- Research article
- Open Access
Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process
https://doi.org/10.1186/1471-2164-8-449
© Schuster et al; licensee BioMed Central Ltd. 2007
- Received: 21 August 2007
- Accepted: 04 December 2007
- Published: 04 December 2007
Abstract
Background
In fungi, light is primarily known to influence general morphogenesis and both sexual and asexual sporulation. In order to expand the knowledge on the effect of light in fungi and to determine the role of the light regulatory protein ENVOY in the implementation of this effect, we performed a global screen for genes, which are specifically effected by light in the fungus Hypocrea jecorina (anamorph Trichoderma reesei) using Rapid Subtraction Hybridization (RaSH). Based on these data, we analyzed whether these genes are influenced by ENVOY and if overexpression of ENVOY in darkness would be sufficient to execute its function.
Results
The cellular functions of the detected light responsive genes comprised a variety of roles in transcription, translation, signal transduction, metabolism, and transport. Their response to light with respect to the involvement of ENVOY could be classified as follows: (i) ENVOY-mediated upregulation by light; (ii) ENVOY-independent upregulation by light; (iii) ENVOY-antagonized upregulation by light; ENVOY-dependent repression by light; (iv) ENVOY-independent repression by light; and (v) both positive and negative regulation by ENVOY of genes not responsive to light in the wild-type. ENVOY was found to be crucial for normal growth in light on various carbon sources and is not able to execute its regulatory function if overexpressed in the darkness.
Conclusion
The different responses indicate that light impacts fungi like H. jecorina at several cellular processes, and that it has both positive and negative effects. The data also emphasize that ENVOY has an apparently more widespread cellular role in this process than only in modulating the response to light.
Keywords
- Light Response
- Quinic Acid
- Constant Darkness
- Peptidyl Arginine Deiminase
- Reverse Northern Blot
Background
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.
Results
Isolation of expressed sequence tags which are differentially expressed in H. jecorina after transfer from dark to illumination by light
We used mRNAs from H. jecorina QM 9414 pregrown in the dark, and mRNAs from the same strain after subjection to illumination to screen for mRNAs which are more abundant under the latter conditions and thus upregulated by light applying Rapid Subtraction Hybridization [27]. To prevent missing transcripts with only transient accumulation or slower response upon receipt of the light pulse, mRNAs were isolated from mycelia after 15 and 30 min of incubation in light, and combined. As we applied a relatively low stringency, we expected to be able to detect not only genes which are absent during darkness but generally such genes which exhibit a different abundance under these both conditions.
Additional genes added to the analysis
Gene(s) | Encoded protein | Function |
---|---|---|
tre34179 and tre37417 | S-adenosyl methionine dependent methyl transferase | Increased methylation of DNA in response to stress leads to decreased transcription [69]; |
tmk3 | MAPkinase | Involved in signal transduction; yeast homologue HOG1 regulates glycogen phosphorylase [70]. Glycogen content of H. jecorina decreases upon illumination [71] |
hac1 | Transcription factor | Transcription factor involved in regulation of unfolded protein response [34] |
thi4 | Thiazole biosynthetic enzyme | Involved in the biosynthesis of thiazols and in DNA damage response, Fusarium homologue is induced under stress conditions [32] |
gph1 | Glycogen phosphorylase | Involved in degradation of glycogen; glycogen content is decreased upon illumination in H. jecorina [71] |
cpc1 | Transcription factor | Cross pathway control protein 1; component of the cross pathway control machinery, involved in activation of amino acid biosynthesis, induced under secretion stress [55] |
Since the Reverse Northern blot provides only preliminary information (e.g. some plasmids could contain more than one insert etc.), we assessed the response of expression of all genes to light by Northern blotting. This investigation proved that among the genes analyzed, 20 were indeed significantly (> 40% change in signal intensity) upregulated upon illumination. Interestingly, four genes were shown to be actually repressed by light, one of them representing a false positive result of RaSH regarding the aim of the assay, while the others were three of the genes which – because of their roles in signaling – were intentionally included in the analysis. Since we will present these data in a broader context below, they are not given at this place. For three genes neither significant light- nor ENVOY dependent regulation was detected and therefore they are not discussed further. We also noted that the fluctuations in transcript abundance, which were also seen earlier with the light regulatory gene env1 upon cultivation after onset of illumination in minimal medium with 1% glycerol as carbon source [22] or N. crassa ccg-2 and NC2B7 (see Figure 4A in [8]) also occur for many of the genes investigated here.
