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      Verticillium longisporum Infection Affects the Leaf Apoplastic Proteome, Metabolome, and Cell Wall Properties in Arabidopsis thaliana

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          Abstract

          Verticillium longisporum (VL) is one of the most devastating diseases in important oil crops from the family of Brassicaceae. The fungus resides for much time of its life cycle in the extracellular fluid of the vascular system, where it cannot be controlled by conventional fungicides. To obtain insights into the biology of VL-plant interaction in the apoplast, the secretome consisting of the extracellular proteome and metabolome as well as cell wall properties were studied in the model Brassicaceae, Arabidopsis thaliana. VL infection resulted in increased production of cell wall material with an altered composition of carbohydrate polymers and increased lignification. The abundance of several hundred soluble metabolites changed in the apoplast of VL-infected plants including signalling and defence compounds such as glycosides of salicylic acid, lignans and dihydroxybenzoic acid as well as oxylipins. The extracellular proteome of healthy leaves was enriched in antifungal proteins. VL caused specific increases in six apoplast proteins (three peroxidases PRX52, PRX34, P37, serine carboxypeptidase SCPL20, α-galactosidase AGAL2 and a germin-like protein GLP3), which have functions in defence and cell wall modification. The abundance of a lectin-like, chitin-inducible protein (CILLP) was reduced. Since the transcript levels of most of the induced proteins were not elevated until late infection time points (>20 dpi), whereas those of CILLP and GLP3 were reduced at earlier time points, our results may suggest that VL enhances its virulence by rapid down-regulation and delay of induction of plant defence genes.

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          Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics.

          Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes.
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            Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics.

            Forward genetic screens have led to the isolation of several genes involved in secondary cell wall formation. A variety of evidence, however, suggests that the list of genes identified is not exhaustive. To address this problem, microarray data have been generated from tissue undergoing secondary cell wall formation and used to identify genes that exhibit a similar expression pattern to the secondary cell wall-specific cellulose synthase genes IRREGULAR XYLEM1 (IRX1) and IRX3. Cross-referencing this analysis with publicly available microarray data resulted in the selection of 16 genes for reverse genetic analysis. Lines containing an insertion in seven of these genes exhibited a clear irx phenotype characteristic of a secondary cell wall defect. Only one line, containing an insertion in a member of the COBRA gene family, exhibited a large decrease in cellulose content. Five of the genes identified as being essential for secondary cell wall biosynthesis have not been previously characterized. These genes are likely to define entirely novel processes in secondary cell wall formation and illustrate the success of combining expression data with reverse genetics to address gene function.
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              The current state of two-dimensional electrophoresis with immobilized pH gradients.

              The original protocol of two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt; Gorg et al., Electrophoresis 1988, 9, 531-546) is updated. Merits and limits of different methods for sample solubilization, sample application (by cup-loading or ingel rehydration) with respect to the pH interval used for IPG-isoelectric focusing are critically discussed. Guidelines for running conditions of analytical and micropreparative IPG-Dalt, using wide IPGs up to pH 12 for overview patterns, or narrow IPGs for zoom-in gels for optimum resolution and detection of minor components, are stated. Results with extended separation distances as well as automated procedures are demonstrated, and a comparison between protein detection by silver staining and fluorescent dyes is given. A brief trouble shooting guide is also included.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                20 February 2012
                : 7
                : 2
                : e31435
                Affiliations
                [1 ]Department of Forest Botany and Tree Physiology, Büsgen-Institute, Georg August University, Göttingen, Germany
                [2 ]Department of Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, Georg August University, Göttingen, Germany
                [3 ]Department of Plant Biochemistry, Albrecht von Haller Institute, Georg August University, Göttingen, Germany
                [4 ]Department of Molecular Biology and Physiology of Plants, Albrecht von Haller Institute, Georg August University, Göttingen, Germany
                Friedrich-Alexander-University Erlangen-Nurenberg, Germany
                Author notes

                Conceived and designed the experiments: AP. Performed the experiments: SF AM MP KF CG HT. Analyzed the data: AP SF AM MP KF IF UK CG. Contributed reagents/materials/analysis tools: AP SF AM MP KF IF UK CG HT. Wrote the paper: AP SF AM MP KF IF UK CG.

                Article
                PONE-D-11-18522
                10.1371/journal.pone.0031435
                3282744
                22363647
                f0331eb7-0145-41d8-bcbb-5d9f0a89e08d
                Floerl et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 19 September 2011
                : 7 January 2012
                Page count
                Pages: 14
                Categories
                Research Article
                Agriculture
                Crops
                Biology
                Model Organisms
                Plant and Algal Models
                Plant Science
                Plant Biochemistry
                Plant Cell Biology
                Plant Pathology
                Plants
                Proteomics

                Uncategorized
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