Multifunctional regulation of NADPH oxidase in growth, microsclerotia formation and virulence in Metarhizium rileyi

Abstract

Microsclerotia (MS), the anti-stress structures produced by many filamentous fungi, have been proved to be a great substitute for conidia in production of insecticides in entomogenous fungi. NADPH oxidase (Nox) constitutes ROS-response protein family widespread in eukaryotes and plays distinct roles in environmental fitness among various filamentous fungi. However, it is not clear whether Nox is required in the pathogenicity and formation of MS in insect pathogenic fungi. In this study, we reported the presence of NADPH oxidase homologs, which contain three different subunits (NoxAp/NoxBp catalytic subunit and NoxRp regulatory subunit), and further showed biological function in the entomopathogenic fungus, Metarhizium rileyi. Three Nox genes all showed high expression levels in the entire process of MS formation. Targeted deletion of MrNoxA, MrNoxB and MrNoxR all led to a decrease in MS yield and impaired morphology. Moreover, our anti-adversity results showed that they are indispensable to regulate the growth, tolerate the osmotic pressure and oxidative stress in Metarhizium rileyi. Most importantly, △MrNoxR and △MrNoxA but not △MrNoxB showed a dramatic reduction in virulence via inoculation. The normality of appressoria was impervious since there are no striking differences in virulence compared with WT and all mutants by topical injections. In general, our results revealed that NADPH oxidase may be involved in growth regulation, MS formation and pathogenicity by regulating the ROS metabolism and hyphal polarity.