Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes.

Fruiting bodies (sporocarps, sporophores or basidiomata) of mushroom-forming fungi (Agaricomycetes) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates their growth, tissue differentiation and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is still limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim at a comprehensive identification of conserved genes related to fruiting body morphogenesis and distil novel functional hypotheses for functionally poorly characterised ones. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported to be involved in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defence, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1 480 genes of Coprinopsis cinerea, and their orthologs in Agaricus bisporus, Cyclocybe aegerita, Armillaria ostoyae, Auriculariopsis ampla, Laccaria bicolor, Lentinula edodes, Lentinus tigrinus, Mycena kentingensis, Phanerochaete chrysosporium, Pleurotus ostreatus, and Schizophyllum commune, providing functional hypotheses for ~10 % of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes. We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi. Citation: Nagy LG, Vonk PJ, Künzler M, Földi C, Virágh M, Ohm RA, Hennicke F, Bálint B, Csernetics Á, Hegedüs B, Hou Z, Liu XB, Nan S, M. Pareek M, Sahu N, Szathmári B, Varga T, Wu W, Yang X, Merényi Z (2023). Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes. Studies in Mycology 104: 1-85. doi: 10.3114/sim.2022.104.01.

A lectin-mediated resistance of higher fungi against predators and parasites.

Fruiting body lectins are ubiquitous in higher fungi and characterized by being synthesized in the cytoplasm and up-regulated during sexual development. The function of these lectins is unclear. A lack of phenotype in sexual development upon inactivation of the respective genes argues against a function in this process. We tested a series of characterized fruiting body lectins from different fungi for toxicity towards the nematode Caenorhabditis elegans, the mosquito Aedes aegypti and the amoeba Acanthamoeba castellanii. Most of the fungal lectins were found to be toxic towards at least one of the three target organisms. By altering either the fungal lectin or the glycans of the target organisms, or by including soluble carbohydrate ligands as competitors, we demonstrate that the observed toxicity is dependent on the interaction between the fungal lectins and specific glycans in the target organisms. The toxicity was found to be dose-dependent such that low levels of lectin were no longer toxic but still led to food avoidance by C. elegans. Finally, we show, in an ecologically more relevant scenario, that challenging the vegetative mycelium of Coprinopsis cinerea with the fungal-feeding nematode Aphelenchus avenae induces the expression of the nematotoxic fruiting body lectins CGL1 and CGL2. Based on these findings, we propose that filamentous fungi possess an inducible resistance against predators and parasites mediated by lectins that are specific for glycans of these antagonists.

Nuclear import of yeast Gcn4p requires karyopherins Srp1p and Kap95p.

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the alpha-importin Srp1p and the beta-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.

Targeting of Ran: variation on a common theme?

The Ran GTPase plays a key role in nucleocytoplasmic transport. In its GTP-bound form, it directly interacts with members of the importin beta family of nuclear transport receptors and modulates their association with cargo. Work in cell-free higher-eukaryote systems has demonstrated additional roles for Ran in spindle and nuclear envelope formation during mitosis. However, until recently, no Ran-target proteins in these cellular processes were known. Several groups have now identified importin beta as one important target of Ran during mitotic spindle formation. This finding suggests that Ran uses the same effectors to regulate different cellular processes.

Mutations in the YRB1 gene encoding yeast ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects.

We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.

Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p.

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.

Yeast Ran-binding protein 1 (Yrb1) shuttles between the nucleus and cytoplasm and is exported from the nucleus via a CRM1 (XPO1)-dependent pathway.

The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.

Purification of protein A-tagged yeast ran reveals association with a novel karyopherin beta family member, Pdr6p.

The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo. Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.

Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for tRNA.

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-beta-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.

Cse1p functions as the nuclear export receptor for importin alpha in yeast.

CSE1 is essential for yeast cell viability and has been implicated in chromosome segregation. Based on its sequence similarity, Cse1p has been grouped into the family of importin beta-like nucleocytoplasmic transport receptors with highest homology to the recently identified human nuclear export receptor for importin alpha, CAS. We demonstrate here that Cse1p physically interacts with yeast Ran and yeast importin alpha (Srp1p) in the yeast two-hybrid system and that recombinant Cse1p, Srp1p and Ran-GTP form a trimeric complex in vitro. Re-export of Srp1p from the nucleus into the cytoplasm and nuclear uptake of a reporter protein containing a classical NLS are inhibited in a cse1 mutant strain. These findings suggest that Cse1p is the exportin of importin alpha in yeast.

The two 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition.

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 microM for phosphoenolpyruvate and 500 microM for erythrose 4-phosphate. The rate constant was calculated as 6 s-1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (Ki of 0.9 microM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate.

Regulation of the yeast HIS7 gene by the global transcription factor Abf1p.

The HIS7 gene of Saccharomyces cerevisiae encodes a bifunctional glutamine amidotransferase: cyclase that catalyzes the formation of biosynthetic precursors for histidine and adenine. HIS7 is activated by Gcn4p upon amino acid starvation and by the Bas1/2p complex in response to adenine limitation. Mutation analysis of the HIS7 promoter in a deltagcn4 background revealed a polyd(A/T) stretch and a d(CT) repeat as essential elements for Gcn4p-independent basal HIS7 transcription. The protein binding this element was enriched and identified as the multifunctional DNA-binding protein Abf1p. Abf1p binds specifically to the d(CT) repeat sequence, which represents a novel Abf1p-binding motif, and protects 17 nucleotides from digestion by DNase I. In addition, Abf1p binding causes bending of the HIS7 promoter structure.

