Inositol Signaling in the Basidiomycete Fungus Schizophyllum commune

Intracellular signaling is conserved in eukaryotes to allow for response to extracellular signals and to regulate development and cellular functions. In fungi, inositol phosphate signaling has been shown to be involved in growth, sexual reproduction, and metabolic adaptation. However, reports on mushroom-forming fungi are lacking so far. In Schizophyllum commune, an inositol monophosphatase has been found up-regulated during sexual development. The enzyme is crucial for inositol cycling, where it catalyzes the last step of inositol phosphate metabolism, restoring the inositol pool from the monophosphorylated inositol monophosphate. We overexpressed the gene in this model basidiomycete and verified its involvement in cell wall integrity and intracellular trafficking. Strong phenotypes in mushroom formation and cell metabolism were evidenced by proteome analyses. In addition, altered inositol signaling was shown to be involved in tolerance towards cesium and zinc, and increased metal tolerance towards cadmium, associated with induced expression of kinases and repression of phosphatases within the inositol cycle. The presence of the heavy metals Sr, Cs, Cd, and Zn lowered intracellular calcium levels. We could develop a model integrating inositol signaling in the known signal transduction pathways governed by Ras, G-protein coupled receptors, and cAMP, and elucidate their different roles in development.


Introduction
Schizophyllum commune is a saprotrophic white-rot fungus that can complete its life cycle within two weeks on artificial media. It has a long haploid growth phase, and the full genome of S. commune has been published [1]. The basidiomycete is accessible to transformation and genetic modification [2][3][4]. With regard to mating, S. commune has been used extensively to study signaling pathways [5,6] involving inositol signaling [7][8][9][10].
Inositol phosphate signaling has been intensively studied since the 1980s [11,12]. In ascomycete and yeast-forming fungi, inositol phosphate signaling plays a major role in a wide range of biological processes, including metabolic adaptation, apoptosis, fungal virulence, vesicle trafficking, and sexual development [13][14][15][16]. The filamentous basidiomycete fungus S. commune could be shown to include phosphate signaling in its life cycle. The first evidence for inositol and Ras signaling cross-talk was suggested to be in phosphate storage [8,10]. The tetrapolar mating system of basidiomycetes is controlled by two non-linked loci. The A locus encoding several pairs of homeodomain transcription factors with multiple mating specificities in each allele, and the B loci with a pheromone receptor system allow for the recognition of multiple non-self-specificity pheromones with each of the two pheromone receptors. This elaborate mating system controls the formation of the dikaryon that is capable of forming fruiting bodies in response to extracellular signals under natural growth conditions. Since mushroom development is of importance for food production and ecosystem functions, the investigation of signal perception and transduction with its integration into sexual development is of broad interest (see [1]).
The enzyme IMPase is quite well studied and known to require magnesium and to be non-competitively inhibited by lithium [26]. Therefore, in humans, the enzyme is addressed in the therapy of manic-depressive disorder with lithium-based drugs [27]. In euryhaline eel, IMPase plays a role in the adaptation to hypertonic conditions [28], while in plants, it contributes to drought and salt resistance, as well as response to pathogens and symbionts [29]. In ascomycetes, yeasts and the filamentous fungus Podospora anserina have been studied [16]. IMPase is highly conserved among basidiomycetes, including the yeast-forming Cryptococcus neoformans [13]. To understand the role in S. commune, we used overexpression of IMPase and discovered potential roles in cell wall integrity and cellular trafficking. Since it has been reported that inositol signaling is also impaired under metal stress [30], we were interested in inositol signaling in basidiomycete fungi, using the model organism S. commune. For this purpose, IMPase overexpression mutants were compared with wildtypes for their metal tolerance against Sr, Cs, Cd, and Zn. Regulation of the genes belonging to inositol signaling was assessed with respect to metal stress using microarray and proteome analyses [31].

Phylogenetic Analysis and Chromosomal Organization of imp1
To construct a phylogenetic tree, the genome sequence [1]  The chromosomal organization based on predicted genes, size, and functional data from the genome sequence was displayed with ChromoMapper.

