Sint-Truiden, Belgium
Sint-Truiden, Belgium

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Holtappels M.,Hasselt University | Noben J.-.P.,Hasselt University | Valcke R.,Hasselt University | Vrancken K.,PC Fruit Research Station | And 2 more authors.
Acta Horticulturae | Year: 2015

Erwinia amylovora is considered to be a homogeneous species although different strains show differences in virulence. In this investigation, two wild type strains with differences in virulence, a high virulent strain PFB5 and a low virulent strain LMG2024, were compared using a proteomic approach. Hereby, proteins were identified which were important for this difference in virulence between both strains. Bacteria were grown in a minimal medium and at mid-exponential phase, samples were taken. The complete proteome was extracted and a 2D differential in-gel electrophoresis (DIGE) approach was used. Differently abundant spots were excised, trypsinized and identified. Eventually 31 spots were identified as being important in the difference between the two strains. Flagellin (FliC) and a chemotaxis regulatory protein (CheY) were more abundant in the low virulent strain. A protein important in amylovoran synthesis, UTP-glucose-1-phosphate uridylyltransferase (GalF), was upregulated in the high virulent strain. Afterwards, phenotypic experiments were performed to validate the proteomic results.


Vrancken K.,Hasselt University | Schoofs H.,PC Fruit Research Station | Deckers T.,PC Fruit Research Station | Valcke R.,Hasselt University
Trees - Structure and Function | Year: 2012

Hydrogen peroxide plays a critical role in the expression of disease resistance in several plant/pathogen interactions. It serves as a substrate for oxidative cross-linking of various plant cell wall components leading to the reinforcement of the cell structure, as a direct toxin against the pathogen and as a signal molecule for the induction of defence-related genes in the adjacent, still healthy tissues. In plant cells, enzymes and redox metabolites act in synergy to carry out the detoxification of hydrogen peroxide and other reactive oxygen species (ROS). Superoxide dismutase (SOD) catalyses the dismutation of superoxide to hydrogen peroxide, catalase (CAT) dismutates hydrogen peroxide to oxygen and water, and ascorbate peroxidase (APX) reduces hydrogen peroxide to water by utilising ascorbate as specific electron donor. These are considered some of the main enzymatic systems for protecting cells against oxidative damage. These enzymes are present in various isozyme forms in several cell compartments and their expression is genetically controlled and regulated both by developmental and environmental stimuli, according to the necessity to remove ROS produced in cells. The aim of this study was to determine the possible role of these antioxidants in the defence mechanism of Pyrus communis cv. Conférence leaf tissue after an infection with Erwinia amylovora. Shoots of 2-year-old pear trees cv. Conférence were infected with E. amylovora strain SGB 225/12, were mock infected or left untreated. To account for structural changes, not only a difference was made between control, infected and mock-infected leaves, but we also included a distinction between young and old leaves, because it is known that older leaves are less susceptible for fire blight infections. Leaf samples were taken at specific time points after infection and the expression pattern of not necrotic tissue close to the infection site was analysed for their diverse isoforms of SOD, APX and CAT by using real time qPCR. In this study, no striking differences in transcription patterns of these enzymes between control, mock infected and E. amylovora infected leaves were observed. However, a significant difference between the expression levels of some genes in young and old leaves was noticed. These differences could partially explain the different progression rate by which E. amylovora infects, invades and causes necrosis in young and old leaves. © 2011 Springer-Verlag.


Vrancken K.,Hasselt University | Holtappels M.,Hasselt University | Schoofs H.,PC Fruit Research Station | Deckers T.,PC Fruit Research Station | And 2 more authors.
Plant Physiology and Biochemistry | Year: 2013

Flavonoids, which are synthesized by the phenylpropanoid-flavonoid pathway, not only contribute to fruit colour and photoprotection, they also may provide antimicrobial and structural components during interaction with micro-organisms. A possible response of this pathway was assessed in both mature and immature leaves of shoots of 2-year-old pear trees cv. Conférence, which were inoculated with the gram-negative bacterium Erwinia amylovora strain SGB 225/12, were mock-inoculated or were left untreated. The phenylpropanoid-flavonoid pathway was analysed by histological studies, by gene expression using RT-qPCR and by HPLC analyses of the metabolites at different time intervals after infection.Transcription patterns of two key genes anthocyanidin reductase (ANR) and chalcone synthase (CHS) related to the phenylpropanoid-flavonoid pathway showed differences between control, mock-inoculated and E.amylovora-inoculated mature leaves, with the strongest reaction 48h after inoculation. The impact of E.amylovora was also visualised in histological sections, and confirmed by HPLC, as epicatechin -which is produced via ANR- augmented 72h after inoculation in infected leaf tissue. Besides the effect of treatments, ontogenesis-related differences were found as well.The increase of certain key genes, the rise in epicatechin and the visualisation in several histological sections in this study suggest a non-negligible impact on the phenylpropanoid-flavonoid pathway in Pyrus communis due to inoculation with E.amylovora. In this study, we propose a potential role of this pathway in defence mechanisms, providing a detailed analysis of the response of this system attributable to inoculation with E.amylovora. © 2013 Elsevier Masson SAS.

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