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Research Area of Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9-1665, A-1060 Vienna, Austria
Correspondence
Irina S. Druzhinina
druzhini{at}mail.zserv.tuwien.ac.at
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Cerdá-Olmedo & Corrochano, 2001; Berrocal-Tito et al., 1999; Nagahashi & Douds, 2004). It also enhances the formation of pigments in order to protect the organisms against the deleterious effects of UV light (Li & Schmidhauser, 1995; Arrach et al., 2001). In Neurospora crassa, all light-induced phenotypes are dependent on at least one of the two regulators white-collar-1 (WC-1; Linden, 2002) and white-collar-2 (WC-2; Linden et al., 1997). These two proteins contain a zinc finger domain and a PAS domain through which they interact physically to form the heteromultimeric ‘white-collar complex’, which can then recruit further protein components (Corrochano, 2007). The WC-1 protein also functions as a blue light receptor via its LOV domain and by its binding of an FAD chromophore (Liu et al., 2003). Idnurm & Heitman (2005) recently demonstrated that the WC-1/WC-2 proteins of N. crassa are present in all asco- and basidiomycetous taxa, and represent an evolutionarily ancient and conserved system to control light-dependent processes.
Apart from the aforementioned studies, knowledge on other physiological properties of fungi that are influenced by light is scarce. Some authors have described effects of light on hyphal growth or branching, thereby mostly referring to inhibitory effects (Lauter et al., 1998; Chen & Dickman, 2002; Ambra et al., 2004; Casas-Flores et al., 2004; Flaherty & Dunkle, 2005; Miyake et al., 2005; Idnurm & Heitman, 2005; Brasch & Kay, 2006). For arbuscular mycorrhizal fungi, low-intensity light is also known to induce hyphal branching and the consequent increase of mycelial biomass (Nagahashi & Douds, 2003). This physiological response is explained by the improved probability of colonizing roots of young seedlings and establishing new arbuscular mycorrhizal relations. The same authors later showed the essential increase of hypal branching of Gigaspora gigantean due to synergetic effect of light and root exudate compounds (Nagahashi & Douds, 2004).
The photostimulation of the sporulation of soil fungus Trichoderma (mitosporic Hypocrea, Hypocreales, Ascomycota) has been known for many years. Gressel et al. (1971) showed that in Hypocrea atroviridis (Trichoderma viride in the original publication) the animal neurotransmitter acetylcholine stimulated asexual sporulation as efficiently as light. This fungus (incorrectly named T. viride or Trichoderma harzianum in many earlier papers) has been used as a simple experimental model to study photoinduced conidiation (for review see Betina & Farkas, 1998). As in other fungi, in Hypocrea/Trichoderma this process also depends on the function of WC-1/WC-2 orthologues (blr-1 and blr-2; Casas-Flores et al., 2004). Gresik et al. (1989, 1991) demonstrated that the stimulation of conidiation by light in H. atroviridis involves the cAMP/protein kinase signalling pathway. A cross-talk between a fungal blue light perception system and the cAMP signalling pathway has indeed recently been demonstrated in H. atroviridis (Casas-Flores et al., 2006).
The influence of light on fungal development may not be restricted to sporulation: we have recently observed that in another Hypocrea/Trichoderma species – Hypocrea jecorina (=Trichoderma reesei) – light stimulates growth of the fungus on cellulose (Schmoll et al., 2005). Also, Chovanec et al. (2001) reported that light stimulated hyphal growth of H. atroviridis on several hexoses as carbon sources. The objective of the present paper was (i) to test these findings with a broad range of carbon sources to arrive at a general picture; (ii) to investigate whether the two blue light receptors BLR-1 and BLR-2 are involved in this phenomenon; and (iii) to obtain insights into the physiological relevance of this finding.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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blr-1 and blr-2 mutants derived from it (Casas-Flores et al., 2004) were used throughout this study. All cultures were maintained on plates containing malt extract agar (MEA) and were subcultured monthly.
Tests for growth of H. atroviridis on 95 different carbon sources.
