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1 Departamento de Ingeniería Genética, Cinvestav Campus Guanajuato, Km 9.6 Libramiento Norte Carretera Irapuato-León, A.P. 629, Irapuato 36500, Guanajuato, Mexico
2 Laboratorio Nacional de Genómica para la Biodiversidad, Cinvestav Campus Guanajuato, Km 9.6 Libramiento Norte Carretera Irapuato-León, A.P. 629, Irapuato 36500, Guanajuato, Mexico
Correspondence
A. Herrera-Estrella
aherrera{at}ira.cinvestav.mx
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Franklin et al., 2005). In particular, the processes of blue-light perception, signal transduction and related responses have been extensively studied in bacteria, algae, plants and fungi.
In fungi, the best-studied responses to light are carotenogenesis, development, morphogenesis, tropism and resetting of the circadian clock (Idnurm et al., 2006; Linden et al., 1997; Liu et al., 2003; Lu et al., 2005; Silva et al., 2006). In Neurospora crassa, the proteins white collar 1 and 2 (WC-1 and WC-2) are essential for all its described light responses (Ballario et al., 1996; Linden & Macino, 1997). Analysis of the deduced amino acid sequences of these proteins revealed that they are PAS (Per-Arnt-Sim) domain-containing transcriptional factors with GATA type, zinc finger, DNA-binding domains. The WC-2 protein has only one PAS domain, whereas WC-1 contains three PAS domains (A–C). WC-1 and WC-2 form complexes through the interaction of the PAS-C domain of WC-1 and the PAS domain of WC-2 (Cheng et al., 2002, 2003). These complexes bind to the light-response element (LRE) found in the promoters of light-regulated genes even in the dark (Froehlich et al., 2002; He & Liu, 2005; Káldi et al., 2006). In the WC-1 protein, a specialized PAS domain essential for blue/UVA light perception, called LOV (light, oxygen, voltage), that binds FAD, similar to the first LOV domains described in plant phototropins, has been identified (Froehlich et al., 2002; He et al., 2002). WC-1 acts as a photoreceptor that, in combination with WC-2, tunes light input directly by activating the expression of genes required for the different responses described in N. crassa (Froehlich et al., 2002; He et al., 2002). A group of early light-induced genes showing transient expression patterns, typical of adaptive responses, have been identified (Lewis et al., 2002). Among these genes, one encodes a second blue-light photoreceptor, VIVID, necessary for adaptation to constant illumination and to detect changes in light intensity (Schwerdtfeger & Linden, 2003). Although other photoreceptors have been found in the N. crassa genome, it has not been possible to assign any function to them. Furthermore, genes homologous to wc-1 and wc-2 have been found in different fungi and they all encode proteins containing PAS domains, presumably used to form complexes that regulate light responses (Casas-Flores et al., 2004; Lu et al., 2005; Idnurm et al., 2006; Silva et al., 2006).
Trichoderma atroviride is a common soil fungus widely used as a biocontrol agent due to its capacity to parasitize phytopathogenic fungi of agricultural importance. It has been shown that its asexual reproduction (conidiation) is tightly regulated by light and nutrient availability (Horwitz et al., 1985). In T. atroviride, a pulse of blue/UVA light induces the synchronous production of conidia situated at the colony perimeter where the pulse was received. Two genes, blr-1 and blr-2, orthologous to wc-1 and wc-2 from N. crassa, have been identified and shown to be essential regulators of the expression of blue-light-responsive genes and photoconidiation (Casas-Flores et al., 2004; Rosales-Saavedra et al., 2006). The BLR proteins contain PAS- and GATA-type DNA-binding domains that indicate their participation in a transcriptional factor complex, but seem to lack activation domains. PAS- and GATA-type binding domains have been found in WC-like proteins in other fungi, suggesting that they are sufficient for their role in light perception and subsequent transduction of the signal (Ambra et al., 2004; Lu et al., 2005; Idnurm et al., 2006; Silva et al., 2006). This is also supported by data demonstrating that a truncated form of WC-1, lacking its N-terminal activation domain, is functional as a photoreceptor (Káldi et al., 2006). In addition, BLR-1 contains the LOV domain essential for binding FAD. All blue-light-regulated genes that are BLR-dependent strictly require both functional proteins, indicating that the BLR proteins act as a complex. By analogy with N. crassa, it is assumed that this complex regulates the expression of early blue-light-responsive genes through putative GATA type cis-elements present in the promoter region of blue-light-responsive genes, similar to the LREs defined in N. crassa (Berrocal-Tito et al., 1999; Casas-Flores et al., 2004; Rosales-Saavedra et al., 2006). Interestingly, the BLR proteins participate in the light-dependent transcriptional activation and repression of early gene expression, a dual role previously not described in any other fungus (Rosales-Saavedra et al., 2006). Additionally, a number of biochemical and molecular data have provided support for the existence of yet another blue-light perception pathway. It appears that activation of both blue-light perception pathways is necessary for the regulation of photoconidiation (Berrocal-Tito et al., 2000; Rocha-Ramírez et al., 2002; Casas-Flores et al., 2006). Furthermore, the identification of light-regulated genes allowed us to identify red-light-regulated genes (Rosales-Saavedra et al., 2006). These data clearly indicate that multiple light perception pathways are functional in T. atroviride, in agreement with the different photoreceptors recently identified in fungal genomes.
