Dataset Information

Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor.

ABSTRACT: Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. Neurospora crassa shares all six components of the Aspergillus nidulans pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the N. crassa pal mutant strains, with the exception of ?pal-9, which is the A. nidulans palI homolog, exhibit low conidiation and are unable to grow at alkaline pH. Moreover, they accumulate the pigment melanin, most likely via regulation of the tyrosinase gene by the pH signaling components. The PAC-3 transcription factor binds to the tyrosinase promoter and negatively regulates its gene expression. PAC-3 also binds to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH, which indicates a feedback regulation of PAC-3 in the pal gene expression. In addition, PAC-3 binds to the pac-3 promoter only at alkaline pH, most likely influencing the pac-3 expression at this pH suggesting that the activation of PAC-3 in N. crassa results from proteolytic processing and gene expression regulation by the pH signaling components. In N. crassa, PAC-3 is proteolytically processed in a single cleavage step predominately at alkaline pH; however, low levels of the processed protein can be observed at normal growth pH. We also demonstrate that PAC-3 preferentially localizes in the nucleus at alkaline pH stress and that the translocation may require the N. crassa importin-? since the PAC-3 nuclear localization signal (NLS) has a strong in vitro affinity with importin-?. The data presented here show that the pH signaling pathway in N. crassa shares all the components with the A. nidulans and S. cerevisiae pathways; however, it exhibits some properties not previously described in either organism.

SUBMITTER: Virgilio S

PROVIDER: S-EPMC4996508 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor.

Virgilio Stela S Cupertino Fernanda Barbosa FB Bernardes Natália Elisa NE Freitas Fernanda Zanolli FZ Takeda Agnes Alessandra Sekijima AA Fontes Marcos Roberto de Mattos MR Bertolini Maria Célia MC

PloS one 20160824 8

Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. Neurospora crassa shares all six components of the Aspergillus nidulans pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the N. crassa pal mutant strains, with the exception of Δpal-9, which is the A. nidulans palI homolog, exhibit low conidiation and are unable to grow at alkaline pH. ...[more]

PMID: 27557053

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Project description:Glycogen and trehalose are storage carbohydrates and their levels in microorganisms vary according to environmental conditions. In Neurospora crassa, alkaline pH stress highly influences glycogen levels, and in Saccharomyces cerevisiae, the response to pH stress also involves the calcineurin signaling pathway mediated by the Crz1 transcription factor. Recently, in yeast, pH stress response genes were identified as targets of Crz1 including genes involved in glycogen and trehalose metabolism. In this work, we present evidence that in N. crassa the glycogen and trehalose metabolism is modulated by alkaline pH and calcium stresses.We demonstrated that the pH signaling pathway in N. crassa controls the accumulation of the reserve carbohydrates glycogen and trehalose via the PAC-3 transcription factor, which is the central regulator of the signaling pathway. The protein binds to the promoters of most of the genes encoding enzymes of glycogen and trehalose metabolism and regulates their expression. We also demonstrated that the reserve carbohydrate levels and gene expression are both modulated under calcium stress and that the response to calcium stress may involve the concerted action of PAC-3. Calcium activates growth of the ?pac-3 strain and influences its glycogen and trehalose accumulation. In addition, calcium stress differently regulates glycogen and trehalose metabolism in the mutant strain compared to the wild-type strain. While glycogen levels are decreased in both strains, the trehalose levels are significantly increased in the wild-type strain and not affected by calcium in the mutant strain when compared to mycelium not exposed to calcium.We previously reported the role of PAC-3 as a transcription factor involved in glycogen metabolism regulation by controlling the expression of the gsn gene, which encodes an enzyme of glycogen synthesis. In this work, we extended the investigation by studying in greater detail the effects of pH on the metabolism of the reserve carbohydrate glycogen and trehalose. We also demonstrated that calcium stress affects the reserve carbohydrate levels and the response to calcium stress may require PAC-3. Considering that the reserve carbohydrate metabolism may be subjected to different signaling pathways control, our data contribute to the understanding of the N. crassa responses under pH and calcium stresses.

Project description:Purpose: We utilized high-throughput genomic approach to investigate transcriptional changes in two Neurospora crassa strains, the Δpac-3 knockout strain, and the Δmus-52 strain, under limited and sufficient Pi availability conditions, to evaluate the effects of the Δpac-3 deletion in gene modulation, from a plant-pathogen perspective. Methods: All strains were maintained on solid Vogel’s Minimal (VM) medium supplemented with 2 % sucrose at 30 °C and pH 5.8. Conidia were germinated in an orbital shaker for 5 h at 30°C (200 rpm) in low- and high-Pi media (final concentrations, 10 μM or 10 mM Pi, respectively). Total RNA was isolated from both strains and a total of 4 cDNA libraries were sequenced, with their respective biological triplicates corresponding to the paired libraries, using an Illumina HiSeq2000 sequencer, to generate 100 bp paired-end reads. To assess the library quality prior to and after trimming, we used the FastQC software. To remove the sequencing bases from the end of the reads, we applied a minimum Phred score of 20. Filtered reads were mapped onto the N. crassa genome using Bowtie2 software. The reads count values were obtained and used to calculate the expression variation of the transcripts from different conditions, considering the statistical significance of the differential gene expression. qRT–PCR validation was performed using SYBR Green assays. Results: Results from Bowtie2 analysis revealed that approximately 84% - 86% of the total high-quality reads were found to align with the reference genome of N. crassa. Furthermore, we identified 145 DEGs that were modulated regardless of the Pi concentration, however, were responsive to deletion of pac-3; while 171 DEGs were found to be modulated concomitantly with the deletion of pac-3 and with low-Pi availability. An additional 111 DEGs were found to be responsive to pac-3 deletion and high-Pi concentration. Extracellular Pi availability influenced the expression of genes involved in several biological functions. Conclusions: We have generated a robust dataset, which advances our knowledge on regulatory mechanisms of PAC-3 within a fungus-host system. We found that in a mutually beneficial, symbiotic fungus-plant relationship (as occurs during interactions in mycorrhizal symbiosis) or during a pathogenic interaction, PAC-3 functions to regulate ambient pH while also affecting myriad of physiological functions, including adaptation to nutritional conditions, regulation of virulence, or regulating the transcription of genes associated with structural and metabolic features. Herein, we highlight the role of PAC-3 in Pi adaptation, acting as a critical regulator of environmental challenges. The widespread regulatory activity of PAC-3 in fungal physiology confirms its role in the propagation of successful infections within hosts.

Dataset Information

Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor.

Publications

Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor.

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