Spore Killer Research Articles

Characterization of mating type, spore killing, and pathogenicity of Fusarium verticillioides populations from maize in China

Sexual reproduction in fungi promotes genetic diversity and helps the fungus to adapt to environmental stresses. Fusarium verticillioides is a heterothallic filamentous ascomycete that is a major cause of maize ear and stalk rot worldwide, while also posing a threat to human and animal health by producing various mycotoxins. Sexual reproduction in F. verticillioides is controlled by the MAT-1 and MAT-2 loci, which mandate that only strains of opposite mating types can mate to yield perithecia and ascospores. Nevertheless, there exists a phenomenon called ‘spore killing’, in which only four typical ascospores appear in the asci following a cross between a strain carrying the spore killer allele (SkK) and one with the spore killer sensitive allele (SkS). In this study, 31 isolates of F. verticillioides collected from eight provinces in China during the maize growing season from 2014 to 2020 were compared based on their mating type, spore killing genotype, and pathogenicity. To determine the mating types and spore killing genotypes of these isolates, partial sequences were amplified from the MAT loci and the SKC1 gene, respectively. The PCR results showed that out of the 31 isolates, 18 were MAT-1 and 13 were MAT-2, and that 25 had SkK genotypes and 6 had SkS genotypes. Genetic crosses between LNF15-11 (MAT-2) and 18 MAT-1 isolates produced normal perithecia with varying numbers. However, crosses between LNF15-11 and the 3 isolates (SDF18-36, HNF14-8, and GSF19-6) produced only four ascospores per ascus, while the remaining isolates except SDF18-28, yielded eight ascospores per ascus. These findings suggest that the SKC1 amplicon variation can be used to differentiate SkK and SkS genotypes in the field and that the 3 isolates are truly SkS genotypes with the MAT-1 allele. Altogether, this study contributes to our knowledge of the mating type and spore killing genotype of F. verticillioides in China and offers valuable strain resources for investigating heterothallic sexual reproduction.

A Natural Fungal Gene Drive Enacts Killing via DNA Disruption.

Fungal spore killers are a class of selfish genetic elements that positively bias their own inheritance by killing non-inheriting gametes following meiosis. As killing takes place specifically within the developing fungal ascus, a tissue which is experimentally difficult to isolate, our understanding of the mechanisms underlying spore killers are limited. In particular, how these loci kill other spores within the fungal ascus is largely unknown. Here, we overcome these experimental barriers by developing model systems in 2 evolutionary distant organisms, Escherichia coli (bacterium) and Saccharomyces cerevisiae (yeast), similar to previous approaches taken to examine the wtf spore killers. Using these systems, we show that the Podospora anserina spore killer protein SPOK1 enacts killing through targeting DNA. IMPORTANCE Natural gene drives have shaped the genomes of many eukaryotes and recently have been considered for applications to control undesirable species. In fungi, these loci are called spore killers. Despite their importance in evolutionary processes and possible applications, our understanding of how they enact killing is limited. We show that the spore killer protein Spok1, which has homologues throughout the fungal tree of life, acts via DNA disruption. Spok1 is only the second spore killer locus in which the cellular target of killing has been identified and is the first known to target DNA. We also show that the DNA disrupting activity of Spok1 is functional in both bacteria and yeast suggesting a highly conserved mode of action.

On the Mechanistic Basis of Killer Meiotic Drive in Fungi.

Spore killers are specific genetic elements in fungi that kill sexual spores that do not contain them. A range of studies in the last few years have provided the long-awaited first insights into the molecular mechanistic aspects of spore killing in different fungal models, including both yeast-forming and filamentous Ascomycota. Here we describe these recent advances, focusing on the wtf system in the fission yeast Schizosaccharomyces pombe; the Sk spore killers of Neurospora species; and two spore-killer systems in Podospora anserina, Spok and [Het-s]. The spore killers appear thus far mechanistically unrelated. They can involve large genomic rearrangements but most often rely on the action of just a single gene. Data gathered so far show that the protein domains involved in the killing and resistance processes differ among the systems and are not homologous. The emerging picture sketched by these studies is thus one of great mechanistic and evolutionary diversity of elements that cheat during meiosis and are thereby preferentially inherited over sexual generations.

A-to-I mRNA editing controls spore death induced by a fungal meiotic drive gene in homologous and heterologous expression systems.