Druzhinina et al. (2006) developed a method using Biolog Phenotype MicroArrays to characterize carbon source utilization profiles (CSUPs) of wild-type and mutant strains of H. jecorina. Here we applied this technique to investigate the effect of constant and rhythmic light on CSUPs of H. atroviridis. For inoculum preparation, conidia of the parental strain IMI 206040 were obtained by cultivation on 3 % MEA plates for 5 days. In the mutant strains blr-1 and blr-2, conidiation was stimulated by the injury of mycelia with a cold sterile scalpel (Casas-Flores et al., 2004). Conidial inocula were prepared by rolling a sterile, wetted cotton swab over sporulating areas of the plates. The conidia were then suspended in sterile Biolog FF inoculating fluid (0.25 % Phytagel, 0.03 % Tween 40), gently mixed and adjusted to a transmission of 70 % at 590 nm (using a Biolog standard turbidimeter calibrated to the Biolog standard for filamentous fungi). Ninety microlitres of the conidial suspension was dispensed into each of the wells of the Biolog FF microplates, which were incubated at 28 °C under either constant white light (1800 lx), constant darkness or 12 h white light/darkness rhythm. The optical density (OD) at 750 nm (mycelial growth) and 490 nm (mitochondrial activity) was measured after 12, 18, 24, 36, 42, 48, 60, 66, 72, 96 and 168 h using a Biolog microplate reader. Each assay was repeated three to six times in series of independent experiments. Due to a generally slower growth rate of the two blr mutants, OD750 at 66 h (not 48 h as used by Druzhinina et al., 2006) was chosen as a reference for all three strains. At this time, all strains were in the linear phase of growth on the majority of assimilated carbon sources and the respective value was therefore proportional to the growth rate.
To study the effects of addition of dibutyryl cAMP and menadione, stock solutions in sterile distilled water were prepared separately and added to the corresponding inoculation fluids at a concentration of 1 mM for both.
Data analysis.
Data from all experiments were combined in a single matrix, and analysed with the STATISTICA 6.1 (StatSoft) software package. All data were first subjected to descriptive statistical evaluations (mean, minimum, maximum and standard deviation values) and checked for outliers. Secondly, we used cluster analysis to detect possible groupings in our results. The significance of correlations was identified using product-moment or partial correlation algorithms. The significance of a hypothesis was tested by analysis of variance (ANOVA); post-hoc comparisons were done using Tukey's honest significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The resultant profile and the respective growth rates essentially resembled those obtained with H. atroviridis strain ATCC 74058 (‘P1’) (Seidl et al., 2006) (ANOVA main effect, P=0.49), indicating that the data are representative for this species. The carbon sources were divided by joining cluster analysis into four principal groups that coincided with the shape of the corresponding growth curves. Thus clusters I, II and III contain carbon sources that allow fast, moderate and slow growth, respectively (see supplementary Table S1, available with the online version of this paper), while cluster IV contains carbon sources on which H. atroviridis grows very poorly or does not grow at all. To identify a possible effect of light on growth, we used both constant illumination by intense white light (1800 lx) and alternating cycles of light and darkness (12 h each). We also verified that short exposure to light (<1 min), which was unavoidable during the reading of the microplates, did not reveal any significant effect (data not shown). Temperature was kept constant at 28 °C and was unaffected by these treatments.
The results presented in Fig. 1 show the comparisons of growth rates in complete darkness, under alternating cycles of light and darkness (12 h each) and under constant illumination. Values shown reflect growth rates and were calculated from the difference in OD750 between 24 and 66 h of incubation, where a linear increase in OD750 took place on the majority of carbon sources (absolute OD750 values for 66 h of growth on individual carbon sources are given in supplementary Table S1). The average growth rate of H. atroviridis IMI 206040 was enhanced in the presence of light (ANOVA, P=0.005 for rhythmic light and P=0.002 for constant illumination). The Tukey HSD post-hoc analysis showed that this was due to a significant stimulation of growth on 17 of the 95 carbon sources, i.e. the hexoses D-fructose, D-mannose (Fig. 1b), D-galactose and 
-D-glucose; the pentoses D-xylose (Fig. 1c) and D-ribose; the polyols D-arabinitol and D-mannitol; the -linked homoglucosides/glucans maltotriose, glycogen and D-trehalose; the -1,3-glucosyl fructoside turanose; and the β-linked disaccharides D-cellobiose, sucrose, lactulose, gentiobiose and -D-lactose (ANOVA main effect, P<0.05). Comparing these carbon sources with their position in the four clusters (I–IV) shows that most of them belonged to those which are well utilized by H. atroviridis; however, the fungus did not show a positive response towards light on all carbon sources of clusters I and II, indicating that fast growth is not a determinant of this effect. For example, growth on glycerol, the best carbon source for this fungus, was independent of light (Fig. 1d, ANOVA, P>0.05). Similarly, growth on N-acetyl-D-glucosamine, 
-aminobutyric acid (Fig. 1e), erythritol, and β-methyl-D-glucoside (all from cluster I), or on maltose, quinic acid and L-arabinose (all from cluster II) was not influenced by light. Growth on D-sorbitol was even significantly reduced by illumination (ANOVA main effect, P=0.003).
