Molecular Identification and Phylogenetic Analysis of a Group 16SrI-B Phytoplasma Associated with Sugarcane Yellow Leaf Syndrome in Brazil

Yellow leaf syndrome was a serious problem in the beginning of the 1990s in Brazil, when yield losses were estimated to be around 50%. The disease is currently endemic, but it is considered potentially important. Previous studies have revealed only the presence of a luteovirus associated with the disease in Brazil. We report that a phytoplasma of 16SrI-B is also associated with this disease. This is the first demonstration of the presence of a group 16SrI-B phytoplasma in association with sugarcane yellow leaf in Brazil.

Molecular and morphological markers for rapid distinction between 2 Colletotrichum species

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia (espinheira-santa) in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense. Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Coll and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.

Molecular evidence of phytoplasma associated with witches` broom on passion fruit in five Brazilian States

Phytoplasmas are cell wall-less prokaryotes and phloem-inhabitants associated with diseases that affect several crops. Passion fruit is a tropical plant species cultivated in various Brazilian regions. Diseases are among the factors that may cause damage to this crop, and witches` broom is a very important one. This disease, associated with a phytoplasma, has been reported only in Brazil, where it was described in Rio de Janeiro and Pernambuco States at the beginning of the 1980`s. In 2005-2006, symptomatic plants Suspected of phytoplasma infection were sampled in areas located in Sao Paulo, Parana, Rio de Janeiro, Sergipe and Bahia. Amplification of DNA fragments of 1.2kb from nested PCR primed by the pairs 16mF2/mR1 and 16F2n/R2 revealed the presence of phytoplasma in the tissue of plants from all sampled regions. Molecular detection of the agent allowed confirmation of the diagnosis based on the symptomatology; demonstrated the strong association between diseased plants and a phytoplasma, confirming previous investigations based on electron microscopy;, and revealed the present occurrence of the agent in the States of Bahia, Parana, Rio de Janeiro, Sergipe, and Sao Paulo.

Localization of Pantoea ananatis inside lesions of maize White Spot Disease using transmission electron microscopy and molecular techniques

The etiological agent of maize white spot (MWS) disease has been a subject of controversy and discussion. Initially the disease was described as Phaeosphaeria leaf spot caused by Phaeosphaeria maydis. Other authors have Suggested the existence of different fungal species causing similar symptoms. Recently, a bacterium, Pantoea ananatis, was described as the causal agent of this disease. The purpose of this Study was to offer additional information on the correct etiology of this disease by providing visual evidence of the presence of the bacterium in the interior of the MWS lesions by using transmission electron microscopy (TEM) and molecular techniques. The TEM allowed Visualization of a large amount of bacteria in the intercellular spaces of lesions collected from both artificially and naturally infected plants. Fungal structures were not visualized in young lesions. Bacterial primers for the 16S rRNA and rpoB genes were used in PCR reactions to amplify DNA extracted from water-soaked (young) and necrotic lesions. The universal fungal oligonucleotide ITS4 was also included to identity the possible presence of fungal structures inside lesions. Positive PCR products from water-soaked lesions, both from naturally and artificially inoculated plants, were produced with bacterial primers, whereas no amplification was observed when ITS4 oligonucleotide was used. On the other hand, DNA amplification with ITS4 primer was observed when DNA was isolated from necrotic (old) lesions. These results reinforced previous report of P. ananatis as the primary pathogen and the hypothesis that fungal species may colonize lesions pre-established by P. ananatis.

Histological, physiological and molecular investigations of Fagus sylvatica seedlings infected with Phytophthora citricola

P>The aim of the work was to shed light into histological, physiological and molecular changes of Fagus sylvatica seedlings infected with the root pathogen Phytophthora citricola with the final goal to distinguish between local and systemic responses. Real-time quantitative PCR analysis proved that P. citricola was able to grow from infected roots into hypocotyl and epicotyl tissue of F. sylvatica seedlings. Light microscopy showed many collapsed parenchyma cells of the cortex without being penetrated by the pathogen. Hyphae were mainly growing intracellular in parenchyma and xylem tissue. Transmission electron microscopy displayed disintegration of xylem vessels and of parenchyma cells. Inhibition of water uptake of infected beech seedlings was positively correlated with the concentration of zoospores used in the experiment. In addition, a split root experiment indicated that invertases were possibly involved locally and systemically in the conversion of sucrose of P. citricola infected roots. During the growth of the pathogen in roots, a transient expression of the 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidase gene was quantified in leaves which was detected in parallel with the first peak of a biphasic ethylene outburst. Additionally a systemic upregulation of aquaporin transcripts was mainly detected in leaves of beech seedlings infected with P. citricola.

