Geographic distribution of neurospora spore killer strains and strains resistant to killing

Spore killer strains, found in Neurospora, provided the first recognized example of meiotic drive in fungi. In the present study, natural populations throughout the world were examined for the presence of killer strains and strains that are resistant to killing. In N. intermedia, Sk-2 and Sk-3 are present but are rare. Killer strains were found at only five sites, in Borneo, Java, and Papua New Guinea. Nonkiller strains that are resistant to killing by Sk-2 or Sk-3 are frequent in that part of the world where the killer strains are present, but resistant stains were not found in regions where killers are absent. In N. sitophila, Sk-1 killer strains are common in nature, but only 1 of 392 nonkiller strains was resistant. In N. crassa, no killer strain was found among >500, but widely scattered Sk-2-resistant strains were present, suggesting the past or present existence of killers.     

Spore killing in the fungus Podospora anserina: a connection between meiotic drive and vegetative incompatibility?

Fungi in which the haploid nuclei resulting from meiosis are linearly arranged in asci provide unique opportunities to analyse abnormal segregation. Any meiotic drive system in such fungi will be observed in a cross between a driving and a sensitive strain as spore killing: the degeneration of half the ascospores in a certain proportion of the asci. In a sample of some 100 strains isolated from a single natural population we have discovered at least six different meiotic drive elements (van der Gaag et al., 2000). Here we report results of research that was aimed at elucidating a possible correlation between meiotic drive and vegetative incompatibility in eight different Spore killer strains from this population. We show that there is a strong correlation between these two phenotypes, although the precise genetic nature of the correlation is not yet clear. We discuss the implications of our results for the understanding of the population genetics of meiotic drive in Podospora.     

Evolutionary Dynamics of Spore Killers

Spore killing in ascomycetes is a special form of segregation distortion. When a strain with the Killer genotype is crossed to a Sensitive type, spore killing is expressed by asci with only half the number of ascospores as usual, all surviving ascospores being of the Killer type. Using population genetic modeling, this paper explores conditions for invasion of Spore killers and for polymorphism of Killers, Sensitives and Resistants (which neither kill, nor get killed), as found in natural populations. The models show that a population with only Killers and Sensitives can never be stable. The invasion of Killers and stable polymorphism only occur if Killers have some additional advantage during the process of spore killing. This may be due to the effects of local sib competition or some kind of ``heterozygous' advantage in the stage of ascospore formation or in the short diploid stage of the life cycle. This form of segregation distortion appears to be essentially different from other, well-investigated forms, and more field data are needed for a better understanding of spore killing.     

Expression of Meiotic Drive Elements Spore Killer-2 and Spore Killer-3 in Asci of Neurospora Tetrasperma

It was shown previously that when a chromosomal Spore killer factor is heterozygous in Neurospora species with eight-spored asci, the four sensitive ascospores in each ascus die and the four survivors are all killers. Sk-2K and Sk-3K are nonrecombining haplotypes that segregate with the centromere of linkage group III. No killing occurs when either one of these killers is homozygous, but each is sensitive to killing by the other in crosses of Sk-2K x Sk-3K. In the present study, Sk-2K and Sk-3K were transferred by recurrent backcrosses from the eight-spored species Neurospora crassa into Neurospora tetrasperma, a pseudohomothallic species which normally makes asci with four large spores, each heterokaryotic for mating type and for any other centromere-linked genes that are heterozygous in the cross. The action of Sk-2K and Sk-3K in N. tetrasperma is that predicted from their behavior in eight-spored species. A sensitive nucleus is protected from killing if it is enclosed in the same ascospore with a killer nucleus. Crosses of Sk-2K x Sk-2S, Sk-3K x Sk-3S, and Sk-sK x Sk-3K all produce four-spored asci that are wild type in appearance, with the ascospores heterokaryotic and viable. The Eight-spore gene E, which shows variable penetrance, was used to obtain N. tetrasperma asci in which two to eight spores are small and homokaryotic. When killer and sensitive alleles are segregating in the presence of E, only those ascospores that contain a killer allele survive. Half of the small ascospores are killed. In crosses of Sk-2K x Sk-3K (with E heterozygous), effectively all small ascospores are killed. The ability of N. tetrasperma to carry killer elements in cryptic condition suggests a possible role for Spore killers in the origin of pseudohomothallism, with adoption of the four-spored mode restoring ascospore viability of crosses in which killing would otherwise occur.     