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). However, we noted that the constant presence of light also stimulated growth on a small set of carbon sources (glycogen, maltotriose, turanose, dextrin) (factorial ANOVA, P<0.05), which did not show this effect under rhythmic illumination.
Light stimulation of mycelial growth requires functional blr-1 and blr-2 genes
Having demonstrated a photostimulation of H. atroviridis growth on selected carbohydrates and related carbon sources, we tested whether the blue light receptors BLR-1 and BLR-2 are involved in this response. We first studied the CSUPs of the two knockout mutants blr-1 and blr-2 of H. atroviridis IMI 206040. With the exception that both mutants grew slightly slower than IMI 206040 on all carbon sources (see supplementary Table S1), the CSUPs in darkness were essentially the same for both mutants (r=0.9917, P<0.05, product-moment correlation coefficient), and also very similar to those of the parent strain (r=0.9140 for blr-1 and r=0.9058 for blr-2). In order to investigate whether the blr mutants lack the photostimulation of growth, we plotted the growth rates of the parent strain and the two mutants in darkness and alternating illumination, respectively, against each other (Fig. 2a). In this plot, photostimulation is evident if the respective data points are clearly above the 4 ° straight line. If the mean value of the observed ratio between rhythmic illumination and darkness is calculated for the two mutant strains, they are not sensitive to light, but in contrast are inhibited by it, and this is even stronger in blr-2 (Fig. 2a; ANOVA, main effect, P=0.76). However, a detailed inspection of the data revealed that the lack of stimulation by light is not complete, and different exceptions are noted for the two mutants. Rhythmic light clearly enhanced the growth rate of blr-1 on some carbon sources while the effect was not observed under constant illumination (Fig. 2b). Interestingly, photostimulation in blr-1 was also detected on N-acetyl-D-glucosamine, maltose, i-erythritol, L-proline and L-ornithine, on which the parent strain showed no photostimulation, indicating a different response of this mutant to light. A different pattern was observed for the blr-2 mutant, which did not differentiate between rhythmic light and darkness but was sensitive to the effect of constant illumination (Fig. 2c). Moreover, the effect was detected mainly on those carbon sources on which the parental strain had the strongest response to light: D-mannose, sucrose, D-ribose, D-trehalose, -D-glucose, D-cellobiose and lactulose. Exceptions to this rule were i-erythritol and D-raffinose, on which growth of the mutant strain blr-2 but not of the parent strain was slightly stimulated by constant light. This finding suggests that BLR-1 is responsible for carbon source selectivity, whereas the intensity of this response requires both BLR-1 and BLR-2.
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blr-1 and blr-2 mutants found that illumination results in a rise in intracellular cAMP levels in H. atroviridis, and Casas-Flores et al. (2006) have recently identified a cross-talk between light response and cAMP. We therefore investigated whether the reduced photosensitivity of blr-1 and blr-2 mutants could be rescued by an artificial increase in the intracellular pool of cAMP. Unfortunately, mutants overproducing adenylate cyclase or with a constitutively activated protein kinase display pleiotropic phenotypes (Saudohar et al., 2002; Zhao et al., 2006), and were thus not considered useful for investigation by Phenotype MicroArrays. We therefore chose to manipulate the internal cAMP pool of the wild-type strain and blr mutants by addition of the analogue dibutyryl-cAMP which is able to penetrate the hyphae (Terenzi et al., 1976) and which is also taken up by H. atroviridis (Reithner et al., 2005; Casas-Flores et al., 2006). The results (Fig. 3) show that the addition of cAMP to cultures growing in darkness does not influence the growth rate of the parent strain in general (ANOVA main effect, P=0.09). However, on four of the carbon sources – which all belonged to the group for which we previously detected an increased growth in the presence of light (i.e. -D-glucose, gentiobiose, D-cellobiose and D-xylose, ANOVA main effect, P=0.0004, P=0.002, P=0.0000, P=0.0035, respectively) – stimulation was indeed observed (Fig. 3b, c, d). This effect did not occur in the blr-1 and blr-2 mutants (ANOVA, P>0.05; data not plotted, but shown in supplementary Table S1), indicating that the involvement of cAMP in light stimulation of growth on these carbon sources is dependent on the function of BLR-1 and BLR-2.
