D ESCRIPCIÓN Y FORMULACIÓN DEL P ROBLEMA

Protein fraction were isolated from the S. commune in different development stage. The abundance of proteins in dikaryon, primordia, and fruiting body was compared to monokaryon 12-43 and 4-39 respectively. Low and high abundant proteins in every developmental stages are classified based on EuKaryotic Orthologous Groups/ KOG (Figure 13 and 14).

Phosphatidylinositol-4-phosphate 5-kinase was found to be repressed in dikaryon vs. monokaryon 12-43 within 2,45-folds and in primordia vs. monokaryon 12-43 within 2,48-folds. On the other hand, phosphatidylinositol 3- and 4-kinase was repressed in fruiting body in comparison to monokaryon 12x43 within 2,10-folds and to monokaryon 4-39 within 2,42-folds. Phosphatidylinositol-4-phosphate 5-kinase generates phosphatidylinositol-4,5-bisphosphatase from phosphatidylinositol-4-phosphatase and phosphatidylinositol 3- and 4-kinase generates phosphatidylinositol-4-phosphatase and phosphatidylinositol-3-phosphatase from phosphatidylinositol. This evidence shows that phosphatidylinositol signaling is involved in the sexual development of S. commune and the phosphoinositides might act as negative regulators for fruiting body development.

Figure 13. Classification of low abundant signal proteins based on EuKaryotic Orthologous Groups (KOG). MK: monokaryon, DK: dikaryon, PR: primordia, FB: fruiting bodies.

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Figure 14. Classification of high abundant signal proteins based on EuKaryotic Orthologous Groups (KOG). MK: monokaryon, DK: dikaryon, PR: primordia, FB: fruiting bodies.

Repression of phosphatidylinositol signaling might affect calcium signaling which was reflected on the repression of calcium-binding EF-hand protein. Calcium-binding EF-hand protein which requires activation from IP3 was found to be down-regulated as well in dikaryon

and in primordia. Multicopper oxidase (schco3 Protein ID 2179879) and peptidase M35 were found to be induced in dikaryon compared to both monokaryon 12-43 and 4-39, showing that at least one of the multicopper oxidase is required for establishing dikaryon. Signal transduction response regulator was repressed in dikaryon in comparison to both monokaryons. This protein has a function in detecting and responding some changes in surrounding environment, including stressors. Interestingly, polysaccharide deacetylase that has been known to play a crucial role in the alteration of cell shape, was induced in primordia and fruiting bodies. As it has been known that in primordia and fruiting body, hyphae alter its structure to become thicker hyphae since the dikaryotic hyphae form aggregates.

Protein that possesses histidine acid phosphatases (HAP) domain was repressed in the fruiting bodies and low abundant of putative inositol phosphatase was observed also in the fruiting bodies. In line with the repression of lipid inositol phosphate kinase, it is found that myo- inositol-1-phosphate synthase was also repressed only in the fruiting bodies compared to monokaryon 12-43. The repression of myo-inositol-1-phosphate synthase indicates that the production of inositol is reduced and therefore, limiting also the lipid inositol phosphate (phosphatidylinositol/ PI) which was reflected on the repression of phosphatidylinositol 3- and 4-kinase in fruiting bodies. Several ATPases were also repressed in fruiting body, in agreement with the repression of HAP domain which is responsible for inositol pyrophosphate generation. Inositol pyrophosphates are capable to regulate many biological processes most probably by controlling energetic metabolism and ATP production. Less abundant of ATPase in the proteome seems to be an effect from the low abundance of inositol pyrophosphate in fruiting body or vice versa.

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Multicopper oxidase (schco3 Protein ID 2179879) as well as glycosyltransferases were induced in primordia formation. Glycosyltransferases are another large multigene family in eukaryote that responsible for biosynthesis of secondary metabolites, and sugar conjugation results in increased stability and water solubility.

Aldehyde dehydrogenase and spermine synthase were induced in primordia. On the other hand, beta tubulin and actinin was repressed in the primordia within 2,03-folds and 2,63-folds, respectively. Glycoside hydrolase family were repressed in primordia but elevated in fruiting bodies, as fungal glycoside hydrolases (GH) is known to act on cell wall components and are responsible for morphological changes, possibly from primordia to fruiting body. Furthermore, several GMC oxidoreductases, peptidases, protein kinases, and ribosomal protein family were also repressed in primordia.

According to the proteome analysis, there are 161 and 215 proteins that are induced in fruiting bodies compared to monokaryon 12-43 and 4-39, respectively. In other basidiomycetes, cytochrome P450 is induced in fruiting body, indicating that this oxidative enzyme might play a role in mushroom development. Moreover, several proteins that are important in carbohydrate metabolism are elevated in fruiting body including glycolysis transferase (GT) family, glyoxalase I, glycoside hydrolase (GH) family, polysaccharide deacetylase, and esterase/ lipase/ thiosterase. Metallophosphoesterase that is required for GPI-anchor proteins transport from the organelle endoplasmic reticulum, found to be induced and repressed in fruiting bodies.

There are 483 and 583 proteins that are repressed in the fruiting body stage compared to monokaryon 12-43 and 4-39, respectively. Mostly repressed proteins are metabolism associated proteins and cellular processes and signaling associated proteins (Table S11-S22). Several microtubules and microfilaments associated proteins were repressed in fruiting body stage, presumably the growth of hyphae is only required in the mycelial expansion but not necessarily needed in fruiting bodies. In fruiting bodies, several proteins that are involved in motor activity such as actin, actin-binding, Arp2/3 complex, alpha tubulin, beta tubulin, F-actin capping protein, kinesin, and myosin, were found lower in abundance. Additionally, dynamin, importin- beta, NSF attachment protein, clathrin propeller, and clathrin adaptor complex were also repressed in fruiting bodies (Table S21 and S22). This evidence shows that the transport activity in fruiting bodies is deactivated. Further, several proteins related to signaling such as Ras small GTPase family, GTP-binding nuclear protein Ran, calcium-binding EF-hand, regulator of G- protein signaling, fungal G-protein alpha subunit, and signal transduction histidine kinase, were repressed as well in fruiting bodies.