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1.
Biomolecules ; 10(1)2020 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-31940882

RESUMO

Verticillium wilt, caused by the ascomycete fungus Verticillium dahliae (Vd), is a devastating disease of numerous plant species. However, the pathogenicity/virulence-related genes in this fungus, which may be potential targets for improving plant resistance, remain poorly elucidated. For the study of these genes in Vd, we used a well-established host-induced gene silencing (HIGS) approach and identified 16 candidate genes, including a putative adenylate kinase gene (VdAK). Transiently VdAK-silenced plants developed milder wilt symptoms than control plants did. VdAK-knockout mutants were more sensitive to abiotic stresses and had reduced germination and virulence on host plants. Transgenic Nicotiana benthamiana and Arabidopsis thaliana plants that overexpressed VdAK dsRNAs had improved Vd resistance than the wild-type. RT-qPCR results showed that VdAK was also crucial for energy metabolism. Importantly, in an analysis of total small RNAs from Vd strains isolated from the transgenic plants, a small interfering RNA (siRNA) targeting VdAK was identified in transgenic N. benthamiana. Our results demonstrate that HIGS is a promising strategy for efficiently screening pathogenicity/virulence-related genes of Vd and that VdAK is a potential target to control this fungus.

2.
Pest Manag Sci ; 2019 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-31762173

RESUMO

BACKGROUD: Volatile terpenes can act as ecological signals to affect insect behavior. It has been proposed that the manipulation of terpenes in plants can help to control herbivore pests. In order to investigate the potential pest management function of (E)-ß-caryophyllene in cotton plants, the (E)-ß-caryophyllene synthase gene (GhTPS1) was inserted into Gossypium hirsutum variety R15 to generate overexpression lines. RESULTS: Four GhTPS1-transgenic lines were generated, and GhTPS1 expression in transgenic L18 and L46 lines was 3-5-fold higher than in R15 plants. The transgenic L18 and L46 lines also emitted significantly more (E)-ß-caryophyllene than R15. In laboratory bioassays, L18 and L46 plants reduced pests Apolygus lucorum, Aphis gossypii and Helicoverpa armigera, and attracted parasitoids Peristenus spretus and Aphidius gifuensis, but not Microplitis mediator. In open-field trials, L18 and L46 plants reduced A. lucorum, Adelphocoris suturalis and H. armigera, but had no significant effects on predators. CONCLUSION: Our findings suggest that L18 and L46 plants reduce several major hemipteran and lepidopteran cotton pests, whereas, two parasitoids P. spretus and A. gifuensis, were attracted by L18 and L46 plants. This study shows that overexpressing GhTPS1 in cotton may help to improve pest management in cotton fields. © 2019 Society of Chemical Industry.

3.
Curr Genet ; 2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31422448

RESUMO

Verticillium dahliae, a soil-borne fungus, can invade plant vascular tissue and cause Verticillium wilt. The enzyme α-oxoglutarate dehydrogenase (OGDH), catalyzing the oxidation of α-oxoglutarate in the tricarboxylic acid cycle (TCA), is vital for energy metabolism in the fungi. Here, we identified the OGDH gene in V. dahliae (VdOGDH, VDAG_10018) and investigated its function in virulence by generating gene deletion mutants (ΔVdOGDH) and complementary mutants (ΔVdOGDH-C). When the ΔVdOGDH mutants were supplemented with different carbon sources, vegetative growth on Czapek Dox medium was significantly impaired, suggesting that VdOGDH is crucial for vegetative growth and carbon utilization. Conidia of the ΔVdOGDH mutants were atypically rounded or spherical, and hyphae were irregularly branched and lacked typical whorled branches. Mutants ΔVdOGDH-1 and ΔVdOGDH-2 were highly sensitive to H2O2 in the medium plates and had higher intracellular ROS levels. ΔVdOGDH mutants also had elevated expression of oxidative response-related genes, indicating that VdOGDH is involved in response to oxidative stress. In addition, the disruption of VdOGDH caused a significant increase in the expression of energy metabolism-related genes VdICL, VdICDH, VdMDH, and VdPDH and melanin-related genes Vayg1, VdSCD, VdLAC, VT4HR, and VaflM in the ΔVdOGDH mutants; thus, VdOGDH is also important for energy metabolism and melanin accumulation. Cotton plants inoculated with ΔVdOGDH mutants exhibited mild leaf chlorosis and the disease index was lower compared with wild type and ΔVdOGDH-C strains. These results together show that VdOGDH involved in energy metabolism of V. dahliae, is also essential for full virulence by regulating multiple fungal developmental factors.