Spore killers are meiotic drive elements that can block the development of sexual spores in fungi. In the maize ear rot and mycotoxin-producing fungus Fusarium verticillioides, a spore killer called SkK has been mapped to a 102-kb interval of chromosome V. Here, we show that a gene within this interval, SKC1, is required for SkK-mediated spore killing and meiotic drive. We also demonstrate that SKC1 is associated with at least 4 transcripts, 2 sense (sense-SKC1a and sense-SKC1b) and 2 antisense (antisense-SKC1a and antisense-SKC1b). Both antisense SKC1 transcripts lack obvious protein-coding sequences and thus appear to be noncoding RNAs. In contrast, sense-SKC1a is a protein-coding transcript that undergoes A-to-I editing to sense-SKC1b in sexual tissue. Translation of sense-SKC1a produces a 70-amino-acid protein (Skc1a), whereas the translation of sense-SKC1b produces an 84-amino-acid protein (Skc1b). Heterologous expression analysis of SKC1 transcripts shows that sense-SKC1a also undergoes A-to-I editing to sense-SKC1b during the Neurospora crassa sexual cycle. Site-directed mutagenesis studies indicate that Skc1b is responsible for spore killing in Fusarium verticillioides and that it induces most meiotic cells to die in Neurospora crassa. Finally, we report that SKC1 homologs are present in over 20 Fusarium species. Overall, our results demonstrate that fungal meiotic drive elements like SKC1 can influence the outcome of meiosis by hijacking a cell's A-to-I editing machinery and that the involvement of A-to-I editing in a fungal meiotic drive system does not preclude its horizontal transfer to a distantly related species.

Molecular Mechanisms and Evolutionary Consequences of Spore Killers in Ascomycetes.

In this review, we examine the fungal spore killers. These are meiotic drive elements that cheat during sexual reproduction to increase their transmission into the next generation. Spore killing has been detected in a number of ascomycete genera, including Podospora, Neurospora, Schizosaccharomyces, Bipolaris, and Fusarium. There have been major recent advances in spore killer research that have increased our understanding of the molecular identity, function, and evolutionary history of the known killers. The spore killers vary in the mechanism by which they kill and are divided into killer-target and poison-antidote drivers. In killer-target systems, the drive locus encodes an element that can be described as a killer, while the target is an allele found tightly linked to the drive locus but on the nondriving haplotype. The poison-antidote drive systems encode both a poison and an antidote element within the drive locus. The key to drive in this system is the restricted distribution of the antidote: only the spores that inherit the drive locus receive the antidote and are rescued from the toxicity of the poison. Spore killers also vary in their genome architecture and can consist of a single gene or multiple linked genes. Due to their ability to distort meiosis, spore killers gain a selective advantage at the gene level that allows them to increase in frequency in a population over time, even if they reduce host fitness, and they may have significant impact on genome architecture and macroevolutionary processes such as speciation.

An introgressed gene causes meiotic drive in Neurospora sitophila

Meiotic drive elements cause their own preferential transmission following meiosis. In fungi, this phenomenon takes the shape of spore killing, and in the filamentous ascomycete Neurospora sitophila, the Sk-1 spore killer element is found in many natural populations. In this study, we identify the gene responsible for spore killing in Sk-1 by generating both long- and short-read genomic data and by using these data to perform a genome-wide association test. We name this gene Spk-1 Through molecular dissection, we show that a single 405-nt-long open reading frame generates a product that both acts as a poison capable of killing sibling spores and as an antidote that rescues spores that produce it. By phylogenetic analysis, we demonstrate that the gene has likely been introgressed from the closely related species Neurospora hispaniola, and we identify three subclades of N. sitophila, one where Sk-1 is fixed, another where Sk-1 is absent, and a third where both killer and sensitive strain are found. Finally, we show that spore killing can be suppressed through an RNA interference-based genome defense pathway known as meiotic silencing by unpaired DNA. Spk-1 is not related to other known meiotic drive genes, and similar sequences are only found within Neurospora These results shed light on the diversity of genes capable of causing meiotic drive, their origin and evolution, and their interaction with the host genome.

Meiotic drive

In this Quick Guide, Srinivasa and Zanders provide an overview of meiotic drivers and the diverse mechanisms these genetic elements use to bias their transmission to the next generation.

Combinations of Spok genes create multiple meiotic drivers in Podospora.

Meiotic drive is the preferential transmission of a particular allele during sexual reproduction. The phenomenon is observed as spore killing in multiple fungi. In natural populations of Podospora anserina, seven spore killer types (Psks) have been identified through classical genetic analyses. Here we show that the Spok gene family underlies the Psks. The combination of Spok genes at different chromosomal locations defines the spore killer types and creates a killing hierarchy within a population. We identify two novel Spok homologs located within a large (74-167 kbp) region (the Spok block) that resides in different chromosomal locations in different strains. We confirm that the SPOK protein performs both killing and resistance functions and show that these activities are dependent on distinct domains, a predicted nuclease and kinase domain. Genomic and phylogenetic analyses across ascomycetes suggest that the Spok genes disperse through cross-species transfer, and evolve by duplication and diversification within lineages.

A large gene family in fission yeast encodes spore killers that subvert Mendel's law.

Spore killers in fungi are selfish genetic elements that distort Mendelian segregation in their favor. It remains unclear how many species harbor them and how diverse their mechanisms are. Here, we discover two spore killers from a natural isolate of the fission yeast Schizosaccharomyces pombe. Both killers belong to the previously uncharacterized wtf gene family with 25 members in the reference genome. These two killers act in strain-background-independent and genome-location-independent manners to perturb the maturation of spores not inheriting them. Spores carrying one killer are protected from its killing effect but not that of the other killer. The killing and protecting activities can be uncoupled by mutation. The numbers and sequences of wtf genes vary considerably between S. pombe isolates, indicating rapid divergence. We propose that wtf genes contribute to the extensive intraspecific reproductive isolation in S. pombe, and represent ideal models for understanding how segregation-distorting elements act and evolve.

