Enriched HeK4me3 marks at Pm-0 resistance-related genes prime courgette against Podosphaera xanthii.

Powdery mildew (PM) disease, caused by the obligate biotrophic fungal pathogen Podosphaera xanthii, is the most reported and destructive disease on cultivated Cucurbita species all over the world. Recently, the appearance of highly aggressive P. xanthii isolates has led to PM outbreaks even in resistant crops, making disease management a very difficult task. To challenge this, breeders rely on genetic characteristics for PM control. Analysis of commercially available intermediate resistance courgette (Cucurbita pepo L. var. cylindrica) varieties using cytological, molecular, and biochemical approaches showed that the plants were under a primed state and induced systemic acquired resistance (SAR) responses, exhibiting enhanced callose production, upregulation of salicylic acid (SA) defense signaling pathway genes, and accumulation of SA and defense metabolites. Additionally, the intermediate resistant varieties showed an altered epigenetic landscape in histone marks that affect transcriptional activation. We demonstrated that courgette plants had enriched H3K4me3 marks on SA-BINDING PROTEIN 2 and YODA (YDA) genes of the Pm-0 interval introgression, a genomic region that confers resistant to Cucurbits against P. xanthii. The open chromatin of SA-BINDING PROTEIN 2 and YDA genes was consistent with genes' differential expression, induced SA pathway, altered stomata characteristics, and activated SAR responses. These findings demonstrate that the altered epigenetic landscape of the intermediate resistant varieties modulates the activation of SA-BINDING PROTEIN 2 and YDA genes leading to induced gene transcription that primes courgette plants.

Rapid and accurate identification of black aspergilli from grapes using high-resolution melting (HRM) analysis.

BACKGROUND: Aspergillus is a diverse genus of fungi with high economic and social impact. Various species that belong to section Nigri (black aspergilli) are common agents of grape spoilage and potent producers of ochratoxin A (OTA), a mycotoxin associated with various nephrotoxic and immunotoxic effects in humans. Black aspergilli are difficult to classify following only phenotypic criteria; thus chemotaxonomic and molecular methods are employed in parallel with phenotypic ones for species characterization. These approaches, though accurate and replicable, require more than one individual step and are to a certain extent laborious when a rapid identification of these species is required. RESULTS: The aim of this study was to develop a high-resolution melting polymerase chain reaction (HRM-PCR) assay as a rapid method for identification of Aspergillus spp. section Nigri isolates and their detection in grape samples. Melt curve analysis of amplicons originating from the internal transcribed spacer 2 (ITS2) ribosomal region generated species-specific HRM curve profiles, enabling the accurate differentiation of the analyzed genotypes. Furthermore, the assay was able to identify A. carbonarius, A. tubingensis, A. niger, A. ibericus and A. japonicus in grape samples artificially inoculated with conidia of these fungi. CONCLUSION: To our knowledge this is the first report on the development of an HRM-PCR assay for the identification of black Aspergillus species in grape samples. © 2018 Society of Chemical Industry.

Quantification of Aspergillus carbonarius in grapes using a real time PCR assay.

A study on the representation of Aspergillus carbonarius in the vineyards of the Mesogeia geographical region of Attica, Greece, was conducted. One hundred and twenty five samples of the indigenous drought and disease resistant Savatiano wine grape variety, the most widely planted in Greece, were collected. The sample's total DNA extracts were initially tested for fungal DNA presence by targeting the Internal Transcribed Spacer (ITS) region in end-point Polymerase Chain Reactions. Samples which were proved positive were further subjected to PCR analysis using specific primers targeting an A. carbonarius polycetide synthase (pks) gene. Among ITS positive samples (70%), A. carbonarius was represented in 42% of them. Furthermore, a SYBR Green I Real Time PCR method was used to quantify the amount of this species in the grape samples. The values of the positive samples were estimated in the range of 13 to 50 × 10(3) fungal haploid genomes/g grapes. The significance of this study lies in the applicability of a rapid and culture-independent method to detect and quantify A. carbonarius on grapes.

The RNA binding protein Tudor-SN is essential for stress tolerance and stabilizes levels of stress-responsive mRNAs encoding secreted proteins in Arabidopsis.

Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death-associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway.

Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (Olea europaea) Twigs.

