Identification of Specificity Determinants and Generation of Alleles with Novel Specificity at the het-cHeterokaryon Incompatibility Locus of Neurospora crassa

ABSTRACT The capacity for nonself recognition is a ubiquitous and essential aspect of biology. In filamentous fungi, nonself recognition during vegetative growth is believed to be mediated by genetic differences at heterokaryon incompatibility (het) loci. Filamentous fungi are capable of undergoing hyphal fusion to form mycelial networks and with other individuals to form vegetative heterokaryons, in which genetically distinct nuclei occupy a common cytoplasm. InNeurospora crassa, 11 het loci have been identified that affect the viability of such vegetative heterokaryons. The het-c locus has at least three mutually incompatible alleles, termed het-cOR, het-cPA , and het-cGR . Hyphal fusion between strains that are of alternative het-c specificity results in vegetative heterokaryons that are aconidial and which show growth inhibition and hyphal compartmentation and death. A 34- to 48-amino-acid variable domain, which is dissimilar in HET-COR, HET-CPA, and HET-CGR, confers allelic specificity. To assess requirements for allelic specificity, we constructed chimeras between the het-c variable domain from 24 different isolates that displayed amino acid and insertion or deletion variations and determined their het-c specificity by introduction into N. crassa. We also constructed a number of artificial alleles that contained novel het-cspecificity domains. By this method, we identified four additional and novel het-c specificities. Our results indicate that amino acid and length variations within the insertion or deletion motif are the primary determinants for conferring het-c allelic specificity. These results provide a molecular model for nonself recognition in multicellular eucaryotes.

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VIB-1 Is Required for Expression of Genes Necessary for Programmed Cell Death in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00253-06 ◽

2006 ◽

Vol 5 (12) ◽

pp. 2161-2173 ◽

Cited By ~ 59

Author(s):

Karine Dementhon ◽

Gopal Iyer ◽

N. Louise Glass

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Neurospora Crassa ◽

Filamentous Fungi ◽

Somatic Growth ◽

Genetic Differences ◽

Extracellular Proteases ◽

Hyphal Fusion ◽

Expression Of Genes ◽

Nonself Recognition

ABSTRACT Nonself recognition during somatic growth is an essential and ubiquitous phenomenon in both prokaryotic and eukaryotic species. In filamentous fungi, nonself recognition is also important during vegetative growth. Hyphal fusion between genetically dissimilar individuals results in rejection of heterokaryon formation and in programmed cell death of the fusion compartment. In filamentous fungi, such as Neurospora crassa, nonself recognition and heterokaryon incompatibility (HI) are regulated by genetic differences at het loci. In N. crassa, mutations at the vib-1 locus suppress nonself recognition and HI mediated by genetic differences at het-c/pin-c, mat, and un-24/het-6. vib-1 is a homolog of Saccharomyces cerevisiae NDT80, which is a transcriptional activator of genes during meiosis. For this study, we determined that vib-1 encodes a nuclear protein and showed that VIB-1 localization varies during asexual reproduction and during HI. vib-1 is required for the expression of genes involved in nonself recognition and HI, including pin-c, tol, and het-6; all of these genes encode proteins containing a HET domain. vib-1 is also required for the production of downstream effectors associated with HI, including the production of extracellular proteases upon carbon and nitrogen starvation. Our data support a model in which mechanisms associated with starvation and nonself recognition/HI are interconnected. VIB-1 is a major regulator of responses to nitrogen and carbon starvation and is essential for the expression of genes involved in nonself recognition and death in N. crassa.

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Programmed Cell Death in Neurospora crassa Is Controlled by the Allorecognition Determinant rcd-1

Genetics ◽

10.1534/genetics.119.302617 ◽

2019 ◽

Vol 213 (4) ◽

pp. 1387-1400 ◽

Cited By ~ 4

Author(s):