Gene identification
Protein domains of genes identified by RaSH
Gene | Amino acids | Protein domain | E-value | % aligned | Position |
---|---|---|---|---|---|
cpc2 | 292 | WD40 | 2.0E-60 | 93.1% | 3–286 |
phr1 | 591 | Deoxyribodipyrimidine photolyase | 7.0E-124 | 99.3% | 96–584 |
FAD_binding_7 | 2.0E-97 | 100% | 316–587 | ||
rpl7 | 248 | Ribosomal_L7 | 5.0E-66 | 100% | 88–247 |
Ribosomal_L30 | 2.0E-14 | 100% | 87–139 | ||
ubi4 | 305 | Ubiquitin | 7.0E-33 | 100% | 1–76 |
Ubiquitin | 7.0E-33 | 100% | 77–152 | ||
Ubiquitin | 7.0E-33 | 100% | 153–228 | ||
Ubiquitin | 7.0E-33 | 100% | 229–304 | ||
tre9347 | 700 | NAD_synthase | 3.0E-59 | 99.2% | 328–653 |
Carbon-nitrogen hydrolase | 1.0E-18 | 100% | 6–201 | ||
Predicted amidohydrolase | 4.0E-27 | 91.6% | 5–282 | ||
tre10571 | 534 | Major facilitator superfamily MFS_1 | 4.0E-12 | 100% | 31–443 |
Arabinose efflux permease | 7.0E-13 | 46.7% | 26–208 | ||
Fungal trichothecene efflux pump (TRI12) | 2.0E-06 | 34.3% | 102–303 | ||
tre16112 | 304 | Hydroxybenzoate polyprenyltransferase | 2.0E-32 | 99.7% | 2–289 |
UbiA prenyltransferase family | 5.0E-23 | 100% | 24–301 | ||
tre20863 | 648 | Succinate dehydrogenase/fumarate reductase | 2.0E-168 | 100% | 56–631 |
FAD binding domain | 2.0E-140 | 99.4% | 162–492 | ||
Aspartate oxidase | 1.0E-92 | 93.8% | 85–618 | ||
Fumarate reductase/succinate dehydrogenase flavoprotein C-terminal domain | 4.0E-44 | 100% | 513–648 | ||
tre22454 | 180 | NTPase/HAM1 | 1.0E-54 | 100% | 5–178 |
Xanthosine triphosphate pyrophosphatase | 2.0E-45 | 96.9% | 5–180 | ||
tre22667 | 193 | Ribosomal protein L6P/L9E | 4.0E-30 | 97.2% | 1–187 |
Ribosomal protein L6 | 7.0E-07 | 100% | 97–180 | ||
tre31929 | 270 | Adenylate kinase | 3.0E-75 | 100% | 44–231 |
Adenylate kinase, active site lid | 2.0E-12 | 100% | 167–202 | ||
tre35050 | 104 | - | - | - | - |
tre39031 | 711 | Peptidase_M49 | 6.0E-170 | 98.8% | 143–709 |
tre39397 | 465 | Sugar (and other) transporter | 7.0E-13 | 87.1% | 44–462 |
tre40105 | 331 | - | - | - | - |
tre41865 | 183 | Perilipin | 2.0E-04 | 27.4% | 15–110 |
tre42719 | 537 | IMP dehydrogenase/GMP reductase domain | 1.0E-159 | 99.4% | 42–526 |
CBS domain | 3.0E-13 | 93.2% | 130–236 | ||
Predicted transcriptional regulator | 7.0E-09 | 36.7% | 130–238 | ||
NAD(P)H-dependent flavin oxidoreductase (oxidored) FMN-binding superfamily domain | 1.0E-04 | 99.1% | 195–397 | ||
tre45629 | 356 | Porphyromonas-type peptidyl-arginine deiminase | 1.0E-67 | 100% | 6–353 |
tre72859 | 114 | - | - | - | - |
Blast analysis of genes identified by RaSH
Gene | Best Hit | E-Value | Fusarium spp. | E-Value | Neurospora crassa | E-Value | Saccharomyces cerevisiae | E-Value |
---|---|---|---|---|---|---|---|---|
cpc2 | XP_390046.1 Guanine nucleotide-binding protein beta subunit [G. zeae] | 2.00E-169 | XP_390046.1 Guanine nucleotide-binding protein beta subunit | 2.00E-169 | Q01369| GBLP_NEUCR WD-repeat protein cpc-2 | 1.00E-166 | NP_013834.1 Asc1p | 2.00E-94 |
phr1 | CAA08916.1 DNA photolyase [Hypocrea lixii] | 0.0 | XP_380973.1 hypothetical protein FG00797.1 | 0.0 | P27526| PHR_NEUCR Deoxyribodipyrimidine photolyase | 0.0 | P05066| PHR_YEAST Deoxyribodipyrimidine photo-lyase | 2.00E-89 |
rpl7 | XP_382718.1 conserved hypothetical protein [G. zeae] | 1.00E-112 | XP_382718.1 conserved hypothetical protein | 1.00E-112 | XP_962950.1 hypothetical protein | 4.00E-108 | NP_011439.1 Rpl7ap | 2.00E-77 |
tre10571 | XP_388925.1 hypothetical protein FG08749.1 [G. zeae] | 0.0 | XP_388925.1 hypothetical protein FG08749.1 | 0.0 | XP_330290.1 hypothetical protein | 4.00E-162 | NP_011740.1 Azr1p | 2.00E-44 |
tre16112 | XP_327992.1 h. p. (AL451012) related to para-hypolyprenyltransferase precursor [N. crassa] | 1.00E-124 | XP_390908.1 hypothetical protein FG10732.1 | 2.00E-110 | - | - | NP_014439.1 Coq2p | 1.00E-56 |
tre20683 | EAQ93406.1 conserved hypothetical protein [Chaetomium globosum CBS ] | 0.0 | XP_387537.1 hypothetical protein FG07361.1 | 0.0 | XP_965239.1 hypothetical protein | 0.0 | Q00711| DHSA_YEAST Succinate dehydrogenase | 0.0 |
tre22454 | XP_955963.1 hypothetical protein [N. crassa N150] | 7.00E-76 | XP_387647.1 hypothetical protein FG07471.1 | 4.00E-73 | XP_955963.1 hypothetical protein [Neurospora crassa N150] | 7.00E-76 | NP_012603.1 Ham1p | 2.00E-33 |
tre22667 | XP_381330.1 hypothetical protein FG01154.1 [G. zeae] | 2.00E-88 | XP_381330.1 hypothetical protein FG01154.1 | 2.00E-88 | XP_965129.1 hypothetical protein | 1.00E-87 | NP_014332.1 Rpl9bp | 2.00E-63 |
tre31929 | XP_390913.1 Probable adenylate kinase (ATP-AMP transph [G. zeae] | 1.00E-121 | XP_390913.1 Probable adenylate kinase | 1.00E-121 | XP_956253.1 probable adenylate kinase [MIPS] | 2.00E-116 | NP_010512.1 Adk1p | 4.00E-89 |
tre35050 | XP_001230117.1 hypothetical protein CHGG_03601 [Chaetomium globosum] | 1.00E-23 | XP_386761.1 hypothetical protein FG06585.1 | 1.00E-15 | XP_956091.1 hypothetical protein | 7.00E-21 | AAB50692.1 Paf1p | 1.7 |
tre39031 | XP_381193.1 hypothetical protein FG01017.1 [G. zeae] | 0.0 | XP_381193.1 hypothetical protein FG01017.1 | 0.0 | CAE76510.1 probable dipeptidylpeptidase III | 0.0 | Q08225| DPP3_YEAST Dipeptidyl aminopeptidase III | 2.00E-163 |
tre39397 | XP_369043.1hypothetical protein MG00201.4 [Magnaporthe grisea] | 0.0 | XP_388057.1 hypothetical protein FG07881.1 | 0.0 | XP_326778.1 hypothetical protein | 1.00E-86 | - | - |
