Developing a Temperature-Inducible Transcriptional Rheostat in Neurospora crassa

Abstract

Heat shock protein (HSP)-encoding genes (hsp), part of the highly conserved heat shock response (HSR), are known to be induced by thermal stress in several organisms. In Neurospora crassa, three hsp genes, hsp30, hsp70, and hsp80, have been characterized; however, the role of defined cis elements in their responses to discrete changes in temperature remains largely unexplored. To fill this gap, while also aiming to obtain a reliable fungal heat shock-inducible system, we analyzed different sections of each hsp promoter by assessing the expression of real-time transcriptional reporters. Whereas all three promoters and their resected versions were acutely induced by high temperatures, only hsp30 displayed a broad range of expression and high tunability, amply exceeding other inducible promoter systems existing in Neurospora, such as quinic acid- or light-inducible ones. As proof of concept, we employed one of these promoters to control the expression of clr-2, which encodes the master regulator of Neurospora cellulolytic capabilities. The resulting strain fails to grow on cellulose at 25°C, whereas it grows robustly if heat shock pulses are delivered daily. Additionally, we designed two hsp30 synthetic promoters and characterized them, as well as the native promoters, using a gradient of high temperatures, yielding a wide range of responses to thermal stimuli. Thus, Neurospora hsp30-based promoters represent a new set of modular elements that can be used as transcriptional rheostats to adjust the expression of a gene of interest or for the implementation of regulated circuitries for synthetic biology and biotechnological strategies. IMPORTANCE A timely and dynamic response to strong temperature fluctuations is paramount for organismal biology. At the same time, inducible promoters are a powerful tool for fungal biotechnological and synthetic biology endeavors. In this work, we analyzed the activity of several N. crassa heat shock protein (hsp) promoters at a wide range of temperatures, observing that hsp30 exhibits remarkable sensitivity and a dynamic range of expression as we charted the response of this promoter to subtle increases in temperature, and also as we built and analyzed synthetic promoters based on hsp30 cis elements. As proof of concept, we tested the ability of hsp30 to provide tight control of a central process, cellulose degradation. While this study provides an unprecedented description of the regulation of the N. crassa hsp genes, it also contributes a noteworthy addition to the molecular toolset of transcriptional controllers in filamentous fungi.

Keywords: HSP; Neurospora; Neurospora crassa; heat shock; hsp promoters; inducible promoter; synthetic biology; synthetic promoter; transcription.

FIG 1

Putative transcriptional heat shock regulatory elements present in the hsp promoters. (a to c) Schemes of hsp30, hsp70, and hsp80 promoters with the putative transcriptional regulatory elements indicated (49–51, 54). The hsp30 and hsp70 promoters contain putative heat shock elements (hse, red boxes), while the hsp80 promoter bears putative temperature-responsive elements (tre, purple boxes). We analyzed the indicated sections, upstream from the ORF (arrowhead). The dimensions of the boxes and lines represent the sizes of the transcriptional regulatory elements and the promoter regions, respectively.

FIG 2

Luciferase activity profiles conferred by hsp promoters and resected sections upon heat shock treatment. (a) Description of the experimental setup. The bent arrows represent the start of the bioluminescence measurements using the CCD camera. The strains grew for 24 h at 25°C under constant light conditions (LL), and then we measured the luminescence at 25°C in constant darkness (DD). The heat shock treatments (30°C, 35°C, and 45°C) were delivered for 1 h using an incubator, after which luminescence was monitored for additional 48 h. (b to i) Luminescence levels are shown in arbitrary units (A.U.). Boxes in gray dotted lines above each chart represent the area of the promoter region being analyzed, whereas the red and purple boxes represent the putative hse and tre, respectively. Each curve corresponds to the average values from four to six biological clones with three independent wells each ± standard deviations (SD) and represents the behavior in two independent experiments.

FIG 3

Fold induction achieved by the hsp promoter regions after heat shock treatments. (a to c) The fold induction (fold change) was calculated with the maximum luciferase expression, based on the average of the three highest consecutive values with respect to the background values before heat shock treatment of each promoter region. The data were obtained from the experiments whose results are shown in Fig. 2b to i. Average fold inductions are shown.

