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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.

SUBMITTER: Cano-Dominguez N

PROVIDER: S-EPMC2519770 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa.

Cano-Domínguez Nallely N Alvarez-Delfín Karen K Hansberg Wilhelm W Aguirre Jesús J

Eukaryotic cell 20080620 8

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 hy ...[more]

PMID: 18567788

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Project description:The NADPH oxidases (NOX) catalyze the production of superoxide by transferring electrons from NADPH to O2, 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 Ca2+-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.

Project description:Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential substrate for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs). Neurospora crassa, a model filamentous fungus, expresses and secretes enzymes required for plant cell wall deconstruction. To better understand genes specifically associated with degradation of hemicellulose, we applied secretome and transcriptome analysis to N. crassa grown on beechwood xylan. We identified 34 secreted proteins and 353 genes with elevated transcription on xylan. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of the wild type was assessed, revealing functions for known and unknown proteins associated with hemicellulose degradation. By evaluating phenotypes of strains containing deletions of predicted TF genes in N. crassa, we identified a TF (XLR-1; xylan degradation regulator 1) essential for hemicellulose degradation that is an ortholog to XlnR/XYR1 in Aspergillus and Trichoderma species, respectively, a major transcriptional regulator of genes encoding both cellulases and hemicellulases. Deletion of xlr-1 in N. crassa abolished growth on xylan and xylose, but growth on cellulose and cellulolytic activity were only slightly affected. To determine the regulatory mechanisms for hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose, xylanolytic, and cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in N. crassa and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. This systematic analysis illustrates the similarities and differences in regulation of hemicellulose degradation among filamentous fungi.

Dataset Information

NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa.

Publications

NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa.

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