Project description:Aedes aegypti SP strain vs. SMK strain. Aedes aegypti is the major vector of yellow fever and dengue/dengue hemorrhagic fever. Starting with a population collected from Singapore, we established a pyrethroid-resistant A. aegypti strain (SP) and investigated three major possible mechanisms of insecticide resistance. After 10 generations of adult selection, an A. aegypti strain developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel genes (Vssc) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can contribute more to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than the susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over-expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2, and confirmed that they were capable of detoxifying permethrin to 4'-HO-permethrin. Over-expression of CYP9M6 was partially due to gene amplification. Association analysis demonstrated that CYP9M6 and CYP6BB2 complementarily conferred permethrin resistance. Two other P450s (CYP9J26 and CYP9J28), which are capable of metabolizing permethrin, were also over-expressed in the SP strain, indicating that at least four P450 isoforms are likely involved in resistance development. Our data show that it is unlikely that reduced cuticle penetration of permethrin contributes to resistance.

Project description:BACKGROUND:Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection. RESULTS:In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar. CONCLUSIONS:Taken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture.

Project description:BackgroundThe Culex quinquefasciatus mosquito, a major pest and vector of filariasis and arboviruses in the tropics, has developed multiple resistance mechanisms to the main insecticide classes currently available in public health. Among them, the insensitive acetylcholinesterase (ace-1(R) allele) is widespread worldwide and confers cross-resistance to organophosphates and carbamates. Fortunately, in an insecticide-free environment, this mutation is associated with a severe genetic cost that can affect various life history traits. Salivary proteins are directly involved in human-vector contact during biting and therefore play a key role in pathogen transmission.Methods and resultsAn original proteomic approach combining 2D-electrophoresis and mass spectrometry was adopted to compare the salivary expression profiles of two strains of C. quinquefasciatus with the same genetic background but carrying either the ace-1(R) resistance allele or not (wild type). Four salivary proteins were differentially expressed (>2 fold, P<0.05) in susceptible (SLAB) and resistant (SR) mosquito strains. Protein identification indicated that the D7 long form, a major salivary protein involved in blood feeding success, presented lower expression in the resistant strain than the susceptible strain. In contrast, three other proteins, including metabolic enzymes (endoplasmin, triosephosphate isomerase) were significantly over-expressed in the salivary gland of ace-1(R) resistant mosquitoes. A catalogue of 67 salivary proteins of C. quinquefasciatus sialotranscriptome was also identified and described.ConclusionThe "resistance"-dependent expression of salivary proteins in mosquitoes may have considerable impact on biting behaviour and hence on the capacity to transmit parasites/viruses to humans. The behaviour of susceptible and insecticide-resistant mosquitoes in the presence of vertebrate hosts and its impact on pathogen transmission urgently requires further investigation.Data depositionAll proteomic data will be deposited at PRIDE (http://www.ebi.ac.uk/pride/).

Project description:Aedes aegypti SP strain vs. SMK strain. Aedes aegypti is the major vector of yellow fever and dengue/dengue hemorrhagic fever. Starting with a population collected from Singapore, we established a pyrethroid-resistant A. aegypti strain (SP) and investigated three major possible mechanisms of insecticide resistance. After 10 generations of adult selection, an A. aegypti strain developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel genes (Vssc) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can contribute more to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than the susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over-expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2, and confirmed that they were capable of detoxifying permethrin to 4'-HO-permethrin. Over-expression of CYP9M6 was partially due to gene amplification. Association analysis demonstrated that CYP9M6 and CYP6BB2 complementarily conferred permethrin resistance. Two other P450s (CYP9J26 and CYP9J28), which are capable of metabolizing permethrin, were also over-expressed in the SP strain, indicating that at least four P450 isoforms are likely involved in resistance development. Our data show that it is unlikely that reduced cuticle penetration of permethrin contributes to resistance. One-color experiment with two strains (SP, SMK) and 3 developmental stages/genders (larvae, adult males, and adult females), 4 biological replicates each.

