Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process

Light is a fundamental abiotic factor and therefore represents a central environmental signal which influences not only phototrophic but in fact rather the majority of living organisms. Light is thereby sensed by chromophore-binding proteins that act as photoreceptors, which transduce the signal to the expression of the genes involved in the respective response [1, 2]. In mitosporic fungi, light is primarily known to stimulate morphogenetic functions and processes of reproduction such as phototropism, spore discharge, the development of sexual and asexual structures [3, 4], as well as pigmentation which protects against the deleterious effects of UV-light [5, 6]. The molecular responses and mechanisms of adaptation to light, especially with respect to circadian rhythmicity are best documented in Neurospora crassa [7–9]. In this fungus, all light-induced phenotypes are dependent on at least one of the two regulators white-collar-1 (WC-1; [10]) or white-collar-2 (WC-2; [11]). These two genes encode proteins, which contain a zinc finger domain and a PAS-domain through which they interact physically to form the "white collar complex [12]." The WC-1 protein also functions as a blue light receptor via its LOV domain and by its binding of an FAD flavin chromophore [13]. Idnurm and Heitman [14] have recently demonstrated that orthologues of the WC-1/WC-2 proteins of N. crassa are present in ascomycetes and basidiomycetes, and thus represent an evolutionary ancient conserved system for the control of light-dependent processes.

The light perception system of N. crassa also comprises the small PAS/LOV domain protein VIVID which is believed to act as a modulator of the light response in N. crassa. [15–17]. It is a member of the LOV-domain subfamily of PER, ARNT and SIM (PAS)-domain proteins which mediate both ligand binding and protein-protein interactions [18]. VIVID is capable of binding a flavin chromophore [17, 19, 20]. It has been shown to be localized in the cytoplasm and influences the transient phosphorylation of WC-1 [15, 16, 21]. The predominant influence of VVD is on the speed with which a transcriptional response to light decays. A loss of VVD causes the clock to be more responsive to light and consequently, circadian gating – the action of the clock to reduce the responses at certain times of day – is muted without VVD [15].

While orthologues of WC-1 and WC-2 have been identified and characterized from various fungi [14], information about possible orthologues of VIVID in other organisms is scarce. Only its orthologue in the ascomycete Hypocrea jecorina, Envoy – which has a high similarity to VIVID but is unable to replace it – has recently been characterized [22]. Comparably to N. crassa vvd, env1 shows a fast and strong transcriptional response to illumination on several carbon sources.

H. jecorina is well known to science because of the use of its anamorph Trichoderma reesei as an industrial producer of cellulases and hemicellulases [23–25]. The expression of its cellulase genes depends on the presence of an inducer such as cellulose, lactose, sophorose or L-sorbose, but is otherwise independent of most other nutrients except for a susceptibility of some – but not all of its – genes to partial carbon catabolite repression [26]. Interestingly, however, light stimulates cellulase gene expression in H. jecorina, and this stimulation is regulated by ENVOY: a mutant lacking the PAS-domain of ENVOY (env1) exhibits an altered cellulase gene transcription pattern both in the presence and absence of light, thus showing that env1 is directly or indirectly impacting cbh1 gene expression in the darkness [22]. In addition, the loss of the PAS-domain of ENVOY led to an altered transcriptional response of the truncated transcript of env1, thus suggesting a regulatory feedback being operative.

Our detection of a light-dependence of cellulase gene transcription of the PAS/LOV domain protein ENVOY raised the question whether there would be more cellular functions in fungi which are controlled by either light and/or ENVOY. To address this question we have performed a genome wide screening for genes regulated by the presence of light, using Rapid Subtraction Hybridization (RaSH). Genes thereby identified were investigated for whether their response would be dependent on a functional env1 gene. We will show that light affects transcription of genes of H. jecorina both positively as well as negatively, and that for both effects env1-independent variants are found. In addition, we will show that ENVOY also acts as a light-independent repressor for several genes, is crucial for normal growth in light on several carbon sources, but is not able to fully execute its regulatory function when overexpressed in darkness. Our data suggest a function of ENVOY in coordination of the light signal with other environmental signals, which is comparable to the gating function shown for VVD of Neurospora crassa.

