Are small secreted proteins (SSPs) regulators of secondary metabolism in the white rot fungus Pleurotus ostreatus ?

Daria Feldman daria.feldman@mail.huji.ac.il 1 David J. Kowbel 2 N. Louise Glass 2 Oded Yarden 1 Yitzhak Hadar 1
1Plant Pathology and Microbiology, The R.H. Smith Faculty Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
2Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, California, USA

Understanding the functions of small-secreted proteins (SSPs) in fungi is at its early stages, and considered the least characterized component of the fungal secretome. The production of SSPs is associated with pathogens and symbionts, who usually have a high proportion of species-specific SSPs, many of which have been shown to function as effectors. SSPs have also been identified in saprophytic fungi, suggesting alternative, yet unknown, roles. We have identified 3 genes (ssp1, 2 and 3) in Pleurotus ostreatus encoding proteins that have been annotated as SSPs. These genes exhibited a ~4,500- fold increase in expression, 24 hr following exposure to 5-hydroxymethylfurfural (HMF), a compound inhibitory to yeasts, which is formed during pre-treatment of plant biomass. HMF is efficiently degraded by the P. ostreatus wild type (PC9) strain. SSPs, aryl-alcohol oxidases (AAOs) and the intracellular aryl-alcohol dehydrogenases (AADs) were also induced after exposure to other aryl-alcohols, known substrates and inducers of AAOs, and during idiophase (after the onset of secondary metabolism). A knockdown strain of ssp1 (KDssp1) exhibited reduced expression of AAO-and AAD-encoding genes after exposure to HMF. Conversely, a strain overexpressing ssp1 (OEssp1) exhibited elevated expression of genes encoding AAOs and ADD, and a 3-fold increase in enzymatic activity of AAOs. Quantitative secretome analysis of the KDssp1, OEssp1 and the parental PC9 strain, in 8, 10 and 13-day-old cultures were used to monitor the effect of SSP levels on protein accumulation over time. The genetic manipulations introduced conferred a time shift in the secretion pattern: OEssp1 entered the idiophase earlier than PC9, while the converse was observed for KDssp1. We propose that SSPs have roles in saprophytes, and suggest that in P. ostreatus they function as part of the regulation of fungal transition from primary to secondary metabolism during the idiophase, such as occurs with the ligninolytic system.









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