RESUMO
Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.
Assuntos
Arabidopsis , Betaína , Peptídeo Sintases , Tylenchoidea , Betaína/metabolismo , Animais , Tylenchoidea/metabolismo , Tylenchoidea/genética , Arabidopsis/parasitologia , Arabidopsis/metabolismo , Arabidopsis/genética , Peptídeo Sintases/metabolismo , Peptídeo Sintases/genética , Interações Hospedeiro-Parasita , Doenças das Plantas/parasitologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Nematoides/metabolismo , Nematoides/genéticaRESUMO
Root-knot nematodes (RKNs) are infamous plant pathogens in tomato production, causing considerable losses in agriculture worldwide. Mi-1 is the only commercially available RKN-resistance gene; however, the resistance is inactivated when the soil temperature is over 28 °C. Mi-9 in wild tomato (Solanum arcanum LA2157) has stable resistance to RKNs under high temperature but has not been cloned and applied. In this study, a chromosome-scale genome assembly of S. arcanum LA2157 was constructed through Nanopore and Hi-C sequencing. Based on molecular markers of Mi-9 and comparative genomic analysis, the localization region and candidate Mi-9 genes cluster consisting of seven nucleotide-binding sites and leucine-rich repeat (NBS-LRR) genes were located. Transcriptional expression profiles confirmed that five of the seven candidate genes were expressed in root tissue. Moreover, virus-induced gene silencing of the Sarc_034200 gene resulted in increased susceptibility of S. arcanum LA2157 to Meloidogyne incognita, and genetic transformation of the Sarc_034200 gene in susceptible Solanum pimpinellifolium conferred significant resistance to M. incognita at 25 °C and 30 °C and showed hypersensitive responses at nematode infection sites. This suggested that Sarc_034200 is the Mi-9 gene. In summary, we cloned, confirmed and applied the heat-stable RKN-resistance gene Mi-9, which is of great significance to tomato breeding for nematode resistance.
Assuntos
Solanum lycopersicum , Solanum , Tylenchoidea , Animais , Solanum/genética , Temperatura Alta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Melhoramento Vegetal , Solanum lycopersicum/genética , Cromossomos/metabolismo , Raízes de Plantas/genética , Doenças das Plantas/genéticaRESUMO
AIMS: Root-knot nematodes (RKNs) are plant pathogens that cause huge economic losses worldwide. The biological management of RKNs may be a sustainable alternative to chemical control methods. Here, the biocontrol potential of Methylorubrum rhodesianum M520 against the RKN Meloidogyne incognita was investigated to theoretically support its application as a biocontrol agent in field production. METHODS AND RESULTS: In-vitro assays showed 91.9% mortality of M. incognita second-stage juveniles in the presence of strain M520 and that the hatching rate of M. incognita eggs was 21.7% lower than that of eggs treated with sterile water. In pot experiments, the M520 treatment caused 70.8% reduction in root-knots and increased plant shoot length and stem and root fresh weights, compared to control plant values. In split-root experiments, cucumber roots treated with M520 showed 25.6% decrease in root gall number, compared to that in control roots. CONCLUSION: M520 has multiple mechanisms against RKNs and might be used as a biocontrol agent against M. incognita in cucumber, laying a foundation for further studying M520 biocontrol against RKNs.
Assuntos
Cucumis sativus , Methylobacteriaceae , Tylenchida , Tylenchoidea , Animais , Raízes de PlantasRESUMO
Fusarium oxysporum f. sp. phaseoli, the causal agent of cowpea fusarium wilt, is a serious threat to cowpea production in China. In this study, a sample of cowpea fusarium wilt was identified as Fusarium oxysporum f. sp. phaseoli using the methods of morphological characters and molecular detection. We further reported the first genome assembly for Fusarium oxysporum f. sp. phaseoli, with 53.7 Mb genome sequence comprising 14,694 genes. Comparative genomic analysis among five Fusarium oxysporum genomes showed that four accessory chromosomes in the five Fusarium oxysporum display similar characteristics, with low sequence similarity (55.35%, vs. overall average of 81.76%), low gene density (2.18 genes/10 kb vs. 3.02 genes/Mb) and highly transposable element density (TEs) (15.01/100 kb vs. 4.89/100 kb), indicating that variable accessory chromosomes are the main source of Fusarium oxysporum evolution. We identified a total of 100 Fusarium oxysporum f. sp. phaseoli-specific effectors in the genome and found 13 specific effector genes located in large insertion or deletion regions, suggesting that insertion or deletion events can cause the emergence of species-specific effectors in Fusarium oxysporum. Our genome assembly of Fusarium oxysporum f. sp. phaseoli provides a valuable resource for the study of cowpea fusarium wilt, and the comparative genomic study of Fusarium oxysporum could contribute to the knowledge of genome and effector-associated pathogenicity evolution in Fusarium oxysporum study.
