Genetic analyses of reddish-brown polyoxin-resistant mutants of Bipolaris maydis

In the southern corn blight fungus, Bipolaris maydis, five polyoxin-resistance genes (Pol1 to Pol5) have been reported. Pol2 and Pol5 are pleiotropic for not only polyoxin resistance but also reddish brown colonies. Here, we used a comparative genomics approach to identify Pol2 and Pol5 at a molecular genetics level. Our analysis revealed that nucleotide sequence variations in the genes for hydroxymethylbilane synthase (HMBS) and ferrochelatase (FECH) were linked to the phenotypes of Pol2 and Pol5, respectively. Further variations in the nucleotide sequences of these genes were also found in other strains of Pol2 and Pol5. Complementation tests with the wild type genes confirmed that these mutations at Hmbs and Fech were responsible for the polyoxin resistance in the Pol2 and Pol5 mutants. The deletion mutants of these genes (ΔHMBS and ΔFECH) were conditionally lethal without exogenous heme. The heme contents of Pol2 and Pol5 mutants were lower than that in the wild type, suggesting that the mutations in hmbs and fech reduced the functions of HMBS and FECH, although neither was completely inactivated. These results suggested Pol2 and Pol5 encode HMBS and FECH, members of enzymes in the heme-biosynthetic pathway of this fungus.