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1.
Pest Manag Sci ; 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31347787

RESUMO

BACKGROUND: Aspergillus fumigatus, the causal agent of aspergillosis in humans, is commonly present as a saprophyte in various organic substrates, such as spoiled silages. Aspergillosis is generally combated with demethylation inhibitor (DMI) fungicides, but the recent appearance of resistant medical and environmental strains made current treatment strategies less reliable. The goal of this study was to determine the evolution of A. fumigatus populations during the ensiling process of whole-crop corn, high moisture corn and wet grain corn, and to monitor the sensitivity of isolates from treated and untreated fields to one medical and one agricultural DMI fungicide. RESULTS: A. fumigatus was isolated from fresh forage at harvest at rather low concentrations (102 cfu g-1 ). The low frequency lingered during the silage process (at 60 and 160 days), whereas it significantly increased during air exposure (at 7 and 14 days of air exposure). Field treatment of corn with a mixture of prothioconazole and tebuconazole did not affect the sensitivity of A. fumigatus isolates. One of 29 isolates from the untreated plot was resistant to voriconazole. A unique amino acid substitution (E427K) was detected in the cyp51A gene of 10 of 12 sequenced isolates, but was not associated with DMI resistance. CONCLUSION: A. fumigatus significantly increased during aerobic deterioration of ensilaged corn after silo opening, compared with the low presence in fresh corn and during ensiling. Field treatment of corn with DMI fungicides did not affect the sensitivity of A. fumigatus isolates collected from fresh and ensiled corn. © 2019 Society of Chemical Industry.

2.
Phytochemistry ; 162: 99-108, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30877900

RESUMO

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.


Assuntos
Especificidade de Anticorpos , Cucumovirus/fisiologia , Engenharia Genética/métodos , RNA Replicase/imunologia , Anticorpos de Cadeia Única/imunologia , Tabaco/genética , Tabaco/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Cucumovirus/enzimologia , Plantas Geneticamente Modificadas , Anticorpos de Cadeia Única/química , Transformação Genética
3.
Phytopathology ; 109(6): 1053-1061, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30667339

RESUMO

The genera Paramyrothecium and Albifimbria have been established from the former genus Myrothecium and they generally comprise common soil-inhabiting and saprophytic fungi. Within these genera, only two fungi have been recognized as phytopathogenic thus far: P. roridum and A. verrucaria, both of which cause necrotic leaf spots and plant collapse. Severe leaf necrosis and plant decay have been observed in Northern and Southern Italy on leafy vegetable crops. Thirty-six strains of Paramyrothecium- and Albifimbria-like fungi were isolated from affected plants belonging to eight different species. Based on morphological characteristics, 19 strains were assigned to A. verrucaria, whereas the remaining strains, which mostly resembled Paramyrothecium-like fungi, could not be identified precisely. Molecular characterization of six loci (internal transcribed spacer [ITS], ß-tubulin [tub2], calmodulin [cmdA], translation elongation factor 1-alpha [tef1], large subunit ribosomal RNA [LSU], and mitochondrial ATP 6synthase 6 [ATP6]) of the 36 new isolates and three previously ITS-characterized isolates assigned all strains to four species: A. verrucaria, P. roridum, P. foliicola, and P. nigrum. Single and concatenated phylogenetic analyses were conducted, and they clearly distinguished the isolated fungi into four different groups. A. verrucaria, P. roridum, P. foliicola, and P. nigrum were able to induce leaf necrosis singly, and they were confirmed to be the causal agents of the leaf spot disease through pathogenicity assays. The involvement of fungi previously considered saprophytic (i.e., P. foliicola and P. nigrum) in the development of plant disease for the first time deserves particular attention because of the possibility of their transmission by seeds and the limited knowledge of their management with chemicals.


Assuntos
Ascomicetos , Doenças das Plantas/microbiologia , Verduras , Itália , Filogenia
4.
Nat Commun ; 9(1): 5308, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30546019

RESUMO

The propensity of viruses to acquire genetic material from relatives and possibly from infected hosts makes them excellent candidates as vectors for horizontal gene transfer. However, virus-mediated acquisition of host genetic material, as deduced from historical events, appears to be rare. Here, we report spontaneous and surprisingly efficient generation of hybrid virus/host DNA molecules in the form of minicircles during infection of Beta vulgaris by Beet curly top Iran virus (BCTIV), a single-stranded DNA virus. The hybrid minicircles replicate, become encapsidated into viral particles, and spread systemically throughout infected plants in parallel with the viral infection. Importantly, when co-infected with BCTIV, B. vulgaris DNA captured in minicircles replicates and is transcribed in other plant species that are sensitive to BCTIV infection. Thus, we have likely documented in real time the initial steps of a possible path of virus-mediated horizontal transfer of chromosomal DNA between plant species.


