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1.
Food Chem ; 337: 127761, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777565

RESUMO

Amino and thiolated aptamers are the main aptamers used to construct label-free electrochemical impedimetric aptasensors. In this study, the modification performance and electrochemical properties of amino aptamers and thiolated aptamers were studied in the construction of label-free impedimetric sensors. The results showed that the initial modification density of amino aptamers was higher than that of thiol aptamers. Aptamers can recognize and bind OTA to generate electrical signals. The higher the density of aptamer modification was, the better the electric signals were. If only considering the initial modification density, amino aptamers were more suitable for the preparation of aptasensors than thiolated aptamers. However, the modification density of the amino aptamer decreased with the prolonged immersion time in 1 mM HCl solution, which suggests that the stability of this sensor was poor. However, the thiolated aptamer maintained relatively constant density and could be reused. Thus, the thiolated aptasensor had a wide range and good reproducibility and stability for the determination of ochratoxin A (OTA). In addition, this study proved that gold nanoparticles play an important role in signal amplification by increasing the effective gold surface to fix more aptamers in the process of sensor preparation.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Impedância Elétrica , Eletroquímica , Eletrodos , Ouro/química , Nanopartículas Metálicas/química , Ocratoxinas/análise , Reprodutibilidade dos Testes
2.
Mol Plant Pathol ; 2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33169928

RESUMO

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks-knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

3.
Mol Plant Microbe Interact ; 33(9): 1103-1107, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32552519

RESUMO

Botrytis cinerea is a foliar necrotrophic fungal-pathogen capable of infecting >580 genera of plants, is often used as model organism for studying fungal-host interactions. We used RNAseq to study transcriptome of B. cinerea infection on a major (worldwide) vegetable crop, tomato (Solanum lycopersicum). Most previous works explored only few infection stages, using RNA extracted from entire leaf-organ diluting the expression of studied infected region. Many studied B. cinerea infection, on detached organs assuming that similar defense/physiological reactions occurs in the intact plant. We analyzed transcriptome of the pathogen and host in 5 infection stages of whole-plant leaves at the infection site. We supply high quality, pathogen-enriched gene count that facilitates future research of the molecular processes regulating the infection process.


Assuntos
Botrytis/genética , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Doenças das Plantas/microbiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Humanos
4.
Environ Microbiol ; 22(7): 2870-2891, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32323444

RESUMO

Colletotrichum gloeosporioides and Penicillium expansum cause postharvest diseases in tropical and deciduous fruit. During colonization, C. gloeosporioides and P. expansum secrete ammonia in hosts with low sugar content (LowSC) and gluconic acid in hosts with high sugar content (HighSC), respectively, as a mechanism to modulate enhanced pathogenicity. We studied the pathogens interactions with tomato lines of similar genetic background but differing in their sugar content. Colletotrichum gloeosporioides showed enhanced colonization of the LowSC line with differential expression response of 15% of its genes including enhanced relative expression of glycosyl hydrolases, glucanase and MFS-transporter genes. Enhanced colonization of P. expansum occurred in the HighSC line, accompanied by an increase in carbohydrate metabolic processes mainly phosphoenolpyruvate carboxykinase, and only 4% of differentially expressed genes. Gene response of the two host lines strongly differed depending on the sugar level. Limited colonization of HighSC line by C. gloeosporioides was accompanied by a marked alteration of gene expression compared the LowSC response to the same pathogen; while colonization by P. expansum resulted in a similar response of the two different hosts. We suggest that this differential pattern of fungal/host responses may be the basis for the differential of host range of both pathogens in nature.

5.
Food Chem ; 317: 126416, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087519

RESUMO

T-2 toxin at low concentrations can induce ROS accumulation and modulate host resistance in plants. NOX plays crucial roles in ROS production and is regulated by Ca2+via direct binding to EF-hand motifs. In this study, the effect of EGTA (Ca2+ chelating agent) on the expression and enzymatic activity of NOX, as well as the activities and corresponding gene expressions involved in ROS metabolism and cell membrane integrity, were investigated in treated slices. Results indicated that EGTA treatment significantly affected gene expression and activity of NOX, and reduced ROS accumulation and cell membrane integrity and the enzymatic activities and gene expression involved in ROS metabolism when exposed to treatment. The addition of exogenous Ca2+ restored the initial relative transcript abundance, ROS accumulation and their activities. Results suggest that Ca2+ affected by EGTA plays a crucial role in NOX activity regulation, ultimately affecting ROS metabolism in slices induced by T-2 toxin.


