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Fungal Genetics and Biology
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Review
Trichoderma in the light of day - Physiology and development
Monika Schmolla, Edgardo Ulises Esquivel-Naranjob, Alfredo Herrera-Estrella b'*
a Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/166-5, 1060 Vienna, Austria b Laboratorio Nacional de Genómica para la Biodiversidad, CINVESTAV Irapuato, Km 9.6 Libramiento Norte Carretera Irapuato-León, CP 36821, Irapuato, Gto., Mexico
ARTICLE INFO
Article history: Received 22 January 2010 Accepted 28 April 2010 Available online 11 May 2010
Keywords: Light
Conidiation
Mycoparasite
Cellulase
Gene expression
Signal-transduction
White-Collar Phytochrome Cryptochrome Photolyase
ABSTRACT
In recent years, considerable progress has been made in the elucidation of photoresponses and the mechanisms responsible for their induction in species of the genus Trichoderma. Although an influence of light on these fungi had already been reported five decades ago, their response is not limited to photoconidi-ation. While early studies on the molecular level concentrated on signaling via the secondary messenger cAMP, a more comprehensive scheme is available today. The photoreceptor-orthologs BLR1 and BLR2 are known to mediate almost all known light responses in these fungi and another light-regulatory protein, ENVOY, is suggested to establish the connection between light response and nutrient signaling. As a central regulatory mechanism, this light signaling machinery impacts diverse downstream pathways including vegetative growth, reproduction, carbon and sulfur metabolism, response to oxidative stress and biosynthesis of peptaibols. These responses involve several signaling cascades, for example the heterotri-meric G-protein and MAP-kinase cascades, resulting in an integrated response to environmental conditions.
© 2010 Elsevier Inc. All rights reserved.
Contents
1. Introduction..................................................................................................................................................................................................................909
2. The discovery of light responses and the initial experiments....................................................................................................................................910
3. The early studies of Trichoderma photoresponses......................................................................................................................................................910
4. The search for photoreceptors......................................................................................................................................................................................911
5. Genome based discovery..............................................................................................................................................................................................912
6. Effects on gene expression............................................................................................................................................................................................912
7. Impact of light on cellulase production......................................................................................................................................................................913
8. Utilization of different carbon sources........................................................................................................................................................................913
9. Sulfur metabolism........................................................................................................................................................................................................913
10. Biosynthesis of secondary metabolites........................................................................................................................................................................913
11. Links to other signaling pathways..............................................................................................................................................................................913
11.1. Cyclic AMP....................................................................................................913
11.2. Heterotrimeric G protein signaling................................................................................914
12. Perspectives..................................................................................................................................................................................................................914
Acknowledgements......................................................................................................................................................................................................915
References....................................................................................................................................................................................................................915
* Corresponding author. E-mail address: aherrera@ira.cinvestav.mx (A. Herrera-Estrella).
1087-1845/$ - see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2010.04.010
1. Introduction
During evolution nearly all forms of life have been exposed to the electromagnetic radiation emitted by the sun, which for our purpose we will call light. Given the optic properties of light, it may be considered that it is non-randomly structured in time
and space (low entropy), and such properties have to important consequences for living organisms: it can be used to produce thermodynamic work and carries information. In order to survive and compete in their natural habitat all forms of life are continuously obtaining and decoding information from their environment (including that contained in light), which they use for their own benefit.
Our sun emits light in a wide wavelength range, of which the radiation of longer wavelength is called infrared, and is mostly transformed in molecular movement (heat). Radiation of shorter wavelength, corresponds to the ultraviolet (UV), and can initiate photochemical reactions. Among the molecules that can be affected by UV, DNA must be highlighted, since the result of one of such reactions can be transmitted as a mutation to the next generation. UV radiation can also damage molecules through its capacity to initiate uncontrolled free radical reactions, in most cases involving reactive oxygen species (ROS). Additionally, visible light can indirectly act in photosensitive reactions in which ROS may be produced through energy transfer from a molecule that can be activated by light such as flavin or porphyrin. In this way, blue light is potentially harmful (Lledias and Hansberg, 2000). In this context, it is understandable that sunlight is a significant element for life, and that besides the utilization of its energy and information, during evolution many mechanisms to resist its negative effects have been selected for. Thus, light has contrasting roles in relation to life, on one side all organisms depend on its energy and information, and on the other it is potentially harmful, and even deadly. For fungi life in light requires significant adjustments in numerous regulatory processes, a fact reflected in the widespread effects on their behavior (Herrera-Estrella and Horwitz, 2007; Tisch and Schmoll, 2009).
2. The discovery of light responses and the initial experiments
Even though Phycomyces was probably the first fungus in which the effect of light was analyzed, the study of the "informational" use of light by plants started much earlier. Darwin became interested in plant movements such as orientation towards the sun or the escape from the excess of light provoked or influenced by blue-light, and dedicated a complete volume to them, in which he described the use of a yellow-orange solution of potassium dichromate as a filter to eliminate phototropism (Darwin, 1880). This phenomenon was considered as key to solve the identity of the blue-light photoreceptor. The similarity of the action spectra for various biological responses to blue light in organisms as diverse as plants, bacteria, ferns and fungi, was intriguing. This led to the proposal that all such responses should be controlled by the same type of photoreceptor of ancestral origin (Bergman et al., 1969), which was named simply the ''near UV/blue" receptor or ''Blue Light-Receptor" (BLR). Other authors adopted the nickname ''cryptochrome"; a term coined by Jonathan Gressel, while studying in detail the effects of blue light in Trichoderma to highlight its hidden absorption (cryptic), and its preponderance in lower plants (cryptogams) and fungi (Gressel, 1979).
In several species of the genus Trichoderma a brief pulse of light triggers conidiation. In contrast to the organisms mentioned above this was the only obvious response of Trichoderma to light and hence the reason, which led to the use of this fungus as a simple photomorphogenic model.