The transcriptional apparatus required for mRNA encoding genes in the yeast Saccharomyces cerevisiae emerges from a jigsaw puzzle of transcription factors.

The number of identified yeast factors involved in transcription has dramatically increased in recent years and the understanding of the interplay between the different factors has become more and more puzzling. Transcription initiation at the core promoter of mRNA encoding genes consisting of upstream, TATA and initiator elements requires an approximately ribosome-sized complex of more than 50 polypeptides. The recent identification and isolation of an RNA polymerase holoenzyme which seems to be preassembled before interacting with a promoter allowed a better understanding of the roles, assignments and interplays of the various constituents of the basal transcription machinery. Recruitment of this complex to the promoter is achieved by numerous interactions with a variety of DNA-bound proteins. These interactions can be direct or mediated by additional adaptor proteins. Other proteins negatively affect transcription by interrupting the recruitment process through protein-protein or protein-DNA interactions. Some basic features of cis-acting elements, the transcriptional apparatus and various trans-acting factors involved in the initiation of mRNA synthesis in yeast are summarized.

Amino acid and adenine cross-pathway regulation act through the same 5'-TGACTC-3' motif in the yeast HIS7 promoter.

The HIS7 gene of Saccharomyces cerevisiae encodes a bifunctional glutamine amidotransferase:cyclase catalyzing two reactions that lead to the formation of biosynthetic intermediates of the amino acid histidine and the purine adenine. The HIS7 gene is activated by GCN4p under environmental conditions of amino acid starvation through two synergistic upstream sites GCRE1 and GCRE2. The BAS1p-BAS2p complex activates the HIS7 gene in response to adenine limitation. For this activation the proximal GCN4p-binding site GCRE2 is required. GCN4p and BAS1p bind to GCRE2 in vitro. Under conditions of simultaneous amino acid starvation and adenine limitation the effects of GCN4p and BAS1/2p are additive and both factors are necessary for maximal HIS7 transcription. These results suggest that GCN4p and BAS1/2p are able to act simultaneously through the same DNA sequence in vivo and use this site independently from each other in a non-exclusive manner.

Molecular analysis of the yeast SER1 gene encoding 3-phosphoserine aminotransferase: regulation by general control and serine repression.

Although serine and glycine are ubiquitous amino acids the genetic and biochemical regulation of their synthesis has not been studied in detail. The SER1 gene encodes 3-phosphoserine aminotransferase which catalyzes the formation of phosphoserine from 3-phosphohydroxy-pyruvate, which is obtained by oxidation of 3-phosphoglycerate, an intermediate of glycolysis. Saccharomyces cerevisiae cells provided with fermentable carbon sources mainly use this pathway (glycolytic pathway) to synthesize serine and glycine. We report the isolation of the SER1 gene by complementation and the disruption of the chromosomal locus. Sequence analysis revealed an open reading frame encoding a protein with a predicted molecular weight of 43,401 Da. A previously described mammalian progesterone-induced protein shares 47% similarity with SER1 over the entire protein, indicating a common function for both proteins. We demonstrate that SER1 transcription is regulated by the general control of amino-acid biosynthesis mediated by GCN4. Additionally, DNaseI protection experiments proved the binding of GCN4 protein to the SER1 promoter in vitro and three GCN4 recognition elements (GCREs) were identified. Furthermore, there is evidence for an additional regulation by serine end product repression.

Activation and repression of the yeast ARO3 gene by global transcription factors.

The ARO3 gene of Saccharomyces cerevisiae codes for the phenylalanine-inhibited 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 4.1.2.15) and is regulated by the general control system of amino acid biosynthesis through a single GCN4-binding site in its promoter. A combined deletion and mutation analysis of the ARO3 promoter region in a delta gcn4-background revealed two additional regulatory systems involved in ARO3 transcription. The ARO3 gene is (i) activated through a sequence element which binds the multifunctional DNA-binding protein ABF1 in vitro and (ii) repressed through an URS1 element, which binds the same protein in vitro as the URS1 element in the CAR1 promoter. Since both the ABF1-binding site and the URS1 element represent cis-acting elements of global transcription regulatory systems in yeast, the ARO3 gene is the first example of a GCN4-regulated gene which is both activated and repressed by global transcription factors. Activation of the ARO3 gene through the ABF1-binding site and repression through the URS1 element seem to be independent of each other and independent of activation by the GCN4 protein.

Functional differences between mammalian transcription activation domains at the yeast GAL1 promoter.

We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.

Cloning, primary structure and regulation of the ARO4 gene, encoding the tyrosine-inhibited 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway. Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells. The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene. Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine. The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli. ARO4 gene expression is regulated by the general control system of aa biosynthesis. As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions. However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions.

Cloning of the LEU2 gene of Saccharomyces cerevisiae by in vivo recombination.

We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites. A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene. In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene. In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.