NADPH Oxidases NOX-1 and NOX-2 Require the Regulatory Subunit NOR-1 To Control Cell Differentiation and Growth in Neurospora crassa

Abstract

We have proposed that reactive oxygen species (ROS) play essential roles in cell differentiation. Enzymes belonging to the NADPH oxidase (NOX) family produce superoxide in a regulated manner. We have identified three distinct NOX subfamilies in the fungal kingdom and have shown that NoxA is required for sexual cell differentiation in Aspergillus nidulans. Here we show that Neurospora crassa NOX-1 elimination results in complete female sterility, decreased asexual development, and reduction of hyphal growth. The lack of NOX-2 did not affect any of these processes but led instead to the production of sexual spores that failed to germinate, even in the presence of exogenous oxidants. The elimination of NOR-1, an ortholog of the mammalian Nox2 regulatory subunit gp67 phox , also caused female sterility, the production of unviable sexual spores, and a decrease in asexual development and hyphal growth. These results indicate that NOR-1 is required for NOX-1 and NOX-2 functions at different developmental stages and establish a link between NOX-generated ROS and the regulation of growth. Indeed, NOX-1 was required for the increased asexual sporulation previously observed in mutants without catalase CAT-3. We also analyzed the function of the penta-EF calcium-binding domain protein PEF-1 in N. crassa. Deletion of pef-1 resulted in increased conidiation but, in contrast to what occurs in Dictyostelium discoideum, the mutation of this peflin did not suppress the phenotypes caused by the lack of NOX-1. Our results support the role of ROS as critical cell differentiation signals and highlight a novel role for ROS in regulation of fungal growth.A significant amount of recent research has established that reactive oxygen species (ROS), long considered as harmful byproducts, can play cell signaling roles (1,7,14,18,23). For many years, we have used the model organism Neurospora crassa to investigate the role of ROS in the regulation of asexual development (conidiation). In this fungus, synchronous conidiation is started when a liquid culture is filtered and exposed to the air. The hyphal cells in contact with the air aggregate and adhere to each other within 40 min and grow aerial hyphae after 2 h, and then asexual spores (conidia) are formed at the tips of branched aerial hyphae (aerial mycelium) after 8 to 9 h of air exposure (37,43). The occurrence of a hyperoxidant state at the start of each of these morphogenetic transitions (hyphal adhesion, formation of aerial mycelium, and conidium formation) has been documented (1, 2, 19-21, 30, 44, 45). In addition, N. crassa develops multicellular fruiting bodies called perithecia, which contain the sexual spores or ascospores. Under nitrogen limitation conditions, a strain with either mating type (A or a) can differentiate a multicellular structure called a protoperithecium and function as a "female" or acceptor strain. A protoperithecium is fertilized through a specialized hypha called the "trichogyne" which fuses with a cell, usually a conidium, from the opposite mating type. Fert...

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

“…In full agreement, our analyses using antioxidants show an important decrease in conidiation in response to injury, which, together with the production of ROS by Nox1, indicate that mechanical injury generates oxidative stress, leading to conidiation in T. atroviride. In this regard, various reports in fungi demonstrated that NoxA is involved in sexual development (11,12), whereas NoxB does not affect this process, and mutants in noxR present the phenotypes observed in both noxA and noxB deletion mutants (12). Interestingly, our results indicate that conidiation in response to injury is mainly attributable to Nox1, and ROS production is an early, transient event.…”

“…Lara-Ortíz et al (11) first reported a direct link between Nox-derived ROS and sexual differentiation. Accordingly, in N. crassa, nox1 elimination results in complete female sterility, slightly decreased asexual development, and reduction of hyphal growth (12).…”

Mn(II) oxidation by an ascomycete fungus is linked to superoxide production during asexual reproduction

“…22, 23, and 31). In particular, deletion of nox genes in a variety of fungi blocks cell differentiation, including asexual and sexual development, which correlates to the inability of the nox mutants to generate superoxide (28,(32)(33)(34).…”

Trichoderma: sensing the environment for survival and dispersal

“…Deletion of nox1 in N. crassa results in complete female sterility and a marked reduction in hyphal growth and asexual development (CanoDomínguez et al, 2008). In both N. crassa and Botrytis cinerea, NoxR is required for both NoxA and NoxB function in cellular differentiation (Cano-Domínguez et al, 2008;Segmüller et al, 2008). In contrast, A. nidulans noxR deletion mutants are defective in both asexual and sexual development (Semighini & Harris, 2008).…”