4.
Sci Rep ; 8(1): 15404, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337674

RESUMO

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae is a common, devastating plant vascular disease notorious for causing economic losses. Despite considerable research on plant resistance genes, there has been little progress in modeling the effects of this fungus owing to its complicated pathogenesis. Here, we analyzed the transcriptional and metabolic responses of Arabidopsis thaliana to V. dahliae inoculation by Illumina-based RNA sequencing (RNA-seq) and nuclear magnetic resonance (NMR) spectroscopy. We identified 13,916 differentially expressed genes (DEGs) in infected compared with mock-treated plants. Gene ontology analysis yielded 11,055 annotated DEGs, including 2,308 for response to stress and 2,234 for response to abiotic or biotic stimulus. Pathway classification revealed involvement of the metabolic, biosynthesis of secondary metabolites, plant-pathogen interaction, and plant hormone signal transduction pathways. In addition, 401 transcription factors, mainly in the MYB, bHLH, AP2-EREBP, NAC, and WRKY families, were up- or downregulated. NMR analysis found decreased tyrosine, asparagine, glutamate, glutamine, and arginine and increased alanine and threonine levels following inoculation, along with a significant increase in the glucosinolate sinigrin and a decrease in the flavonoid quercetin glycoside. Our data reveal corresponding changes in the global transcriptomic and metabolic profiles that provide insights into the complex gene-regulatory networks mediating the plant's response to V. dahliae infection.


Assuntos
Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Metabolômica , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Transcriptoma , Verticillium/fisiologia , Arabidopsis/genética , Arabidopsis/microbiologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno , Doenças das Plantas/genética , Proteínas de Plantas/genética
5.
Genes (Basel) ; 9(10)2018 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-30340423

RESUMO

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes a destructive vascular wilt, but details of the molecular mechanism behind its pathogenicity are not very clear. Here, we generated a red fluorescent isolate of V. dahliae by protoplast transformation to explore its pathogenicity mechanism, including colonization, invasion, and extension in Nicotiana benthamiana, using confocal microscopy. The nucleotide sequences of mCherry were optimized for fungal expression and cloned into pCT-HM plasmid, which was inserted into V. dahliae protoplasts. The transformant (Vd-m) shows strong red fluorescence and its phenotype, growth rate, and pathogenicity did not differ significantly from the wild type V. dahliae (Vd-wt). Between one and three days post inoculation (dpi), the Vd-m successfully colonized and invaded epidermal cells of the roots. From four to six dpi, hyphae grew on root wounds and lateral root primordium and entered xylem vessels. From seven to nine dpi, hyphae extended along the surface of the cell wall and massively grew in the xylem vessel of roots. At ten dpi, the Vd-m was found in petioles and veins of leaves. Our results distinctly showed the pathway of V. dahliae infection and colonization in N. benthamiana, and the optimized expression can be used to deepen our understanding of the molecular mechanism of pathogenicity.