To better understand the role of blr-2 in the light perception and transduction pathways of T. atroviride, we analysed its transcriptional regulation and the effect of its overexpression on the light responses of this organism. We found that expression of blr-2 is a limiting factor for light signal transduction and its overexpression leads to exacerbated responses to this stimulus. Furthermore, our results indicate that the expression of blr-2 is induced by light and autoregulated at the post-transcriptional level. Finally, blr-2-overexpressing strains showed higher sensitivity to light. In contrast, strains overexpressing blr-1 were less responsive to light than the wild-type (WT). Our results indicate that BLR-2 plays key roles both in the perception and in the transduction of blue light in this fungus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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blr-1 and blr-2 mutants were grown on PDA medium (Difco) at 27 °C. Conidiation of the blr-1 and blr-2 mutants was induced by mechanical injury to obtain inocula. Escherichia coli strain TOP10F' (Invitrogen) was used for plasmid DNA transformation. To analyse growth in the dark, in photoperiods of 12 h dark/12 h light and in constant illumination with white light, the strains were grown on PDA in a controlled environment growth chamber operating at 27 °C (constant temperature) with a fluence of 230 µmol m–2 s–1, provided by cool-white fluorescent tubes. Petri plates (15 cm diameter) containing PDA were inoculated in the centre with plugs of mycelia (0.5 cm diam.) and incubated for 72 h at 27 °C (±1 °C). The radial growth rate of the strains was determined by measuring colony diameter every 12 h. The experiment was carried out with six replicates for each strain.
Overexpression of blr genes in T. atroviride.
Plasmid pCB1004, carrying the hygromycin B resistance marker (FGSC), was used as the backbone for the construction of a constitutive expression vector. A 550 bp fragment of the Aspergillus nidulans trpC terminator and a 750 bp fragment containing the promoter of the Trichoderma reesei pki1 gene were cloned by PCR using the primers: TtrpC-f (5'-GGTACCTAGTGATTTAATAGCTCC-3') and TtrpC-r (5'-GGTACCTGTGCATTCTGG-3') for the terminator, and Ppki1-f (5'-CCGCGGCTCGAGATAACGGTG-3') and Ppki1-r (5'-CCGCGGTTAAGAGGGTTCTTC-3') for the promoter. KpnI (terminator) and SstII (promoter) sites in the primers (underlined) were added to facilitate cloning into plasmid pCB1004. Plasmids pHat
(Herrera-Estrella et al., 1990) and pZEGA (Mach et al., 1994) were used as templates for the amplification of the trpC terminator (TtrpC) and pki1 promoter (Ppki1), respectively, under the following PCR conditions: one initial cycle of 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 68 °C for 45 s, and one final cycle of 68 °C for 7 min. The trpC terminator and pki1 promoter were sequenced and cloned into the KpnI and SstII restriction sites of pCB1004, respectively, obtaining the expression vector pUE08. The correct orientation of the regulatory regions in pUE08 was confirmed by PCR using universal and reverse primers, in combination with the primers described above.
To overexpress the blr genes, the corresponding complete blr-2 cDNA (1.6 kb) containing the UTR-5' and 3' ends (Casas-Flores et al., 2004) was cloned into the EcoRI site of pUE08, and the blr-1 ORF was cloned into the BamHI and HindIII sites of the same plasmid. The correct orientation of the blr-2 cDNA was corroborated by restriction with different endonucleases. The plasmids were purified using the the Qiagen plasmid Midi Kit and used to transform T. atroviride protoplasts using the PEG-CaCl2 method described by Baek & Kenerley (1998). All transformants were subjected to three rounds of monosporic culture. The identification and selection of the transformants was performed by PCR using primers pki1-f and trpC-r.
Northern and Southern analysis.
Genomic DNA was isolated following the procedure described by Raeder & Broda (1985). Total RNA was isolated according to the protocol described by Jones et al. (1985). Southern and Northern blotting was performed using Hybond-N+ membranes (Amersham), according to the manufacturer's recommendations. Filters were hybridized with probes labelled by random priming with [-32P]dCTP and processed using standard procedures (Sambrook et al., 1989).
Analysis of light responses.
T. atroviride cultures were grown in the dark for 48 h at 27 °C on PDA plates and used as pre-inoculum. Mycelial plugs (0.5 cm diam.) were taken from the colony growth front and placed on the centre of PDA plates with (for RNA extraction) or without (for photoconidiation analysis) a cellophane overlay. Cultures were allowed to grow for further 36 h under these conditions, and then photoinduced as described by Berrocal-Tito et al. (1999).
