Envoy, a PAS/LOV domain protein of Hypocrea jecorina (Anamorph Trichoderma reesei), modulates cellulase gene transcription in response to light

Abstract

Envoy, a PAS/LOV domain protein with similarity to the Neurospora light regulator Vivid, which has been cloned due to its lack of expression in a cellulase-negative mutant, links cellulase induction by cellulose to light signaling in Hypocrea jecorina. Despite their similarity, env1 could not compensate for the lack of vvd function. Besides the effect of light on sporulation, we observed a reduced growth rate in constant light. An env1(PAS-) mutant of H. jecorina grows significantly slower in the presence of light but remains unaffected in darkness compared to the wild-type strain QM9414. env1 rapidly responds to a light pulse, with this response being different upon growth on glucose or glycerol, and it encodes a regulator essential for H. jecorina light tolerance. The induction of cellulase transcription in H. jecorina by cellulose is enhanced by light in the wild-type strain QM9414 compared to that in constant darkness, whereas a delayed induction in light and only a transient up-regulation in constant darkness of cbh1 was observed in the env1(PAS-) mutant. However, light does not lead to cellulase expression in the absence of an inducer. We conclude that Envoy connects the light response to carbon source signaling and thus that light must be considered an additional external factor influencing gene expression analysis in this fungus.

FIG. 1.

H. jecorina env1 locus and strategy for deletion of the PAS domain of env1 and complementation with env1. The location of env1 between genes encoding two hypothetical proteins (as predicted by a homology search within the Trichoderma draft genome database [http://gsphere.lanl.gov/trire1/trire1.home.html]) is shown. The transcription start points, as determined by RACE-PCR, are shown by arrows. The region deleted in env1^PAS− is indicated, and the XbaI sites given show the region used for complementation of the mutant.

FIG. 2.

Phylogenetic tree and multiple sequence alignment of PAS domain proteins. (A) Phylogenetic tree obtained by MEGA 2.1, using the minimum evolution method. The sequences used include all sequences present in the alignment in panel B. Numbers at branches indicate their bootstrap support values. (B) Multiple sequence alignment of PAS domain-containing proteins sharing similarity with env1. Residues similar to those interacting with the chromophore in the phy3 LOV2 crystal structure (13) are marked with an arrow, and residues that form the conserved salt bridge therein are marked with “S.” PAS/LOV sequences in the alignment include the following: Hypocrea jecorina blue light regulator 1 (BLR-1; accession no. AY823264), Gibberella zeae hypothetical protein FG07941.1 (EAA77313.1), unnamed Podospora anserina protein product (CAD60767.1), Neurospora crassa white collar 1 protein (WC-1; Q01371), Magnaporthe grisea hypothetical protein MG03538.4 (EAA51943), Gibberella zeae hypothetical protein FG08456.1 (XP_388632), Hypocrea jecorina Envoy (Env1; AY551084), Magnaporthe grisea hypothetical protein MG01041.4 (EAA49383), Neurospora crassa vivid PAS protein VVD (CAF06140), Aspergillus nidulans hypothetical protein AN3435.2 (EAA62912), Neurospora crassa white collar 2 (WC-2; P78714), Nectria hematococca cutinase gene palindrome binding protein (Q00858), Hypocrea jecorina blue light regulator 2 (BLR-2; AY823265), and Magnaporthe grisea hypothetical protein MG04521.4 (EAA50762.1).

FIG. 3.

Hypocrea jecorina env1 does not complement a Neurospora crassa nonfunctional vvd mutant but shares two putative UASs with vvd. (A) Northern analysis of al-1 and env1 transcript accumulation in the Neurospora crassa wild-type strain 74OR23-1a (WT), in the vvd mutant strain vvd^SS692, and in vvd^SS692 mutant strains complemented with the original env1 gene, including its promoter and terminator, indicated by NCvenv1⁺1 and NCvenv1⁺2. After being precultured in the dark, the cultures were illuminated for 10 min, incubated in the dark for 1 h, and again subjected to a light pulse of 10 min. For controls, the cultivations were done in constant darkness. Twenty micrograms of RNA was loaded per lane, and hybridizations were performed with a 0.5-kb fragment of env1 spanning the region containing the PAS domain and with a 0.7-kb PCR fragment of al-1 from Neurospora crassa. Hybridization with an 18S rRNA probe is shown as a control. (B) Analysis of GC content within the promoter regions of env1 and vvd. One thousand nucleotides of the regions upstream of the respective start codon were screened, and the areas of low GC content were searched for shared motifs. Approximate positions of the identified shared UAS motif (5′ ACCTTGAC 3′; EUM2) in env1 and vvd are given schematically.

