Cotransformation and targeted gene inactivation in the maize anthracnose fungus, Glomerella graminicola

Cotransformation of Glomerella graminicola was achieved with the G. graminicola genes TUB1R1 (encoding a β-tubulin which confers resistance to the fungicide benomyl) and PYR1 (encoding orotate phosphoribosyl transferase, which confers pyrimidine prototrophy). The cotransformation frequency was about 30% when selection was for pyrimidine prototrophy (Pyr⁺) and 87% when selection was for benomyl-resistant (Bml(r)) transformants. Southern blots confirmed that both transforming DNAs had integrated into the genomes of transformants which were expressing both Pyr⁺ and Bml(r) phenotypes. A plasmid, p23, which contained a truncated 500-bp segment representing the central region of the PYR1 gene was constructed. The plasmid was introduced with pCG7, containing TUB1R1, into G. graminicola M1.001 (Pyr⁺ Bml(s)), and Bml(r) transformants were selected. The Bml(r) transformants were screened on medium which did not contain uridine in order to identify Pyr^- mutants created by integration of p23 at the PYR1 locus. None of the primary transformants were Pyr^-, but 0.2% of uninucleate conidia collected from the pooled primary transformants gave rise to Pyr^- auxotrophs. Southern blots representing two of these Pyr^- mutants confirmed that they had the expected homologous integration of p23 at the PYR1 locus. This suggested that integration resulted in production of two nonfunctional copies of the gene, one lacking the 5' sequences and the other lacking the 3' sequences. This study demonstrates the feasibility of using cotransformation to perform targeted gene disruptions in G. graminicola.