Metabarcoding approaches for the identification of plant disease pathogens and characterization of plant microbial populations constitute a rapidly evolving research field. Fungal plant diseases are of major phytopathological concern; thus, the development of metabarcoding approaches for the detection of phytopathogenic fungi is becoming increasingly imperative in the context of plant disease prognosis. We developed a multiplex metabarcoding method for the identification of fungal phytopathogens and endophytes in olive young shoots, using the MinION sequencing platform (Oxford Nanopore Technologies). Selected fungal-specific primers were used to amplify three different genomic DNA loci (ITS, beta-tubulin, and 28S LSU) originating from olive twigs. A multiplex metabarcoding approach was initially evaluated using healthy olive twigs, and further assessed with naturally infected olive twig samples. Bioinformatic analysis of basecalled reads was carried out using MinKNOW, BLAST+ and R programming, and results were also evaluated using the BugSeq cloud platform. Data analysis highlighted the approaches based on ITS and their combination with beta-tubulin as the most informative ones according to diversity estimations. Subsequent implementation of the method on symptomatic samples identified major olive pathogens and endophytes including genera such as Cladosporium, Didymosphaeria, Paraconiothyrium, Penicillium, Phoma, Verticillium, and others.

High level of heterogeneity among Listeria monocytogenes isolates from clinical and food origin specimens in Greece.

In order to examine the genetic variation of clinical and food isolates of Listeria monocytogenes in Greece, a total of 61 L. monocytogenes non-duplicate isolates, recovered from clinical specimens (n=19) and food (n=42), were serotyped and genotyped using two different Random Amplification of Polymorphic DNA (RAPD) protocols and Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Serotype group 4b, 4d, 4e prevailed (39.4%), among both clinical and food isolates, followed by serotype group 1/2a, 3a (23.0%), which nevertheless was detected only among food isolates. The most discriminatory typing protocol was MLVA, which grouped four isolates into two pairs, while the remaining isolates produced unique fingerprints. Similar results were obtained when taking into account the combination of the two RAPD protocols (Simpson index 0.999); six isolates were grouped into three pairs, two of which were the pairs that were identified also by MLVA. Single use of each RAPD protocol resulted in inferior discrimination (Simpson index 0.978 and 0.997, respectively). In conclusion, the two molecular procedures, MLVA, and the combined RAPD protocols, produced similar results, showing that L. monocytogenes isolates from clinical and food specimens were highly heterogenous and that clustering was very uncommon.

Fast and Accurate Determination of Minute Ochratoxin A Levels in Cereal Flours and Wine with the Label-Free White Light Reflectance Spectroscopy Biosensing Platform.

Ochratoxin A (OTA) is one of the most toxic naturally encountered contaminants and is found in a variety of foods and beverages, including cereals and wine. Driven by the strict regulations regarding the maximum allowable OTA concentration in foodstuff and the necessity for on-site determination, the development of fast and sensitive methods for the OTA determination in cereal flours and wine samples, based on white light reflectance spectroscopy, is presented. The method relied on appropriately engineered silicon chips, on top of which an OTA-protein conjugate was immobilized. A polyclonal antibody against OTA was then employed to detect the analyte in the framework of a competitive immunoassay; followed by the subsequent addition of a biotinylated secondary antibody and streptavidin for signal enhancement. A small size instrument performed all assay steps automatically and the bioreactions were monitored in real time as the software converted the spectral shifts into effective biomolecular adlayer thickness increase. The assay developed had a detection limit of 0.03 ng/mL and a working range up to 200 ng/mL. The assay lasted 25 min (less than 1h, including calibrators/antibody pre-incubation) and was accomplished following a simple sample preparation protocol. The method was applied to corn and wheat flour samples and white and red wines with recovery values ranging from 87.2 to 111%. The simplicity of the overall assay protocol and convenient instrumentation demonstrates the potential of the immunosensor developed for OTA detection at the point of need.

AgtA, the dicarboxylic amino acid transporter of Aspergillus nidulans, is concertedly down-regulated by exquisite sensitivity to nitrogen metabolite repression and ammonium-elicited endocytosis.

We identified agtA, a gene that encodes the specific dicarboxylic amino acid transporter of Aspergillus nidulans. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.