Assuntos
Beta vulgaris/genética , Beta vulgaris/virologia , DNA Circular/genética , DNA de Plantas/genética , DNA Viral/genética , Geminiviridae/genética , Transferência Genética Horizontal/genética , Arabidopsis/virologia , DNA de Cadeia Simples/genética , Doenças das Plantas/virologia , Tabaco/virologia
5.
J Appl Microbiol ; 2018 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-30417496

RESUMO

AIMS: The response of rhizosphere and bulk soil indigenous microbial communities focusing on nitrifiers was evaluated after the application of different biological control agents (BCAs) (Bacillus, Trichoderma, Pseudomonas) and compost in controlling lettuce Fusarium wilt. METHODS AND RESULTS: Experiments were conducted "in situ" over two lettuce cropping seasons. Total fungal, bacterial and archaeal populations and the nitrifiers were analysed using quantitative polymerase chain reaction (qPCR) method. The pathogen, Fusarium oxysporum forma specialis lactucae (FOL), Bacillus, Trichoderma and Pseudomonas and three antifungal genes (chiA, 2,4 - diacetylphloroglucinol - phlD and HCN synthase - hcnAB genes) were also assessed. Quantitative data were corroborated with disease severity, potential nitrification activity and soil chemical parameters. The application of BCAs and compost resulted in the disease reduction by as much as 69%, confirmed by significant negative correlations between Bacillus subtilis, Trichoderma and Pseudomonas spp. abundances and disease severity. The FOL presence in the untreated control resulted in the nitrifiers niche differentiation. CONCLUSIONS: The used treatments were efficient against Fusarium wilt and did not influence negatively the non - target microbial communities. SIGNIFICANCE AND IMPACT OF STUDY: The use of BCAs and compost appears as an effective and safe strategy to implement sustainable agricultural practices. This article is protected by copyright. All rights reserved.

6.
Plant Pathol J ; 34(4): 316-326, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30140185

RESUMO

The effect of simulated climate changes by applying different temperatures and CO2 levels was investigated in the Blumeria graminis f. sp. tritici/wheat pathosystem. Healthy and inoculated plants were exposed in single phytotrons to six CO2+temperature combinations: (1) 450 ppm CO2/18-22°C (ambient CO2 and low temperature), (2) 850 ppm CO2/18-22°C (elevated CO2 and low temperature), (3) 450 ppm CO2/22-26°C (ambient CO2 and medium temperature), (4) 850 ppm CO2/22-26°C (elevated CO2 and medium temperature), (5) 450 ppm CO2/26-30°C (ambient CO2 and high temperature), and (6) 850 ppm CO2/26-30°C (elevated CO2 and high temperature). Powdery mildew disease index, fungal DNA quantity, plant death incidence, plant expression of pathogenesis-related (PR) genes, plant growth parameters, carbohydrate and chlorophyll content were evaluated. Both CO2 and temperature, and their interaction significantly influenced powdery mildew development. The most advantageous conditions for the progress of powdery mildew on wheat were low temperature and ambient CO2. High temperatures inhibited pathogen growth independent of CO2 conditions, and no typical powdery mildew symptoms were observed. Elevated CO2 did not stimulate powdery mildew development, but was detrimental for plant vitality. Similar abundance of three PR transcripts was found, and the level of their expression was different between six phytotron conditions. Real time PCR quantification of Bgt was in line with the disease index results, but this technique succeeded to detect the pathogen also in asymptomatic plants. Overall, future global warming scenarios may limit the development of powdery mildew on wheat in Mediterranean area, unless the pathogen will adapt to higher temperatures.