tre40105 | BAE58733.1 unnamed protein product [Aspergillus oryzae] | 3.00E-54 | XP_384339.1 hypothetical protein FG04163.1 | 8.00E-38 | XP_960170.1 hypothetical protein | 7.00E-26 | - | - |
tre41025 | EAS32414.1 predicted protein [Coccidioides immitis RS] | 2.00E-21 | XP_384237.1 hypothetical protein FG04061.1 | 5.00E-16 | XP_959109.1 hypothetical protein | 0.001 | NP_012284.1 Muc1p | 0.038 |
tre41865 | XP_385353.1hypothetical protein FG05177.1 [G. zeae] | 2.00E-62 | XP_385353.1hypothetical protein FG05177.1 | 2.00E-62 | CAD70317.1 probable CAP20-virulence factor | 4.00E-35 | - | - |
tre42719 | XP_964976.1 hypothetical protein [N. crassa] | 0.0 | XP_381037.1 conserved hypothetical protein | 0.0 | XP_964976.1 hypothetical protein | 0.0 | NP_013656.1 Imd4p | 0.0 |
tre45629 | XP_748505.1 Porphyromonas-type peptidyl-arginine deiminase superfamily [Aspergillus fumigatus Af293] | 2.00E-62 | - | - | - | - | - | - |
tre72859 | XP_381443.1 hypothetical protein FG01267.1 [G. zeae] | 7.00E-28 | XP_381443.1 hypothetical protein FG01267.1 | 7.00E-28 | XP_964260.1 hypothetical protein | 1.00E-23 | Q07953| YL022_YEAST UPF0023 protein YLR022C | 0.014 |
tre9347 | XP_387574.1 hypothetical protein FG07398.1 [G. zeae] | 0.0 | XP_387574.1 hypothetical protein FG07398.1 | 0.0 | XP_959191.1 hypothetical protein | 0.0 | NP_011941.1 Qns1p | 0.0 |
ubi4 | XP_460488.1 protein DEHA0F03157g [Debaryomyces hansenii CBS767] | 2.00E-168 | XP_388944.1 protein FG08768.1 | 2.00E-124 | XP_958803.1 polyubiquitin | 2.00E-166 | NP_013061.1 Ubi4p | 9.00E-168 |
Light-dependent upregulation of gene expression can occur in env1-dependent, env1-independent, and env1-antagonized ways
Northern analysis of light- and env1 -responsive genes. Strains were grown on Mandels Andreotti minimal medium with 1% (w/v) glycerol as carbon source for 24 h in darkness (DD) and harvested after the indicated time (DL) of illumination (1800 lux, 25 μmol photons m-2s-1). A representative hybridization with 18S rRNA for every set of Northerns is given below the respective series. Transcript abundance is given below the blots and was measured for wild-type QM9414 (Q) and env1PAS- by densitometry to verify up-regulation until 60 min of illumination, related to 18S rRNA and normalized to the dark control of the wild-type strain (QM9414, 24 h, DD). If no transcript was detected in QM9414 in darkness, the values represent signal strength above background. (A) Transcription of genes upregulated by light but not in the env1PAS- strain. (B) Transcription of genes upregulated both by light and in the env1PAS- strain. (C) Transcription of genes upregulated by light, which show increased upregulation in the env1PAS- strain.
In contrast, five genes (rpl7, tre22667, tre35050, tre45629 and tre72859), while also showing this upregulation by light, did so also in the env1PAS- mutant. Despite the fact that ENVOY seems to be involved in their regulation due to the altered transcription pattern in env1PAS-their response to light by increased transcription is not exclusively dependent on ENVOY (Fig. 1B).
In addition, three other genes (ubi4, tre10571, tre41025) also exhibited significant upregulation of gene expression upon exposure to light, but this upregulation was even stronger in the env1PAS- mutant, indicating that ENVOY antagonizes this activation in the wild-type strain (Fig. 1C). Since this enhanced transcription in the mutant strain also occurs in darkness with ubi4 and tre41025, these genes seem to be subject to a general repression by ENVOY.
Light repression of gene expression can occur in env1-dependent and env1-independent manners
Northern analysis of genes showing decreased transcription upon illumination. Strains were grown on Mandels Andreotti minimal medium with 1% (w/v) glycerol as carbon source for 24 h in darkness (DD) and harvested after the indicated time (DL) of illumination (1800 lux, 25 μmol photons m-2s-1). A representative hybridization with 18S rRNA for every set of Northerns is given below the respective series. Transcript abundance is given below the blots and was measured for wild-type QM9414 (Q) and env1PAS- by densitometry to verify up-regulation until 60 min of illumination, related to 18S rRNA and normalized to the dark control of the wild-type strain (QM9414, 24 h, DD). If no transcript was detected in QM9414 in darkness, the values represent signal strength above background.
env1 also regulates expression of genes which do not respond to light
Northern analysis of genes lacking response to light, but whose transcription is impacted by env1. Strains were grown on Mandels Andreotti minimal medium with 1% (w/v) glycerol as carbon source for 24 h in darkness (DD) and harvested after the indicated time (DL) of illumination (1800 lux, 25 μmol photons m-2s-1). A representative hybridization with 18S rRNA for every set of Northerns is given below the respective series. Transcript abundance is given below the blots and was measured for wild-type QM9414 (Q) and env1PAS- by densitometry to verify up-regulation until 60 min of illumination, related to 18S rRNA and normalized to the dark control of the wild-type strain (QM9414, 24 h, DD). If no transcript was detected in QM9414 in darkness, the values represent signal strength above background.