Two action spectra of photoconidiation, which depict the relative effectiveness of different wavelengths of light in eliciting the physiological response, were determined (Gressel and Galun, 1967; Kumagai and Oda, 1969). Both action spectra show the characteristic shape attributed to the ''cryptochrome", including a sharp peak in the near UV 350-380 nm, and a wider peak in the
B wild Type Aenvl
Fig. 1. Effect of blue light on Trichoderma. (A) The pictures show the effect of a short (5 min) pulse of blue light (BL) on T. atroviride. The upper photographs show a colony of Trichoderma growing in the dark (D). The lower photograph shows a colony of Trichoderma 36 h after exposure to light, with the characteristic ring of green conidia at the what was the colony perimeter at the time of exposure. Photographs at the right correspond to scanning electron micrographs of the indicated area (fine lines), hyphae (top), and hyphae and conidiophores (bottom). (B) The pictures show the dramatic effect of light on colony growth in the Denv1 mutant of T. reesei (left), as compared to the wild type strain (right). T. reesei was grown under continuous exposure to blue light (BL) for 72 h.
blue with a maximum at 440-450 nm. Accordingly, experiments with the riboflavin structural analog roseoflavin indicated the participation of a flavin as the photoreceptive pigment (Horwitz et al., 1984a).
3. The early studies of Trichoderma photoresponses
The first description of the effect of light on conidiation of Trichoderma was made in 1957 (Gressel and Galun, 1967; Gutter, 1957). In the dark Trichoderma grows indefinitely as mycelium, and a brief pulse of light applied to the actively growing zone of the mycelium leads to the formation of dark green mature conidia, forming a ring at what was the edge of the colony when light was applied (Fig. 1A). The first event induced by light is a fast, first-order, photochemical reaction that does not require the presence of molecular oxygen and is independent of temperature. The fungus appears to be responsive to light (competent) only after 16 h of growth (Gressel and Galun, 1967). Three to seven hours after the induction abundant branching of aerial hyphae with an increased number of septa can be observed, as well as the formation of new aerial hy-phae, leading to conidiophore development (Galun, 1971).
This developmental program can be suppressed using RNA synthesis inhibitors, such as 5-fluorouracil, once it was triggered by light, but only in a time-window of approximately 7 h after illumination (Galun and Gressel, 1966; Gressel and Galun, 1967). T. atro-viride photoconidiation obeys the Bunsen-Roscoe law of reciprocity for pulses of blue light lasting from nanoseconds to minutes. Based on these data, it would appear that in Trichoderma photoconidiation is triggered by a single receptor system that is neither recycled to the photoreceptive form nor counted by enzymatic processes during or immediately following irradiation (Horwitz et al., 1990). This conclusion is supported by the observation that photoinduction is ''remembered" while the culture is maintained in conditions that do not allow cellular growth (cold or absence of oxygen), as soon as growth is resumed, under optimal conditions, the colony conidiates (Gressel et al., 1975; Horwitz et al., 1990).
Fig. 2. Schematic representation of the eight potential photoreceptors found in Trichoderma species and their domain structure. Conserved domains in all putative photoreceptors were determined using Pfam (http://pfam.sanger.ac.uk/search) and SMART (http://smart.embl-heidelberg.de/). Nomenclature: HDAC histone deacetylase interaction domain; LOV, Light, Oxygen, and Voltage; PAS, domain first found in the Per - period circadian protein, Arnt - Ah receptor nuclear translocator protein, and Sim -single-minded protein; GATA, GATA-type Zinc finger; PHY, characteristic phytochrome domain; GAF, cGMP phosphodiesterase/Adenylate cyclase/FhlA; HIS kinase, Histidine kinase domain.
Recently, sexual development has been discovered in T. reesei (teleomorph Hypocrea jecorina), but so far in no other Trichoderma species (Kuhls et al., 1996; Seidl et al., 2009). Both, asexual sporulation and sexual development, in T. reesei require light (Castellanos et al., 2010; Eveleigh, 1985 Seidl et al., 2009).
4. The search for photoreceptors
Using a mutagenesis approach, Horwitz and coworkers (Hor-witz et al., 1985) first attempted to isolate the photoreceptor involved in photoconidiation. They screened for T. atroviride mutants that did not conidiate in response to light but did in response to starvation. Despite thorough screenings they could not obtain the desired type of mutants, which in their view suggested that the photoreceptor could be essential. Interestingly, they isolated mutants that required more light than the wild type strain to get an equivalent response and overproduced a yellow pigment (dimY, for dimsighted & Yellow). It was then suggested that the mutants were affected in the photoreceptor (Horwitz et al., 1985, 1986). However, the molecular nature of the dimY mutants still remains to be determined.
A second strategy used to clone a blue-light photoreceptor from T. atroviride in the 1990s was based on the early proposal made by Benjamin Horwitz on the likelihood that such a photoreceptor could have evolved from a common ancestor for the receptor and DNA photolyases (Sancar, 1996). The approach led to the cloning and characterization of phr1, a gene with photolyase activity that regulates its own expression (Berrocal-Tito et al., 1999, 2007), and the closest homolog of which is the A. nidulans cryptochrome (cryA), recently shown to control sexual development (Bayram et al., 2008).
Finally, a few years ago two genes (blr-1 and blr-2) from T. atroviride were identified (Casas-Flores et al., 2004). The genes encode transcription factors homologous to the N. crassa White-Collar (WC) proteins (Ballario et al., 1996; Linden and Macino, 1997; Liu et al., 2003). BLR1 has a DNA binding domain and three PAS domains (Fig. 2). The first PAS domain is considered a LOV (Light-Oxygen-Voltage) sensory domain that has all amino acids necessary to interact with the chromophore FAD, including the cysteine
that forms a photoaduct with the flavin. Both BLR1 and BLR2 are essential for photoconidiation and gene expression regulated by blue light (Casas-Flores et al., 2004; Rosales-Saavedra et al., 2006). Consequently, it has been postulated that in T. atroviride, BLR-1 acts as the photoreceptor, in association with BLR-2. Recently, mutants in the T. reesei homologs of the blr genes have been obtained and shown to behave similarly to the T. atroviride corresponding mutants (Castellanos et al., 2010). Interestingly, overexpression of BLR2 resulted in enhanced sensitivity to light (Esquivel-Naranjo and Herrera-Estrella, 2007). Although BLR1 and BLR2 form a blue/UV-A receptor, these proteins also have light-independent functions. The BLR proteins are required for con-idiation induced by a sudden carbon deprivation and by addition of cAMP in darkness (Casas-Flores et al., 2006). However, although sexual development requires light, it is not known yet, whether BLR1 and BLR2 are essential for this process.
