BACKGROUND
most organisms can perceive light as a signal and the response to this stimulus depends on the length of exposure and light quality, serving as a cue of environmental conditions  <cit> .
fungi can perceive a wide range of light wavelengths, from far red to ultraviolet  light .
many reports on light responses in fungi are related to reproduction, which determines whether they should enter asexual or sexual reproduction or neither .
the best-characterized photoreceptor in fungi is the white collar complex  of neurospora crassa.
this blue light photoreceptor wcc, formed by the white collar  proteins, regulates pigmentation, circadian rhythm, conidiation and phototropism of perithecial beaks .
the wcc of n. crassa controls all light responses characterized so far in this fungus, despite the presence of red , uv  or green  photoreceptors in its genome .
however, a cryptochrome dependent oscillator  driving rhythmic spore development under constant light, where the frequency/wcc oscillator does not operate, was recently described  <cit> .
the cog- <dig>  mutation uncovered this cry  dependent oscillator in n. crassa, which regulates light responsive genes independently of the wcc.
similarly, as no obvious biological functions for phy-1/phy- <dig> or nop- <dig> are reported, their role might be masked by the wcc function.
moreover, in aspergillus nidulans the phytochrome  is involved in repression of sexual development and mycotoxin production by red light, whereas the lrea and lreb proteins  stimulate both  <cit> .
asexual reproduction induced by light in t. atroviride results in the formation of the so called “conidiation ring” in the periphery of the colony.
the blrc, formed by the blr <dig> and blr <dig> proteins, is responsible for the perception of blue light  <cit> .
like their n. crassa counterparts, the blr proteins have gata zinc finger dna binding domains, that allows them to act as transcription factors, and pas domains to form a protein complex.
the blrc controls the transcriptional response to light and photoconidiation .
in t. atroviride a set of genes targeted by the blrc was identified by microarray analysis after a 30 min pulse of white light  <cit> .
rosales et al.
<cit>  reported only  <dig> differentially expressed genes,  <dig> blue light up-regulated  and  <dig> blue light down-regulated .
from the  <dig> light regulated genes, only the blu <dig> gene was predicted as a putative transcription factor  encoding a c2h <dig> zinc finger dna binding domain protein.
in this sense, microarray analyses covering the complete genome of the fungus n. crassa revealed the activation of six genes encoding transcription factors in response to light  <cit> .
however, recently, high-throughput rna sequencing showed that there are  <dig> light responsive transcription factors in n. crassa  <cit> .
furthermore, transcriptional regulation by the n. crassa’s wcc in response to a brief pulse  of white light was recently analyzed by chip-seq, uncovering more than  <dig> genes regulated by this complex, including  <dig> transcription factors  as direct targets  <cit> .
currently, only six of the transcription factors, ,  <dig> c2h <dig>  and  <dig> zn2cys <dig> ) reported by chen et al.,  <cit>  have been studied in response to constant white light exposure.
these transcription factors regulate genes after illumination at early or late stages  <cit> .
however, only the absence of the gata factor sub- <dig>  showed the expected lack of expression in late light regulated genes after illumination with white light, as a component of a second transcriptional cascade  <cit> .
despite the lack of late light regulated genes in ∆sub- <dig>  asexual reproduction and carotenoid accumulation after illumination is similar to that of the wild type ; suggesting a main role of sub- <dig> in sexual reproduction, acting as a repressor of protoperithecia formation  <cit> .
the recent findings by rna-seq analysis of the light response in n. crassa revealed a complex regulation of gene expression during illumination, unveiling down-regulated genes previously not found by microarrary analysis.
in addition, tfs negatively regulated after the light treatment were discovered, integrating more pieces to the puzzle of the light response in this fungus  <cit> .
in a. nidulans the lrea and lreb proteins regulate  <dig> genes positively and  <dig> genes negatively in response to a brief pulse of light, representing 5 % of the genome  <cit> .
expression of the flbc gene, a c2h <dig> tf involved in conidiation, depends on the complex photoreceptor system integrated by the light response proteins lrea and lreb as well as the red light photoreceptor fpha.
flbc activation turns on the expression of the transcription factor brla, a well-known c2h <dig> tf regulator of conidiation  <cit> .
the photoreceptor complex also activates the expression of flbb and flbd encoding bzip and cmyc tfs, which also regulate the expression of brla, to promote the morphological transition of vegetative growth to conidiophores in a. nidulans  <cit> .
brla regulates the transition of the elongated hyphae to metulae, which in turn activates abaa expression  <cit> .
abaa controls the correct formation of conidial beaks, whose maturation is reached upon activation of weta  <cit> .
t. atroviride has one orthologue of the flbc gene as putative transcriptional activator but its expression does not appear to be affected by light, similarly to what is observed in a. fumigatus  <cit> .
despite the fact that there is no obvious brla orthologue in the genome of t. atroviride, homologues of abaa  and weta  have been identified.
overall, it is clear that there are undiscovered light transcriptional response pathways in t. atroviride.
at least part of such pathways must be blrc targets, such as the putative c2h <dig> transcription factor blu <dig>  acting downstream in response to light.
the rapid activation of blu <dig> expression, by the blr proteins, showed a maximum at 15 min both under constant illumination and after a pulse of blue light, suggesting a role in the control of early light regulated genes  <cit> .
the camp signaling pathway is involved in several processes in fungi, such as growth, reproduction and nutrient utilization  <cit> .
in t. atroviride light stimulates camp synthesis and asexual reproduction is stimulated by addition of extracellular camp both in the dark and in light on rich medium, but requires the presence of the blr <dig> and blr <dig> proteins.
intriguingly, the induction of asexual sporulation by sudden carbon starvation also requires the presence of the blr <dig> and blr <dig> proteins, but the addition of extracellular camp triggers conidiation even in the absence of the blr <dig> or blr <dig> genes  <cit> .
in addition, extracellular camp changes the degree of stimulation of conidiation provoked by different carbon sources in the blr mutants of t. atroviride, both in a positive and negative way  <cit> .
furthermore, the carbon source available and the blr <dig> or blr <dig> proteins act together to stimulate growth and conidiation in the presence of light or in the dark  <cit> .
these data led to the proposal that the camp-signaling pathway regulates conidiation genes through the action of the camp dependent kinase  in coordination with the blrc in response to light  <cit> .
on top of that, the blrc dependent pas domain protein envoy of trichoderma reesei, another photoreceptor, regulates camp production in the presence of light mainly by inhibition of the corresponding phosphodiesterase, linking regulation of asexual reproduction and nutrient signaling by modulating the expression of the g-proteins gna <dig> and gna <dig>  <cit> .
the present work describes the role of the putative c2h <dig> zinc finger transcription factor blu <dig> in the response to light.
rna-seq analysis of the blu <dig> gene replacement mutants was carried out after a pulse of blue light  to evaluate its role in photoconidiation.
the rapid accumulation of the blu <dig> mrna in response to light led us to hypothesize that it could be part of a transcriptional cascade resulting in asexual reproduction.
the blr <dig> and blr <dig> dependent induction of asexual reproduction by glucose starvation led us ask if the blu <dig> c2h <dig> zinc finger protein is involved in this process.
hence, we evaluated the transcriptional response of the ∆blu <dig> mutant when exposed to constant white light on glucose as a carbon source.
surprisingly, the transcriptional analysis of the light response uncovered a role of the blu <dig> gene in nitrogen regulation in a glucose dependent way.
RESULTS
blu <dig> is commonly found among hypocreales
blue light perception in t. atroviride through the blr <dig> and blr <dig> proteins activates transcription factors to control subsequent events of a transcriptional cascade.
one of this putative blr dependent transcription factors is encoded by the up-regulated gene blu <dig>  <cit> .
the previously reported blu <dig> cdna   <cit> , differed from the gene prediction based on genome sequencing , in that it contained a shorter open reading frame .
to establish what was the actual gene, we sequenced the cdna, which corresponded to a  <dig> bases long mrna .
the cds corresponded with that of another predicted gene , and encodes a  <dig> amino acid protein with a c2h <dig> zinc finger dna binding domain at the c-terminal region, which also contains a nuclear localization signal , two proline rich  motifs at the n-terminal region, and a glutamine rich region predicted by motif-scan  as a putative activation domain  <cit> .
to get insight into the putative function of the blu <dig> protein we searched for homologues by blast against the non-redundant database  of the ncbi.
orthologues of the blu <dig> protein are present in many, but not all ascomycetes, and they are more commonly found in the hypocreales, being trichoderma spp., fusarium, metarhizium and colletotrichum spp.
the most representative.
interestingly, we did not find orthologues of blu <dig> in the n. crassa or a. nidulans genomes.
absence of the blu <dig> gene results in reduced photoconidiation in t. atroviride
we replaced part of the coding sequence of blu <dig>  covering the polyq, prorich, and zinc finger c2h <dig> type domain at the c-terminal region of the originally predicted  <dig> amino acid protein  <cit> , by a cassette containing the selectable marker hph using the pcr double-joint protocol  <cit> .
replacement of this locus was validated by pcrs flanking the replacement cassette and confirmed by southern blot in six independent transformants .
although deletion of the blu <dig> locus was not complete, given that the replacement eliminated the most relevant motifs, we expected to generate a loss of function allele for this putative transcription factor .
the growth rate of the mutants was not affected in the dark, neither under constant white light illumination , as compared to the wt .
however, a clear growth delay of the three independent ∆blu <dig> mutants tested was observed when the strains were grown under constant blue light .
noticeably, during the first 36 h continuous illumination provoked the strongest effect on growth but by the end of the assay all colonies had the same diameter, suggesting that the mutants do not adapt to or tolerate light normally, and might, upon prolonged exposure to light, be able to compensate for this defect.
in contrast, when grown under photoperiods of 12 h blue light-darkness  during 72 h, the ∆blu <dig> mutants showed the same growth as the wt .
we also observed that 90 % more conidia were produced under constant blue  compared to white light  in the wt, whereas in the ∆blu <dig> mutants the level of conidiation in blue or white light was similar .
production of conidia by the ∆blu <dig> mutants under constant white light was 50 % of that observed for the wt, whereas when exposed to blue light conidia production of the mutant dropped to 33 % of that of the wt .
since the blrc is required to induce the formation of a conidiation ring after a pulse of blue light, and blu <dig> expression is blr dependent, we evaluated the production of asexual spores of the mutants when exposed to varying blue light fluence.
the conidiation ring was not formed at light fluences lower than  <dig> μmolm− <dig> in the ∆blu <dig> mutants.
nevertheless, at higher blue light fluences the conidiation ring is observed, but the mutants never reach the yield of conidia of the wt .
production of conidia is already detectable upon exposure to  <dig> μmolm− <dig> of blue light in the wt, whereas the ∆blu <dig> mutants require  <dig> μmolm− <dig> to trigger the conidiation process, pointing to either a light perception or downstream signaling defect .
however, the two strains appear to require approximately the same amount of photons to reach half-saturation of the response, suggesting that there is no such a defect in light perception.
we then analyzed the expression of the blu <dig>  env <dig> and, short aerial hyphae  <dig>  genes after a pulse of  <dig> μmolm− <dig> of blue light by semiquantitative pcr .
mrna of blu <dig> was observed even after 120 min of light induction in the wt and, as expected, it was not detected in the ∆blu <dig> mutant.
expression of the sah <dig> and env <dig> genes had the same profile in the wt and the ∆blu <dig> strain after light induction, suggesting that light perception by the blrc is functional.
thus, the absence of conidiation after exposure to  <dig> μmolm− <dig> of blue light is possibly due to the control of a particular set of genes by blu <dig> fig.
