Synthesis of C-glycosyl flavonoid phytoalexins as a site-specific response to fungal penetration in cucumber

Leaf tissue harvested from cucumber plants ( Cucumis sativus L.) expressing induced resistance against the powdery mildew fungus Podosphaera xanthii (syn. Sphaerotheca fuliginea, Castagne; Braun and Shishkoff) was extracted and analyzed for phytoalexin compounds. Fluorescence microscopy was then used to observe the production of these compounds in planta, and laser scanning confocal microscopy observations were made to locate the subcellular sites of phytoalexin accumulation. Phytochemical analyses and fluorescence microscopy observations revealed the production of autofluorescent C-glycosyl flavonoid phytoalexins within the epidermal tissues of disease-resistant plants undergoing fungal ingress. Phytoalexin production was triggered by the combination of an eliciting/inoculation treatment, and tissue autofluorescence of color characteristic of the phytoalexins reached a maximum 48 h after elicitation prior to subsiding following the collapse of the pathogen. After a second eliciting treatment, disease-resistant plants produced phytoalexins more rapidly in response to fungal challenge. At the cellular level, autofluorescent C-glycosyl flavonoid phytoalexins were observed associated with the plasma membrane of infected epidermal cells immediately following elicitation. In the hours that preceded the collapse of conidial chains, phytoalexins accumulated inside the haustorial complexes of the pathogen within the epidermal cells of disease-resistant plants. Taken together, the results of this study show the timely synthesis of C-glycosyl flavonoid phytoalexins at precise subcellular locations as a key defense reaction used by cucumber to create incompatible interactions with powdery mildew.