In contrast to the blr-genes, identification of the Trichoderma ortholog of the second blue-light photoreceptor described in N. crassa, VIVID (Heintzen et al., 2001; Schwerdtfeger and Linden, 2001, 2003) was not the result of a study targeted at elucidation of photoreceptors. It was initially found as a signaling factor puta-tively involved in cellulase gene expression in T. reesei (Schmoll et al., 2004). Although not yet proven to act as a photoreceptor in Trichoderma species, the PAS/LOV domain protein ENVOY (encoded by env1) plays an important role in light responses. In fact, Denv1 mutants express light-induced genes for a long time under constant illumination, indicating that ENVOY is a negative regulator of the light input, switching off the expression of genes regulated by BLR1 and BLR2 (Castellanos et al., 2010). Thus, ENVOY regulates photoadaptation similarly to the function performed by VIVID in N. crassa. Despite considerable sequence similarity, and comparable induction and regulatory characteristics, ENVOY cannot complement a VIVID non-functional mutant, suggesting that the mechanism used by ENVOY to transmit the light signal to the BLR complex may differ from that used by VIVID. The vvd nonfunctional mutant accumulates high amounts of carotenoids, and is not affected in growth. In contrast growth of the env1 mutant is severely affected by light with reduced hyphal extension rate and loss of polar growth (Fig. 1B), indicating a function of envl in light tolerance (Castellanos et al., 2010; Schmoll et al., 2005). On
the other hand, mutants in blrl and blr2 that do not express envl are not severely affected in growth (Casas-Flores et al., 2004; Castellanos et al., 2010).
In darkness, envl is transcribed at a very low basal level. Upon illumination, strong induction of transcription within minutes results in 50- and 500-fold increase in transcript abundance in both T. atroviride and T. reesei, respectively, which is mediated by the photoreceptors BLR1 and BLR2 (Castellanos et al., 2010; A. Herrera-Estrella et al., unpublished results). Interestingly, it was found that a light-dependent complex binds to the promoter of envl (Schuster and Schmoll, 2009). This observation might reflect that in addition to its transcriptional control by the BLR proteins, the recently described ENVOY upstream motif 1 (Schmoll et al., 2005) may play a relevant role in its regulation. Despite its function as a putative photoreceptor, ENVOY was shown to also have a regulatory function in darkness. Moreover, the signaling function of ENVOY must involve additional, light dependent auxiliary components or it could be post-translationally regulated by light, since overexpression of ENVOY in darkness did not eliminate the requirement of light (Schuster et al., 2007).
5. Genome based discovery
Nowadays three Trichoderma genome sequences are available, that of T. reesei (Martinez et al., 2008; http://genome.jgi-psf.org/ Trire2/Trire2.home.html), T. atroviride (http://genome.jgi-psf.org/ Triat1/Triat1.home.html) and T. virens (http://genome.jgi-psf.org/ Trive1/Trive1.home.html). In all three fungi homologs of the photoreceptors blrl and blr2, and envl are present (Fig. 2). It is noteworthy that basidiomycetes and zygomycetes have no homologous of envl/vvd, indicating that another mechanism for photoadaptation is operating in them. It has been observed that there are light responses even in Dblr mutants (see Sections 6 & 11.1), which suggests that Trichoderma species should have additional genes encoding functional photoreceptors. Indeed, there is a CPD photolyase (PHR1), a DASH-cryptochrome (Ta-12806, Tv-28006 and Tr-59726) and a cryptochrome/6-4 photolyase (Ta-86846, Tv-37166 and Tr-77473) encoded in its genome (Fig. 2). In contrast, in the N. crassa genome there is one CPD photolyase and a DASH cryptochrome, and A. nidulans has only one CPD photolyase but with cryptochrome like roles. The cryptochromes/6-4 photolyases of Trichoderma form part of the animal cryptochromes and 6-4 photolyases subfamily and have a COOH-terminal extension of 79 aa with a similar size to animal cryptochromes and the recently described 6-4 photolyase of Cercospora zeae-maydis (Bluhm and Dunkle, 2008). Another notable difference is that the Trichoderma DASH cryptochromes have longer COOH-termini (Fig. 2) than those described in animals, plants, bacteria and other fungi. The T. atroviride Cry-DASH has a relatively short extension (391 amino acids) compared to those of T. virens and T. reesei, 710 and 661 amino acids long, respectively. This region is highly variable among all three Trichoderma species and it showed no homology to any other known protein in the databases, suggesting that the Trichoderma cryptochromes might form a subdivision with novel features within DASH-cryptochromes.
In Trichoderma, red light provokes a reduction in mycelial growth and has also an impact on the transcriptional regulation of some genes, suggesting the participation of a phytochrome in these responses (Casas-Flores et al., 2004; Rosales-Saavedra et al., 2006). As found in many fungal genomes, Trichoderma species, have a phytochrome containing putative GAF, phytochrome, histi-dine kinase, and receiver domains, characteristic of these type of photoreceptors (Fig. 2).
The impact of light on the cAMP-signaling pathway suggests that a GPCR (G-Protein Coupled Receptor) photoreceptor could be
involved in this signaling pathway. Surprisingly, among Tricho-derma species only the T. atroviride genome contains a gene encoding a putative sensory opsin, containing a bacteriorhodopsin superfamily domain (Ta-83833; Fig. 2), with homology to opsins ORP-1 (NCU01735.3; opsin related protein, 46% identity) and NOP-1 (NCU10055; 27% identity) from N. crassa (Borkovich et al., 2004), among others. However, the known light responses of T. atroviride and T. reesei are very similar, suggesting that opsin plays only a minor role, if any, in the photobiology of these species.
Homologs of other major components of the light response machinery in N. crassa and D. melanogaster (FRQ1, FWD1, BLI1, SHAGGY, TIMELESS) appear to be present in T. reesei, T. virens and T. atroviride. Even though homologs of FREQUENCY (frq) are present in the Trichoderma species genomes, and the T. atroviride gene is expressed, no obvious sign of circadian rhythms (Brunner and Kaldi, 2008) or their regulation by BLR1/BLR2 or ENVOY has been reported in these fungi.