<dig> photoconidiation of the wt and ∆blu <dig> strains after a pulse of blue light.
a induction of conidiation ring in wt and ∆blu <dig> mutant strains in response to increasing blue light fluence, as indicated.
b conidia production of the wt  and the ∆blu <dig>  mutant at different blue light fluence.
the inset is a zoom view of the induction of conidiation at fluences lower than  <dig> μmolm− <dig>  the strains were cultivated during 36 h in the dark, then exposed to a pulse of blue light of  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> μmolm− <dig>  c semiquantitative rt-pcr of the blu <dig>  env <dig> and sah <dig> genes after a pulse of blue light in the wt and the ∆blu <dig> mutant; gpd was used as a control.
replicates of three independent assays were used to evaluate conidia production by the mutant and wt strains.
a t-test was applied to data with significant differences set at p <  <dig> .
a logarithmic base  <dig> tendency line was obtained for the data of the wt  and ∆blu <dig> mutant
the transcriptional response to light of the ∆blu <dig> mutant
given that the ∆blu <dig> mutants are unable to form the conidiation ring at  <dig> μmolm− <dig> blue light, we decided to analyze the transcriptomes of the wt and a ∆blu <dig> mutant under this condition to identify genes whose expression could be affected in the mutant, and that could be involved in asexual reproduction.
a total of  <dig> genes were differentially expressed in the ∆blu <dig> and  <dig> in the wt in light as compared to controls kept in the dark .
the wt strain showed more up-regulated genes  than the ∆blu <dig> mutant , while the ∆blu <dig> mutant presented more repressed genes  than the wt .
the expression levels of the main light responsive genes in the wt and ∆blu <dig> showed similar degree of induction or repression.
however, in the wt the light regulated genes in average are more strongly induced .fig.
<dig> transcriptional response of the wt and ∆blu <dig> strains 30 min after a pulse of blue light.
a boxplot of log2fc of the total differential light regulated genes in the wt and ∆blu <dig> strains, compared to dark control of each strain.
b plot of the wt vs ∆blu <dig> by log2fc, the resulting  <dig> clusters of the common regulated genes are indicated with roman numbers.
the light induced genes present only in the mutant are localized at the positive y-axis depicted in cyan and the repressed genes in dark orange in the negative y-axis.
the light induced genes present only in the wt are observed in green at the positive x-axis and the repressed genes are in red at the negative x-axis.
c blast2go annotation by biological process of the light regulated genes in the wt or ∆blu <dig> mutant strain.
d venn diagram showing the overlap in up-regulated  and down-regulated  genes that respond in the wt and ∆blu <dig> mutant strain
hierarchical clustering of the differentially expressed genes, based on expression level, results in six main clusters for the total light responsive genes, as compared to their corresponding dark control .
the most responsive genes, up- or down-regulated, were grouped in clusters i and vi .
cluster ii contained induced genes ranging from fc  <dig>  to  <dig>  cluster iii contained genes up-regulated in ∆blu <dig> in a fc range between  <dig> and  <dig> , whereas in the wt ranged from fc  <dig> to  <dig> .
in cluster iv we found the less repressed genes.
in cluster v the level of repression was similar in both strains, although not in the same genes.
homologues of previously reported light responsive genes in fungi were found distributed in all clusters.
among the genes regulated in common in both strains we found six genes more strongly induced in ∆blu <dig>  and  <dig> genes with higher induction in the wt .
on the other hand, among this set of genes we found  <dig> more strongly repressed in ∆blu <dig>  and  <dig> genes with stronger repression in the wt .
in cluster i we found two alleles  orthologous to the n. crassa ccg-1/grg <dig> gene , one of them  showed the highest level of expression in both the wt  and the ∆blu <dig>  mutant .
interestingly env <dig>  a gene related to photoadaptation of light regulated genes, was more strongly induced in the wt  than in the ∆blu <dig>  mutant .
the remaining  <dig> genes in cluster i were related to catalytic activity, protein binding and nucleotide binding with similar expression profile in the wt and the ∆blu <dig> strain.
in cluster ii we found an orthologue of the frq gene of n. crassa , involved in circadian clock regulation, and the blu <dig> gene , an orthologue of the n. crassa al- <dig> gene .
the frq gene had the same level of expression in both strains; but the orthologue of al- <dig> was slightly more strongly induced in the wt .
looking into the transcription factor encoding genes, we detected four in this cluster, three of them corresponding to zn2cys <dig> zinc finger proteins not yet studied, and the cp <dig> gene , an orthologue of the grh-like gene of n. crassa involved in the release of spores  <cit> .
only cp <dig> showed a slightly stronger induction in the ∆blu <dig>  than in the wt .
interestingly, the dna photolyase phr <dig>  and a putative base excision dna repair  protein encoding genes had stronger induction in the wt than in the mutant, both genes related to dna damage response .
in cluster iii we detected two transcription factors, the c2h <dig> zinc finger protein encoded by azf <dig>  and the gata type sub <dig> gene .
induction of the azf <dig> gene, involved in glucose growth regulation by cyclin control in saccharomyces cerevisiae  <cit>  was stronger in the wt  than in ∆blu <dig> .
induction of sub <dig> was higher in the ∆blu <dig> mutant  than in the wt .
in cluster iv we found nine genes that were more repressed in the wt than in the mutant, and  <dig> genes more repressed in the mutant than in the wt .
the genes encoding a calcipresin family protein , a ste <dig> like protein kinase  were among the most repressed ones in the wt.
calcipresin is a negative regulator of the calcineurin-calmodulin phosphatase in yeast and mammals  <cit> .
in aspergillus fumigatus and botrytis cinerea, calcipresin participates as a positive regulator of hyphal growth  <cit> .
in cluster v,  <dig> genes were more repressed in the wt than in the mutant, and  <dig> genes more repressed in ∆blu <dig> than in the wt.
the genes with stronger repression in both strains were detected in cluster vi .
the remaining genes were repressed to a similar extent in the ∆blu <dig> and the wt .
most of the induced genes in the wt and ∆blu <dig> mutant belong to the go-term metabolic process .
we observed the same subcategories of the metabolic process in the wt and ∆blu <dig> strains.
however, genes belonging to the lipid metabolic process were more abundant in the wt than in the mutant, whereas in ∆blu <dig> genes belonging to the protein metabolic process were more abundant.
the ∆blu <dig> mutant lacked two go biological process categories  among the induced genes compared to the wt, whereas the signaling process category was found as induced only in the ∆blu <dig> mutant.
in contrast, both the mutant and wt strain down regulated genes showed the same go categories in biological process, however more genes constituted these categories in the case of the ∆blu <dig> mutant .
even though the number of total light regulated genes in each strain is similar, the ∆blu <dig> mutant has less than half of uniquely induced genes  of those found for the wt , and almost twice the number of unique down-regulated genes .
blast2go annotation by biological process showed that the response to stress, nitrogen metabolism, localization and biosynthetic process were up-regulated only in the wt , while processes related to single organism signaling and catabolic process were up-regulated only in the ∆blu <dig> mutant .
although the same go biological processes were present in the down-regulated genes of both strains , those genes down regulated by light only in the ∆blu <dig> strain showed  <dig> repressed genes whose expression in the wt remained almost unchanged.
interestingly, more genes repressed only in ∆blu <dig> mutant integrated the go molecular process hydrolase activity, reported in t. reesei as regulated by the blr proteins and envoy  <cit> .
the expression profile of the genes  <dig>  and  <dig> , annotated in single organism signaling process, were induced only in the ∆blu <dig> mutant.
those genes annotated as belonging to response to stress, were induced in the wt, and showed low expression levels in the mutant.
aside, genes related to correct protein folding were more strongly repressed in the wt .
however the hsp <dig> gene, also related to correct protein folding was induced only in the wt .
furthermore, we observed several genes encoding a ribonuclease p-mrp, an hsp <dig> protein, a trichotecene c- <dig> hydrolase, a glycerol hydrolase, and a peroxisomal catalase with more than two fold induction in the wt that in the mutant did not show a change in expression after the light pulse , making them potential targets of blu <dig> regulation .fig.
<dig> blast2go annotation of the light regulated genes.
a go annotation by biological processes of the up- or down- regulated genes present only in wt or ∆blu <dig>  b transcriptional profile of selected genes from the transcriptome related to stress and signaling.
c light regulated genes present only in wt, not responsive in ∆blu <dig> , and light regulated genes present only in ∆blu <dig>  not responsive in wt  with hit description by blast2go.
d heatmap of the transcription factors detected among the light regulated genes of both strains
the molecular function of the most repressed genes  in the mutant or the wt strains was related to hydrolase activity, protein binding, transferase activity, and catalytic activity .
previous microarray studies in t. atroviride showed light induced expression of only one gene encoding a c2h <dig> zinc finger tf   <cit> .
here, we found  <dig> tfs differentially regulated by light in both strains .
twelve  belong to the zn2cys <dig> zinc finger family, five  to the c2h <dig> family, including blu <dig>  and the remaining 32 % to other families .
we detected six genes encoding tfs  repressed more than  <dig> fold only in the wt .
on the other hand, we identified four transcription factor encoding genes repressed more than two fold only in ∆blu <dig>
the blu <dig> protein is involved in tolerance to continuous exposure to light
the reduction of colony growth of the ∆blu <dig> mutant when grown under constant illumination prompted us to evaluate the response of the mutants and the wt under constant blue light.
radial growth and mycelial mass of the wt in darkness and constant exposure to blue light  was the same on pda.
however, a slight radial growth reduction at  <dig> and  <dig> μmolm−2s− <dig> of blue-light was noticed in the wt, although not statistically significant .
in contrast, the ∆blu <dig> mutant strain showed a stronger growth reduction under constant blue light as fluence increased .
the growth delay of the mutant strain was observed already at  <dig>  μmolm−2s− <dig> , and was more evident at  <dig> and  <dig> μmolm−2s− <dig> of blue light .
growth inhibition by light in t. atroviride has been reported to be dependent on the blr proteins and the carbon source available  <cit> .
since the blr complex induces blu <dig> gene expression, we evaluated the growth of the mutant under constant white light using glucose, glycerol, mannitol or fructose as sole carbon sources on minimal media and on pda .
we observed growth inhibition under constant white light on all carbon sources tested for both the wt and the ∆blu <dig> mutant strains .
however, the growth reduction of the mutants on medium containing glucose, glycerol or fructose was 40 % more pronounced than that of the wt, whereas the growth rate on mannitol was similar to that of the wt .
in this regard, it has been clearly established that primary and secondary metabolism processes are regulated by light in fungi  <cit> , but how are they regulated is still largely an open question.fig.
<dig> reduced growth of the ∆blu <dig> mutant cultivated under constant light.
a colony growth rate of the ∆blu <dig> and wt strains registered every 12 h during 72 h when grown under exposure to  <dig> ,  <dig> or  <dig> μmolm−2s− <dig> blue-light on pda.
b colonies of the wt and ∆blu <dig> strains grown on minimal media with glucose , glycerol , mannitol  or fructose  as a sole carbon source, pda was used as control.
colonies were photographed after 72 h of growth in the carbon source indicated, grown either in the dark  or under illumination with white light of  <dig>  μmolm−2s− <dig> .
c colony diameters measured after 72 h of growth.
one-way anova and pairwise t-test was applied to the data.
different letters indicate statistically significant differences.
three independent biological replicates were used for each strain
to gain deeper understanding of the function of blu <dig> in the light response, we obtained an overexpressing strain  of the blu <dig> gene reported by rosales et al.