Regulatory elements putatively responsible for light response
Regulatory motifs within the promoters of the genes analyzed in this study
Gene | EUM1 | EUM2 | APE | GATA | AGGGG | LRE |
---|---|---|---|---|---|---|
cpc1 | 0 | 0 | 1 | 2 | 2 | 0 |
cpc2 | 0 | 0 | 1 | 4 | 0 | 0 |
gph1 | 0 | 0 | 0 | 3 | 6 | 2 |
hac1 | 1 | 0 | 0 | 1 | 7 | 0 |
phr1 | 1 | 0 | 3 | 2 | 1 | 0 |
rpl7 | 1 | 0 | 0 | 0 | 3 | 0 |
thi4 | 0 | 0 | 0 | 1 | 7 | 0 |
tmk3 | 2 | 0 | 0 | 2 | 2 | 1 |
ubi4 | 0 | 0 | 2 | 2 | 5 | 0 |
tre9347 | 1 | 0 | 0 | 2 | 1 | 0 |
tre10571 | 0 | 0 | 0 | 0 | 6 | 1 |
tre16112 | 0 | 0 | 0 | 2 | 0 | 1 |
tre20683 | 1 | 0 | 2 | 3 | 2 | 0 |
tre22454 | 0 | 0 | 0 | 1 | 1 | 2 |
tre22667 | 0 | 0 | 0 | 3 | 1 | 1 |
tre31929 | 0 | 0 | 0 | 2 | 1 | 0 |
tre34179 | 1 | 0 | 0 | 5 | 2 | 0 |
tre35050 | 0 | 0 | 0 | 3 | 0 | 0 |
tre37414 | 0 | 0 | 0 | 4 | 1 | 2 |
tre39031 | 1 | 0 | 0 | 1 | 2 | 0 |
tre39397 | 0 | 1 | 0 | 2 | 0 | 1 |
tre40105 | 1 | 1 | 0 | 2 | 0 | 0 |
tre41025 | 2 | 0 | 0 | 1 | 0 | 0 |
tre41865 | 1 | 0 | 1 | 3 | 3 | 1 |
tre42719 | 0 | 0 | 0 | 4 | 2 | 1 |
tre45629 | 1 | 0 | 1 | 5 | 0 | 0 |
tre72859 | 0 | 0 | 0 | 1 | 2 | 0 |
Analysis of promoter motifs present in light- and/or env1 -responsive genes. (A) Alignment of LRE-motifs; the extension of the motifs was limited to 50 bp, only true LRE motifs comprising the GATNC – CGATN consensus with N being the same nucleotide in both repeats were included. (B) Distribution of the respective promoter motifs among the regulatory characteristics of ENVOY as determined by Northern analysis (Figures 1 – 3). The total number of motifs present in one group was related to the number of genes of this group. A: genes upregulated by light but not in the env1PAS- strain; B: genes upregulated both by light and in the env1PAS- strain; C: genes upregulated by light, which show increased upregulation in the env1PAS- strain; D: genes showing decreased transcription upon illumination; E: genes lacking response to light, but whose transcription is impacted by env1.
Stimulation of growth on various carbon sources by light and ENVOY
Light stimulation of growth of H. jecorina on selected carbon sources, and the impact of ENVOY. Biomass formation of wild-type strain QM9414 (open circles) or env1PAS- (full diamonds) has been analyzed by the BIOLOG microplate assay. Biomass equivalents (OD750) after 72 hrs of growth are given. This time was chosen because then both strains were still in the phase of active growth on all carbon sources tested. The y-axes shows values obtained under constant light of 1800 lux, whereas the x-axes shows those obtained in constant darkness. Consequently, carbon sources on which no difference between growth in light or darkness occurs lie on the border between the shaded area (indicating light stimulation) and the open area (light inhibition). The two lines indicate the mean values for all carbon sources. All experiments were done in triplicates, standard deviation is indicated by bars.
Up-regulation of env1 is not sufficient for regulation of its target genes in darkness
Northern analysis of ENVOY overexpressing strains. Strains were grown on Mandels Andreotti minimal medium with 1% (w/v) glycerol as carbon source in constant darkness and transcription of env1 in 4env1qa+ was induced by adding quinic acid to a final concentration of 0.6%. The parent strain QM9414 was used as control and treated equally. Mycelia were harvested after 1, 2, 3, 5 and 7 hours in darkness.
As a second model case, we wanted to test whether expression of env1 would be sufficient for the up-regulation of tre39031 in darkness and if the effect of the mutation of env1 in env1PAS- (i.e. detectable transcript in darkness and decreased transcript levels in light; figure 1A) could be reversed by overexpression of env1 in constant darkness. Also in this case no transcript of tre39031 was detected in darkness and after induction of env1-transcription by quinic acid (data not shown). These results are in agreement with the assumption that ENV1 does not directly regulate the transcription of its target genes (at least not generally), but executes its function indirectly by interaction with transcriptional regulators, which are not available or inactive in darkness.
Discussion
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.
Conclusion
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.
Methods
Microbial strains and culture conditions
The H. jecorina (T. reesei) wild-type strain QM9414 (ATCC 26921) and the env1 recombinant mutant lacking the PAS-domain (env1PAS-[22]) were used throughout this study. H. jecorina was grown in liquid culture in 1-L Erlenmeyer flasks on a rotary shaker (200 rpm) at 28°C in 200 ml of medium as described by [58] with 1% (w/v) glycerol as sole carbon source using 108 conidia/L (final concentration) as inoculum in constant darkness and harvested with red safety light or after the time of illumination (1800 lux; 25 μmol photons m-2 s-1) as indicated with the figures. Cultivations of wild-type and mutant strain were done in parallel to ensure equal conditions.
E. coli JM109 [59] was used for the propagation of vector molecules and DNA manipulations.
Preparation of PCR-Based cDNA Libraries
The experiment was performed essentially as described by Schmoll et al.[60] according to the RaSH method as published by [27]. For the driver cDNA mycelia were grown in constant darkness on minimal medium as described above for 24 hours, tester cDNA was prepared from mycelia exposed to light for 15 and 30 minutes and pooled.