Reynoutria sachalinensis extract elicits SA-dependent defense responses in courgette genotypes against powdery mildew caused by Podosphaera xanthii.

Powdery mildew (PM) caused by Podosphaera xanthii is one of the most important courgette diseases with high yield losses and is currently controlled by fungicides and sulphur applications in conventional and organic production. Plant derived elicitors/inducers of resistance are natural compounds that induce resistance to pathogen attack and promote a faster and/or more robust activation of plant defense responses. Giant knotweed (Reynoutria sachalinensis, RS) extract is a known elicitor of plant defenses but its mode of action remains elusive. The aim of this study was to investigate the mechanisms of foliar RS applications and how these affect PM severity and crop performance when used alone or in combination with genetic resistance. RS foliar treatments significantly reduced conidial germination and PM severity on both an intermediate resistance (IR) and a susceptible (S) genotype. RS application triggered plant defense responses, which induced the formation of callose papillae, hydrogen peroxide accumulation and the Salicylic acid (SA) - dependent pathway. Increased SA production was detected along with increased p-coumaric and caffeic acid concentrations. These findings clearly indicate that RS elicits plant defenses notably as a consequence of SA pathway induction.

Cultivar-Dependent Responses of Eggplant (Solanum melongena L.) to Simultaneous Verticillium dahliae Infection and Drought.

Several studies regarding the imposition of stresses simultaneously in plants have shown that plant responses are different under individual and combined stress. Pathogen infection in combination with drought can act both additively and antagonistically, suggesting a tailored-made plant response to these stresses. The aforementioned combination of stresses can be considered as one of the most important factors affecting global crop production. In the present research we studied eggplant responses to simultaneous Verticillium dahliae infection and drought with respect to the application of the individual stresses alone and investigated the extent to which these responses were cultivar dependent. Two eggplant cultivars (Skoutari and EMI) with intermediate resistance to V. dahliae were subjected to combined stress for a 3-week period. Significant differences in plant growth, several physiological and biochemical parameters (photosynthesis rate, leaf gas exchanges, Malondialdehyde, Proline) and gene expression, were found between plants subjected to combined and individual stresses. Furthermore, plant growth and molecular (lipid peroxidation, hydrogen peroxide, gene expression levels) changes highlight a clear discrimination between the two cultivars in response to simultaneous V. dahliae infection and drought. Our results showed that combined stress affects significantly plants responses compared to the application of individual stresses alone and that these responses are cultivar dependent.

Monitoring the Temporal Expression of Genes Involved in Ochratoxin A Production of Aspergillus carbonarius under the Influence of Temperature and Water Activity.

The objective of this study was to investigate the effect of environmental factors, namely temperature and water activity, on genes involved in the regulation of ochratoxin A (OTA) production over time. For this purpose, the previously characterized toxigenic Aspergilluscarbonarius Ac29 isolate from Greek vineyards and the A. carbonarius ITEM 5010 reference strain were subjected to combined temperature and water activity (aw) treatments to study OTA production and relative gene expression. The fungal isolates were grown on a synthetic grape juice liquid medium (SGM) under different temperature (20 °C, 25 °C and 30 °C) and aw (0.94 and 0.98) regimes. The expression of the AcOTApks, AcOTAnrps, and laeA OTA related genes was investigated using real time PCR. Gene expression was monitored at the same time points, along with fungal biomass and OTA accumulation at three, six and nine days of incubation. In gene expression analysis, stimulation of the biosynthetic genes was observed a few days before any toxin could be detected. This fact may underline a possible early indicator of potential toxin contamination of grapes. However, the transcript levels varied with respect to the different combinations of ecophysiological conditions and time, highlighting a complex regulation of OTA related gene expression of A. carbonarius in the specific medium.

Real-Time PCR Assay Targeting the veA Gene for Quantification of Aspergillus carbonarius in Grapes.