<cit> .
for this purpose the 642 bp cds was placed under the control of the pyruvate kinase constitutive promoter.
the  <dig> aa long overexpressed version of blu <dig>  was enough to increase more than twice conidiation after a pulse of blue light compared to wt levels .
growth of the oeblu <dig> strain in darkness or in constant light  did not differ from that of the wt on minimal media on glucose .
however in constant light colonies of the oeblu <dig> strain appear to be greener, apparently due to higher production of conidia .
indeed, the oeblu <dig> strain produced 76 % more conidia than the wt under continuous exposure to white light, and 110 % more conidia when exposed to blue light , and no inhibition of growth by light was observed .
the fact that the overexpressing transformant still required light to conidiate indicates that expression of blu <dig> is not sufficient to trigger conidiation, and that other factors are required to achieve photoconidiation or that the blu <dig> protein needs to be post-translationally modified in a light dependent manner.fig.
<dig> effect of constant white and blue light effects on conidiation and growth of t. atroviride wt, ∆blu <dig> and oeblu <dig> strains using glucose as sole carbon source.
a colony growth after 72 h of growth under constant white  <dig>  μmolm−2s− <dig> or blue light  <dig>  μmolm−2s− <dig> of the ∆blu <dig>  wt and oeblu <dig> strains.
b colony diameter measured after 72 h. c conidia production of the ∆blu <dig>  wt and oeblu <dig> strains grown under constant white  or blue light .
b, c one-way anova and pairwise t-test was applied to the data.
different letters indicate statistically significant differences.
three independent biological replicates were used for each strain
carbon starvation induces asexual reproduction in t. atroviride  <cit> .
interestingly, in the absence of the blr proteins there is no response to this stimulus  <cit> , suggesting that down stream targets of the blr proteins regulate conidiation and growth under carbon starvation.
the fact that growth of the ∆blu <dig> mutants is reduced when compared to that of the wt, but only in the presence of light, indicates an alteration in their capacity to respond to light rather than a defect on carbon uptake.
consequently, we decided to analyze the transcriptional response of the ∆blu <dig> mutant and the wt strain under constant light using glucose as sole carbon source to identify the genes that are deregulated and involved in growth under such conditions.
blu <dig> is involved in glucose metabolism under constant light
a ∆blu <dig> mutant strain and the wt were cultivated under constant white light or in darkness with 2 % of glucose as sole carbon source during 60 h, and their transcriptome analyzed by rnaseq.
genes showing differential expression under these conditions may include direct targets of blr, genes regulated by blu <dig>  and genes regulated by events taking place further downstream in the signaling cascade.
global expression of light regulated genes ranged from  <dig> to  <dig>  fc for the wt, and between  <dig> and  <dig>  fc for the ∆blu <dig> mutant .
a total of  <dig> light regulated genes were identified in both strains , representing 16 % of the genome.
in the wt strain,  <dig> light regulated genes were identified and  <dig> in ∆blu <dig>  as compared to the corresponding controls grown in the dark.
interestingly we found five genes  induced in wt but repressed in the mutant and two genes  repressed in the wt but induced in the mutant.
twice as many uniquely induced genes were found in the wt  as in the mutant , and  <dig> genes overlapped between the two strains.
in contrast, unique down-regulated genes were almost the same number in the wt  and the ∆blu <dig> strain , and  <dig> common down-regulated genes were identified .fig.
<dig> comparison of the transcriptional response of the ∆blu <dig> and wt strains under constant white light.
a boxplot of differential light regulated genes with |log2fc| >  <dig> for the ∆blu <dig> and wt strains.
b overall plot of log2fc of the light regulated genes in wt vs ∆blu <dig>  hierarchical clustering results in  <dig> clusters .
horizontal lines of ± <dig> contain the genes differentially expressed only in the wt strain; vertical lines of ± <dig> contain the genes differentially expressed only in the ∆blu <dig> mutant.
c blast2go annotation of the light regulated genes in wt or ∆blu <dig> by biological process , molecular function  or cellular component .
d venn diagram showing the overlap in up-regulated  and down-regulated  genes that respond in the wt and ∆blu <dig> mutant strain
hierarchical clustering of the light regulated genes results in eight main groups depicted by colors and roman numbers .
in cluster i ,  <dig> genes integrated the highly induced genes in both strains, which included the conidiation marker gene con- <dig>  an orthologue of n. crassa  <cit> .
cluster ii also has genes highly up-regulated in both strains, however  <dig> genes were more induced in the wt and  <dig> were more induced in the ∆blu <dig> mutant .
interestingly, the env <dig> gene was more strongly induced in ∆blu <dig>  than in the wt , suggesting an altered photoadaptation response.
previously reported light responsive genes of fungi were also found in this cluster, such as the orthologues of the n. crassa clock controlled genes  <dig> and  <dig> , and con- <dig>  showing similar induction levels in both strains .
in cluster iii more genes of the wt showed stronger induction, compared to that found in the ∆blu <dig> strain.
go-terms of those genes indicate that they are involved in metabolism , stress responses  and transport .
this cluster also encompasses genes that respond more strongly in the ∆blu <dig> mutant than in the wt strain, two genes were involved in transport  and  <dig> genes were annotated in metabolism .
cluster iv contained genes more strongly induced in the wt than in the ∆blu <dig> mutant .
in this case, the main go terms enriched by biological process were related to biosynthetic, metabolic and nucleobase-containing compound metabolic process.
in this cluster we found the orthologues of the n. crassa clock-controlled gene  <dig>  and regulator of conidiation- <dig>  induced to a higher extent in the wt than in ∆blu <dig>  additionally, we observed  <dig> genes in the wt with more than  <dig> fold induction compared to the ∆blu <dig> mutant .
cluster v  contains genes with the lowest induction in the wt that in the ∆blu <dig> mutant were more induced.
the main genes in this group were annotated in transport and carbohydrate metabolic process.
mainly transporter encoding genes annotated as amino acid permeases and mfs multidrug transporters with more than two fold higher induction in the mutant.
cluster vi contains slightly repressed genes under continuous illumination .
in this case, we observed  <dig> genes repressed more than two fold in ∆blu7; whereas in the wt strain,  <dig> of the genes detected showed twice stronger repression compared to the mutant.
genes more strongly repressed in ∆blu <dig> were related mainly to metabolic process, and in the wt to metabolic process and transport.
in general, cluster vii contains genes repressed to the same extent in the wt and the ∆blu <dig> mutant.
however we detected  <dig> genes with more than two fold repression in ∆blu <dig>  two of them with peptidase activity  and the remaining genes were related to catalytic activity and hydrolase activity.
in the case of the wt, we observed  <dig> genes repressed more than two fold, as compared to the mutant.
in cluster viii we found the five most repressed genes of the light regulated set.
the benzoate 4-monooxygenase cytochrome p <dig> encoding gene was more strongly repressed in the wt  than in the mutant  and the cyclohexanone monooxygenase encoding gene  was more strongly repressed in the ∆blu <dig>  compared to wt .
while the tricothecene c15-hydroxylase and two cyclohexanone monooxygenase encoding genes were repressed to the same extent in the wt and the ∆blu <dig> strains.
most of the go-terms by biological process , molecular function  or cellular component  enriched in the up- or down- regulated genes were present in both strains .
however, go-terms enriched of the biological process categories related to localization, establishment of localization and transport were induced in the ∆blu <dig> mutant but repressed in wt .
interestingly, we observed that the molecular function  related to hydrolase activity  was enriched only in the case of the wt induced genes.
furthermore, the term transporter activity was enriched in the wt repressed genes, and in the ∆blu <dig> mutant induced ones.
these data prompted us to analyze the genes that are light regulated only in one of the strains.
enrichment analysis of the unique up- or down- regulated genes in the wt or ∆blu <dig> are shown in fig.
7a.
go-terms: localization, establishment of localization and transport were enriched in down-regulated genes of the wt and enriched in the up-regulated genes of the ∆blu <dig> mutant.
we also observed go-terms  of induced genes enriched only in wt and go-terms  of induced genes enriched only in the ∆blu <dig> mutant.
on the other hand, down-regulated genes were enriched in the go-terms carbohydrate metabolic process and catalytic activity in both strains .
looking into transport and transporter activity, go-terms enriched in the genes repressed in the wt and those induced in ∆blu <dig>  we observed that these categories were mainly represented by general nitrogen transporter and sugar transporter encoding genes .
interestingly, the ∆blu <dig> mutant presented general amino acid transporter and general monosaccharide transporter encoding genes as induced; whereas in the wt methionine transporter and general sugar transporter genes were repressed .
these data indicate a nitrogen metabolism defect in the mutants, so we cultivated the wt and ∆blu <dig> strains on minimal media supplemented with  <dig>  % peptone under constant light during 60 h .
radial growth of the wt and ∆blu <dig> mutant was the same under constant light or in darkness when grown on peptone-supplemented media.
however, production of conidia in the ∆blu <dig> mutant was much lower than in the wt , indicating that nitrogen sufficiency is enough to tolerate prolonged light exposure but not to support conidiation in ∆blu <dig> mutants.fig.
<dig> analysis of genes differentially expressed only in the wt or the ∆blu <dig> strains.
a enrichment analysis by biological process and molecular function of wt or ∆blu <dig> mutant of unique light regulated genes.
b heatmap of annotated genes in localization, establishment of localization and transport process in the wt and the ∆blu <dig> strains
fig.
<dig> growth under continuous exposure to light is rescued by a nitrogen rich medium.
growth of the wild type  and ∆blu <dig> mutant strain under constant white light  or in the dark , supplemented or not with  <dig>  % of peptone, as indicated
discussion
asexual reproduction of t. atroviride is induced by light, nutrient limitation and mechanical damage.
the blr complex does not only regulate photoconidiation, but also conidiation in response to sudden carbon deprivation  <cit> .
notwithstanding, downstream components of the signaling pathway initiated through the blr proteins are largely unknown.
a cdna microarray analysis identified a single putative transcription factor induced by a pulse of white light.
based on that microarray approach, covering only 12 % of the genes in the genome,  <dig> genes  were found to be light regulated.
to extend our knowledge of the transcriptional response of t. atroviride to light to a genome wide scale, we used rna-seq to analyze the role of the putative transcription factor blu <dig> in the control of asexual development, light sensitivity, and response to light on glucose as carbon source.
the transcriptional response of the wt and ∆blu <dig> mutant, 30 min after a pulse of 100 μmolm- <dig> of blue light, showed  <dig>  % of the genome responsive with more than 2-fold change, similarly to n. crassa, a. nidulans and a. fumigatus  <cit> .
interestingly we observed a significant correlation  of the up-regulated genes shared between the Δblu <dig> mutant and the wt, in contrast the down-regulated genes were not correlated , showing more negative values in log2fc of the ∆blu <dig> responsive genes.
these data indicate that blu <dig> plays a particularly relevant role in tuning the regulation of light repressed genes.
the go-terms  induced after the pulse of light only in the wt were related to hypothetical genes, whereas in the signaling category present only in the ∆blu <dig> mutant the genes encoded envoy <dig>  a 3’–5’ cyclic nucleotide phosphodiesterase and an arrestin domain-containing protein.
in this regard, it has been established that env <dig> is an orthologue of the n. crassa vivid involved in the negative regulation of light responsive genes in a process called photoadaptation.
while the cyclic nucleotide phosphodiesterase controls camp levels by hydrolyzing the phosphodiester bond in camp.
in trichoderma it is known that there is a transient biphasic oscillation in intracellular camp levels, activation of adenylyl cyclase, and phosphorylation of proteins upon exposure to a pulse of blue light.
in fact, addition of exogenous camp to trichoderma promotes sporulation even in the dark  <cit> , whereas atropine, a compound known to inhibit adenylyl cyclase in neurospora  <cit> , prevents sporulation even after photoinduction  <cit> .
in addition, the arrestin gene, which encodes a protein that blocks coupling of the gpcr to g proteins, was more strongly induced in the mutant.
thus, changes in arrestin levels could contribute to lower camp production by impairing gpcrs gna <dig> or gna <dig> signaling.