Fungal Growth and Microscopy
S. commune (strains listed in Supplementary Table S1) was used to construct imp1 overexpressing strains OEIMP4 and OEIMP6 (primers, Supplementary Table S2; for construction, Supplementary Figure S1) and an empty vector control EVC. The gene imp1 under control of the constitutive, strong tef1 promotor was cloned using yeast recombination of amplified sequences (Q5 High-Fidelity polymerase, New England Biolabs, Ipswich, USA) using primers with 3 and 5 overlap (Supplementary Table S2). Shuttle plasmid pRS415 was linearized with SacI and SacII, the HindIII/PciI fragment (2.9 kb) ligated into pSKtrp resulting in pOEIMP. Circular plasmid pOEIMP was introduced in S. commune T33 according to [32]. Transformants were screened by PCR using primers 5F and 5R (see Supplementary Table S2).
To visualize cellular trafficking, SynaptoRed FM 4-64 (3.2 µM for 40 min, washed twice with PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) was used after 3 days of cultivation in 24 well plates (Greiner Bio One, Frickenhausen, Germany) at room temperature with 150 µL agar and 150 µL liquid medium to keep the culture moist. The sample was directly observed (CLSM Zeiss LSM 780, Jena, Germany; 40×/1.30 NA EC Plan-Neofluar objective and transmission light or fluorescence Ar/ML 488/514 nm laser with 0.2% intensity). The signal from 622 nm to 759 nm was detected with GaAsP detector. Pinhole size and laser dwell time were minimized. Finalization and intensity measurements of images were carried out with the program Zen2012 (Zeiss, Jena, Germany).

Calcium Levels under Metal Stress
S. commune 12-43 was grown on MM + ura with either 100 mM SrCl 2 , 75 mM CsCl, 10 mM ZnCl 2 , or 0.5 mM CdCl 2 topped with a cellophane foil. After 14 days at 28 • C, the mycelium was harvested with a plastic spatula and dried for 18 h at 70 • C until constant weight and the mycelium homogenized in an agate mortar. The experiment was performed in five replicates. Ca as well as metal contents were measured in a mixed sample of five biological replicates after microwave extraction using ICP-MS (Inductively Coupled Plasma -Mass Spectrometry, XSeries II, Thermo Fisher Scientific, Bremen, Germany; for details compare [35]).
The obtained tandem mass spectra were searched against the JGI database of S. commune H4-8 v3.0 (https://mycocosm.jgi.doe.gov/Schco3/Schco3.home.html; accessed on 2 February 2017) using Proteome Discoverer PD 2.1 and the algorithms of Mascot 2.4, Sequest HT, and MS Amanda. Two missed cleavages were allowed for trypsin digestion. The precursor mass tolerance was set to 10 ppm and the fragment mass tolerance was set to 0.02 Da. Modifications were defined as dynamic Met oxidation and static Cys carbamidomethylation. At least two peptides per protein and a strict false discovery rate < 1% (reverse decoy) were required. Label-free quantification of the Top 3 unique peptides per protein was based on the precursor ion area detector approach implemented in PD 2.1. For quantification, data were normalized using the total peptide amount approach. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [38] partner repository with the dataset identifier PXD026465.

Transcriptome Analyses
An mRNA sequencing approach was analyzed and enzymes of inositol signaling were found to be significantly regulated under metal stress [39]. The two significantly regulated genes, myo-inositol oxygenase (XM_003027475.1) and a hypothetical inositol polyphosphate phosphatase (XM_003036036.1) were selected to confirm regulation under metal stress using qPCR.
Microarray data (GEO omnibus acc. no. GSE172373) were evaluated for regulation of enzymes involved in inositol signaling. In the microarray, the gene regulation of S. commune 12-43 grown on complex yeast medium [33] was compared with growth in presence of multi-heavy metal containing seepage water and to co-isogenic S. commune W22 grown with 0.01 mM cadmium nitrate. All enzymes of inositol signaling that were regulated under metal stress were selected from this microarray data.

