Introduction

Elucidating a gene’s function rests heavily on the ability to impact its expression. The simplest form of this involves blocking protein synthesis wholesale—either through mutagenesis of the genome or knockdown of the message—and then observing the loss-of-function phenotype. Essential genes cannot be scrutinized in this way for the obvious reason, and knockout/knockdown mutants often display little or no phenotype due to homeostatic buffering by redundant genes or pathways (Giaever et al. 2002; Hartman et al. 2001). In these cases, informative phenotypes may be achieved by overexpressing the gene-of-interest to mimic a gain-of-function allele, or titrating down expression to confirm essentiality. This approach involves replacing the endogenous promoter with one that provides the desired level of expression and experimental control, and such systems have been useful in inferring gene function in several eukaryotes (Rørth et al. 1998; Zhang 2003; Sopko et al. 2006). Regulatable promoter systems are highly desirable in bioindustry as well. Whereas constitutive and/or robust overexpression of a heterologous protein may be toxic to the cell, for example, a more tightly controllable system may be desired to keep expression levels low or induce sharply once sufficient biomass has accrued.

This review will focus on regulatable promoter systems in filamentous fungi, an important group of organisms whose members include agriculturally and medically important pathogens (Möller and Stukenbrock 2017; Powers-Fletcher et al. 2016), essential genetic models (Roche et al. 2014; Osmani and Mirabito 2004), and prolific cell factories in industrial fermentations (Druzhinina and Kubicek 2017; Meyer et al. 2015). We will begin with a brief overview of promoter systems already in place for fungal research and industry, which will lead into our main topic concerning the exploitation of fungal photobiology to those ends.

Current regulatable systems: benefits and limitations

The ideal regulatable promoter has the following traits: (1) tightness, i.e., not leaky in the “off-state,” (2) robustness, allowing for many folds expression above or below endogenous levels, (3) tunability, such that expression changes linearly with the concentration of the signal (inducer/repressor), (4) temporal controllability, i.e., the signal can be applied and removed from the system at any given time, and (5) cost-effectiveness, such that the signal is inexpensive on both a small and large scale. Several promoter systems have been developed for use in molds and are generally regulated either by a primary nutrient or some chemical inducer. A brief overview of these strategies, including a mention of their strengths and weaknesses, will be provided.

Most regulatable systems in fungi utilize conditional metabolic promoters, meaning that the expression state (on/off) varies with qualitative changes in the nutrient composition of the media. This includes several that are induced on alternative carbon sources and repressed by glucose (e.g., PalcA, Pcbh-1, Pcrg1, PxlnA), as well as orthologs of the nitrite reductase (PniiA/nit-6) which is repressed by ammonium and induced by nitrate (Waring et al. 1989; Liu et al. 2008; Bottin et al. 1996; Amaar and Moore 1998; Exley et al. 1993; de Graaff et al. 2005). These promoters are generally robust under inducing conditions and tight under repressing ones and have indeed been employed to study essential genes and to create over-expression libraries to screen for phenotypes of interest (Hu et al. 2007; Lee et al. 2005; Romero et al. 2003). Their major limitations, however, stem from the constraints they place on the medium that can be used (i.e., they are metabolically restrictive). This is problematic from a research perspective if the global metabolic influence of the medium masks the phenotype otherwise associated with overexpression of the target-gene (Ouyang et al. 2015). Furthermore, as many of these promoters are repressed by glucose, they exclude the use of most low-cost and complex (nutrient rich) media for use in biofermenters. These promoters are further limited by the fact that they are essentially a binary switch (on or off) and consequently do not allow for finely tunable expression. Nor do they allow for easy temporal controllability, as the entire medium would have to be changed to facilitate a timed induction. Thoroughly removing repressive media from a mycelial mat is laborious and undesirable in a large-scale grow-ups, and trace carryover nutrients may impact induction levels and kinetics. Thus, while primary nutritional promoters may be robust and tight, other desirable features such as tunability, temporal controllability, and we can now add metabolic flexibility to the list of considerations, are best achieved with a signal that can be spiked into the culture (e.g., a chemical) and operate irrespective of the medium.