6.
J Insect Sci ; 18(4)2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30137436

RESUMO

To determine cellular changes caused by the chimeric protein Vip3AcAa against Helicoverpa armigera, we used transmission electron microscopy to examine ultrastructural changes in midgut cells of third-instar larvae of Cry1Ac-susceptible H. armigera after feeding on an artificial diet containing the Vip3AcAa toxin. Midgut epithelial cells of Cry1Ac-resistant H. armigera larvae that had fed on an artificial diet containing Vip3AcAa or on Bt cotton expressing Vip3AcAa+Cry1Ac were also examined using optical microscopy and hematoxylin-eosin staining. In the midgut cells of H. armigera larvae fed with Vip3AcAa, microvilli were swollen and broken; inner cristae of the mitochondria were indistinct and vacuolated; endoplasmic reticulum was swollen, fractured, and disordered; boundaries of karyotheca in the nucleus were indistinct and chromatin underwent pyknosis and was pressed close to the karyotheca. Histopathological changes and the time of onset in midgut tissues of H. armigera larvae fed on Vip3AcAa or Cry1Ac were similar. Vip3AcAa and transgenic cotton expressing Vip3AcAa+Cry1Ac caused the goblet cell cavity and microvilli pathological changes in the midgut epithelial cells of the Cry1Ac-susceptible and Cry1Ac-resistant H. armigera larvae that eventually killed the larvae.


Assuntos
Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Gossypium/química , Proteínas Hemolisinas/farmacologia , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Animais , Dieta , Sistema Digestório/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Mariposas/crescimento & desenvolvimento , Mariposas/fisiologia , Plantas Geneticamente Modificadas/química
7.
Genes (Basel) ; 9(3)2018 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-29534051

RESUMO

The soil-borne ascomycete fungus Verticillium dahliae causes vascular wilt disease and can seriously diminish the yield and quality of important crops. Functional analysis of growth- and pathogenicity-related genes is essential for revealing the pathogenic molecular mechanism of V. dahliae. Phospholipase is an important virulence factor in fungi that hydrolyzes phospholipids into fatty acid and other lipophilic substances and is involved in hyphal development. Thus far, only a few V. dahliae phospholipases have been identified, and their involvement in V. dahliae development and pathogenicity remains unknown. In this study, the function of the patatin-like phospholipase gene in V. dahliae (VdPLP, VDAG_00942) is characterized by generating gene knockout and complementary mutants. Vegetative growth and conidiation of VdPLP deletion mutants (ΔVdPLP) were significantly reduced compared with wild type and complementary strains, but more microsclerotia formed. The ΔVdPLP mutants were very sensitive to the cell-wall-perturbing agents: calcofluor white (CFW) and Congo red (CR). The transcriptional level of genes related to the cell wall integrity (CWI) pathway and chitin synthesis were downregulated, suggesting that VdPLP has a pivotal role in the CWI pathway and chitin synthesis in V. dahliae. ΔVdPLP strains were distinctly impaired in in their virulence and ability to colonize Nicotiana benthamiana roots. Our results demonstrate that VdPLP regulates hyphal growth and conidial production and is involved in stabilizing the cell wall, thus mediating the pathogenicity of V. dahliae.

8.
Curr Genet ; 64(3): 645-659, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29177887

RESUMO

Ferric reductases are integral membrane proteins involved in the reduction of environmental ferric iron into the biologically available ferrous iron. In the most overwhelming phytopathogenic fungus, Verticillium dahliae, these ferric reductase are not studied in details. In this study we explored the role of FreB gene (VDAG_06616) in the ferric reduction and virulence of V. dahliae by generating the knockout mutants (ΔFreB) and complementary strains (ΔFreB-C) using protoplast transformation. When cultured on media supplemented with FeSO4, FeCl3 and no iron, ΔFreB exhibited significantly reduced growth and spore production especially on media with no iron. Transmembrane ferric reductase activity of ΔFreB was decreased up to 50% than wild type strains (Vd-wt). The activity was fully restored in ΔFreB-C. Meanwhile, the expression levels of other related genes (Frect-4, Frect-5, Frect-6 and Met) were obviously increased in ΔFreB. Compared with the Vd-wt and ΔFreB-C, ΔFreB-1 and ΔFreB-2 were impaired in colony diameter and spore number on different carbon sources (starch, sucrose, galactose and xylose). ΔFreB-1 and ΔFreB-2 were also highly sensitive to oxidative stress as revealed by the plate diffusion assay when 100 µM H2O2 was applied to the fungal culture. When Nicotiana benthamiana plants were inoculated, ΔFreB exhibited less disease symptoms than Vd-wt and ΔFreB-C. In conclusion, the present findings not only indicate that FreB mediates the ferric metabolism and is required for the full virulence in V. dahliae, but would also accelerate future investigation to uncover the pathogenic mechanism of this fungus.