Reverse Northern Hybridization
For the Reverse Northern Hybridization, PCR products were loaded onto duplicate agarose gels and blotted with 0.4 N NaOH onto Hybond N membranes (Pall, New York, USA). Hybridization was performed using 2.5 μg of PCR amplified and subsequently radioactively labeled cDNA from tester or driver, respectively, as probes after Eco RII digestion and purification. The candidates for a more detailed analysis were chosen by visual inspection first, then this decision was cross-checked by quantitative measurements using the BIORAD Geldoc Imaging system (Bio-Rad, Hercules, California, US) and BIORAD Quantity One software, both for three different expositions of the blot.
Nucleic acid isolation and hybridization
Fungal mycelia were harvested by filtration, washed with tap water, frozen and ground in liquid nitrogen. For extraction of DNA, mycelial powder was suspended in buffer A (1.2 M NaCl, 5 mM EDTA, 0.1 M Tris-HCl, pH 8.0), incubated for 20 min at 65°C, cooled down on ice, mixed with 1 vol. phenol:chloroform:isoamylalcohol 49:49:2 (v/v/v) and centrifuged (21000 g, 15 min). Following an extraction with 1 vol. of chloroform:isoamylalcohol 24:1 (v/v), the DNA was precipitated with 1 vol. of isopropanol and washed with 70% (v/v) ethanol. Total RNA was isolated by the guanidinium thiocyanate method [60, 61]. Standard methods [62] were used for electrophoresis, blotting and hybridization of nucleic acids. The transcription pattern of env1 [22] under the respective conditions was used as a control hybridization for appropriate conditions with every cultivation. In case of unclear results or small signal differences, the hybridizations were repeated with samples from different, independent cultivations.
Normalization of gene expression was performed according to the following formula:
{(transcript abundance of gene x at time point)/(transcript abundance of gene x in wild-type in darkness)}/{(control 18SrRNA, time point)/(18S rRNA wild-type, darkness)}. The quantitative measurements were performed using the BIORAD Geldoc Imaging system and BIORAD Quantity One software from different expositions of the respective film. For every set of Northern blots one 18S rRNA hybridization was included as a loading and blotting control and used for quantification of the respective films.
Sequence analysis and identification
The most promising candidates for light responsive genes showing clear differential transcription in the Reverse Northern Blot were selected for sequencing. PCR products as used for reverse Northern blotting were sequenced using primer RaSH1R, which binds within pBluescript immediately upstream of the inserts to be analyzed [60]. The respective sequences were used for BLASTX searches of the T. reesei genome database v2.0 [63]. For the genes identified thereby, protein sequences as provided by this database were used for a search for conserved domains in CDD [64, 65] and for the nearest neighbour with NCBI Blastp [66, 66, 68]. If an E-value below 1E-30 for the Blastp result or 1E-10 for the result of the CDD search was obtained for a certain gene, the result was considered to assign a putative function. 1000 bp of the promoter sequence upstream of the first ATG of the respective open reading frame as predicted in the genome database were analyzed for known regulatory motifs.
Biolog Phenotype Array analysis
Growth rates on selected carbon sources were investigated by means of the Biolog FF MicroPlate™ assay (Biolog Inc., Hayward, CA) as described by Druzhinina et al.[40]. Inoculated microplates were incubated in constant light (1800 lux, 25 μmol photons m-2 s-1) or in the dark at 28°C, and percent absorbance at 750 nm determined in 12 h intervals between 36 and 72 h. Analyses were repeated at least three times for each strain.
Construction of strains overexpressing env1
For inducible expression of ENV1 we introduced the open reading frame of env1 into the Sma I-site of the vector pmyx2 [69], resulting in env1 being under the control of the N. crassa qa-2 promoter which can be induced by addition of quinic acid to the culture medium to a final concentration of 0.6%. The resulting construct was transformed into the wild-type strain QM9414. Two positive strains were selected by PCR screening and Southern blotting, pregrown for 24 h on 1% (w/v) glycerol in darkness before adding quinic acid and transcript abundance was analyzed at several time points after addition of quinic acid in constant darkness. The wild-type strain was used as a control in parallel to those strains and was treated equally.
Declarations
Acknowledgements
This work has been supported by grants from the Austrian Science Foundation (FWF-P17325) to CPK. The T. reesei, T. atroviride and T. virens genome sequencing project was funded by the Department of Energy. We gratefully acknowledge the permission to use sequence data of T. atroviride and T. virens prior to publication of the sequence. MS is a recipient of an APART fellowship of the Austrian Academy of Sciences (grant 11212).
