Archives of Biochemistry and Biophysics 432 (2004) 145–151 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.09.017 A photoaYnity probe designed for host-speciWc signal Xavonoid receptors in phytopathogenic Peronosporomycete zoospores of Aphanomyces cochlioides Yasuko Sakihama a , Takashi Shimai a , Mitsuyoshi Sakasai a , Toshiaki Ito b , Yukiharu Fukushi a , Yasuyuki Hashidoko a , Satoshi Tahara a,¤ a Laboratory of Ecological Chemistry, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan b Laboratory of Electron Microscopy, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan Received 7 June 2004, and in revised form 15 September 2004 Available online 22 October 2004 Abstract Aphanomyces cochlioides zoospores show chemotaxis to cochliophilin A (5-hydroxy-6,7-methylenedioxyXavone, 1), a host derived attractant, and also respond to 5,7-dihydroxyXavone (2) known as an equivalent chemoattractant. To investigate the chemotactic receptors in the zoospores, we designed photoaYnity probes 4'-azido-5,7-dihydroxyXavone (3) and 4'-azido-7-O-biotinyl-5- hydroxyXavone (4) considering chemical structure of 2. Both 3 and 4 had zoospore attractant activity which was competitive with that of 1. When zoospores were treated with the biotinylated photoaYnity probe followed by UV irradiation and streptavidin–gold or peroxidase-conjugated streptavidin, probe-labeled proteins were detected on the cell membrane. This result indicated that the 1- speciWc-binding proteins, a candidate for hypothetical cochliophilin A receptor, were localized on the cell membrane of the zoosp- ores. This is the Wrst experimental evidence of Xavonoid-binding proteins being present in zoospores, using chemically synthesized azidoXavone as photoaYnity-labeling reagent.  2004 Elsevier Inc. All rights reserved. Keywords: Flavonoidal photoaYnity probe; Biotinylated Xavone; Flavonoid-binding protein; Aphanomyces cochlioides; Chemotactic receptor As part of its life cycle, the Peronosporomycete (pre- viously known as Oomycete) Aphanomyces cochlioides, causative agent of spinach root rot and sugar beet damp- ing-oV diseases, goes through a motile zoospore stage. During this stage the zoospore recognizes host-speciWc signals and swims in soil water directly to the host plant [1]. The host-speciWc attractant exuded from spinach roots has been identiWed as cochliophilin A (5-hydroxy- 6,7-methylenedioxyXavone, 1) [2]. The A. cochlioides zoospores are strongly attracted to a Chromosorb W AW particle coated with 1 (10 ¡9 M). The attracted zoospores encyst in a few minutes [3] and Wnally the cys- tospores germinate within 30–60 min. When they germi- nate on spinach roots, the hyphae penetrate into the plant tissue via clear or non-clear appressorium formed on the germ tube [3,4]. On the contrary, cystospores induced by mechanical stimulation or nicotinamide do not become mature but rather regenerate to zoospores, so these stimuli resulted rarely in germination of the spores [4,5]. Thus, compound 1 is revealed to be inevita- bly correlated with the establishment of infection in the relationship between A. cochlioides life-cycle develop- ment and signal transduction in the zoospores stimu- lated by the host plant, spinach [1]. In fact, 5.3 g of 1 in 1 g fresh roots of spinach was found and exuding amount/plant/day was 34 ng [6]. Compound 1 was * Corresponding author. Fax: +81 11 706 4182. E-mail address: tahara@abs.agr.hokudai.ac.jp (S. Tahara).