Cloning, expression, molecular modelling and docking analysis of glutathione transferase from Saccharum officinarum

Sugarcane yield and quality are affected by a number of biotic and abiotic stresses. In response to such stresses, plants may increase the activities of some enzymes such as glutathione transferase (GST), which are involved in the detoxification of xenobiotics. Thus, a sugarcane GST was modelled and molecular docked using the program LIGIN to investigate the contributions of the active site residues towards the binding of reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). As a result, W13 and I119 were identified as key residues for the specificity of sugarcane GSTF1 (SoGSTF1) towards CDNB. To obtain a better understanding of the catalytic specificity of sugarcane GST (SoGSTF1), two mutants were designed, W13L and I119F. Tertiary structure models and the same docking procedure were performed to explain the interactions between sugarcane GSTs with GSH and CDNB. An electron-sharing network for GSH interaction was also proposed. The SoGSTF1 and the mutated gene constructions were cloned and expressed in Escherichia coli and the expressed protein purified. Kinetic analyses revealed different Km values not only for CDNB, but also for GSH. The Km values were 0.2, 1.3 and 0.3 mM for GSH, and 0.9, 1.2 and 0.5 mM for CDNB, for the wild type, W13L mutant and I119F mutant, respectively. The V(max) values were 297.6, 224.5 and 171.8 mu mol min(-1) mg(-1) protein for GSH, and 372.3, 170.6 and 160.4 mu mol min(-1) mg(-1) protein for CDNB.

Molecular and morphological diversity in Japanese rice germplasm

Germplasm molecular and phenotypic characterization is instrumental to its utilization and to genetic variability incorporation into rice breeding programmes. The diversity within 192 Japanese rice accessions was analysed for 22 agro-morphological traits and 24 single sequence repeat markers. A total of 181 alleles were detected, 38 of which were exclusive. The number of alleles/marker ranged from 2 to 16, with an average of 7.54 alleles/locus and the H(e) value ranged from 0.01 to 0.82, with an average of 0.46. The accessions showed diversity at molecular and phenotypic level and few showed also good agronomic performance. Tocher`s method applied on a total-dissimilarity matrix was used to determine cluster formation of 13 diversity groups. Most of the accessions (81%) were clustered within a group, whereas eight accessions (Kyuushuu, Eika Ine, Ishiwari Mochi, Col/Fukui/1965, Ookuma Nishiki, Suzume Shirazu, Iwate Ryoon and Toga) did not cluster with other accessions.

An integrated molecular map of yellow passion fruit based on simultaneous maximum-likelihood estimation of linkage and linkage phases

The development of genetic maps for auto-incompatible species, such as the yellow passion fruit (Passiflora edulis Sims f.flavicarpa Deg.) is restricted due to the unfeasibility of obtaining traditional mapping populations based on inbred lines. For this reason, yellow passion fruit linkage maps were generally constructed using a strategy known as two-way pseudo-testeross, based on monoparental dominant markers segregating in a 1:1 fashion. Due to the lack of information from these markers in one of the parents, two individual (parental) maps were obtained. However, integration of these maps is essential, and biparental markers can be used for such an operation. The objective of our study was to construct an integrated molecular map for a full-sib population of yellow passion fruit combining different loci configuration generated from amplified fragment length polymorphisms (AFLPs) and microsatellite markers and using a novel approach based on simultaneous maximum-likelihood estimation of linkage and linkage phases, specially designed for outcrossing species. Of the total number of loci, approximate to 76%, 21%, 0.7%, and 2.3% did segregate in 1:1, 3:1, 1:2:1, and 1:1:1:1 ratios, respectively. Ten linkage groups (LGs) were established with a logarithm of the odds (LOD) score >= 5.0 assuming a recombination fraction : <= 0.35. On average, 24 markers were assigned per LG, representing a total map length of 1687 cM, with a marker density of 6.9 cM. No markers were placed as accessories on the map as was done with previously constructed individual maps.