Several chemically regulated promoter systems that function in standard (i.e., glucose-containing) media exist for fungi. The human estrogen receptor system, for example, has remarkably been adapted for use in Aspergillus niger, which requires the heterologous expression of the human estrogen receptor along with the gene-of-interest being placed downstream of a URA3 promoter modified to contain several estrogen response elements (EREs). The system is exquisitely sensitive to estrogen (picomolar range) and tunable; however, depending upon the exact promoter construct utilized, it is either robust or tight, but not both (Pachlinger et al. 2005). Additional concerns stem from the inhibitory effect of estrogen on the engineered strains at concentrations as low as 10 nM (Pachlinger et al. 2005). Endogenous fungal promoters may also be used, including PthiaA from Aspergillus oryzae, which is repressed by thiamine and is tunable; however, the system does not function at neutral or weakly alkaline conditions (pH = 7), likely because the loss of thiamine’s charge (it is a weak base) affects its import (Shoji et al. 2005). The qa-2 promoter from Neurospora is induced by and tunable across four to five log-orders of quinic acid. This promoter may, however, lack sufficient robustness for high degrees of overexpression and is moreover leaky under low glucose (Giles et al. 1985; Shi et al. 2010; Larrondo et al. 2009). This is a good place to note that promoter leakiness can be exploited in certain experimental contexts. Our group, for example, could assess phenotypes associated with the essential gene casein kinase-1 (ck-1) due to weak expression from pqa-2 in the absence of quinic acid (Mehra et al. 2009).

Three systems that have emerged recently utilize apparently non-toxic signals that also provide tight and tunable expression. The copper-repressible promoter of tuc-1 from Neurospora is notable because copper can be spiked into the culture for a timed repression or, alternatively, the copper chelator BCS can be added into a copper-containing culture for a timed induction (Lamb et al. 2013; Ouyang et al. 2015). Importantly, Ouyang and colleagues demonstrated that copper has no discernible impact on the metabolite profile of Neurospora when analyzed by proton nuclear magnetic resonance spectroscopy (Ouyang et al. 2015). The tetracycline resistance operon (both the tet-on and tet-off variations) was first developed in Aspergillus fumigatus (Vogt et al. 2005; Helmschrott et al. 2013) and has since been adapted for use in A. niger, where it is tight and responsive within minutes of adding doxycycline to the culture (Meyer et al. 2011; Wanka et al. 2016). Finally, the benzoate para-hydroyxylase (bphA) promoter from A. niger is tightly repressed in the absence of benzoic acid and induced within 10 min upon its addition to the medium (Antunes et al. 2016). That the fungus can grow on benzoic acid as the sole carbon source indicates that toxicity is a minimal concern in the system.

In summary, chemically induced promoter systems offer a tunability and temporal controllability that are typically lacking with nutritional/conditional promoters. General considerations may include the cost of the inducer as well as its potential toxicity to the fungus. In all cases, the ability to remove the chemical inducer once it is applied requires a wash out step, which can be both laborious and inefficient. Thus, a signal that can be applied and removed instantaneously would be ideal, which is where we can now shed light on the story! While recent work and reviews have focused on the development of optogenetics in yeast and filamentous fungi, which involves the expression of heterologous/synthetic light sensing modules (Drepper and Krauss 2011; Wang et al. 2014; Zhang et al. 2016; Salinas et al. 2017), we will instead focus on promoters that function downstream of endogenous photosensory pathways in your fungus of interest.

