| 1. |
- Bello, M. A., et al.
(författare)
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Scanning electron microscopy (SEM) protocols for problematic plant, oomycete, and fungal samples
- 2017
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Ingår i: Journal of Visualized Experiments. - : Journal of Visualized Experiments. - 1940-087X. ; 2017:120
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Tidskriftsartikel (refereegranskat)abstract
- Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricalesas examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.
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| 2. |
- Rezinciuc, Svetlana, et al.
(författare)
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Specialized attachment structure of the fish pathogenic oomycete Saprolegnia parasitica
- 2018
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The secondary cysts of the fish pathogen oomycete Saprolegnia parasitica possess bundles of long hooked hairs that are characteristic to this economically important pathogenic species. Few studies have been carried out on elucidating their specific role in the S. parasitica life cycle and the role they may have in the infection process. We show here their function by employing several strategies that focus on descriptive, developmental and predictive approaches. The strength of attachment of the secondary cysts of this pathogen was compared to other closely related species where bundles of long hooked hairs are absent. We found that the attachment of the S. parasitica cysts was around three times stronger than that of other species. The time sequence and influence of selected factors on morphology and the number of the bundles of long hooked hairs conducted by scanning electron microscopy study revealed that these are dynamic structures. They are deployed early after encystment, i.e., within 30 sec of zoospore encystment, and the length, but not the number, of the bundles steadily increased over the encystment period. We also observed that the number and length of the bundles was influenced by the type of substrate and encystment treatment applied, suggesting that these structures can adapt to different substrates (glass or fish scales) and can be modulated by different signals (i.e., protein media, 50 mM CaCl2 concentrations, carbon particles). Immunolocalization studies evidenced the presence of an adhesive extracellular matrix. The bioinformatic analyses of the S. parasitica secreted proteins showed that there is a high expression of genes encoding domains of putative proteins related to the attachment process and cell adhesion (fibronectin and thrombospondin) coinciding with the deployment stage of the bundles of long hooked hairs formation. This suggests that the bundles are structures that might contribute to the adhesion of the cysts to the host because they are composed of these adhesive proteins and/or by increasing the surface of attachment of this extracellular matrix.
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| 3. |
- Srivastava, Vaibhav, et al.
(författare)
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Quantitative proteomic analysis of four developmental stages of Saprolegnia parasitica
- 2018
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 8:Jan
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Tidskriftsartikel (refereegranskat)abstract
- Several water mold species from the Saprolegnia genus infect fish, amphibians, and crustaceans in natural ecosystems and aquaculture farms. Saprolegnia parasitica is one of the most severe fish pathogens. It is responsible for millions of dollars of losses to the aquaculture industry worldwide. Here, we have performed a proteomic analysis, using gel-based and solution (iTRAQ) approaches, of four defined developmental stages of S. parasitica grown in vitro, i.e., the mycelium, primary cysts, secondary cysts and germinated cysts, to gain greater insight into the types of proteins linked to the different stages. A relatively high number of kinases as well as virulence proteins, including the ricin B lectin, disintegrins, and proteases were identified in the S. parasitica proteome. Many proteins associated with various biological processes were significantly enriched in different life cycle stages of S. parasitica. Compared to the mycelium, most of the proteins in the different cyst stages showed similar enrichment patterns and were mainly related to energy metabolism, signal transduction, protein synthesis, and post-translational modifications. The proteins most enriched in the mycelium compared to the cyst stages were associated with amino acid metabolism, carbohydrate metabolism, and mitochondrial energy production. The data presented expand our knowledge of metabolic pathways specifically linked to each developmental stage of this pathogen.
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