Recombination block in the Spore killer region of Neurospora

Spore killers Sk-2K and Sk-3K are chromosomal meiotic drive factors in Neurospora. In heterozygous crosses, ascospores not containing the Spore killer die. Sk-2K and Sk-3K, which differ in killing specificity, were found to be associated with suppression of recombination in a centromere-spanning region of linkage group III, and investigation of that recombination block is reported here. The block covers a region that is normally 30 to 40 map units long. A locus (r(Sk-2)) conferring resistance to Sk-2K maps to the left end of the recombination block. Recombination is normal in r(Sk-2) X Sk sensitive but blocked in Sk-2K X r(Sk-2); so the block does not depend upon killing. By selective plating, SkK stocks carrying genetic markers within the block were obtained at frequencies on the order of 10(-5) or 10(-6). Since this tight block is far beyond what has been observed for genetic reduction of recombination, a structural basis is assumed. No evidence of chromosome rearrangement was obtained. Crosses homozygous for Sk-2K show normal crossing-over and map order for the flanking markers cum and his-7 and three included markers (acr-7, acr-2, and leu-1). Results would be consistent with a divergence of sequence great enough to interfere with homologous pairing.     

Genetic analysis of spore killing in the filamentous ascomycete Podospora anserina

In the present study, we analysed different Podospora anserina strains for their ability to induce spore killing and identified three new killer strains. Test crosses of killer strains with different sensitive strains revealed different second division segregation ratios suggesting an influence of the sensitive strain on the crossing-over frequency. In crosses of killer strain O with a sensitive strain, the frequency of two-spored asci was found to vary extremely from perithecium to perithecium. Furthermore, crosses of strain O with sensitive strain Us5 led to a significant proportion of asci containing an unexpected high number of surviving spores as the result of gene conversion. Finally, for the first time, we present data demonstrating that in a number of ascospores the killer and the corresponding sensitive allele is located in one individual nucleus. Mycelia derived from such ascospores display a "sensitive killer" phenotype. Crosses of these mycelia with a killer strain as well as with a sensitive strain result in spore killing. Strikingly, heterokaryotic spores containing the recombined "sensitive/killer" allele and a nucleus with a killer allele give rise to mycelia protected against spore killing during selfing.     

Genes That Bias Mendelian Segregation

Mendel laws of inheritance can be cheated by Meiotic Drive Elements (MDs), complex nuclear genetic loci found in various eukaryotic genomes and distorting segregation in their favor. Here, we identify and characterize in the model fungus Podospora anserina Spok1 and Spok2, two MDs known as Spore Killers. We show that they are related genes with both spore-killing distorter and spore-protecting responder activities carried out by the same allele. These alleles act as autonomous elements, exert their effects independently of their location in the genome and can act as MDs in other fungi. Additionally, Spok1 acts as a resistance factor to Spok2 killing. Genetical data and cytological analysis of Spok1 and Spok2 localization during the killing process suggest a complex mode of action for Spok proteins. Spok1 and Spok2 belong to a multigene family prevalent in the genomes of many ascomycetes. As they have no obvious cellular role, Spok1 and Spok2 Spore Killer genes represent a novel kind of selfish genetic elements prevalent in fungal genome that proliferate through meiotic distortion.     