A primer on light and fungi

Visible light is a ubiquitous environmental signal that can provide important environmental information to the fungal cell. Sporulation is photoinducible in many fungi, for example, because light presumably signals the surface-to-air interface for optimal dispersal. Other species use visible light primarily as a proxy for co-occurring stresses (e.g., genotoxic ultraviolet radiation) and therefore repress growth and differentiation. Regardless of the response, light perception is owed to highly sensitive and wavelength-specific photoreceptor proteins that transduce the signal either through some biochemical activity (e.g., a kinase) or an altered intermolecular interaction (e.g., protein-protein, protein-DNA) (recently reviewed in Fischer et al. 2016; Fuller et al. 2015). Fungal phytochromes, for example, are histidine kinases that bind a tetrapyrrole chromophore that imparts a sensitivity to the near-infrared/red range (600–850 nm wavelength) (Yu et al. 2016); opsins are transmembrane proteins (ion channels or cyclases) that bind retinal and have a peak sensitivity to green light (495–570 nm) (Avelar et al. 2014; García-Martínez et al. 2015); and the cryptochromes and LOV-domain proteins both bind flavin to detect light in the near-ultraviolet/blue range (400–490 nm) (Losi and Gärtner 2011). In this way, the combined effort of multiple photoreceptor types can incur a visible spectrum to fungi that approximates that of mammals, and their activities may be coordinated to achieve an optimal response to changing light qualities across the day.

The mechanisms of fungal phototransduction are best characterized for the white collar-1 (WC-1) family of proteins, which are GATA-type transcription factors that contain both a Zn-finger DNA-binding domain as well as a specialized PAS (Per-Arnt-Sim) domain called the LOV domain (for light, oxygen, and voltage) which binds flavin-adenine dinucleotide (FAD) as a chromophore. First shown to be a photoreceptor in Neurospora (Froehlich et al. 2002)—a foundational model for fungal photobiology—WC-1 obligately exists as a heterodimer with another Zn finger protein called WC-2 to form the white collar complex (WCC) (Ballario et al. 1998; Cheng et al. 2002). Upon the absorption of blue light (~ 465 nm wavelength) by FAD, a conformational change alters the ability of the heterodimer to drive expression of genes containing WCC binding sites called LREs (for light-response element) (Froehlich et al. 2002; Smith et al. 2010; Wang et al. 2015). In addition to being the most-well studied photoreceptor in fungi, the WCC is also the most well-conserved, with orthologs distributed across the Ascomycota, Basidiomycota (Kamada et al. 2010; Brych et al. 2016; Idnurm and Heitman 2005b), Mucormycotina (formerly called Zygomycota) (Idnurm et al. 2006; Sanz et al. 2009; Corrochano et al. 2016; Corrochano and Garre 2010; Stajich 2016), and even the anciently diverged chytrids (Idnurm et al. 2010; Dunlap and Loros 2006). Corresponding to the presence of these genes, blue light responses manifest in key research models (Neurospora crassa, Aspergillus nidulans), plant pathogens (Botrytis cinerea, Magnaporthe oryzae), human pathogens (A. fumigatus, Cryptococcus neoformans), and industrial workhorses (A. niger, Trichoderma reesei) (reviewed broadly in Purschwitz et al. 2006; Rodriguez-Romero et al. 2010; Fuller et al. 2016). As these physiological responses may be fast, robust, and fluency dependent (titratable), the promoters of light-responsive genes may serve as useful regulatable systems in these diverse and important organisms. We will explore this concept, first through a test case in the form of the Neuropora vvd promoter, and then speculate upon the utility of the approach in other fungal systems.

The vvd promoter in Neurospora: a case study for light-regulated expression systems in fungi

In Neurospora, the expression of many light-induced genes begins returning to baseline (dark level) after a few hours in constant illumination. This process, termed “photoadaptation,” is due to a negative feedback on the WCC and is facilitated by an additional blue light receptor called VVD. Briefly summarized, the WCC drives strong expression of the vvd promoter within minutes of culture illumination. The VVD protein, which also binds FAD as a chromophore, becomes light activated, dimerizes with WC-1, and inhibits the transcriptional activity of the WCC (Chen et al. 2010; Hunt et al. 2010; Malzahn et al. 2010) (Fig. 1a). The upshot of this negative feedback is that the fungus does not constitutively synthesize light-output genes during constant illumination at a fixed intensity but instead remains responsive to increasing intensities over time (Heintzen et al. 2001; Schwerdtfeger and Linden 2001, 2003; Dasgupta et al. 2015). This is evident in vvd mutants that hyperaccumulate carotenoids in light, thus giving the mycelium a bright (“vivid”) orange color from which the gene derives its name (Heintzen et al. 2001).