Assuntos
Fusarium , Fusarium/genética , Doenças das Plantas , Genoma FúngicoRESUMO
Cucumis metuliferus (African horned cucumber), a wild relative of Cucumis sativus (cucumber) and Cucumis melo (melon), displays high-level resistance to several important plant pathogens (e.g., root-knot nematodes and several viruses). Here, we report a chromosome-level genome assembly for C. metuliferus, with a 316 Mb genome sequence comprising 29 039 genes. Phylogenetic analysis of related species in family Cucurbitaceae indicated that the divergence time between C. metuliferus and melon was 17.8 million years ago. Comparisons between the C. metuliferus and melon genomes revealed large structural variations (inversions and translocations >1 Mb) in eight chromosomes of these two species. Gene family comparison showed that C. metuliferus has the largest number of resistance-related nucleotide-binding site leucine-rich repeat (NBS-LRR) genes in Cucurbitaceae. The loss of NBS-LRR loci caused by large insertions or deletions (indels) and pseudogenization caused by small indels explained the loss of NBS-LRR genes in Cucurbitaceae. Population structure analysis suggested that C. metuliferus originated in Zimbabwe, then spread to other southern African regions where it likely underwent similar domestic selection as melon. This C. metuliferus reference sequence will accelerate the understanding of the molecular evolution of resistance-related genes and enhance cucurbit crop improvement efforts.
Assuntos
Cucumis/genética , Genes de Plantas , Genoma de Planta , Filogenia , África , Cromossomos de Plantas , Cucumis melo/genética , Evolução Molecular , Variação Genética , Genética Populacional , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Seleção Genética , ZimbábueRESUMO
Peptidases are very important to parasites, which have central roles in parasite biology and pathogenesis. In this study, by comparative genome analysis, genome-wide peptidase diversities among plant-parasitic nematodes are estimated. We find that genes encoding cysteine peptidases in family C13 (legumain) are significantly abundant in pine wood nematodes Bursaphelenchus genomes, compared to those in other plant-parasitic nematodes. By phylogenetic analysis, a clade of B. xylophilus-specific legumain is identified. RT-qPCR detection shows that these genes are highly expressed at early stage during the nematode infection process. Utilizing transgene technology, cDNAs of three species-specific legumain were introduced into the Arabidopsis γvpe mutant. Functional complementation assay shows that these B. xylophilus legumains can fully complement the activity of Arabidopsis γVPE to mediate plant cell death triggered by the fungal toxin FB1. Secretory activities of these legumains are experimentally validated. By comparative transcriptome analysis, genes involved in plant cell death mediated by legumains are identified, which enrich in GO terms related to ubiquitin protein transferase activity in category molecular function, and response to stimuli in category biological process. Our results suggest that B. xylophilu-specific legumains have potential as effectors to be involved in nematode-plant interaction and can be related to host cell death.
