Neurospora Crassa: Tunable Light-Induced Protein Expression

Can the light-inducible promoters found in Neurospora Crassa be used to control endogenous and exogenous gene expression?

INTRODUCTION: Neurospora Crassa is a type of filamentous fungus model organism which has been used for studying the effects of light on higher eukaryotes for many years. Additionally, it is both easy to grow and possesses a  haploid life cycle which allows for the study of recessive genes, making it a great model organism (2). Filamentous fungi are important model systems which provide insight into the study of expression of different proteins as well as other biological functions. A useful feature of these fungi is the ability of the organism’s own protein-processing machinery to perform all necessary post-translational modifications. This allows for an excellent host organism in the production of various eukaryotic proteins. Neurospora Crassa have several previously identified promoters which can be regulated, however these are still not perfected for all types of gene expression. Harnessing of the light-induced vvd promoter in Neurospora provides an opportunity for better control and increased protein expression through their use.

Neurospora Crassa can be used to further the investigation through the two known blue light receptors that they possess: White Collar-1 (WC-1) and Vivid (VVD). WC-1 forms a complex referred to as the White Collar Complex (WCC) with a transcriptional co-activator, WC-2. When activated by light, WCC binds to and activates promoters of several light-responsive genes, including VVD. Binding of WCC and the vvd promoter leads to the expression of VVD and acts to negatively regulate the light response initiated by the WCC through direct interaction with the complex. The interaction between VVD and the WCC plays a integral role in the fungi’s ability to adapt to varying intensities of light.

The vvd gene is rapidly induced, by as much as 300-fold, within minutes of light exposure and is weakly expressed in the absence of light. WCC-mediated transcription repressors work together with VVD inhibition of WCC activity to repress vvd transcription within an hour of light exposure.

GOAL AND MAIN CONCLUSION: Based on the properties of vvd, it was postulated that these promoters could be used as a tool to drive expression of other endogenous proteins as well as exogenous proteins. Additionally, by expressing the gene of interest in a vvd knockout, protein expression could be further increased from the vvd promoter. Three different genes under the regulation of the vvd promoter (wc-1, gfp, and gh5-1) were expressed at 50-fold to 150-fold of the induced level. The heterologous gfp gene was driven at levels similar to the native wc-1 and gh5-1, showing a graded response to light duration as well as intensity and a quick return to the inactive state after the exposure to light ended. Finally, the vvd promoter produced three-fold more protein than the ccg-1 promoter, a promoter which is commonly used for constitutive over-expression. These findings led to the confirmation of the possibility for use of the vvd promoter as a novel tunable and controllable system to express both endogenous and exogenous genes of the Neurospora Crassa.

RESULTS: First, GFP was used to demonstrate that the vvd promoter was capable of expressing exogenous genes. A vvd knockout strain was produced to contain pvvdgfp and the levels of gfp mRNA and GFP protein were tracked with RT-qPCR and western blot analysis once the strains were exposed to light to induce gene expression.

Figure 1. Tunable expression driven by the vvd promoter determined at the transcriptional and translational levels in the knockout background by measuring the induction of gfp at high light (A), very low light (B), and light exposure for h hours at in light exposure (C).

The “tunability”, or regulatory efficiency, of the vvd promoter was evaluated using gfp at different levels of light exposure as seen in Figure 1. Very low light exposure resulted in a 10-fold increase of gfp mRNA and a 60-fold increase was observed from the high light exposure. This experiment resulted in the conclusion that GFP protein expression increased in a direct and proportional relationship with the amount of light exposure. Upon return to the dark conditions, there was a rapid decrease in activity of the promoter. The promoter returned to the completely inactive state after a 3 hour period  and protein levels neared zero after an 8 hour period following the light exposure.

Figure 2. Expression of wc-1 driven by the vvd promoter in the knockout background is greater than in wild-type. DD is dark conditions, and LL is light. Transcriptional levels were determined measuring the tagged wc-1 in wild-type (light grey) and knockout (dark grey) strains using real-time PCR analysis compared with actin.