A Basidiomycete Clade of IMPases
The gene coding for IMPase, imp1, is located in scaffold 2 of the genome of S. commune H4-8 and has a coding region of 1247 bp interrupted by five introns. IMPase is highly conserved among basidiomycetes, and the single gene imp1 in S. commune encodes 340 aa containing all three motifs for substrate and metal binding, and nucleophilic activation. Alignments and phylogenetic analysis including human, yeast, and basidiomycete sequences revealed the existence of different evolutionary clades in IMPases (Figure 2). The highest aa identity was shared between the S. commune, Laccaria amethystina (67%), Laccaria bicolor (67%), Termitomyces (66.5%), and Fistulina hepatica (64%) sequences, all of basidiomycete origin. Surrounding genes included a conserved zinc finger transcription factor and a chaperone of the DnaJ superfamily. This direct neighborhood is conserved with a zinc finger protein, zfand1, and also with the human gene coding for inositol monophosphatase 1.

Imp1 Overexpression Leads to Improved Cell Wall Integrity
Overexpression was achieved with the gene imp1 under the control of the strong promotor of tef1, originally leading to constitutive expression of translation elongation factor EF1α. The overexpression was verified by RT-qPCR (Supplementary Figure S2). A remarkable increase of 42-and 67-fold up-regulation was confirmed for the two independent transformants, OEIMP4 and OEIMP6, respectively. Growth was only slightly retarded upon long term incubation for imp1 overexpressing strains as compared to a wildtype and the empty vector control without obvious changes in morphology of mycelia (Figure 3). A more detailed analysis of cellular effects of overexpression revealed a role in cell wall integrity, as decreased susceptibility against calcofluor white, binding to chitin, Congo red for specific polysaccharide staining, and SDS as membrane-active detergent were observed (Supplementary Figure S4). The results indicate that Imp1 and inositol signaling might play a role in biosynthesis and organization of the cell wall of S. commune. Since stability against cell wall stress was observed, growth with heavy metals exerting such stress was screened.

Influence of Altered Inositol Signaling on Metal Tolerance
The influence of altered inositol signals on metal tolerance was investigated by exposing IMPase-overexpressing S. commune OEIMP4 to heavy metal stress. For all four tested metals, the strain S. commune 12-43 performed inferior as compared to S. commune T33 and its derivatives, S. commune EVC1 and S. commune OEIMP4 (Figure 3). In addition to higher inhibition through metals, S. commune 12-43 formed more aerial mycelium. The remaining three strains all showed, unexpectedly, improved growth with Sr. However, this is due to the fact that aerial mycelium was reduced in favor of more (and thus faster) substrate hyphae formation. The inositol monophosphatase overexpression led to a significantly higher inhibition by Cs and Zn, which was not seen with Cd.
The intracellular calcium levels were distinctly reduced in S. commune 12-43 grown in the presence of heavy metals. The difference was most evident in the presence of Sr, but clearly visible also for Cs and Cd, where calcium levels were reduced (Table 1).

Inositol Signaling Related Gene Regulation in Metal Stress
The general direction of the gene regulation of the two genes of interest could be confirmed using qPCR. Exceptions were the comparisons of minimal medium versus Chernobyl soil for the hypothetical inositol polyphosphate phosphatase gene and heavy metal medium versus Paradies park soil for the myo-inositol oxygenase gene (Figure 4). The two microarray comparisons (Supplementary Table S3) showed that increased metal stress lead to a higher expression of both genes. This also tended to be the case for the first three comparisons of the mRNA sequencing, but not for the comparison minimal medium versus heavy metal medium. The qPCR investigation confirmed that the hypothetical polyphosphate phosphatase gene was highly up-regulated under the influence of all metals tested. The Myo-inositol oxygenase gene just tended to be up-regulated under metal stress. However, in the presence of zinc and cadmium, no regulation could be recognized and clear down-regulation in the case of strontium.
The two comparisons of the microarray data mostly reacted under metal stress with gene regulation in the same direction for the same genes. A difference was most likely to be seen in the regulation of protein kinase C, which occurs outside of the immediate inositol cycle (see Figure 1). It could also be seen that under metal stress, kinases tended to be up and phosphatase tended to be down-regulated (Table S3). Down-regulation was clearly observed for inositol triphosphate phosphatase, which catalyzes the phosphorylation of inositol triphosphate to inositol diphosphate.

