Cotransformation andTargeted GeneInactivation intheMaize Anthracnose Fungus, Glomerella graminicolat

(Bmlr) transformants. Southern blots confirmed thatbothtransforming DNAshadintegrated intothegenomes of transformants whichwere expressing bothPyr+andBmlrphenotypes. A plasmid, p23,whichcontained a truncated 500-bp segmentrepresenting thecentral region ofthePYRIgene was constructed. Theplasmid was introduced withpCG7,containing TUBIR1,into G.graminicola M1.001(Pyr+Bmls), andBmlrtransformants were selected. TheBmlrtransformants were screened on mediumwhichdidnotcontain uridine inorder to identify Pyr-mutantscreated byintegration ofp23atthePYRIlocus. Noneoftheprimary transformants were Pyr-,but0.2%ofuninucleate conidia collected fromthepooled primary transformants gaverise toPyrauxotrophs. Southern blots representing twoofthese Pyr-mutantsconfirmed thattheyhadtheexpected homologous integration ofp23atthePYRIlocus. Thissuggested that integration resulted inproduction oftwo nonfunctional copies ofthegene,one lacking the5'sequencesandtheotherlacking the3'sequences.This studydemonstrates thefeasibility ofusingcotransformation toperform targeted gene disruptions inG. graminicola.