Assuntos
Arabidopsis , Micotoxinas , Parasitos , Pinus , Rabditídios , Tylenchida , Animais , Arabidopsis/genética , Cisteína/genética , Cisteína Endopeptidases , Peptídeo Hidrolases/genética , Filogenia , Pinus/parasitologia , Doenças das Plantas/parasitologia , Plantas/parasitologia , Transferases/genética , Tylenchida/genética , Ubiquitinas/genética , Virulência , XylophilusRESUMO
Root-knot nematodes, Meloidogyne spp., secrete effectors to modulate plant immune responses and establish a parasitic relationship with host plants. However, the functions and plant targets of C-type lectin (CTL)-like effectors of Meloidogyne incognita remain unknown. Here, we characterized a CTL-like effector of M. incognita, MiCTL1a, and identified its target and role in nematode parasitism. In situ hybridization demonstrated the expression of MiCTL1 in the subventral glands; and in planta, immunolocalization showed its secretion during M. incognita parasitism. Virus-induced gene silencing of the MiCTL1 reduced the infection ability of M. incognita in Nicotiana benthamiana. The ectopic expression in Arabidopsis not only increased susceptibility to M. incognita but also promoted root growth. Yeast two-hybrid and co-immunoprecipitation assays revealed that MiCTL1a interacts with Arabidopsis catalases, which play essential roles in hydrogen peroxide homeostasis. Knockout or overexpression of catalases showed either increased or reduced susceptibility to M. incognita, respectively. Moreover, MiCTL1a not only reduced catalase activity in vitro and in planta but also modulated stress-related gene expressions in Arabidopsis. Our data suggest that MiCTL1a interacts with plant catalases and interferes with catalase activity, allowing M. incognita to establish a parasitic relationship with its host by fine-tuning responses mediated by reactive oxygen species.
Assuntos
Tylenchoidea , Animais , Catalase , Proteínas de Helminto , Lectinas Tipo C , Doenças das PlantasRESUMO
Root-knot nematodes (Meloidogyne spp.) are soilborne pathogens that infect vegetable crops and cause major economic losses worldwide annually. Therefore, there is an urgent need for novel nematicides or biological control agents to reduce the damage caused by root-knot nematodes. In this study, we tested efficacy of the Bacillus cereus strain Bc-cm103, isolated from the rhizoplane of Cucumis metuliferus, against Meloidogyne incognita. Strain Bc-cm103 fermentation broth caused 100% mortality of the nematode second-stage juveniles within 12 h and decreased the egg hatching rate by 40.06% within 72 h compared with sterile water. Confocal laser-scanning microscopy revealed that strain Bc-cm103 formed a biofilm on cucumber (C. sativus) roots, which protected the roots from the infection of M. incognita. Additionally, strain Bc-cm103 activated the defense-responsive genes PR1, PR2, LOX1, and CTR1 in cucumber. Furthermore, strain Bc-cm103 significantly reduced the appearance of root galls in pot, split-root, and field tests. These results indicated that B. cereus strain Bc-cm103 had a strong suppressive effect on M. incognita and therefore could be used as a potential biocontrol agent against this pathogen.
Assuntos
Solanum lycopersicum , Tylenchoidea , Animais , Antinematódeos , Bacillus cereus , Agentes de Controle BiológicoRESUMO
Matricaria chamomilla flower extract was used as a biocompatible material for synthesis of zinc oxide nanoparticles (ZnONPs). The synthesized NPs were evaluated for their antibacterial potential in vitro and in vivo against the Gram-negative bacterium Ralstonia solanacearum, which causes devastating bacterial wilt disease in tomato and other crops. Synthesized ZnONPs were further analyzed by UV-visible spectroscopy, Fourier transform infrared spectroscopy, x-ray diffraction, transmission electron microscopy, and scanning electron microscopy with energy-dispersive spectroscopy. The synthesized polydisperse ZnONPs were found to be in the size range of 8.9 to 32.6 nm, and at 18.0 µg ml-1 exhibited maximum in vitro growth inhibition of the pathogen R. solanacearum. Scanning electron microscopy analysis of affected bacterial cells showed morphological deformation such as disruption of the cell membrane and wall, and the leakage of cell contents. Results of in vivo studies also showed that application of ZnONPs to the artificially inoculated tomato plants with the pathogen R. solanacearum significantly enhanced the plant growth by reducing bacterial soil population and disease severity as compared with the untreated control. Biosynthesized ZnONPs could be an effective approach to control the bacterium R. solanacearum.