For analysis of the expression of light-responsive genes after a light pulse, colonies were exposed to 1200 µmol m–2 (unless otherwise indicated) using a light source consisting of two cool-white fluorescent tubes filtered with LEE filter #183 (fluence rate 5 µmol m–2 s–1) and placed back in the dark at 27 °C. Mycelial samples were subsequently collected at different times after exposure to light. At the indicated times, mycelia were scraped from the surface of the cellophane overlaying the PDA medium under low red safe-light [LEE filter #106 (fluence rate 0.1 µmol m–2 s–1)] and immediately frozen in liquid nitrogen for RNA extraction.
For photoadaptation analysis, we followed the procedure described above, except that mycelia were kept under constant illumination.
Sensitivity to blue light was investigated following the procedure described above, except that colonies were exposed to a different fluence of blue light as indicated. Colonies were then placed back in the dark at 27 °C for 15 min, and mycelia were collected and frozen in liquid nitrogen for RNA extraction.
Analysis of photoconidiation.
Colonies were incubated for 48 h in the dark after photoinduction and conidia were collected in 8 ml distilled water. Quantification of conidia was performed using an Axiostar Plus microscope (Zeiss) in a Newbauer chamber.
Analyses of blr gene expression by real-time PCR.
Primers for the analysis of blr-1 (blr1-f 5'-GAATGGCGGAGGGGGCCGAGT-3' and blr1-r 5'-CCGTTGCTGGGGATTGGATTTGGA-3') and blr-2 (blr2-f 5'-GTAACCGCAGCCCTACCCTCATC-3' and blr2-r 5'-GCCCACCGCAACCCGCAGGC-3') gene expression by real-time PCR were designed. The primers gpd-f (5'-GGCTGCCGATGGTGAGCTCAAGGG-3') and gpd-r (5'-GAGGTCGAGGACACGGCGGGA-3') were designed to use the gpd gene (encoding glyceraldehyde-3-phosphate dehydrogenase) as an internal loading and normalization control (Puyesky et al., 1997). Twenty micrograms of total RNA was obtained and, as described above, treated with amplification-grade RNase-free DNaseI (Invitrogen) and cleaned using RNeasy mini kit columns (Qiagen). cDNA synthesis was performed using 10 µg RNA, a reverse primer mixture (gpd-r, blr1-r and blr2-r, 20 pmol each) and SuperScript RT III (Invitrogen), following the manufacturer's recommendations. After synthesis of the cDNA, the mixture was treated with 0.2 M NaOH for 15 min at 37 °C to eliminate RNA and then neutralized with 0.7 M HEPES before purification of the cDNA by using QIAquick Spin columns (Qiagen). The optimal conditions and specificity for amplification of the three genes were determined by PCR using a Corbett Research thermocycler (version 2.2). Standard and relative efficiency curves were performed using the cDNA sample obtained from the WT strain grown in the dark to determine the dynamic range and to validate our reaction conditions for these genes. To this end, five serial dilutions (1 : 3) were made starting from 50 ng to 0.61 ng cDNA. The detection of blr-2 transcript in amounts below 0.1 ng DNA was inconsistent. PCR amplifications were performed under the following conditions: 1 cycle of 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 64 °C for 30 s and 72 °C for 35 s, using SYBR Green PCR Master Mix (Applied Biosystems). Five replicates were made for each experimental condition and all PCR reactions were performed in a 25 µl final volume using a 7500 Real-Time PCR system (Applied Biosystems). The specificity of amplification was monitored and evaluated by the corresponding dissociation curves. The relative quantification of blr expression levels was performed with 6 ng cDNA per reaction and their CT was determined as a function of the calibrator condition (growth in the dark) to obtain comparative levels of relative expression for both blr genes after treatment with light, using the 2–CT method (Livak & Schmittgen, 2001). To compare the expression levels between blr-1 and blr-2, the CT values were determined as a function of the CT obtained in the dark for the blr-2 gene.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In contrast, the expression of blr-1 decreased by 20 % at 60 min after exposure to the light, coincident with the reduction of blr-2 expression. Upon comparison of the expression levels of blr-1 and blr-2, it was evident that the level of blr-1 transcripts was threefold higher than that detected for blr-2 (Fig. 1), suggesting that BLR-2 protein levels could be a limiting factor in blue light perception in T. atroviride. Thus, we decided to explore the effect of overexpression of blr-2 on the blue light response.
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). The 7.1 kb band can only be explained if all integrations occurred at the same place. Indeed, homologous integration appears to be a highly frequent event in T. atroviride (unpublished data). Densitometric analysis of the hybridizing bands obtained indicated tandem integration of two copies of the vector in transformants OEblr2-1, -2, -3, -4, -6, -8, -12 and -14, and three copies in OEblr2-13. To demonstrate the overexpression of blr-2 in the transformants, we carried out Northern blot analysis. All transformants carrying the construct, as confirmed previously by the Southern blot assays, showed high levels of blr-2 transcripts, whereas in the WT strain and in strain T-9, used as an additional control, no detectable levels of blr-2 transcripts were observed (Fig. 2c).
