A photoaffinity probe designed for host-specific signal flavonoid receptors in phytopathogenic Peronosporomycete zoospores of Aphanomyces cochlioides

Abstract

Aphanomyces cochlioides zoospores show chemotaxis to cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone, 1), a host derived attractant, and also respond to 5,7-dihydroxyflavone ( 2) known as an equivalent chemoattractant. To investigate the chemotactic receptors in the zoospores, we designed photoaffinity probes 4′-azido-5,7-dihydroxyflavone ( 3) and 4′-azido-7- O-biotinyl-5-hydroxyflavone ( 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 photoaffinity 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-specific-binding proteins, a candidate for hypothetical cochliophilin A receptor, were localized on the cell membrane of the zoospores. This is the first experimental evidence of flavonoid-binding proteins being present in zoospores, using chemically synthesized azidoflavone as photoaffinity-labeling reagent.