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blr-1 and blr-2 mutants). In order to test whether the observed photostimulation of H. atroviridis may be mimicked by exposure to conditions of oxidative stress, we decided to expose the cultures to menadione, a superoxide generator (Pocsi et al., 2005). Fig. 4(a) shows a comparison of growth rates under rhythmic light and in complete darkness with menadione added. In general, H. atroviridis IMI 206040 was able to grow in the presence of menadione on only 16 of the 95 carbon sources, and growth was completely inhibited on the others.
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). The most pronounced menadione stimulation (group I) was observed on dextrin (232 % compared to growth rate in darkness), where photostimulation by constant light was significantly stronger than the effect of rhythmic light (208 and 132 %, respectively; see also Fig. 1a). The second group (II) contained five sugars (D-cellobiose, gentiobiose, D-xylose, D-mannose and -D-glucose) on which photostimulation by light was fully mimicked by addition of menadione (Fig. 4a, b). Interestingly, four of these carbon sources (i.e. all except D-mannose) were identical to those on which growth in darkness was also stimulated by addition of cAMP (cf. Fig. 3). This may hint at a carbon-source-dependent cross-talk between photostimulation, intracellular level of cAMP and response to oxidative stress. The third group (III) contained ‘cluster I’ carbon sources, which showed strong photostimulation, but on which menadione inhibited growth rather than stimulating it (i.e. -aminobutyric acid, D-trehalose, D-fructose, D-ribose, sucrose, D-galactose, D-mannitol and D-arabitol; Fig. 4b). Groups IV, V and VII are characterized by weak photostimulation and nearly complete inhibition of growth by menadione. The statistical difference between these groups was only due to different growth rates in darkness. Interestingly, menadione completely blocked growth on glycerol – the best carbon source for H. atroviridis. The effect of menadione was the same under illumination and in darkness for all carbon sources (ANOVA, P=0.55), indicating that the operation of oxidative stress is not affected by light.
Stimulation of growth by menadione was not detected in the blr-1 and blr-2 mutants. In contrast, growth of both was inhibited by menadione on the same carbon sources as the parental strain, and was unaffected on those on which growth of the parent strain was stimulated by menadione (data not shown). However, addition of menadione completely blocked growth of both mutants on sucrose on which IMI 206040 grew in all conditions tested. When the two mutants were compared with each other, clear differences were noted on D-trehalose, D-fructose, D-arabitol, maltotriose and glycogen, where mutant blr-1 was more strongly inhibited by menadione than mutant blr-2 (Fig. 5). However, on D-cellobiose, gentiobiose and D-galactose this difference did not occur when menadione-added plates were incubated under constant illumination (Fig. 5). Thus, the resistance of both blr mutants to conditions of oxidative stress is carbon-source dependent and is notably weaker compared to the wild-type strain. Moreover, the blr-2 mutant exhibited higher menadione resistance than the blr-1 mutant
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DISCUSSION |
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Ecological aspects of photostimulation
Vegetative growth of fungi has been reported to be influenced by light, both negatively (Casas-Flores et al., 2006; Ambra et al., 2004; Flaherty & Dunkle, 2005; Schmoll et al., 2005) and positively (Lauter et al., 1998; Chen & Dickman, 2002; Chovanec et al., 2001). On a biochemical level, exposure of Aspergillus ornatus to a pulse of light led to protein phosphorylation and decreased glucose uptake (Hill, 1976), and in the slime mould Physarum polycephalum it stopped glucose consumption, and decreased the accumulation of glucans and D-trehalose (Schreckenbach et al., 1981). On the other hand, photoinduction or photostimulation of conidiation has been known for a long time and is well studied in fungi (Sulová & Farkás, 1991; Lauter et al., 1997). Chovanec et al. (2001) further noted that photostimulation or photoinhibition of growth and conidiation of H. atroviridis is dependent on the carbon sources.