8.
PLoS One ; 13(7): e0200569, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30001414

RESUMO

Aspergillus fumigatus, the causal agent of human aspergilloses, is known to be non-pathogenic in plants. It is present as saprophyte in different types of organic matter and develops rapidly during the high-temperature phase of the composting process. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides and resistant isolates have been recently reported. The present study aims to estimate the abundance, genetic diversity and DMI sensitivity of A. fumigatus during the composting process of orange fruits. Composting of orange fruits resulted in a 100-fold increase in A. fumigatus frequency already after 1 week, demonstrating that the degradation of orange fruits favoured the growth of A. fumigatus in compost. Most of A. fumigatus isolates belonged to mating type 2, including those initially isolated from the orange peel, whereas mating type 1 evolved towards the end of the composting process. None of the A. fumigatus isolates expressed simultaneously both mating types. The 52 investigated isolates exhibited moderate SSR polymorphisms by formation of one major (47 isolates) and one minor cluster (5 isolates). The latter included mating type 1 isolates from the last sampling and the DMI-resistant reference strains. Only few isolates showed cyp51A polymorphisms but were sensitive to DMIs as all the other isolates. None of the A. fumigatus isolates owned any of the mutations associated with DMI resistance. This study documents a high reproduction rate of A. fumigatus during the composting process of orange fruits, requesting specific safety precautions in compost handling. Furthermore, azole residue concentrations in orange-based compost were not sufficient to select A. fumigatus resistant genotypes.

9.
Pest Manag Sci ; 2018 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-29888848

RESUMO

BACKGROUND: Pythium species attack various vegetable crops causing seed, stem and root rot, and 'damping-off' after germination. Pythium diseases are prevalently controlled by two classes of fungicides, QoIs with azoxystrobin and phenlyamides with mefenoxam as representatives. The present study aimed to test the sensitivity of six Pythium species from different vegetable crops to azoxystrobin and mefenoxam and differentiating species based on ITS, cytochrome b and RNA polymerase I gene sequences. RESULTS: The inter- and intra-species sensitivity to azoxystrobin was found to be stable, with the exception of one Pythium paroecandrum isolate, which showed reduced sensitivity and two cytochrome b amino acid changes. For mefenoxam, the inter-species sensitivity was quite variable and many resistant isolates were found in all six Pythium species, but no RNA polymerase I amino acid changes were observed in them. ITS and cytochrome b phylogenetic analyses permitted a clear separation of Pythium species corresponding to globose- and filamentous-sporangia clusters. CONCLUSION: The results document the necessity of well-defined chemical control strategies adapted to different Pythium species. Since the intrinsic activity of azoxystrobin among species was stable and no resistant isolates were found, it may be applied without species differentiation, provided it is used preventatively to also control highly aggressive isolates. For a reliable use of mefenoxam, precise identification and sensitivity tests of Pythium species are crucial because its intrinsic activity is variable and resistant isolates may exist. Appropriate mixtures and/or alternation of products may help to further delay resistance development. © 2018 Society of Chemical Industry.

10.
Mol Biotechnol ; 59(9-10): 425-434, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28801830

RESUMO

Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons. Both genes yielded approximately 500 µg of total amelogenin per gram of fresh leaf tissue. Two purification procedures based on affinity chromatography or on intrinsic solubility properties of the protein were followed, yielding from 12 to 150 µg of amelogenin per gram of fresh leaf tissue, respectively, at different purity. The identity of the plant-made human amelogenin was confirmed by MALDI-TOF-MS analysis of peptides generated following chymotrypsin digestion. Using dynamic light scattering, we showed that plant extracts made in acetic acid containing human amelogenin have a bimodal distribution of agglomerates, with hydrodynamic diameters of 22.8 ± 3.8 and 389.5 ± 86.6 nm. To the best of our knowledge, this is the first report of expression of human amelogenin in plants, offering the possibility to use this plant-made protein for nanotechnological applications.


Assuntos
Amelogenina/genética , Clonagem Molecular , Nanotecnologia/métodos , Tabaco/genética , Amelogenina/biossíntese , Amelogenina/isolamento & purificação , Sequência de Aminoácidos/genética , Regulação da Expressão Gênica de Plantas/genética , Humanos , Espectrometria de Massas , Peptídeos/química , Peptídeos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Pest Manag Sci ; 73(12): 2481-2494, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28618166

