Namboori B. Raju

Meiotic Silencing by Unpaired DNA

The silencing of gene expression by segments of DNA present in excess of the normal number is called cosuppression in plants and quelling in fungi. We describe a related process, meiotic silencing by unpaired DNA (MSUD). DNA unpaired in meiosis causes silencing of all DNA homologous to it, including genes that are themselves paired. A semidominant Neurospora mutant, Sad-1, fails to perform MSUD. Sad-1 suppresses the sexual phenotypes of many ascus-dominant mutants. MSUD may provide insights into the function of genes necessary for meiosis, including genes for which ablation in vegetative life would be lethal. It may also contribute to reproductive isolation of species within the genus Neurospora. The wild-type allele, sad-1(+), encodes a putative RNA-directed RNA polymerase.

Genetic control of the sexual cycle in Neurospora

Over 200 mutants that affect sexual development have been isolated and analysed in Neurospora crassa . Mutational, recombinational, and molecular analyses on the mating type region show that the mating type genes A and a specify both the mating function and a vegetative (heterokaryon) incompatibility function. The two functions are inseparable by recombination but separable by a mutational event, as in a m 33 . An unlinked tol mutation suppresses the vegetative incompatibility associated with mating type genes A and a without affecting their mating type function. Mating type mutants (except a m 33 ), as well as male- and female-sterile mutants, are usually defective in haploid sexual functions or in early peritheical development. Many mutants that affect post-fertilization events produce normal-sized perithecia that are barren. Some mutants show distinct blocks during ascus development from karyogamy through ascospore formation, leading to complete or partial abortion of asci. Other mutants are variable in their expression, with development blocked at different stages from ascus to ascus, and a few asci capable of producing viable ascospores. Another category shows dramatic effects on ascus development without drastically reducing fertility. These mutations affect ascus or ascospore morphology, the size and number of ascospores, or ascospore pigmentation. Spore killer strains show meiotic drive, causing the death of ascospores not carrying the Spore killer allele. Although many applications have been found for specific mutants, as described in the text, their use for analysing the molecular mechanisms that underlie morphogenesis and development remain at best rudimentary. Judging from the recent history of developmental genetics in organisms such as Drosophila , the wide array of developmental mutations described for Neurospora in this review should be a valuable resource for future research into the control of sexual development.

Homothallic Sordariaceae from nature: The absence of strains containing only thea mating type sequence

Abstract An efficient procedure is described for the isolation of heterothallic and homothallic Sordariaceae from soil. New strains obtained by this method were classified to genus on the basis of ascospore morphology and ornamentation. Hetero- and homothallic Neurospora spp. were most often isolated from soils collected in the tropics, whereas Gelasinospora spp. were obtained from both tropical and temperate zone soils. The use of molecular probes on genomic DNA of the isolates showed that all of the new homothallic Neurospora strains contained a sequence similar to the A mating type sequence of N. crassa , but none contained a sequence similar to the a mating type. In contrast, all of the homothallic Gelasinospora strains contained sequences similar to both the A and a mating type sequences of N. crassa . Genomic DNA from type cultures of homothallic species of Gelasinospora ( G. calospora, G. reticulospora , and Gelasinospora S23), Sordaria ( S. fimicola and S. macrospora ), and Anixiella ( A. sublineata ) also contained sequences similar to the N. crassa A and a sequences. Unlike the homothallic Gelasinospora , heterothallic Gelasinospora isolates contained a sequence corresponding to either the A mating type or the a mating type, but not both. Hybridization with additional molecular probes was performed to characterize the new isolates further on the basis of restriction fragment length polymorphisms (RFLPs). Surprisingly, many of the homothallic strains could not be distinguished from one another either morphologically or on the basis of several molecular probes, although they were isolated from distinct geographical regions. This is in contrast to numerous RFLPs detected between heterothallic Gelasinospora and Neurospora isolates.

Functional heterothallism resulting from homokaryotic conidia and ascospores in Neurospora tetrasperma

Each ascospore of Neurospora tetrasperma normally contains haploid nuclei of both mating types, A and a , and produces a self-fertile culture. A majority of cultures from single conidia are also self-fertile, because most conidia are multinucleate and contain nuclei of both mating types. It has long been known that some single-mating type, self-sterile cultures are produced, either from exceptional homokaryotic ascospores or from the minority of conidia that are homokaryotic. The homokaryotic, self-sterile cultures are crossfertile with strains of opposite mating type, and can be fertilized by compatible nuclei from either homokaryotic or heterokaryotic cultures. The present study shows that the spontaneous proportion of self-sterile propagules from nonmutant strains can be as high as 10% for ascospores and 20% for conidia. Thus, although N. tetrasperma is predominantly inbred, the species is nevertheless capable of significant heterothallic behaviour and outcrossing. As shown by B. O. Dodge, the Eight-spore mutation E causes most of the asci to produce five to eight ascospores by affecting spindle behaviour, pairwise association of nuclei, and ascospore delimitation. Cytological events in crosses heterozygous for E have been re-examined and crosses have also been examined that involve two wild strains which superficially resemble E in producing many homokaryotic ascospores by a somewhat different mechanism. Events in the developing wild type and mutant asci are documented photographically for the first time.