6. Effects on gene expression
The use of cDNA microarrays representing 1438 T. atroviride genes allowed the discovery of 40 light-regulated genes, 30 of which were up-regulated and ten down-regulated. Surprisingly, not all light responsive genes are regulated by the BLR proteins, but all those regulated through them contain in their promoter regions GATA elements, similar to the LRE (light response element) consensus sequence described in light-regulated genes in N. crassa (He and Liu, 2005; Rosales-Saavedra et al., 2006). Although the participation of LREs in Trichoderma light responses has not been demonstrated, it would seem that those cis-elements might be functionally conserved among fungi. In fact, the envl gene driven by its own promoter was regulated by light in N. crassa, in a similar manner as in Trichoderma (Schmoll et al., 2005). The fact that the promoter region of the blue light down-regulated genes of T. atro-viride also contains LRE-like elements, suggest a more complex mechanism for the control of transcriptional activity by the BLR proteins in this fungus, where combined arrangements of cis acting elements and additional trans-factors (i.e., co-activators or co-repressors) can be decisive for the regulatory output (Rosales-Saavedra et al., 2006).
In a recent genome wide analysis of gene expression using pyrosequencing, 331 early light-regulated genes were identified, 70 of which appear to be blr-independent, supporting the existence of additional, functional, light receptors. This set includes genes encoding transcription factors, DNA repair enzymes, and metabolic enzymes. Interestingly, 39 out of the 178 light-induced genes are related to stress responses. Significantly, seventeen of these stress-induced genes are related to oxidative stress. This set of genes includes key elements such as components of the MAPK (p38/Hog1) cascade. In N. crassa, the corresponding gene (os-2) is also induced by light and its phosphorylation shows circadian oscillations, regulating rhythmic expression of output genes (de Paula et al., 2008). These data suggest that light is perceived as a stress signal impacting a MAPK cascade, perhaps anticipating harmful effects of light, to provide protection against them.
In T. reesei, ENVOY influences gene expression not only in the light but also in darkness (Schuster et al., 2007). Nevertheless, ENVOY is dependent on the presence of additional - presumably light responsive - factors to perform its regulatory function. The variety of genes found to be regulated by light and partially by ENVOY in T. reesei ranges from those involved in transcription, translation and signal transduction to genes involved in metabolism and transport (Schuster et al., 2007).
Hydrophobins are also induced by light in Trichoderma species. In T. reesei it was shown that transcription of hfb2 is influenced by
light (Nakari-Setala et al., 1997) and that the G-protein alpha subunit GNA1 is involved in this regulation (Seibel et al., 2009). Moreover, in T. atroviride the photoreceptors BLR1 and BLR2 contribute to adjustment of hydrophobin levels to environmental conditions (Mikus et al., 2009). The mechanism triggering hydrophobin gene expression in Trichoderma is likely to be highly sophisticated and involves light dependent splicing of pre-mRNA (Vargovic et al., 2006).
7. Impact of light on cellulase production
The first report identifying the signal transduction pathways that are involved in regulating cellulase gene expression in T. reesei was published only in 2005. Surprisingly, the first signaling component involved in regulation of cellulase gene expression was the light-regulatory protein ENVOY (Schmoll et al., 2005, 2004). It was demonstrated that transcription of cellulase genes increased during growth on cellulose in constant light compared to constant darkness. Subsequently, ENVOY was shown to be involved in the regulation of cellulase formation as well as in the light signal transduction process (Schmoll et al., 2005). Later, it was found that the T. reesei photoreceptor complex BLR1/BLR2 regulates cellulase gene expression and hence corroborates the link between cellulase gene expression and light response (Castellanos et al., 2010).
8. Utilization of different carbon sources
If fungi are grown under suboptimal conditions, differences in growth in light and darkness can be observed (Carlisle, 1965). For T. atroviride this phenomenon is confirmed by experiments analyzing growth of this fungus on numerous carbon sources in light and darkness (Friedl et al., 2008a,b). Hence the presence or absence of light appears to be relevant for utilization and/or uptake of a specific carbon source (Tisch and Schmoll, 2009) also in Trich-oderma species.
The initial evidence of the direct influence of light on carbon metabolism came from the fact that T. atroviride mutants in either blrl or blr2 were unable to produce conidia in response to a sudden deprivation of carbon source (Casas-Flores et al., 2004). Analysis of conidiation of T. atroviride on various carbon sources revealed that this process is strongly carbon source dependent both in light and darkness and that light only plays a catalytic role enhancing the extent of conidiation. Interestingly, conidiation was not completely repressed in the largely blind photoreceptor mutants Ablr1 and Ablr2, but still occurred on several carbon sources (Friedl et al., 2008a). These different conidiation patterns are suggested to be due to different redox potentials upon catabolism of the various carbon sources used. This suggestion is in agreement with the hypothesis that BLR1 and BLR2 could act as redox and oxygen sensors (Casas-Flores et al., 2006).
However, not only conidiation, but also vegetative growth on different carbon sources is influenced by light in T. atroviride and here mutants in blrl and blr2 show a different residual response to light. From the growth patterns on these carbon sources it can be concluded that BLR1 is responsible for carbon source selectivity, but that the intensity of the response requires both BLR proteins (Friedl et al., 2008b). Consistent with these data, a comparable study in T. reesei also revealed an enhanced growth rate as well as indications of an oxidative stress response in light on several carbon sources. However, in contrast to the photoreceptor mutants studied in T. atroviride, which still showed a residual light response with respect to growth and no apparent growth defect in light, similar experiments with the T. reesei envl deletion strain showed a severe growth phenotype on all but one carbon source (Schuster et al., 2007).
9. Sulfur metabolism
Many sulfur compounds; especially cysteine, methionine and S-adenosylmethionine are essential for the viability of most cells. Thus, many organisms have developed a complex regulatory circuit that governs the expression of enzymes involved in sulfur assimilation and metabolism (reviewed by Marzluf (1997)). Nevertheless, a relevance of light for this process has only been investigated in T. reesei so far. Intriguingly, it was shown that the uptake of sulfate is dependent on the light status, and crucial for growth on cellulose in light and cannot be compensated by addition of the organic sulfur source methionine.