Assuntos
Cálcio/metabolismo , NADPH Oxidases/metabolismo , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Solanum tuberosum/metabolismo , Toxina T-2/metabolismo , Animais , Cálcio/química , Membrana Celular/metabolismo , Ácido Egtázico/química , Malondialdeído/metabolismo , NADPH Oxidases/genética , Proteínas de Plantas/genética , Tubérculos/metabolismo
6.
Food Chem ; 309: 125608, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31678673

RESUMO

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) can improve wound healing of potato tubers; however, how the chemical regulates reactive oxygen species (ROS) generation and scavenging during wound healing is not completely understood. BTH at 100 mg·L-1 regulated changes in ROS generation and scavenging in healing tissues of potato tubers. A higher H2O2 content was presented in healing tissues of potato tubers, while cell membrane permeability and malondialdehyde content declined due to BTH treatment. Additionally, the activities and transcript level of enzymes related with ROS generation, including NADPH oxidase, peroxidase and polyamine oxidase, as well as enzymes involved in ROS scavenging, such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, were significantly enhanced by BTH treatment. It is suggested that ROS metabolism might play a crucial role in wound healing of potato tubers mediated by BTH during postharvest.


Assuntos
Tubérculos/efeitos dos fármacos , Tubérculos/metabolismo , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/metabolismo , Tiadiazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Food Chem ; 302: 125288, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419774

RESUMO

The effects of benzothiadiazole (BTH) on Penicillium expansum development, mitochondria energy metabolism, and changes in the number and structure of mitochondria in apple fruit were investigated after the fruit were immersed in 100 mg L-1 BTH for 10 min and then stored at 22 °C. The results indicated that BTH treatment significantly decreased the lesion diameter of fruit challenged with P. expansum; further, treatment enhanced the activities of mitochondrial respiratory metabolism-related enzymes, such as succinate dehydrogenase, cytochrome oxidase, H+-ATPase and Ca2+-ATPase, along with high ATP level and energy status in apple fruit during storage. Moreover, transmission electron microscopy results indicated that BTH treatment was beneficial for maintaining the number and structure of mitochondria during storage. The results suggested that BTH treatment enhanced ATP levels via mitochondrial energy metabolism, which might contribute to the induced resistance in apple fruit during storage.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Armazenamento de Alimentos , Frutas/metabolismo , Malus/efeitos dos fármacos , Malus/metabolismo , Mitocôndrias/efeitos dos fármacos , Tiadiazóis/farmacologia , Frutas/microbiologia , Malus/microbiologia , Mitocôndrias/metabolismo , Penicillium/fisiologia
8.
BMC Genomics ; 20(1): 1020, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31878885

RESUMO

BACKGROUND: Genomic studies demonstrate that components of virulence mechanisms in filamentous eukaryotic pathogens (FEPs, fungi and oomycetes) of plants are often highly conserved, or found in gene families that include secreted hydrolytic enzymes (e.g., cellulases and proteases) and secondary metabolites (e.g., toxins), central to the pathogenicity process. However, very few large-scale genomic comparisons have utilized complete proteomes from dozens of FEPs to reveal lifestyle-associated virulence mechanisms. Providing a powerful means for exploration, and the discovery of trends in large-scale datasets, network analysis has been used to identify core functions of the primordial cyanobacteria, and ancient evolutionary signatures in oxidoreductases. RESULTS: We used a sequence-similarity network to study components of virulence mechanisms of major pathogenic lifestyles (necrotroph (ic), N; biotroph (ic), B; hemibiotroph (ic), H) in complete pan-proteomes of 65 FEPs and 17 saprobes. Our comparative analysis highlights approximately 190 core functions found in 70% of the genomes of these pathogenic lifestyles. Core functions were found mainly in: transport (in H, N, B cores); carbohydrate metabolism, secondary metabolite synthesis, and protease (H and N cores); nucleic acid metabolism and signal transduction (B core); and amino acid metabolism (H core). Taken together, the necrotrophic core contains functions such as cell wall-associated degrading enzymes, toxin metabolism, and transport, which are likely to support their lifestyle of killing prior to feeding. The biotrophic stealth growth on living tissues is potentially controlled by a core of regulatory functions, such as: small G-protein family of GTPases, RNA modification, and cryptochrome-based light sensing. Regulatory mechanisms found in the hemibiotrophic core contain light- and CO2-sensing functions that could mediate important roles of this group, such as transition between lifestyles. CONCLUSIONS: The selected set of enriched core functions identified in our work can facilitate future studies aimed at controlling FEPs. One interesting example would be to facilitate the identification of the pathogenic potential of samples analyzed by metagenomics. Finally, our analysis offers potential evolutionary scenarios, suggesting that an early-branching saprobe (identified in previous studies) has probably evolved a necrotrophic lifestyle as illustrated by the highest number of shared gene families between saprobes and necrotrophs.


