Overexpression of the Capebp2 Gene Encoding the PEBP-like Protein Promotes the Cap Redifferentiation in Cyclocybe aegerita.

Phosphatidylethanolamine-binding protein (PEBP) is widely involved in various physiological behaviors, such as the transition from vegetative growth to reproductive growth in plants, tumorigenesis in the human, etc. However, few functional studies have examined pebp genes affecting the development of fungi. In this study, Capebp2 was cloned from Cyclocybe aegerita AC0007 strains based on the genome sequence and gene prediction, and the sequence alignment of CaPEBP2 with other PEBP proteins from other biological sources including plant, animal, fungi, and bacteria indicated that PEBP had low sequence similarity in fungi, whereas all protein sequences had some conserved motifs such as DPDAP and HRY. Expression analysis showed the transcription level of Capebp2 increased approximately 20-fold in fruiting bodies compared with mycelia. To uncover the function of Capebp2 in C. aegetita development, Capebp2 was cloned into a pATH vector driven by the actin promoter for obtaining overexpression transformant lines. Fruiting experiments showed the transformed strains overexpressing Capebp2 exhibited redifferentiation of the cap on their surface, including intact fruiting bodies or partial lamella during fruiting development stage, and the longitudinal section indicated that all regenerated bodies or lamella sprouted from the flesh and shared the epidermis with the mother fruiting bodies. In summary, the sequence characterization of Capebp2, expression level during different development stages, and function on fruiting body development were documented in this study, and these findings provided a reference to study the role of pebp in the development process of basidiomycetes. Importantly, gene mining of pebp, function characterization, and the regulating pathways involved need to be uncovered in further studies.

A Putative Guanosine Triphosphate Cyclohydrolase I Named CaGCH1 Is Involved in Hyphal Branching and Fruiting Development in Cyclocybe aegerita.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) is the limiting enzyme of the tetrahydrobiopterin (BH4) synthesis pathway. The disruption of gch1 gene may cause conditional lethality due to folic acid auxotrophy in microorganisms, although the function of gch1 in basidiomycetes has not been deciphered so far. In the present study, gch1 expression in Cyclocybe aegerita (cagch1) was downregulated using the RNAi method, which resulted in growth retardation in both solid and liquid medium, with the hyphal tips exhibiting increased branching compared to that in the wild strain. The development of fruiting bodies in the mutant strains was significantly blocked, and there were short and bottle-shaped stipes. The transcriptional profile revealed that the genes of the MAPK pathway may be involved in the regulation of these effects caused by cagch1 knockdown, which provided an opportunity to study the role of gch1 in the development process of basidiomycetes.