In this work, a SYBR Green I real-time PCR method has been developed for the detection and quantification of Aspergillus carbonarius in grapes by targeting the veA gene with a primer pair (veAF4/veAR4) that specifically amplifies a 91-bp PCR product. The quantification of the fungal DNA was performed by generation of standard curves for two A. carbonarius strains, using spectrophotometrically measured DNA quantities (Log) with a linearity range from 50 to 5 × 10(-4) ng of DNA. A high positive correlation (R(2) > 0.99) between exponential increases of DNA and real-time PCR threshold cycles showed a high amplification efficiency for the assay (E values 100.06 and 101.51%, respectively). Quantification of the fungal genomic DNA in grape samples artificially inoculated with A. carbonarius conidia was successfully performed with a minimum threshold of 10(4) conidia per g of grape berry. The assay developed would allow reliable, specific, and efficient detection and quantification of A. carbonarius in grapes.

Comparative study of growth responses and screening of inter-specific OTA production kinetics by A. carbonarius isolated from grapes.

The aim of this work was to assess OchratoxinA (OTA) production of different Aspergillus carbonarius isolates, evaluate their growth profile through different growth measurements, and reveal any underlying correlation between them. Ten different isolates of A. carbonarius isolated from Greek vineyards located in different geographical regions were examined in vitro for their OTA production potential after an incubation period of up to 11 days. All fungal isolates grew on a synthetic grape juice medium (SGM) similar to grape composition at optimum conditions of temperature and water activity (25°C and 0.98 aw). Samples for OTA determination were removed at 3, 5, 7, 9, and 11 days of growth and analyzed by HPLC. Based on OTA measurements the isolates were characterized by diverse OTA production ranging from 50 to 2000 ppb at day 11. The different fungal growth responses (colony diameter, colony area, biomass, biomass dry weight, and colony density) have been measured and correlated with toxin production by means of principal components analysis (PCA), confirming satisfactory correlation and explained over 99% of data variability. Leudeking-Piret model was also used to study OTA production with time, revealing a mixed-growth associated trend and pointing a fail-safe model with slightly better prediction through colony area. This approach contributes to the assessment of correlation between mycotoxin production and different methods of fungal growth determination in relation to time.

Biodiversity and ITS-RFLP characterisation of Aspergillus section Nigri isolates in grapes from four traditional grape-producing areas in Greece.

A study on the occurrence of Aspergillus section Nigri species on grapes from four traditional grape-producing areas in Greece during the 2011/2012 vintage, and their capability to produce OTA was conducted. One hundred and twenty-eight black aspergilli isolates were characterised at the species level initially by the use of morphological criteria in accordance with appropriate keys, followed by molecular characterisation performed with Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 5.8 ribosomal RNA gene Internal Transcribed Spacer region (5.8 rRNA ITS). Restriction enzyme digestion of the ITS amplicons using the HhaI, HinfI and RsaI, endonucleases distinguished eleven different patterns of restriction fragment length polymorphism (RFLP), four for each of the HhaI and RsaI digests and three for HinfI. From a total number of 128 individual isolates, 124 were classified into four Aspergillus species corresponding to A. carbonarius, A. tubingensis, A. japonicus and A. ibericus, and the remaining 4 were classified as members of the A. niger aggregate. A. carbonarius and A. tubingensis being the main representative species were equally counted, with higher geographical representation of the former in southern and the latter in northern regions, respectively. All isolates were tested for their ochratoxigenic potential by use of High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immuno Sorbent Assay (ELISA), resulting in significant interspecies differences in OTA production.

The effect of iron and fat in a diet containing green tea extract (Camellia sinensis) on the antioxidant capacity of some organs and the mRNA expression of specific genes in mice.

The hypothesis that iron and fat in the diet may affect green tea extract (GTE) bioactivity, in particular antioxidant capacity and gene expression, was proposed and tested in mice. Thirty mice were randomly assigned to have for 37 days free access to standard or high-fat diets with or without GTE and ferrous lactate. Mice were euthanized and specific organs were removed. Total antioxidant capacity (TAC) was measured using the ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity assays. Polymerase chain reaction was performed on liver and heart mRNA extracts. The FRAP assay showed that GTE from the standard diet did not affect plasma TAC but increased TAC of heart, aorta, and duodenum. GTE from diets enriched with iron resulted to lower TAC of liver and heart than diets with GTE alone. GTE from the fatty diet did not have any effect on TAC compared with fatty control diet, but increased TAC in heart and aorta compared with standard control diet. An effect on expression of the mapk-1 and NF-kB genes in heart was observed in the presence of GTE. These results suggest that GTE may exhibit bioactivity in some organs affected by dietary fat and iron. The findings of this study contribute to the elucidation of the role of dietary components on tea bioactivity.

