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Eukaryotic Cell, December 2005, p. 2140-2152, Vol. 4, No. 12
1535-9778/05/$08.00+0     doi:10.1128/EC.4.12.2140-2152.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic and Molecular Analysis of Phytochromes from the Filamentous Fungus Neurospora crassa

Allan C. Froehlich,1,{dagger} Bosl Noh,2,{ddagger} Richard D. Vierstra,2 Jennifer Loros,1,3 and Jay C. Dunlap1*

Departments of Genetics,1 Biochemistry,Dartmouth Medical School, Hanover, New Hampshire 03755,3 Department of Genetics, 425-G Henry Mall, University of Wisconsin—Madison, Madison, Wisconsin 537062

Received 16 July 2005/ Accepted 29 September 2005

Phytochromes (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 fungus Neurospora 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 of phy-1 and phy-2 revealed two splice isoforms for each gene. The levels of the phy transcripts are not regulated by light, but the abundance of the phy-1 mRNAs 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 of phy-1 and phy-2, either singly or in tandem, were not compromised in any known photoresponses in Neurospora, leaving their function(s) unclear.

* Corresponding author. Mailing address: Department of Genetics, Dartmouth Medical School, Hanover, New Hampshire 03755. Phone: (603) 650-1108. Fax: (603) 650-1233. E-mail: jay.c.dunlap{at}dartmouth.edu.

{dagger} Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

{ddagger} Present address: Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701, Korea.

Eukaryotic Cell, December 2005, p. 2140-2152, Vol. 4, No. 12
1535-9778/05/$08.00+0     doi:10.1128/EC.4.12.2140-2152.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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