Generation of disruption constructs and subsequent identification of mutants of Mycosphaerella graminicola. (A) (i) The cDNA fragments of MgGpa1, MgGpa2, MgGpa3, and MgGpb1 were excised from the pSport1 vector, using KpnI/XbaI restriction enzymes, and inserted into the same site of the binary vector of Agrobacterium tumefaciens (pCGN1589), generating the target constructs pCGN1589MgGpa1, pCGN1589MgGpa2, pCGN1589-MgGpa3, and pCGN1589MgGpb1, respectively. The target constructs were transposed using the customized donor construct pGPS3HygKan (ii), resulting in the disruption constructs pCGN-1598ΔMgGpa1 (iii), pCGN1598ΔMgGpa2 (iv), pCGN1598 ΔMgGpa3 (v), and pCGN1598ΔMgGpb1 (vi). (B) Multiplex PCR screening to identify homologous recombinants in MgGpa1 (i), MgGpa2 (ii), MgGpa3 (iii), and MgGpb1 (iv), using two gene-specific primers (indicated in panel A) together with either primer N or primer S, located at the right or left border of the transposon, respectively. Homologous recombinants (lanes 1 and 2) amplified a small amplicon identical in size to the amplicon generated from the disruption construct (lane 3), whereas in ectopic transformants (lane 4) two bands were amplified, including a small band corresponding to the disruptant strains and a larger band of equal size to the amplicon generated from the WT strain M. graminicola IPO323 (lane 5). Lane M shows One Kb Plus markers.