furthermore, envoy <dig> has been postulated as a regulator of camp levels in t. reesei trough repression of the camp phosphodiesterase  <cit> , and the g-protein coupled receptors gna <dig> and gna <dig> direct that control of camp levels by regulating env <dig> and their own expression.
in agreement with our observations of the light induction of blu <dig>  the corresponding mutants are less responsive to light, hence to conidiation, possibly due to reduced phosphodiesterase repression and partial loss of camp signaling of gna <dig> or gna <dig> by the presence of the arrestin protein.
although, the camp accumulation in the ∆blu <dig> mutant after the pulse of blue light requires further investigation in order to validate this hypothesis.
taking together these data, we suggest that in the t. atroviride wt strain camp might allow asexual reproduction after the pulse of light, but in the ∆blu <dig> strain the missing signaling impairs conidiation at low light fluence.
at high blue light fluence another level of regulation may exist, such as the accumulation of ros or dna damage, to induce conidiation in order to survive to extreme environments.
in agreement with these observations, a peroxisomal catalase and one heat shock protein were induced only in the wt, and another catalase was more strongly induced in the mutant than in the wt.
thus, the typical activation of stress responses observed in several fungi after exposure to light  <cit>  is reduced in the trichoderma ∆blu <dig> mutants.
it has been observed that oxidative stress stimulates differentiation in n. crassa, aspergillus and trichoderma , and ros produced upon light exposure induces conidiation in n. crassa  <cit> .
in this sense, catalases and superoxide dismutases reduce ros after the light pulse to re-establish cell homeostasis.
furthermore, it was recently shown that in b. cinerea the white collar proteins are required to deal with ros produced under constant illumination, and that the bcltf <dig> gata tf, a homologue of sub- <dig> of n. crassa, is important to cope with oxidative stress  <cit> .
in the ∆blu <dig> mutant described here we observed a higher expression of sub- <dig>  which might, consequently result in lower levels of ros after exposure to light, and might play a particularly relevant role at low light fluences.
another possible explanation for the reduced catalases transcript levels and lower conidiation levels in the ∆blu <dig> mutants is that there is a higher reducing power in these mutants than in the wt.
in this regard, we observed  <dig> genes with oxidoreductase activity repressed only in the mutant.
also, we observed  <dig> genes encoding enzymes with reductase activity  more strongly induced in ∆blu <dig> and  <dig> genes  repressed only in the wt.
in addition, there were  <dig> genes  repressed to a lower extent in the ∆blu <dig> mutant  than in the wt strain .
therefore, the reduced levels of ros signaling and camp production would not be sufficient to trigger conidiation in the ∆blu <dig> mutants at low light fluence.
this defect might be compensated at high light fluence by higher production of ros, although under those conditions these signals might be regulated through another mechanism.
in n. crassa and a. nidulans the wcc controls several transcription factors that in turn regulate asexual reproduction  <cit> .
among the transcription factors regulated by a pulse of blue light only  <dig> have go annotation.
we observed two tf  more strongly induced in the ∆blu <dig> mutant.
grhy-like or cp <dig> has been found in several fungal transcriptomes of light treatments  <cit> .
recently a cp2/grhy-like was identified as a conidial separation- <dig>  allele in n. crassa and shown to be involved in conidial separation and spore release by cell wall remodeling  <cit> .
reports on the study of a sub- <dig> deletion mutant showed that its presence is required to activate late light regulated genes in n. crassa.
however, the mutant presents defects only in sexual development  <cit> .
it might be that some of the genes more strongly induced in the ∆blu <dig> mutant are not directly under its control, but at least are in part regulated by cp <dig> or sub- <dig>
under constant illumination, we observed a growth delay in the ∆blu <dig> mutant at high light intensities .
this is in agreement with the requirement of the blr proteins to grow in the presence of blue or red light in t. atroviride  <cit> .
under constant illumination the blrc must regulate genes to deal with the constant exposure to light .
the growth delay of the mutants under constant blue light suggests that blu <dig> participates in light tolerance.
reduced tolerance of the ∆blu <dig> mutants was much more evident when glucose was used as sole carbon source compared to rich media, such as pda, suggesting a role of blu <dig> in carbon metabolism to tolerate continuous light exposure.
the combination of light with different carbon sources differentially stimulates conidiation and growth, and this response depends on the blr proteins  <cit> .
in addition, the downstream target of the blrc, envoy <dig> of t. reesei is involved in carbon dependent growth in the presence of light.
since light only temporarily delayed growth of the mutants, we suggest that they are affected in their capacity to tolerate light.
these phenotypes resemble those of the ∆env <dig> mutants of t. reesei and t. atroviride in constant light on pda  <cit> .
in the env <dig> mutants of t. reesei the lack of the negative feedback loop, over the light regulated genes controlled by the blrc, leads to a reduced growth  <cit> .
in this loop envoy acts as a repressor of negative regulators of growth activated by the blr proteins.
accordingly, the stronger induction of env <dig> in ∆blu <dig> suggests that blu <dig> participates as a positive regulator of growth in this negative feedback loop; however, further investigation is required to test this hypothesis.
conidiation of the ∆blu <dig> mutant was not completely impaired but reduced in constant light.
this is also in agreement with the phenotype of ∆env <dig> mutants of t. atroviride which produce more conidia than the wt strain in response to light, likely due to a longer permanence of mrnas of photoconidiation genes, such as blu <dig>  <cit> .
in the ∆blu <dig> mutant, the increased induction of env <dig> may lead to fast shut down of the photoconidiation genes and the absence of blu <dig> in reduced camp levels, due to increased transcript levels of the camp phosphodiesterase gene, and thereof of its enzymatic activity.
the differentially expressed genes observed in the ∆blu <dig> and the wt grown on glucose containing medium under continuous exposure to white light reflected major metabolic changes in both strains compared to darkness.
since in darkness, growth of the wt and the ∆blu <dig> mutant was similar, glucose uptake or metabolism is not deficient or impaired.
this points to a light activation of the blu <dig> protein or its light regulation through an interacting protein to support metabolic changes.
interestingly, go-terms related to localization and transport categories were repressed in the wt and induced in the ∆blu <dig> mutant.
these categories contained genes encoding sugar, amino acid and multidrug transporters.
in addition, we observed two-fold higher induction of the nmra gene  in the wt  compared to the ∆blu <dig> mutant .
in a. nidulans, nmra is a negative regulator of nitrogen metabolite repression, which controls expression of enzymes and permeases necessary for the use of non-preferred nitrogen sources  <cit> .
thus, the partial nitrogen metabolism deregulation observed in the ∆blu <dig> mutant might be due to nmra repression, leading to the induction of several amino acid transporter genes in ∆blu <dig>  in m. oryzae the tps <dig> gene , a glucose-6-phosphate sensor, negatively regulates nmr inactivating nmr1- <dig>  also the multidrug and toxin extrusion -family pump mdt <dig>  a downstream target of tps <dig>  is involved in glucose assimilation, conidiation and virulence  <cit> .
similarly, we observed several monosaccharide and general sugar transporters induced in ∆blu <dig> and repressed in the wt, as well as multidrug transporters induced in both strains.
these observations point to the light regulation of carbon and nitrogen metabolism to stimulate conidiation and growth in t. atroviride, respectively.
carbon or nitrogen starvation results in the formation of less branched compact hyphae than those observed in rich media, in addition the sudden lack of nitrogen or carbon source in t. atroviride resembles the ring of conidiation triggered by a pulse of light  <cit> .
interestingly, in the absence of the blr <dig> or blr <dig> genes the induction of conidiation by carbon  starvation is lost, in contrast nitrogen starvation can still stimulate conidiation, suggesting an independent regulation of asexual reproduction by the blr proteins  <cit> .
thus we hypothesized that nitrogen metabolism should be involved in hyphal growth under constant light.
several genes encoding enzymes with peptidase activity , including an intracellular serine protease, were induced in the wt strain, suggesting that protein degradation or recycling of nitrogen resources is activated in response to light.
nitrogen metabolism related genes have also been found among the light regulated genes of n. crassa  <cit> .
in accordance with these observations, we found an allantoinase-encoding gene , involved in purine metabolism, induced only in the wt.
besides two allantoate permeases  were repressed in the wt, but not in the ∆blu <dig> strain, and two allantoate transporters  were induced only in the mutant - allantoate is used as nitrogen storage in plants  <cit> .
transport of amino acids was repressed in the wt, mainly methionine transport.
in contrast, in ∆blu <dig> higher induction levels of genes encoding amino acid transporters and allantoate transporters were observed.
these data are highly coincident with the transcriptional response to nitrogen starvation reported in the phytopathogenic fungus magnaporthe grisea  <cit> .
taken together, our data appear to indicate that the absence of blu <dig> mimics a nitrogen starvation condition when trichoderma is grown under constant exposure to light, which results in clearly reduced growth.
restoration of growth rate and radial colony size of the wt and ∆blu <dig> mutants by the addition of peptone to the media suggests that light growth inhibition is mainly due to nitrogen intracellular metabolism, and that blu <dig> indirectly participates in the regulation of nitrogen metabolism in the presence of light under limited nitrogen supply .
as a consequence of the lack of blu <dig>  the mutants have lower capacity to recycle nitrogen sources, requiring the induction of general amino acid transporters, which in the presence of peptides from peptone are taken up and metabolized, resulting in much better growth.
we also observed misregulation of the ras gtpase , which was induced to higher levels in the ∆blu <dig>  mutant than in the wt .
activation of this pathway in yeast involves a g-protein coupled receptor and ras signaling to regulate glucose availability  <cit> .
from the gpcrs regulated in both strains only the predicted gpcr gene id.
<dig> is more strongly induced in the ∆blu <dig> mutant, which might act as an upstream regulator.
gpcrs are involved in amino acid and carbon source sensing as well as in camp perception.
the gpr <dig> gene , encoding a homolog of the n. crassa camp receptor gpr <dig>  <cit> , is induced only in the wt strain.
silencing of gpr <dig> in t. atroviride p <dig> reduces growth, conidiation and secondary metabolism  <cit> .
in yeast the gpr1-gpa <dig> gpcr system is involved in the regulation of camp signaling  <cit> .
the absence of gpr <dig> in the ∆blu <dig> mutants in conjunction with the increase in camp phosphodiesterase might be reflected in the lack of protein kinase a  activity resulting in the loss of this signaling pathway to regulate the light response.
pka signaling after a pulse of light is required to regulate up or down responsive genes, also regulated by the blr proteins  <cit> .
we further observed  <dig> serine-threonine protein kinase encoding genes induced only in the wt, which might exert posttranslational modifications in transcription factors, consequently affecting regulation of gene expression in response to light, or might directly affect enzyme activity, resulting in altered metabolism.
an overview of the data presented here is depicted in fig.
<dig>  the blrc perceives light and induces blu <dig> expression.
transient increase of camp by inhibition of the 3’–5’ camp phosphodiesterase leads to activation of photoconidiation genes by pka.
growth rate under constant light depends on nitrogen availability, controlling the uptake from the media or mobilization of stored nitrogen .fig.