Fig. 1
figure 1

The use of pvvd to drive heterologous gene expression in Neurospora. a Scheme of Neurospora photoadaptation, whereby the WC-1/WC-2 heterodimer (the WCC) drives expression vvd in response to light. The VVD protein itself becomes photoactivated and negatively regulates the WCC through a direct interaction, thus attenuating the light response. b The left graph depicts the kinetics of heterologous gene expression in a Neurospora wild-type background, in which photoadaptation results in reduced expression after an initial induction. The right graft depicts the kinetics of the same gene in a Δvvd background, in which photoadaptation is ablated and gene expression remains constitutively high under constant illumination. The protein blot demonstrates a linear accumulation of GFP when driven by pvvd in the Δvvd mutant (adapted from Hurley et al. 2012)

Full size image

In addition to the rapid onset of induction, which can be as high as 300-fold within minutes of light exposure, vvd expression is effectively undetectable in constant darkness. Given the tight and robust nature of this response, the vvd promoter was investigated by our group as a putative system to drive autologous and heterologous proteins in Neurospora (Hurley et al. 2012). As a proof-of-principle, the 3000 bp fragment that immediately precedes the vvd translational start (pvvd) was placed upstream of the gene encoding green fluorescent protein (gfp). Moreover, this construct was expressed ectopically and in a vvd knockout background (Δvvd) in order to attenuate the photoadaptation response and increase the degree and duration of target-gene overexpression. The tightness of the system was evident in that GFP protein remained undetectable by western blot when the fungus was cultured in constant darkness. After 1 h of light exposure, however, GFP was detectable and steady-state protein increased linearly for up to 48 h incubation in constant illumination (Fig. 1b). At the transcript level, gfp message was already 50-fold above background (dark) after 1 h of induction, and levels were maintained as high as 80-fold for the duration of the 48 h time course. Notably, similar degrees of tightness and robustness were observed when the same vvd promoter fragment drove the Neurospora cellulase gene, gh5-1, which is used in the production of cellulosic biofuels (Hurley et al. 2012; Sun et al. 2011). Thus, the ability to conditionally and strongly express genes in Neurospora to basic research or industrial ends can be achieved with light-regulated systems, and the use of strains deficient in photoadaptation can further prolong overexpression in this organism (Hurley et al. 2012).

To better characterize the kinetics of vvd promoter activity, the pvvd-gfp strain was exposed to a 1 h light-pulse and then returned to constant darkness for further incubation. In this experiment, a 160-fold increase in gfp mRNA was observable directly after the light pulse, but these levels had returned to baseline 7 h after light was removed. This underscores perhaps the greatest advantage to using light as opposed to chemical inducers: It can be applied and removed rapidly (and repeatedly) without manipulation of the culture. This temporal control is unmatched compared to nutritionally or chemically driven systems, which require a complete change of the medium (and washing of the mycelium) to achieve multiple cycles of induction/repression. Moreover, gfp mRNA and protein displayed an attenuated (but still linear) induction when Neurospora was exposed to lower light intensities, thereby demonstrating the tunability of the promoter (Hurley et al. 2012).

In summary, work with the Neurospora vvd promoter demonstrates generally that the intrinsic photosensing capabilities of fungi can be exploited to control genes-of-interest. In the case of pvvd itself, the system was tight, robust, tunable, temporally controllable, and presumably, as it was not addressed experimentally, nutritionally flexible. We will now set a wider gaze and discuss the use of light-driven expression systems in other fungi, focusing on the identification of putative promoters that might be developed in one’s organism of interest.

Light-regulatable promoters in your favorite fungus?

