RESUMO
There is a limited number of studies analyzing the molecular and biochemical processes regulating the metabolism of the maturation of Cocos nucifera L. zygotic embryos. Our research focused on the regulation of carbohydrate and lipid metabolic pathways occurring at three developmental stages of embryos from the Mexican Pacific tall (MPT) and the Yucatan green dwarf (YGD) cultivars. We used the TMT-synchronous precursor selection (SPS)-MS3 strategy to analyze the dynamics of proteomes from both embryos; 1044 and 540 proteins were determined for the MPT and YGD, respectively. A comparison of the differentially accumulated proteins (DAPs) revealed that the biological processes (BP) enriched in the MPT embryo included the glyoxylate and dicarboxylate metabolism along with fatty acid degradation, while in YGD, the nitrogen metabolism and pentose phosphate pathway were the most enriched BPs. Findings suggest that the MPT embryos use fatty acids to sustain a higher glycolytic/gluconeogenic metabolism than the YGD embryos. Moreover, the YGD proteome was enriched with proteins associated with biotic or abiotic stresses, e.g., peroxidase and catalase. The goal of this study was to highlight the differences in the regulation of carbohydrate and lipid metabolic pathways during the maturation of coconut YGD and MPT zygotic embryos.
Assuntos
Metabolismo dos Carboidratos , Cocos , Ácidos Graxos , Proteínas de Plantas , Sementes , Ácidos Graxos/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Sementes/crescimento & desenvolvimento , Cocos/metabolismo , Proteômica/métodos , Proteoma/metabolismo , Metabolismo dos Lipídeos , Regulação da Expressão Gênica de PlantasRESUMO
Conidia play a vital role in the survival and rapid spread of fungi. Many biological processes of conidia, such as adhesion, signal transduction, the regulation of oxidative stress, and autophagy, have been well studied. In contrast, the contribution of pathogenicity factors during the development of conidia in fungal phytopathogens has been poorly investigated. To date, few reports have centered on the pathogenicity functions of fungal phytopathogen conidia. Pseudocercospora fijiensis is a hemibiotrophic fungus and the causal agent of the black Sigatoka disease in bananas and plantains. Here, a conidial transcriptome of P. fijiensis was characterized computationally. Carbohydrates, amino acids, and lipid metabolisms presented the highest number of annotations in Gene Ontology. Common conidial functions were found, but interestingly, pathogenicity factors and effectors were also identified. Upon analysis of the resulting proteins against the Pathogen-Host Interaction (PHI) database, 754 hits were identified. WideEffHunter and EffHunter effector predictors identified 618 effectors, 265 of them were shared with the PHI database. A total of 1107 conidial functions devoted to pathogenesis were found after our analysis. Regarding the conidial effectorome, it was found to comprise 40 canonical and 578 non-canonical effectors. Effectorome characterization revealed that RXLR, LysM, and Y/F/WxC are the largest effector families in the P. fijiensis conidial effectorome. Gene Ontology classification suggests that they are involved in many biological processes and metabolisms, expanding our current knowledge of fungal effectors.
RESUMO
Cocos nucifera L. is a crop grown in the humid tropics. It is grouped into two classes of varieties: dwarf and tall; regardless of the variety, the endosperm of the coconut accumulates carbohydrates in the early stages of maturation and fatty acids in the later stages, although the biochemical factors that determine such behavior remain unknown. We used tandem mass tagging with synchronous precursor selection (TMT-SPS-MS3) to analyze the proteomes of solid endosperms from Yucatan green dwarf (YGD) and Mexican pacific tall (MPT) coconut cultivars. The analysis was conducted at immature, intermediate, and mature development stages to better understand the regulation of carbohydrate and lipid metabolisms. Proteomic analyses showed 244 proteins in YGD and 347 in MPT; from these, 155 proteins were shared between both cultivars. Furthermore, the proteomes related to glycolysis, photosynthesis, and gluconeogenesis, and those associated with the biosynthesis and elongation of fatty acids, were up-accumulated in the solid endosperm of MPT, while in YGD, they were down-accumulated. These results support that carbohydrate and fatty acid metabolisms differ among the developmental stages of the solid endosperm and between the dwarf and tall cultivars. This is the first proteomics study comparing different stages of maturity in two contrasting coconut cultivars and may help in understanding the maturity process in other palms.