Assuntos
Matricaria , Nanopartículas , Ralstonia solanacearum , Solanum lycopersicum , Óxido de Zinco , Testes de Sensibilidade Microbiana , Óxido de Zinco/farmacologiaRESUMO
Bacillus cereus strain Bc-cm103 shows nematicidal activity and, therefore, has been used as a biological control agent to control the root-knot nematode Meloidogyne incognita. However, it remains unknown whether volatile organic compounds (VOCs) produced by B. cereus strain Bc-cm103 are effective in biocontrol against M. incognita. Therefore, in this study, we investigated the activity of Bc-cm103 VOCs against M. incognita. The B. cereus strain Bc-cm103 significantly repelled the second-stage juveniles (J2s) of M. incognita. In vitro evaluation of VOCs produced by the fermentation of Bc-cm103 in a three-compartment Petri dish revealed the mortality rates of M. incognita J2s as 90.8% at 24 h and 97.2% at 48 h. Additionally, evaluation of the ability of Bc-cm103 VOCs to suppress M. incognita infection in a double-layered pot test showed that root galls on cucumber roots decreased by 46.1%. Furthermore, 21 VOCs were identified from strain Bc-cm103 by solid-phase microextraction gas chromatography-mass spectrometry, including alkanes, alkenes, esters, and sulfides. Among them, dimethyl disulfide (30.63%) and S-methyl ester butanethioic acid (30.29%) were reported to have strong nematicidal activity. Together, these results suggest that B. cereus strain Bc-cm103 exhibits fumigation activity against M. incognita.
Assuntos
Solanum lycopersicum , Tylenchoidea , Compostos Orgânicos Voláteis , Animais , Bacillus cereus , Fumigação , Compostos Orgânicos Voláteis/farmacologiaRESUMO
Fungi are considered to be rich in biologically active natural products for agricultural and medicinal purposes. The discovery and accurate identification of the bioactive fungal natural products is important for their efficient utilization. During the course of our continuing search for the new natural products from the fungal agents, we found the well-known bio-control fungus Purpureocillium lilacinum showed in vitro activity against Botrytis cinerea, an airborne plant pathogenic fungus causing gray mold disease in many vegetables and fruits. The co-culture of two fungi on agar plate showed that P. lilacinum inhibited the growth of B. cinerea which means P. lilacinum has potential to produce some bioactive secondary metabolites against B. cinerea. In this study, we applied matrix-assisted laser desorption ionization-time of flight mass spectrometry imaging mass spectrometry (MALDI-TOF-IMS), as a fast identification tool, for the discovery of a new antifungal lipopeptaibol (leucinostatin Z) from P. lilacinum against B. cinerea. The planar structure of leucinostatin Z was further established by using the LC-HRESI-MS-MS analysis. MALDI-TOF-IMS is becoming a new approach that allows us to observe the bioactive natural products directly on growth media between the colonies of two fungi, which is faster and more effective than the traditional techniques to discover new bioactive compounds in fungi.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Agentes de Controle Biológico/química , Agentes de Controle Biológico/farmacologia , Botrytis/efeitos dos fármacos , Hypocreales/química , Antifúngicos/isolamento & purificação , Agentes de Controle Biológico/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Botrytis/crescimento & desenvolvimento , Técnicas de Cocultura , Hypocreales/crescimento & desenvolvimento , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Large amounts of effectors are secreted by the oesophageal glands of plant-parasitic nematodes, but their molecular mode of action remains largely unknown. We characterized a Meloidogyne incognita protein disulphide isomerase (PDI)-like effector protein (MiPDI1) that facilitates nematode parasitism. In situ hybridization showed that MiPDI1 was expressed specifically in the subventral glands of M. incognita. It was significantly upregulated during parasitic stages. Immunolocalization demonstrated MiPDI1 secretion in planta during nematode migration and within the feeding cells. Host-induced silencing of the MiPDI1 gene affected the ability of the nematode to infect the host, whereas MiPDI1 expression in Arabidopsis increased susceptibility to M. incognita, providing evidence for a key role of MiPDI1 in M. incognita parasitism. Yeast two-hybrid, bimolecular fluorescence complementation and coimmunoprecipitation assays showed that MiPDI1 interacted with a tomato stress-associated protein (SlSAP12) orthologous to the redox-regulated AtSAP12, which plays an important role in plant responses to abiotic and biotic stresses. SAP12 silencing or knocking out in Nicotiana benthamiana and Arabidopsis increased susceptibility to M. incognita. Our results suggest that MiPDI1 acts as a pathogenicity factor promoting disease by fine-tuning SAP-mediated responses at the interface of redox signalling, defence and stress acclimation in Solanaceae and Arabidopsis.