In principle, the vvd promoter from Neurospora could be used to drive gene expression in heterologous fungal systems so long as (1) the organism contains orthologs to the white collar proteins and (2) those proteins recognize the promoter elements of pvvd. The reverse experiment has been tried, in which the apparent vvd-ortholog of T. reesei, called env1, was expressed using its own promoter in a Neurospora Δvvd mutant. Although the env1 transgene was (intriguingly) unable to rescue the Δvvd photoadaptation phenotype, its expression pattern was conserved: env1 mRNA was undetectable in the dark but strongly accumulated in Neurospora after 10-min light pulses (Schmoll et al. 2005). Neurospora and Trichoderma are relatively closely relatives, however, as they are both in the Sordariomycetes clade (phylum Ascomycota). Although it is assumed that WCC recognition would be similar across more distantly related fungi (Weirauch et al. 2014), direct experimental evidence—by either sequence analysis of known light-regulated genes, or by WCC chromatin immunoprecipitation (ChIP)—will be greatly informative (Chen et al. 2009; Smith et al. 2010). Even if binding sites are conserved in distant Neurospora relatives, the mode of action of the WCC on the promoter may differ. For example, the WC-1 ortholog in Aspergillus nidulans, LreA, has been demonstrated to operate in a repressive fashion; that is, it binds DNA in the dark and represses gene expression and then is released from the DNA upon illumination (Hedtke et al. 2015). Moreover, and in contrast to Neurospora (Chen et al. 2009), the proper induction of light-regulated genes in A. nidulans is not solely dependent on LreA/B (the WCC); rather, proper induction of light-regulated genes depends upon an interplay of LreA/B with the red-light sensing phytochrome FphA (Purschwitz et al. 2008; Purschwitz et al. 2009; Bayram et al. 2010; Hedtke et al. 2015). Thus, the peculiarities of the light sensing mechanisms across divergently related species may impact the tightness or robustness of heterologous light-driven promoters such as pvvd.

It instead may be more feasible to turn to the organism-of-interest itself, i.e., identifying light-regulated genes in your fungus and cloning the promoters of those that display the desired photo-kinetics. Sticking with vvd as an example, orthologs in both T. reesei and Fusarium fujikuroi (a model for fungal secondary metabolism and toxin production) are rapidly and robustly induced by light in a manner dependent upon the respective wc-1 orthologs (Schmoll et al. 2005; Castellanos et al. 2010; Castrillo and Avalos 2014). In a general way, therefore, orthologs of Neurospora light-regulated genes may be a good place to start in the search for photoregulatable promoters in a different species. Such an analysis will likely have to extend beyond vvd unfortunately, as this family of proteins is restricted to the Sordariomycetes (including Neurospora, Trichoderma, Fusarium, Botrytis, Magnaporthe), so those working on other fungi, such as any one of the important Aspergillus species, will have to look elsewhere. We will let genome-wide studies of light-regulated fungal genes guide the search.

Putative light-inducible promoters

To date, light-regulated transcriptomic studies have been published for only a handful of fungi and include (1) Neurospora microarray and RNA-seq, both of which compare dark against multiple timepoints in white light (Chen et al. 2009; Wu et al. 2014); (2) Botrytis cinerea microarray comparing dark against 60 min white light illumination (Schumacher et al. 2014); T. reesei microarrays comparing 72 h dark against 72 h white light illumination (Tisch et al. 2011, 2014; Tisch and Schmoll 2013); Trichoderma atroviride microarray and RNA-seq, both comparing dark against a 4–5 min white light pulse (Rosales-Saavedra et al. 2006; García-Esquivel et al. 2016); A. nidulans microarray, comparing dark against 30 min white light illumination (Ruger-Herreros 2011); and A. fumigatus microarray, comparing dark against several timepoints in white light (Fuller et al. 2013). Table 1 is a list of the 10 most robustly light-induced genes from each of these organisms; in the case of Neurospora and T. atroviride, the RNA-seq datasets were preferentially used over the microarray studies. For those interested in developing a light-based expression system in the organisms listed, Table 1 may be useful for selecting genes that provide the greatest robustness. For example, vvd appears on the list of all Sordariomycetes in the table (i.e., all those except the two Aspergilli); however, even greater induction may be achieved by the con-6 promoter in Neurospora or the hypothetical protein, Ta_131348, promoter in T. atroviride. In the case of con-6, expression had been previously shown to be undetectable in the dark by northern blot but became abundant within 15 min of white light illumination (Navarro-Sampedro et al. 2008), suggesting that its promoter may be a suitable alternative to pvvd when more robust overexpression is required. In general, however, the tightness of each candidate gene in the dark, as well as the overall photoinduction kinetics, will have to be assessed independently, e.g., by qRT-PCR or western blot.