Assuntos
Cocos , Endosperma , Endosperma/metabolismo , Cocos/metabolismo , Ácidos Graxos/metabolismo , Proteoma/metabolismo , Proteômica , Carboidratos , Redes e Vias MetabólicasRESUMO
Effectors are small, secreted molecules that mediate the establishment of interactions in nature. While some concepts of effector biology have stood the test of time, this area of study is ever-evolving as new effectors and associated characteristics are being revealed. In the present review, the different characteristics that underly effector classifications are discussed, contrasting past and present knowledge regarding these molecules to foster a more comprehensive understanding of effectors for the reader. Research gaps in effector identification and perspectives for effector application in plant disease management are also presented, with a focus on fungal effectors in the plant-microbe interaction and interactions beyond the plant host. In summary, the review provides an amenable yet thorough introduction to fungal effector biology, presenting noteworthy examples of effectors and effector studies that have shaped our present understanding of the field.
Assuntos
Proteínas Fúngicas , Doenças das Plantas , Doenças das Plantas/microbiologia , Plantas/microbiologia , Interações Hospedeiro-PatógenoRESUMO
Newer effectorome prediction algorithms are considering effectors that may not comply with the canonical characteristics of small, secreted, cysteine-rich proteins. The use of effector-related motifs and domains is an emerging strategy for effector identification, but its use has been limited to individual species, whether oomycete or fungal, and certain domains and motifs have only been associated with one or the other. The use of these strategies is important for the identification of novel, non-canonical effectors (NCEs) which we have found to constitute approximately 90% of the effectoromes. We produced an algorithm in Bash called WideEffHunter that is founded on integrating three key characteristics: the presence of effector motifs, effector domains and homology to validated existing effectors. Interestingly, we found similar numbers of effectors with motifs and domains within two different taxonomic kingdoms: fungi and oomycetes, indicating that with respect to their effector content, the two organisms may be more similar than previously believed. WideEffHunter can identify the entire effectorome (non-canonical and canonical effectors) of oomycetes and fungi whether pathogenic or non-pathogenic, unifying effector prediction in these two kingdoms as well as the two different lifestyles. The elucidation of complete effectoromes is a crucial step towards advancing effectoromics and disease management in agriculture.
Assuntos
Oomicetos , Doenças das Plantas , Doenças das Plantas/microbiologia , Plantas/metabolismo , Oomicetos/metabolismo , Fungos , AlgoritmosRESUMO
Effectors are small, secreted molecules that alter host cell structure and function, thereby facilitating infection or triggering a defense response. Effectoromics studies have focused on effectors in plant-pathogen interactions, where their contributions to virulence are determined in the plant host, i.e., whether the effector induces resistance or susceptibility to plant disease. Effector molecules from plant pathogenic microorganisms such as fungi, oomycetes and bacteria are major disease determinants. Interestingly, the effectors of non-pathogenic plant organisms such as endophytes display similar functions but have different outcomes for plant health. Endophyte effectors commonly aid in the establishment of mutualistic interactions with the plant and contribute to plant health through the induction of systemic resistance against pathogens, while pathogenic effectors mainly debilitate the plant's immune response, resulting in the establishment of disease. Effectors of plant pathogens as well as plant endophytes are tools to be considered in effectoromics for the development of novel strategies for disease management. This review aims to present effectors in their roles as promotors of health or disease for the plant host.