<dig> model of the role of the transcriptional regulator blu <dig> in the molecular response of t. atroviride to light.
a molecular responses to a pulse of blue light.
exposure of t. atroviride to low fluence blue light perceived by the blrc results in the transcriptional activation of blu <dig>  sub <dig>  azf <dig>  xlnr and  <dig> other tf encoding genes, as well as the induction of envoy.
blu <dig> tunes photoconidiation by repressing the expression of the 5’-3’ camp phosphodiesterase encoding gene .
likewise, the induction of env <dig> enhances pd repression, while transitory accumulation of camp activates pka that in turn regulates photoconidiation genes  <cit> .
nitrogen mobilization  is indirectly regulated by blu <dig> and modulates the balance between conidiation and mycelial growth.
b molecular responses to continuous light exposure.
under constant white light, blu <dig> induction by the blrc is regulated by the carbon source available in the medium.
env <dig> negatively regulates blrc target genes for adaptation to constant light.
when t. atroviride is grown on glucose containing medium, blu <dig> limits the induction of env <dig>  blu <dig> is required for nitrogen mobilization through the inhibition of the orthologue of area by the negative regulator nmra up-regulation.
in addition, hyphal growth and conidiation are tightly regulated by the amount of flbc to induce the sequential expression of the transcription factors c2h <dig> ➜abaa ➜ weta.
black lines indicate control points supported by our rna-seq data and gray lines are based on previously reported experimental evidence
CONCLUSIONS
the present work describes the transcriptional response to light of the mutant in the putative transcription factor encoding gene blu <dig>  a blrc dependent light regulated gene in t. atroviride.
the blu <dig> gene is required for photoconidiation at low but not at high blue light fluence.
the absence of blu <dig> resulted in increased levels of expression of the 3’-5’ camp phosphodiesterase encoding gene, which could explain the reduced conidiation observed in the mutant.
furthermore, the increased expression of env <dig> observed in the ∆blu <dig> strain under constant illumination suggests linked roles in photoadaptation between these two regulators.
interestingly, the growth inhibition response of the blu <dig> replacement mutant to light was reduced in rich media.
the decreased expression of energy and metabolism related genes in the ∆blu <dig> mutant under constant light may explain the slower growth, rendered by defective nitrogen metabolism.
ethics
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 most organisms can perceive light as a signal and the response to this stimulus depends on the length of exposure and light quality, serving as a cue of environmental conditions. fungi can perceive a wide range of light wavelengths, from far red to ultraviolet light. many reports on light responses in fungi are related to reproduction, which determines whether they should enter asexual or sexual reproduction or neither. the best-characterized photoreceptor in fungi is the white collar complex of neurospora crassa. this blue light photoreceptor wcc, formed by the white collar proteins, regulates pigmentation, circadian rhythm, conidiation and phototropism of perithecial beaks. the wcc of n. crassa controls all light responses characterized so far in this fungus, despite the presence of red, uv or green photoreceptors in its genome. however, a cryptochrome dependent oscillator driving rhythmic spore development under constant light, where the frequency/wcc oscillator does not operate, was recently described. the cog- mutation uncovered this cry dependent oscillator in n. crassa, which regulates light responsive genes independently of the wcc. similarly, as no obvious biological functions for phy-1/phy- or nop- are reported, their role might be masked by the wcc function. moreover, in aspergillus nidulans the phytochrome is involved in repression of sexual development and mycotoxin production by red light, whereas the lrea and lreb proteins stimulate both. asexual reproduction induced by light in t. atroviride results in the formation of the so called “conidiation ring” in the periphery of the colony. the blrc, formed by the blr and blr proteins, is responsible for the perception of blue light. like their n. crassa counterparts, the blr proteins have gata zinc finger dna binding domains, that allows them to act as transcription factors, and pas domains to form a protein complex. the blrc controls the transcriptional response to light and photoconidiation. in t. atroviride a set of genes targeted by the blrc was identified by microarray analysis after a 30 min pulse of white light. rosales et al. reported only differentially expressed genes, blue light up-regulated and blue light down-regulated. from the light regulated genes, only the blu gene was predicted as a putative transcription factor encoding a c2h zinc finger dna binding domain protein. in this sense, microarray analyses covering the complete genome of the fungus n. crassa revealed the activation of six genes encoding transcription factors in response to light. however, recently, high-throughput rna sequencing showed that there are light responsive transcription factors in n. crassa. furthermore, transcriptional regulation by the n. crassa’s wcc in response to a brief pulse of white light was recently analyzed by chip-seq, uncovering more than genes regulated by this complex, including transcription factors as direct targets. currently, only six of the transcription factors,, c2h and zn2cys ) reported by chen et al., have been studied in response to constant white light exposure. these transcription factors regulate genes after illumination at early or late stages. however, only the absence of the gata factor sub- showed the expected lack of expression in late light regulated genes after illumination with white light, as a component of a second transcriptional cascade. despite the lack of late light regulated genes in ∆sub- asexual reproduction and carotenoid accumulation after illumination is similar to that of the wild type; suggesting a main role of sub- in sexual reproduction, acting as a repressor of protoperithecia formation. the recent findings by rna-seq analysis of the light response in n. crassa revealed a complex regulation of gene expression during illumination, unveiling down-regulated genes previously not found by microarrary analysis. in addition, tfs negatively regulated after the light treatment were discovered, integrating more pieces to the puzzle of the light response in this fungus. in a. nidulans the lrea and lreb proteins regulate genes positively and genes negatively in response to a brief pulse of light, representing 5 % of the genome. expression of the flbc gene, a c2h tf involved in conidiation, depends on the complex photoreceptor system integrated by the light response proteins lrea and lreb as well as the red light photoreceptor fpha. flbc activation turns on the expression of the transcription factor brla, a well-known c2h tf regulator of conidiation. the photoreceptor complex also activates the expression of flbb and flbd encoding bzip and cmyc tfs, which also regulate the expression of brla, to promote the morphological transition of vegetative growth to conidiophores in a. nidulans. brla regulates the transition of the elongated hyphae to metulae, which in turn activates abaa expression. abaa controls the correct formation of conidial beaks, whose maturation is reached upon activation of weta. t. atroviride has one orthologue of the flbc gene as putative transcriptional activator but its expression does not appear to be affected by light, similarly to what is observed in a. fumigatus. despite the fact that there is no obvious brla orthologue in the genome of t. atroviride, homologues of abaa and weta have been identified. overall, it is clear that there are undiscovered light transcriptional response pathways in t. atroviride. at least part of such pathways must be blrc targets, such as the putative c2h transcription factor blu acting downstream in response to light. the rapid activation of blu expression, by the blr proteins, showed a maximum at 15 min both under constant illumination and after a pulse of blue light, suggesting a role in the control of early light regulated genes. the camp signaling pathway is involved in several processes in fungi, such as growth, reproduction and nutrient utilization. in t. atroviride light stimulates camp synthesis and asexual reproduction is stimulated by addition of extracellular camp both in the dark and in light on rich medium, but requires the presence of the blr and blr proteins. intriguingly, the induction of asexual sporulation by sudden carbon starvation also requires the presence of the blr and blr proteins, but the addition of extracellular camp triggers conidiation even in the absence of the blr or blr genes. in addition, extracellular camp changes the degree of stimulation of conidiation provoked by different carbon sources in the blr mutants of t. atroviride, both in a positive and negative way. furthermore, the carbon source available and the blr or blr proteins act together to stimulate growth and conidiation in the presence of light or in the dark. these data led to the proposal that the camp-signaling pathway regulates conidiation genes through the action of the camp dependent kinase in coordination with the blrc in response to light. on top of that, the blrc dependent pas domain protein envoy of trichoderma reesei, another photoreceptor, regulates camp production in the presence of light mainly by inhibition of the corresponding phosphodiesterase, linking regulation of asexual reproduction and nutrient signaling by modulating the expression of the g-proteins gna and gna. the present work describes the role of the putative c2h zinc finger transcription factor blu in the response to light. rna-seq analysis of the blu gene replacement mutants was carried out after a pulse of blue light to evaluate its role in photoconidiation. the rapid accumulation of the blu mrna in response to light led us to hypothesize that it could be part of a transcriptional cascade resulting in asexual reproduction. the blr and blr dependent induction of asexual reproduction by glucose starvation led us ask if the blu c2h zinc finger protein is involved in this process. hence, we evaluated the transcriptional response of the ∆blu mutant when exposed to constant white light on glucose as a carbon source. surprisingly, the transcriptional analysis of the light response uncovered a role of the blu gene in nitrogen regulation in a glucose dependent way.  blu is commonly found among hypocreales blue light perception in t. atroviride through the blr and blr proteins activates transcription factors to control subsequent events of a transcriptional cascade. one of this putative blr dependent transcription factors is encoded by the up-regulated gene blu. the previously reported blu cdna, differed from the gene prediction based on genome sequencing, in that it contained a shorter open reading frame. to establish what was the actual gene, we sequenced the cdna, which corresponded to a bases long mrna. the cds corresponded with that of another predicted gene, and encodes a amino acid protein with a c2h zinc finger dna binding domain at the c-terminal region, which also contains a nuclear localization signal, two proline rich motifs at the n-terminal region, and a glutamine rich region predicted by motif-scan as a putative activation domain. to get insight into the putative function of the blu protein we searched for homologues by blast against the non-redundant database of the ncbi. orthologues of the blu protein are present in many, but not all ascomycetes, and they are more commonly found in the hypocreales, being trichoderma spp., fusarium, metarhizium and colletotrichum spp. the most representative. interestingly, we did not find orthologues of blu in the n. crassa or a. nidulans genomes. absence of the blu gene results in reduced photoconidiation in t. atroviride we replaced part of the coding sequence of blu covering the polyq, prorich, and zinc finger c2h type domain at the c-terminal region of the originally predicted amino acid protein, by a cassette containing the selectable marker hph using the pcr double-joint protocol. replacement of this locus was validated by pcrs flanking the replacement cassette and confirmed by southern blot in six independent transformants. although deletion of the blu locus was not complete, given that the replacement eliminated the most relevant motifs, we expected to generate a loss of function allele for this putative transcription factor. the growth rate of the mutants was not affected in the dark, neither under constant white light illumination, as compared to the wt. however, a clear growth delay of the three independent ∆blu mutants tested was observed when the strains were grown under constant blue light. noticeably, during the first 36 h continuous illumination provoked the strongest effect on growth but by the end of the assay all colonies had the same diameter, suggesting that the mutants do not adapt to or tolerate light normally, and might, upon prolonged exposure to light, be able to compensate for this defect. in contrast, when grown under photoperiods of 12 h blue light-darkness during 72 h, the ∆blu mutants showed the same growth as the wt. we also observed that 90 % more conidia were produced under constant blue compared to white light in the wt, whereas in the ∆blu mutants the level of conidiation in blue or white light was similar. production of conidia by the ∆blu mutants under constant white light was 50 % of that observed for the wt, whereas when exposed to blue light conidia production of the mutant dropped to 33 % of that of the wt. since the blrc is required to induce the formation of a conidiation ring after a pulse of blue light, and blu expression is blr dependent, we evaluated the production of asexual spores of the mutants when exposed to varying blue light fluence. the conidiation ring was not formed at light fluences lower than μmolm− in the ∆blu mutants. nevertheless, at higher blue light fluences the conidiation ring is observed, but the mutants never reach the yield of conidia of the wt. production of conidia is already detectable upon exposure to μmolm− of blue light in the wt, whereas the ∆blu mutants require μmolm− to trigger the conidiation process, pointing to either a light perception or downstream signaling defect. however, the two strains appear to require approximately the same amount of photons to reach half-saturation of the response, suggesting that there is no such a defect in light perception. we then analyzed the expression of the blu env and, short aerial hyphae genes after a pulse of μmolm− of blue light by