RESUMO

BACKGROUND: Aspergillus fumigatus is a widespread fungus that colonizes dead organic substrates but it can also cause fatal human diseases. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides; however, resistant isolates appeared recently in the medical and also environmental area. The present study aims at molecular characterizing and quantifying A. fumigatus in major environmental habitats and determining its sensitivity to medical and agricultural DMI fungicides. RESULTS: A. fumigatus was isolated only rarely from soil and meadow/forest organic matter but high concentrations (103 to 107 cfu/g) were detected in substrates subjected to elevated temperatures, such as compost and silage. High genetic diversity of A. fumigatus from compost was found based on SSR markers, distinguishing among fungal isolates even when coming from the same substrate sample, while subclustering was observed based on mutations in cyp51A gene. Several cyp51A amino acid substitutions were found in 15 isolates, although all isolates were fully sensitive to the tested DMI fungicides, with exception of one isolate in combination with one fungicide. CONCLUSION: This study suggests that the tested A. fumigatus isolates collected in Italy, Spain and Hungary from the fungus' major living habitats (compost) and commercial growing substrates are not potential carriers for DMI resistance in the environment. © 2017 Society of Chemical Industry.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Microbiologia do Solo , Antifúngicos/química , Aspergillus fumigatus/classificação , Aspergillus fumigatus/isolamento & purificação , Compostagem , Desmetilação , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Variação Genética , Pradaria
12.
Rice (N Y) ; 10(1): 29, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28597326

RESUMO

BACKGROUND: Bakanae disease, caused by seed-borne Fusarium species, mainly F. fujikuroi, is a rice disease whose importance is considerably increasing in several rice growing countries, leading to incremental production losses. RESULTS: A germplasm collection of japonica rice was screened for F. fujikuroi resistance, allowing the identification of accessions with high-to-moderate levels of resistance to bakanae. A GWAS approach uncovered two genomic regions highly associated with the observed phenotypic variation for response to bakanae infection on the short arm of chromosome 1 (named as qBK1_628091) and on the long arm of chromosome 4 (named as qBK4_31750955). High levels of phenotypic resistance to bakanae were associated to the cumulated presence of the resistant alleles at the two resistance loci, suggesting that they can provide useful levels of disease protection in resistance breeding. A fine comparison with the genomic positions of qBK1_628091 and qBK4_31750955 with respect to the QTLs for bakanae resistance reported in the literature suggests that the resistant loci here described represent new genomic regions associated to F. fujikuroi resistance. A search for candidate genes with a putative role in bakanae resistance was conducted considering all the annotated genes and F. fujikuroi-related DEGs included in the two genomic regions highlighting several gene functions that could be involved in resistance, thus paving the way to the functional characterization of the resistance loci. CONCLUSIONS: New effective sources for bakanae resistance were identified on rice chromosomes 1 and 4 and tools for resistance breeding are provided.

13.
Front Biosci (Elite Ed) ; 9: 333-344, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28410155

RESUMO

Bakanae disease, one of the most noteworthy seedborne rice diseases, is caused by Fusarium fujikuroi, a member of the Gibberella fujikuroi species complex. The decreasing availability of chemical seed-dressing products over the last few years has raised the concerns of rice seed companies regarding bakanae disease. Therefore, new research trends require a deeper investigation into the main aspects of bakanae disease through interactions between rice and F. fujikuroi, in order to find new resistant or tolerant cultivars and alternative bakanae disease control strategies, as well as to develop more sensitive molecular diagnostic techniques. Here, some new aspects of F. fujikuroi epidemiology and pathogenicity, as well as its interactions with rice, are reported, and recent approaches applied to control bakanae disease are summarized.


Assuntos
Fusarium/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia
14.
Phytopathology ; 107(7): 885-892, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28398878

RESUMO

Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.


Assuntos
Fusarium/isolamento & purificação , Oryza/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sementes/microbiologia , Doenças das Plantas/microbiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
15.
BMC Genomics ; 17(1): 608, 2016 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-27515776