Table 1 Top light-induced genes across whole-genome transcriptional datasets
Full size table

For those working in species for which transcriptomic data are currently not available, candidate promoters may be selected on the basis of their homology to ones listed in Table 1. For each gene in the table (each row), homologs from the other datasets that display similar light-regulation are noted. Gene AN8640 of A. nidulans, for example, is listed in the row corresponding to Neurospora’s con-6 because the two genes are orthologs and they are similarly light-induced in their respective whole-genome experiments; notably, AN8640 was not among the top 10 most induced in A. nidulans (Ruger-Herreros et al. 2011). In principle then, those genes with the largest degree of conserved regulation across the species represented in Table 1 are more likely to be light-induced in fungi not in the table. To illustrate, the ortholog of the T. atroviride grg-1 may be a better candidate in your fungus (as it is up-regulated in the other five species in Table 1) than is the ortholog to T. atroviride transcription factor Ta_138208 (which only appears in the T. atroviride dataset). Indeed, orthologs of the A. fumigatus photolyase, phr1, are not only upregulated by light in all the species represented in Table 1 but also demonstrate similar patterns of robust light induction in various other ascomycetes (Trichoderma harzianum, Fusarium oxysporum), the basidiomycete pathogen Ustilago maydis, as even the Mucromycotina Phycomyces blakesleeanus (Tagua et al. 2015; Brych et al. 2016; Alejandre-Durán et al. 2003; Berrocal-Tito et al. 1999).

Although informative, care must be taken when assessing the transcriptomic studies described above. First, microarray-based studies are relatively insensitive compared to RNA-seq and consequently may have failed to detect subsets of light-induced genes. This is perhaps best exemplified in the basidiomycete yeast Cryptococcus neoformans, in which microarrays detected only a single light-induced gene at the 2-fold cutoff, hem15 (Idnurm and Heitman 2010). For that reason, this study was omitted from Table 1. However, the hem15 promoter identified may prove to be an excellent candidate in your fungus, as it was demonstrated to be light induced in the ascomycete Neurospora and two Mucormycotina species, Phycomyces blakesleeanus and Rhizopus oryzae (Idnurm and Heitman 2010). A second consideration is that the nutritional source and mode of growth (i.e., liquid versus solid culture) are important variables across the transcriptomic experiments described in Table 1. In one way, this perhaps places greater emphasis on those genes that are conserved across them, i.e., their light induction transcends those variables and makes them more likely to be conservatively regulated in other fungi. However, those experimental variables also represent an important caveat in interpreting the data. For example, the asexual developmental genes induced in the A. nidulans array were not found in A. fumigatus, but this may reflect the fact that A. fumigatus was grown in submerged liquid culture (Fuller et al. 2013), a condition that may suppress sporulation (Lee et al. 2016). Finally, one must consider the length of the light treatment employed in each of the studies. Most involve acute light inductions (minutes to a couple of hours), which likely reveal those genes that are direct targets of the white collar orthologs, or are perhaps a target of another transcription factor directly downstream of the WCC (Chen et al. 2009). The T. reesei study in Table 1, by contrast, assesses gene expression changes following 72 h light exposure. While some genes identified in the study may be WCC targets (e.g., env1), others may be involved in the long-term adaptation to light and, consequently, may be unresponsive during the early photoresponse (Wolfers et al. 2015). Arguably, an ideal photoregulatable promoter is one that provides both an acute and prolonged light induction, but studies that track gene expression over an extended timecourse (from minutes to days post-induction) in a single organism are currently lacking.