semiquantitative pcr. mrna of blu was observed even after 120 min of light induction in the wt and, as expected, it was not detected in the ∆blu mutant. expression of the sah and env genes had the same profile in the wt and the ∆blu strain after light induction, suggesting that light perception by the blrc is functional. thus, the absence of conidiation after exposure to μmolm− of blue light is possibly due to the control of a particular set of genes by blu fig. photoconidiation of the wt and ∆blu strains after a pulse of blue light. a induction of conidiation ring in wt and ∆blu mutant strains in response to increasing blue light fluence, as indicated. b conidia production of the wt and the ∆blu mutant at different blue light fluence. the inset is a zoom view of the induction of conidiation at fluences lower than μmolm− the strains were cultivated during 36 h in the dark, then exposed to a pulse of blue light of and μmolm− c semiquantitative rt-pcr of the blu env and sah genes after a pulse of blue light in the wt and the ∆blu mutant; gpd was used as a control. replicates of three independent assays were used to evaluate conidia production by the mutant and wt strains. a t-test was applied to data with significant differences set at p < . a logarithmic base tendency line was obtained for the data of the wt and ∆blu mutant the transcriptional response to light of the ∆blu mutant given that the ∆blu mutants are unable to form the conidiation ring at μmolm− blue light, we decided to analyze the transcriptomes of the wt and a ∆blu mutant under this condition to identify genes whose expression could be affected in the mutant, and that could be involved in asexual reproduction. a total of genes were differentially expressed in the ∆blu and in the wt in light as compared to controls kept in the dark. the wt strain showed more up-regulated genes than the ∆blu mutant, while the ∆blu mutant presented more repressed genes than the wt. the expression levels of the main light responsive genes in the wt and ∆blu showed similar degree of induction or repression. however, in the wt the light regulated genes in average are more strongly induced.fig. transcriptional response of the wt and ∆blu strains 30 min after a pulse of blue light. a boxplot of log2fc of the total differential light regulated genes in the wt and ∆blu strains, compared to dark control of each strain. b plot of the wt vs ∆blu by log2fc, the resulting clusters of the common regulated genes are indicated with roman numbers. the light induced genes present only in the mutant are localized at the positive y-axis depicted in cyan and the repressed genes in dark orange in the negative y-axis. the light induced genes present only in the wt are observed in green at the positive x-axis and the repressed genes are in red at the negative x-axis. c blast2go annotation by biological process of the light regulated genes in the wt or ∆blu mutant strain. d venn diagram showing the overlap in up-regulated and down-regulated genes that respond in the wt and ∆blu mutant strain hierarchical clustering of the differentially expressed genes, based on expression level, results in six main clusters for the total light responsive genes, as compared to their corresponding dark control. the most responsive genes, up- or down-regulated, were grouped in clusters i and vi. cluster ii contained induced genes ranging from fc to cluster iii contained genes up-regulated in ∆blu in a fc range between and, whereas in the wt ranged from fc to. in cluster iv we found the less repressed genes. in cluster v the level of repression was similar in both strains, although not in the same genes. homologues of previously reported light responsive genes in fungi were found distributed in all clusters. among the genes regulated in common in both strains we found six genes more strongly induced in ∆blu and genes with higher induction in the wt. on the other hand, among this set of genes we found more strongly repressed in ∆blu and genes with stronger repression in the wt. in cluster i we found two alleles orthologous to the n. crassa ccg-1/grg gene, one of them showed the highest level of expression in both the wt and the ∆blu mutant. interestingly env a gene related to photoadaptation of light regulated genes, was more strongly induced in the wt than in the ∆blu mutant. the remaining genes in cluster i were related to catalytic activity, protein binding and nucleotide binding with similar expression profile in the wt and the ∆blu strain. in cluster ii we found an orthologue of the frq gene of n. crassa, involved in circadian clock regulation, and the blu gene, an orthologue of the n. crassa al- gene. the frq gene had the same level of expression in both strains; but the orthologue of al- was slightly more strongly induced in the wt. looking into the transcription factor encoding genes, we detected four in this cluster, three of them corresponding to zn2cys zinc finger proteins not yet studied, and the cp gene, an orthologue of the grh-like gene of n. crassa involved in the release of spores. only cp showed a slightly stronger induction in the ∆blu than in the wt. interestingly, the dna photolyase phr and a putative base excision dna repair protein encoding genes had stronger induction in the wt than in the mutant, both genes related to dna damage response. in cluster iii we detected two transcription factors, the c2h zinc finger protein encoded by azf and the gata type sub gene. induction of the azf gene, involved in glucose growth regulation by cyclin control in saccharomyces cerevisiae was stronger in the wt than in ∆blu. induction of sub was higher in the ∆blu mutant than in the wt. in cluster iv we found nine genes that were more repressed in the wt than in the mutant, and genes more repressed in the mutant than in the wt. the genes encoding a calcipresin family protein, a ste like protein kinase were among the most repressed ones in the wt. calcipresin is a negative regulator of the calcineurin-calmodulin phosphatase in yeast and mammals. in aspergillus fumigatus and botrytis cinerea, calcipresin participates as a positive regulator of hyphal growth. in cluster v, genes were more repressed in the wt than in the mutant, and genes more repressed in ∆blu than in the wt. the genes with stronger repression in both strains were detected in cluster vi. the remaining genes were repressed to a similar extent in the ∆blu and the wt. most of the induced genes in the wt and ∆blu mutant belong to the go-term metabolic process. we observed the same subcategories of the metabolic process in the wt and ∆blu strains. however, genes belonging to the lipid metabolic process were more abundant in the wt than in the mutant, whereas in ∆blu genes belonging to the protein metabolic process were more abundant. the ∆blu mutant lacked two go biological process categories among the induced genes compared to the wt, whereas the signaling process category was found as induced only in the ∆blu mutant. in contrast, both the mutant and wt strain down regulated genes showed the same go categories in biological process, however more genes constituted these categories in the case of the ∆blu mutant. even though the number of total light regulated genes in each strain is similar, the ∆blu mutant has less than half of uniquely induced genes of those found for the wt, and almost twice the number of unique down-regulated genes. blast2go annotation by biological process showed that the response to stress, nitrogen metabolism, localization and biosynthetic process were up-regulated only in the wt, while processes related to single organism signaling and catabolic process were up-regulated only in the ∆blu mutant. although the same go biological processes were present in the down-regulated genes of both strains, those genes down regulated by light only in the ∆blu strain showed repressed genes whose expression in the wt remained almost unchanged. interestingly, more genes repressed only in ∆blu mutant integrated the go molecular process hydrolase activity, reported in t. reesei as regulated by the blr proteins and envoy. the expression profile of the genes and, annotated in single organism signaling process, were induced only in the ∆blu mutant. those genes annotated as belonging to response to stress, were induced in the wt, and showed low expression levels in the mutant. aside, genes related to correct protein folding were more strongly repressed in the wt. however the hsp gene, also related to correct protein folding was induced only in the wt. furthermore, we observed several genes encoding a ribonuclease p-mrp, an hsp protein, a trichotecene c- hydrolase, a glycerol hydrolase, and a peroxisomal catalase with more than two fold induction in the wt that in the mutant did not show a change in expression after the light pulse, making them potential targets of blu regulation.fig. blast2go annotation of the light regulated genes. a go annotation by biological processes of the up- or down- regulated genes present only in wt or ∆blu b transcriptional profile of selected genes from the transcriptome related to stress and signaling. c light regulated genes present only in wt, not responsive in ∆blu, and light regulated genes present only in ∆blu not responsive in wt with hit description by blast2go. d heatmap of the transcription factors detected among the light regulated genes of both strains the molecular function of the most repressed genes in the mutant or the wt strains was related to hydrolase activity, protein binding, transferase activity, and catalytic activity. previous microarray studies in t. atroviride showed light induced expression of only one gene encoding a c2h zinc finger tf. here, we found tfs differentially regulated by light in both strains. twelve belong to the zn2cys zinc finger family, five to the c2h family, including blu and the remaining 32 % to other families. we detected six genes encoding tfs repressed more than fold only in the wt. on the other hand, we identified four transcription factor encoding genes repressed more than two fold only in ∆blu the blu protein is involved in tolerance to continuous exposure to light the reduction of colony growth of the ∆blu mutant when grown under constant illumination prompted us to evaluate the response of the mutants and the wt under constant blue light. radial growth and mycelial mass of the wt in darkness and constant exposure to blue light was the same on pda. however, a slight radial growth reduction at and μmolm−2s− of blue-light was noticed in the wt, although not statistically significant. in contrast, the ∆blu mutant strain showed a stronger growth reduction under constant blue light as fluence increased. the growth delay of the mutant strain was observed already at μmolm−2s−, and was more evident at and μmolm−2s− of blue light. growth inhibition by light in t. atroviride has been reported to be dependent on the blr proteins and the carbon source available. since the blr complex induces blu gene expression, we evaluated the growth of the mutant under constant white light using glucose, glycerol, mannitol or fructose as sole carbon sources on minimal media and on pda. we observed growth inhibition under constant white light on all carbon sources tested for both the wt and the ∆blu mutant strains. however, the growth reduction of the mutants on medium containing glucose, glycerol or fructose was 40 % more pronounced than that of the wt, whereas the growth rate on mannitol was similar to that of the wt. in this regard, it has been clearly established that primary and secondary metabolism processes are regulated by light in fungi, but how are they regulated is still largely an open question.fig. reduced growth of the ∆blu mutant cultivated under constant light. a colony growth rate of the ∆blu and wt strains registered every 12 h during 72 h when grown under exposure to, or μmolm−2s− blue-light on pda. b colonies of the wt and ∆blu strains grown on minimal media with glucose, glycerol, mannitol or fructose as a sole carbon source, pda was used as control. colonies were photographed after 72 h of growth in the carbon source indicated, grown either in the dark or under illumination with white light of μmolm−2s−. c colony diameters measured after 72 h of growth. one-way anova and pairwise t-test was applied to the data. different letters indicate statistically significant differences. three independent biological replicates were used for each strain to gain deeper understanding of the function of blu in the light response, we obtained an overexpressing strain of the blu gene reported by rosales et al.. for this purpose the 642 bp cds was placed under the control of the pyruvate kinase constitutive promoter. the aa long overexpressed version of blu was enough to increase more than twice conidiation after a pulse of blue light compared to wt levels. growth of the oeblu strain in darkness or in constant light did not differ from that of the wt on minimal media on glucose. however in constant light colonies of the oeblu strain appear to be greener, apparently due to higher production of conidia. indeed, the oeblu strain produced 76 % more conidia than the wt under continuous exposure to white light, and 110 % more conidia when exposed to blue light, and no inhibition of growth by light was observed. the fact that the overexpressing transformant still required light to conidiate indicates that expression of blu is not sufficient to trigger conidiation, and that other factors are required to achieve photoconidiation or that the blu protein needs to be post-translationally modified in a light dependent manner.fig. effect of constant white and blue light effects on conidiation and growth of t. atroviride wt, ∆blu and oeblu strains using glucose as sole carbon source. a colony growth after 72 h of growth under constant white μmolm−2s− or blue light μmolm−2s− of the ∆blu wt and oeblu strains. b colony diameter measured after 72 h. c conidia production of the ∆blu wt and oeblu strains grown under constant white or blue light. b, c one-way anova and pairwise t-test was applied to the data. different letters indicate statistically significant differences. three independent biological replicates were used for each strain carbon starvation induces asexual reproduction in t. atroviride. interestingly, in the absence of the blr proteins there is no response to this stimulus, suggesting that down stream targets of the blr proteins regulate conidiation and growth under carbon starvation. the fact that growth of the ∆blu mutants is reduced when compared to that of the wt, but only in the presence of light, indicates an alteration in their capacity to respond to light rather than a defect on carbon uptake. consequently, we decided to analyze the transcriptional response of the ∆blu mutant and the wt strain under constant light using glucose as sole carbon source to identify the genes that are deregulated