Disruption, Replacement, and Cosuppression of Nitrate Assimilation Genes inStagonospora nodorum

Abstract

We usedStagonospora (Septoria) nodorumto explore gene disruption as a general method of fungicide target validation. Nitrate reductase was chosen as a model target because the gene (NIA1) has been cloned fromS. nodorumand disruptants should have a readily detectable phenotype (chlorate resistant and nitrate nonutilizing). We have succeeded in disrupting theNIA1gene by both integration of an unselected vector during cotransformation and one-step gene replacement. Around 2% of transformants from the cotransformation approach became nitrate nonutilizing and Southern analysis confirmed disruption of the residentNIA1gene. Half of the transformants with the gene replacement vector showed the nitrate nonutilizing phenotype expected from disruption. However, Southern analyses of 14 of these transformants showed that only 6 contained the expectedNIA1gene replacement. Of the remaining transformants, 6 had integrated multiple copies of the vector elsewhere in their genome and still had a functional nitrate reductase gene. Their inability to utilize nitrate was due to a lack of nitrite reductase activity. How this phenotype arose is not clear, but it might involve cosuppression of the nitrite reductase gene as the vector carried 1.1 kb of the coding region and the complete 5′ region of this gene which is adjacent toNIA1.Mutants of both types retained full pathogenicity in detached leaf assays, thereby invalidating both nitrate and nitrite reductase as fungicide targets.