Giant ascospores and abnormal croziers in a mutant of Neurospora crassa

In crosses heterozygous for a dominant mutant, Banana ( Ban ), nearly all asci delimit a single large banana-shaped ascospore instead of eight normal spores. Ascus development and its nuclear divisions are normal until after the third division. In the Ban + / Ban ascus, the eight nuclei do not become realigned in single file but remain across the axis, and a single large ascospore wall is delimited enclosing them all. During wall formation, the spindle pole bodies greatly decrease in size. The nuclei then divide once, or rarely twice, so that the Banana ascospores contain 16 or 32 nuclei. After the first 40 to 60 asci per perithecium are formed, the croziers stop making new asci. Instead, the nuclei in most ascogenous cells divide several times mitotically and synchronously; all nuclei in an affected crozier are found at the same stage of mitosis. Some ascogenous cells, however, make very large nuclei, sometimes with more than one nucleolus. The multinucleate and giant-nucleate cells eventually degenerate, and new asci subsequently develop and make Banana ascospores. It has been shown, with the aid of mei-3 which blocks meiosis, that the supernumerary mitoses in the croziers do not depend on the delimitation of Banana ascopores. Ban is linked to mating type and is probably left of leu-3. Ban strains have abnormal vegetative morphology and are female sterile.

A SIMPLE FLUORESCENT STAINING METHOD FOR MEIOTIC CHROMOSOMES OF NEUROSPORA

The DNA-specific fluorochrome acriflavin has been used to stain meiotic chromosomes in Neurospora. The acriflavin method is simple and highly reliable. Acriflavin-stained nuclei show good contrast and excellent resolution for detailed chromosome analysis. The nucleolus organizer region can be seen as an attenuated strand embedded within the unstained nucleolus ghost. Since spindles and spindle pole bodies do not fluoresce at all, the method is useful for determining chromosome numbers during condensed division stages in the ascus.

Neurospora discreta, a new heterothallic species defined by its crossing behavior

Abstract The species is described and named Neurospora discreta sp. nov. because of its stringent reproductive isolation. Isolates collected from burned vegetation at a single site near Kirbyville, Texas, include both mating types (Aanda). Experimental criteria based on cross-fertility were used for assigning species status. Crosses between isolates of opposite mating type are highly fertile, producing abundant eightspored asci. In contrast, when the Kirbyville strains are crossed to sexually compatible speciestester strains representing N. crassa, N. intermedia, N. sitophila , and N. tetrasperma , perithecia are rudimentary and no ascospores are produced. The haploid chromosome number is 7. Chromosomes at pachytene resemble those of other Neurospora species. Biotin is required. Linear growth is slower than for other heterothallic species. When A and a strains from Kirbyville grow toward one another and intersect on crossing medium, there is no barrage. A single homogeneous band of perithecia is formed where they meet, indicating that opposite mating types are vegetatively compatible. The Kirbyville population differs from other heterothallic Neurospora species in ascospore morphology and vegetative traits. Ascospores from Kirbyville parents are larger, and the ribs between confluent parallel grooves are ornamented with dot-like pits. Vegetative cultures from Kirbyville are yellowish rather than orange, and large empty barren protoperithecia or false perithecia are produced abundantly in unfertilized haploid cultures. Isolates from two other N. discreta populations resemble other Neurospora species more closely with respect to these morphological traits but are clearly conspecific with the Kirbyville strains on the basis of fertility in crosses.

Ascomycete Spore killers: Chromosomal elements that distort genetic ratios among the products of meiosis

Spore killers (Sk), studied most extensively in Neurospora, are also known in Podospora, Gibberella and Cochliobolus. Spore killers are no doubt present in natural populations of other fungi. Crite...

Neurospora Spore Killers Sk-2 and Sk-3 Suppress Meiotic Silencing by Unpaired DNA

In Neurospora crassa, pairing of homologous DNA segments is monitored during meiotic prophase I. Any genes not paired with a homolog, as well as any paired homologs of that gene, are silenced during the sexual phase by a mechanism known as meiotic silencing by unpaired DNA (MSUD). Two genes required for MSUD have been described previously: sad-1 (suppressor of ascus dominance), encoding an RNA-directed RNA polymerase, and sad-2, encoding a protein that controls the perinuclear localization of SAD-1. Inactivation of either sad-1 or sad-2 suppresses MSUD. We have now shown that MSUD is also suppressed by either of two Spore killer strains, Sk-2 and Sk-3. These were both known to contain a haplotype segment that behaves as a meiotic drive element in heterozygous crosses of killer × sensitive. Progeny ascospores not carrying the killer element fail to mature and are inviable. Crosses homozygous for either of the killer haplotypes suppress MSUD even though ascospores are not killed. The killer activity maps to the same 30-unit-long region within which recombination is suppressed in killer × sensitive crosses. We suggest that the region contains a suppressor of MSUD.

Meiotic silencing in the homothallic fungus Gibberella zeae

The homothallic ascomycete fungus Gibberella zeae is an important pathogen on major cereal crops. The objective of this study was to determine whether meiotic silencing occurs in G. zeae. Cytological studies demonstrated that GFP and RFP-fusion proteins were not detected during meiosis, both in heterozygous outcrosses and homozygous selfings. The deletion of rsp-1, a homologue used for studies on meiotic silencing of Neurospora crassa, triggered abnormal ascospores from selfing, but outcrosses between the mutant and wild-type strain resulted in some ascospores with mutant phenotype (low occurrence of ascus dominance). When the ectopic mutants that carried an additional copy of rsp-1 were selfed, they primarily produced ascospores with normal shape but a few ascospores (0.23 %) were abnormal, in which both endogenous and ectopically integrated genes contained numerous point mutations. The ectopic mutants showed low occurrence of ascus dominance in outcrosses with strains that carried the wild-type allele. Approximately 10 % of ascospores were abnormal but all of the single-ascospore isolates produced normal-shaped ascospores from selfing. However, no ascus dominance was observed when the mutants were outcrossed with a sad-1 deletion mutant, which lacks the putative RNA-dependent RNA polymerase essential for meiotic silencing in N. crassa. All results were consistent with those generated from an additional gene, roa, required for ascospore morphogenesis. This study demonstrated that G. zeae possesses a functional meiotic silencing mechanism which is triggered by unpaired DNA, as in N. crassa.