Distribution and Sequestration of Cercosporin by Cercospora cf. flagellaris

Plant-pathogenic fungi produce toxins as virulence factors in many plant diseases. In Cercospora leaf blight of soybean caused by Cercospora cf. flagellaris, symptoms are a consequence of the production of a perylenequinone toxin, cercosporin, which is light-activated to produce damaging reactive oxygen species. Cercosporin is universally toxic to cells, except to the cells of the producer. The current model of self-resistance to cercosporin is largely attributed to the maintenance of cercosporin in a chemically reduced state inside hyphae, unassociated with cellular organelles. However, in another perylenequinone-producing fungus, Phaeosphaeria sp., the toxin was specifically sequestered inside lipid droplets (LDs) to prevent reactive oxygen species production. This study hypothesized that LD-based sequestration of cercosporin occurred in C. cf. flagellaris and that lipid-inhibiting fungicides could inhibit toxin production. Confocal microscopy using light-cultured C. cf. flagellaris indicated that 3-day-old hyphae contained two forms of cercosporin distributed in two types of hyphae. Reduced cercosporin was uniformly distributed in the cytoplasm of thick, primary hyphae, and, contrary to previous studies, active cercosporin was observed specifically in the LDs of thin, secondary hyphae. The production of hyphae of two different thicknesses, a characteristic of hemibiotrophic plant pathogens, has not been documented in C. cf. flagellaris. No correlation was observed between cercosporin production and total lipid extracted, and two lipid-inhibiting fungicides had little effect on fungal growth in growth-inhibition assays. This study lays a foundation for exploring the importance of pathogen lifestyle, toxin production, and LD content in the pathogenicity and symptomology of Cercospora.