Defining the transcriptional responses of Aspergillus nidulans to cation/alkaline pH stress and the role of the transcription factor SltA

Abstract

Fungi have developed the ability to overcome extreme growth conditions and thrive in hostile environments. The model fungus Aspergillus nidulans tolerates, for example, ambient alkalinity up to pH 10 or molar concentrations of multiple cations. The ability to grow under alkaline pH or saline stress depends on the effective function of at least three regulatory pathways mediated by the zinc-finger transcription factor PacC, which mediates the ambient pH regulatory pathway, the calcineurin-dependent CrzA and the cation homeostasis responsive factor SltA. Using RNA sequencing, we determined the effect of external pH alkalinization or sodium stress on gene expression. The data show that each condition triggers transcriptional responses with a low degree of overlap. By sequencing the transcriptomes of the null mutant, the role of SltA in the above-mentioned homeostasis mechanisms was also studied. The results show that the transcriptional role of SltA is wider than initially expected and implies, for example, the positive control of the PacC-dependent ambient pH regulatory pathway. Overall, our data strongly suggest that the stress response pathways in fungi include some common but mostly exclusive constituents, and that there is a hierarchical relationship among the main regulators of stress response, with SltA controlling pacC expression, at least in A. nidulans.

Tolerance to alkaline ambient pH in Aspergillus nidulans depends on the activity of ENA proteins

“…A minor positive regulatory role for the salt activated transcription factor SltA on expression of ena genes has been shown in this and previous works 16 . This is in agreement with our RNA-seq results 10 , in which both enaA and enaB (but not enaC) were among the most intensely upregulated genes at pH 8 in a wild-type background (Top45 and Top2 genes with the highest log2FC values, respectively), but none of them was significantly downregulated in the sltAΔ mutant under these stress conditions.…”

“…the regulation of expression of ena genes in total RNAs isolated from mycelia grown at alkaline pH, in excess of Na + or Li + , and the mixture of high levels of either Na + or Li + with alkalinity. In agreement with previous results from Northern analyses 16,18 and RNA sequencing 10 , we confirmed that the expression of both enaA and enaB is regulated by these stresses. Under non stressing conditions (C, control condition), expression of enaA was detected and a positive effect was observed after addition of large amounts of Na + or Li + , or by alkalinizing the cultures (Fig.…”

“…However, this synergic response was not observed when combining alkalinity and 0.3 M Li + . Despite the fact that reads mapping at the enaA locus in our RNA-seq experiments 10 suggest the existence of alternative splicing events at the three introns predicted in the coding region (i.e. the processed form seems to be more abundant in the wild-type at pH 8 than under standard culture conditions), Northern hybridization did not indicate the generation of transcripts of different sizes (Fig.…”

Transcription factors in the fungus Aspergillus nidulans: Markers of genetic innovation, network rewiring and conflict between genomics and transcriptomics

“…Thus, I wondered what percentage of the potential A. nidulans TFome is transcribed as predicted and if there was a subset of genes rendering TF forms different from the predicted full-length forms or even non-functional. With that aim, RNA-seq experiments published by us previously [31–33] and transcriptomic data available in the FungiDB database were used as a reference to compare genomic annotations and the most probable transcript sequences (Figure 2).…”

“…B-E) Density of reads mapping to AN5078 (B), AN0396 (C), AN2392 (D) and AN2811 (E) loci . Images were exported using the IGV software (top; [31]) or from the JBrowse genome browser linked to the FungiDB database ( middle ). Bottom pictures correspond to BLAST searches at the NCBI website (panels B-D) or a Clustal alignment visualized with Genedoc (panel E).…”