Increased levels of cell wall degrading enzymes and peptidases are associated with aggressiveness in a virulent isolate of Pyrenophora teres f. maculata

Pyrenophora teres f. maculata (Ptm) is a fungal pathogen that causes the spot form of net blotch on barley and leads to economic losses in many of the world's barley-growing regions. Isolates of Ptm exhibit varying levels of aggressiveness that result in quantifiable changes in the severity of the disease. Previous research on plant -pathogen interactions has shown that such divergence is reflected in the proteome and secretome of the path-ogen, with certain classes of proteins more prominent in aggressive isolates. Here we have made a detailed comparative analysis of the secretomes of two Ptm isolates, GPS79 and E35 (highly and mildly aggressive, respectively) using a proteomics-based approach. The secretomes were obtained in vitro using media amended with barley leaf sections. Secreted proteins therein were harvested, digested with trypsin, and fractionated offline by HPLC prior to LC-MS in a high-resolution instrument to obtain deep coverage of the proteome. The subsequent analysis used a label-free quantitative proteomics approach with relative quantification of proteins based on precursor ion intensities. A total of 1175 proteins were identified, 931 from Ptm and 244 from barley. Further analysis revealed 160 differentially abundant proteins with at least a two-fold abundance difference between the isolates, with the most enriched in the aggressive GPS79 secretome. These proteins were mainly cell-wall (carbohydrate) degrading enzymes and peptidases, with some oxidoreductases and other pathogenesis-related proteins also identified, suggesting that aggressiveness is associated with an improved ability of GPS79 to overcome cell wall barriers and neutralize host defense responses

Temperature-induced changes in the wheat phosphoproteome reveal temperature-regulated interconversion of phosphoforms

Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower, and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental, and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term and mild increase in temperature on the wheat leaf and spikelet phosphoproteome. In total, 3822 (containing 5178 phosphosites) and 5581 phosphopeptides (containing 7023 phosphosites) were identified in leaf and spikelet samples, respectively. Following statistical analysis, the resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature. This community resource on the high temperature-mediated wheat phosphoproteome will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which probably impacts protein activity

The complete nucleotide sequence of prune dwarf ilarvirus RNA1 and virus detection by reverse transcription PCR and triple-antibody sandwich ELISA

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) with a monoclonal antibody was developed and evaluated for the detection of prune dwarf ilarvirus (PDV) in sweet cherry trees {Prunus avium). A reverse transcribed polymerase chain reaction test was also developed to establish the incidence of PDV in 40 sweet cherry trees and to confirm the absence of virus in 15 control trees. Trees with two-thirds of their leaves positive for PDV by TAS-ELISA would be identified with 99% probability by testing four leaves per tree. The monoclonal antibody did not cross- react with Prunus necrotic ringspot ilarvirus in the TASELISA. The nucleotide sequence of PDV RNA1 was determined. The RNA consists of 3374 nucleotides and encodes a single open reading frame of 3168 nucleotides. The putative translation product is 1055 amino acids in length with a calculated molecular mass of 118.9 kDa. Both the nucleic acid and the translated amino acid sequences show stronger homology to RNA1 and the corresponding translation product (ORF1) of alfalfa mosaic alfamovirus (AMV) than' to citrus leaf rugose ilarvirus, the only other ilarvirus for which RNA1 sequence data is available. There is extensive sequence homology in the 3'-untranslated regions of PDV RNA1 and the 3'-regions of other ilarvirus and AMV RNAs. The reported sequence and its single open reading frame conform to the genomic organization typical of the Bromoviridae genus. Clones representing sequence from the 5'and 3'-end of RNA1 were used to construct a deletion-type defective interfering particle and its ability to replicate in vivo was assessed.Land and Food Systems, Faculty ofGraduat

Biodiversitätsschäden im Rahmen der Umwelthaftungsrichtlinie 2004/35/EG

Die Diplomarbeit über Biodiversitätsschäden im Rahmen der Umwelthaftungsrichtlinie 2004/35/EG behandelt das Themengebiet der Biodiversität - als das Wichtigste der drei Schutzgüter aus der Umwelthaftungsrichtlinie - sowie die Vermeidung und Sanierung von Umweltschäden und zeigt nebenbei Methoden und Probleme auf, die bei der Bewertung und Sanierung von ökologischen Schäden an Biodiversitätsgütern entstehen und relevant sein können.eingereicht von Melanie Elisabeth RampitschUniversität Linz, Diplomarbeit, 2017(VLID)224642

Proteome of monoclonal antibody-purified haustoria from Puccinia triticina Race-1

WOS: 000352512100016PubMed ID: 25546510Puccinia triticina causes leaf rust, a disease that causes annual yield losses in wheat. It is an obligate parasite that invades the host leaf and forms intracellular structures called haustoria, which obtain nutrients and suppress host immunity using secreted proteins called effectors. Since effector proteins act at the frontier between plant and pathogen and help determine the outcome of the interaction, it is critical to understand their functions. Here, we used a direct proteomics approach to identify effector candidates from P. triticina Race 1 haustoria isolated with a specific monoclonal antibody. Haustoria were >95% pure and free of host contaminants. Using high resolution MS we have identified 1192 haustoria proteins. These were quantified using normalized spectral counts and spanned a dynamic range of three orders of magnitude, with unknown proteins and metabolic enzymes as the most highly represented. The dataset contained 140 candidate effector proteins, based on the presence of a signal peptide and the absence of a known function for the protein. Some of these candidates were significantly enriched with cysteine, with up to 13 residues per protein and up to 6.8% cysteine in composition.Agriculture and Agrifood CanadaAgriculture & Agri Food Canada; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK)The authors thank J. Chong, B. McCallum, X. Wang, C. Hiebert, G. Bakkeren, and F. You. This project was funded by a grant from Agriculture and Agrifood Canada to C.R., and a grant from TUBITAK to A.G

Map kinase MGV1: a potential shared control point of butenolide and deoxynivalenol biosynthesis.