Meiotic drive in fungi: Chromosomal elements that cause fratricide and distort genetic ratios

Fungal Spore killers (Sk), studied most extensively inNeurospora and to a lesser extent inPodospora, Gibberella andCochliobolus, cause the death of ascospores (= meiospores) that do not contain the killer (Skk) element. When a Spore killer is heterozygous (SkK× Sks) inNeurospora, every ascus (= meiocyte) contains four normal-sized, black, viable ascospores (SkK), and four ascospores that are tiny, unpigmented and unviable (SKs). Killing of sensitive nuclei is expressed postmeiotically, and results in gross distortion of segregation ratios forSk-linked genes. A sensitive nucleus that would otherwise die is rescued if a killer nucleus is also enclosed in the same ascospore. InNeurospora, Sk is centromere-linked (linkage group III), and when heterozygous, shows a recombination block in a 30-map-unit region spanning the centromere of linkage group III. There is no ascospore death or recombination block in killer×killer or sensitive×sensitive crosses. Spore killers are fairly common inGibberella fujikuroi andNeurospora sitophila but extremely rare inN. intermedia, and have not yet been found among natural isolates ofN. crassa.     

Analysis of two additional loci in Neurospora crassa related to Spore killer-2

Two new loci found in one strain of Neurospora crassa (P2604) collected in Malaya are related to the meiotic drive system Spore killer Sk-2. Sk-2 was found in Neurospora intermedia and introgressed into N. crassa. P2604 showed high resistance to killing when crossed to Sk-2. This resistance was found to be linked to, but not allelic to, resistance locus r(Sk-2) on LGIIIL. Analysis showed that the high resistance phenotype of P2604 requires resistance alleles at two different loci on LGIIIR. Strains carrying a resistance allele at only the proximal or the distal locus, respectively, were obtained and intercrossed. Highly resistant strains were obtained by rejoining the two genes. The proximal locus alone confers a low level of resistance. This locus was named pr(Sk-2) for partial resistance to Sk-2. The distal locus was named mod(pr) because its only known phenotype is to modify pr(Sk-2).     

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 x 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 x sensitive crosses. We suggest that the region contains a suppressor of MSUD.     

A Critical Component of Meiotic Drive in Neurospora Is Located Near a Chromosome Rearrangement

Neurospora fungi harbor a group of meiotic drive elements known as Spore killers (Sk). Spore killer-2 (Sk-2) and Spore killer-3 (Sk-3) are two Sk elements that map to a region of suppressed recombination. Although this recombination block is limited to crosses between Sk and Sk-sensitive (Sk^S) strains, its existence has hindered Sk characterization. Here we report the circumvention of this obstacle by combining a classical genetic screen with next-generation sequencing technology and three-point crossing assays. This approach has allowed us to identify a novel locus called rfk-1, mutation of which disrupts spore killing by Sk-2. We have mapped rfk-1 to a 45-kb region near the right border of the Sk-2 element, a location that also harbors an 11-kb insertion (Sk-2^INS1) and part of a >220-kb inversion (Sk-2^INV1). These are the first two chromosome rearrangements to be formally identified in a Neurospora Sk element, providing evidence that they are at least partially responsible for Sk-based recombination suppression. Additionally, the proximity of these chromosome rearrangements to rfk-1 (a critical component of the spore-killing mechanism) suggests that they have played a key role in the evolution of meiotic drive in Neurospora.     

Use of Neurospora spore killer strains to obtain centromere linkage data without dissecting asci

Use of a centromere-linked Spore killer gene Sk reduces manyfold the labor involved in obtaining tetrad data that would otherwise require ordered dissection of intact linear eight-spored asci. Heterozygous crosses are made for Spore killer (SkK X SkS) and for markers to be tested. In such crosses only SkK ascospores survive. The four viable (SkK) and four aborted (SkS) ascospores of each ascus are ejected from the perithecium as a physically disordered group. The four surviving SkK ascospores of individual asci are germinated and scored. SkK segregates from SkS at the first meiotic division. If both marker alleles are represented in the surviving products, they must therefore have segregated from one another at the second division. Four-spore (Fsp) genes have been used to eliminate one postmeiotic nuclear division, so that only two ascospores per ascus need to be scored. The Spore killer method has been useful for mapping closely linked genes in centromere regions, for identifying genes that are far out on chromosome arms, for obtaining information on meiotic crossing-over, and for comparing linkages in different species.     