Imp1 Overexpression and Changed Intracellular Trafficking
Fungal endocytosis and exocytosis are key processes in growth and cell wall synthesis at the growing tip. SynaptoRed FM 4-64 staining of vesicles and vacuoles in the hyphal apex showed altered vesicle size ( Figure 5). In addition, overexpression of imp1 in conjunction with Brefeldin A treatment, which is a potent inhibitor of exocytosis, increased size and variance in organelles, supporting the notion of involvement of Imp1 in vesicle trafficking. Wavy hyphae in the treated wildtype and imp1 overexpressing strains show that directional growth might be co-afflicted. Since a strong phenotype thus could be assigned to imp1 overexpression, changes in proteome were investigated to identify proteins with changed expression profiles in the transformant OEIMP4 as compared to EVC1.

Proteome Analysis Verifies a Function of imp1 in Cellular Trafficking
KOG classification of the 287 proteins with higher abundance upon imp1 overexpression revealed 43 proteins involved in cellular processes and signaling, 41 in information storage and processing, and 58 in cell metabolism (31 were poorly and 114 not well characterized). Of the 127 proteins with lower abundance, 12 were classified into cellular processes and signaling, 7 in the information storage and processing, and 35 in cell metabolism, with 21 poorly and 52 not well characterized (Supplementary Table S4).
The highest regulation in the membrane-associated proteome was recognized with respect to cellular trafficking ( Figure 6). Among those were membrane AAA + -type ATPase containing the peptidase M41 domain, isopentenyl-diphosphate delta-isomerase involved in the mevalonate pathway, and the antioxidant thioredoxin. Another 14 proteins (signal recognition particle SRP19, ABC transporter, C5 cytosine-specific DNA methylase, acyltransferase, signal recognition particle SRP72, mitochondrial import inner membrane translocase, Tim8 containing zinc finger domain, armadillo type-fold Sec7-like, VPS5/SNX1 containing a PhoX domain, cysteine protease required for autophagy Apg4p/Aut2p, nuclear transport factor 2, t-SNARE, dynamin/VPS1, and two SNF7 proteins) decreased in abundance.
Higher levels were observed for cytoskeleton-associated proteins, three actin binding protein-related proteins and a kinesin-like protein. This supports a function in vesicle trafficking, since vesicles are cargo to the cytoskeleton and moved by motor proteins on the cytoskeletal tracks for directed intracellular movement.