On the basis of the present results, we offer the following speculation as to why these two different physiological processes are both affected by light. Species of Hypocrea/Trichoderma are soil saprotrophs and/or facultative mycoparasites, which can also be found in the rhizosphere or become plant symbionts (endophytes). They are frequently detected on the surface of decaying plant debris in a litter layer (Klein & Eveleigh, 1998). Thus, the open air environment of these fungi almost exclusively consists of pre-digested celluloses and hemicelluloses of decaying plant materials (Albersheim et al., 1994). For many soil fungi, aerial conidiation appears to be the most successful strategy for reproduction, dispersal and general survival. Therefore these fungi require a reliable mechanism to sense the most favourable environmental conditions to initiate conidiation. The soil litter layer, the optimal niche for sporulation, is usually a very heterogeneous substratum composed mainly of dead plant materials of various degradation stages and usually essentially mixed by soil fauna. This implies that the availability of one or another single nutrient compound alone can not guarantee that the hyphae will reach the most successful area for spore dispersion. Thus, light may serve as a second necessary criterion by which the fungus senses the optimal conditions for sporulation. Therefore, we reason that when at least two abiotic factors (availability of certain carbon sources and illumination) signal the presence of an aerial environment to the fungus, it will increase its rate of growth and branching in order to reach the state of conidiophore production and sporulation in the shortest possible time. This interpretation is also supported by the findings that conidiation and its photostimulation are also carbon-source dependent (Chovanec et al., 2001; M. A. Friedl & I. S. Druzhinina, unpublished data).
A comparison of individual carbon sources for which photostimulation of growth was observed with the potential chemical composition of this ‘aerial’ niche of Hypocrea/Trichoderma shows that they coincide. Growth stimulation was observed on (i) monosaccharides, which occur as major constituents of the plant polysaccharides (Albersheim et al., 1994); (ii) polyols, which accumulate from the metabolism of the pentoses in hemicelluloses (Seiboth et al., 2003); and (iii) oligosaccharides, which are degraded by enzymes secreted for complete degradation of the aforementioned polysaccharides (e.g. Aro et al., 2005). The assumption that this correlation is not accidental is supported by the fact that the growth rate on other carbon sources, which are well assimilated but usually not easily available in the soil litter layer, is not stimulated by light: a notable example is N-acetyl-D-glucosamine, the monomer of chitin. It becomes available for H. atroviridis nutrition in the form of the cell wall of other soil fungi, which it can attack as a mycoparasite, or from the exoskeleton of dead arthropoda. Another example is glycerol, which arises from triacylglycerides from various materials but only rarely from dead plant material.
Hence, we conclude that the nature of the carbon sources for which photostimulation of growth was observed reflects the composition of the potential niche for rapid growth of H. atroviridis, with the aim of accelerating subsequent conidiation.
Role of BLR proteins in photostimulation of growth
The blue light photoreceptors BLR-1 and BLR-2 have previously been shown to regulate photostimulation of conidiation in H. atroviridis (Casas-Flores et al., 2004), and it was therefore plausible to expect their involvement also in the photostimulation of hyphal growth. This expectation could indeed be confirmed. However, the responses in the two blr mutants were significantly different: the blr-1 mutant strain still showed photostimulation of growth on some carbon sources – even on ones where the parental strain exhibited no response – whereas growth of the blr-2 strain in the presence of rhythmic light was essentially the same as in darkness. These findings imply that the two BLR proteins must have different functions in the sensing of light: assuming that the residual sensing of light by the blr-1 mutant is due to the function of BLR-2, the latter must be involved in determining the intensity of this stimulation. In this context it is noteworthy that the stimulatory effects in the blr-2 strain were only observed in the presence of constant light, and were almost absent in the presence of alternating cycles of light and darkness, thus suggesting that BLR-1 only senses high, probably deleterious doses of light in the absence of BLR-2. No such finding was obtained with the blr-1 mutant, suggesting that in the wild-type genotype, BLR-1 plays the major role in the photostimulation of growth. This is consistent with the recent demonstration that blr-2 expression is a limiting factor for photo-perception and photo-transduction (Esquivel-Naranjo & Herrera-Estrella, 2007). We note that this conclusion is in contrast to findings in N. crassa, where the BLR-1 orthologue WC-1 has been reported to be the limiting factor for the formation of the WC-1/WC-2 photoreceptor complex (He et al., 2005).