Assuntos
Fungos/genética , Fungos/fisiologia , Redes Reguladoras de Genes , Genômica , Oomicetos/genética , Oomicetos/fisiologia , Plantas/microbiologia , Evolução Molecular , Fungos/metabolismo , Oomicetos/metabolismo
9.
Food Chem ; 301: 125303, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387045

RESUMO

The effects of exogenous polyamines treatment on reactive oxygen species (ROS) metabolism in apricot fruits were systematically analyzed through the investigation of their curative and preventive effects on black spot disease. Results showed that 1.5 mM spermine (Spm), 1.5 mM spermidine (Spd) and 10 mM putrescine (Put) treatment significantly inhibited black spot development, additionally, the efficacy of this control was dependent upon the type of polyamines used and concentration level applied. Further studies have shown that exogenous polyamines treatments significantly improved production of O2- and H2O2, and increased the activities and gene expression levels of NADPH oxidase (NOX), super oxide dismutase (SOD), catalase (CAT) ascorbate peroxidase (AXP) and glutathione reductase (GR) in apricot fruit. Increased ascorbic acid (AsA) and reduced glutathione (GSH) content were also observed after exogenous polyamines treatment. These results have revealed that postharvest polyamines treatment effectively enhanced disease resistance through the maintenance of homeostasis in apricot fruits.


Assuntos
Alternaria/patogenicidade , Frutas/efeitos dos fármacos , Doenças das Plantas/microbiologia , Poliaminas/farmacologia , Prunus armeniaca/efeitos dos fármacos , Ascorbato Peroxidases/metabolismo , Catalase/metabolismo , Resistência à Doença/efeitos dos fármacos , Frutas/metabolismo , Frutas/microbiologia , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Homeostase/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Prunus armeniaca/metabolismo , Prunus armeniaca/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
10.
Front Plant Sci ; 10: 338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30949192

RESUMO

Blue mold and core rot caused by Penicillium expansum and Trichothecium roseum are major diseases of apple fruit in China; however, their differential aggressiveness in apples and effect on fruit postharvest physiology are unclear. The effects of colonization of apples cv. Red Delicious by both pathogens were compared to physiological parameters of ripening and release of volatile compounds (VOCs). P. expansum colonization showed increased aggressiveness compared to T. roesum colonization of apple fruits. P. expansum enhanced colonization occurred with differential higher ethylene production and respiratory rate evolution, lower membrane integrity and fruit firmness in correspondence with the colonization pattern of inoculated apples. Moreover, P. expansum caused lower contents of total soluble solid and titratable acid, and higher malondialdehyde compared with T. roesum colonization. While both pathogen infections enhanced VOCs release, compared with T. roseum inoculated apples, P. expansum inoculated apple showed a higher total VOCs production including alcohols, aldehydes and esters, being the C6 alcohols, aldehydes and esters amount. PLS-DA analysis indicated that hexanoic acid was the most important factor to distinguish the inoculated fruits from the controls. Interestingly, propyl acetate and hexyl benzoate, and undecylenic acid and hexadecane were only identified in the P. expansum and T. roseum inoculated fruits, respectively. Taken together, our findings indicate that both fungi inoculations promote apple fruit ripening and release specific VOCs; moreover, apple fruits are more susceptible to P. expansum colonization than T. roesum.

