Disruption, replacement, and cosuppression of nitrate assimilation genes in Stagonospora nodorum

We used Stagonospora (Septoria) nodorum to 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 from S. nodorum and disruptants should have a readily detectable phenotype (chlorate resistant...

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Bibliographic Details
Published in:Fungal genetics and biology 1999-03, Vol.26 (2), p.152-162
Main Authors: Howard, K, Foster, S.G, Cooley, R.N, Caten, C.E
Format: Article
Language:English
Subjects:
Blotting, Southern
enzyme activity
excretion
Gene Deletion
Gene Targeting
Genes, Fungal
genetic transformation
homologous recombination
leaves
Leptosphaeria nodorum
Mitosporic Fungi - enzymology
Mitosporic Fungi - genetics
Mitosporic Fungi - growth & development
Mitosporic Fungi - pathogenicity
mutagenesis
nia1 gene
Nitrate Reductase
Nitrate Reductases - genetics
Nitrate Reductases - metabolism
nitrates
Nitrates - metabolism
nitrites
Nitrites - metabolism
pathogenicity
phenotype
plasmid vectors
Recombination, Genetic
reduction
Restriction Mapping
Stagonospora nodorum
structural genes
Transformation, Genetic
Triticum - microbiology
Triticum aestivum
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Summary:We used Stagonospora (Septoria) nodorum to 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 from S. nodorum and disruptants should have a readily detectable phenotype (chlorate resistant and nitrate nonutilizing). We have succeeded in disrupting the NIA1 gene 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 resident NIA1 gene. 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 expected NIA1 gene 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 to NIA1. Mutants of both types retained full pathogenicity in detached leaf assays, thereby invalidating both nitrate and nitrite reductase as fungicide targets.
ISSN:1087-1845
1096-0937
DOI:10.1006/fgbi.1998.1113