ABSTRACT: 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.