and involved in growth under such conditions. blu is involved in glucose metabolism under constant light a ∆blu mutant strain and the wt were cultivated under constant white light or in darkness with 2 % of glucose as sole carbon source during 60 h, and their transcriptome analyzed by rnaseq. genes showing differential expression under these conditions may include direct targets of blr, genes regulated by blu and genes regulated by events taking place further downstream in the signaling cascade. global expression of light regulated genes ranged from to fc for the wt, and between and fc for the ∆blu mutant. a total of light regulated genes were identified in both strains, representing 16 % of the genome. in the wt strain, light regulated genes were identified and in ∆blu as compared to the corresponding controls grown in the dark. interestingly we found five genes induced in wt but repressed in the mutant and two genes repressed in the wt but induced in the mutant. twice as many uniquely induced genes were found in the wt as in the mutant, and genes overlapped between the two strains. in contrast, unique down-regulated genes were almost the same number in the wt and the ∆blu strain, and common down-regulated genes were identified.fig. comparison of the transcriptional response of the ∆blu and wt strains under constant white light. a boxplot of differential light regulated genes with |log2fc| >  for the ∆blu and wt strains. b overall plot of log2fc of the light regulated genes in wt vs ∆blu hierarchical clustering results in clusters. horizontal lines of ± contain the genes differentially expressed only in the wt strain; vertical lines of ± contain the genes differentially expressed only in the ∆blu mutant. c blast2go annotation of the light regulated genes in wt or ∆blu by biological process, molecular function or cellular component. d venn diagram showing the overlap in up-regulated and down-regulated genes that respond in the wt and ∆blu mutant strain hierarchical clustering of the light regulated genes results in eight main groups depicted by colors and roman numbers. in cluster i, genes integrated the highly induced genes in both strains, which included the conidiation marker gene con- an orthologue of n. crassa. cluster ii also has genes highly up-regulated in both strains, however genes were more induced in the wt and were more induced in the ∆blu mutant. interestingly, the env gene was more strongly induced in ∆blu than in the wt, suggesting an altered photoadaptation response. previously reported light responsive genes of fungi were also found in this cluster, such as the orthologues of the n. crassa clock controlled genes and, and con- showing similar induction levels in both strains. in cluster iii more genes of the wt showed stronger induction, compared to that found in the ∆blu strain. go-terms of those genes indicate that they are involved in metabolism, stress responses and transport. this cluster also encompasses genes that respond more strongly in the ∆blu mutant than in the wt strain, two genes were involved in transport and genes were annotated in metabolism. cluster iv contained genes more strongly induced in the wt than in the ∆blu mutant. in this case, the main go terms enriched by biological process were related to biosynthetic, metabolic and nucleobase-containing compound metabolic process. in this cluster we found the orthologues of the n. crassa clock-controlled gene and regulator of conidiation- induced to a higher extent in the wt than in ∆blu additionally, we observed genes in the wt with more than fold induction compared to the ∆blu mutant. cluster v contains genes with the lowest induction in the wt that in the ∆blu mutant were more induced. the main genes in this group were annotated in transport and carbohydrate metabolic process. mainly transporter encoding genes annotated as amino acid permeases and mfs multidrug transporters with more than two fold higher induction in the mutant. cluster vi contains slightly repressed genes under continuous illumination. in this case, we observed genes repressed more than two fold in ∆blu7; whereas in the wt strain, of the genes detected showed twice stronger repression compared to the mutant. genes more strongly repressed in ∆blu were related mainly to metabolic process, and in the wt to metabolic process and transport. in general, cluster vii contains genes repressed to the same extent in the wt and the ∆blu mutant. however we detected genes with more than two fold repression in ∆blu two of them with peptidase activity and the remaining genes were related to catalytic activity and hydrolase activity. in the case of the wt, we observed genes repressed more than two fold, as compared to the mutant. in cluster viii we found the five most repressed genes of the light regulated set. the benzoate 4-monooxygenase cytochrome p encoding gene was more strongly repressed in the wt than in the mutant and the cyclohexanone monooxygenase encoding gene was more strongly repressed in the ∆blu compared to wt. while the tricothecene c15-hydroxylase and two cyclohexanone monooxygenase encoding genes were repressed to the same extent in the wt and the ∆blu strains. most of the go-terms by biological process, molecular function or cellular component enriched in the up- or down- regulated genes were present in both strains. however, go-terms enriched of the biological process categories related to localization, establishment of localization and transport were induced in the ∆blu mutant but repressed in wt. interestingly, we observed that the molecular function related to hydrolase activity was enriched only in the case of the wt induced genes. furthermore, the term transporter activity was enriched in the wt repressed genes, and in the ∆blu mutant induced ones. these data prompted us to analyze the genes that are light regulated only in one of the strains. enrichment analysis of the unique up- or down- regulated genes in the wt or ∆blu are shown in fig. 7a. go-terms: localization, establishment of localization and transport were enriched in down-regulated genes of the wt and enriched in the up-regulated genes of the ∆blu mutant. we also observed go-terms of induced genes enriched only in wt and go-terms of induced genes enriched only in the ∆blu mutant. on the other hand, down-regulated genes were enriched in the go-terms carbohydrate metabolic process and catalytic activity in both strains. looking into transport and transporter activity, go-terms enriched in the genes repressed in the wt and those induced in ∆blu we observed that these categories were mainly represented by general nitrogen transporter and sugar transporter encoding genes. interestingly, the ∆blu mutant presented general amino acid transporter and general monosaccharide transporter encoding genes as induced; whereas in the wt methionine transporter and general sugar transporter genes were repressed. these data indicate a nitrogen metabolism defect in the mutants, so we cultivated the wt and ∆blu strains on minimal media supplemented with  % peptone under constant light during 60 h. radial growth of the wt and ∆blu mutant was the same under constant light or in darkness when grown on peptone-supplemented media. however, production of conidia in the ∆blu mutant was much lower than in the wt, indicating that nitrogen sufficiency is enough to tolerate prolonged light exposure but not to support conidiation in ∆blu mutants.fig. analysis of genes differentially expressed only in the wt or the ∆blu strains. a enrichment analysis by biological process and molecular function of wt or ∆blu mutant of unique light regulated genes. b heatmap of annotated genes in localization, establishment of localization and transport process in the wt and the ∆blu strains fig. growth under continuous exposure to light is rescued by a nitrogen rich medium. growth of the wild type and ∆blu mutant strain under constant white light or in the dark, supplemented or not with  % of peptone, as indicated discussion asexual reproduction of t. atroviride is induced by light, nutrient limitation and mechanical damage. the blr complex does not only regulate photoconidiation, but also conidiation in response to sudden carbon deprivation. notwithstanding, downstream components of the signaling pathway initiated through the blr proteins are largely unknown. a cdna microarray analysis identified a single putative transcription factor induced by a pulse of white light. based on that microarray approach, covering only 12 % of the genes in the genome, genes were found to be light regulated. to extend our knowledge of the transcriptional response of t. atroviride to light to a genome wide scale, we used rna-seq to analyze the role of the putative transcription factor blu in the control of asexual development, light sensitivity, and response to light on glucose as carbon source. the transcriptional response of the wt and ∆blu mutant, 30 min after a pulse of 100 μmolm- of blue light, showed  % of the genome responsive with more than 2-fold change, similarly to n. crassa, a. nidulans and a. fumigatus. interestingly we observed a significant correlation of the up-regulated genes shared between the Δblu mutant and the wt, in contrast the down-regulated genes were not correlated, showing more negative values in log2fc of the ∆blu responsive genes. these data indicate that blu plays a particularly relevant role in tuning the regulation of light repressed genes. the go-terms induced after the pulse of light only in the wt were related to hypothetical genes, whereas in the signaling category present only in the ∆blu mutant the genes encoded envoy a 3’–5’ cyclic nucleotide phosphodiesterase and an arrestin domain-containing protein. in this regard, it has been established that env is an orthologue of the n. crassa vivid involved in the negative regulation of light responsive genes in a process called photoadaptation. while the cyclic nucleotide phosphodiesterase controls camp levels by hydrolyzing the phosphodiester bond in camp. in trichoderma it is known that there is a transient biphasic oscillation in intracellular camp levels, activation of adenylyl cyclase, and phosphorylation of proteins upon exposure to a pulse of blue light. in fact, addition of exogenous camp to trichoderma promotes sporulation even in the dark, whereas atropine, a compound known to inhibit adenylyl cyclase in neurospora, prevents sporulation even after photoinduction. in addition, the arrestin gene, which encodes a protein that blocks coupling of the gpcr to g proteins, was more strongly induced in the mutant. thus, changes in arrestin levels could contribute to lower camp production by impairing gpcrs gna or gna signaling. furthermore, envoy has been postulated as a regulator of camp levels in t. reesei trough repression of the camp phosphodiesterase, and the g-protein coupled receptors gna and gna direct that control of camp levels by regulating env and their own expression. in agreement with our observations of the light induction of blu the corresponding mutants are less responsive to light, hence to conidiation, possibly due to reduced phosphodiesterase repression and partial loss of camp signaling of gna or gna by the presence of the arrestin protein. although, the camp accumulation in the ∆blu mutant after the pulse of blue light requires further investigation in order to validate this hypothesis. taking together these data, we suggest that in the t. atroviride wt strain camp might allow asexual reproduction after the pulse of light, but in the ∆blu strain the missing signaling impairs conidiation at low light fluence. at high blue light fluence another level of regulation may exist, such as the accumulation of ros or dna damage, to induce conidiation in order to survive to extreme environments. in agreement with these observations, a peroxisomal catalase and one heat shock protein were induced only in the wt, and another catalase was more strongly induced in the mutant than in the wt. thus, the typical activation of stress responses observed in several fungi after exposure to light is reduced in the trichoderma ∆blu mutants. it has been observed that oxidative stress stimulates differentiation in n. crassa, aspergillus and trichoderma, and ros produced upon light exposure induces conidiation in n. crassa. in this sense, catalases and superoxide dismutases reduce ros after the light pulse to re-establish cell homeostasis. furthermore, it was recently shown that in b. cinerea the white collar proteins are required to deal with ros produced under constant illumination, and that the bcltf gata tf, a homologue of sub- of n. crassa, is important to cope with oxidative stress. in the ∆blu mutant described here we observed a higher expression of sub- which might, consequently result in lower levels of ros after exposure to light, and might play a particularly relevant role at low light fluences. another possible explanation for the reduced catalases transcript levels and lower conidiation levels in the ∆blu mutants is that there is a higher reducing power in these mutants than in the wt. in this regard, we observed genes with oxidoreductase activity repressed only in the mutant. also, we observed genes encoding enzymes with reductase activity more strongly induced in ∆blu and genes repressed only in the wt. in addition, there were genes repressed to a lower extent in the ∆blu mutant than in the wt strain. therefore, the reduced levels of ros signaling and camp production would not be sufficient to trigger conidiation in the ∆blu mutants at low light fluence. this defect might be compensated at high light fluence by higher production of ros, although under those conditions these signals might be regulated through another mechanism. in n. crassa and a. nidulans the wcc controls several transcription factors that in turn regulate asexual reproduction. among the transcription factors regulated by a pulse of blue light only have go annotation. we observed two tf more strongly induced in the ∆blu mutant. grhy-like or cp has been found in several fungal transcriptomes of light treatments. recently a cp2/grhy-like was identified as a conidial separation- allele in n. crassa and shown to be involved in conidial separation and spore release by cell wall remodeling. reports on the study of a sub- deletion mutant showed that its presence is required to activate late light regulated genes in n. crassa. however, the mutant presents defects only in sexual development. it might be that some of the genes more strongly induced in the ∆blu mutant are not directly under its control, but at least are in part regulated by cp or sub- under constant illumination, we observed a growth delay in the ∆blu mutant at high light intensities. this is in agreement with the requirement of the blr proteins to grow in the presence of blue or red light in t. atroviride. under constant illumination the blrc