Transcription of the Neurospora crassa 70-kDa class heat shock protein genes is modulated in response to extracellular pH changes

Abstract

Heat shock proteins belong to a conserved superfamily of molecular chaperones found in prokaryotes and eukaryotes. These proteins are linked to a myriad of physiological functions. In this study, we show that the N. crassa hsp70-1 (NCU09602.3) and hsp70-2 (NCU08693.3) genes are preferentially expressed in an acidic milieu after 15 h of cell growth in sufficient phosphate at 30 degrees C. No significant accumulation of these transcripts was detected at alkaline pH values. Both genes accumulated to a high level in mycelia that were incubated for 1 h at 45 degrees C, regardless of the phosphate concentration and extracellular pH changes. Transcription of the hsp70-1 and hsp70-2 genes was dependent on the pacC (+) background in mycelia cultured under optimal growth conditions or at 45 degrees C. The pacC gene encodes a Zn-finger transcription factor that is involved in the regulation of gene expression by pH. Heat shock induction of these two hsp genes in mycelia incubated in low-phosphate medium was almost not altered in the nuc-1 (-) background under both acidic and alkaline pH conditions. The NUC-1 transcriptional regulator is involved in the derepression of nucleases, phosphatases, and transporters that are necessary for fulfilling the cell's phosphate requirements. Transcription of the hsp70-3 (NCU01499.3) gene followed a different pattern of induction-the gene was depressed under insufficient phosphate conditions but was apparently unaffected by alkalinization of the culture medium. Moreover, this gene was not induced by heat shock. These results reveal novel aspects of the heat-sensing network of N. crassa.

Fig. 1

Phylogenetic analysis of Neurospora crassa hsp70 genes. Multiple alignments of 10 HSP70 amino acid sequences using the N. crassa genome database was performed using ClustalW (http://www.ebi.ac.uk/clustalw). A phylogenetic tree was constructed from these alignments using the neighbor-joining method with the software package MEGA version 3.1 (http://www.megasoftware.net). Three of them were chosen for transcription analysis (filled diamonds). The number at the nodes represents the percentage of bootstrap values (1,000 replicates). The scale bar represents the phylogenetic distance of 0.1 amino acid substitution per site

Fig. 2

Gene expression analysis. Northern blot analysis of hsp70-1 (NCU09602.3), hsp70-2 (NCU08693.3), and hsp70-3 (NCU01499.3) transcripts using total RNA obtained from N. crassa mycelia. The wild-type strain was cultured for 15 h at 30°C in low- or high-Pi liquid medium (0.1 and 10 mM Pi, respectively) at pH 5.4 or 7.8. The effect of heat shock was assayed in mycelia incubated for 1 or 2 h at 45°C. The ethidium bromide-stained rRNA band is shown as a loading control. The β-tubulin gene was hybridized as additional loading control on the northern blot. Bars show fold expression determined from the intensity measured by densitometric analysis. Data are average values ± standard deviation (SD) obtained from two independent experiments

Fig. 3

Relative gene expression levels of the three hsp70 gene family members in response to low-Pi cultures at pH 5.4 (northern blots are shown in Fig. 2). The images were captured by the Cyclone Storage Phosphor System (Packard) at 600 DPI resolution and analyzed with the OptiQuant program version 3.0 (Packard). The signal intensities of these image areas were expressed in Digital Light Units (DLU) per mm and normalized for the screen background. We assumed identical DLU values for both hsp70-1 and hsp70-2 transcripts expressed in mycelia incubated for 15 h at 30°C. The bars indicate the fold expression determined from the intensities measured by densitometric analysis. Data are average values ± standard deviation (SD) obtained from two independent experiments

Fig. 4

Gene expression analysis. Northern blot analysis of hsp70-1 (NCU09602.3), hsp70-2 (NCU08693.3), and hsp70-3 (NCU01499.3) transcripts using total RNA obtained from N. crassa mycelia. The wild-type (wt) and pacC^KO strains were cultured for 15 h at 30°C in high-Pi liquid medium (10 mM Pi) at pH 5.4 or 7.8. The effect of heat shock was assayed in mycelia incubated for 1 or 2 h at 45°C. The ethidium bromide-stained rRNA band is a loading control. The β-tubulin gene acted as additional loading control for the northern blots. Bars show fold expression, determined from the intensity measured by densitometric analysis. Data are average values ± standard deviation (SD) obtained from two independent experiments

Fig. 5

Gene expression analysis. Northern blot analysis of hsp70-1 (NCU09602.3), hsp70-2 (NCU08693.3), and hsp70-3 (NCU01499.3) transcripts using total RNA obtained from N. crassa mycelia. The wild-type (wt) and nuc-1^RIP strains were cultured for 15 h at 30°C in a low-Pi liquid medium (0.1 mM Pi) at pH 5.4 or 7.8. The effect of heat shock was assayed in mycelia incubated for 1 or 2 h at 45°C. The ethidium bromide-stained rRNA band is a loading control. The β-tubulin gene acted as additional loading control for the northern blots. The bars show fold expression determined from the intensity measured by densitometric analysis. Data are average values ± standard deviation (SD) obtained from two independent experiments