Outcrossing rate in sweet passion fruit based on molecular markers

P>Yellow and sweet passion fruit are insect-pollinated species native to the tropics. Fruits are used commercially for human consumption worldwide. The yellow passion fruit is an outcrossing species with self-incompatible flowers. However, the reproductive system of the sweet passion fruit (Passiflora alata) has not been well elucidated. The objective of this work was to characterize aspects of the mating system in the sweet passion fruit using random amplified polymorphic DNA (RAPD) and microsatellite markers, particularly the rate of outcrossing in P. alata progenies. A multilocus outcrossing rate of t(m) = 0.994 was determined from RAPD and t(m) = 0.940 from microsatellites, supporting P. alata as an outcrossing species. The fixation indices of the maternal generation (F(m)) were -0.200 and 0.071 with RAPD and microsatellite loci, respectively, indicating the absence of inbreeding in the maternal generation. The paternity correlation (r(p)) varied from -0.008 with RAPD markers to 0.208 with microsatellite markers, suggesting a low probability of finding full sibs within the progenies. The results demonstrated that all progenies assessed in this study were derived from outcrossing.

A conserved dibasic site is essential for correct processing of the peptide hormone AtRALF1 in Arabidopsis thaliana

Prohormone proteins in animals and yeast are typically processed at dibasic sites by convertases. Propeptide hormones are also found in plants but little is known about processing. We show for the first time that a dibasic site upstream of a plant peptide hormone, AtRALF1, is essential for processing. Overexpression of preproAtRALF1 causes semidwarfism whereas overexpression of preproAtRALF1(R69A), the propeptide with a mutation in the dibasic site, shows a normal phenotype. RALF1(R69A) plants accumulate only the mutated proprotein and not the processed peptide. In vitro processing using microsomal fractions suggests that processing is carried out by a kexin-like convertase. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

HFE gene mutations in patients with primary iron overload: Is there a significant improvement in molecular diagnosis yield with HFE sequencing?

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation >50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and beta 2-microglobulin ((beta 2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations. (C) 2010 Elsevier Inc. All rights reserved.

Chlorogenic Acid UVA-UVB Photostability

Chlorogenic acid is a natural potent antioxidant. It can be used in cosmetics formulations, but for this purpose its photochemical stability should be determined to ensure that the compound will not be degraded after UV radiation exposure. To evaluate this possibility, the concentration of a chlorogenic acid solution was determined by HPLC before and after UVA and UVB irradiation. The results indicate that chlorogenic acid is not degraded under UVA or UVB irradiation.

A lipocalin sequence signature modulates cell survival

Lipocalins are beta-barrel proteins, which share three conserved motifs in their amino acid sequence. In this study, we identified by a peptide mapping approach, a seven-amino acid sequence related to one of these motifs (motif 2) that modulates cell survival. A synthetic peptide based on an insect lipocalin displayed cytoprotective activity in serum-deprived endothelial cells and leucocytes. This activity was dependent on nitric oxide synthase. This sequence was found within several lipocalins, including apolipoprotein D, retinol binding protein, lipocalin-type prostaglandin D synthase, and many unknown proteins, suggesting that it is a sequence signature and a lipocalin conserved property. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Cu and Fe metallic ions-mediated oxidation of low-density lipoproteins studied by NMR, TEM and Z-scan technique

In this work we report on a study of the morphological changes of LDL induced in vitro by metallic ions (Cu(2+) and Fe(3+)). These modifications were characterized by transmission electron microscopy, nuclear magnetic resonance and the Z-scan technique. The degree of oxidative modification of LDL was determined by the TBARS and lipid hydroperoxides assays. It is shown that distinct pathways for modifying lipoproteins lead to different morphological transformations of the particles characterized by changes in size and/or shape of the resulting particles, and by the tendency to induce aggregation of the particles. There were no evidence of melting of particles promoted by oxidative processes with Cu and Fe. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Electronegative LDL induction of apoptosis in macrophages: Involvement of Nrf2

The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(-)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264 7 macrophages with LDL(-) for 24 11 resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(-): incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(-)-treated cells CD95 (Fas), CD95 ligand (FasL). CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(-)-treated cells However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(-) treatment. LDL(-) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(-), together with an increase in the production of ROS in the absence of alterations in CD36 expression These results provide evidence that CD36 expression induced by LDL(-) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(-)-induced apoptosis in macrophages. (C) 2009 Elsevier B V. All rights reserved