Project description:Knowledge of regulation of genes associated with metal resistance in higher plants is very limited. Many plant species have developed different genetic mechanisms and metabolic pathways to cope with metal toxicity. The main objectives of this study were to 1) assess gene expression dynamics of A. rubrum in response to nickel (Ni) stress and 2) describe gene function based on ontology. Certified A. rubrum genotypes were treated with 1,600 mg of Ni per 1 Kg of soil corresponding to a soil total nickel content in a metal-contaminated region in Ontario, Canada. Nickel resistant and susceptible genotypes were selected and used for transcriptome analysis. Overall, 223,610,443 bases were generated. Trinity reads were assembled to trinity transcripts. The transcripts were mapped to protein sequences and after quality controls and appropriate trimmings, 66,783 annotated transcripts were selected as expressed among the libraries. The study reveals that nickel treatment at a high dose of 1,600 mg/kg triggers regulation of several genes. When nickel-resistant genotypes were compared to water controls, 6,263 genes were upregulated and 3,142 were downregulated. These values were 3,308 and 2,176, respectively, when susceptible genotypes were compared to water control. The coping mechanism of A. rubrum to Ni toxicity was elucidated. Upregulation of genes associated with transport in cytosol was prevalent in resistant genotypes compared to controls while upregulation of genes associated with translation in the ribosome was higher in susceptible genotypes when compared to water. The analysis revealed no major gene associated with Ni resistance in A. rubrum. Overall, the results of this study suggest that the genetic mechanism controlling the resistance of this species to nickel is controlled by genes with limited expression. The subtle differences between resistant and susceptible genotypes in gene regulation were detected using water-treated genotypes as references.

Project description:BackgroundIn higher plants, salicylic acid (SA) plays important roles in inducing resistance to biotic and abiotic stresses. Tomato yellow leaf curl virus (TYLCV) causes a highly devastating viral disease in plants, particularly in tomato. However, the roles of SA in inducing tomato plant resistance to TYLCV remain unclear.ResultsIn this study, we investigated whether the exogenous application of SA can improve the resistance of tomato plants to TYLCV in two tomato cultivars, resistant 'Zhefen-702' and susceptible 'Jinpeng-1'. The impacts of SA on the accumulation of ascorbic acid (AsA) and biosynthetic gene expression, the activity of some important reactive oxygen species (ROS)-scavenging enzymes, and the expression patterns of stress-related genes were also determined. Results indicated that SA can effectively regulate the accumulation of AsA, especially in 'Jinpeng-1'. Similarly, the expression patterns of most of the AsA biosynthetic genes showed a negative relationship with AsA accumulation in the resistant and susceptible tomato cultivars. In the two tomato cultivars, the activities of ascorbate peroxidase (APX) and peroxidase (POD) in the SA + TYLCV treated plants were increased during the experiment period except at 14 days (APX in 'Jinpeng-1' was also at 4 days) post infected (dpi) with TYLCV. Simultaneously, the activity of SOD was reduced in 'Jinpeng-1' and increased in 'Zhefen-702' after treatment with SA + TYLCV. SA can substantially induce the expression of ROS-scavenging genes at different extents. From 2 to 10 dpi, the virus content in the SA + TYLCV treated plants was remarkably lower than those in the TYLCV treated plants in 'Jinpeng-1'and Zhefen-702'.ConclusionsThe above results suggest that SA can enhance tomato plant resistance by modulating the expression of genes encoding for ROS-scavenging players, altering the activity of resistance-related enzymes, and inducing the expression of pathogenesis-related genes to produce systemic acquired resistance. Simultaneously, these results confirm that SA is a resistance-inducing factor against TYLCV infection that can be effectively applied in tomato plants.

Project description:BackgroundDowny mildew, caused by Plasmopara viticola, is one of the most severe diseases of grapevine and is commonly controlled by fungicide treatments. The beneficial microorganism Trichoderma harzianum T39 (T39) can induce resistance to downy mildew, although the molecular events associated with this process have not yet been elucidated in grapevine. A next generation RNA sequencing (RNA-Seq) approach was used to study global transcriptional changes associated with resistance induced by T39 in Vitis vinifera Pinot Noir leaves. The long-term aim was to develop strategies to optimize the use of this agent for downy mildew control.ResultsMore than 14.8 million paired-end reads were obtained for each biological replicate of T39-treated and control leaf samples collected before and 24 h after P. viticola inoculation. RNA-Seq analysis resulted in the identification of 7,024 differentially expressed genes, highlighting the complex transcriptional reprogramming of grapevine leaves during resistance induction and in response to pathogen inoculation. Our data show that T39 has a dual effect: it directly modulates genes related to the microbial recognition machinery, and it enhances the expression of defence-related processes after pathogen inoculation. Whereas several genes were commonly affected by P. viticola in control and T39-treated plants, opposing modulation of genes related to responses to stress and protein metabolism was found. T39-induced resistance partially inhibited some disease-related processes and specifically activated defence responses after P. viticola inoculation, causing a significant reduction of downy mildew symptoms.ConclusionsThe global transcriptional analysis revealed that defence processes known to be implicated in the reaction of resistant genotypes to downy mildew were partially activated by T39-induced resistance in susceptible grapevines. Genes identified in this work are an important source of markers for selecting novel resistance inducers and for the analysis of environmental conditions that might affect induced resistance mechanisms.

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Transcriptional Comparison Reveals Differential Resistance Mechanisms between CMV-Resistant PBC688 and CMV-Susceptible G29

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