Authors’ Affiliations
References
- Corrochano LM: Fungal photoreceptors: sensory molecules for fungal development and behaviour. Photochem Photobiol Sci. 2007, 6 (7): 725-736. 10.1039/b702155k.PubMedView ArticleGoogle Scholar
- Herrera-Estrella A, Horwitz BA: Looking through the eyes of fungi: molecular genetics of photoreception. Mol Microbiol. 2007, 64 (1): 5-15. 10.1111/j.1365-2958.2007.05632.x.PubMedView ArticleGoogle Scholar
- Betina V, Farkas V: Sporulation and light-induced development in Trichoderma. Trichoderma & Gliocladium. Edited by: Harman, G. E., P. KC. 1998, London , Taylor & Francis, 1: 75 -794.Google Scholar
- Gressel JARW: Photomorphogenesis. Encyclopedia of Plant Physiology. Edited by: Shropshire JAMH. 1983, Berlin , Springer, 16B: 603 -6639.Google Scholar
- Arrach N, Fernandez-Martin R, Cerda-Olmedo E, Avalos J: A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces. Proc Natl Acad Sci U S A. 2001, 98 (4): 1687-1692. 10.1073/pnas.021555298.PubMed CentralPubMedView ArticleGoogle Scholar
- Li C, Schmidhauser TJ: Developmental and photoregulation of al-1 and al-2, structural genes for two enzymes essential for carotenoid biosynthesis in Neurospora. Dev Biol. 1995, 169 (1): 90-95. 10.1006/dbio.1995.1129.PubMedView ArticleGoogle Scholar
- Dunlap JC, Loros JJ: The neurospora circadian system. J Biol Rhythms. 2004, 19 (5): 414-424. 10.1177/0748730404269116.PubMedView ArticleGoogle Scholar
- Lewis ZA, Correa A, Schwerdtfeger C, Link KL, Xie X, Gomer RH, Thomas T, Ebbole DJ, Bell-Pedersen D: Overexpression of White Collar-1 (WC-1) activates circadian clock- associated genes, but is not sufficient to induce most light-regulated gene expression in Neurospora crassa. Mol Microbiol. 2002, 45 (4): 917-931. 10.1046/j.1365-2958.2002.03074.x.PubMedView ArticleGoogle Scholar
- Vitalini MW, de Paula RM, Park WD, Bell-Pedersen D: The rhythms of life: circadian output pathways in Neurospora. J Biol Rhythms. 2006, 21 (6): 432-444. 10.1177/0748730406294396.PubMedView ArticleGoogle Scholar
- Ballario P, Vittorioso P, Magrelli A, Talora C, Cabibbo A, Macino G: White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein. Embo J. 1996, 15 (7): 1650-1657.PubMed CentralPubMedGoogle Scholar
- Linden H, Macino G: White collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa. Embo J. 1997, 16 (1): 98-109. 10.1093/emboj/16.1.98.PubMed CentralPubMedView ArticleGoogle Scholar
- He Q, Liu Y: Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation. Genes Dev. 2005, 19 (23): 2888-2899. 10.1101/gad.1369605.PubMed CentralPubMedView ArticleGoogle Scholar
- Liu Y, He Q, Cheng P: Photoreception in Neurospora: a tale of two White Collar proteins. Cell Mol Life Sci. 2003, 60 (10): 2131-2138. 10.1007/s00018-003-3109-5.PubMedView ArticleGoogle Scholar
- Idnurm A, Heitman J: Light controls growth and development via a conserved pathway in the fungal kingdom. PLoS Biol. 2005, 3 (4): e95-10.1371/journal.pbio.0030095.PubMed CentralPubMedView ArticleGoogle Scholar
- Heintzen C, Loros JJ, Dunlap JC: The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting. Cell. 2001, 104 (3): 453-464. 10.1016/S0092-8674(01)00232-X.PubMedView ArticleGoogle Scholar
- Schwerdtfeger C, Linden H: Blue light adaptation and desensitization of light signal transduction in Neurospora crassa. Mol Microbiol. 2001, 39 (4): 1080-1087. 10.1046/j.1365-2958.2001.02306.x.PubMedView ArticleGoogle Scholar
- Schwerdtfeger C, Linden H: VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. Embo J. 2003, 22 (18): 4846-4855. 10.1093/emboj/cdg451.PubMed CentralPubMedView ArticleGoogle Scholar
- Taylor BL, Zhulin IB: PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol Mol Biol Rev. 1999, 63 (2): 479-506.PubMed CentralPubMedGoogle Scholar
- Crosson S, Rajagopal S, Moffat K: The LOV Domain Family: Photoresponsive Signaling Modules Coupled to Diverse Output Domains. Biochemistry. 2003, 42 (1): 2-10. 10.1021/bi026978l.PubMedView ArticleGoogle Scholar
- Zoltowski BD, Schwerdtfeger C, Widom J, Loros JJ, Bilwes AM, Dunlap JC, Crane BR: Conformational switching in the fungal light sensor Vivid. Science. 2007, 316 (5827): 1054-1057. 10.1126/science.1137128.PubMed CentralPubMedView ArticleGoogle Scholar
- Schwerdtfeger C, Linden H: Localization and light-dependent phosphorylation of white collar 1 and 2, the two central components of blue light signaling in Neurospora crassa. Eur J Biochem. 2000, 267 (2): 414-422. 10.1046/j.1432-1327.2000.01016.x.PubMedView ArticleGoogle Scholar
- Schmoll M, Franchi L, Kubicek CP: Envoy, a PAS/LOV domain protein of Hypocrea jecorina (Anamorph Trichoderma reesei), modulates cellulase gene transcription in response to light. Eukaryot Cell. 2005, 4 (12): 1998-2007. 10.1128/EC.4.12.1998-2007.2005.PubMed CentralPubMedView ArticleGoogle Scholar
- Buchert J, Oksanen T, Pere J, Siika-Aho M, Suurnäkki A, Viikari L: Applications of Trichoderma reesei enzymes in the pulp and paper industry. Trichoderma & Gliocladium. Edited by: Harman GE, CP K. 1998, London , Taylor & Francis, 2: 343 -3363.Google Scholar
- Galante YM, De Conti A, Monteverdi R: Application of Trichoderma enzymes in the textile industry. Trichoderma & Gliocladium. Edited by: Harman GE KCP. 1998, London , Taylor & Francis, 311 -3325.Google Scholar