must regulate genes to deal with the constant exposure to light. the growth delay of the mutants under constant blue light suggests that blu participates in light tolerance. reduced tolerance of the ∆blu mutants was much more evident when glucose was used as sole carbon source compared to rich media, such as pda, suggesting a role of blu in carbon metabolism to tolerate continuous light exposure. the combination of light with different carbon sources differentially stimulates conidiation and growth, and this response depends on the blr proteins. in addition, the downstream target of the blrc, envoy of t. reesei is involved in carbon dependent growth in the presence of light. since light only temporarily delayed growth of the mutants, we suggest that they are affected in their capacity to tolerate light. these phenotypes resemble those of the ∆env mutants of t. reesei and t. atroviride in constant light on pda. in the env mutants of t. reesei the lack of the negative feedback loop, over the light regulated genes controlled by the blrc, leads to a reduced growth. in this loop envoy acts as a repressor of negative regulators of growth activated by the blr proteins. accordingly, the stronger induction of env in ∆blu suggests that blu participates as a positive regulator of growth in this negative feedback loop; however, further investigation is required to test this hypothesis. conidiation of the ∆blu mutant was not completely impaired but reduced in constant light. this is also in agreement with the phenotype of ∆env mutants of t. atroviride which produce more conidia than the wt strain in response to light, likely due to a longer permanence of mrnas of photoconidiation genes, such as blu. in the ∆blu mutant, the increased induction of env may lead to fast shut down of the photoconidiation genes and the absence of blu in reduced camp levels, due to increased transcript levels of the camp phosphodiesterase gene, and thereof of its enzymatic activity. the differentially expressed genes observed in the ∆blu and the wt grown on glucose containing medium under continuous exposure to white light reflected major metabolic changes in both strains compared to darkness. since in darkness, growth of the wt and the ∆blu mutant was similar, glucose uptake or metabolism is not deficient or impaired. this points to a light activation of the blu protein or its light regulation through an interacting protein to support metabolic changes. interestingly, go-terms related to localization and transport categories were repressed in the wt and induced in the ∆blu mutant. these categories contained genes encoding sugar, amino acid and multidrug transporters. in addition, we observed two-fold higher induction of the nmra gene in the wt compared to the ∆blu mutant. in a. nidulans, nmra is a negative regulator of nitrogen metabolite repression, which controls expression of enzymes and permeases necessary for the use of non-preferred nitrogen sources. thus, the partial nitrogen metabolism deregulation observed in the ∆blu mutant might be due to nmra repression, leading to the induction of several amino acid transporter genes in ∆blu in m. oryzae the tps gene, a glucose-6-phosphate sensor, negatively regulates nmr inactivating nmr1- also the multidrug and toxin extrusion -family pump mdt a downstream target of tps is involved in glucose assimilation, conidiation and virulence. similarly, we observed several monosaccharide and general sugar transporters induced in ∆blu and repressed in the wt, as well as multidrug transporters induced in both strains. these observations point to the light regulation of carbon and nitrogen metabolism to stimulate conidiation and growth in t. atroviride, respectively. carbon or nitrogen starvation results in the formation of less branched compact hyphae than those observed in rich media, in addition the sudden lack of nitrogen or carbon source in t. atroviride resembles the ring of conidiation triggered by a pulse of light. interestingly, in the absence of the blr or blr genes the induction of conidiation by carbon starvation is lost, in contrast nitrogen starvation can still stimulate conidiation, suggesting an independent regulation of asexual reproduction by the blr proteins. thus we hypothesized that nitrogen metabolism should be involved in hyphal growth under constant light. several genes encoding enzymes with peptidase activity, including an intracellular serine protease, were induced in the wt strain, suggesting that protein degradation or recycling of nitrogen resources is activated in response to light. nitrogen metabolism related genes have also been found among the light regulated genes of n. crassa. in accordance with these observations, we found an allantoinase-encoding gene, involved in purine metabolism, induced only in the wt. besides two allantoate permeases were repressed in the wt, but not in the ∆blu strain, and two allantoate transporters were induced only in the mutant - allantoate is used as nitrogen storage in plants. transport of amino acids was repressed in the wt, mainly methionine transport. in contrast, in ∆blu higher induction levels of genes encoding amino acid transporters and allantoate transporters were observed. these data are highly coincident with the transcriptional response to nitrogen starvation reported in the phytopathogenic fungus magnaporthe grisea. taken together, our data appear to indicate that the absence of blu mimics a nitrogen starvation condition when trichoderma is grown under constant exposure to light, which results in clearly reduced growth. restoration of growth rate and radial colony size of the wt and ∆blu mutants by the addition of peptone to the media suggests that light growth inhibition is mainly due to nitrogen intracellular metabolism, and that blu indirectly participates in the regulation of nitrogen metabolism in the presence of light under limited nitrogen supply. as a consequence of the lack of blu the mutants have lower capacity to recycle nitrogen sources, requiring the induction of general amino acid transporters, which in the presence of peptides from peptone are taken up and metabolized, resulting in much better growth. we also observed misregulation of the ras gtpase, which was induced to higher levels in the ∆blu mutant than in the wt. activation of this pathway in yeast involves a g-protein coupled receptor and ras signaling to regulate glucose availability. from the gpcrs regulated in both strains only the predicted gpcr gene id. is more strongly induced in the ∆blu mutant, which might act as an upstream regulator. gpcrs are involved in amino acid and carbon source sensing as well as in camp perception. the gpr gene, encoding a homolog of the n. crassa camp receptor gpr, is induced only in the wt strain. silencing of gpr in t. atroviride p reduces growth, conidiation and secondary metabolism. in yeast the gpr1-gpa gpcr system is involved in the regulation of camp signaling. the absence of gpr in the ∆blu mutants in conjunction with the increase in camp phosphodiesterase might be reflected in the lack of protein kinase a activity resulting in the loss of this signaling pathway to regulate the light response. pka signaling after a pulse of light is required to regulate up or down responsive genes, also regulated by the blr proteins. we further observed serine-threonine protein kinase encoding genes induced only in the wt, which might exert posttranslational modifications in transcription factors, consequently affecting regulation of gene expression in response to light, or might directly affect enzyme activity, resulting in altered metabolism. an overview of the data presented here is depicted in fig.  the blrc perceives light and induces blu expression. transient increase of camp by inhibition of the 3’–5’ camp phosphodiesterase leads to activation of photoconidiation genes by pka. growth rate under constant light depends on nitrogen availability, controlling the uptake from the media or mobilization of stored nitrogen.fig. model of the role of the transcriptional regulator blu in the molecular response of t. atroviride to light. a molecular responses to a pulse of blue light. exposure of t. atroviride to low fluence blue light perceived by the blrc results in the transcriptional activation of blu sub azf xlnr and other tf encoding genes, as well as the induction of envoy. blu tunes photoconidiation by repressing the expression of the 5’-3’ camp phosphodiesterase encoding gene. likewise, the induction of env enhances pd repression, while transitory accumulation of camp activates pka that in turn regulates photoconidiation genes. nitrogen mobilization is indirectly regulated by blu and modulates the balance between conidiation and mycelial growth. b molecular responses to continuous light exposure. under constant white light, blu induction by the blrc is regulated by the carbon source available in the medium. env negatively regulates blrc target genes for adaptation to constant light. when t. atroviride is grown on glucose containing medium, blu limits the induction of env blu is required for nitrogen mobilization through the inhibition of the orthologue of area by the negative regulator nmra up-regulation. in addition, hyphal growth and conidiation are tightly regulated by the amount of flbc to induce the sequential expression of the transcription factors c2h ➜abaa ➜ weta. black lines indicate control points supported by our rna-seq data and gray lines are based on previously reported experimental evidence  the present work describes the transcriptional response to light of the mutant in the putative transcription factor encoding gene blu a blrc dependent light regulated gene in t. atroviride. the blu gene is required for photoconidiation at low but not at high blue light fluence. the absence of blu resulted in increased levels of expression of the 3’-5’ camp phosphodiesterase encoding gene, which could explain the reduced conidiation observed in the mutant. furthermore, the increased expression of env observed in the ∆blu strain under constant illumination suggests linked roles in photoadaptation between these two regulators. interestingly, the growth inhibition response of the blu replacement mutant to light was reduced in rich media. the decreased expression of energy and metabolism related genes in the ∆blu mutant under constant light may explain the slower growth, rendered by defective nitrogen metabolism. ethics not applicable. consent to publish not applicable. 
genes related to amino acid, sugar and general transporters were enriched in the induced genes in the mutant and the repressed genes of the wild type. blu appears to regulate light sensitivity in terms of induction of conidiation, and to play a major role in supporting growth under continuous exposure to light. rna-seq analyses under constant white light using glucose as sole carbon source revealed that localization and transport process present the opposite regulation pattern in the ∆blu and wild type strains. for the first time we show a genome wide survey of light regulated gene expression in t. atroviride, including rna-seq analyses of the wild type and the Δblu strains after brief exposure to blue-light. light activates stress responses and several metabolic processes in the wild type strain that are no longer activated in the mutant. consequently, they have developed mechanisms to sense light quality and quantity. peptone supplemented in the media restored growth of the ∆blu mutant in constant light, suggesting a role of blu in the regulation of nitrogen metabolism in the presence of light. blrrna-seqphotoconidiationnitrogen metabolismglucosegrowthlightconacytfordecyt-2012-02-193512herrera-estrella alfredo issue-copyright-statement© the author 2016 in agreement with the misregulation of metabolic processes, continuous exposure to white light strongly inhibited growth of the ∆blu mutant, in a carbon source dependent fashion. the diminished conidiation observed in ∆blu mutants is likely due to misregulation of the camp signaling pathway and ros production, whereas their low tolerance to continuous exposure to light indicates that blu is required for adaptation. in the fungus trichoderma atroviride blue-light is perceived through the blue light regulator complex, which in turn up-regulates a set of genes and down-regulates another set, triggering asexual reproduction. Δblu mutants show reduced conidiation at low light fluences, which is still clear even when exposed to saturating light.
 most living organisms use sunlight as a source of energy and/or information about their environment. consequently, they have developed mechanisms to sense light quality and quantity. in the fungus trichoderma atroviride blue-light is perceived through the blue light regulator complex, which in turn up-regulates a set of genes and down-regulates another set, triggering asexual reproduction. to gain insight into this process, we characterized the blu gene, which encodes a protein containing a c2h zinc finger domain.  Δblu mutants show reduced conidiation at low light fluences, which is still clear even when exposed to saturating light. for the first time we show a genome wide survey of light regulated gene expression in t. atroviride, including rna-seq analyses of the wild type and the Δblu strains after brief exposure to blue-light. our data show a reduction in the number of induced genes and an increase in down-regulated genes in the mutant. light activates stress responses and several metabolic processes in the wild type strain that are no longer activated in the mutant. in agreement with the misregulation of metabolic processes, continuous exposure to white light strongly inhibited growth of the ∆blu mutant, in a carbon source dependent fashion. rna-seq analyses under constant white light using glucose as sole carbon source revealed that localization and transport process present the opposite regulation pattern in the ∆blu and wild type strains. genes related to amino acid, sugar and general transporters were enriched in the induced genes in the mutant and the repressed genes of the wild type. peptone supplemented in the media restored growth of the ∆blu mutant in constant light, suggesting a role of blu in the regulation of nitrogen metabolism in the presence of light.  blu appears to regulate light sensitivity in terms of induction of conidiation, and to play a major role in supporting growth under continuous exposure to light. the diminished conidiation observed in ∆blu mutants is likely due to misregulation of the camp signaling pathway and ros production, whereas their low tolerance to continuous exposure to light indicates that blu is required for adaptation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords blrrna-seqphotoconidiationnitrogen metabolismglucosegrowthlightconacytfordecyt-2012-02-193512herrera-estrella alfredo issue-copyright-statement© the author 2016 