11.
Food Chem ; 289: 369-376, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955625

RESUMO

The effects of postharvest treatment with sodium silicate (Si) (100 mM) on mitochondrial ROS production and energy metabolism of the muskmelon fruits (cv. Yujinxiang) on development of defense responses to Trichothecium roseum were studied. Si treatment decreased decay severity of inoculated muskmelons, enhanced the activities of energy metabolism of key enzymes and kept the intracellular ATP at a higher level; meanwhile, Si also induced the mtROS accumulation such as H2O2 and superoxide anion. TMT-based quantitative proteomics analysis revealed that a total of 24 proteins with significant differences in abundance involved in energy metabolism, defense and stress responses, glycolytic and TCA cycle, and oxidation-reduction process. It is suggested by our study that melon fruit mitochondria, when induced by Si treatments, play a key role in priming of host resistance against T. roseum infection through the regulation of energy metabolism and ROS production in the pathogen infected muskmelon fruits.


Assuntos
Cucumis , Metabolismo Energético/efeitos dos fármacos , Frutas/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Silicatos/farmacologia , Trifosfato de Adenosina/análise , Frutas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Doenças das Plantas/prevenção & controle , Proteínas/metabolismo , Superóxidos/metabolismo
12.
J Agric Food Chem ; 67(15): 4337-4345, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30865450

RESUMO

Mitochondria play an essential part in fighting against pathogen infection in the defense responses of fruits. In this study, we investigated the reactive oxygen species (ROS) production, energy metabolism, and changes of mitochondrial proteins in harvested muskmelon fruits ( Cucumis melo cv. Yujinxiang) inoculated with Trichothecium roseum. The results indicated that the fungal infection obviously induced the H2O2 accumulation in mitochondria. Enzyme activities were inhibited in the first 6 h postinoculation (hpi), including succinic dehydrogenase, cytochrome c oxidase, H+-ATPase, and Ca2+-ATPase. However, the activities of Ca2+-ATPase and H+-ATPase and the contents of intracellular adenosine triphosphate (ATP) were improved to a higher level at 12 hpi. A total of 42 differentially expressed proteins were identified through tandem mass tags-based proteomic analyses, which are mainly involved in energy metabolism, stress responses and redox homeostasis, glycolysis and tricarboxylic acid cycle, and transporter and mitochondria dysfunction. Taken together, our results suggest that mitochondria play crucial roles in the early defense responses of muskmelons against T. roseum infection through regulation of ROS production and energy metabolism.


Assuntos
Cucumis melo/metabolismo , Cucumis melo/microbiologia , Metabolismo Energético , Hypocreales/fisiologia , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Cucumis melo/enzimologia , Cucumis melo/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo
13.
Front Plant Sci ; 9: 1094, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30100914

RESUMO

The mycotoxin patulin is produced in colonized tissue by Penicillium expansum during storage of apples and is significantly affected by environmental factors that contribute to its accumulation. Few reports have, however, examined the effect of natural intrinsic factors associated with the fruit on the production of patulin. Here, we find that with advancing maturity, Golden Delicious apples show increased concentrations of total soluble solids (TSS) from 14 to 17% associated with the increased expression of the global transcription factor involved in regulation of secondary metabolite biosynthesis in filamentous fungi, laeA expression and patulin accumulation. However, the apple cultivar Granny Smith, with similar TSS values but differing in pH levels and malic acid concentrations, showed reduced expression levels of laeA and the patulin biosynthesis gene cluster (pat genes) and patulin accumulation, suggesting a complexity of host factors contribution to patulin accumulation during P. expansum colonization. To start elucidating these apple intrinsic factors, we examined their in vitro impact on laeA and pat gene expression concomitant with patulin synthesis. Increasing sucrose concentrations from 15 to 175 mM repressed laeA and pat gene expression and patulin production. However, this affect was modified and often reversed and sometimes accentuated by changes in pH, or the addition of malic acid or the major apple phenolic compounds, chlorogenic acid and epicatechin. While the increase in malic acid from 0 to 1% increased laeA and pat gene expression, the decrease in pH from 3.5 to 2.5 reduced their expression. Also the increased laeA and pat genes expressions at increasing epicatechin concentrations from 0 to 1 mM, was reversed by increasing sucrose concentrations, all together suggesting the complexity of the interactions in vivo.