Assuntos
Compostos Férricos/metabolismo , Proteínas Fúngicas/metabolismo , Verticillium/metabolismo , Adaptação Fisiológica , Carbono/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Teste de Complementação Genética , Estresse Oxidativo , Filogenia , Verticillium/genética , Verticillium/crescimento & desenvolvimento , Verticillium/patogenicidade , Virulência
9.
Curr Genet ; 64(1): 235-246, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28798981

RESUMO

Verticillium dahliae is the most overwhelming plant pathogen, causing Verticillium wilt in a number of economic crops. The molecular mechanism is still unclear and identification of the genes involved in the pathogenicity or virulence of this fungus would benefit to uncover such mechanism. STT3 is a catalytic subunit of the multi-subunit oligosaccharyl transferase (OST) and plays an essential role in glycoprotein modification. Here, we characterized STT3 gene (VDAG_03232.1) of V. dahliae to explore its regulatory role in the development and virulence by deletion and complementation of this gene, as well as its silence in transgenic plants. The expression of the STT3 gene increased at the stage of conidia germination and reached its peak level with germ tube formation and elongation. We generated the knockout mutants (ΔSTT3) using protoplast transformation. Mycelial growth, sporulation ability and glycoprotein secretion were impaired when ΔSTT3 mutants were grown on media supplemented with different carbon sources. Moreover, ΔSTT3 mutants exhibited distinctly decreased germination ratio and reduction in virulence compared with the wild type (Vd wt) and complementary (ΔSTT3-C) strains. We also generated transgenic Nicotiana benthamiana (Trans-1 and -2) plants by expressing dsRNA against the STT3 gene. Transgenic plants showed significant reduction in the disease index and fungal biomass resulting in elevated resistance to V. dahliae compared with the wild-type plants when inoculated with Vd wt. Our results indicated that STT3 mediates the full virulence through the regulation in fungal development, hyphal growth, glycoprotein secretion of V. dahliae and merits further study as a potential RNAi target to control this fungus.


Assuntos
Hexosiltransferases/metabolismo , Subunidades Proteicas/metabolismo , Verticillium/fisiologia , Carbono/metabolismo , Hexosiltransferases/química , Hexosiltransferases/genética , Mutação , Fenótipo , Doenças das Plantas/microbiologia , Plantas/microbiologia , Subunidades Proteicas/genética , Análise de Sequência de DNA , Verticillium/patogenicidade , Virulência/genética
10.
Transgenic Res ; 26(6): 763-774, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29143178

RESUMO

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for pest resistance management in China.


Assuntos
Proteínas de Bactérias/genética , Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Mariposas/efeitos dos fármacos , Animais , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Regulação da Expressão Gênica de Plantas , Gossypium/fisiologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Larva , Mariposas/fisiologia , Controle Biológico de Vetores , Plantas Geneticamente Modificadas
11.
J Invertebr Pathol ; 149: 59-65, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28782511

RESUMO

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for integrated pest management in China.