- Galante YM, De Conti A, Monteverdi R: Application of Trichoderma enzymes in the food and feed industries. Trichoderma and Gliocladium. Edited by: Harman, G. E., Kubicek, C.P. 1998, London , Taylor & Francis, 327 -3342.Google Scholar
- Schmoll M, Kubicek CP: Regulation of Trichoderma cellulase formation: lessons in molecular biology from an industrial fungus. A review. Acta Microbiol Immunol Hung. 2003, 50 (2-3): 125-145. 10.1556/AMicr.50.2003.2-3.3.PubMedView ArticleGoogle Scholar
- Jiang H, Kang DC, Alexandre D, Fisher PB: RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes. Proc Natl Acad Sci U S A. 2000, 97 (23): 12684-12689. 10.1073/pnas.220431297.PubMed CentralPubMedView ArticleGoogle Scholar
- Huang F, Adelman J, Jiang H, Goldstein NI, Fisher PB: Differentiation induction subtraction hybridization (DISH): a strategy for cloning genes displaying differential expression during growth arrest and terminal differentiation. Gene. 1999, 236 (1): 125-131. 10.1016/S0378-1119(99)00244-9.PubMedView ArticleGoogle Scholar
- Kang DC, LaFrance R, Su ZZ, Fisher PB: Reciprocal subtraction differential RNA display: an efficient and rapid procedure for isolating differentially expressed gene sequences. Proc Natl Acad Sci U S A. 1998, 95 (23): 13788-13793. 10.1073/pnas.95.23.13788.PubMed CentralPubMedView ArticleGoogle Scholar
- Berrocal-Tito GM, Rosales-Saavedra T, Herrera-Estrella A, Horwitz BA: Characterization of blue-light and developmental regulation of the photolyase gene phr1 in Trichoderma harzianum. Photochem Photobiol. 2000, 71 (5): 662-668. 10.1562/0031-8655(2000)071<0662:COBLAD>2.0.CO;2.PubMedView ArticleGoogle Scholar
- Akiyama K, Thanonkeo P, Ogawa H, Ohguchi T, Takata R: Detection and cloning of the gene encoding a protein produced by nonpathogenic mutants of Fusarium oxysporum. J Biosci Bioeng. 2000, 90 (3): 302-307.PubMedView ArticleGoogle Scholar
- Choi GH, Marek ET, Schardl CL, Richey MG, Chang SY, Smith DA: sti35, a stress-responsive gene in Fusarium spp. J Bacteriol. 1990, 172 (8): 4522-4528.PubMed CentralPubMedGoogle Scholar
- Hwang CS, Flaishman MA, Kolattukudy PE: Cloning of a gene expressed during appressorium formation by Colletotrichum gloeosporioides and a marked decrease in virulence by disruption of this gene. Plant Cell. 1995, 7 (2): 183-193. 10.1105/tpc.7.2.183.PubMed CentralPubMedView ArticleGoogle Scholar
- Saloheimo M, Valkonen M, Penttila M: Activation mechanisms of the HAC1-mediated unfolded protein response in filamentous fungi. Mol Microbiol. 2003, 47 (4): 1149-1161. 10.1046/j.1365-2958.2003.03363.x.PubMedView ArticleGoogle Scholar
- Carattoli A, Cogoni C, Morelli G, Macino G: Molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino-3 gene of Neurospora crassa. Mol Microbiol. 1994, 13 (5): 787-795. 10.1111/j.1365-2958.1994.tb00471.x.PubMedView ArticleGoogle Scholar
- Scazzocchio C: The fungal GATA factors. Curr Opin Microbiol. 2000, 3 (2): 126-131. 10.1016/S1369-5274(00)00063-1.PubMedView ArticleGoogle Scholar
- Froehlich AC, Liu Y, Loros JJ, Dunlap JC: White Collar-1, a circadian blue light photoreceptor, binding to the frequency promoter. Science. 2002, 297 (5582): 815-819. 10.1126/science.1073681.PubMedView ArticleGoogle Scholar
- Ruis H, Schuller C: Stress signaling in yeast. Bioessays. 1995, 17 (11): 959-965. 10.1002/bies.950171109.PubMedView ArticleGoogle Scholar
- Seidl V, Seiboth B, Karaffa L, Kubicek CP: The fungal STRE-element-binding protein Seb1 is involved but not essential for glycerol dehydrogenase (gld1) gene expression and glycerol accumulation in Trichoderma atroviride during osmotic stress. Fungal Genet Biol. 2004, 41 (12): 1132-1140. 10.1016/j.fgb.2004.09.002.PubMedView ArticleGoogle Scholar
- Druzhinina IS, Schmoll M, Seiboth B, Kubicek CP: Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina. Appl Environ Microbiol. 2006, 72 (3): 2126-2133. 10.1128/AEM.72.3.2126-2133.2006.PubMed CentralPubMedView ArticleGoogle Scholar
- Rosales-Saavedra T, Esquivel-Naranjo EU, Casas-Flores S, Martinez-Hernandez P, Ibarra-Laclette E, Cortes-Penagos C, Herrera-Estrella A: Novel light-regulated genes in Trichoderma atroviride: a dissection by cDNA microarrays. Microbiology. 2006, 152 (Pt 11): 3305-3317. 10.1099/mic.0.29000-0.PubMedView ArticleGoogle Scholar
- Padovan AC, Sanson GF, Brunstein A, Briones MR: Fungi evolution revisited: application of the penalized likelihood method to a Bayesian fungal phylogeny provides a new perspective on phylogenetic relationships and divergence dates of Ascomycota groups. J Mol Evol. 2005, 60 (6): 726-735. 10.1007/s00239-004-0164-y.PubMedView ArticleGoogle Scholar
- Kozak M: Initiation of translation in prokaryotes and eukaryotes. Gene. 1999, 234 (2): 187-208. 10.1016/S0378-1119(99)00210-3.PubMedView ArticleGoogle Scholar
- Sancar GB, Smith FW: Construction of plasmids which lead to overproduction of yeast PHR1 photolyase in Saccharomyces cerevisiae and Escherichia coli. Gene. 1988, 64 (1): 87-96. 10.1016/0378-1119(88)90483-0.PubMedView ArticleGoogle Scholar
- Nakabeppu Y, Tsuchimoto D, Ichinoe A, Ohno M, Ide Y, Hirano S, Yoshimura D, Tominaga Y, Furuichi M, Sakumi K: Biological significance of the defense mechanisms against oxidative damage in nucleic acids caused by reactive oxygen species: from mitochondria to nuclei. Ann N Y Acad Sci. 2004, 1011: 101-111. 10.1196/annals.1293.011.PubMedView ArticleGoogle Scholar