Discussion
The inositol cycle depends on IMPase activity maintaining a moderate level of the cellular inositol pool [41]. The imp1 gene encoding IMPase had been found to be up-regulated upon Ras1 activation, suggesting cross-talk between Ras and inositol signaling [8,10]. In addition, lithium as IMPase inhibitor induced a shift to inositol polyphosphates. The phylogenetic analysis obtained now shows conservation between basidiomycete IMPases, including the presence of only one gene for IMPase and linkage to zinc finger (see [42]) and chaperone genes [43]. The conservation is also true for the three strains of S. commune with genome sequences available (Supplementary Figure S5).
Overexpression could not only be verified by qPCR, but also independently with the proteome study. A functional role in cell wall structure could be assigned in cell wall integrity and membrane stability, which confirms earlier studies showing particularly amino sugars contents being affected by changes in phosphoinositide in Neurospora [44]. Thus, cell wall biosynthesis through formation and membrane fusion of excretory vesicles involved in cell wall biosynthesis during tip growth of fungi is supported with this study.
Downstream of inositol signaling, cellular processes, and signaling related proteins were predominantly affected through imp1 overexpression, which over-represented intracellular trafficking machinery and cytoskeleton associated proteins. This finding supports the notion that inositol signaling is involved in vesicle trafficking.
The up-regulated signal recognition particle subunits, SRP19 and SRP72, are involved in targeting secretory vesicles in eukaryotes [45][46][47]. Receptor-mediated activation of SRP results in signal peptide cleavage and release of proteins meant for secretion at the endoplasmic reticulum [48,49]. Another protein with elevated abundance, Snf7, is a component of the ESCRT (endosomal sorting complex required for transport)-III complex that is needed for the sorting of proteins into invaginating vesicles of the multivesicular body. The ESCRT complexes are released from the endosomal membrane with the help of an AAA-motif containing ATPase for further rounds of membrane invaginating [50]. Such an ATPase containing an M41 peptidase domain, Snf7, co-regulated with an ABC transporter responsible for export/import of a wide range of substrates including ions and macromolecules, was identified.
Heavy metals are omnipresent in the environment and exert toxic effects at higher concentrations involving oxidative stress [51]. In plants, inositol signaling has been shown to be involved in the reaction to oxidative stress. Here, S. commune kinases in inositol signaling were shown to be elevated under metal stress, while phosphatases mainly decreased in abundance. This would shift the cycle to more phosphoinositols like PIP 2 and less myo-inositol. This is in accordance with the finding that H 2 O 2 -induced cell death is associated with decreased myo-inositol amounts in Arabidopsis thaliana and brown algae [30,52]. The up-regulated inositol polyphosphate phosphatase has been shown to be involved in response to ROS and abiotic stress in both plants and animals [53,54]. We could show altered inositol signaling to influence metal tolerance in S. commune.
Our investigations showed a down-regulation of inositol multikinases under the influence of metals, which probably leads to lower amounts of highly phosphorylated inositol phosphates and pyrophosphates (IP 7 and IP 8 ; compare [23,55].
The calcium levels of S. commune were decreased 4-to 7-fold in the presence of different heavy metals. This could be on one hand due to the presence of extra amounts of metals which were taken up by the hyphae instead of calcium by calcium channels [56] or, on the other hand, could be in a relationship with altered inositol signaling and thus calcium as a second messenger [57]. Calcium has a protective role against ROS and supplemental added calcium leads to increased Cd tolerance [58].
As for vesicle transport, t-SNARE and Sec7-like proteins were induced upon imp1 overexpression. SEC-7 is involved in vesicular budding and intracellular trafficking between compartments of the Golgi apparatus, and target membrane bound SNARE (t-SNARE) plays an important role in facilitating vesicle fusion in cargo trafficking [59]. For cargo transport, the cytoskeleton is needed that was up-regulated at the proteome level as well.
Long-distance transport along microtubules is suggested by the up-regulation of kinesin, while a plus-end directed microtubule-dependent motor protein [60], dynamin, connects the long-distance and short-distance transport, e.g., at Golgi compartments. Here, again the activity of small GTPases is involved [61,62]. Filamentous actin is organized by the activity of actin-bundling proteins, two of which were detected among the proteins with increased abundances under imp1 overexpression. Finally, actin-binding proteins and cofilin/tropomyosin permit transport of cargo along actin filaments. This is connected to exocytosis, where vesicles are brought to the vesicle supply center at the growing tip before fusing with the cell membrane to release cell wall components needed for tip growth. The role of actin transport in endocytosis in older hyphal compartments has been discussed controversially for fungi, but seems likely.
To further investigate the role of inositol signaling in intracellular trafficking, we further examined the effect of the strong protein secretion inhibitor brefeldin A in imp1 overexpressing transformants [63]. Indeed, larger vacuoles were more abundant in the overexpressing strains, supporting the view that vesicle fusion is increased through IMPase activation, and hence the result of inositol signaling. With this study, the view on signal transduction could be expanded (Figure 7) to include inositol phosphate signaling in mushroom forming basidiomycetes.