The pH Signaling Transcription Factor PAC-3 Regulates Metabolic and Developmental Processes in Pathogenic Fungi

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. Transcriptional profile of N. crassa Δpac-3 strains using RNA-sequencing technique highlight the role of PAC-3 in Pi adaptation, acting as a critical regulator of environmental challenges.

研究目的：本研究采用高通量基因组学方法，探究两株粗糙脉孢菌（Neurospora crassa）——Δpac-3敲除菌株与Δmus-52菌株——在低磷与高磷（Pi）条件下的转录组变化，从植物-病原体互作视角评估Δpac-3缺失对基因调控的影响。
研究方法：所有菌株均接种于添加2%蔗糖的固体沃格尔基础培养基（Vogel’s Minimal Medium，VM）上，于30℃、pH 5.8条件下培养。将分生孢子置于摇床（200 rpm、30℃）中于低磷与高磷培养基（终浓度分别为10 μM和10 mM Pi）中萌发5小时。提取两株菌株的总RNA，构建共计4个cDNA文库，每个文库设置3次生物学重复，采用Illumina HiSeq2000测序仪进行测序，生成100 bp双端读段。在对测序读段进行修剪前后，均使用FastQC软件评估文库质量；为去除读段末端的低质量测序碱基，设置最低Phred质量值为20进行过滤。利用Bowtie2软件将过滤后的读段比对至粗糙脉孢菌参考基因组。获取读段计数值，结合差异基因表达的统计学显著性，计算不同条件下转录本的表达差异。采用SYBR Green法进行qRT-PCR验证。
研究结果：Bowtie2分析结果显示，总计高质量读段中约84%~86%可比对至粗糙脉孢菌参考基因组。本研究共鉴定得到145个差异表达基因（Differentially Expressed Genes，DEGs），其表达调控不受Pi浓度影响，但对pac-3缺失产生响应；另有171个DEGs的表达同时受pac-3缺失与低Pi条件调控。此外还鉴定到111个DEGs，其表达受pac-3缺失与高Pi条件共同影响。细胞外Pi有效性可调控多种生物学功能相关基因的表达。
研究结论：本研究生成了高质量的数据集，加深了我们对真菌-宿主系统中PAC-3调控机制的认知。研究发现，在互利共生的菌根真菌-植物互作体系，或是病原互作过程中，PAC-3不仅可调控环境pH，还可影响众多生理功能，包括营养条件适应、毒力调控，以及结构与代谢相关基因的转录调控。本研究着重阐明了PAC-3在Pi适应中的核心作用，其作为环境胁迫的关键调控因子发挥功能。PAC-3在真菌生理过程中广泛的调控活性，证实了其在宿主内成功侵染增殖中的关键作用。本研究通过RNA测序技术解析了粗糙脉孢菌Δpac-3菌株的转录组特征，明确了PAC-3在Pi适应中的核心调控功能，揭示其作为环境胁迫关键调控因子的作用。