Assuntos
Proteínas de Bactérias/genética , Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Animais , Resistência a Inseticidas , Mariposas , Controle Biológico de Vetores , Plantas Geneticamente Modificadas
12.
Planta ; 246(3): 453-469, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28474114

RESUMO

MAIN CONCLUSION: ABP9 , encoding a bZIP transcription factor from maize, enhances tolerance to multiple stresses and may participate in the ABA signaling pathway in transgenic cotton by altering physiological and biochemical processes and stress-related gene expression. Abiotic stresses, such as soil salinity and drought, negatively affect growth, development, and yield in cotton. Gene ABP9, which encodes a bZIP transcription factor, binds to the abscisic acid (ABA)-responsive-element (ABRE2) motif of the maize catalase1 gene. Its expression significantly improves tolerance in Arabidopsis to multiple abiotic stresses, but little is known about its role in cotton. In the present study, the ABP9 gene was introduced into upland cotton (Gossypium hirsutum L.) cultivar R15 by Agrobacterium tumefaciens-mediated transformation, and 12 independent transgenic cotton lines were obtained. Cotton plants over-expressing ABP9 have enhanced tolerance to salt and osmotic stress. Under stress, they developed better root systems in a greenhouse and higher germination, reduced stomatal aperture, and stomatal density in a growth chamber. Under drought conditions, survival rate and relative water content (RWC) of transgenic cotton were higher than those of R15 plants. Under salt and osmotic stresses, chlorophyll, proline, and soluble sugar contents significantly increased in transgenic cotton leaves and the malondialdehyde (MDA) content was lower than in R15. Overexpression of ABP9 also enhanced oxidative stress tolerance, reduced cellular levels of reactive oxygen species (ROS) through increased activities of antioxidative enzymes, and alleviated oxidative damage to cell. Interestingly, ABP9 over-expressing cotton was more sensitive to exogenous ABA than R15 at seed germination, root growth, stomatal aperture, and stomatal density. Moreover, ABP9 overexpression upregulated significantly the transcription levels of stress-related genes such as GhDBP2, GhNCED2, GhZFP1, GhERF1, GhHB1, and GhSAP1 under salt treatment. Conjointly, these results showed that overexpression of ABP9 conferred enhanced tolerance to multiple abiotic stresses in cotton. The stress-tolerant transgenic lines provide valuable resources for cotton breeding.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Gossypium/genética , Plantas Tolerantes a Sal/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Desidratação/genética , Desidratação/metabolismo , Gossypium/metabolismo , Gossypium/fisiologia , Estresse Oxidativo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/genética , Tolerância ao Sal/fisiologia , Plantas Tolerantes a Sal/metabolismo , Plantas Tolerantes a Sal/fisiologia , Zea mays/genética
13.
Genes (Basel) ; 8(1)2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-28075391

RESUMO

Verticillium dahliae invades the roots of host plants and causes vascular wilt, which seriously diminishes the yield of cotton and other important crops. The protein AAC (ADP, ATP carrier) is responsible for transferring ATP from the mitochondria into the cytoplasm. When V. dahliae protoplasts were transformed with short interfering RNAs (siRNAs) targeting the VdAAC gene, fungal growth and sporulation were significantly inhibited. To further confirm a role for VdAAC in fungal development, we generated knockout mutants (ΔVdACC). Compared with wild-type V. dahliae (Vd wt), ΔVdAAC was impaired in germination and virulence; these impairments were rescued in the complementary strains (ΔVdAAC-C). Moreover, when an RNAi construct of VdAAC under the control of the 35S promoter was used to transform Nicotiana benthamiana, the expression of VdAAC was downregulated in the transgenic seedlings, and they had elevated resistance against V. dahliae. The results of this study suggest that VdAAC contributes to fungal development, virulence and is a promising candidate gene to control V. dahliae. In addition, RNAi is a highly efficient way to silence fungal genes and provides a novel strategy to improve disease resistance in plants.