The mitogen-activated protein kinase (MAPK) MGV1 has been knocked out in Fusarium graminearum to produce the mutant Fg∆MGV1.  The mutant displays complementary phenotypes concerning deoxynivalenol (DON) and butenolide (BT) biosynthesis in vitro.   In the rich medium 15-ADON accumulates are at low levels as detected by HPLC, whereas the accumulation of BT is substantial in Fg∆MGV1.  This is supported by the high expression of butenolide cluster genes in the mutant compared to the wild-type strain.  Under nutrient-limiting conditions, where DON biosynthesis is normally favoured, the expression of genes of the trichothecene cluster does not differ between the two strains.  However, the accumulation of 15-ADON is vastly different in Fg∆MGV1.  There is a reduction of 15-ADON accumulation with a concomitant accumula- tion of a novel compound. Although gene clusters comprising the synthesis of DON and BT have been identi- fied, their regulation at the molecular level has not been fully elucidated.  Since the expression levels of two regulatory genes from the trichothecene gene cluster and three regulatory genes from the butenolide gene cluster remained unchanged between WT and Fg∆MGV1, we suggest that differential accumulation of both BT and DON biosynthesis is at least partially under post-transcriptional and/or post-translational control

Secretome Analysis of Clavibacter nebraskensis Strains Treated with Natural Xylem Sap In Vitro Predicts Involvement of Glycosyl Hydrolases and Proteases in Bacterial Aggressiveness

The Gram-positive bacterium Clavibacter nebraskensis (Cn) causes Goss’s wilt and leaf blight on corn in the North American Central Plains with yield losses as high as 30%. Cn strains vary in aggressiveness on corn, with highly aggressive strains causing much more serious symptoms and damage to crops. Since Cn inhabits the host xylem, we investigated differences in the secreted proteomes of Cn strains to determine whether these could account for phenotypic differences in aggressiveness. Highly and a weakly aggressive Cn strains (Cn14-15-1 and DOAB232, respectively) were cultured, in vitro, in the xylem sap of corn (CXS; host) and tomato (TXS; non-host). The secretome of the Cn strains were extracted and processed, and a comparative bottom-up proteomics approach with liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to determine their identities and concentration. Relative quantitation of peptides was based on precursor ion intensities to measure protein abundances. In total, 745 proteins were identified in xylem sap media. In CXS, a total of 658 and 396 proteins were identified in strains Cn14-5-1 and DOAB232, respectively. The unique and the differentially abundant proteins in the secretome of strain Cn14-5-1 were higher in either sap medium compared to DOAB232. These proteins were sorted using BLAST2GO and assigned to 12 cellular functional processes. Virulence factors, e.g., cellulase, β-glucosidase, β-galactosidase, chitinase, β-1,4-xylanase, and proteases were generally higher in abundance in the aggressive Cn isolate. This was corroborated by enzymatic activity assays of cellulase and protease in CXS. These proteins were either not detected or detected at significantly lower abundance levels in Cn strains grown in non-host xylem sap (tomato), suggesting potential factors involved in Cn–host (corn) interactions

Genome-wide analysis of Fusarium graminearum’s nucleosome landscape

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Proteomic analysis of Aspergillus niger type strain (MUM 03.01) after accelerated freeze-drying preservation

Industrial processes based on biotechnology require a reliable source of microbial cultures, raising a need for the microbial safe long-term storage. The expanding biotechnological applications of Aspergillus niger requires effective long-term preservation that maintains their viability and also their physiological and genetic features [I]. From the best of our knowledge, proteomic analysis for monitoring the effect of preservation techniques on fungai intra- and extracellular protein expression by filamentous fungi has not been studied. Proteomic analysis is a powerful methodology for evaluation of proteins in complex mixtures and for the study of alteration of protein expression in an organism under varying environmental conditions. Proteomics can also be used as an important monitoring tool on the fungai cellular structure and enzymatic expression before and after filamentous fungi long-term preservation. ln this study, a comparative proteomic analysis of intra- and extracellular protein secretion expressed by A. niger type strain (MUM 03.01) preserved on freeze-drying ampoules and then subjected to accelerated shelf-life aging through the temperature increasing was assessed. Proteomic analysis based on 2D gel electrophoresis of A. niger (MUM 03.01) was assessed in 5 different liquid media. According to the results obtained the intracellular fungai proteomic profile was not modified by the accelerated shelf-life aging. It was also observed that the extracellular fungai proteomic profile was negligible which makes this A. niger type strain a weak fungallineage for biotechnological applications