Transcription of the sulfur control gene liml, which encodes an E3 ubiquitin ligase, is responsive to light as well as cellulase inducing conditions and regulated by ENVOY. In correlation with this result, the organic sulfur source methionine impacts cellulase gene transcription positively in darkness, but negatively in light. Therefore the presence and nature of the sulfur source must have a light dependent significance beyond just signaling the availability of a nutrient (Gremel et al., 2008).
10. Biosynthesis of secondary metabolites
Trichoderma species produce peptaibols, which are important for its antagonistic activity and represent a potential alternative to conventional antibiotics (Duclohier, 2007; Kubicek et al., 2007; Szekeres et al., 2005). The biosynthesis of peptaibols is associated with initiation of sporulation and influenced by light. Biosynthesis of secondary metabolites by fungi is dependent on the light status in many cases (Tisch and Schmoll, 2009). Therefore it is not surprising that no peptaibols have been detected in T. atroviride mutants of blrl or blr2, hence indicating that the photoreceptors of Trichoderma play an important role in regulation of peptaibol production. However, this response is also observed upon starvation and in this case independent of light or the BLR proteins. Consequently, they are not essential for this process (Komon-Zelazowska et al., 2007).
11. Links to other signaling pathways
ll.l. Cyclic AMP
The effects of light in the physiology of Trichoderma include the hyperpolarization of the plasma membrane (Gresik et al., 1991; Horwitz et al., 1984b), increase in intracellular ATP levels, increase in the activity of adenylate cyclase, and a transient biphasic oscillation in intracellular cAMP levels, and protein phosphorylation. Such changes are associated with the activation of a signaling pathway modulated by cAMP (Gresik et al., 1988; Kolarova et al., 1992). The rapid changes in membrane potential and increase of both metabolites suggested the participation of a transmembrane receptor (Gresik et al., 1988), perhaps associated with heterotri-meric G proteins.
The addition of an analog of cAMP (dibutyryl-cAMP) to a colony growing in the dark, triggers conidiation, while that of an adenyl-ate cyclase inhibitor (atropin) blocks light induced conidiation (Berrocal-Tito et al., 2000). Neither of these inhibitors alters the transcriptional activation of phr-l, suggesting the existence of two at least partially independent signaling pathways that are activated by blue light. In this sense, Casas-Flores and coworkers (Casas-Flores et al., 2006) observed an increase in cAMP dependent protein kinase (PKA) activity after a pulse of blue light. This activation occurred even in the Dblr-1 and Dblr-2 mutant strains, confirming the existence of an alternative system for light perception linked to cAMP. They also showed that transformants
expressing an antisense version of pkr-1, a gene encoding the regulatory subunit ofPKA, which have increased levels of PKA activity, did not produce conidia when a pulse of blue light was applied. In contrast, decreased levels of PKA activity achieved by overexpression of the pkr-1 gene, result in the production of conidia even in the dark. This evidence suggests complex mechanisms involved in the cAMP-signaling pathway that regulate asexual reproduction in T. atroviride. However, the light receptor responsible for the activation of the cAMP-pathway has not been identified.
In T. atroviride PKA activity has a direct impact not only on photoconidiation, but also on the expression of genes regulated by blue light through the BLR proteins (Casas-Flores et al., 2006). Transformants with low levels of PKA activity are blocked in the activation of gene expression regulated by blue light. These results are clear evidence that PKA is involved in light perception, and it has been postulated that the BLR proteins could be a substrate for PKA, or that this kinase could regulate a factor necessary for the transcriptional activity of the BLR complex (Casas-Flores et al., 2006).
11.2. Heterotrimeric G protein signaling
In all Trichoderma species investigated so far, three G-protein a-subunits and one p-subunit as well as one G-protein y subunit have been detected (Schmoll, 2008); Schmoll, unpublished data). In the course of studies on light dependent cellulase regulation, a clear impact of light on signaling via the heterotrimeric G-protein pathway became obvious. The most promising candidate for a function not only in cellulase gene expression, but also light response was GNA3, because of its putative interrelationship with cAMP signaling, which plays a role in both processes in Tricho-derma (Sestak and Farkas, 1993; Tisch and Schmoll, 2009).
Transcription of gna3 was indeed found to be significantly stimulated by light. Analysis of a recombinant strain expressing a con-stitutively active version of GNA3 resulted in strong light dependent up-regulation of the major cellulase cbhl (cellobiohy-drolase 1) transcript. Nevertheless, this G-alpha subunit is not solely responsible for cellulase induction, since the presence of an
inducer is still required (Schmoll et al., 2009). In addition to the light dependent function of GNA3 in cellulase gene expression in T. reesei also a function in peptaibol biosynthesis was observed in T. atroviride. Deletion of gna3 abolished peptaibol formation, despite illumination or addition of cAMP and irrespective of conidia-tion (Komon-Zelazowska et al., 2007). Hence the signal transmitted by GNA3 is essential for peptaibol production in contrast to the light signal.
The second G-protein alpha subunit investigated in T. reesei (GNA1), is also involved in the light dependent regulation of cellu-lase gene transcription albeit in a different way than GNA3. In a T. reesei mutant strain lacking the gnal gene no transcription of cbhl in light on cellulose could be observed, whereas a strong increase in transcript abundance was detected under the same conditions in darkness (Seibel et al., 2009). Nevertheless, also in the case of this G-alpha subunit, expression of a constitutively active version of GNA1 did not lead to inducer independent expression of the cel-lulase genes on the repressing carbon sources glucose and glycerol or cause relieve from carbon catabolite repression. Hence, GNA1 is involved in light dependent regulation of cellulase gene expression, but not essential for induction of this process. In T. atroviride, the ortholog of gnal (tgal) was shown to play a role as negative regulator of conidiation. Transformants silenced in this gene formed conidia constitutively. In contrast, transformants over-expressing the gene or expressing a constitutively active allele were unable to produce conidia in response to light (Rocha-Ramirez et al., 2002). Intriguingly, these effects on conidiation were not observed in T. reesei (Seibel et al., 2009).
