Gene expression in Phycomyces blakesleeanus after light and gravity stimulation

Light is one of the most powerful abiotic factors triggering a multitude of developmental key processes of fungal biology and lifecycle. The filamentous fungus Phycomyces blakesleeanus has been a model organism in sensory physiology since more than 50 years. Therefore a linear flow of light information from sensors to effectors was assumed. Since the isolation and characterization of behavioral mad mutant strains and even more since the recent identification of the set of wc-photoreceptor genes (madA, madB) molecular approaches of photophysiology were able to advance. The counterparts of N. crassa White Collar-1/-2 photoreceptors/transcription factors in P. blakesleeanus are the MADA and MADB proteins which form a photosensitive transcription factor complex (MAD complex) to govern many light-controlled processes in the fungus (Sanz et al., 2009). In this work 25 genes encoding mainly five different functional protein groups, the wc-type photoreceptors, heat shock proteins, RNA-/chromatin-modulating enzymes, the ß-carotene enzymes and constituents of the cytoskeleton were used to move a step further in light dependent gene expression to gain new insights about the regulatory role of the wc-type photoreceptor mutants madA and madB. Therefore, a protocol was developed that consisted of a pulse of actinic light, which gave a non saturating transcriptional response, measured by the method of qRT-PCR. Not only the characterization of the blue light responses in both wildtype and mad mutant strains came into the focus of these approaches, but also the search for red light and gravity responses on a transcriptional level. The gene expression in dark grown mycelia of the wildtype of P. blakesleeanus measured during an 11.5 hour time course shows different expression levels for each gene. The levels of expression are not correlated with a functional group of genes, indicating that the genes are individually regulated even in darkness. In the mad mutant strains the gene expression levels of more than half of the genes are different to the ones in the wildtype supporting this hypothesis and demonstrating the importance of the photosensitive transcription factor complex MAD. In contrast to the findings of N. crassa none of the genes show an endogenous rhythmicity of their expression. In the wildtype of P. blakesleeanus many of the analyzed genes (60 %) show a response after exposure to continuous or a pulse of blue light. These photoresponses are mainly a transcriptional activation but a suppression of gene expression was detected as well. Continuous light conditions can enhance the expression of 11 genes moderately, about three to 20-fold. After a pulse of it, the transcription of 15 genes is increased. The photoactivation after a pulse of blue light can be divided into a strong (20-80-fold) or moderate increase of mRNA in the wildtype which do not correlate with a functional group of genes, indicating that the genes are individually regulated. As an empirical rule, the increase of transcript after blue light exposure is higher, the lower the dark expression level is. Four genes show a moderate decrease of their transcript after both blue light treatments. After a pulse of blue light none of the single mad mutant strains show a complete suppression of the photoactivation but strongly diverse mRNA levels of the light induced genes compared to the wildtype. Only in the double madAB or triple madABC mutant strains an abolished photoactivation of transcription could be detected. It shows the dominant role of the MAD complex on the one hand and suggests a more complex regulatory mechanism on the other. In addition to the blue light effects the influence of red light on transcription was investigated. Red light moderately modulates the gene expression, both positively and negatively. Some genes show photoreversible expression patterns after a pulse and continuous blue and red light. The time dependent red light effects on transcriptional level are different to the ones of blue light and for some genes show reverse patterns in the wildtype. For some genes mutation of the wc-type photoreceptors results in a more pronounced suppression after red than activation after blue light in the wildtype or increased transcription in the mutant strains while unaffected in the wildtype after a pulse of red light. The results demonstrate the complex regulatory mechanisms of gene expression in P. blakesleeanus suggesting the interaction of more than only the madAB photoreceptors. The expression patterns after bichromatic light treatments support the notation of other than wc-type photoreceptors being involved in transcriptional regulation. Gravity stimulation, for some genes, results in specific or transient expression changes in the wildtype, dramatically different to the ones in the madC and madABC triple mutant strains. These results underlie the central role of the madAB photoreceptors/transcription factors not only by integrating the light, but also the gravity signal as well and suggest the interaction of more than only the madAB photoreceptors in a gene regulation complex. A model of the gene regulation in P. blakesleeanus is presented with a set of proposal, as other photoreceptors and gene specific regulating elements