Cross talk between a fungal blue-light perception system and the cyclic AMP signaling pathway

Abstract

Blue light regulates many physiological and developmental processes in fungi. In Trichoderma atroviride the complex formed by the BLR-1 and BLR-2 proteins appears to play an essential role as a sensor and transcriptional regulator in photoconidiation. Here we demonstrate that the BLR proteins are necessary for carbon deprivation induced conidiation, even in the absence of light, pointing to the existence of an unprecedented cross talk between light and carbon sensing. Further, in contrast to what has been found in all other fungal systems, clear BLR-independent blue-light responses, including the activation of protein kinase A (PKA) and the regulation of gene expression, were found. Expression of an antisense version of the pkr-1 gene, encoding the regulatory subunit of PKA, resulted in a nonsporulating phenotype, whereas overexpression of the gene produced colonies that conidiate even in the dark. In addition, overexpression of pkr-1 blocked the induction of early light response genes. Thus, our data demonstrate that PKA plays an important role in the regulation of light responses in Trichoderma. Together, these observations suggest that the BLR complex plays a general role in sensing environmental cues that trigger conidiation and that such a role can be separated from its function as a transcription factor.

FIG. 1.

Effect of glucose and nitrogen deprivation on the conidiation phenotype. Pictures show dark-grown colonies of the wild-type and blr mutant strains 24 h after a change of growth medium from 2% glucose to medium without nitrogen (A) or without glucose (B). A 10 mM concentration of cAMP was added to the medium as indicated. The hyaline mycelium is not visible on the white filter paper.

FIG. 2.

Effect of dB-cAMP on the conidiation of the blr mutants and wild-type strains. The pictures show dark-grown colonies 24 h after exposure to 200 μM dB-cAMP, a 5-min blue-light pulse (Light), or a combination of both. Control colonies were treated identically except for the fact that the light pulse was not applied (Dark) or no dB-cAMP was added.

FIG. 3.

(A) PKA activity in the wild type and in blr mutants. Total protein was prepared from different strains from dark grown colonies and exposed to a 5-min pulse of blue light, and activities were determined subsequently at the indicated times. Experiments were made in triplicate and the figure shows the results of a representative experiment. (B) Northern blot analysis of total RNA extracted from blr mutants and wild-type colonies, hybridized with the blu1, blu6, blu17, blu8, phr-1, tpk-1, and c51 cDNAs. 28S was included as loading control. RNA was extracted at the indicated times (in minutes) after a 5-min light pulse. D, dark control.

FIG. 4.

(A) PKA activity in wild type and pkr-1 AS and OE transformants. Total protein was prepared from different strains from dark-grown colonies and exposed to a 5-min pulse of blue light. Activities were subsequently determined at the indicated times. Experiments were made in triplicate, and the figure shows the results of a representative experiment. (B) Photoconidiation assay for the wild-type, AS, and OE strains. The pictures show dark-grown colonies 24 h after a 5-min blue-light pulse (Light) and colonies treated identically except for the absence of the light pulse (Dark). (C) Effect of cAMP on the conidiation phenotypes of the AS, OE, and wild-type strains. The pictures show dark-grown colonies 24 h after transfer to medium with no glucose with or without 10 mM cAMP, as indicated.

FIG. 5.

Northern blot analysis of total RNA extracted from AS and OE transformants and the wild-type strains, hybridized to the blu1, blu6, blu17, blu8, phr-1, tpk-1, c51, and acl cDNAs. The acl gene was included as a light-independent transcription control, and 28S was included as a loading control. RNA was extracted at the indicated times after a 5-min light pulse. D, dark control.

FIG. 6.

(A) Hypothetical model showing the function of the BLR complex in carbon sensing. The BLR-1 and/or BLR-2 could perceive or transduce the signal originated from the lack of glucose detected by a sensor/receptor (CS) or due the lack of reducing power in the cell, via reactive oxygen species (ROS) originated from the mitochondria (M), resulting in the induction of gene expression. (B) Hypothetical model integrating the distinct elements that participate in blue-light perception in Trichoderma atroviride. Two independent light inputs are necessary induce conidiation. The BLR independent pathway could activate adenylyl cyclase (AC), leading to the production cAMP, which in turn binds to the PKA regulatory subunit (R), resulting in the activation of the catalytic subunit (C). Phosphodiesterase (PD) would regulate the levels of cAMP, exerting a negative control on photoconidiation. The increase in PKA activity would activate the BLR complex, triggering the expression of the blue-light-responsive genes; such a function may involve direct phosphorylation of either of the BLR proteins or phosphorylation of an as-yet-unidentified regulatory partner (TA). Alternatively, PKA may phosphorylate a putative transcription factor (TF), whose modification is necessary for gene activation. Noncontinuous lines indicate hypothetical steps.