RESUMO
Lipases are enzymes that hydrolyze triglycerides to fatty acids and glycerol. A typical element in lipases is a conserved motif of five amino acids (the pentapeptide), most commonly G-X-S-X-G. Lipases with the pentapeptide A-X-S-X-G are present in species of Bacillus, Paucimonas lemoignei, and the yeast Trichosporon asahii; they are usually thermotolerant and solvent resistant. Recently, while searching for true lipases in the Trichoderma harzianum genome, one lipase containing the pentapeptide AHSMG was identified. In this study, we cloned from T. harzianum strain B13-1 the lipase ID135964, renamed here as ThaL, which is 97.65% identical with the reference. We found that ThaL is a lid-containing true lipase of cluster III that belongs to a large family comprising highly conserved proteins in filamentous fungi in the orders Hypocreales and Glomerellales, in which predominantly pathogenic fungi are found. ThaL was expressed in conidia, as well as in T. harzianum mycelium, where it was cultured in liquid minimal medium. These results-together with the amino acid composition, absence of a signal peptide, mitochondrial sorting prediction, disordered regions in the protein, and lineage-specific phylogenetic distribution of its homologs-suggest that ThaL is a non-canonical effector. In summary, AHSMG-lipase is a novel lipase family in filamentous fungi, and is probably involved in pathogenicity.
Assuntos
Bacillus , Hypocreales , Bacillus/metabolismo , Fungos/metabolismo , Hypocreales/metabolismo , Lipase/metabolismo , Filogenia , Pseudomonas/metabolismoRESUMO
Zygotic and somatic embryogenesis in plants is a fascinating event that is finely regulated through the expression of a specific group of genes and dynamic levels of plant hormones whose concerted action determines the fate that specific cells follow towards zygotic or somatic embryo development. This work studied different stages of Capsicum chinense Jacq. zygotic and somatic embryogenesis. HPLC quantification determined that the levels of indole-3-acetic acid (IAA) increase as the zygotic or somatic embryogenesis progresses, being higher at maturity, thus supporting a positive correlation between embryo cell differentiation and IAA increase. A monoclonal anti-IAA-antibody was used to detect IAA levels. Findings revealed a dynamic pattern of auxin distribution along the different embryogenic embryonic stages. In the early stages of zygotic embryos, the IAA gradient was observed in the basal cells of the suspensor and the hypostases, suggesting that they are the initial source of the IAA hormone. As embryogenesis proceeds, the dynamic of the IAA gradient is displaced to the embryo and endosperm cells. In the case of induced somatic embryogenesis, the IAA gradient was detected in the dividing cells of the endodermis, from where pre-embryogenic cells emerge. However, the analysis of somatic embryos revealed that IAA was homogeneously distributed. This study shows evidence supporting a correlation between IAA levels during zygotic or somatic embryogenesis in Capsicum chinense species.