In this study, we used RaSH to isolate early light-responding genes from H. jecorina, which led to the identification of a total of 20 genes which were upregulated and 4 which were downregulated shortly after illumination. In addition, several of these genes were differentially effected by a mutation in the light regulatory protein ENVOY [22], whose closest neighbour N. crassa VIVID, is known to be a photoreceptor [17]. Besides ENVOY, VIVID is the only characterized PAS-domain protein of this type in fungi to date. To put the data obtained in this study in a genomic perspective: Rosales-Saveedra et al.[41] have recently used microarrays containing approximately one fifth of the genome of Hypocrea atroviridis (anamorph Trichoderma atroviride) for the screening of genes regulated by light, and identified 30 genes to be upregulated. This corresponds to 2.8% of the genes contained in the array used, and compares well to a value of 3% obtained for a similar study in N. crassa [8]. Only two of the genes which were found to be upregulated by blue light in H. atroviridis [41] were also upregulated in N. crassa [8], which may be explained by a significant difference in the physiology of these two fungi and the long phylogenetic distance between Neurospora and Hypocrea/Trichoderma spp. [42]. It is therefore interesting to note that only one of the genes (photolyase phr1) identified in this study was also found by Rosales-Saveedra et al. [41], although H. jecorina and H. atroviridis belong to the same fungal genus. The authors mentioned unpublished data that "several of the genes identified by them were also light upregulated in H. jecorina." This difference may be due to the missing of 80% of the genome in this study. On the contrary, the method of subtraction hybridization used in our study includes the whole transcriptome of the respective conditions to be compared, but from our experience also yields only a subset of all genes regulated under the conditions of interest. Hence we consider the present study complementary to that of Rosales Saveedra et al.[41]. Yet another explanation for the difference in the set of genes which were found to respond to light in these two fungi could be the fact that the ENVOY-homologue of H. atroviridis may not be functional: this assumption is supported by the following findings: first, its N-terminus is truncated at amino acids 1–6; second, it contains two upstream open reading frames close to the ATG (M. Schmoll, unpublished), which can profoundly influence the translation of the main ORF [43]. Finally, we could not detect the transcript of H. atroviridis env1 under several conditions where env1 is strongly transcribed in H. jecorina (data not shown). Taken together, this could reflect a different light regulatory machinery in these two closely related fungi.

Genes, which were actually upregulated by light and which required ENVOY for this process to function properly were the largest sample detected in this study (11 genes). One of them was the photolyase gene phr1, which has also been isolated from H. atroviridis, and which plays a role in the protection of genes against UV-light by photoreactivation of cyclobutan dimers of the pyrimidine nucleotides [44]. Rosales-Saveedra et al. [41] also reported the identification of a gene (blu3), which encodes a protein with an endonuclease III-type domain and which could function in excision repair. This gene was not identified in this study. However, another gene identified in this study (tre22454) encodes an ITP triphosphatepyrophosphatase, an enzyme responsible for the degradation of IMP and XMP, which accumulate as a result of cellular degradation of nucleotides which were modified by oxidative stress [46]. These data indicate that the early light response of H. jecorina involves reactions both against UV-light as well as oxidative stress. This is supported by the upregulation of tre42719 (encoding IMP dehydrogenase, an enzyme involved in the biosynthesis of nucleotide phosphates). Yoshida et al. [47] have shown that exposure of N. crassa to light evokes an oxidative stress response, in which nucleoside diphosphate kinase 1 plays a essential role e.g. by associating with a G-protein &-subunit for transmission of the light signal [48]. Finally, an increased demand for protection of the cell against a major threat is also evident from the upregulation of genes encoding components of cellular protein turnover such as tre16112 (a prenyltransferase required for ubiquitin biosynthesis), tre39031 (encoding a dipeptidyl peptidase III), and ubi4 (encoding polyubiquitin). The upregulation of an MSF toxin efflux pump (tre10571) with homology to proteins providing tolerance against fungicides in Botrytinia fuckeliana (DHA14 like major facilitator protein, AAF64435, E-value 3E-130; [49, 50]) and Mycosphaerella graminicola (Mfs1, ABG57045, E-value 5E-127; [51]) upon illumination raises an intriguing question: is this defense-mechanism predominantly active in the presence of light i. e. during the day? If so, the efficiency of fungicides could be increased by carefully timing their application. However, we do not yet know whether the light regulation of this efflux pump also occurs in plant pathogenic fungi. In agreement with H. atroviridis [41], several of the genes which are upregulated by light encode genes involved in energy metabolism (tre9347, NAD synthase; tre20863, succinate dehydrogenase; tre39397, glucose transport), and regulation of all of them was influenced by env1. This is reminiscent of the findings by Kolarova et al.[52] that exposure of T. viride to light leads to increments in ATP levels and respiratory activity. Although this increased energy production could be required for the onset of photoconidiation upon exposition to light, this explanation is rather not applicable to H. jecorina, because this fungus does not need illumination for the induction of formation of conidia, and conidiates well in darkness. The detection of several genes involved in protein turnover to be responsive to light rather suggests that this enhanced energy demand reflects the physiological change in gene expression which is needed to adapt to light.

