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Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway

ABSTRACT: Transcriptional profiling of Neurospora crassa ∆mak-2 strain grown under phosphate-shortage were compared the transcription profile of the control strain grown in both low- and high-Pi cultures. The filamentous fungus Neurospora crassa provides an excellent model system for the study of molecular responses to ambient signaling in eukaryotic microorganisms. Inorganic phosphate is an essential growth-limiting nutrient in nature and is crucial in genetic information. Numerous ambient signals activate the recruitment of mitogen-activated protein kinase (MAPK) cascades. Thus, in an attempt to identify genes involved in metabolic responses to exogenous phosphate sensing and in the functioning of a mitogen-activated protein kinase coding gene (mak-2) in N. crassa, we performed microarray experiments with the strain carrying the mak-2 gene knockout (Δmak-2) grown under phosphate-shortage by comparing the transcription profile to that of the control strain grown in both low- and high-phosphate cultures. Here we provide evidence that the mak-2 gene is an element of the adaptive response to extracellular Pi changes revealing novel aspects of phosphorus-sensing network in N. crassa.

ORGANISM(S): Neurospora crassa

PROVIDER: GSE41806 | GEO | 2013/07/31

SECONDARY ACCESSION(S): PRJNA178221

REPOSITORIES: GEO

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Project description:Purpose: We utilised high-throughput genomic approache to investigate transcriptional changes in two Neurospora strains (74A [wild-type] and mutant Δmus-52), under different phosphate availability conditions to evaluate the effects of mus-52 deletion in gene transcriptional modulation, and thus, infer its influence regarding metabolic response to changing extracellular levels of inorganic phosphate (Pi). Methods: All N. crassa strains were maintained on Vogel’s minimal medium, pH 5.8, at 30°C. Conidia from each strain were germinated for 5 h at 30°C in an orbital shaker (200 rpm), in low-Pi (10µM) and high-Pi (10mM) media. 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. FastQC software was used to visualize the library quality before and after trimming. For quality and sequence filtering, a Phred score lower than 20 was employed to remove sequencing bases from the read ends. 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: extracellular Pi availability influenced the expression of genes involved in several biological functions. The main affected gene groups were those associated with integral components of the membrane, such as transport, regulation, and cell signalling pathways, as well as genes involved with the nucleolus and protein synthesis. The absence of mus-52 affected global gene transcription in all conditions tested, highlighting the expression of some specific gene groups, such as transcription factors, kinase proteins, circadian clock-related genes, oxi-reduction balance, phosphate pathways, and general metabolism. Conclusions: The consequences of N. crassa mus-52 disruption to the cell may be deeper than the obvious phenotypic aspect. It may affect canonical and non-canonical pathways, leading to alternative adaptive processes, altering the perception and response to the environmental changes.

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.

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Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway

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