RESUMO

BACKGROUND: Fusarium fujikuroi is the causal agent of bakanae, the most significant seed-borne disease of rice. Molecular mechanisms regulating defence responses of rice towards this fungus are not yet fully known. To identify transcriptional mechanisms underpinning rice resistance, a RNA-seq comparative transcriptome profiling was conducted on infected seedlings of selected rice genotypes at one and three weeks post germination (wpg). RESULTS: Twelve rice genotypes were screened against bakanae disease leading to the identification of Selenio and Dorella as the most resistant and susceptible cultivars, respectively. Transcriptional changes were more appreciable at 3 wpg, suggesting that this infection stage is essential to study the resistance mechanisms: 3,119 DEGs were found in Selenio and 5,095 in Dorella. PR1, germin-like proteins, glycoside hydrolases, MAP kinases, and WRKY transcriptional factors were up-regulated in the resistant genotype upon infection with F. fujikuroi. Up-regulation of chitinases and down-regulation of MAP kinases and WRKY transcriptional factors were observed in the susceptible genotype. Gene ontology (GO) enrichment analyses detected in Selenio GO terms specific to response to F. fujikuroi: 'response to chitin', 'jasmonic acid biosynthetic process', and 'plant-type hypersensitive response', while Dorella activated different mechanisms, such as 'response to salicylic acid stimulus' and 'gibberellin metabolic process', which was in agreement with the production of gibberellin A3 in Dorella plants. CONCLUSIONS: RNA-seq profiling was performed for the first time to analyse response of rice to F. fujikuroi infection. Our findings allowed the identification of genes activated in one- and three- week-old rice seedlings of two genotypes infected with F. fujikuroi. Furthermore, we found the pathways involved in bakanae resistance, such as response to chitin, JA-dependent signalling and hypersensitive response. Collectively, this provides important information to elucidate the molecular and cellular processes occurring in rice during F. fujikuroi infection and to develop bakanae resistant rice germplasm.


Assuntos
Resistência à Doença/genética , Fusarium/patogenicidade , Oryza/genética , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Proteínas de Plantas/imunologia , Quitina/metabolismo , Fusarium/fisiologia , Perfilação da Expressão Gênica , Ontologia Genética , Genótipo , Giberelinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Anotação de Sequência Molecular , Oryza/imunologia , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Ácido Salicílico/metabolismo , Plântula/genética , Plântula/imunologia , Plântula/microbiologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Transcriptoma
16.
Virus Res ; 215: 12-9, 2016 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-26826600

RESUMO

The role of the C2 protein in the pathogenicity of tomato yellow leaf curl Sardinia virus (TYLCSV) was investigated. Here we report that Agrobacterium-mediated transient expression of TYLCSV C2 resulted in a strong hypersensitive response (HR) in Nicotiana benthamiana, N. tabacum, and Arabidopsis thaliana, with induction of plant cell death and production of H2O2. Since HR is not evident in plants infected by TYLCSV, it is expected that TYLCSV encodes a gene (or genes) that counters this response. HR was partially counteracted by co-agroinfiltration of TYLCSV V2 and Rep, leading to chlorotic reaction, with no HR development. Considering that the corresponding C2 protein of the closely related tomato yellow leaf curl virus (TYLCV) did not induce HR, alignment of the C2 proteins of TYLCSV and TYLCV were carried out and a hypervariable region of 16 amino acids was identified. Its role in the induction of HR was demonstrated using TYLCSV-TYLCV C2 chimeric genes, encoding two TYLCSV C2 variants with a complete (16 aa) or a partial (10 aa only) swap of the corresponding sequence of TYLCV C2. Furthermore, using NahG transgenic N. benthamiana lines compromised in the accumulation of salicylic acid (SA), a key regulator of HR, only a chlorotic response occurred in TYLCSV C2-infiltrated tissue, indicating that SA participates in such plant defense process. These findings demonstrate that TYLCSV C2 acts as a pathogenicity determinant and induces host defense responses controlled by the SA pathway.


Assuntos
Arabidopsis/imunologia , Begomovirus/patogenicidade , Interações Hospedeiro-Patógeno , Doenças das Plantas/virologia , Tabaco/imunologia , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Agrobacterium/genética , Arabidopsis/virologia , Morte Celular , Expressão Gênica , Células Vegetais/fisiologia , Células Vegetais/virologia , Doenças das Plantas/imunologia , Tabaco/virologia , Transformação Genética , Proteínas Virais/genética , Fatores de Virulência/genética
17.
Plant Biotechnol J ; 14(1): 153-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25865255

RESUMO

The rat ErbB2 (rErbB2) protein is a 185-kDa glycoprotein belonging to the epidermal growth factor-related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2-pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ-HT expression vector as 6X His tag fusions. All rErbB2 variants (72-74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2(+) mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.