One light-responsive but not env1-dependent gene shows intriguing characteristics – tre45629: although the primary structure of the encoded protein is only poorly conserved, it contains all the signature sequences of a peptidyl arginine deiminase, an enzyme which converts arginine residues in proteins to citrulline, thereby altering the positive charge and hence the proteins ability to interact with other proteins and membranes. To the best of our knowledge, the role of this deimination has not yet been investigated in fungi. Moscarello et al.[53] have recently proposed that citrullinylation of myeline basic protein from brain is an important event in the pathogenesis of multiple sclerosis.

Another interesting finding was the detection that the cross pathway control protein CPC2 is regulated by light. Cross pathway control (CPC) of amino acid biosynthetic pathways is activated during amino acid starvation and also controls sexual development in A. nidulans. This activation is executed by the transcription factor CPC1/CPCA. In the presence of amino acids the pathway is repressed by the transcription factor CPC2/CPCB [54]. We therefore tested whether H. jecorina cpc1 would also respond to light. Interestingly, cpc1 did not respond to light, but is influenced by the presence of ENVOY. Thus, in Hypocrea jecorina the repressing factor is regulated by light but the activating factor is modulated by ENVOY. The coregulation of cpc1 and hac1 by ENVOY is also interesting in the context that Gcn4p (the S. cerevisiae orthologue of CPC1) is involved in the unfolded protein response [55], and that CPC1 was found to be upregulated during UPR in H. jecorina [56]. This suggests that ENVOY may be involved in the control of UPR.

Envoy – and particularly its putative counterpart in N. crassa, VIVID – have been described as proteins modulating the cellular response to light. However, we have shown here that this is only one of several roles which ENVOY apparently plays. Only eleven of the nineteen genes upregulated by light needed the function of env1 for this process. Five other genes showed an upregulation by light independently of env1, and in three genes the light-dependent upregulation was even stimulated in an env1-negative background. The nature of the proteins encoded by these genes did not yet provide us with an explanation for the specific role of these env1-independent and env1-repressed upregulations.

The results of this study point at an involvement of Envoy in the regulation of various cellular processes. Although based on this study we cannot differentiate between direct and indirect influences of ENVOY, it is obvious that this protein plays an important regulatory role at a central junction of signaling pathways. The finding that ENVOY is at least in some cases – as exemplified by the influence on phr1 and tre39031 – not able to execute its light-dependent function in darkness suggests that the presence of its putative interaction partners is required for a proper function of this regulatory mechanism. Similarly, also for N. crassa White collar-1 (WC-1) Lewis et al.[8] showed, that increased levels of WC-1 in darkness are not sufficient to activate all aspects of the phototransduction pathway. Since ENVOY comprises no known DNA-binding domain, it likely does not directly bind to DNA, and therefore executes its function via interaction with downstream regulatory proteins targeting the respective pathways. Thereby it could interact with either positive as well as negative regulatory factors, which would explain its positive and negative influences as shown in this paper. This interaction could also be influenced by the phosphorylation state of the casein kinase II phosphorylation sites in ENVOY (M. Schmoll, unpublished), and/or binding of the ligands to the PAS-domain of ENVOY. Since PAS domains are well known to be able to bind different ligands [57], ENVOY could thus execute its regulatory function both at a qualitative (conformational change due to bound ligand) and quantitative (expression efficiency) level. While the well characterized photoreceptors BLR1 and BLR2 (putatively as BLR1–BLR2 photoreceptor-complex) are predicted to mediate the reception of the light signal, this study reveals that ENVOY is involved in the conditional adaptation to light, because lack of functional ENVOY does not result in blindness but leads to an altered gene expression pattern of light-regulated genes. Such a function would well correspond with the finding of a gating function for the N. crassa orthologue VIVID [15]. In other words, Envoy most likely determines the significance of the light signal for a given cellular process under the current environmental conditions.

The different responses to light, as demonstrated in this study, stress that light plays a role in several cellular processes of fungi, thereby displaying both positive and negative effects. Our data also emphasize that ENVOY has an apparently more widespread cellular role in this process than only in modulating the response to light. The importance of such a coordinator becomes apparent when it is considered that sunlight causes subsequent changes such as a rise in temperatures, decrease in humidity, and increase in UV light intensity. The adaptation to these environmental cues is of crucial importance in the evolution of every organism.