The STRIPAK complex and its role in fruiting-body development of the filamentous fungus Sordaria macrospora

Abstract

The mammalian Striatin-interacting phosphatase and kinase (STRIPAK) complex consist of many proteins, among them Striatin as scaffold, the putative kinase activator monopolar spindle-one-binder 3 (MOB3), serine/threonine phosphatase PP2A subunits A and C, the Striatin-interacting protein (STRIP)1 and STRIP2, sarcolemmal membrane-associated protein (SLMAP) and the germinal center kinases (GCK) MST4, STK24, STK25 and MINK1. In this study, we used the filamentous ascomycete Sordaria macrospora as model organism to analyze the role of the STRIPAK complex in fruiting-body development. S. macrospora is coprophytic fungus which solely undergoes a sexually lifecycle and does not require a mating partner for sexual reproduction. In S. macrospora, the STRIPAK complex is required for fruiting-body development and hyphal fusion. Hyphal fusion is a process that results in mixed cell contents of involved cells without lysis. In filamentous ascomycetes, hyphal fusion occurs at different stages of vegetative growth and sexual reproduction. The STRIPAK complex in S. macrospora contains homologs to mammalian Striatin (PRO11), MOB3 (SmMOB3), subunits A and C of PP2A (SmPP2AA and C), STRIP1/2 (PRO22) and SLMAP (PRO45). However, fungal STRIPAK-associated kinases have not been characterized to date. This study is divided into two parts, one comprises the characterization of SmGPI1, a GPI-anchored protein, identified as interaction partner of SmMOB3 in cross-species microarrays, and the other part is about identification of potential STRIPAK-associated kinases. Interaction between STRIPAK-associated SmMOB3 and SmGPI1 was successfully verified by co-Immunoprecipitation (co-IP) and yeast two-hybrid (Y2H) using S. macrospora cDNA. Deletion of Smgpi1 was the next step to investigate its impact on fruiting-body development; in contrast to Δpro11, Δ pro22, ΔSmMOB3 and Δpro45, ΔSmgpi1 underwent hyphal fusion and was fertile, but generated more fruiting bodies, which were smaller but normal in shape compared to wt. Interestingly deletion of Smgpi1 in a sterile ΔSmmob3 deletion background restored the phenotypes caused by Smmob3 deletion. As already mentioned, ΔSmmob3 is sterile and not capable of hyphal fusion. In contrast, the double-deletion strain ΔSmgpi1/ΔSmmob3 is fertile and underwent hyphal fusion. This effect was Smmob3 specific and did not occur in other STRIPAK-specific double-deletion strains, e.g. ΔSmgpi1/Δpro11, ΔSmgpi1/Δpro22 and ΔSmgpi1/Δpro45. Moreover, fluorescence microscopy and differential centrifugation of SmGPI1 revealed a dual targeting; SmGPI1 localizes at the cell wall and the mitochondria. Regarding the identification of STRIPAK-associated kinases in S. macrospora, two kinases were identified to be homologous to the mammalian STRIPAK-associated kinases MST4, STK24, STK25 and MINK1 by BLASTP search and were named SmKIN3 and SmKIN24. Phylogenetic analysis revealed a conservation of these kinases among ascomycetes. Interaction of SmKIN3 and SmKIN24 with S. macrospora Striatin homolog PRO11 was shown via Y2H and for SmKIN3 and PRO11 also by means of co-IP. Fluorescence microscopy of SmKIN3 and SmKIN24 revealed localization to the septa, whereas SmKIN3 localizes at the outer part of the septum and SmKIN24 to the septal pore. Deletion of Smkin3 or Smkin24 to analyze their impact on fruiting-body development showed only ΔSmkin24 to be sterile. However, ΔSmkin3 and ΔSmkin24 were reduced in vegetative growth and exhibited impaired septa formation; whereas ΔSmkin3 displayed greater distances between adjacent septa compared to wt, deletion of Smkin24 resulted in numerous closely-packed septal bundles of abnormal shape. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain. Moreover, we discovered that protoplasts harboring the ΔSmkin3 deletion background recover faster than protoplasts obtained from wt. Based on the results of this study and findings in N. crassa that homologs to SmKIN3 and SmKIN24 are implicated in septation initiation network (SIN) we assume STRIPAK to function aside from sexual development and hyphal fusion, also in regulation of fruiting-body number via SmMOB3-SmGPI1 interaction and suggest a crosstalk between SIN and STRIPAK in S. macrospora.