Homothallic Ascomycete Sordaria Macrospora Research Articles

Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora.

In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.

The small serine-threonine protein SIP2 interacts with STE12 and is involved in ascospore germination in Sordaria macrospora

In fungi, the homoeodomain protein STE12 controls diverse developmental processes, and derives its regulatory specificity from different protein interactions. We recently showed that in the homothallic ascomycete Sordaria macrospora, STE12 is essential for ascospore development, and is able to interact with the alpha-domain mating-type protein SMTA-1 and the MADS box protein MCM1. To further evaluate the functional roles of STE12, we used the yeast two-hybrid approach to identify new STE12-interacting partners. Using STE12 as bait, a small, serine-threonine-rich protein (designated STE12- interacting protein 2, SIP2) was identified. SIP2 is conserved among members of the fungal class Sordariomycetes. In vivo localization studies revealed that SIP2 was targeted to the nucleus and cytoplasm. The STE12/SIP2 interaction was further confirmed in vivo by bimolecular fluorescence complementation. Nuclear localization of SIP2 was apparently mediated by STE12. Unlike deletion of ste12, deletion of sip2 in S. macrospora led to only a slight decrease in ascospore germination, and no other obvious morphological phenotype. In comparison to the Δste12 single knockout strain, ascospore germination was significantly increased in a Δsip2/ste12 double knockout strain. Our data provide evidence for a regulatory role of the novel fungal protein SIP2 in ascospore germination.

Functional Characterization of MAT1 - 1 -Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria macrospora Provides New Insights into Essential and Nonessential Sexual Regulators

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the alpha domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes.

SmATG7 is required for viability in the homothallic ascomycete Sordaria macrospora

In filamentous ascomycetes, autophagy is involved in several developmental processes. Nevertheless, until now little is known about its role in fruiting-body development. We therefore isolated a gene of the homothallic ascomycete Sordaria macrospora with high sequence similarity to the Saccharomyces cerevisiae autophagy-related gene ATG7, encoding a core autophagy regulator. This is the first characterization of an ATG7 homolog in filamentous ascomycetes. A S. cerevisiae complementation assay demonstrated that the S. macrospora Smatg7 gene functionally replaces the yeast homolog. We were not able to generate a homokaryotic knock-out mutant in S. macrospora, suggesting that Smatg7 is required for viability. However, a heterokaryotic ΔSmatg7/ Smatg7 strain and transformants generated by RNA interference showed considerable morphological phenotypes during fruiting-body development. Using real-time PCR, we demonstrated that in the wild type, the transcriptional expression of Smatg7 is markedly up-regulated under amino acid starvation conditions and at late stages during sexual development. Moreover, we showed that transcriptionally down-regulation of Smatg7 disturbs autophagy in S. macrospora.

Detection of hyphal fusion in filamentous fungi using differently fluorescence-labeled histones

Cell fusion occurs regularly during the vegetative and sexual phases of the life cycle in filamentous fungi. Here, we present a simple and efficient method that can detect even rare hyphal fusion events. Using the homothallic ascomycete Sordaria macrospora as an experimental system, we developed a histone-assisted merged fluorescence (HAMF) assay for the investigation of hyphal fusion between vegetative mycelia. For this purpose, two reporter vectors were constructed encoding the histone proteins HH2B or HH2A fused at their C terminus either with the cyan or yellow fluorescent protein, respectively. The chimeric proteins generate fluorescently labeled nuclei and thus enable the distinction between different strains in a mycelial mixture. For example, hyphae with nuclei that show both cyan as well as yellow fluorescence indicate the formation of a heterokaryon as a result of hyphal fusion. To test the applicability of our HAMF assay, we used two S. macrospora developmental mutants that are supposed to have reduced hyphal fusion rates. The simple and efficient HAMF assay described here could detect even rare fusion events and should be applicable to a broad range of diverse fungal species including those lacking male or female reproductive structures or asexual spores.

Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi

Here, we report the construction of two transformation vectors, pD-NAT1 and pG-NAT1, carrying the nat1 gene encoding the nourseothricin acetyltransferase. The nat1 gene is expressed under the control of the Aspergillus nidulans trpC promoter and thus can be used as a dominant drug-resistance marker for the DNA-mediated transformation of filamentous fungi. The successful application of both vectors was demonstrated by transforming the homothallic ascomycete Sordaria macrospora as well as the β-lactam producer Acremonium chrysogenum. For both fungi and for both vectors, transformation frequencies were between 10 and 40 transformants per 10 µg of plasmid DNA.