Asen Daskalov ◽

Pierre Gladieux ◽

Jens Heller ◽

N. Louise Glass

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Neurospora Crassa ◽

Filamentous Fungi ◽

Fungal Pathogens ◽

Defense Mechanism ◽

Balancing Selection ◽

Nonself Recognition ◽

Fusion Cell ◽

Necessary And Sufficient

Nonself recognition following cell fusion between genetically distinct individuals of the same species in filamentous fungi often results in a programmed cell death (PCD) reaction, where the heterokaryotic fusion cell is compartmentalized and rapidly killed. The allorecognition process plays a key role as a defense mechanism that restricts genome exploitation, resource plundering, and the spread of deleterious senescence plasmids and mycoviruses. Although a number of incompatibility systems have been described that function in mature hyphae, less is known about the PCD pathways in asexual spores, which represent the main infectious unit in various human and plant fungal pathogens. Here, we report the identification of regulator of cell death-1 (rcd-1), a novel allorecognition gene, controlling PCD in germinating asexual spores of Neurospora crassa; rcd-1 is one of the most polymorphic genes in the genomes of wild N. crassa isolates. The coexpression of two antagonistic rcd-1-1 and rcd-1-2 alleles was necessary and sufficient to trigger cell death in fused germlings and in hyphae. Based on analysis of wild populations of N. crassa and N. discreta, rcd-1 alleles appeared to be under balancing selection and associated with trans-species polymorphisms. We shed light on genomic rearrangements that could have led to the emergence of the incompatibility system in Neurospora and show that rcd-1 belongs to a much larger gene family in fungi. Overall, our work contributes toward a better understanding of allorecognition and PCD in an underexplored developmental stage of filamentous fungi.

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Allelic Specificity at the het-c Heterokaryon Incompatibility Locus of Neurospora crassa Is Determined by a Highly Variable Domain

Genetics ◽

10.1093/genetics/146.4.1299 ◽

1997 ◽

Vol 146 (4) ◽

pp. 1299-1309

Author(s):

Sven J Saupe ◽

N Louise Glass

Keyword(s):

Neurospora Crassa ◽

Variable Domain ◽

Type Locus ◽

Multiple Alleles ◽

Mhc Genes ◽

Histocompatibility Complex ◽

Sexual Compatibility ◽

Incompatibility Locus ◽

Glycine Rich Protein

In filamentous fungi, the ability to form a productive heterokaryon with a genetically dissimilar individual is controlled by specific loci termed het loci. Only strains homozygous for all het loci can establish a heterokaryon. In Neurospora crassa, 11 loci, including the mating-type locus, regulate the capacity to form heterokaryons. An allele of the het-c locus (het-cOR) of N. crassa has been previously characterized and encodes a nonessential 966 amino acid glycine-rich protein. Herein, we describe the genetic and molecular characterization of two het-c alleles, het-cPA and het-cGR, that have a different specificity from that of het-cOR, showing that vegetative incompatibility is mediated by multiple alleles at het-c. By constructing chimeric alleles, we show that het-c specificity is determined by a highly variable domain of 34–48 amino acids in length. In this regard, het-c is similar to loci that regulate recognition in other species, such as the (S) self-incompatibility locus in plants, the sexual compatibility locus in basidiomycetes and the major histocompatibility complex (MHC) genes in vertebrates.

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Transcriptional profiling and functional analysis of heterokaryon incompatibility in Neurospora crassa reveals that reactive oxygen species, but not metacaspases, are associated with programmed cell death

Microbiology ◽

10.1099/mic.0.032284-0 ◽

2009 ◽

Vol 155 (12) ◽

pp. 3957-3970 ◽

Cited By ~ 32

Author(s):

Elizabeth Hutchison ◽

Sarah Brown ◽

Chaoguang Tian ◽

N. Louise Glass

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Programmed Cell Death ◽

Neurospora Crassa ◽

Fungal Infections ◽

Reactive Oxygen ◽

Oxygen Species ◽

Hyphal Fusion ◽

Heterokaryon Incompatibility ◽

Nonself Recognition

Heterokaryon incompatibility (HI) is a nonself recognition phenomenon occurring in filamentous fungi that is important for limiting resource plundering and restricting viral transfer between strains. Nonself recognition and HI occurs during hyphal fusion between strains that differ at het loci. If two strains undergo hyphal fusion, but differ in allelic specificity at a het locus, the fusion cell is compartmentalized and undergoes a rapid programmed cell death (PCD). Incompatible heterokaryons show a macroscopic phenotype of slow growth and diminished conidiation, and a microscopic phenotype of hyphal compartmentation and cell death. To understand processes associated with HI and PCD, we used whole-genome microarrays for Neurospora crassa to assess transcriptional differences associated with induction of HI mediated by differences in het-c pin-c haplotype. Our data show that HI is a dynamic and transcriptionally active process. The production of reactive oxygen species is implicated in the execution of HI and PCD in N. crassa, as are several genes involved in phosphatidylinositol and calcium signalling pathways. However, genes encoding mammalian homologues of caspases or apoptosis-inducing factor (AIF) are not required for HI or programmed cell death. These data indicate that PCD during HI occurs via a novel and possibly fungal-specific mechanism, making this pathway an attractive drug target for control of fungal infections.

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Arcopilus aureus MaC7A as a New Source of Resveratrol: Assessment of Amino Acid Precursors, Volatiles, and Fungal Enzymes for Boosting Resveratrol Production in Batch Cultures

Applied Sciences ◽

10.3390/app11104583 ◽

2021 ◽

Vol 11 (10) ◽

pp. 4583

Author(s):

Nemesio Villa-Ruano ◽

Luis Ángel Morales-Mora ◽

Jenaro Leocadio Varela-Caselis ◽

Antonio Rivera ◽

María de los Ángeles Valencia de Ita ◽

...