12. Perspectives
Although considerable progress has been made in elucidating the mechanisms of light response in Trichoderma and other fungi, we still lack a comprehensive understanding on how this machinery works and how its function is modulated under different environmental conditions. Our first insights into the light signaling process and its implications in Trichoderma species as integrated from the data reviewed above are summarized in Fig. 3. It will be
Fig. 3. Diagram of the signaling network involved in regulation of light responses in Trichoderma species. The diagram has been divided in two sections for simplicity. (A) The photoreceptor complex BLR1/BLR2 controls expression of blue light up-regulated (blu) and down-regulated (bld) genes. The BLR proteins control transcription of the envl gene. ENV1 in turn establishes a feedback regulatory loop with the BLR proteins, and is involved in photoadaptation. Blu genes impact stress tolerance and DNA repair. Both blu and bld genes regulate growth. The photoreceptor BLR1/BLR2 is also crucial for light dependent carbon source utilization. In addition, light causes oxidative stress and influences peptaibol biosynthesis, which is also regulated by the G-protein alpha subunit GNA3. (B) The BLR1/BLR2 complex regulates photoconidiation, which is also influenced by a second light input involving the cAMP-pathway, constituted by adenylate cyclase (ACY), cAMP, and protein kinase A (PKA). Light enhanced cellulase gene expression involves the function of the photoreceptors BLR1/BLR2 and ENV1, the sulfur signaling pathway, the heterotrimeric G-protein alpha subunits GNA1 and GNA3 and the cAMP-pathway. Arrows indicate positive regulation and lines with a bar at the end indicate negative regulation. Solid lines are used when there is direct experimental evidence and dotted lines when the role is still hypothetical. Overlapping lines appear in gray for clarity.
important to delineate the pathways that directly or indirectly interact with components of the light signaling machinery in order to establish how a light signal is transmitted and integrated into other physiological pathways. Genome wide analyses of gene expression suggest that light may be a cue for the fungus to protect itself from the harmful effects of light, the fungus would in turn activate a variety of mechanisms to ensure its preservation. However, the link of the light signal to other stress responses requires further investigation. Similarly, it will be important to determine whether light-induced ROS have detrimental consequences or function as signaling molecules in light responses (Aguirre et al., 2005). Knowledge on the underlying processes will not only enable us to exploit these properties for the countless applications of Trichoderma species, but we will also gain a deeper insight into how fungi manage to survive and succeed in their natural habitat.
Acknowledgements
This work was supported by Grant P20004 of the Austrian Science Fund (FWF) to MS. MS is recipient of an APART fellowship of the Austrian Academy of Sciences. The research lines of AH-E described in this manuscript are currently supported by Conacyt project 83798.
References
Aguirre, J., Rios-Momberg, M., Hewitt, D., Hansberg, W., 2005. Reactive oxygen species and development in microbial eukaryotes. Trends Microbiol. 13, 111— 118.
Ballario, P., Vittorioso, P., Magrelli, A., Talora, C., Cabibbo, A., Macino, G., 1996. White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein. EMBO J. 15, 1650-1657. Bayram, O., Biesemann, C., Krappmann, S., Galland, P., Braus, G.H., 2008. More than a repair enzyme: Aspergillus nidulans photolyase-like CryA is a regulator of sexual development. Mol. Biol. Cell 19, 3254-3262. Bergman, K., Burke, P.V., Cerda-Olmedo, E., David, C.N., Delbruck, M., Foster, K.W., Goodell, E.W., Heisenberg, M., Meissner, G., Zalokar, M., Dennison, D.S., Shropshire Jr., W., 1969. Phycomyces. Bacteriol. Rev. 33, 99-157. Berrocal-Tito, G., Sametz-Baron, L., Eichenberg, K., Horwitz, B.A., Herrera-Estrella, A., 1999. Rapid blue light regulation of a Trichoderma harzianum photolyase gene. J. Biol. Chem. 274, 14288-14294. Berrocal-Tito, G.M., Rosales-Saavedra, T., Herrera-Estrella, A., Horwitz, B.A., 2000. Characterization of blue-light and developmental regulation of the photolyase gene phrl in Trichoderma harzianum. Photochem. Photobiol. 71, 662-668. Berrocal-Tito, G.M., Esquivel-Naranjo, E.U., Horwitz, B.A., Herrera-Estrella, A., 2007. Trichoderma atroviride PHR1, a fungal photolyase responsible for DNA repair, autoregulates its own photoinduction. Eukaryot. Cell 6, 1682-1692. Bluhm, B.H., Dunkle, L.D., 2008. PHL1 of Cercospora zeae-maydis encodes a member of the photolyase/cryptochrome family involved in UV protection and fungal development. Fungal Genet. Biol. 45,1364-1372. Borkovich, K.A., Alex, L.A., Yarden, O., Freitag, M., Turner, G.E., Read, N.D., Seiler, S., Bell-Pedersen, D., Paietta, J., Plesofsky, N., Plamann, M., Goodrich-Tanrikulu, M., Schulte, U., Mannhaupt, G., Nargang, F.E., Radford, A., Selitrennikoff, C., Galagan, J.E., Dunlap, J.C., Loros, J.J., Catcheside, D., Inoue, H., Aramayo, R., Polymenis, M., Selker, E.U., Sachs, M.S., Marzluf, G.A., Paulsen, I., Davis, R., Ebbole, D.J., Zelter, A., Kalkman, E.R., O'Rourke, R., Bowring, F., Yeadon, J., Ishii, C., Suzuki, K., Sakai, W., Pratt, R., 2004. Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol. Mol. Biol. Rev. 68,1-108.