Subtle proteome differences identified between post-dormant vegetative and floral peach buds.

Proper development of deciduous tree species, including peach, is accomplished through an annual growth cycle. Freezing avoidance during winter is necessary for tree survival and is achieved by the enclosure of meristems in floral and vegetative buds. To elucidate the role of developmentally regulated protein networks in bud break, proteins of the two bud-types were extracted and analyzed by two-dimensional gel electrophoresis (2-DE). Of the 1107 protein spots that were picked, 475 were identified and annotated assembling the peach bud proteome reference map. The majority of these proteins are involved in stress-response, detoxification, defense, carbohydrate metabolism and energy production. The protein profiles of both bud-types bear high similarity, whereas only 11 proteins were differentially expressed. These proteins were mainly involved in carbon-nitrogen homeostasis/metabolism and certain developmental processes to sustain rapid growth of the newly emerging organs. Among these are enzymes that differentially regulate the levels of H(2)O(2) between floral and vegetative buds, potentially promoting sequential bud-break. Distinct Nucleoside Diphosphate Kinase (NDPK) variants in floral and vegetative buds were detected suggesting the potential role of NDPKs in H(2)O(2)-mediated signaling for post-dormant bud break. This study provides data towards a better understanding of dormancy release and bud break.

Maize DBF1-interactor protein 1 containing an R3H domain is a potential regulator of DBF1 activity in stress responses.

The maize dehydration-responsive element (DRE)-binding factor, DBF1, is a member of the Apetala 2/Ethylene Response Factor transcription factors family and is involved in the regulation of the ABA-responsive gene rab17 through the DRE in an ABA-dependent pathway. In this study we analysed the functionality of DBF1 in abiotic stress responses and found that Arabidopsis plants over-expressing DBF1 were more tolerant to osmotic stress than control plants. In yeast two-hybrid analyses, DBF1 interacted with DBF1-interactor protein 1 (DIP1), a protein containing a conserved R3H single-strand DNA-binding domain. Subcellular localization of DIP1 showed that the protein fusion DIP1-Red Flourescent Protein (RFP) was mainly localized in the cytoplasm. However, after co-transformation of DBF1-GFP and DIP1-RFP, both proteins co-localized in the nucleus. Interestingly, when the N-terminal DBF1-GFP was co-expressed with DIP1-RFP, both proteins co-localized predominantly in the cytoplasmic speckles observed for N-terminal DBF1-GFP fusion protein. These results clearly show in vivo interaction of DBF1 with DIP1 in the cell and that this interaction is necessary for the nuclear localization of DIP1 protein. Analysis of the regulatory effect of the DBF1 and DIP1 interaction on the maize rab17 promoter activity indicated that co-transfection of DBF1 with DIP1 enhances promoter activity in the absence of ABA treatment. We suggest that the regulated association of DBF1 and DIP1 may control the levels of target gene expression during stress conditions.

Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABA-dependent pathway.

The abscisic acid-responsive gene rab17 of maize is expressed during late embryogenesis, and is induced by ABA and desiccation in embryo and vegetative tissues. ABRE and DRE cis-elements are involved in regulation of the gene by ABA and drought. Using yeast one-hybrid screening, we isolated two cDNAs encoding two new DRE-binding proteins, designated DBF1 and DBF2, that are members of the AP2/EREBP transcription factor family. Analysis of mRNA accumulation profiles showed that DBF1 is induced during maize embryogenesis and after desiccation, NaCl and ABA treatments in plant seedlings, whereas the DBF2 mRNA is not induced. DNA-binding preferences of DBFs were analysed by electrophoretic mobility shift assays, and showed that both DBF1 and DBF2 bound to the wild-type DRE2 element, but not to the DRE2 mutant or to the DRE1 element which differs only in a single nucleotide. Transactivation activity using particle bombardment showed that DBF1 functioned as activator of DRE2-dependent transcription of rab17 promoter by ABA, whereas DBF2 overexpression had a repression action downregulating not only the basal promoter activity, but also the ABA effect. These results show that ABA plays a role in the regulation of DBF activity, and suggests the existence of an ABA-dependent pathway for the regulation of genes through the C-repeat/DRE element.