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). In contrast, strain T-9 showed a phenotype identical to that of the non-transformed strain, indicating that the transformation process did not affect its capacity to respond to light. The production of conidia increased significantly in the blr-2 overexpressers, with strain OEblr2-13 yielding the highest levels of conidia, producing 11.9-fold more conidia than the WT strain (Fig. 2e). Consistently, this strain was shown to carry three copies of the vector and to express the highest levels of the blr-2 transcript. These results are in agreement with the expression levels found for the blr genes and support our proposal that BLR-2 is a limiting factor in the phototransduction pathway of T. atroviride. Based on the results shown above, we selected the OEblr2-4 and OEblr2-13 strains, which represent all blr-2 overexpressers, for further analyses of light-regulated responses in T. atroviride.
The BLR proteins modulate mycelial growth in the presence of light
Previously, Casas-Flores et al. (2004) described a negative effect of light on mycelial growth of T. atroviride. Thus, we decided to investigate the effect of light on the growth of the blr-2-overexpressing strains compared to the WT strain, the transformation control strain T-9, and the gene replacement mutants blr-1 and blr-2. Under all conditions of growth analysed, we determined the radial growth every 12 h (radial growth rate). In the dark, the growth rate was similar among all strains, reaching a maximum after 24 h. Moreover, a constant radial growth rate was maintained after 24 h of growth, without further apparent changes. Furthermore, the total radial growth was similar among all the strains (Fig. 3a, d). However, under constant illumination, the pattern of radial growth rate was modified in all the strains when compared to the corresponding dark controls. The blr strains reached a maximum growth rate by 24 h, whereas the WT and T-9 strains reached their maximum between 36 and 48 h, and OEblr2-4 and -13 reached it only at 48 h (Fig. 3b). The growth rate of all strains decreased gradually to a minimum by 72 h. The growth pattern of the blr and OEblr2 strains showed an early maximum for the gene disruptants and a delayed maximum for the overexpressers, in contrast to the pattern observed for the WT strain. Under precise photoperiods, the growth rate was cyclic for the WT, T-9 and blr strains with faster growth rates in the dark and lower rates when exposed to light (Fig. 3c). Interestingly, OEblr2-4 and -13 showed an arrhythmic growth in the first four dark–light periods, developing an altered colony morphology, which recovered to a growth comparable to that of the rest of the strains by 60 h (Fig. 3c, e). Noticeably, the first light period provoked the strongest decrease in radial growth rate and the recovery was also slower in the next dark period. Interestingly, the blr-2-overexpressing strains were apparently insensitive to a second light period as suggested by the constant radial growth rate observed during this period (Fig. 3c). This behaviour can be observed in Fig. 3(e), where the formation of two nearly continuous rings of conidia, corresponding to the first two light periods (central part with dense conidiation), was observed. In subsequent periods the spacing between the conidiation rings became similar among the different strains. The decrease in total radial growth could have been directly related to the arrhythmic growth provoked by the first two periods of exposure to light. This behaviour could not be associated with the higher photoconidiation of the blr-2 overexpressers, because the blr strains, which did not conidiate, had a rhythmic growth similar to that of the WT and T-9 strains (Fig. 3c, e).
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). We analysed the expression of some of these genes in the blr-2-overexpressing and WT strains. The expression of all the light-inducible genes (blu-1, -4, -7, -17 and phr-1) was clearly higher in the OEblr2 strains (Fig. 4a). These genes showed different patterns of expression and could be divided into three well defined groups. In group 1, blu-7 showed rapid expression with a maximum at 15 min, decreasing rapidly by 30 min after the light pulse, thus indicating a strong transient upregulation. In group 2, phr-1 and blu-17 showed an intermediate pattern of expression, reaching a maximum by 15–30 min and returning to basal levels of expression by 60 min. In group 3, blu-1 and blu-4 showed a peak between 30 and 60 min after the light pulse, which decreased by 2 h after the stimulus (Fig. 4a–c). In all cases, the expression was faster and sustained for longer periods of time in the OEblr2-4 and OEblr2-13 strains, compared to the WT (Fig. 4a–c). Interestingly, the expression of blu-4, which was previously considered independent of or partially dependent on the BLR proteins (Rosales-Saavedra et al., 2006), was clearly affected by the overexpression of blr-2 (Fig. 4a).
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). These results provide further evidence of the role of the BLR proteins in the transcriptional activation and repression of blue-light-responsive genes.