Assuntos
Arabidopsis , Tylenchoidea , Animais , Arabidopsis/genética , Proteínas de Choque Térmico , Doenças das Plantas , NicotianaRESUMO
Plant-parasitic nematodes secrete a series of effectors to promote parasitism by modulating host immunity, but the detailed molecular mechanism is ambiguous. Animal parasites secrete macrophage migration inhibitory factor (MIF)-like proteins for evasion of host immune systems, in which their biochemical activities play essential roles. Previous research demonstrated that MiMIF-2 effector was secreted by Meloidogyne incognita and modulated host immunity by interacting with annexins. In this study, we show that MiMIF-2 had tautomerase activity and protected nematodes against H2O2 damage. MiMIF-2 expression not only decreased the amount of H2O2 generation during nematode infection in Arabidopsis, but also suppressed Bax-induced cell death by inhibiting reactive oxygen species burst in Nicotiana benthamiana. Further, RNA-seq transcriptome analysis and RT-qPCR showed that the expression of some heat-shock proteins was down regulated in MiMIF-2 transgenic Arabidopsis. After treatment with flg22, RNA-seq transcriptome analysis indicated that the differentially expressed genes in MiMIF-2 expressing Arabidopsis were pointed to plant hormone signal transduction, compound metabolism and plant defense. RT-qPCR and metabolomic results confirmed that salicylic acid (SA) related marker genes and SA content were significantly decreased. Our results provide a comprehensive understanding of how MiMIF-2 modulates plant immunity and broaden knowledge of the intricate relationship between M. incognita and host plants.
Assuntos
Proteínas de Helminto/metabolismo , Ácido Salicílico/metabolismo , Tylenchoidea/enzimologia , Animais , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/parasitologia , Regulação para Baixo/efeitos dos fármacos , Escherichia coli , Flagelina/farmacologia , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas/efeitos dos fármacos , Parasitos/metabolismo , Raízes de Plantas/parasitologia , Plantas Geneticamente Modificadas , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Purpureocillium lilacinum is a promising commercial agent for controlling plant-parasitic nematodes and plant pathogens. Leucinostatins are a family of lipopeptides produced by P. lilacinum that are synthesized, modified, and regulated by a gene cluster consisting of 20 genes. Sequence analyses have indicated that lcsL, a gene in the lcs cluster, is a putative bZIP transcription factor. In this study, the CRISPR-Cas9 system was introduced to increase the efficiency of homologous recombination for the disruption of lcsL. The expression of genes in the cluster was significantly reduced in lcsL disruption mutants, and the output of leucinostatins was decreased to undetectable levels. In the lcsL overexpression strain, the expression of genes in the cluster and the yield of leucinostatins were all increased. The antagonism of both the wild type and mutant against Phytophthora infestans was also consistent with the gene expression and the output of leucinostatins. These results indicate that the gene lcsL is crucial for the regulating the synthesis of leucinostatins.
Assuntos
Vias Biossintéticas/genética , Regulação Fúngica da Expressão Gênica , Hypocreales/metabolismo , Família Multigênica , Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Peptídeos Catiônicos Antimicrobianos , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Deleção de Genes , Técnicas de Inativação de Genes , Recombinação Homóloga , Hypocreales/genética , Phytophthora infestans/efeitos dos fármacos , Phytophthora infestans/crescimento & desenvolvimento , Fatores de Transcrição/genéticaRESUMO
The fungus Isaria javanica is an important entomopathogen that parasitizes various insects and is effective for pest control. In this study, we sequenced and assembled the genomes (IJ1G and IJ2G) of two I. javanica strains isolated from different insects. The genomes were approximately 35 Mb in size with 11,441 and 11,143 protein-coding genes, respectively. Using a phylogenomic approach, we evaluated genome evolution across five entomopathogenic fungi in Cordycipitaceae. By comparative genome analysis, it was found that family S53 serine peptidases were expanded in Cordycipitaceae entomopathogens, particularly in I. javanica. Gene duplication events were identified based on phylogenetic relationships inferred from 82 S53 peptidases within six entomopathogenic fungal genomes. Moreover, we found that carbohydrate-active enzymes and proteinases were the largest secretory protein groups encoded in the I. javanica genome, especially chitinases (GH18), serine and aspartic peptidases (S53, S08, S10, A01). Pathogenesis-related genes and genes for bacterial-like toxins and secondary metabolites were also identified. By comparative transcriptome analysis, differentially expressed genes in response to insect nutrients (in vitro) were identified. Moreover, most S53 peptidases were detected to be significantly upregulated during the initial fungal infection process in insects (in vivo) by RT-qPCR. Our results provide new clues about understanding evolution of pathogenic proteases and may suggest that abundant S53 peptidases in the I. javanica genome may contribute to its effective parasitism on various insects.