Keyword(s):

Amino Acid ◽

Filamentous Fungi ◽

Hydrolytic Enzymes ◽

Glucanase Activity ◽

Fungal Enzymes ◽

Activity Increase ◽

Batch Cultures ◽

Low Concentrations ◽

Chemical Factors ◽

Amino Acid Precursors

The chemical factors that regulate the synthesis of resveratrol (RV) in filamentous fungi are still unknown. This work reports on the RV production by Arcopilus aureus MaC7A under controlled conditions and the effect of amino acid precursors (PHE and TYR), monoterpenes (limonone, camphor, citral, thymol, menthol), and mixtures of hydrolytic enzymes (Glucanex) as elicitors for boosting fungal RV. Batch cultures with variable concentrations of PHE and TYR (50–500 mg L−1) stimulated RV production from 127.9 ± 4.6 to 221.8 ± 5.2 mg L−1 in basic cultures developed in PDB (pH 7) added with 10 g L−1 peptone at 30 °C. Maximum levels of RV and biomass were maintained during days 6–8 under these conditions, whereas a dramatic RV decrease was observed from days 10–12 without any loss of biomass. Among the tested volatiles, citral (50 mg L−1) enhanced RV production until 187.8 ± 2.2 mg L−1 in basic cultures, but better results were obtained with Glucanex (100 mg L−1; 198.3 ± 7.6 mg L−1 RV). Optimized batch cultures containing TYR (200 mg L−1), citral (50 mg L−1), thymol (50 mg L−1), and Glucanex (100 mg L−1) produced up to 237.6 ± 4.7 mg L−1 of RV. Our results suggest that low concentrations of volatiles and mixtures of isoenzymes with β-1, 3 glucanase activity increase the biosynthesis of fungal RV produced by A. aureus MaC7A in batch cultures.

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Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽

10.1139/g91-098 ◽

1991 ◽

Vol 34 (4) ◽

pp. 644-651 ◽

Cited By ~ 16

Author(s):

Kenneth Koo ◽

W. Dorsey Stuart

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurospora Crassa ◽

Rna Binding ◽

Signal Sequence ◽

Average Length ◽

Open Reading Frame ◽

Mrna Transcript ◽

S1 Nuclease ◽

Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

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Cell Biology of Conidial Anastomosis Tubes in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.4.5.911-919.2005 ◽

2005 ◽

Vol 4 (5) ◽

pp. 911-919 ◽

Cited By ~ 116

Author(s):

M. Gabriela Roca ◽

Jochen Arlt ◽

Chris E. Jeffree ◽

Nick D. Read

Keyword(s):

Neurospora Crassa ◽

Optical Tweezers ◽

Cell Biology ◽

Transmembrane Protein ◽

Mitogen Activated Protein Kinase ◽

Cellular Element ◽

Hyphal Fusion ◽

Self Recognition ◽

Mitogen Activated Protein ◽

Germ Tubes

ABSTRACT Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.

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Nitrogen regulation of amino acid catabolism in Neurospora crassa

Biochemical Genetics ◽

10.1007/bf00484090 ◽

1978 ◽

Vol 16 (3-4) ◽

pp. 343-354 ◽

Cited By ~ 25

Author(s):

Thomas J. Facklam ◽

George A. Marzluf

Keyword(s):

Amino Acid ◽

Neurospora Crassa ◽

Nitrogen Regulation ◽

Amino Acid Catabolism

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The ham-2 Locus, Encoding a Putative Transmembrane Protein, Is Required for Hyphal Fusion in Neurospora crassa

Genetics ◽

10.1093/genetics/160.1.169 ◽

2002 ◽

Vol 160 (1) ◽

pp. 169-180

Author(s):

Qijun Xiang ◽

Carolyn Rasmussen ◽

N Louise Glass

Keyword(s):

Neurospora Crassa ◽

Somatic Cell ◽

Molecular Mechanism ◽

Cell Fusion ◽

Deletion Mutant ◽

Transmembrane Protein ◽

Wild Type ◽

Vegetative Incompatibility ◽

Hyphal Fusion ◽

Aerial Hyphae

Abstract Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.

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The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.11.8.4022-4035.1991 ◽

1991 ◽

Vol 11 (8) ◽

pp. 4022-4035

Author(s):

A R Kubelik ◽

B Turcq ◽

A M Lambowitz

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Neurospora Crassa ◽

Temperature Sensitivity ◽

Temperature Sensitive ◽

Cellular Transport ◽

Missense Mutations ◽

Reading Frame ◽

Trna Synthetases ◽

Cytosolic Protein

The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.

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