The expression of blr-2 is regulated through the BLR complex in both the dark and the light
In N. crassa, the expression of the wc genes is mainly regulated at the transcriptional level by means of the WC proteins. This WC protein dependence has been found both in the dark and after light induction (Ballario et al., 1996; Cheng et al., 2003; Káldi et al., 2006). Thus, we investigated, first, if blr-2 overexpression had any effect on the expression of the blr genes and, second, if they were involved in regulating their own expression. In this regard, we found that blr-2 expression levels in the dark were 12.4-fold higher in the OEblr2-13 strain than in the WT strain, and up to 21.6-fold higher upon light induction (Fig. 5a), thus demonstrating that blr-2-overexpressing strains express higher levels of blr-2 than of blr-1, an inverted situation to the behaviour found in the WT strain (Fig. 5a). Interestingly, the transcript levels of both blr genes increased similarly in response to blue light, showing a 75 % increase for both blr-1 and blr-2 in the OEblr2-13 strain, and the transcript level of blr-1 fluctuated over time (Fig. 5a). Furthermore, light-induced accumulation of blr-2 transcripts in the OEblr2-13 strain was faster and stronger, reaching its maximum level 15 min after a light pulse. This change in blr-2 expression is similar to the expression pattern found in the blue-light-upregulated genes (Fig. 4a), indicating that it regulates its own expression (Fig. 5a). Noticeably, the 75 % increase in the blr-2 transcript levels upon light induction in the blr-2 overexpresser represents a 9.2-fold increase compared to the level found in the WT strain. Northern blot analysis of a T. atroviride transformant carrying a transcriptional fusion of the gene encoding the green fluorescent protein (gfp) with the pki-1 promoter indicated that there were no significant changes in gfp expression in response to light (data not shown). Furthermore, conidiation and the expression of blu genes induced by light in that strain were similar to those of the WT. Given the fact that the overexpression of blr-2 was achieved using a constitutive promoter, these data suggest autoregulation at the post-transcriptional level. In the dark, the levels of blr-1 expression were not altered in the blr-2 overexpresser (Fig. 5a), directly linking the observed phenotypes to blr-2 overexpression. Additionally, we analysed the expression of blr-2 in the blr-1 strain to investigate the dependence of blr-2 expression on BLR-1. We found approximately 3.5-fold lower levels of blr-2 expression in the mutant compared to the expression levels in the WT strain, even in dark (Fig. 5b), indicating that BLR-1 is necessary to maintain the expression of blr-2 in the absence of light. Furthermore, the light induction of the steady-state levels of blr-2 transcripts in the blr-1 strain was not observed. Thus, our data suggest that the BLR complex regulates the induction of blr-2 transcripts occasioned by light at the post-transcriptional level.
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). Therefore, we analysed the expression of the blr genes under constant illumination. The expression of blr-2 in the WT strain under constant blue light was upregulated by 40 % and showed an expression pattern identical to the induction provoked by a light pulse, with transcript levels decreasing to those found in the dark (Fig. 6a) after 60 min. However, the expression pattern of blr-2 was modified in the OEblr2-13 strain, losing the typical transitory expression found in the WT strain (compare Fig. 6a and Fig. 6b). Light induction was only detected after 30–60 min of continuous exposure to light, showing a similar increase in transcript levels of 40 % in both blr genes, but no decrease was observed in the time frame analysed (Fig. 6b, c). Furthermore, expression of blr-1 under constant illumination was different in both strains. In the WT strain, light had a modest negative effect on blr-1 expression (25 % decrease) that apparently coincided with a decrease in blr-2 transcript levels (data not shown), whereas in the OEblr2-13 strain light stimulated blr-1 expression (Fig. 6c). These results indicate altered light regulation of the blr genes under constant illumination occasioned by the overexpression of blr-2.
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). In addition, repression of the bld-2 gene was also comparable in both strains, even though recovery of the transcript levels observed in the dark was not evident in the time frame analysed here, considering that its expression starts increasing only after 6 h of growth under these conditions (data not shown). These results suggest that blr-2 overexpression does not alter the photoadaptative response of the light-regulated genes of Trichoderma and that a different mechanism regulates the temporary expression of the blr genes under constant illumination.
A high level of blr-2 expression enhances sensitivity to blue light in T. atroviride
Based on the possibility that the BLR proteins act as a complex, we rationalized that the formation of such complexes in the dark should be important for appropriate light perception. Therefore, blr-2 overexpression should result in changes in the sensitivity to blue light. In this context, we decided to investigate if the blr-2 overexpressers were altered in their photosensitivity. The WT strain, and the OEblr2-4 and -13 strains were exposed to different fluences of blue light. As shown in Fig. 7(a), both transformants showed a conidiation ring when exposed to 225 µmol blue light m–2, comparable to the ring observed in the WT strain when exposed to 1200 µmol m–2 using the same light source. Statistical analysis of the fluence response allowed us to estimate a saturation dose of 2400 and 1200 µmol m–2 for the WT and OEblr2 strains, respectively (Fig. 7b). The half-saturating doses for photoconidiation were 1859 µmol m–2 for the WT strain, and 612 and 560 µmol m–2 for OEblr2-4 and -13, respectively, indicating a greater than threefold higher sensitivity of both transformants than that detected in the WT strain (Fig. 7b). The difference between the two transformants was statistically not significant, indicating that the BLR-2 levels were saturating, even in the OEblr2-4 strain, which had lower blr-2 expression. Additionally, the yield of conidia was evidently higher in the blr-2-overexpressing strains than that in the WT strain (Fig. 7a, b).
