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

Abstract

The fungal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) 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 Alternaria alternata. Loss-of-function genetics revealed that all three Nox components are required for the accumulation of cellular hydrogen peroxide (H₂O₂). Alternaria alternata strains lacking NoxA, NoxB or NoxR also displayed an increased sensitivity to H₂O₂ and many ROS-generating oxidants. These phenotypes are highly similar to those previously seen for the Δyap1 mutant lacking a YAP1 transcriptional regulator and for the Δhog1 mutant lacking a HOG1 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 Nox components. The ΔnoxB mutant strain failed to produce any conidia; both ΔnoxA and ΔnoxR 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, Congo red and the fungicides vinclozolin and fludioxonil. Virulence assays revealed that all three Nox 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 YAP1 and HOG1 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 Nox in ROS resistance and provide insights into its critical role in regulating both YAP1 and HOG1 in A. alternata.