Assuntos
Capsicum/embriologia , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Sementes/embriologia , Zigoto/crescimento & desenvolvimentoRESUMO
INTRODUCTION: Dirofilaria immitis is a nematode that affects human health in several countries of the world. This study was conducted to examine whether serum samples from the owners of microfilaremic dogs present immunoreactivity to parasite proteins. METHODOLOGY: Eight serum samples from the owners of microfilaremic dogs were examined. Total proteins were extracted from adult worms and 12% SDS-PAGE was performed. The gel was electroblotted to a nitrocellulose membrane, and a Western blot (WB) was performed. Reactive bands of 22, 33, 39, 49, and 63 kDa in WB were excised from the gel and analyzed by mass spectrometry (MS). RESULTS: The MS results showed the presence of 10 different proteins of D. immitis recognized by the human serum samples. CONCLUSIONS: These results indicate that in endemic areas of D. immitis, owners of infected dogs recognize specific proteins of the parasite, suggesting a possible infection.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Dirofilaria immitis/química , Dirofilariose/imunologia , Doenças do Cão/parasitologia , Proteínas de Helminto/imunologia , Propriedade , Adulto , Idoso , Animais , Western Blotting , Dirofilaria immitis/genética , Dirofilaria immitis/imunologia , Dirofilariose/transmissão , Cães , Feminino , Humanos , Masculino , México , Microfilárias/genética , Microfilárias/imunologia , Pessoa de Meia-Idade , Animais de Estimação/parasitologiaRESUMO
Pseudocercospora fijiensis causes black Sigatoka disease, the most important threat to banana. The cell wall is crucial for fungal biological processes, including pathogenesis. Here, we performed cell wall proteomics analyses of two P. fijiensis strains, the highly virulent Oz2b, and the less virulent C1233 strains. Strains were starved from nitrogen to mimic the host environment. Interestingly, in vitro cultures of the C1233 strain grew faster than Oz2b in PDB medium, suggesting that C1233 survives outside the host better than the highly virulent Oz2b strain. Both strains were submitted to nitrogen starvation and the cell wall proteins were isolated and subjected to nano-HPLC-MS/MS. A total of 2686 proteins were obtained from which only 240 had a known function and thus, bioinformatics analyses were performed on this group. We found that 90 cell wall proteins were shared by both strains, 21 were unique for Oz2b and 39 for C1233. Shared proteins comprised 24 pathogenicity factors, including Avr4 and Ecp6, two effectors from P. fijiensis, while the unique proteins comprised 16 virulence factors in C1233 and 11 in Oz2b. The P. fijiensis cell wall proteome comprised canonical proteins, but thirty percent were atypical, a feature which in other phytopathogens has been interpreted as contamination. However, a comparison with the identities of atypical proteins in other reports suggests that the P. fijiensis proteins we detected were not contaminants. This is the first proteomics analysis of the P. fijiensis cell wall and our results expands the understanding of the fundamental biology of fungal phytopathogens and will help to decipher the molecular mechanisms of pathogenesis and virulence in P. fijiensis.
Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Proteoma , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Ascomicetos/isolamento & purificação , Ascomicetos/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Genoma Fúngico , Musa/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Espectrometria de Massas em Tandem , VirulênciaRESUMO
Black Sigatoka is a disease that occurs in banana plantations worldwide. This disease is caused by the hemibiotrophic fungus Pseudocercospora fijiensis, whose infection results in a significant reduction in both product quality and yield. Therefore, detection and identification in the early stages of this pathogen in plants could help minimize losses, as well as prevent the spread of the disease to neighboring cultures. To achieve this, a highly sensitive SPR immunosensor was developed to detect P. fijiensis in real samples of leaf extracts in early stages of the disease. A polyclonal antibody (anti-HF1), produced against HF1 (cell wall protein of P. fijiensis) was covalently immobilized on a gold-coated chip via a mixed self-assembled monolayer (SAM) of alkanethiols using the EDC/NHS method. The analytical parameters of the biosensor were established, obtaining a limit of detection of 11.7 µg mL-1, a sensitivity of 0.0021 units of reflectance per ng mL-1 and a linear response range for the antigen from 39.1 to 122 µg mL-1. No matrix effects were observed during the measurements of real leaf banana extracts by the immunosensor. To the best of our knowledge, this is the first research into the development of an SPR biosensor for the detection of P. fijiensis, which demonstrates its potential as an alternative analytical tool for in-field monitoring of black Sigatoka disease.