Assuntos
Cordyceps/genética , Proteínas Fúngicas/genética , Genoma Fúngico/genética , Insetos/microbiologia , Peptídeo Hidrolases/genética , Animais , Cordyceps/classificação , Proteínas Fúngicas/metabolismo , Duplicação Gênica , Regulação Fúngica da Expressão Gênica , Hypocreales/classificação , Hypocreales/genética , Peptídeo Hidrolases/metabolismo , Filogenia , TranscriptomaRESUMO
Applications that use Bacterial Artificial Chromosome (BAC) libraries often require paired-end sequences and knowledge of the physical location of each clone in plates. To facilitate obtaining this information in high-throughput, we generated pBACode vectors: a pool of BAC cloning vectors, each with a pair of random barcodes flanking its cloning site. In a pBACode BAC library, the BAC ends and their linked barcodes can be sequenced in bulk. Barcode pairs are determined by sequencing the empty pBACode vectors, which allows BAC ends to be paired according to their barcodes. For physical clone mapping, the barcodes are used as unique markers for their linked genomic sequence. After multi-dimensional pooling of BAC clones, the barcodes are sequenced and deconvoluted to locate each clone. We generated a pBACode library of 94,464 clones for the flounder Paralichthys olivaceus and obtained paired-end sequence from 95.4% of the clones. Incorporating BAC paired-ends into the genome preassembly improved its continuity by over 10-fold. Furthermore, we were able to use the barcodes to map the physical locations of each clone in just 50 pools, with up to 11 808 clones per pool. Our physical clone mapping located 90.2% of BAC clones, enabling targeted characterization of chromosomal rearrangements.
Assuntos
Cromossomos Artificiais Bacterianos , Clonagem Molecular , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mapeamento Físico do Cromossomo/métodos , Análise de Sequência de DNA/métodos , Animais , Linguado/genética , Biblioteca Gênica , Genoma , Saccharomyces cerevisiae/genéticaRESUMO
Purpureocillium lilacinum of Ophiocordycipitaceae is one of the most promising and commercialized agents for controlling plant parasitic nematodes, as well as other insects and plant pathogens. However, how the fungus functions at the molecular level remains unknown. Here, we sequenced two isolates (PLBJ-1 and PLFJ-1) of P. lilacinum from different places Beijing and Fujian. Genomic analysis showed high synteny of the two isolates, and the phylogenetic analysis indicated they were most related to the insect pathogen Tolypocladium inflatum. A comparison with other species revealed that this fungus was enriched in carbohydrate-active enzymes (CAZymes), proteases and pathogenesis related genes. Whole genome search revealed a rich repertoire of secondary metabolites (SMs) encoding genes. The non-ribosomal peptide synthetase LcsA, which is comprised of ten C-A-PCP modules, was identified as the core biosynthetic gene of lipopeptide leucinostatins, which was specific to P. lilacinum and T. ophioglossoides, as confirmed by phylogenetic analysis. Furthermore, gene expression level was analyzed when PLBJ-1 was grown in leucinostatin-inducing and non-inducing medium, and 20 genes involved in the biosynthesis of leucionostatins were identified. Disruption mutants allowed us to propose a putative biosynthetic pathway of leucinostatin A. Moreover, overexpression of the transcription factor lcsF increased the production (1.5-fold) of leucinostatins A and B compared to wild type. Bioassays explored a new bioactivity of leucinostatins and P. lilacinum: inhibiting the growth of Phytophthora infestans and P. capsici. These results contribute to our understanding of the biosynthetic mechanism of leucinostatins and may allow us to utilize P. lilacinum better as bio-control agent.