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). Furthermore, blu-1 and phr-1 reached their maximum expression, in the OEblr2 strains, with a fluence of 600 µmol m–2, whereas in the WT strain their expression was still increasing when 1200 µmol m–2 was applied. In the case of blu-7 and blu-17, the response was saturated at 300 µmol m–2 in the OEblr2 strains, whereas saturation in the WT strain was reached using 600 µmol m–2. The expression of blu-4 did not appear to reach saturation even when exposed to the maximum fluence used in this analysis (Fig. 7c). Taken together, these results show that the higher photosensitivity and early saturation with light are directly associated with the overexpression of blr-2.
Effect of blr-1 overexpression in photoconidiation and gene expression
To provide further support to our hypothesis of blr-2 expression being a limiting factor in light signal transduction, we cloned the complete cDNA of blr-1 into plasmid pUE08. The resulting construct (pUE08-OEblr1) was used for transformation of T. atroviride. Out of 40 patitive transformants analysed by PCR, only eight carried the transformation vector and were confirmed by Southern analysis (data not shown). Northern analysis of the transformants carrying the construct showed, in all cases, high levels of blr-1 transcript even in the dark, whereas in the WT and OEblr2-13 strains no detectable levels of blr-1 transcript could be observed (Fig. 8a). Unexpectedly, overexpression of blr-1 caused a strong decrease in light-induced conidiation compared to the WT and OEblr2 strains (Fig. 8b). Additionally, photoconidiation of the blr-1-overexpressing strains induced by different doses of light was always lower than for the WT and OEblr2-13 strains. However, conidiation induced by mycelial injury was similar among all strains (data not shown), indicating a light-specific alteration.
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). These results suggest that the regulation and levels of the BLR proteins are very important for appropriate induction of gene expression. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Casas-Flores et al., 2004; Idnurm et al., 2006; Lu et al., 2005; Silva et al., 2006). In T. atroviride, blr-1 and blr-2 encode essential proteins for photoconidiation and regulation of a set of blue-light-responsive genes (Casas-Flores et al., 2004; Rosales-Saavedra et al., 2006). The expression of these genes is apparently very low, similar to the level found for the Cryptococcus neoformans cwc genes (Lu et al., 2005). The blr-2 transcript levels were lower than those detected for blr-1. T. atroviride strains overexpressing blr-2 had a higher yield of conidia in response to blue light. Our results are consistent with the hypothesis that blr-2 expression is a limiting factor in the transduction of the light signal. In N. crassa, WC-1 is limiting, compared to WC-2, and its overexpression enhanced the expression levels of blue-light-regulated genes and the rhythmic expression of frq (Cheng et al., 2001; Franchi et al., 2005). Consistent with the higher levels of WC-2 compared to WC-1, overexpression of wc-2 did not cause any obvious photosensorial alteration in N. crassa.
Exposure of T. atroviride to continuous white light results in a reduction in growth, even in blr deletion mutants. This effect was intensified when only the blue or red regions of the spectrum were applied (Casas-Flores et al., 2004). Additionally, growth of a T. reesei strain with a mutation in envoy, encoding a putative secondary light receptor (ENVOY), was strongly affected by white light (Schmoll et al., 2005). In Tuber borchii and N. crassa, light also affects radial growth during light–dark periods which is partially modulated through WC-1 (Ambra et al., 2004). We did not detect significant differences among the different strains analysed when grown in the dark. However, notable changes were observed during growth under constant illumination and during photoperiods. In all cases, light had a negative effect on growth, being stronger in the blr-2 overexpressers. Under constant illumination, we found different growth patterns with consistent radial growth rate profiles among the phenotypes analysed (WT, OEblr2 and blr), therefore suggesting that the BLR proteins modulate T. atroviride growth as proposed by Casas-Flores et al. (2004). Under defined photoperiods, we found a rhythmic growth rate in the WT strain and the blr mutants. Interestingly, the blr-2 overexpressers showed a temporary arrhythmic growth in the first period of exposure to light. This growth alteration was slightly stronger in OEblr2-13 than in OEblr2-4, directly correlating the observed phenotype with the level of expression of blr-2. Furthermore, there was a clear state of insensitivity in the second light period, suggesting an alteration of photoadaptation in the blr-2-overexpressing strains. However, we did not find significant changes in the expression pattern of light-regulated genes under continuous light exposure, indicating that various mechanisms may be operating in photoadaptation of T. atroviride. Although the envoy mutant is defective in growth under constant illumination, and the corresponding gene is clearly light-regulated (Schmoll et al., 2005) through the action of BLR proteins (unpublished data), we did not observe such strong inhibition in the blr mutants, indicating that, at least in part, the BLR proteins are negative modulators of mycelial growth and that ENVOY may participate in a negative feedback loop that desensitizes the light input through the BLR complex, as indicated in Fig. 9(a). Furthermore, the fact that this growth disturbance could be observed even in the blr strains strongly suggests the participation of an alternate light perception system, which at some point must establish cross-talk with the BLR-dependent system (Fig. 9a). Noticeably, this growth disturbance is temporary and the recovery after the fourth photoperiod suggests the existence of interrelated feedback loops among different light perception systems.