Assuntos
Ascomicetos/isolamento & purificação , Ascomicetos/patogenicidade , Técnicas Biossensoriais/métodos , Ressonância de Plasmônio de Superfície/métodosRESUMO
Ovule and seed development in plants has long fascinated the scientific community given the complex cell coordination implicated in these processes. These cell events are highly conserved but are not necessarily representative of all plants. In this study, with the aim of obtaining information regarding the cellular patterns that follow the usual development of the ovule and the zygotic embryo, we carried out an integral anatomical study of the Capsicum chinense Jacq., floral buds and seeds at various days during maturation. This study allowed us to identify the main histo-morphological stages accompanying the transition of somatic cells into the macrospore, female gamete, and the zygotic embryogenesis. This knowledge is fundamental for future biotechnological research focused on solving the morphological recalcitrance observed during the in vitro induction of somatic or microspore embryogenesis in Capsicum. For the first time in C. chinense, we have described the hypostases, a putative source of plant growth regulators, and "the corrosion cavity", a space around the embryo. Additionally, the cell wall pectin-esterification status was investigated by immunohistology. At early stages of morphogenesis, the pectin is highly methyl-esterified; however, methyl-esterification decreases gradually throughout the process. A comparison of the results obtained here, together with the histo- and immunological changes occurring during the somatic and microspore embryogenesis, should help to elucidate the biochemical mechanisms that trigger the morphogenic events in Capsicum spp.
Assuntos
Capsicum/crescimento & desenvolvimento , Óvulo Vegetal/crescimento & desenvolvimento , Pectinas/metabolismo , Sementes/crescimento & desenvolvimento , Capsicum/anatomia & histologia , Capsicum/metabolismo , Esterificação , Flores/anatomia & histologia , Flores/crescimento & desenvolvimento , Flores/metabolismo , Imunofluorescência , Óvulo Vegetal/anatomia & histologia , Óvulo Vegetal/metabolismo , Sementes/anatomia & histologia , Sementes/metabolismoRESUMO
A lipolytic screening with fungal strains isolated from lignocellulosic waste collected in banana plantation dumps was carried out. A Trichoderma harzianum strain (B13-1) showed good extracellular lipolytic activity (205 UmL-1). Subsequently, functional screening of the lipolytic activity on Rhodamine B enriched with olive oil as the only carbon source was performed. The successful growth of the strain allows us to suggest that a true lipase is responsible for the lipolytic activity in the B13-1 strain. In order to identify the gene(s) encoding the protein responsible for the lipolytic activity, in silico identification and characterization of triacylglycerol lipases from T. harzianum is reported for the first time. A survey in the genome of this fungus retrieved 50 lipases; however, bioinformatic analyses and putative functional descriptions in different databases allowed us to choose seven lipases as candidates. Suitability of the bioinformatic screening to select the candidates was confirmed by reverse transcription polymerase chain reaction (RT-PCR). The gene codifying 526309 was expressed when the fungus grew in a medium with olive oil as carbon source. This protein shares homology with commercial lipases, making it a candidate for further applications. The success in identifying a lipase gene inducible with olive oil and the suitability of the functional screening and bioinformatic survey carried out herein, support the premise that the strategy can be used in other microorganisms with sequenced genomes to search for true lipases, or other enzymes belonging to large protein families.
RESUMO
The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr â¼7.57 kDa and pI â¼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the â¼7.57 kDa polypeptide, immunodetected an â¼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the â¼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the â¼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the â¼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense.
Assuntos
Capsicum/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Capsicum/genética , Capsicum/crescimento & desenvolvimento , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Focalização Isoelétrica , Peso Molecular , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L(-1) for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for ß-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L(-1) for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.
Assuntos
Ascomicetos/efeitos dos fármacos , Farmacorresistência Fúngica , Fungicidas Industriais/farmacologia , Musa/microbiologia , Doenças das Plantas/microbiologia , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Uso de Medicamentos , México , Mutação de Sentido Incorreto , Doenças das Plantas/prevenção & controle , Doenças das Plantas/terapia , Tubulina (Proteína)/genéticaRESUMO
The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.