Space and the persistence of male-killing endosymbionts in insect populations

Male-killing bacteria are bacteria that are transmitted vertically through the females of their insect hosts. They can distort the sex ratio of their hosts by killing infected male offspring. In nature, male-killing endosymbionts (male killers) often have a 100% efficient vertical transmission, and multiple male-killing bacteria infecting a single population are observed. We use different model formalisms to study these observations. In mean-field models a male killer with perfect transmission drives the host population to extinction, and coexistence between multiple male killers within one population is impossible; however, in spatially explicit models, both phenomena are readily observed. We show how the spatial pattern formation underlies these results. In the case of high transmission efficiencies, waves with a high density of male killers alternate with waves of mainly wild-type hosts. The male killers cause local extinction, but this creates an opportunity for uninfected hosts to re-invade these areas. Spatial pattern formation also creates an opportunity for two male killers to coexist within one population: different strains create spatial regions that are qualitatively different; these areas then serve as different niches, making coexistence possible.     

Non-mendelian inheritance of the HET-s prion or HET-s prion domains determines the het-S spore killing system in Podospora anserina

Two alleles of the het-s/S locus occur naturally in the filamentous fungus Podospora anserina, het-s and het-S. The het-s encoded protein can form a prion that propagates a self-perpetuating amyloid aggregate, resulting in two phenotypes for the het-s strains. The prion-infected [Het-s] shows an antagonistic interaction to het-S whereas the prion-free [Het-s*] is neutral in interaction to het-S. The antagonism between [Het-s] and het-S is seen as heterokaryon incompatibility at the somatic level and as het-S spore killing in the sexual cycle. Two different domains of the HET-s and HET-S proteins have been identified, and a structure-function relationship has been established for interactions at the somatic level. In this study, we correlate accumulation of the HET-s and HET-S proteins (visualized using GFP) during the sexual cycle with timing of het-S spore abortion. Also, we present the structure-function relationship of the HET-s domains for interactions in the sexual cycle. We show that the constructs that ensure het-s incompatibility function in somatic mycelium are also active in het-S spore killing in the sexual cycle. In addition, paternal prion transmission and het-S spore killing has been found with the HET-s(157-289) truncated protein. The consequences of the unique transition from a coenocytic to a cellular state in the sexual phase and the timing, and localization of paternal and maternal HET-s and HET-S expression that are pertinent to prion transmission, and het-S spore killing are elaborated. These data further support our previously proposed model for het-S spore killing.     

A Study of the Transmission and Structure of Double Stranded Rnas Associated with the Killer Phenomenon in SACCHAROMYCES CEREVISIAE

Killer strains contain two double stranded RNAs, L and M. The M dsRNA appears to be necessary for production of a toxin and for resistance to that toxin. Mutant strains have been found that are defective in their ability to kill and in their resistance to toxin. These sensitive, non-killer strains have altered dsRNA composition. One class has no M dsRNA. Another class of sensitive, non-killers called suppressives has no M dsRNA but instead has smaller dsRNAs called S. In diploids resulting from a cross of a wild-type killer by a suppressive the transmission of the M dsRNA is suppressed by the S dsRNA. When a suppressive is crossed by a strain with no M dsRNA, the diploids and all four meiotic spores have the S dsRNA characteristic of the parental suppressive strain. Suppressive strains do not suppress each other. Intercrosses between two different suppressives yields diploids with both parental S dsRNAs. These two S dsRNAs are transmitted to all 4 meiotic progeny. Another class of mutants has been found which is defective for one of the traits but retains the other. One type, temperature-sensitive killers, has a normal dsRNA composition but is unable to kill at 30°. The other type, immunity-minus, has a complex dsRNA pattern. The immunity-minus strain is extremely unstable during mitotic growth and segregates several different types of non-killers. Analysis of the dsRNAs from wild type and the mutants by electron microscopy shows that the L, M, and S dsRNAs are linear. All strains regardless of killer phenotype appear to have the same size L dsRNA.     

