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.

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A gene unpaired during the meiotic homolog pairing stage in Neurospora generates a sequence-specific signal that silences the expression of all copies of that gene. This process is called Meiotic Silencing by Unpaired DNA (MSUD). Previously, we have shown that SAD-1, an RNA-directed RNA polymerase (RdRP), is required for MSUD. We isolated a second gene involved in this process, sad-2. Mutated Sad-2 RIP alleles, like those of Sad-1, are dominant and suppress MSUD. Crosses homozygous for Sad-2 are blocked at meiotic prophase. SAD-2 colocalizes with SAD-1 in the perinuclear region, where small interfering RNAs have been shown to reside in mammalian cells. A functional sad-2 ؉ gene is necessary for SAD-1 localization, but the converse is not true. The data suggest that SAD-2 may function to recruit SAD-1 to the perinuclear region, and that the proper localization of SAD-1 is important for its activity.epigenetics ͉ meiosis ͉ MSUD ͉ Neurospora ͉ RNA interference C oenocytic organisms, in which nuclei coexist in a common cytoplasm, are probably at especially high risk from proliferation of detrimental retrotransposons. In the haploid ascomycete Neurospora crassa, several gene-silencing mechanisms exist to maintain its genome integrity. Quelling, which defends the organism during the vegetative phase, is an RNA interference (RNAi) system that suppresses the expression of transgenes occurring in more than one copy (1, 2). Another surveillance system, known as repeat-induced point mutation (RIP), is a premeiotic process that scans the genome for duplicated sequences and targets them for C to T mutations (3). A third silencing mechanism, named meiotic silencing by unpaired DNA, scans the genome and monitors the pairing of DNA segments with their homologs during meiotic prophase (4-6). This mechanism probably prevents the expression and transposition of invasive sequences, and serves the organism in its need to counter exogenous elements and perhaps to regulate endogenous elements. Deletion or extensive mutation in an RdRPencoding gene, sad-1 (suppressor of ascus dominance), reduces meiotic silencing to a low level. RdRP plays an important role in some RNAi systems (2). For example, if foreign nucleic acids trigger the production of aberrant RNA (aRNA), the singlestranded aRNA can be replicated into a double-stranded species (dsRNA) via the activity of an RdRP. The dsRNA is then processed into small interfering RNA (siRNA) duplexes by Dicer. The siRNAs subsequently guide the cleavage of mRNA via the RNA-induced silencing complex. The fact that an RdRP is required for meiotic silencing suggests that the synthesis of dsRNA, its amplification, or both, are essential for the process.We have now identified an additional gene, sad-2, which is also required for meiotic silencing. Dominant mutations (Sad-2) can suppress the meiotic silencing of unpaired loci with efficiency comparable to that of Sad-1. A Sad-2 mutation does not give any obvious abnormal phenotype during vegetative growth and, correspondingly, sad-2 ϩ mRNA can only...

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Meiosis and ascospore development in the four-spored pseudohomothallic ascomycetes Neurospora tetrasperma, Gelasinospora tetrasperma, Podospora anserina, and P. tetraspora have been reexamined, highlighting differences that reflect independent origins of the four-spored condition in the different genera. In these species, as in the heterothallic eight-spored N. crassa, fusion of haploid nuclei is followed directly by meiosis and a postmeiotic mitosis. These divisions take place within a single unpartitioned giant cell, the ascus, which attains a length of > 0.1 mm before nuclei are enclosed by ascospore walls. Two basically different modes underlie the delivery of opposite mating type nuclei into each of the four ascospores in the different genera. In N. tetrasperma on the one hand, the mating type locus is closely centromere-linked. Mating types therefore segregate at the first meiotic division. The second-division spindles of N. tetrasperma overlap and are usually parallel to one another, in contrast to the their tandem arrangement in N. crassa. As a result, nonsister nuclei of opposite mating type are placed close together in each half-ascus and a pair is enclosed in each ascospore. In the Podospora and Gelasinospora species on the other hand, the second-division spindles are in tandem, with sister nuclei of opposite mating type associated as a pair in each half-ascus. It is established for P. anserina and inferred for P. tetraspora and G. tetrasperma that a single reciprocal crossing over almost always occurs in the mating type-centromere interval, ensuring that mating types segregate at the second meiotic division and that nuclei of opposite mating type are enclosed in each ascospore. Other differences are also seen that are less fundamental. Neurospora tetrasperma differs from the other species in the orientation of chromosomes and spindle pole body plaques at interphase II. Third-division spindles are oriented parallel to the ascus wall in Gelasinospora but across the ascus in Podospora and Neurospora. The two Podospora species differ from one another in nuclear behavior following mitosis in the young ascospores. In P. tetraspora, two of the four nuclei migrate into the tail cell, which degenerates, leaving one functional nucleus of each mating type. In P. anserina, by contrast, only one of the four nuclei moves into the tail cell, leaving the germinating ascospore with two functional nuclei of one mating type and one of the other. The pseudohomothallic condition with its heterokaryotic vegetative phase has significant consequences for both the individual organism and the breeding system. Genetic controls of development and recombination are complex.(ABSTRACT TRUNCATED AT 400 WORDS)