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). All genes analysed showed an earlier and stronger response to a light pulse in OEblr2 strains, compared to the WT strain. These results were consistent with the higher level of photoconidiation observed, directly associated with blr-2 overexpression. Overall, our results clearly indicate that blr-2 expression is a limiting factor in light signal transduction as demonstrated by the lower transcript levels detected compared to those detected for blr-1, as well as the enhanced responsiveness to light. This conclusion was corroborated through blr-1 overexpression. The negative effect caused by blr-1 overexpression was reflected both in the activation and repression of light-regulated genes, and finally in photoconidiation, suggesting a global alteration in the perception and transduction of blue light by the alteration of the balance of the amounts of the BLR proteins. Although these results were unexpected and previously not described in fungi, they suggest that a delicate balance in BLR-1/BLR-2 levels is a major factor for the appropriate sensing and transduction of blue light. An alternative explanation is that overproduction of the blr-1 transcript might trigger a strong negative regulation mechanism, affecting the efficiency of translation of the transcript. The effect of blr-1 overexpression on light signal transduction is certainly interesting and deserves future in-depth analysis.
It is noteworthy that, in addition to its regulation by red light (Rosales-Saavedra et al., 2006), we found that the expression of blu-4 is clearly regulated by blue light through the BLR complex. In agreement with this observation, analysis of the blu-4 promoter region in T. reesei indicated the presence of an LRE (GATTC-N21-CGATC) 725 bp upstream of the putative ATG, as well as other GATA-type elements, as shown for other BLR-dependent light-regulated genes (Rosales-Saavedra et al., 2006). To date, blu-4 is the first gene that represents a point of convergence of the signal transduction cascades involved in the response to two different light inputs in fungi. Furthermore, the cross-talk between these two light perception systems might be, at least in part, responsible for the altered growth provoked by the overexpression of blr-2, probably due to an imbalance in the light regulation between the two (or more) inputs (Fig. 9a). In plants, cross-talk between cryptochromes and phytochromes that regulate plant growth and architecture has been well documented (Franklin et al., 2005).
In N. crassa, the expression of wc genes is mainly regulated at the transcriptional level through WC proteins. This dependence on both WC proteins has been found both in the dark and after light induction (Ballario et al., 1996; Cheng et al., 2003; Káldi et al., 2006). In T. atroviride, the induction of blr-2 by light was faster and higher in the OEblr2-13 strain compared to the WT strain, similar to the expression pattern found for the blue-light-induced genes. We determined a 75 % induction of blr-2 in OEblr2 that represents a 9.2-fold increase with respect to the transcript levels found in the WT strain, strongly suggesting a positive post-transcriptional autoregulation. Analysis of the expression of blr-2 in the blr-1 mutant showed that its expression in the dark and accumulation induced by light depends on BLR-1, providing further support to the notion that the BLR proteins act as a complex in light-activated post-transcriptional regulation (Fig. 9a). The fact that the transcript levels of blr-2 decreased 3.5-fold in the blr-1 strain may be explained by the presence of a putative LRE (GATGC-N23-GATGC-N7-CGATT) 1122 nt upstream of the ATG in the blr-2 promoter, similar to what is found in other light responsive genes, suggesting regulation at the transcriptional level mediated by the BLR complex in the dark (Fig. 9a). Consistently, the WC complex regulates the expression of wc-1 through two different promoters upstream of its ORF, named the distal and proximal promoters. The distal promoter is necessary for expression in the dark and the proximal promoter is essential for light-responsive expression (Káldi et al., 2006). Altogether, our data suggest that the BLR complex is a transcriptional regulator active even in the dark and that after a light pulse it can regulate expression of light-responsive genes at the transcriptional level, and that of blr-2 at the post-transcriptional level (Fig. 9a).