Assuntos
Paecilomyces/genética , Paecilomyces/metabolismo , Peptídeos/metabolismo , Phytophthora/microbiologia , Peptídeos Catiônicos Antimicrobianos , Cromatografia Líquida de Alta Pressão , Perfilação da Expressão Gênica , Genes Fúngicos , Genômica , Análise de Sequência com Séries de Oligonucleotídeos , Controle Biológico de Vetores/métodos , Filogenia , Reação em Cadeia da Polimerase , TranscriptomaRESUMO
BACKGROUND: microRNAs (miRNAs) are endogenous small RNAs that play important regulatory functions in plant development. Genetic variations in miRNAs sequences or their target-binding sites (microRNA-target interaction sites) can alter miRNA targets in animal and human. Whether these single nucleotide polymorphisms (SNPs) in plant are functional have not yet been determined. RESULTS: In this study, we constructed leaf, root, and stem-derived small libraries of cucumber (Cucumis sativus) line 9930 (cultivated China-group cucumber) and C. sativus var. hardwickii (wild India group cucumber). A total of 22 conserved miRNA families, nine less-conserved miRNA families, and 49 cucumber-specific miRNAs were identified in both line 9930 and hardwickii. We employed cucumber resequencing data to perform a genome-wide scan for SNPs in cucumber miRNA-target interaction sites, including miRNA mature sequences and miRNA-target binding sites. As a result, we identified a total of 19 SNPs in mature miRNA sequences and 113 SNPs in miRNA-target binding sites with the potential to affect miRNA-target interactions. Furthermore, we experimentally confirmed that these SNPs produced 14 9930-unique targets mRNAs and 15 hardwickii-unique targets mRNA for cucumber miRNAs. This is the first experimental validation of SNPs in miRNA-target interaction sites affecting miRNA-target binding in plants. CONCLUSIONS: Our results indicate that SNPs can alter miRNA function and produce unique miRNA targets in cultivated and wild cucumbers. Therefore, miRNA-related SNPs may have played important in events that led to the agronomic differences between domestic and wild cucumber.
Assuntos
Cucumis sativus/genética , MicroRNAs/fisiologia , Sítios de Ligação , Cucumis sativus/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNARESUMO
BACKGROUND: The fungus Pochonia chlamydosporia parasitizes nematode eggs and has become one of the most promising biological control agents (BCAs) for plant-parasitic nematodes, which are major agricultural pests that cause tremendous economic losses worldwide. The complete mitochondrial (mt) genome is expected to open new avenues for understanding the phylogenetic relationships and evolution of the invertebrate-pathogenic fungi in Hypocreales. RESULTS: The complete mitogenome sequence of P. chlamydosporia is 25,615 bp in size, containing the 14 typical protein-coding genes, two ribosomal RNA genes, an intronic ORF coding for a putative ribosomal protein (rps3) and a set of 23 transfer RNA genes (trn) which recognize codons for all amino acids. Sequence similarity studies and syntenic gene analyses show that 87.02% and 58.72% of P. chlamydosporia mitogenome sequences match 90.50% of Metarhizium anisopliae sequences and 61.33% of Lecanicillium muscarium sequences with 92.38% and 86.04% identities, respectively. A phylogenetic tree inferred from 14 mt proteins in Pezizomycotina fungi supports that P. chlamydosporia is most closely related to the entomopathogenic fungus M. anisopliae. The invertebrate-pathogenic fungi in Hypocreales cluster together and clearly separate from a cluster comprising plant-pathogenic fungi (Fusarium spp.) and Hypocrea jecorina. A comparison of mitogenome sizes shows that the length of the intergenic regions or the intronic regions is the major size contributor in most of mitogenomes in Sordariomycetes. Evolutionary analysis shows that rps3 is under positive selection, leading to the display of unique evolutionary characteristics in Hypocreales. Moreover, the variability of trn distribution has a clear impact on gene order in mitogenomes. Gene rearrangement analysis shows that operation of transposition drives the rearrangement events in Pezizomycotina, and most events involve in trn position changes, but no rearrangement was found in Clavicipitaceae. CONCLUSIONS: We present the complete annotated mitogenome sequence of P. chlamydosporia. Based on evolutionary and phylogenetic analyses, we have determined the relationships between the invertebrate-pathogenic fungi in Hypocreales. The invertebrate-pathogenic fungi in Hypocreales referred to in this paper form a monophyletic group sharing a most recent common ancestor. Our rps3 and trn gene order results also establish a foundation for further exploration of the evolutionary trajectory of the fungi in Hypocreales.