14.
BMC Biotechnol ; 16(1): 57, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27455996

RESUMO

BACKGROUND: Large efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation. RESULTS: High-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H20), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs-19-nt duplex with 2-nt 3' overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)-into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis. CONCLUSION: Our quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Protoplastos/metabolismo , Transformação Genética/genética , Verticillium/genética , Fatores de Virulência/genética , Proteínas Fúngicas/metabolismo , Especificidade da Espécie , Verticillium/classificação , Verticillium/metabolismo
15.
Mol Plant Microbe Interact ; 29(7): 545-59, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27089469

RESUMO

Verticillium dahliae causes a serious wilt disease of important crops and is difficult to control. Few plasma-membrane transport proteins for nutrient acquisition have been identified for this fungus, and their involvement in the disease process is unknown. Here, a plasma-membrane protein, the V. dahliae thiamine transporter protein VdThit, was characterized functionally by deletion of the VdThit gene in V. dahliae. Disruption strains were viable, but growth and conidial germination and production were reduced and virulence was impaired. Interestingly, by supplementing exogenous thiamine, growth, conidiation, and virulence of the VdΔThit mutants were partially restored. Stress-tolerance assays showed that the VdΔThit mutant strains were markedly more susceptible to oxidative stress and UV damage. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses showed low levels of pyruvate metabolism intermediates acetoin and acetyl coenzyme A (acetyl-CoA) in the VdΔThit mutant strains, suggesting that pyruvate metabolism was suppressed. Expression analysis of VdThit confirmed the importance of VdThit in vegetative growth, reproduction, and invasive hyphal growth. Furthermore, a green fluorescent protein (GFP)-labeled VdΔThit mutant (VdΔThit-7-GFP) was suppressed in initial infection and root colonization, as viewed with light microscopy. Together, these results showed that VdThit plays an indispensable role in the pathogenicity of V. dahliae.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Doenças das Plantas/microbiologia , Plantas/microbiologia , Tiamina/metabolismo , Verticillium/genética , Proteínas Fúngicas/metabolismo , Genes Reporter , Fenótipo , Raízes de Plantas/microbiologia , Deleção de Sequência , Esporos Fúngicos , Verticillium/patogenicidade , Virulência
16.
Int J Mol Sci ; 17(3): 296, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26927087

RESUMO

Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family.


Assuntos
Proteínas de Bactérias/metabolismo , Bifidobacterium/enzimologia , Coenzimas/metabolismo , Isocitrato Desidrogenase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bifidobacterium/metabolismo , Estabilidade Enzimática , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Magnésio/metabolismo , Manganês/metabolismo , Dados de Sequência Molecular , NAD/química , NAD/metabolismo , Especificidade por Substrato
17.
Guang Pu Xue Yu Guang Pu Fen Xi ; 36(6): 1755-60, 2016 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-30052386

RESUMO

A fast and efficient way to synthesize a large number of silver nanowires was developed in this paper, in which the reaction conditions were optimized. Under the protection of Cu(NO3)2 silver nitrate was reduced by polyol with polyvinyl pyrrolidone (PVP) in existence. The silver nanowires with uniform structure and good dispersion were obtained. Surface enhancement activity of the silver nanowires was detected by using RhB as a probe molecule,its surface enhancement factor can reach 6.4×105. The results showed that the nanowires significantly enhance the Raman spectroscopy of RhB. The normal Raman spectroscopy (NRS), Raman spectroscopy of D-carnitine solution and Surface enhanced Raman Spectroscopy of D-carnitine by means of the new base were obtained. There are obvious Raman peaks at 3 100~2 800 and 1 700~200 cm-1, and the peak of 1 700~200 cm-1 in the surface enhanced Raman spectra of the D-carnitine can be obviously enhanced. The analysis showed that the angle between the molecular and silver nanoparticles were 180°. The vibrational peaks were assigned comprehensively. Compared with the NRS and SERS of D-carnitine, the detailed structural information of D-carnitine was obtained. In this paper, the surface enhanced Raman spectra of the D-carnitine absorbed on the synthesized silver nanoparticles were obtained, and the minimum detection concentration was 10-6 mol·L-1. The new method can be a rapid and characteristic way to detect D-carnitine, and it will also provide an important guidance for the studies on pharmacology of D-carnitine.