Recently, we determined that the expression of light-regulated genes in T. atroviride is subjected to photoadaptation (Rosales-Saavedra et al., 2006). Similarly, blr-2 expression under constant illumination showed a typical adaptative response, similar to the expression pattern found after a light pulse, indicating the existence of a turn-off mechanism operating in its post-transcriptional regulation. Conversely, the expression pattern of the blr genes was different in the blr-2-overexpressing strain. However, the expression pattern of light-regulated genes was the same in the WT and OEblr2-13 strains, indicating that different adaptation mechanisms may be operating. In N. crassa, a negative feedback loop involving the flavoprotein VIVID and phosphorylation by different kinases that destabilize WC-1, and block the transcriptional activity of the WC complex, are the main mechanisms that control adaptation under continuous exposure to light (Schwerdtfeger & Linden, 2003; He & Liu, 2005). T. reesei, a close relative of T. atroviride, has a homologue (ENVOY) of VIVID that might play a similar role in photoadaptation (Schmoll et al., 2005). In our view, the mechanisms controlling the expression of blr-2 at the post-transcriptional level and, possibly, mycelial growth under continuous exposure to light differ from those reported for N. crassa. The expression of blr-1 was not altered by light in the WT strain and an increase in the transcript levels was only evident in the blr-2 overexpresser. The increase in the blr-1 transcript steady-state levels observed only in a blr-2-overexpressing strain was comparable to the change found in the blr-2 expression levels. The fluctuations observed in blr-1 transcript levels have been observed in autoregulated genes, and typically arise from interference in the half-life of mRNA and the resulting time gap between transcription and translation, as shown for the zebrafish somitogenesis oscillator (Lewis, 2003; Schmoll et al., 2005). This may be indicative of the existence of a post-transcriptional regulation mechanism for both transcripts. However, since we did not find light-induced changes in the blr-1 mRNA steady-state levels in the WT strain, these results must await further experimental support.
Formation of complexes between the WC proteins (WCC) has been demonstrated (Cheng et al., 2002; Froehlich et al., 2002). The WC proteins have been found in the dark as a heterodimer able to bind LREs and, after a light pulse, a large WCC is formed that transiently binds the LREs of light-regulated genes in vivo (Froehlich et al., 2002; He & Liu, 2005). Additionally, although WC-2 regulates WC-1 steady-state levels, WC-2 overexpression did not seem to alter the levels of WC-1 found in the WT strain (Cheng et al., 2001, 2002). Our data suggest that, similar to the WC proteins, the T. atroviride BLR proteins act as a complex. Additionally, blr-2 overexpression did not alter the transcript levels of blr-1 in the dark. Therefore, we consider that all phenotypes found may be associated directly with higher BLR-2 levels. Overexpression of blr-2 resulted in a threefold higher sensitivity to light of the blr-2-overexpressing strains than that of the WT strain. The fluence required for the light response was lower for the OEblr2 strains than for the WT strain. This parameter, directly associated with photoreceptors, was modified by blr-2 overexpression. The enhanced sensitivity to blue light of the OEblr2 strains can also explain the arrhythmic growth observed in the first light period, which possibly occurred as a result of an imbalance in the photosensitivity of different perception systems that regulate growth under constant illumination with white light. Our data strongly suggest that a BLR-1/BLR-2 complex is necessary for light perception. Although we cannot discard any effect associated with changes in the levels of BLR-1 caused by blr-2 overexpression, our data support the notion that a preformed photoreceptor complex between BLR proteins in the dark is key for the adequate perception and transduction of the light signal, as indicated in Fig. 9(b).
The BLR proteins regulate a set of specific genes, as demonstrated by the deletion of either of them (Rosales-Saavedra et al., 2006), and the overexpression of blr-1 and blr-2. Together, these data clearly demonstrate a role dual for the BLR complex in light-dependent induction and repression of transcription (Fig. 9a). Here, we show evidence of additional roles of the BLR complex in the positive post-transcriptional regulation of blr-2 triggered by light, as well as of transcriptional regulation in the dark (Fig. 9a). The expression analysis of blr and early light-responsive genes under constant illumination clearly indicates that a negative feedback loop is operating in the downregulation of the expression of these genes (Fig. 9a). Our results indicate that the adaptation mechanism to light is different between the blr and light-responsive genes, even though their regulation clearly occurs through the BLR complex. On the other hand, we found that the BLR proteins are dispensable for the regulation of growth of T. atroviride under defined photoperiods. However, there is strong evidence for cross-talk among the different light perception pathways. We demonstrated regulation of blu-4 expression by blue light through the BLR complex, in addition to its regulation by red light as described previously (Rosales-Saavedra et al., 2006), thus supporting cross-talk between the blue and red light transduction pathways. Finally, our results support a key role of BLR-2 in blue light sensing and transduction, and we propose that pre-formation of a BLR photoreceptor complex is key for appropriate light perception, and for subsequent robust regulation of all blue light responses (Fig. 9b).
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ACKNOWLEDGEMENTS |
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Edited by: B. A. Horwitz
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 20 February 2007;
revised 2 July 2007;
accepted 5 July 2007.
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, WT; 
, OEblr2-4; 
, OEblr2-13.
