Genetic and Molecular Analysis of Phytochromes from the Filamentous FungusNeurospora crassa

• DOI: 10.1128/ec.4.12.2140-2152.2005
• Publication Date: 2005-12-08T23:23:25Z
• Authors: Allan C. Froehlich; Bosl Noh; Richard D. Vierstra; Jennifer Loros; Jay C. Dunlap
• Keywords: Cytoplasm; 0303 health sciences; DNA, Complementary; Base Sequence; Histidine Kinase; Genetic Linkage; Amino Acid Motifs; Genes, Fungal; Chromosome Mapping; Exons; Introns; Circadian Rhythm; Fungal Proteins; Alternative Splicing; 03 medical and health sciences; Gene Expression Regulation, Fungal; Escherichia coli; Amino Acid Sequence; Chromosomes, Fungal; Genome, Fungal; DNA, Fungal; Gene Deletion

ABSTRACTPhytochromes (Phys) comprise a superfamily of red-/far-red-light-sensing proteins. Whereas higher-plant Phys that control numerous growth and developmental processes have been well described, the biochemical characteristics and functions of the microbial forms are largely unknown. Here, we describe analyses of the expression, regulation, and activities of two Phys in the filamentous fungusNeurospora crassa. In addition to containing the signature N-terminal domain predicted to covalently associate with a bilin chromophore, PHY-1 and PHY-2 contain C-terminal histidine kinase and response regulator motifs, implying that they function as hybrid two-component sensor kinases activated by light. A bacterially expressed N-terminal fragment of PHY-2 covalently bound either biliverdin or phycocyanobilin in vitro, with the resulting holoprotein displaying red-/far-red-light photochromic absorption spectra and a photocycle in vitro. cDNA analysis ofphy-1andphy-2revealed two splice isoforms for each gene. The levels of thephytranscripts are not regulated by light, but the abundance of thephy-1mRNAs is under the control of the circadian clock. Phosphorylated and unphosphorylated forms of PHY-1 were detected; both species were found exclusively in the cytoplasm, with their relative abundances unaffected by light. Strains containing deletions ofphy-1andphy-2, either singly or in tandem, were not compromised in any known photoresponses inNeurospora, leaving their function(s) unclear.