed 19 xenobiotic metabolisms genes that were upregulated. Stage-specific expression profiling revealed that the most prominent upregulated gene, CYP385C10, in transcriptome data was significantly higher in resistant strains in all stages. Furthermore, functional analysis by RNAi indicated that the mortality caused by spirodiclofen was significantly increased by silencing the P450 gene CYP385C10. The current results suggest that overexpression of the P450 gene, CYP385C10, may be involved in spirodiclofen resistance in P. citri.
RESUMO
Correction for 'Preparation of nanoscale cationic metal-organic framework Nano Mn(III)-TP for theranostics based on valence changes' by Shijiang Yu et al., J. Mater. Chem. B, 2022, 10, 8988-8995, https://doi.org/10.1039/D2TB01619B.
RESUMO
A single treatment strategy to produce a significant effect on cancer treatment is difficult due to the complex variability of the tumor environment. Herein, considering over-expressed glutathione (GSH) in the tumor environment can reduce Mn3+ to Mn2+, and Mn2+ exhibits an excellent T1-weighted magnetic resonance imaging performance and a good Fenton-like effect. A Mn-based nano-cation metal-organic framework (MOF) with a size of about 200 nm was synthesized and labeled as Nano Mn(III)-TP. It showed an outstanding T1-weighted MRI performance and Fenton-like effect due to valence changes of the Mn ions. Meanwhile, MTXNa@Nano Mn(III)-TP was obtained by loading with the anionic anticancer drug methotrexate disodium (MTXNa), and it could release MTXNa specifically in GSH solution with different pH values and temperatures, resulting in an improvement in the tumor suppressive effect of the Nano Mn(III)-TP. Based on the nanoplatform, the effective combination of magnetic resonance imaging (MRI), CDT and chemotherapy could be used as a potential therapeutic agent for multimodal therapy, which is also expected to be applied to clinical tumor treatment.
Assuntos
Antineoplásicos , Estruturas Metalorgânicas , Nanopartículas , Neoplasias , Humanos , Medicina de Precisão , Nanopartículas/uso terapêutico , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologiaRESUMO
BACKGROUND: Amitraz is a broad-spectrum insecticide/acaricide for the control of aphids, psyllids, ticks and mites. Current evidence suggests that ticks and phytophagous mites have developed strong resistance to amitraz. Previous studies have shown that multiple mechanisms are associated with amitraz resistance in ticks, but very few reports have involved Panonychus citri. We therefore used whole genome sequencing and bulked segregant analysis (BSA) to identify the mechanism underlying P. citri's resistance to amitraz. RESULTS: High-quality assembly of the whole P. citri genome was completed, resulting in a genome of approximately 83.97 Mb and a contig N50 of approximately 1.81 Mb. Gene structure predictions revealed 11 577 genes, of which 10 940 genes were annotated. Trait-associated regions in the genome were mapped with bulked segregant analysis and 38 candidate SNPs were obtained, of which T752C had the strongest correlation with the resistant trait, located at the 5' untranslated region (UTR) of the ß-2R adrenergic-like octopamine receptor gene. The mutation resulted in the formation of a short hairpin loop structure in mRNA and gene expression was down-regulated by more than 50% in the amitraz-resistant strain. Validation of the T752C mutation in field populations of P. citri found that the correlation between the resistance ratio and the base mutation was 94.40%. CONCLUSION: Our results suggest that this 5' UTR mutation of the ß-2R octopamine receptor gene, confers amitraz resistance in P. citri. This discovery provides a new explanation for the mechanism of pest resistance: base mutations in the 5' untranslated region of target gene may regulate the susceptibility of pests to pesticides.
Assuntos
Acaricidas , Ácaros , Toluidinas , Animais , Ácaros/genética , Sequenciamento Completo do GenomaRESUMO
A biocompatible Y(III)-based metal-organic framework [Y4(TATB)2]·(DMF)3.5·(H2O) (ZJU-16, H3TATB= 4,4',4''-(1,3,5-triazine-2,4,6-triyl) tribenzoic acid) was synthesized, and it was adopted to load Mn2+ for chemodynamic therapy. Meanwhile, ibuprofen sodium (IBUNa), an anti-inflammatory drug, was introduced to increase the amount of Mn2+ (about 5.66 wt %) due to the low loading capacity of Mn2+. Mn&IBUNa@ZJU-16 which was loaded by Mn2+ and IBUNa exhibited significant effects of chemodynamic therapy and excellent inhibition of the 4T1 tumor cell growth, implying its long-term prospects in chemodynamic therapy and its possibility in bimodal cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Materiais Biocompatíveis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Estruturas Metalorgânicas/farmacologia , Ítrio/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Liberação Controlada de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Estruturas Metalorgânicas/síntese química , Estruturas Metalorgânicas/química , Camundongos , Células Tumorais Cultivadas , Ítrio/químicaRESUMO
Panonychus citri (McGregor) is the most common pest in citrus-producing regions. Special low-toxicity acaricides, such as spirocyclic tetronic acids and mite growth inhibitors, have been used for a long time in China. However, pesticide resistance in mites is a growing problem due to the lack of new acaricide development. Wide-spectrum insecticides, such as amitraz have gained acceptance among fruit growers. An amitraz-resistant strain of P. citri was obtained by indoor screening to examine field resistance monitoring of mites to acaricides and to explore the resistant mechanism of mites against amitraz. The amitraz-resistant strain of P. citri had an LC50 value of 2361.45 mg l-1. The resistance ratio was 81.35 times higher in the resistant strain of P. citri compared with the sensitive strain. Crossing experiments between the sensitive and resistant strains of P. citri were conducted, resulting in a D value of 0.11 for F1 SSâ×RSâ and 0.06 for F1 RSâ×SSâ. Reciprocal cross experiments showed that the dose-mortality curves for the F1 generations coincided, indicating that the resistance trait was not affected by cytoplasmic inheritance. The dose-expected response relationship was evaluated in the backcross generation and a significant difference was observed compared with the actual value. The above results indicate that the inheritance of resistance trait was incompletely dominant, governed by polygenes on the chromosome. Synergism studies demonstrated that cytochrome P450s and esterase may play important roles in the detoxification of amitraz. Based on differential gene analysis, 23 metabolism-related genes of P. citri were identified, consistent with the results of synergism studies. Real-time PCR verification implied that P450s, ABC transporters, and acetylcholinesterase might influence the detoxification of amitraz by P. citri. These results provide the genetic and molecular foundation for the management of pest mite resistance.