FIG 4

Transcriptional responses conferred by subjecting the full or resected hsp30 promoters to a temperature gradient and different exposure times. (a) Activity profile of each hsp30 promoter region in different temperatures after 1 and 15 min of treatment. Closeups of the hsp30_0.5 kb graphs are displayed as insets. Each curve corresponds to the average results from two biological clones with eight independent wells each ± standard deviations (SD) and represents the behavior in two independent experiments. (b and c) Maximum luminescence (b) and fold change (c) values obtained after all the heat shock treatments for each hsp30 promoter region. The maximum luminescence was defined as the average of the highest values. The fold induction was calculated with the maximum luciferase expression (shown in panel b) with respect to the average of the background values of each promoter region before heat shock treatment. The data were obtained from the luciferase activity profiles shown in Fig. S4. Statistical significance was determined using two-way analysis of variance (ANOVA) plus Dunnett’s test (for time treatments, all values were compared to the values obtained with 15-min treatments [*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant], and for temperature treatments, all values were compared to the values obtained with 44°C treatments [Ø, nonsignificant]). Time significance is indicated below each value, whereas for temperature, only nonsignificance (Ø) is shown above the value when it corresponds.

FIG 5

The hsp30_0.5 kb promoter can control a metabolic pathway of biotechnological interest. (a) Conidia (10⁶) from WT (x654-1), Δclr-2, and hsp30_0.5 kb:clr-2 (biological clones 1 and 2) strains were inoculated into Vogel’s medium with crystalline cellulose (Avicel, 2% [wt/vol]) as the carbon source. The flasks were placed under constant light conditions (LL) at 25°C with or without a high-temperature treatment (a pulse at 45°C for 2 h every 6 h [25:45°C 6:2h]). Before imaging, all the flasks were placed for 7 days in a shaker (125 rpm). The image depicts representative phenotypes from three independent experiments. (b and c) Supernatant protein concentrations (b) and total mycelial protein contents (c) were determined from 7-day cultures of WT, Δclr-2, and hsp30_0.5 kb:clr-2 strains grown on 2% Avicel with or without the high-temperature treatment (25:45°C 6:2h) as explained in Material and Methods. The supernatant concentrations were normalized to the total mycelial proteins per condition. The mean values and standard deviations represent three independent measurements and three independent experiments. N.D., not detected. Statistical significance was determined using two-way ANOVA plus Sidak’s test (***, P < 0.001; ****, P < 0.0001).

FIG 6

Design of two inducible synthetic promoters tunable by subtle changes in heat shock treatments. (a and b) Schemes of hsp30 synthetic promoters (SP30), where synthetic promoters with 24-bp (SP30A) (a) or 50-bp (SP30B) (b) spacers between the indicated putative hse were used to generate reporter genes in the context of a minimal hsp30 promoter of 250 bp. (c) Luciferase activity profiles of the synthetic promoters against a temperature gradient for 1 min and 15 min. Closeups of the hsp30_0.5 kb graphs are displayed as insets. Each curve corresponds to the average values from two or three biological clones with four independent wells each ± standard deviations (SD) and represents the behavior in two independent experiments. (b and c) Maximum luminescence (b) and fold change (c) values observed after the heat shock treatments for the indicated hsp30 promoters are indicated. The maximum luminescence was defined as the average of the highest values. Fold induction was calculated based on the maximum luciferase expression (shown in panel b) with respect to the average of the background values of each promoter region before heat shock treatment. The data were obtained from the luciferase activity profiles shown in Fig. S10. Statistical significance was determined using two-way ANOVA plus Dunnett’s test (for time treatments, all values were compared to the values obtained with 15-min treatments [*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns = non-significant], and for temperature treatments, all values were compared to the values obtained with 44°C treatments [Ø, nonsignificant]). Time significance is indicated below each value, whereas for temperature, only nonsignificance (Ø) is shown above the value when it corresponds.