14.
Mol Plant Pathol ; 19(12): 2635-2650, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30047230

RESUMO

Amongst the universal diseases affecting apples, blue mould caused by Penicillium expansum is a major concern, resulting in yield and quality losses as a result of the production of the mycotoxin patulin. Despite the characterization of the patulin biosynthetic gene cluster at both the molecular and chemical levels, the underlying regulation of patulin biosynthesis in P. expansum and the mechanisms of apple colonization remain largely obscure. Recent work has indicated that sucrose, a carbon catabolite repressive metabolite, is a critical factor in the regulation of patulin synthesis. Here, CreA, the global carbon catabolite regulator, was assessed for virulence both in vitro and in vivo. We showed that loss-of-function creA strains were nearly avirulent and did not produce patulin in apples. On the basis of RNA-sequencing (RNA-seq) analysis and physiological experimentation, these mutants were unable to successfully colonize apples for a multitude of potential mechanisms including, on the pathogen side, a decreased ability to produce proteolytic enzymes and to acidify the environment and impaired carbon/nitrogen metabolism and, on the host side, an increase in the oxidative defence pathways. Our study defines CreA and its downstream signalling pathways as promising targets for the development of strategies to fight against the development and virulence of this post-harvest pathogen.


Assuntos
Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno/genética , Malus/microbiologia , Mutação/genética , Penicillium/patogenicidade , Carbono/farmacologia , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Gluconatos/metabolismo , Malus/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Patulina/metabolismo , Penicillium/efeitos dos fármacos , Fenótipo , Reprodutibilidade dos Testes , Sacarose/farmacologia , Virulência/efeitos dos fármacos
16.
Toxins (Basel) ; 9(9)2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28895896

RESUMO

Storage of freshly harvested fruit is a key factor in modulating their supply for several months after harvest; however, their quality can be reduced by pathogen attack. Fruit pathogens may infect their host through damaged surfaces, such as mechanical injuries occurring during growing, harvesting, and packing, leading to increased colonization as the fruit ripens. Of particular concern are fungal pathogens that not only macerate the host tissue but also secrete significant amounts of mycotoxins. Many studies have described the importance of physiological factors, including stage of fruit development, biochemical factors (ripening, C and N content), and environmental factors (humidity, temperature, water deficit) on the occurrence of mycotoxins. However, those factors usually show a correlative effect on fungal growth and mycotoxin accumulation. Recent reports have suggested that host factors can induce fungal metabolism, leading to the synthesis and accumulation of mycotoxins. This review describes the new vision of host-factor impact on the regulation of mycotoxin biosynthetic gene clusters underlying the complex regulation of mycotoxin accumulation in ripening fruit.


Assuntos
Frutas , Fungos , Micotoxinas , Contaminação de Alimentos , Conservação de Alimentos , Armazenamento de Alimentos , Interações Hospedeiro-Patógeno , Fatores de Risco
17.
BMC Genomics ; 18(1): 579, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28778147

RESUMO

BACKGROUND: The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared. RESULTS: C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process. CONCLUSIONS: Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi-SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.


Assuntos
Colletotrichum/genética , Colletotrichum/fisiologia , Frutas/genética , Perfilação da Expressão Gênica , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Interferência de RNA , Ciclopentanos/metabolismo , Frutas/química , Ontologia Genética , Genes Fúngicos/genética , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Lycopersicon esculentum/metabolismo , Oxilipinas/metabolismo , Propanóis/metabolismo , Açúcares/metabolismo
18.
Mol Plant Pathol ; 18(8): 1150-1163, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-27528575

RESUMO

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose-responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175- and five-fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe-21 from Israel and Pe-T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch-Pe-T01 strains to 15%-25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is-Pe-21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.