Wild-type and knockout strains containing a tagged wc-1 driven by the vvd promoter were created in the dark. Upon light exposure, a rapid and distinct increase in transcriptional expression was seen in both the wild-type and knockout strains, as seen in Figure 2. Additionally, the high expression previously observed was sustained for the duration of the experiment. The knockout strain demonstrated a 2- to 3-fold greater expression than the wild-type strain, confirming that the removal of vvd from the genome alleviates repression of active WC-1.

Figure 3. The vvd promoter drives stronger expression than the ccg-1 promoter. (A) Expression of tagged GH5-1-GFP under the vvd and ccg-1 promoters was determined by measuring GFP levels in high light exposure (LL). (B) Purified GFP run on the same gel as a standard. (C) Protein levels of GFP/CBH-1 under the ccg-1 promoter (dark grey) and GFP/CBH-1 under the vvd promoter (black) were compared with the standard GFP control (light grey). (D) Transcriptional levels were determined measuring induction of gfp under vvd (dark grey) and ccg-1 (light grey) promoters using RT-qPCR analysis compared with actin.

The light-induced and glucose-repressed ccg-1 promoter is commonly used for over-expression in various genetics investigations. Expression of gh5-1 driven by pvvd was compared with that driven by induced and de-repressed pccg-1, as seen in Figure 3. The protein expression was not completely repressed under the ccg-1 promoter before induction, however the level of transcription under the vvd promoter was 10-fold greater than that of the fully active ccg-1 and 90-fold above the knockout background. Protein expressed using the vvd promoter was on average 2- to 8-fold greater than that from the ccg-1 promoter.

The light-inducible vvd promoter has a very dynamic range that can be improved another 2-fold with the removal of auto-regulation of VVD in the vvd knockout strain. Expression of both endogenous and exogenous genes under the vvd promoter is greater than expression under ccg-1, even after 32 hours. The vvd promoter is activated with more speed and strength with light. Additionally, vvd is repressed at a faster rate when returned to dark conditions than other inducible promoters such as qa-2. In conclusion, the vvd promoter compares favorably with existing models for regulated promoters currently available for Neurospora Crassa.

CONCLUSIONS AND CRITIQUES: Overall, this paper investigated the use of Neurospora Crassa for light-inducible endogenous and exogenous protein expression. The system was found to be successful and more useful than the existing popular system for protein over-expression, ccg-1. The paper investigated various light exposures to confirm the proposed findings and compared the proposed system to ccg-1 in the various light exposures.

Figure 4. Outcomes of expression conditions from the vvd promoter in Neurospora Crassa.

The schematic above represents the predicted as well as actual output from the vvd promoter in different conditions. In the dark conditions (A), all genes driven by the vvd promoter are not induced. Light conditions in the wild-type strain (B) illustrate that light activates WCC rapidly and strongly turns on expression at the vvd promoter, leading to production of VVD. The VVD acts back on the WCC to inhibit the activation of the vvd promoter, referred to as photoadaptation. Light conditions in the knockout strain (C) demonstrate light activation of the vvd promoter but result in no VVD production, therefore no photoadaptation occurs, and the gene of interest is continually expressed. Very low light conditions in the knockout strain (D) show that lower levels of light cause a lower level of expression which demonstrates that the system can be regulated by manipulating the level of light. Light exposure for 1 hour in the knockout strain (E) indicate that once light is turned off after activation of the vvd promoter, promoter activity is repressed.

Additional genes and proteins should be explored to further confirm the results presented in this paper. Also, the activity of the produced proteins should be investigated before the system can be taken into other research. Over-expression of protein is useful in antibiotic and insulin manufacturing, as well as the biofuel industry, and the discovery of the vvd promoter as a tool for over-expression could prove to be very beneficial for these areas.

References:

Photos courtesy of the paper and here.

(1)Jennifer M. Hurley, Chen-Hui Chen, Jennifer J. Loros, and Jay C. Dunlap. “Light-Inducible System for Tunable Protein Expression in Neurospora Crassa.Genes, Genomics, Genetics. (2012) 1207-1212.

(2) Wikipedia. “Neurospora Crassa.” Accessed online at: http://en.wikipedia.org/wiki/Neurospora_crassa

Additional reading on Neurospora Crassa can be found on NIH and on Benchfly.

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