Putative light-repressed promoters

Table 2 catalogs the most strongly light-repressed genes from the studies described above and, in doing so, highlights two major barriers to the development of a light-repressible promoter system. First, the magnitude to which genes tend to be repressed by light is considerably smaller than that of induction. For example, the greatest degree of light suppression observed in Neurospora and T. atroviride (the RNA-seq studies) was 10-fold and 16-fold, respectively; by comparison, the strongest degrees of induction were, respectively, 362-fold and 266-fold. Indeed, assessment of Neurospora light-regulated genes by microarray failed to detect the light-repressed category altogether due to the lower sensitivity of the technique (Chen et al. 2009; Wu et al. 2014). An exception to this appears to be T. reesei microarray dataset, for which the highest magnitudes of induction and repression were both around 30-fold. Nevertheless, it has yet to be determined if such a promoter can repress transcription enough to be experimentally useful, e.g., studies in which essentiality is to be tested. The analysis of light-repressed genes by RT-PCR and western blotting will be important follow-ups to this end. Table 2 also reveals a lack of correspondence between light-suppressed genes across the datasets. This suggests that selecting putative photorepressible genes in species outside of those in Table 2 may be difficult.

Table 2 Top light-repressed genes across whole-genome transcriptional datasets
Full size table

In summary, the existing transcriptomic datasets provide a good starting point in the search for light-regulatable promoters. Such data are, however, so-far limited in their phylogenetic scope (all are in the Ascomycota, mostly Sordariomycetes), and so comparable studies in representatives of the Basidiomycota or Mucormycotina will be needed for accurate promoter predictions in those clades. Furthermore, whole-genome data are lacking with respect to the differential regulation of genes by distinct light qualities, either directly with blue versus red light, or by proxy using white light against strains bearing knockouts of blue (WCC) vs. red (phytochrome) light photoreceptors. Only for Neurospora do such data exist, and in this case, red light fails to drive detectable transcriptional changes and the response to white light is apparently unaltered in a phytochrome knockout mutant (Froehlich et al. 2005; Chen et al. 2009). In contrast, many fungi that harbor phytochrome orthologs do indeed exhibit red-light-driven responses (Idnurm and Heitman 2005a; Fuller et al. 2015); therefore, one exciting possibility in those fungi is the development of a multichromatic system (e.g., one gene-of-interest is induced in blue light, a different gene by red light, and both in white!) which would be particularly useful for epistasis or synthetic lethality studies.

Is a light-regulatable promoter system right for you?

There are some minimal requirements in place for a light-regulatable promoter system to get off the ground. First and foremost, your fungus must overtly respond to light. The details of the photosensory cascade may not be important, however (e.g., whether the WCC serves as an activator or a repressor), so long as light-responsive promoters meet your experimental need, e.g., they are tight and robust (Dasgupta et al. 2016). Some important fungal lineages have apparently lost their suite of photoreceptors over the course of their evolution, including the hemiascomycete yeasts (including the important model Saccharomyces pathogen Candida sp.) and various ascomycete and basidiomycete dermatophytes (e.g., Trichophyton, Microsporum, Malessezia) (Dunlap and Loros 2006; Idnurm et al. 2010). A few technical requirements exist as well. For example, the experimenter must be able to control the light environment, which may include specialized incubators that are fitted with a programmable light source. White fluorescent lights are typically sufficient as their emissions span the major biologically active spectra (e.g., blue and red wavelengths). However, multiple LEDs may be needed if dual-wavelength systems were developed. Molecular biology experiments may further demand that un-induced (dark) samples be harvested in the absence of stimulating light. In the case of fungi that are unresponsive to the red spectrum (e.g., Neurospora), sample collection can be performed under a red safe light, such as those used for film development; for most fungi, however, samples harvesting should be performed in complete darkness with infrared oculars.