A STE12 homologue of the homothallic ascomycete Sordaria macrospora interacts with the MADS box protein MCM1 and is required for ascosporogenesis

The MADS box protein MCM1 controls diverse developmental processes and is essential for fruiting body formation in the homothallic ascomycete Sordaria macrospora. MADS box proteins derive their regulatory specificity from a wide range of different protein interactions. We have recently shown that the S. macrospora MCM1 is able to interact with the alpha-domain mating-type protein SMTA-1. To further evaluate the functional roles of MCM1, we used the yeast two-hybrid approach to identify MCM1-interacting proteins. From this screen, we isolated a protein with a putative N-terminal homeodomain and C-terminal C2/H2-Zn2+ finger domains. The protein is a member of the highly conserved fungal STE12 transcription factor family of proteins and was therefore termed STE12. Furthermore, we demonstrate by means of two-hybrid and far western analysis that in addition to MCM1, the S. macrospora STE12 protein is able to interact with the mating-type protein SMTA-1. Unlike the situation in the closely related heterothallic ascomycete Neurospora crassa, deletion (Delta) of the ste12 gene in S. macrospora neither affects vegetative growth nor fruiting body formation. However, ascus and ascospore development are highly impaired by the Deltaste12 mutation. Our data provide another example of the functional divergence within the fungal STE12 transcription factor family.

A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora.

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Deltamcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development.

Functional analysis of the C 6 zinc finger gene pro1 involved in fungal sexual development

The pro1 gene, controlling fruiting body development in the homothallic ascomycete Sordaria macrospora, encodes a C 6 zinc finger protein with a typical DNA binding domain of GAL4-like C 6 zinc finger proteins as well as a putative nuclear targeting signal. In the corresponding mutant pro1, the pro1 gene is deleted, and the transition of primordia into mature fruiting bodies is prevented. To further characterize the PRO1 polypeptide, the yeast system was used for identifying a transactivation domain in the N-terminal half of PRO1, which probably also functions in S. macrospora. The functional analysis was extended by using truncated versions of the pro1 gene in complementation transformations of a Δ pro1 mutant. Interestingly, the 5 ′ part of the pro1 gene encoding the DNA binding and transactivation domain as well as putative nuclear targeting signals was sufficient to restore fertility in the sterile pro1 mutant. In vitro mutagenesis verified that the DNA binding domain is essential for normal fruiting body development. This was concluded from transformation experiments with eight pro1 derivatives containing triplet substitutions in conserved codons of the DNA binding domain; some, but not all, failed in restoring the wild-type phenotype in mutant pro1. Using a PCR-based cloning strategy, pro1 homologs from the two related heterothallic species Neurospora crassa and Sordaria brevicollis were isolated, showing similarities in the predicted amino acid sequences of 91 and 90%, respectively. When a N. crassa pro1 cDNA clone was used in complementation transformations, we succeeded in restoring the wild-type phenotype to the S. macrospora pro1 mutant. These data suggest that pro1 homologs from heterothallic species can provide the pro1 function in homothallic ascomycetes. Based on the published sequence of the N. crassa genome, we identified hpro1A, another transcriptionally expressed gene, with a similarity of 40% to the pro1 genes, which is present as a single copy gene in N. crassa as well as in S. macrospora.

Two pheromone precursor genes are transcriptionally expressed in the homothallic ascomycete Sordaria macrospora

In order to analyze the involvement of pheromones in cell recognition and mating in a homothallic fungus, two putative pheromone precursor genes, named ppg1 and ppg2, were isolated from a genomic library of Sordaria macrospora. The ppg1 gene is predicted to encode a precursor pheromone that is processed by a Kex2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. The ppg2 gene encodes a 24-amino-acid polypeptide that contains a putative farnesylated and carboxy methylated C-terminal cysteine residue. The sequences of the predicted pheromones display strong structural similarity to those encoded by putative pheromones of heterothallic filamentous ascomycetes. Both genes are expressed during the life cycle of S. macrospora. This is the first description of pheromone precursor genes encoded by a homothallic fungus. Southern-hybridization experiments indicated that ppg1 and ppg2 homologues are also present in other homothallic ascomycetes.