FIG. 4.

Growth and sporulation of Hypocrea jecorina QM9414 are influenced by light, and the light response is altered in the env1^PAS− mutant. (A) QM9414 shows characteristic “banding” upon growth in daylight (LD, 12-h-12-h cycles; 25°C; malt extract medium). In contrast, the env1^PAS− mutant exhibited only poor growth and a faint mycelium with a decreasing extension rate and completely stopped growing after 10 days, while the wild-type strain QM9414 grew constantly (as shown for three more days). The defect of the non-cellulaseinducible strain QM9978 seems to impact not only cellulase formation but also growth characteristics in daylight, as its extension rate is slightly higher and the banding pattern observed in the wild type is not detectable. (B) Growth and sporulation of env1^PAS− are unaltered in constant darkness. In daylight (LD, 12-h-12-h cycles; 25°C; malt extract medium), env1^PAS− shows secretion of a yellow pigment into the medium. (C) The wild-type strain QM9414 grows significantly slower in daylight than in constant darkness. While growing at approximately the same rate in constant darkness, env1^PAS− exhibits a decreasing extension rate in daylight (measurements were taken from Race tubes, as described in the text). (D) Retransformation of env1^PAS− with an XbaI fragment comprising the wild-type env1 gene, resulting in strain env1RE, reconstitutes the phenotype of the wild-type strain QM9414.

FIG. 5.

env1 responds to light, and this response is dependent on the carbon source. env1 is not transcribed during growth in Mandels-Andreotti minimal medium containing 1% (wt/vol) glucose or glycerol as the carbon source in constant darkness (DD) but is induced within 15 min after a shift to constant light (1,800 lx; 25 μmol photons m⁻² s⁻¹). The Northern data resulted from two sets of separate experiments which revealed complementary and consistent results. An α-³²P-random-labeled PCR fragment spanning the region from the second transcription start point to the start of the region deleted in env1^PAS− was used as a probe, and 20 μg of total RNA was loaded per lane. The results were quantified, and the amount of light-induced transcription of env1 in the specified strain was normalized to the 18S rRNA control hybridization. Graphs below the Northern blots show transcription levels above the background.

FIG. 6.

Cellulase expression is significantly influenced by light during growth on microcrystalline cellulose and is dependent on functional env1. (A) cbh1 is significantly more up-regulated in the wild-type strain QM9414 during cultivation in Mandels-Andreotti minimal medium supplemented with 1% microcrystalline cellulose as the carbon source in constant light (1,800 lx; 25 μmol photons m⁻² s⁻¹), indicated by LL, after 48, 72, and 96 h than in constant darkness (DD). For Northern blotting, 20 μg of total RNA was loaded per lane, and a 1.2-kb α-³²P-radiolabeled PCR fragment of cbh1 was used as a probe. (B) Analysis of env1 light response and cbh1 transcription on lactose. Mycelia were precultured for 25 h in Mandels-Andreotti minimal medium with 1% (wt/vol) lactose as the carbon source, and then the cultures were exposed to light and harvested after the indicated times. Twenty micrograms of total RNA was loaded per lane, and a 1.2-kb α-³²P-radiolabeled PCR fragment of cbh1 was used as a probe. (C) Quantitative analysis of cbh1 signal strength in the wild-type strain QM9414. The transcription of cbh1 after 48 or 72 h on microcrystalline cellulose is decreased roughly 50% in constant light compared to that in constant darkness. While transcription increases further from 72 to 96 h in constant light, autolysis starts in constant darkness, as indicated by sporulation and foaming of the culture and consequently a poor quality of total RNA. Measurements were combined from two independent cultivations; densitometric analysis was done for two expositions of the respective blots.