Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene.
A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5'-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial beta-glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of beta-glucuronidase activity.
Smit
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Allg. Botanik/Mikrobiologie, Institut für Botanik der Westfälischen Wilhelms-Universität, Münster, FRG.
Tudzynski
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D015139:Blotting, Southern; D002967:Claviceps / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D015698:Genomic Library; D009154:Mutation / Q000235:genetics; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011743:Pyrimidines / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic / Q000235:genetics*
D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D009964:Orotidine-5'-Phosphate Decarboxylase; C030986:pyrimidine