Analysis of phytochrome-dependent signal transduction in Aspergillus nidulans

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The fungi Aspergillus nidulans and Alternaria alternata use phytochrome to sense and respond to red light. The signal transduction cascade which leads to differential expression of more than 1000 genes after 15 min of illumination involves the HOG pathway. In the previous funding period, the transcriptional changes were unraveled, and novel functions of phytochrome in far-red and in blue-light sensing were discovered. In A. alternata a long-standing question about the formation of the linear tetrapyrrole was solved and two hemeoxyenases characterized. In A. nidulans the question about heme ring opening is still unsolved. Interestingly, both A. alternata HOs associated to mitochondria where they directly interact with the phytochrome apoprotein until this is loaded with the chromophore. Now we propose to study the role of the C-terminus of HoxA in mitochondrial membrane association and to try to identify the electron donor for the HOs. Ferredoxin and a P450 cytochrome are good candidates. In preliminary experiments, we found that the C-terminus of HoxB appears to also serve a function, namely, to prevent the protein from moving into nuclei. A truncated version was imported efficiently. We have evidence that the nuclear function is related to stress resistance. This apparently novel function of hemeoxygenase HoxB in nuclei will be studied through the analysis of protein interactions with “stress”-transcription factors as well as the study of a transcriptomic response after overexpression of the truncated protein. Lastly, the HO mutants in A. alternata offer the possibility to identify a corresponding enzyme in A. nidulans. Since there are no proteins with similar sequences, we anticipate a very distinct protein. In the previous funding period, we established also methods to produce large amounts of the phytochrome photosensory domain or the full-length protein in E. coli. These proteins will be used for spectroscopic and crystallographic experiments, in collaboration with P. Hildebrandt and P. Scherer (Berlin). In addition, the role of specific amino acid residues in phytochrome will be tested for their contribution to function and conformational changes.