Brunner, M., Kaldi, K., 2008. Interlocked feedback loops of the circadian clock of
Neurospora crassa. Mol. Microbiol. 68, 255-262. Carlisle, M.J., 1965. The photobiology of fungi. Ann. Rev. Plant Physiol. 16,175-202. Casas-Flores, S., Rios-Momberg, M., Bibbins, M., Ponce-Noyola, P., Herrera-Estrella, A., 2004. BLR-1 and BLR-2, key regulatory elements of photoconidiation and mycelial growth in Trichoderma atroviride. Microbiology 150, 3561-3569. Casas-Flores, S., Rios-Momberg, M., Rosales-Saavedra, T., Martinez-Hernandez, P., Olmedo-Monfil, V., Herrera-Estrella, A., 2006. Cross talk between a fungal blue-light perception system and the cyclic AMP signaling pathway. Eukaryot. Cell 5, 499-506.
Castellanos, F., Schmoll, M., Martinez, P., Tisch, D., Kubicek, C.P., Herrera-Estrella, A., Esquivel-Naranjo, E.U., 2010. Crucial factors of the light perception machinery and their impact on growth and cellulase gene transcription in Trichoderma reesei. Fungal Genet. Biol. 47, 468-476. Darwin, C., 1880. The Power of Movement in Plants. John Murria, London. de Paula, R.M., Lamb, T.M., Bennett, L., Bell-Pedersen, D., 2008. A connection
between MAPK pathways and circadian clocks. Cell Cycle 7, 2630-2634. Duclohier, H., 2007. Peptaibiotics and peptaibols: an alternative to classical antibiotics? Chem. Biodivers. 4,1023-1026.
Esquivel-Naranjo, E.U., Herrera-Estrella, A., 2007. Enhanced responsiveness and sensitivity to blue light by blr-2 overexpression in Trichoderma atroviride. Microbiology 153, 3909-3922.
Eveleigh, D., 1985. Trichoderma. In: Demain, A.L., Solomon, N.A. (Eds.), Biology of Industrial Microorganisms. Butterworths, Menlo Park, CA.
Friedl, M.A., Kubicek, C.P., Druzhinina, I.S., 2008a. Carbon source dependence and photostimulation of conidiation in Hypocrea atroviridis. Appl. Environ. Microbiol. 74, 245-250.
Friedl, M.A., Schmoll, M., Kubicek, C.P., Druzhinina, I.S., 2008b. Photostimulation of Hypocrea atroviridis growth occurs due to a cross-talk of carbon metabolism, blue light receptors and response to oxidative stress. Microbiology 154,12291241.
Galun, E., 1971. Scanning electron microscopy of intact Trichoderma colonies. J. Bacteriol. 108, 938-940.
Galun, E., Gressel, J., 1966. Morphogenesis in Trichoderma: suppression of photoinduction by 5-fluorouracil. Science 151, 696-698.
Gremel, G., Dorrer, M., Schmoll, M., 2008. Sulphur metabolism and cellulase gene expression are connected processes in the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei). BMC Microbiol. 8,174.
Gresik, M., Kolarova, N., Farkas, V., 1988. Membrane potential, ATP, and cyclic AMP changes induced by light in Trichoderma viride. Exp. Mycol. 12, 295-301.
Gresik, M., Kolarova, N., Farkas, V., 1991. Hyperpolarization and intracellular acidification in Trichoderma viride as a response to illumination. J. Gen. Microbiol. 137, 2605-2609.
Gressel, J., 1979. Yearly review: blue light photoreception. Photochem. Photobiol. 30, 749-754.
Gressel, J., Galun, E., 1967. Morphogenesis in Trichoderma: photoinduction and RNA. Dev Biol. 15, 575-598.
Gressel, J., Bar-Lev, S., Galun, E., 1975. Blue light induced response in the absence of free oxygen. Plant Cell Physiol. 16, 367-370.
Gutter, Y., 1957. Effect of light in sporulation of Trichoderma viride. Bull. Res. Council Israel Sect. D 5, 273-286.
He, Q., Liu, Y., 2005. Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation. Genes Dev. 19, 2888-2899.
Heintzen, C., Loros, J.J., Dunlap, J.C., 2001. The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting. Cell 104, 453-464.
Herrera-Estrella, A., Horwitz, B.A., 2007. Looking through the eyes of fungi: molecular genetics of photoreception. Mol. Microbiol. 64, 5-15.
Horwitz, B.A., Malkin, S., Gressel, J., 1984a. Roseoflavin inhibition of photoconidiation in a Trichoderma riboflavin auxotroph: indirect evidence for flavin requirement for photoreactions. Photochem. Photobiol. 40, 763-769.
Horwitz, B.A., Weisenseel, M.H., Dorn, A., Gressel, J., 1984b. Electric currents around growing Trichoderma hyphae, before and after photoinduction of conidiation. Plant Physiol. 74, 912-916.
Horwitz, B.A., Gressel, J., Malkin, S., Epel, B.L., 1985. Modified cryptochrome in vivo absorption in dim photosporulation mutants of Trichoderma. Proc. Natl. Acad. Sci. USA 82, 2736-2740.
Horwitz, B.A., Trad, C.H., Lipson, E.D., 1986. Modified light-induced absorbance changes in dim Y photoresponse mutants of Trichoderma. Plant Physiol. 81, 726-730.
Horwitz, B.A., Perlman, A., Gressel, J., 1990. Induction of Trichoderma sporulation by nanosecond laser pulses: evidence against cryptochrome cycling. Photochem. Photobiol. 51, 99-104.
Kolarova, N., Haplova, J., Gresik, M., 1992. Light-activated adenyl cyclase from Trichoderma viride. FEMS Microbiol. Lett. 72, 275-278.
Komon-Zelazowska, M., Neuhof, T., Dieckmann, R., von Dohren, H., Herrera-Estrella, A., Kubicek, C.P., Druzhinina, I.S., 2007. Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated by blue light and the G protein GNA3. Eukaryot. Cell 6, 2332-2342.
Kubicek, C.P., Komon-Zelazowska, M., Sandor, E., Druzhinina, I.S., 2007. Facts and challenges in the understanding of the biosynthesis of peptaibols by Trichoderma. Chem. Biodiv. 4,1068-1082.
Kuhls, K., Lieckfeldt, E., Samuels, G.J., Kovacs, W., Meyer, W., Petrini, O., Gams, W., Borner, T., Kubicek, C.P., 1996. Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. Proc. Natl. Acad. Sci. USA 93, 7755-7760.
