Similar and distinct roles of NADPH oxidase components in the tangerine pathotype of A lternaria alternata

Summary The fungal nicotinamide adenine dinucleotide phosphate ( NADPH ) oxidase ( N ox) complex, which has been implicated in the production of low‐level reactive oxygen species ( ROS ), contains mainly NoxA , NoxB (gp91 phox homologues) and NoxR (p67 phox homologue). Here, we report the developmental and pathological functions of NoxB and NoxR in the tangerine pathotype of A lternaria alternata . Loss‐of‐function genetics revealed that all three N ox components are required for the accumulation of cellular hydrogen peroxide ( H 2 O 2 ). A lternaria alternata strains lacking NoxA , NoxB or NoxR also displayed an increased sensitivity to H 2 O 2 and many ROS ‐generating oxidants. These phenotypes are highly similar to those previously seen for the Δyap1 mutant lacking a YAP 1 transcriptional regulator and for the Δhog1 mutant lacking a HOG 1 mitogen‐activated protein ( MAP ) kinase, implicating a possible link among them. A fungal strain carrying a NoxA NoxB or NoxA NoxR double mutation was more sensitive to the test compounds than the strain mutated at a single gene, implicating a synergistic function among N ox components. The Δnox B mutant strain failed to produce any conidia; both Δnox A and Δnox R mutant strains showed a severe reduction in sporulation. Mutant strains carrying defective NoxB had higher chitin content than the wild‐type and were insensitive to calcofluor white, C ongo red and the fungicides vinclozolin and fludioxonil. Virulence assays revealed that all three N ox components are required for the elaboration of the penetration process. The inability to penetrate the citrus host, observed for Δnox mutants, could be overcome by wounding and by reacquiring a dominant Nox gene. The A . alternata   NoxR did not influence the expression of NoxB , but negatively regulated NoxA . Importantly, the expression of both YAP 1 and HOG 1 genes, whose products are involved in resistance to ROS , was down‐regulated in fungi carrying defective NoxA , NoxB or NoxR . Our results highlight the requirement of N ox in ROS resistance and provide insights into its critical role in regulating both YAP 1 and HOG 1 in A . alternata .