Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation |
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Authors: | Münch Steffen Ludwig Nancy Floss Daniela S Sugui Janyce A Koszucka Anna M Voll Lars M Sonnewald Uwe Deising Holger B |
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Affiliation: | Martin-Luther-University Halle-Wittenberg, Faculty of Agricultural and Nutritional Sciences, Phytopathology and Plant Protection, Betty-Heimann-Str. 3, 06120 Halle (Saale), Germany. |
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Abstract: | A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function. |
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