Photostimulation of mycelial growth due to oxidative stress
While the above considerations support a role for the BLR proteins in the photostimulation of hyphal growth, they do not explain the biochemical mechanism responsible for this effect. From our data, we propose that the observed photostimulation is – at least in part – due to oxidative stress. UV light is known to cause the accumulation of reactive oxygen species and lead to oxidative stress in mammals and fungi (Yoshida & Hasunuma, 2004; Iigusa et al., 2005). Even in N. crassa, which is protected against UV light by formation of carotenoid pigments (Yoshida & Hasunuma, 2004; Iigusa et al., 2005), UV light has been demonstrated to exert oxidative stress. Hyaline hyphae of H. atroviridis lack such pigments, and we consequently conclude that this fungus must perceive light as a signal for a potentially hazardous environment. Support for such an assumption comes from recent findings with N. crassa, where rhythmic activation of the osmotically stress-activated p38-type MAPK by the N. crassa circadian clock allows anticipation and preparation for the hyperosmotic stress and desiccation that begin at sunrise (Vitalini et al., 2007). Moreover, the idea is also supported by our recent observation that in another species of Hypocrea (H. jecorina), the gene encoding the heat-shock factor HSF1 is strongly upregulated by exposure to light (A. Schuster et al., unpublished observations). The heat-shock response mediated by HSF1 is known to cooperate in the response to oxidative stress in fungi (Noventa-Jordao et al., 1999; Hahn et al., 2006; Yamamoto et al., 2007). In further support of our assumption, oxidative stress has been shown to increase the formation of aerial hyphae in N. crassa (Michan et al., 2003) and cause increased hyphal extension in arbuscular mycorrhizal fungi (Pawlowska & Charvat, 2004). In view of these and our own findings that the evocation of oxidative stress by addition of menadione in darkness mimics photostimulation of growth of H. atroviridis on several carbon sources, we hypothesize that the observed effect of light is at least partly due to an oxidative stress response. If this hypothesis is correct, it also identifies a new role of the BLR proteins: our data show clearly that the two BLR proteins are essential for the response to oxidative stress. The fact that the oxidative stress response is only observed on some carbohydrates and not on others, and also not on non-carbohydrate compounds, suggests that the oxidative stress response is dependent on carbon signalling and/or carbon metabolism. While there is no direct proof for this at the moment, Hong & Carlson (2007) showed that the Snf1 kinase (a central carbon-specific signalling component in fungi, which differentiates between carbohydrate and non-carbohydrate carbon sources; Schuller, 2003), is involved in the oxidative stress response. Also, Magherini et al. (2007) identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase as a potential signalling component in oxidative stress. While related findings are not yet available for H. atroviridis, these data are indicative that oxidative stress has a link to carbohydrate metabolism.
A cross-talk between effect of light, oxidative stress response and carbohydrate assimilation
Addition of cAMP to the cultivation medium in darkness stimulated the growth rate only on a narrow range of those carbon sources where photostimulation was also observed, i.e. -D-glucose, gentiobiose, D-cellobiose, D-mannose and D-xylose. These carbon sources can all be considered as the main degradation products from cellulose and hemicellulose, and we conclude that the mechanism of photostimulation of their assimilation involves the cAMP/protein kinase pathway. In agreement with Casas-Flores et al. (2006), the phenotype of blr-1 and blr-2 mutants was not rescued by the addition of cAMP. Interestingly, protein kinase A is known to be involved in the fungal response to oxidative stress (Zhao et al., 2006). Casas-Flores et al. (2006) suggested that BLR-1/BLR-2 may activate proteins upstream of protein kinase A, which would be consistent with the lack of effect of cAMP addition to the two blr mutants. However, growth on most of the carbon sources on which stimulation by light was observed was insensitive to the addition of cAMP. It is possible that this is due to the existence of a cAMP-insensitive response to oxidative stress. Yamamoto et al. (2007) reported that while cAMP generally activates gene expression in response to oxidative stress, it also negatively regulates the expression elicited by superoxide radicals such as menadione. This would be consistent with our findings, because the addition of cAMP was done in the dark, and thus in the absence of oxidative stress. In any case, our data imply that the oxidative stress response caused by light may involve different mechanisms on different carbohydrates.
While some of the conclusions drawn above still require the functional characterization of the components of oxidative stress response in H. atroviridis for full confirmation, our data nevertheless demonstrate the existence of a cross-talk between light, the oxidative stress response and carbohydrate assimilation. They also document the existence of different roles for the two blue light receptor proteins in this process. Given the fact that a deletion in these two proteins is not lethal (Casas-Flores et al., 2004), together with the observation of their role in hyphal growth in the presence of light and in stress response, and owing to their high conservation throughout the fungi (Idnurm & Heitman, 2005), BLR proteins present themselves as promising candidates for targeted control of plant-pathogenic fungi.
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ACKNOWLEDGEMENTS |
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blr-1 and blr-2 mutant strains. This work has been supported by grants from the Austrian Science Foundation (FWF-P17325) to C. P. K. Edited by: N. L. Glass
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Received 20 October 2007;
revised 16 December 2007;
accepted 3 January 2008.
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