Proteomička analiza patogene plijesni Monilinia laxa

Brown rot fungus Monilinia laxa (Aderh. & Ruhl.) Honey is an important plant pathogen in stone and pome fruits in Europe. We applied a proteomic approach in a study of M. laxa isolates obtained from apples and apricots in order to show the host specifity of the isolates and to analyse differentially expressed proteins in terms of host specifity, fungal pathogenicity and identification of candidate proteins for diagnostic marker development. Extracted mycelium proteins were separated by 2-D electrophoresis (2-DE) and visualized by Coomassie staining in a non-linear pH range of 3–11 and Mr of 14–116 kDa. We set up a 2-DE reference map of M. laxa, resolving up to 800 protein spots, and used it for image analysis. The average technical coefficient of variance (13 %) demonstrated a high reproducibility of protein extraction and 2-D polyacrylamide gel electrophoresis (2-DE PAGE), and the average biological coefficient of variance (23 %) enabled differential proteomic analysis of the isolates. Multivariate statistical analysis (principal component analysis) discriminated isolates from two different hosts, providing new data that support the existence of a M. laxa specialized form f. sp. mali, which infects only apples. A total of 50 differentially expressed proteins were further analyzed by LC-MS/MS, yielding 41 positive identifications. The identified mycelial proteins were functionally classified into 6 groups: amino acid and protein metabolism, energy production, carbohydrate metabolism, stress response, fatty acid metabolism and other proteins. Some proteins expressed only in apple isolates have been described as virulence factors in other fungi. The acetolactate synthase was almost 11-fold more abundant in apple-specific isolates than in apricot isolates and it might be implicated in M. laxa host specificity. Ten proteins identified only in apple isolates are potential candidates for the development of M. laxa host-specific diagnostic markers.Smeđa trulež plodova, koju uzrokuje plijesan Monilinia laxa (Aderh. & Ruhl.) Honey, važan je patogen koštičavog i jabučastog voća što raste u Europi. U radu je provedena proteomička analiza te plijesni, izolirane iz plodova jabuka i marelica, da bi se dokazala njezina specifičnost za infekciju domaćina, te odredila ekspresija proteina odgovornih za njezinu specifičnost i patogenost. Također su određeni proteini koji se mogu upotrijebiti kao dijagnostički markeri. Proteini izolirani iz micelija plijesni, molekularne mase od 14 do 116 kDa, odijeljeni su 2-D elektroforezom (2-DE) i određeni bojanjem Coomassie bojom u nelinearnom pH području od 3 do 11. Za analizu dobivenoga grafičkog prikaza upotrijebljena je 2-DE referentna mapa plijesni M. laxa, te određeno 800 točaka što prikazuju proteine. Prosječni tehnički koeficijent varijacije od 13 % pokazuje da ekstrakcija proteina i 2-DE PAGE elektoroforeza imaju odličnu ponovljivost, a prosječni biološki koeficijent varijacije od 23 % omogućio je proteomičku analizu izolata. Multivarijantnom statističkom analizom glavnih komponenata (PCA) razdvojeni su izolati iz dvaju domaćina, što potvrđuje da postoji specijalizirani oblik M. laxa f. sp. mali koji uzrokuje smeđu trulež jabuke. Pomoću LC-MS/MS metode ispitano je 50 eksprimiranih proteina, od kojih je 41 pokazao podudarnost. Identificirani proteini micelija funkcionalno su razdvojeni u 6 skupina: proteini što sudjeluju u metabolizmu aminokiselina i proteina, proizvodnji energije, mehanizmu otpornosti na stres, metabolizmu masnih kiselina, te ostali proteini. Neki proteini izolirani iz plijesni s plodova jabuka djeluju kao virulentni faktori na ostale plijesni. Izolati plijesni specifični za jabuku sadržavali su približno 11 puta više enzima acetolaktat sintaze od onih izoliranih s marelica, što znači da je M. laxa specifična za domaćina. Deset proteina identificiranih samo u izolatima s jabuka mogu se upotrijebiti za razvoj dijagnostičkih markera za specifičnu M. laxa

Proteome Analysis of the Plant Pathogenic Fungus Monilinia laxa Showing Host Specificity

Brown rot fungus Monilinia laxa (Aderh. & Ruhl.) Honey is an important plant pathogen in stone and pome fruits in Europe. We applied a proteomic approach in a study of M. laxa isolates obtained from apples and apricots in order to show the host specifity of the isolates and to analyse differentially expressed proteins in terms of host specifity, fungal pathogenicity and identification of candidate proteins for diagnostic marker development. Extracted mycelium proteins were separated by 2-D electrophoresis (2-DE) and visualized by Coomassie staining in a non-linear pH range of 3–11 and Mr of 14–116 kDa. We set up a 2-DE reference map of M. laxa, resolving up to 800 protein spots, and used it for image analysis. The average technical coefficient of variance (13 %) demonstrated a high reproducibility of protein extraction and 2-D polyacrylamide gel electrophoresis (2-DE PAGE), and the average biological coefficient of variance (23 %) enabled differential proteomic analysis of the isolates. Multivariate statistical analysis (principal component analysis) discriminated isolates from two different hosts, providing new data that support the existence of a M. laxa specialized form f. sp. mali, which infects only apples. A total of 50 differentially expressed proteins were further analyzed by LC-MS/MS, yielding 41 positive identifications. The identified mycelial proteins were functionally classified into 6 groups: amino acid and protein metabolism, energy production, carbohydrate metabolism, stress response, fatty acid metabolism and other proteins. Some proteins expressed only in apple isolates have been described as virulence factors in other fungi. The acetolactate synthase was almost 11-fold more abundant in apple-specific isolates than in apricot isolates and it might be implicated in M. laxa host specificity. Ten proteins identified only in apple isolates are potential candidates for the development of M. laxa host-specific diagnostic markers

Comparative proteomic profiling in compatible and incompatible interactions between hop roots and Verticillium albo-atrum

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Green ham pH value affects proteomic profile of dry-cured ham

In the present study we investigated the effect of green ham pH value on the proteomic profile of m. biceps femoris of the 14 month old ‘Kraški pršut’ dry hams. Two groups (n=12) of samples were chosen according to green ham m. semimembranosus pH (i.e. low pH group with values from 5.51 to 5.60 and high pH group with values from 5.80 to 6.18). Two groups of hams were similar with regard to fat thickness and ham weight. The myofibrillar muscle protein fraction was extracted from dry-cured ham m. biceps femoris and separated using 2-dimensional electrophoresis technique. More than 1,000 protein spots were detected on the gels, out of which 100 spots had significantly different intensity according to pH group. Notable clustering of the spots was observed on the gel images. Namely, the protein spots differentiating low and high pH groups were more intense in the acidic part of the gel for the low pH group, and in the basic part for the high pH group. The proteomic approach proved to be a suitable tool to investigate the influence of green ham pH on the pattern of protein degradation. However, further research (protein spot identification, association with sensory properties) is in progress

Genome sequence of a lethal strain of xylem-invading Verticillium nonalfalfae

Verticillium nonalfalfae, a soilborne vascular phytopathogenic fungus, causes wilt disease in several crop species. Of great concern are outbreaks of highly aggressive V. nonalfalfae strains, which cause a devastating wilt disease in European hops. We report here the genome sequence and annotation of V. nonalfalfae strain T2, providing genomic information that will allow better understanding of the molecular mechanisms underlying the development of highly aggressive strains