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...

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) act as signaling determinants that induce different cellular processes. To characterize NOX function during fungal development, we utilized the genetically tractable ascomycete Sordaria macrospora. Genome sequencing of a sterile mutant led us to identify the NADPH oxidase encoding nox1 as a gene required for fruiting body formation, regular hyphal growth, and hyphal fusion. These phenotypes are shared by Δnor1, lacking the NOX regulator NOR1. Further phenotypic analyses revealed a high correlation between increased ROS production and hyphal fusion deficiencies in Δnox1 and other sterile mutants. A genome-wide transcriptional profiling analysis of mycelia and isolated protoperithecia from wild type and Δnox1 revealed that nox1 inactivation affects the expression of genes related to cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration. Genetic analysis of Δnox2, lacking the NADPH oxidase 2 gene, Δnor1, and transcription factor deletion mutant Δste12, revealed a strict melanin-dependent ascospore germination defect, indicating a common genetic pathway for these three genes. We report that gsa3, encoding a G-protein a-subunit, and sac1, encoding cAMP-generating adenylate cyclase, act in a separate pathway during the germination process. The finding that cAMP inhibits ascospore germination in a melanin-dependent manner supports a model in which cAMP inhibits NOX2 activity, thus suggesting a link between both pathways. Our results expand the current knowledge on the role of NOX enzymes in fungal development and provide a frame to define upstream and downstream components of the NOX signaling pathways in fungi. D URING sexual reproduction, filamentous fungi generate complex fruiting bodies that contain and protect meiosporangia. We used the ascomycetous model fungus Sordaria macrospora to identify genes directly involved in fruiting body development (Kück et al. 2009;Engh et al. 2010;Kück et al. 2009). Due to its homothallic life style, S. macrospora is able to complete the sexual life cycle without the mating of strains with opposite sex, and therefore, fruiting body-deficient mutants can be recognized directly without the need for crossing experiments. In earlier work, we generated sterile mutants showing a developmental block after formation of young fruiting bodies (protoperithecia), but being unable to generate mature perithecia, and referred to these mutants as pro. Recently, we have applied next-generation genome re-sequencing to identify the genes affected in some of these mutants . Based on this approach, we now have characterized mutant pro32 and show that it carries a mutation in the nox1 gene encoding NAPDH oxidase 1 (NOX1).NADPH oxidase (NOX) enzymes are transmembrane proteins that are highly conserved among eukaryotes and produce reactive oxygen species (ROS) through the oxidation of NADPH (Lambeth 2004;Kawahara and Lambeth 2007). ROS have long been recognized as damaging agents due to uncontrolled oxidizing re...

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The NADPH oxidases (NOX) catalyze the production of superoxide by transferring electrons from NADPH to O 2 , in a regulated manner. In Neurospora crassa NOX-1 is required for normal growth of hyphae, development of aerial mycelium and asexual spores, and it is essential for sexual differentiation and cell-cell fusion. Determining the subcellular localization of NOX-1 is a critical step in understanding the mechanisms by which this enzyme can regulate all these different processes. Using fully functional versions of NOX-1 tagged with mCherry, we show that in growing hyphae NOX-1 shows only a minor association with the endoplasmic reticulum (ER) markers Ca 2+ -ATPase NCA-1 and an ER lumen-targeted GFP. Likewise, NOX-1 shows minor co-localization with early endosomes labeled with YPT-52, a GTPase of the Rab5 family. In contrast, NOX-1 shows extensive co-localization with two independent markers of the entire vacuolar system; the vacuolar ATPase subunit VMA-1 and the fluorescent molecule carboxy-DFFDA. In addition, part of NOX-1 was detected at the plasma membrane. The NOX-1 regulatory subunit NOR-1 displays a very different pattern of localization, showing a fine granular distribution along the entire hypha and some accumulation at the hyphal tip. In older hyphal regions, germinating conidia, and conidiophores it forms larger and discrete puncta some of which appear localized at the plasma membrane and septa. Notably, co-localization of NOX-1 and NOR-1 was mainly observed under conidial cell-cell fusion conditions in discrete vesicular structures. NOX functions in fungi have been evaluated mainly in mutants that completely lacked this protein, also eliminating interactions between hyphal growth regulatory proteins NOR-1, the GTPase RAC-1 and the scaffold protein BEM-1. To dissect NOX-1 roles as scaffold and as ROS-producing enzyme, we analyzed the function of NOX-1::mCherry proteins carrying proline 382 by histidine (P382H) or cysteine 524 by arginine (C524R) substitutions, predicted to only affect NADPH-binding. Without notably affecting NOX-1 localization or protein levels, each of these substitutions resulted in lack of function phenotypes, indicating that NOX-1 multiple functions are all dependent on its oxidase activity. Our results open new interpretations to possible NOX functions, as components of the fungal vacuolar system and the plasma membrane, as well as to new vacuolar functions.

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Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing.

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places where good vaccination coverage has been achieved. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing implementation in resource-limited settings. It is therefore important to develop testing protocols that facilitate implementation and are inexpensive, fast, and sufficiently sensitive. In this work, we optimized the composition of a buffer (PKTP) containing a protease, a detergent, and an RNase inhibitor, that is compatible with the RT-qPCR chemistry, allowing for direct testing of SARS-CoV-2 from saliva in an RNA extraction-independent manner. This buffer is compatible with heat-inactivation reducing the biohazard risk of handling the samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (82.2% for the N1 and 84.4% for the N2 target, respectively) and correlations (R=0.77, p<0.001 for N1, and R=0.78, p<0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to reduce the number of PCR reactions per patient and further reduce overall costs while maintaining diagnostic standards in favor of massive testing.