Assuntos
Proteínas Fúngicas/metabolismo , Penicillium/metabolismo , Penicillium/patogenicidade , Metabolismo Secundário , Sacarose/farmacologia , Contagem de Colônia Microbiana , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Malus/microbiologia , Família Multigênica , Mutação/genética , Patulina/biossíntese , Penicillium/efeitos dos fármacos , Penicillium/genética , Virulência/efeitos dos fármacos
19.
Fungal Genet Biol ; 99: 29-39, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28027951

RESUMO

The GATA transcription factor AreA is a global nitrogen regulator that restricts the utilization of complex and poor nitrogen sources in the presence of good nitrogen sources in microorganisms. In this study, we report the biological function of an AreA homolog (the CgareA gene) in the fruit postharvest pathogen Colletotrichum gloeosporioides. Targeted gene deletion mutants of areA exhibited significant reductions in vegetative growth, increases in conidia production, and slight decreases in conidial germination rates. Quantitative RT-PCR (qRT-PCR) analysis revealed that the expression of AreA was highly induced under nitrogen-limiting conditions. Moreover, compared to wild-type and complemented strains, nitrogen metabolism-related genes were misregulated in ΔareA mutant strains. Pathogenicity assays indicated that the virulence of ΔareA mutant strains were affected by the nitrogen content, but not the carbon content, of fruit hosts. Taken together, our results indicate that CgareA plays a critical role in fungal development, conidia production, regulation of nitrogen metabolism and virulence in Colletotrichum gloeosporioides.


Assuntos
Colletotrichum/genética , Proteínas Fúngicas/genética , Fatores de Transcrição GATA/genética , Esporos Fúngicos/genética , Colletotrichum/crescimento & desenvolvimento , Colletotrichum/patogenicidade , Frutas/microbiologia , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Mutação , Nitrogênio/metabolismo , Doenças das Plantas/microbiologia , Deleção de Sequência , Esporos Fúngicos/crescimento & desenvolvimento
20.
PLoS One ; 11(12): e0168561, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28030573

RESUMO

Growth of Colletotrichum gloeosporioides in the presence of cation salts NaCl and KCl inhibited fungal growth and anthracnose symptom of colonization. Previous reports indicate that adaptation of Aspergillus nidulans to salt- and osmotic-stress conditions revealed the role of zinc-finger transcription factors SltA and CrzA in cation homeostasis. Homologs of A. nidulans SltA and CrzA were identified in C. gloeosporioides. The C. gloeosporioides CrzA homolog is a 682-amino acid protein, which contains a C2H2 zinc finger DNA-binding domain that is highly conserved among CrzA proteins from yeast and filamentous fungi. The C. gloeosporioides SltA homolog encodes a 775-amino acid protein with strong similarity to A. nidulans SltA and Trichoderma reesei ACE1, and highest conservation in the three zinc-finger regions with almost no changes compared to ACE1 sequences. Knockout of C. gloeosporioides crzA (ΔcrzA) resulted in a phenotype with inhibited growth, sporulation, germination and appressorium formation, indicating the importance of this calciu006D-activated transcription factor in regulating these morphogenetic processes. In contrast, knockout of C. gloeosporioides sltA (ΔsltA) mainly inhibited appressorium formation. Both mutants had reduced pathogenicity on mango and avocado fruit. Inhibition of the different morphogenetic stages in the ΔcrzA mutant was accompanied by drastic inhibition of chitin synthase A and B and glucan synthase, which was partially restored with Ca2+ supplementation. Inhibition of appressorium formation in ΔsltA mutants was accompanied by downregulation of the MAP kinase pmk1 and carnitine acetyl transferase (cat1), genes involved in appressorium formation and colonization, which was restored by Ca2+ supplementation. Furthermore, exposure of C. gloeosporioides ΔcrzA or ΔsltA mutants to cations such as Na+, K+ and Li+ at concentrations that the wild type C. gloeosporioides is not affected had further adverse morphogenetic effects on C. gloeosporioides which were partially or fully restored by Ca2+. Overall results suggest that both genes modulating alkali cation homeostasis have significant morphogenetic effects that reduce C. gloeosporioides colonization.


Assuntos
Cátions Bivalentes/toxicidade , Colletotrichum/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Morfogênese/fisiologia , Persea/microbiologia , Doenças das Plantas/microbiologia , Fatores de Transcrição/metabolismo , Colletotrichum/genética , Colletotrichum/metabolismo , Proteínas Fúngicas/genética , Morfogênese/efeitos dos fármacos , Fenótipo , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Esporos Fúngicos/fisiologia , Fatores de Transcrição/genética , Virulência/genética , Dedos de Zinco
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