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

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.

At least three families of hyphosphere small secreted cysteine‐rich proteins can optimize surface properties to a moderately hydrophilic state suitable for fungal attachment

“…Probably the most interesting feature of SSCPs is their abundant secretion in liquid medium (up to 20%–30% of the total secretome) (Pellegrin et al ., 2015 ; Feldman et al ., 2020 ; Gao et al ., 2020 ) and the unusually high polymorphism of their primary structures (Gaderer et al ., 2014 ; Krizsán et al ., 2019 ). Some SSCPs have a long evolutionary history of gene duplications (GDs) and lateral gene transfers (LGTs), making them frequent members of the orphomes (the genome fraction comprising orthologue‐less genes) of individual fungal taxa (Przylucka et al ., 2017a ; Feldman et al ., 2019 ; Gao et al ., 2020 ). Paradoxically, despite the abundance of SSCPs in fungal secretomes, functions have been proposed for only a few, while the role of the majority of them remains unknown (Aimanianda and Latge, 2010 ; Feldman et al ., 2017 ; Wang et al ., 2019 ; Gao et al ., 2020 ).…”

Seeking the Roles for Fungal Small-Secreted Proteins in Affecting Saprophytic Lifestyles

“…In contrast, the KDssp1 had a longer tropohase, the strain did not generate pigmentation and no lysis of the colony occurred. Taken together, Ssp1 was shown to be involved in the transition from tropohase to idiophase, aging, and metabolism (Feldman et al, 2019), along with regulating ligninolytic enzyme production (Feldman et al, 2017). Overexpression of Ssp1 also negatively affected fruiting body initiation (Feldman et al, 2019), which is in line with Krizsán et al (2019), who suggested that SSPs function as fruiting body effectors.…”

“…In fungi, QS has been described to regulate processes such as sporulation, secondary metabolite production, morphological transition and enzyme secretion (Barriuso et al, 2018). The effects of Ssp1 in P. ostreatus highly resemble the mentioned characteristics of QS (Feldman et al, 2017(Feldman et al, , 2019. The majority of the known fungal QS molecules are chemical compounds, such as farnesol and alcohol tyrosol, like in Candida albicans (Albuquerque and Casadevall, 2012;Franceschetti et al, 2017;Barriuso et al, 2018).…”

The Conifer Root and Stem Rot Pathogen (Heterobasidion parviporum): Effectome Analysis and Roles in Interspecific Fungal Interactions

“…Diverse strategies utilized by fungal individuals might confer flexibility towards local changes in the environment to increase their fitness, which may be partly derived from elaborately regulated gene expression profiles in response to diverse conditions [26]. Fungi have hundreds or even over a thousand predicted secreted proteins, among which the proportion of SSPs in most fungi is 25% to 60% across all lifestyles [27][28][29]. In this study, H. parviporum was predicted to have 21.4% HpSSPs in its secretome (163/759).…”