Ultrastructure of Aphanomyces cochlioides zoospores and changes during their developmental transitions triggered by the host-specific flavone cochliophilin A.

Aphanomyces cochlioides is a serious damping-off causing pathogen of sugar beet, spinach and some other members of Chenopodiaceae and Amaranthaceae. The biflagellated motile zoospores of the pathogen locate their host roots by perceiving the host-specific flavone cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone), transiently modify into cystospores that germinate prior to penetration. This study for the first time illustrated ultrastructure of the zoospores and morphological modification during their developmental transitions triggered by cochliophilin A using transmission electron microscopy (TEM). TEM revealed that zoospores had two heterokont flagella inserted laterally into a ventral groove of their body where each is attached to a kinetosome. In the cross sections of flagellar axonemes, two single and nine peripheral microtubules in doublets were clearly observed. Mitochondria, the Golgi complexes, finger print vesicles, and vesicles with striated electron opaque inclusion and vesicles containing a granular cortex and center were also detected. The latter vesicles disappeared and two flagella were shed when zoospores converted to spherical cystsopores. The shape, size and number of mitochondria were dynamically changed during the encystment of zoospores presumably through fission and fusion processes. The dynamics of mitochondria observed in this study indicated its distinct role in the signal transduction pathway of the zoospore encystment. This study also revealed the transformation of shape of nuclei from pyriform in zoospores to spherical in cystospores and lanceolate in the hyphae.

Dynamic rearrangement of F-actin organization triggered by host-specific plant signal is linked to morphogenesis of Aphanomyces cochlioides zoospores.

Cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone), a root releasing host-specific plant signal triggers chemotaxis and subsequent morphological changes in pathogenic Aphanomyces cochlioides zoospores before host penetration. The present study illustrates time-course changing patterns of cytoskeletal filamentous actin (F-actin) organization in the zoospores of A. cochlioides during rapid morphological changes (encystment and germination) after exposure to cochliophilin A. Confocal laser scanning microscopic analysis revealed that F-actin microfilaments remained concentrated at ventral groove and diffusely distributed in peripheral cytoplasm of the zoospore. These microfilaments dramatically rearranged and changed into granular F-actin plaques interconnected with fine arrays during encystment. A large patch of actin arrays accumulated at one pole of the cystospores just before germination. Then the actin plaques moved to the emerging germ tube where a distinct cap of microfilaments was seen at the tip of the emerging hypha. Zoospores treated with an inhibitor of F-actin polymerization, latrunculin B or motility halting and regeneration inducing compound nicotinamide, displayed different patterns of F-actin in both zoospores and cystospores than those obtained by the induction of cochliophilin A. Collectively, these results indicate that the host-specific plant signal cochliophilin A triggers a dynamic polymerization/depolymerization of F-actin in pathogenic A. cochlioides zoospores during early events of plant-peronosporomycete interactions.

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

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.

Quantification of the particle method for chemotactic bioassay using Peronosporomycete zoospores.

We estimated the amount of test solution absorbed by each Chromosorb W AW particle (60-80 mesh) using an isotopic technique to quantitate the particle method. 14C-Labeled standard compounds like carbendazim (MBC), 5-O-methylcochliophilin A, sucrose and proline were dissolved in several solvents, and Chromosorb carrier particles were treated with the solution to coat the particle with these test compounds. The ratios of the radioactivity of 5 microl of the test solution to that of 2 mg of carrier particles treated with the solution at some different concentrations were measured. It was found that each carrier particle holds approx. 3.8 nl of the test solution within a range of 2 x 10(-3) to 1 x 10(-7) M concentrations. Accordingly, it is now possible to widely use the particle method as a quantitative procedure to assay chemotaxis of Peronosporomycete zoospores.