Assuntos
Ascomicetos/efeitos dos fármacos , Farmacorresistência Fúngica , Fungicidas Industriais/farmacologia , Musa/microbiologia , Doenças das Plantas/microbiologia , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Uso de Medicamentos , México , Mutação de Sentido Incorreto , Doenças das Plantas/prevenção & controle , Doenças das Plantas/terapia , Tubulina (Proteína)/genéticaRESUMO
The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.(AU)
Assuntos
Ascomicetos/efeitos dos fármacos , Farmacorresistência Fúngica , Fungicidas Industriais/farmacologia , Musa/microbiologia , Doenças das Plantas/microbiologia , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Uso de Medicamentos , México , Mutação de Sentido Incorreto , Doenças das Plantas/prevenção & controle , Doenças das Plantas/terapia , Tubulina (Proteína)/genéticaRESUMO
The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host.
Assuntos
Ascomicetos/patogenicidade , Morte Celular/efeitos dos fármacos , Endopeptidases/farmacologia , Proteínas Fúngicas/farmacologia , Musa/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Carica/efeitos dos fármacos , Carica/microbiologia , Interações Hospedeiro-Patógeno , Hidrólise , Musa/classificação , Musa/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Especificidade da Espécie , Fatores de Virulência/farmacologiaRESUMO
Partial peptide sequence of a 36 kDa protein from common bean embryo axes showed 100% identity with a reported beta-subunit of a heterotrimeric G protein from soybean. Analysis of the full sequence showed 96.6% identity with the reported soybean G(beta)-subunit, 86% with RACK1B and C from Arabidopsis and 66% with human and mouse RACK1, at the amino acid level. In addition, it showed 85.5, 85 and 83% identities with arcA from Solanum lycopersicum, Arabidopsis (RACK1A) and Nicotiana tabacum, respectively. The amino acid sequence displayed seven WD40 domains and two sites for activated protein kinase C binding. The protein showed a constant expression level but the mRNA had a maximum at 32 h post-imbibition. Western immunoblotting showed the protein in vegetative plant tissues, and in both microsomal and soluble fractions from embryo axes. Synthetic auxin treatment during germination delayed the peak of RACK1 mRNA expression to 48 h but did not affect the protein expression level while the polar auxin transport inhibitor, naphtylphtalamic acid had no effect on either mRNA or protein expression levels. Southern blot and genomic DNA amplification revealed a small gene family with at least one member without introns in the genome. Thus, the RACK1/arcA homolog from common bean has the following features: (1) it is highly conserved; (2) it is both soluble and insoluble within the embryo axis; (3) it is encoded by a small gene family; (4) its mRNA has a peak of expression at the time point of germination stop and (5) its expression is only slightly affected by auxin but unaffected by an auxin transport blocker.
Assuntos
Germinação , Phaseolus/genética , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Clonagem Molecular , DNA de Plantas/genética , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica de Plantas , Humanos , Ácidos Indolacéticos/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Neuropeptídeos/genética , Phaseolus/embriologia , Phaseolus/metabolismo , Filogenia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Mitogen activated protein (MAP) kinase-like activity was determined in extracts obtained from transformed Catharanthus roseus hairy roots by the ability to phosphorylate myelin basic protein (MBP). Both in solution and in gel kinase assays showed variation in activity, depending on root developmental stage. In gel kinase assays, using the extract soluble fraction, revealed a 56 kDa polypeptide with phosphorylation activity on MBP. In addition, another 75 kDa polypeptide was observed in the particulate fraction. Immunodetection with monoclonal antibodies against ERK-1, a mammalian MAP kinase, and with anti-phosphotyrosine antibodies cross-reacted with the 56 kDa polypeptide, named SMK56, from the soluble fraction, suggesting that this polypeptide could be related with members of the MAP kinase family. Antibodies against the dually phosphorylated threonine-tyrosine motif, characteristic of active forms of MAP kinases, also cross-reacted with this 56 kDa polypeptide. Changes in the levels of SMK56 were detected within the first 30 min of root exposure to low temperatures or hypo-osmotic shock, suggesting that this protein may be involved in the perception of environmental changes.