Use of insertional mutagenesis to tag putative parasitic fitness genes in the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi

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Abstract:

We used insertional mutagenesis to produce genetically tagged mutants of the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi. We first optimized transformation of O. novo-ulmi protoplasts by the restriction enzyme mediated integration method. A concentration of 80 U of HindIII with 108 fungal protoplasts and 5 g of plasmid DNA was the most efficient for generating a high number of O. novo-ulmi mutants carrying a single insertion in their genome. Mycelium- and yeast-like growth kinetics of 24 O. novo-ulmi mutants were evaluated in vitro. Flanking sequences were successfully recovered in 8% of the transformants analyzed. Some mutant phenotypes appeared to result from gene disruption events, whereas others likely involved modifications of noncoding regions. Several nuclear loci that control vegetative growth and could potentially impact parasitic fitness were successfully tagged.

Nous avons utilisé la mutagénèse insertionnelle pour produire des mutants marqués génétiquement chez le champignon Ophiostoma novo-ulmi subsp. novo-ulmi, agent de la maladie hollandaise de l’orme. En premier lieu, nous avons optimisé la transformation des protoplastes d’O. novo-ulmi par la méthode d’intégration par l’intermédiaire d’une enzyme de restriction. La concentration la plus efficace afin d’induire le plus grand nombre de mutants d’O. novo-ulmi ayant une insertion unique est 80 U d’enzyme HindIII pour 108 protoplastes en présence de 5 g d’ADN plasmidique. Nous avons mesuré in vitro les cinétiques de croissance mycélienne et levuriforme chez 24 mutants d’O. novo-ulmi. On a pu isoler les ADN fongiques bordant le plasmide chez 8 % des transformants analysés. Le phénotype de quelques mutants semble être le résultat d’un événement d’interruption génique. Dans d’autre cas, il pourrait s’agir d’une modification dans une région non-codante de l’ADN fongique. Plusieurs gènes nucléaires contrôlant la croissance végétative et pouvant affecter la fitness parasitaire ont pu être étiquetés.

Document Type: Research Article

Publication date: September 1, 2008

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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