Molecular mechanisms of zoosporogenesis, motility, chemotaxis and differentiation of Peronosporomycete zoospores

The zoosporogenesis, motility and differentiation of zoospores are critical in the disease cycles of Peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. The biflagellate motile zoospores released from sporangia of these phytopathogens are believed to locate their host plants by chemotaxis, after which they undergo a series of morphological changes before penetrating the host tissues to establish the diseases. We conducted series to experiments to understand underlying molecular mechanisms of zoosporogenesis, motility, chemotaxis and differentiation of two important peronosporomycetes Aphanomyces cochlioides and Plasmopara viticola. Bioassay-guided chromatographic investigations using A. cochlioides zoospores identified a host-specific plant signal cochliophilin A (5-hydroxy 6,7-methylenedioxyflavone) in root exudates which are not only responsible for chemotaxis but also trigger developmental transitions (encystment and germination) of zoospores on host surface to initiate infection. Interestingly, the morphological changes of zoospores induced by hostspecific signaling compounds appeared to be linked to polymerization/depolymerization of the filamentous actin in their cells. In contrast, nonhost plants possess diverse secondary metabolites (chemical weapons) such as nicotinamide (motility inhibitor), polyflavonoid tannins (lytic factors) to ward-off phytopathogenic zoospores. We also found that exogenous applications of G protein activator matoparan, primary alcohols (n-BuOH) and pure phospholipase C enzymes triggered differentiation of both A. cochlioides and P. viticola zoospores. Milimolar level of Ca2+ triggered germination of mechanically-induced cystospores that blocked by both Ca2+ channel blockers or calmodulin antagonists. We observed that both differentiations of zoospores into cystospores and germination of cystospores to haphal germ tubes by pharmacological effectors and Ca2+, respectively were associated with induction of 32P-phosphatidic acid (PA) accumulation in the treated cells. Interestingly, an antagonist of PA generation, lisofylline suppressed encystment of zoospores by mastoparan and Ca2+-induced germination of cystospores and remarkably decreased 32P-PA accumulation in the respective cells. Our results suggest that both PLD and PLC pathways are likely to be involved with P. viticola zoospore encystment and PA might act as a second messenger during germination of cystospores by Ca2+. To understand how motility of zoospores is maintained, we searched inhibitory compounds from Streptomyces species. We isolated a broad-spectrum kinase inhibitor, staurosporine from a Streptomyces sp. strain B 5136, which impaired motility of zoospores without causing any lysis. Among the 22 known kinase inhibitors tested, the PKC inhibitor chelerythrine was the most potent to arrest the motility of zoospores. Inhibitors that targeted kinase pathways other than PKC pathways did not practically show any activity in impairing zoospore motility. Interestingly, both staurosporine (5 nM) and chelerythrine (10 nM) also inhibited the release of zoospores from the P. viticola sporangia. In addition, staurosporine completely suppressed downy mildew disease in grapevine leaves at 2 µM, suggesting the potential of small-molecule PKC inhibitors for the control of peronosporomycete phytopathogens. Taken together, these results suggest that PKC is likely to be a key signaling mediator associated with zoosporogenesis and the maintenance of flagellar motility in peronosporomycete zoospores.