Two important considerations, depending upon the experiment, center around the long-term exposure of fungus to light. First is the issue of photoadaptation which, as described above, may limit the ability to constitutively overexpress a protein under constant illumination. This can be bypassed by using photoadaptation-deficient strains as demonstrated with Δvvd in Neurospora (Hurley et al. 2012). However, photoadaptation has been observed in fungi that lack a clear vvd ortholog, including Aspergillus (Fig. 2a) and Phycomyces, and the mechanism by which it occurs remains obscure (Ruger-Herreros et al. 2011; Rodríguez-Romero and Corrochano 2006; Olmedo et al. 2013; Fuller et al. 2013). Second is the potential of phototoxicity mediated by light itself. In S. cerevisiae for instance—a fungus with no intrinsic photosensory capability—high intensity light exposure can modulate cellular respiration and induce oxidative stress (Robertson et al. 2013). In agreement, light reduces the growth rate of the plant fungal pathogen Botrytis cinerea, but this can be overcome through the addition of the antioxidant ascorbate to the medium (Canessa et al. 2013). Therefore, the secondary impact of reactive oxygen species may represent an important experimental variable between light and dark cultures, or may alter cellular metabolism and interfere with the expression of the target gene. Both the photoadaptation and phototoxicity issues may be bypassed by exposing the fungus to periodic light pulses, however, rather than placing it under constant illumination. Figure 2b demonstrates that 3 min of white light is sufficient to induce phr1 expression in A. fumigatus, and this induction remains detectable for several hours (Fuller, Dunlap, and Loros, previously unpublished). Therefore, a short light pulse every 4 h or so may be sufficient to keep expression levels high on a long-term basis (depending on the experimental demand) and effectively minimize off-target effects by light itself (Schwerdtfeger and Linden 2001).

Fig. 2
figure 2

Kinetics of light-regulated gene expression in A. fumigatus. a Photoadaptation of two genes under constant white light illumination. b The initial induction and eventual decline of phr1 (photolyase) transcript after a 3 min white light pulse. Both panels represent previously unpublished qRT-PCR data, with actin serving as a normalizing gene

Full size image

A major advantage of using light as an inducing signal, in principle, is that it allows for metabolic flexibility, i.e., it can be used irrespective of the medium. Indeed, the light induction of certain conserved genes (e.g., the white collar, photolyases, or vvd orthologs) seems to occur on various carbon sources, such as glucose, glycerol, and microcrystalline cellulose (Wu et al. 2014; Tisch et al. 2011). However, in other cases, the light response may vary qualitatively and quantitatively with the medium. In A. nidulans, for example, the influence of light on secondary metabolism depends on the glucose concentration: At 1% glucose, light represses sterigmatocystin production; at 2% glucose, it induces production (Atoui et al. 2010). The degree to which light stimulates conidiation in A. nidulans, and likely the expression of developmental gene promoters, also depends upon the glucose concentration (Atoui et al. 2010). Similarly, Neurospora ccg-1, which is the ortholog of the highly light-induced T. atroviride grg-1 and A. nidulans ccgA genes listed in Table 1, is only weakly light-induced in the Neurospora RNA-seq experiment because the gene is also glucose-repressed (McNally and Free 1988). Broadly speaking, light is well known to influence primary metabolic pathways in fungi, and so long-term/secondary effects of culture illumination may impact phenotypes beyond the direct induction of the target gene (Tisch and Schmoll 2010; Tisch et al. 2014).

In summary, regulatable promoter systems are important tools to probe fungal gene function as well as optimize protein/metabolite production in industry. Perhaps the greatest advantage of a light-regulatable promoter over nutritional or chemical systems is the ease at which the signal can be applied and removed—literally with a flip of a switch! The pvvd system of Neurospora further demonstrates the potential tightness, robustness, and tunability of light-regulatable promoters, but these should be weighed against the need to stringently control the light environment as well as the global impact of light on the metabolism and development on your organism.