Manipulating the Expression of Small Secreted Protein 1 (Ssp1) Alters Patterns of Development and Metabolism in the White-Rot Fungus Pleurotus ostreatus

Small secreted proteins (SSPs) are common in fungal saprotrophs, but their roles remain elusive. As such, they comprise part of a gene pool which may be involved in governing fungal lifestyles not limited to symbiosis and pathogenicity, in which they are commonly referred to as “effectors. ” We propose that Ssp1 in the white-rot fungus Pleurotus ostreatus regulates the transition from primary to secondary metabolism, development, aging, and fruiting body initiation. Our observations uncover a novel regulatory role of effector-like SSPs in a saprotroph, suggesting that they may act in fungal communication as well as in response to environmental cues. The presence of Ssp1 homologues in other fungal species supports a common potential role in environmental sensing and fungal development. ABSTRACT The function of small secreted proteins (SSPs) in saprotrophic fungi is, for the most part, unknown. The white-rot mushroom Pleurotus ostreatus produces considerable amounts of SSPs at the onset of secondary metabolism, during colony development, and in response to chemical compounds such as 5-hydroxymethylfurfural and aryl alcohols. Genetic manipulation of Ssp1, by knockdown (KDssp1) or overexpression (OEssp1), indicated that they are, in fact, involved in the regulation of the ligninolytic system. To elucidate their potential involvement in fungal development, quantitative secretome analysis was performed during the trophophase and the idiophase and at a transition point between the two growth phases. The mutations conferred a time shift in the secretion and expression patterns: OEssp1 preceded the entrance to idiophase and secondary metabolism, while KDssp1 was delayed. This was also correlated with expression patterns of selected genes. The KDssp1 colony aged at a slower pace, accompanied by a slower decline in biomass over time. In contrast, the OEssp1 strain exhibited severe lysis and aging of the colony at the same time point. These phenomena were accompanied by variations in yellow pigment production, characteristic of entrance of the wild type into idiophase. The pigment was produced earlier and in a larger amount in the OEssp1 strain and was absent from the KDssp1 strain. Furthermore, the dikaryon harboring OEssp1 exhibited a delay in the initiation of fruiting body formation as well as earlier aging. We propose that Ssp1 might function as a part of the fungal communication network and regulate the pattern of fungal development and metabolism in P. ostreatus. IMPORTANCE Small secreted proteins (SSPs) are common in fungal saprotrophs, but their roles remain elusive. As such, they comprise part of a gene pool which may be involved in governing fungal lifestyles not limited to symbiosis and pathogenicity, in which they are commonly referred to as “effectors. ” We propose that Ssp1 in the white-rot fungus Pleurotus ostreatus regulates the transition from primary to secondary metabolism, development, aging, and fruiting body initiation. Our observations uncover a novel regulatory role of effector-like SSPs in a saprotroph, suggesting that they may act in fungal communication as well as in response to environmental cues. The presence of Ssp1 homologues in other fungal species supports a common potential role in environmental sensing and fungal development.