18.
J Sep Sci ; 39(2): 272-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26553707

RESUMO

Solid-phase extraction coupled with dispersive liquid-liquid microextraction was developed as an ultra-preconcentration method for the determination of four organophosphorus pesticides (isocarbophos, parathion-methyl, triazophos and fenitrothion) in water samples. The analytes considered in this study were rapidly extracted and concentrated from large volumes of aqueous solutions (100 mL) by solid-phase extraction coupled with dispersive liquid-liquid microextraction and then analyzed using high performance liquid chromatography. Experimental variables including type and volume of elution solvent, volume and flow rate of sample solution, salt concentration, type and volume of extraction solvent and sample solution pH were investigated for the solid-phase extraction coupled with dispersive liquid-liquid microextraction with these analytes, and the best results were obtained using methanol as eluent and ethylene chloride as extraction solvent. Under the optimal conditions, an exhaustive extraction for four analytes (recoveries >86.9%) and high enrichment factors were attained. The limits of detection were between 0.021 and 0.15 µg/L. The relative standard deviations for 0.5 µg/L of the pesticides in water were in the range of 1.9-6.8% (n = 5). The proposed strategy offered the advantages of simple operation, high enrichment factor and sensitivity and was successfully applied to the determination of four organophosphorus pesticides in water samples.

19.
World J Microbiol Biotechnol ; 31(12): 1889-97, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26475327

RESUMO

To overcome the challenges met with gene deletion in the plant pathogen Verticillium dahliae, a mutant strain with impaired non-homologous end joining DNA repair was generated to improve targeted gene replacement frequencies. A V. dahliae 991 ΔVdku70 null mutant strain was generated using Agrobacterium tumefaciens-mediated transformation. Despite having impaired non-homologous end joining DNA repair function, the ΔVdku70 strain exhibited normal growth, reproduction capability, and pathogenicity when compared with the wild-type strain. When the ΔVdku70 strain was used to delete 2-oxoglutarate dehydrogenase E2, ferric reductase transmembrane component 3 precursor, and ferric reductase transmembrane component 6 genes, gene replacement frequencies ranged between 22.8 and 34.7% compared with 0.3 and 0.5 % in the wild-type strain. The ΔVdku70 strain will be a valuable tool to generate deletion strains when studying factors that underlie virulence and pathogenesis in this filamentous fungus.


Assuntos
Antígenos Nucleares/genética , Proteínas de Ligação a DNA/genética , Marcação de Genes , Verticillium/genética , Virulência/genética , Agrobacterium tumefaciens/genética , Reparo do DNA por Junção de Extremidades , DNA Fúngico/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Recombinação Homóloga/genética , Autoantígeno Ku , Mutagênese Insercional , Mutação , Doenças das Plantas/microbiologia , Sementes/microbiologia , Tabaco/microbiologia , Verticillium/crescimento & desenvolvimento
20.
PLoS One ; 10(5): e0125229, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25942017

RESUMO

In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD+-specific (NAD-IDH) or NADP+-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD+-specific and showed no detectable activity with NADP+. The Km values of the enzyme for NAD+ were 262.6±7.4 µM or 309.1±11.2 µM with Mg2+ or Mn2+ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP+ was approximately 24-fold higher than that for NAD+, suggesting that ClIDH is an NAD+-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn2+. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD+-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs.


Assuntos
Gammaproteobacteria/enzimologia , Isocitrato Desidrogenase/metabolismo , Sequência de Aminoácidos , Ativação Enzimática , Gammaproteobacteria/genética , Expressão Gênica , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Cinética , Dados de Sequência Molecular , Mutação , NAD/metabolismo , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
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