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      Use of a non-homologous end-joining-deficient strain (delta- ku70) of the biocontrol fungus Trichoderma virens to investigate the function of the laccase gene lcc1 in sclerotia degradation

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          Abstract

          The aim of this study was to apply a generated Δ tku70 strain with increased homologous recombination efficiency from the mycoparasitic fungus Trichoderma virens for studying the involvement of laccases in the degradation of sclerotia of plant pathogenic fungi. Inactivation of the non-homologous end-joining pathway has become a successful tool in filamentous fungi to overcome poor targeting efficiencies for genetic engineering. Here, we applied this principle to the biocontrol fungus T. virens, strain I10, by deleting its tku70 gene. This strain was subsequently used to delete the laccase gene lcc1, which we found to be expressed after interaction of T. virens with sclerotia of the plant pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Lcc1 was strongly upregulated at early colonization of B. cinerea sclerotia and steadily induced during colonization of S. sclerotiorum sclerotia. The Δ tku70Δ lcc1 mutant was altered in its ability to degrade the sclerotia of B. cinerea and S. sclerotiorum. Interestingly, while the decaying ability for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum sclerotia was even enhanced, suggesting the operation of different mechanisms in the mycoparasitism of these two types of sclerotia by the laccase LCC1.

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Molecular Cloning : A Laboratory Manual

            <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
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              Biocontrol mechanisms of Trichoderma strains.

              The genus Trichoderma comprises a great number of fungal strains that act as biological control agents, the antagonistic properties of which are based on the activation of multiple mechanisms. Trichoderma strains exert biocontrol against fungal phytopathogens either indirectly, by competing for nutrients and space, modifying the environmental conditions, or promoting plant growth and plant defensive mechanisms and antibiosis, or directly, by mechanisms such as mycoparasitism. These indirect and direct mechanisms may act coordinately and their importance in the biocontrol process depends on the Trichoderma strain, the antagonized fungus, the crop plant, and the environmental conditions, including nutrient availability, pH, temperature, and iron concentration. Activation of each mechanism implies the production of specific compounds and metabolites, such as plant growth factors, hydrolytic enzymes, siderophores, antibiotics, and carbon and nitrogen permeases. These metabolites can be either overproduced or combined with appropriate biocontrol strains in order to obtain new formulations for use in more efficient control of plant diseases and postharvest applications.
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                Author and article information

                Contributors
                +43-1-58801166554 , vseidl@mail.tuwien.ac.at
                Journal
                Curr Genet
                Current Genetics
                Springer-Verlag (Berlin/Heidelberg )
                0172-8083
                1432-0983
                26 September 2010
                26 September 2010
                February 2011
                : 57
                : 1
                : 13-23
                Affiliations
                [1 ]Department of Tree Science, Entomology and Plant Pathology G. Scaramuzzi, Plant Pathology Section, Faculty of Agriculture, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy
                [2 ]Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstrasse 1a, 1060 Vienna, Austria
                [3 ]Scuola Normale Superiore di Pisa, Piazza dei Cavalieri 7, 56126 Pisa, Italy
                Author notes

                Communicated by U. Kueck.

                Article
                322
                10.1007/s00294-010-0322-2
                3023040
                20872221
                ff8dd61b-44ce-4c59-b743-f6458eca10d8
                © The Author(s) 2010
                History
                : 13 August 2010
                : 7 September 2010
                : 10 September 2010
                Categories
                Research Article
                Custom metadata
                © Springer-Verlag 2011

                Genetics
                gene targeting,trichoderma virens,sclerotia,laccase,mycoparasitism,ku70
                Genetics
                gene targeting, trichoderma virens, sclerotia, laccase, mycoparasitism, ku70

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