Kumagai, T., Oda, Y., 1969. An action spectrum for photoinduced sporulation in the fungus Trichoderma viride. Plant Cell Physiol. 10, 387-392.
Linden, H., Macino, G., 1997. White collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa. EMBO J. 16, 98-109.
Liu, Y., He, Q., Cheng, P., 2003. Photoreception in Neurospora: a tale of two White Collar proteins. Cell Mol. Life Sci. 60, 2131-2138.
Lledias, F., Hansberg, W., 2000. Catalase modification as a marker for singlet oxygen. Methods Enzymol. 319, 110-119.
Martinez, D., Berka, R.M., Henrissat, B., Saloheimo, M., Arvas, M., Baker, S.E., Chapman, J., Chertkov, O., Coutinho, P.M., Cullen, D., Danchin, E.G., Grigoriev, I.V., Harris, P., Jackson, M., Kubicek, C.P., Han, C.S., Ho, I., Larrondo, L.F., de Leon, A.L., Magnuson, J.K., Merino, S., Misra, M., Nelson, B., Putnam, N., Robbertse, B., Salamov, A.A., Schmoll, M., Terry, A., Thayer, N., Westerholm-Parvinen, A., Schoch, C.L., Yao, J., Barbote, R., Nelson, M.A., Detter, C., Bruce, D., Kuske, C.R., Xie, G., Richardson, P., Rokhsar, D.S., Lucas, S.M., Rubin, E.M., Dunn-Coleman, N., Ward, M., Brettin, T.S., 2008. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat. Biotechnol. 26, 553-560.
Marzluf, G.A., 1997. Molecular genetics of sulfur assimilation in filamentous fungi and yeast. Annu. Rev. Microbiol. 51, 73-96.
Mikus, M., Hatvani, L., Neuhof, T., Komon-Zelazowska, M., Dieckmann, R., Schwecke, T., Druzhinina, I.S., von Dohren, H., Kubicek, C.P., 2009. Differential regulation and posttranslational processing of the class II hydrophobin genes from the biocontrol fungus Hypocrea atroviridis. Appl. Environ. Microbiol. 75,3222-3229.
Nakari-Setala, T., Aro, N., Ilmen, M., Munoz, G., Kalkkinen, N., Penttila, M., 1997. Differential expression of the vegetative and spore-bound hydrophobins of Trichoderma reesei - cloning and characterization of the hfb2 gene. Eur. J. Biochem. 248, 415-423.
Rocha-Ramirez, V., Omero, C., Chet, I., Horwitz, B.A., Herrera-Estrella, A., 2002. Trichoderma atroviride G-protein alpha-subunit gene tgai is involved in mycoparasitic coiling and conidiation. Eukaryot. Cell 1, 594-605.
Rosales-Saavedra, T., Esquivel-Naranjo, E.U., Casas-Flores, S., Martinez-Hernandez, P., Ibarra-Laclette, E., Cortes-Penagos, C., Herrera-Estrella, A., 2006. Novel lightregulated genes in Trichoderma atroviride: a dissection by cDNA microarrays. Microbiology 152, 3305-3317.
Sancar, A., 1996. No ''End of History" for photolyases. Science 272, 48-49.
Schmoll, M., 2008. The information highways of a biotechnological workhorse -signal transduction in Hypocrea jecorina. BMC Genomics 9, 430.
Schmoll, M., Zeilinger, S., Mach, R.L., Kubicek, C.P., 2004. Cloning of genes expressed early during cellulase induction in Hypocrea jecorina by a rapid subtraction hybridization approach. Fungal Genet. Biol. 41, 877-887.
Schmoll, M., Franchi, L., Kubicek, C.P., 2005. Envoy, a PAS/LOV domain protein of Hypocrea jecorina (Anamorph Trichoderma reesei), modulates cellulase gene transcription in response to light. Eukaryot. Cell 4,1998-2007.
Schmoll, M., Schuster, A., do Nascimento Silva, R., Kubicek, C.P., 2009. The G-alpha protein GNA3 of Hypocrea jecorina (anamorph Trichoderma reesei) regulates cellulase gene expression in the presence of light. Eukaryot. Cell 8, 410-420.
Schuster, A., Schmoll, M., 2009. Heterotrimeric G-protein signaling and light response: two signaling pathways coordinated for optimal adjustment to nature. Commun. Integr. Biol. 2, 308-310.
Schuster, A., Kubicek, C.P., Friedl, M.A., Druzhinina, I.S., Schmoll, M., 2007. Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process. BMC Genomics 8, 449.
Schwerdtfeger, C., Linden, H., 2001. Blue light adaptation and desensitization of light signal transduction in Neurospora crassa. Mol. Microbiol. 39,1080-1087.
Schwerdtfeger, C., Linden, H., 2003. VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. EMBO J. 22, 4846-4855.
Seibel, C., Gremel, G., Silva, R.D., Schuster, A., Kubicek, C.P., Schmoll, M., 2009. Light-dependent roles of the G-protein subunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei). BMC Biol. 7, 58.
Seidl, V., Seibel, C., Kubicek, C.P., Schmoll, M., 2009. Sexual development in the industrial workhorse Trichoderma reesei. Proc. Natl. Acad. Sci. USA 106,1390913914.
Sestak, S., Farkas, V., 1993. Metabolic regulation of endoglucanase synthesis in Trichoderma reesei: participation of cyclic AMP and glucose-6-phosphate. Can. J. Microbiol. 39, 342-347.
Szekeres, A., Leitgeb, B., Kredics, L., Antal, Z., Hatvani, L., Manczinger, L., Vagvolgyi, C., 2005. Peptaibols and related peptaibiotics of Trichoderma. A review. Acta Microbiol. Immunol. Hung. 52,137-168.
Tisch, D., Schmoll, M., 2009. Light regulation of metabolic pathways in fungi. Appl. Microbiol. Biotechnol. 85, 1259-1277.
Vargovic, P., Pokorny, R., Holker, U., Hofer, M., Varecka, L., 2006. Light accelerates the splicing of srhl homologue gene transcripts in aerial mycelia of Trichoderma viride. FEMS Microbiol. Lett. 254, 240-244.