Assuntos
Imunidade , Neoplasias Mamárias Animais/imunologia , Receptor ErbB-2/biossíntese , Receptor ErbB-2/imunologia , Tabaco/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade/efeitos dos fármacos , Imunização , Camundongos Endogâmicos C57BL , Plantas Geneticamente Modificadas , Domínios Proteicos , Ratos , Receptor ErbB-2/química , Receptor ErbB-2/isolamento & purificação , Solubilidade , Tabaco/imunologia
18.
Int J Food Microbiol ; 199: 54-61, 2015 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-25632799

RESUMO

Metschnikowia fructicola strain AP47 is a yeast antagonist against postharvest pathogens of fruits. The yeast was able to produce chitinase enzymes in the presence of pathogen cell wall. A novel chitinase gene MfChi (GenBank accession number HQ113461) was amplified from the genomic DNA of Metschnikowia fructicola AP47. Sequence analysis showed lack of introns, an open reading frame (ORF) of 1098 bp encoding a 365 amino acid protein with a calculated molecular weight of 40.9 kDa and a predicted pI of 5.27. MfChi was highly induced in Metschnikowia fructicola after interaction with Monilinia fructicola cell wall, suggesting a primary role of MfChi chitinase in the antagonistic activity of the yeast. The MfChi gene overexpressed in the heterologous expression system of Pichia pastoris KM71 and the recombinant chitinase showed high endochitinase activity towards 4-Nitrophenyl ß-d-N,N',N″-triacetylchitotriose substrate. The antifungal activity of the recombinant chitinase was investigated against Monilinia fructicola and Monilinia laxa in vitro and on peaches. The chitinase significantly controlled the spore germination and the germ tube length of the tested pathogens in PDB medium and the mycelium diameter in PDA. The enzyme, when applied on peaches cv. Redhaven, successfully reduced brown rot severity. This work shows that the chitinase MfChi could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short shelf life, and confirms that P. pastoris KM71 is a suitable microorganism for cost-effective large-scale production of recombinant chitinases.


Assuntos
Ascomicetos/efeitos dos fármacos , Quitinases/genética , Quitinases/metabolismo , Microbiologia de Alimentos , Frutas/microbiologia , Metschnikowia/enzimologia , Prunus/microbiologia , Antifúngicos/farmacologia , Metschnikowia/genética , Micélio/efeitos dos fármacos , Fases de Leitura Aberta , Pichia/genética
19.
Int J Food Microbiol ; 182-183: 1-8, 2014 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-24854386

RESUMO

The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline serine protease could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short storage period.


Assuntos
Ascomicetos/enzimologia , Microbiologia de Alimentos/métodos , Malus/microbiologia , Fungos Mitospóricos/efeitos dos fármacos , Controle Biológico de Vetores/normas , Pichia/genética , Serina Endopeptidases/farmacologia , Antifúngicos/farmacologia , Ascomicetos/genética , Manipulação de Alimentos , Hifas/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Serina Endopeptidases/metabolismo , Esporos Fúngicos/efeitos dos fármacos
20.
Int J Food Microbiol ; 166(3): 515-23, 2013 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-24055868

RESUMO

Three Fusarium species associated with bakanae disease of rice (Fusarium fujikuroi, Fusarium proliferatum, and Fusarium verticillioides) were investigated for their ability to produce fumonisins (FB1 and FB2) under different light conditions, and for pathogenicity. Compared to darkness, the conditions that highly stimulated fumonisin production were yellow and green light in F. verticillioides strains; white and blue light, and light/dark alternation in F. fujikuroi and F. proliferatum strains. In general, all light conditions positively influenced fumonisin production with respect to the dark. Expression of the FUM1 gene, which is necessary for the initiation of fumonisin production, was in accordance with the fumonisin biosynthetic profile. High and low fumonisin-producing F. fujikuroi strains showed typical symptoms of bakanae disease, abundant fumonisin-producing F. verticillioides strains exhibited chlorosis and stunting of rice plants, while fumonisin-producing F. proliferatum strains were asymptomatic on rice. We report that F. fujikuroi might be an abundant fumonisin producer with levels comparable to that of F. verticillioides and F. proliferatum, highlighting the need of deeper mycotoxicological analyses on rice isolates of F. fujikuroi. Our results showed for the first time the influence of light on fumonisin production in isolates of F. fujikuroi, F. proliferatum, and F. verticillioides from rice.


Assuntos
Fusarium , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Oryza/microbiologia , Fumonisinas/análise , Fumonisinas/metabolismo , Fusarium/genética , Fusarium/metabolismo , Fusarium/efeitos da radiação , Perfilação da Expressão Gênica , Gibberella/metabolismo
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