- Yoshida Y, Ogura Y, Hasunuma K: Interaction of nucleoside diphosphate kinase and catalases for stress and light responses in Neurospora crassa. FEBS Lett. 2006, 580 (13): 3282-3286. 10.1016/j.febslet.2006.01.096.PubMedView ArticleGoogle Scholar
- Yoshida Y, Hasunuma K: Light-dependent subcellular localization of nucleoside diphosphate kinase-1 in Neurospora crassa. FEMS Microbiol Lett. 2006, 261 (1): 64-68. 10.1111/j.1574-6968.2006.00329.x.PubMedView ArticleGoogle Scholar
- Hayashi K, Schoonbeek HJ, De Waard MA: Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides. Appl Environ Microbiol. 2002, 68 (10): 4996-5004. 10.1128/AEM.68.10.4996-5004.2002.PubMed CentralPubMedView ArticleGoogle Scholar
- Vermeulen T, Schoonbeek H, De Waard MA: The ABC transporter BcatrB from Botrytis cinerea is a determinant of the activity of the phenylpyrrole fungicide fludioxonil. Pest Manag Sci. 2001, 57 (5): 393-402. 10.1002/ps.309.PubMedView ArticleGoogle Scholar
- Roohparvar R, De Waard MA, Kema GH, Zwiers LH: MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides. Fungal Genet Biol. 2007, 44 (5): 378-388. 10.1016/j.fgb.2006.09.007.PubMedView ArticleGoogle Scholar
- Kolarova N, Haplova J, Gresik M: Light-activated adenyl cyclase from Trichoderma viride. FEMS Microbiol Lett. 1992, 72 (3): 275-278. 10.1111/j.1574-6968.1992.tb05109.x.PubMedView ArticleGoogle Scholar
- Moscarello MA, Mastronardi FG, Wood DD: The role of citrullinated proteins suggests a novel mechanism in the pathogenesis of multiple sclerosis. Neurochem Res. 2007, 32 (2): 251-256. 10.1007/s11064-006-9144-5.PubMed CentralPubMedView ArticleGoogle Scholar
- Braus GH, Pries R, Düvel K, Valerius O: Molecular biology of fungal amino acid biosynthesis regulation. The Mycota II: genetics and biotechnology. Edited by: Kück U. 2004, Berlin Heidelberg New York , Springer, 230 -2269. 2ndGoogle Scholar
- Patil CK, Li H, Walter P: Gcn4p and novel upstream activating sequences regulate targets of the unfolded protein response. PLoS Biol. 2004, 2 (8): E246-10.1371/journal.pbio.0020246.PubMed CentralPubMedView ArticleGoogle Scholar
- Arvas M, Pakula T, Lanthaler K, Saloheimo M, Valkonen M, Suortti T, Robson G, Penttila M: Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae. BMC Genomics. 2006, 7: 32-10.1186/1471-2164-7-32.PubMed CentralPubMedView ArticleGoogle Scholar
- Denison MS, Nagy SR: Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu Rev Pharmacol Toxicol. 2003, 43: 309-334. 10.1146/annurev.pharmtox.43.100901.135828.PubMedView ArticleGoogle Scholar
- Mandels M, Andreotti RE: Problems and challenges in the cellulose to cellulase fermentation. Proc Biochem. 1978, 13: 6 -13.Google Scholar
- Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985, 33 (1): 103-119. 10.1016/0378-1119(85)90120-9.PubMedView ArticleGoogle Scholar
- Schmoll M, Zeilinger S, Mach RL, Kubicek CP: Cloning of genes expressed early during cellulase induction in Hypocrea jecorina by a rapid subtraction hybridization approach. Fungal Genet Biol. 2004, 41 (9): 877-887. 10.1016/j.fgb.2004.06.002.PubMedView ArticleGoogle Scholar
- Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem. 1987, 162 (1): 156-159. 10.1016/0003-2697(87)90021-2.PubMedView ArticleGoogle Scholar
- Sambrook J Fritsch, E. F., and Maniatis, T.: Molecular cloning: a Laboratory Manual. Plainview. 1989, NY , Cold Spring Harbour Laboratory Press, 2nd edn.Google Scholar
- DOE Joint Genome Institute - Trichoderma reesei Genome Database v2.0. [http://genome.jgi-psf.org/Trire2/Trire2.home.html]
- NCBI Conserved Domain Database (CDD). [http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi]
- Marchler-Bauer A, Bryant SH: CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 2004, 32 (Web Server issue): W327-31. 10.1093/nar/gkh454.PubMed CentralPubMedView ArticleGoogle Scholar
- NCBI Blastp. [http://www.ncbi.nlm.nih.gov/BLAST/]
- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25 (17): 3389-3402. 10.1093/nar/25.17.3389.PubMed CentralPubMedView ArticleGoogle Scholar
- Schaffer AA, Aravind L, Madden TL, Shavirin S, Spouge JL, Wolf YI, Koonin EV, Altschul SF: Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res. 2001, 29 (14): 2994-3005. 10.1093/nar/29.14.2994.PubMed CentralPubMedView ArticleGoogle Scholar
- Campbell JW, Enderlin CS, Selitrennikoff CP: Vectors for expression and modification of cDNA sequences in Neurospora crassa. Fungal Genetics News. 1994, 41: 21-22.Google Scholar
- Schubeler D, Lorincz MC, Cimbora DM, Telling A, Feng YQ, Bouhassira EE, Groudine M: Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation. Mol Cell Biol. 2000, 20 (24): 9103-9112. 10.1128/MCB.20.24.9103-9112.2000.PubMed CentralPubMedView ArticleGoogle Scholar
- Sunnarborg SW, Miller SP, Unnikrishnan I, LaPorte DC: Expression of the yeast glycogen phosphorylase gene is regulated by stress-response elements and by the HOG MAP kinase pathway. Yeast. 2001, 18 (16): 1505-1514. 10.1002/yea.752.PubMedView ArticleGoogle Scholar
- Farkas V, Gresik M, Kolarova N, Sulova Z, Sestak S: Biochemical and physiological changes during photo-induced conidiation and derepression of cellulase synthesis in Trichoderma. Trichoderma reesei cellulase: biochemistry, genetics, physiology, and application. Edited by: Kubicek CP, Eveleigh DE, Esterbauer W, Steiner W, Kubicek-Pranz EM. 1990, Cambridge , Graham House, 139 -1155.Google Scholar
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