Assuntos
Inativação Metabólica/genética , Tetranychidae/genética , Tetranychidae/metabolismo , Toluidinas , Acaricidas , Animais , Sistema Enzimático do Citocromo P-450 , Esterases , Tetranychidae/enzimologiaRESUMO
BACKGROUND: Dialeurodes citri is an important pest in citrus-producing areas of the world. Lecanicillium attenuatum parasitizes D. citri and kills it, suggesting a potential approach for the biological control of pests. However, the low virulence of the fungus and its slow rate of killing have limited its commercial competitiveness. The objective reason for these disadvantages is immunological rejection by the host. Our strategy was to use fungi to express the double-stranded RNA (dsRNA) of the host immune genes. The fungal hyphae release siRNA at the time of infection, thus interfering with the expression of immune genes in the host and facilitating fungal invasion. RESULTS: We selected prophenoloxidase (DcPPO), prophenoloxidase-activating factor (DcPPO-AF), and lysozyme (DcLZM) as target genes to construct intron-splicing hairpin RNA expression vectors and to successfully obtain transgenic fungi. Two days after infection, the immune genes of D. citri showed varying degrees of silencing compared with those in the positive control group. The median lethal concentration (LC50 ; spores mL-1 ) values of La::GFP, La::DcPPO, La::DcPPO-AF, and La::DcLZM were 9.63 × 104 , 2.66 × 104 , 1.21 × 105 , and 3.31 × 104 , respectively. The 50% lethal time (LT50 ) values of these fungi were 5.15, 3.60, 5.34, and 4.04 days, respectively. The virulence of La::DcPPO and La::DcLZM increased 3.62- and 2.91-fold, respectively, and their LT50 decreased by 30.10% and 21.55%, respectively. CONCLUSIONS: The results indicate that this method, which uses tens of thousands of hyphae to inject dsRNA to improve the virulence of transgenic fungi, can play a greater role in the prevention and control of pests in the future. © 2018 Society of Chemical Industry.
Assuntos
Hemípteros/microbiologia , Hypocreales/fisiologia , Proteínas de Insetos/genética , Controle Biológico de Vetores , RNA de Cadeia Dupla/genética , Animais , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Hemípteros/enzimologia , Hypocreales/enzimologia , Hypocreales/genética , Controle de Insetos , Proteínas de Insetos/metabolismo , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/fisiologia , Muramidase/genética , Muramidase/metabolismo , RNA de Cadeia Dupla/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Dialeurodes citri is a major pest in citrus producing areas, and large-scale outbreaks have occurred increasingly often in recent years. Lecanicillium attenuatum is an important entomopathogenic fungus that can parasitize and kill D. citri. We separated the fungus from corpses of D. citri larvae. However, the sound immune defense system of pests makes infection by an entomopathogenic fungus difficult. Here we used RNA sequencing technology (RNA-Seq) to build a transcriptome database for D. citri and performed digital gene expression profiling to screen genes that act in the immune defense of D. citri larvae infected with a pathogenic fungus. De novo assembly generated 84,733 unigenes with mean length of 772 nt. All unigenes were searched against GO, Nr, Swiss-Prot, COG, and KEGG databases and a total of 28,190 (33.3%) unigenes were annotated. We identified 129 immunity-related unigenes in transcriptome database that were related to pattern recognition receptors, information transduction factors and response factors. From the digital gene expression profile, we identified 441 unigenes that were differentially expressed in D. citri infected with L. attenuatum. Through calculated Log2Ratio values, we identified genes for which fold changes in expression were obvious, including cuticle protein, vitellogenin, cathepsin, prophenoloxidase, clip-domain serine protease, lysozyme, and others. Subsequent quantitative real-time polymerase chain reaction analysis verified the results. The identified genes may serve as target genes for microbial control of D. citri.