Morphological variation in the sugarcane smut Ustilago scitaminea Syd

Summary A study of the morphological variability among 25 different collections of sugarcane smut fungusUstilago scitaminea from the States of Delhi, Uttar Pradesh, Bihar, Madhya Pradesh, Maharastra, Andhra Pradesh and Madras revealed that considerable variation exists in spore size, colour, spore-wall markings of the isolates. On the basis of statistically significant differences in the spore size, the 25 isolates can be classified into 4 distinct groups. Three types of spore-wall markings viz., verrucose, punctate and echinulate are met within the isolates studied. There is no appreciable difference in the spore wall thickness of the different isolates. Spore colour of the different isolates varied from yellow to brown with several shades in between.Part of a thesis submitted by the senior author in partial fulfilment of the Degree of Doctor of Philosophy, P. G. School, Indian Agricultural Research Institute, New Delhi.     

Cloned "anomalous" killer cells derived from allogeneic mixed leukocyte culture

In addition to allospecific cytotoxic lymphocytes, cytolytic effector cells capable of killing a broad range of targets are generated during mixed leukocyte culture (MLC). These cells, which have been previously called anomalous killer cells, are a distinct functional subset separate from natural killer cells or allospecific cytotoxic lymphocytes but display many characteristics of lymphokine-activated killers. In order to isolate anomalous killer cells for detailed analysis, we generated the cytolytic effectors from an allogeneic MLC using heat-inactivated stimulators. This treatment of the stimulator population abrogated the generation of classical allospecific cytotoxic lymphocytes but allowed the generation of anomalous killer cells which were subsequently cloned via limiting dilution. The clones derived by this method displayed the functional properties of anomalous killers seen in bulk MLCs. The clones demonstrated potent cytolytic activity against both NK-sensitive and NK-resistant tumor targets in vitro and also suppressed tumor growth in vivo. Ultrastructural studies revealed features similar to those of cloned antigen-specific cytolytic cells and clones with NK-like function. The cells expressed surface glycoproteins associated with both NK and T lymphocytes including Thy-1, Ly-2, T200, Qa-5, asialo GM1, and the antigens defined by the NK alloantisera NK-2.1 and NK-3.1. These cells may play an important role during early phases of the immune response, since cytolytic cells of broad specificity may protect the host until classical cytotoxic lymphocytes with restricted specificity are generated.     

Germination and infectivity of ectomycorrhizal fungal spores in relation to their ecological traits during primary succession

The spores of ectomycorrhizal fungi (EMF) play critical roles in the population and community development of EMF. Here, the germination and infectivity of EMF spores are examined with reference to the ecological traits of the EMF species. Spores were collected from 12 EMF species, whose successional patterns have been studied in the volcanic desert on Mount Fuji, Japan. Spore germination experiments were conducted with host plants (Salix reinii), with nonhost plants (Polygonum cuspidatum), and without plants. The mycorrhizal formation ability of spores was also examined in seven EMF using spore inoculation experiments. To determine the effects of the spore preservation period, both experiments were repeated up to 1 yr after spore collection. Spore germination was very low in the absence of host plants. In the presence of hosts, even 30 d after spore collection, spore germination was significantly enhanced in all pioneer EMF (c. 20%) but less so in late-stage EMF (< 5%), except in Hebeloma species. Mycorrhizal formation from spores was also greater in pioneer EMF but was significantly reduced by 1 yr of spore preservation. High spore germination and infectivity of pioneer EMF should enable these species to colonize disturbed and isolated areas in accordance with their ecological traits.     

A seven-day volumetric spore trap for use within buildings

The Burkard Volumetric spore trap, designed to operate for seven days continuously in the field, was modified to sample still air within buildings. The efficiency with which spores of Lycopodium clavatum and Agaricus bisporus were trapped at two rates of suction was determined. Spore distribution within traces and deposition on surfaces not beneath the orifice were assessed. In an appendix catches of four spore types by the Hirst and Burkard (field model) spore traps operating over mown grass were compared.