A Meiotic Drive Element in the Maize Pathogen Fusarium verticillioides Is Located Within a 102 kb Region of Chromosome V.

Fusarium verticillioides is an agriculturally important fungus because of its association with maize and its propensity to contaminate grain with toxic compounds. Some isolates of the fungus harbor a meiotic drive element known as Spore killer (SkK) that causes nearly all surviving meiotic progeny from an SkK × Spore killer-susceptible (SkS) cross to inherit the SkK allele. SkK has been mapped to chromosome V but the genetic element responsible for meiotic drive has yet to be identified. In this study, we used cleaved amplified polymorphic sequence markers to genotype individual progeny from an SkK × SkS mapping population. We also sequenced the genomes of three progeny from the mapping population to determine their single nucleotide polymorphisms. These techniques allowed us to refine the location of SkK to a contiguous 102 kb interval of chromosome V, herein referred to as the Sk region. Relative to SkS genotypes, SkK genotypes have one extra gene within this region for a total of 42 genes. The additional gene in SkK genotypes, herein named SKC1 for Spore Killer Candidate 1, is the most highly expressed gene from the Sk region during early stages of sexual development. The Sk region also has three hyper-variable regions, the longest of which includes SKC1. The possibility that SKC1, or another gene from the Sk region, is an essential component of meiotic drive and spore killing is discussed.

Mating type and spore killing characterization of Fusarium verticillioides strains

Fusarium verticillioides is a heterothallic ascomycete causing maize ear rot, and produces fumonisin mycotoxins harmful to livestock and human health. A meiotic drive phenomenon called spore killing has been reported in several filamentous fungi including F. verticillioides. F. verticillioides reference genome strain M-3125 (FGSC 7600) is spore killer sensitive (SK S ), and genetic crosses of M-3125 with a strain carrying SK K (the killer element) result in only four viable ascospores per ascus instead of the normal eight ascospores. We sought to identify a strain of F. verticillioides that is SK S and MAT1-2 for use in genetic analysis with M-3125. To accomplish this, we screened 50 F. verticillioides strains from the Fusarium Research Center at The Pennsylvania State University, USA for spore killing. To characterize the mating types of these strains, portions of the MAT locus idiomorphs were amplified using polymerase chain reaction, and genetic crosses were performed. The PCR amplification results show that 18 of the 50 strains are MAT1-2 and 32 are MAT1-1. Genetic crosses between M-3125 and 11 of the 18 MAT1-2 strains produced normal perithecia. Crosses between two (M-8024 and M-7815) of the 11 strains and M-3125 produced perithecia with eight ascospores per ascus, and nine others had only four ascospores per ascus, suggesting that M-8024 and M-7815 are SK S and the other nine are SK K . This study expands our knowledge of mating type and spore killing in F. verticillioides and identifies two SK S , MAT1-2 strains for use in genetic crosses with genome reference strain M-3125.

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.

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.

Molecular dissection of Neurospora Spore killer meiotic drive elements

Meiotic drive is a non-Mendelian inheritance phenomenon in which certain selfish genetic elements skew sexual transmission in their own favor. In some cases, progeny or gametes carrying a meiotic drive element can survive preferentially because it causes the death or malfunctioning of those that do not carry it. In Neurospora, meiotic drive can be observed in fungal spore killing. In a cross of Spore killer (Sk) × WT (Sk-sensitive), the ascospores containing the Spore killer allele survive, whereas the ones with the sensitive allele degenerate. Sk-2 and Sk-3 are the most studied meiotic drive elements in Neurospora, and they each theoretically contain two essential components: a killer element and a resistance gene. Here we report the identification and characterization of the Sk resistance gene, rsk (resistant to Spore killer). rsk seems to be a fungal-specific gene, and its deletion in a killer strain leads to self-killing. Sk-2, Sk-3, and naturally resistant isolates all use rsk for resistance. In each killer system, rsk sequences from an Sk strain and a resistant isolate are highly similar, suggesting that they share the same origin. Sk-2, Sk-3, and sensitive rsk alleles differ from each other by their unique indel patterns. Contrary to long-held belief, the killer targets not only late but also early ascospore development. The WT RSK protein is dispensable for ascospore production and is not a target of the spore-killing mechanism. Rather, a resistant version of RSK likely neutralizes the killer element and prevents it from interfering with ascospore development.

Neurospora Spore KillersSk-2andSk-3Suppress 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.

New findings of Neurospora in Europe and comparisons of diversity in temperate climates on continental scales

The life cycles of the conidiating species of Neurospora are adapted to respond to fire, which is reflected in their natural history. Neurospora is found commonly on burned vegetation from the tropic and subtropical regions around the world and through the temperate regions of western North America. In temperate Europe it was unknown whether Neurospora would be as common as it is in North America because it has been reported only occasionally. In 2003 and 2004 a multinational effort surveyed wildfire sites in southern Europe. Neurospora was found commonly from southern Portugal and Spain (37 degrees N) to Switzerland (46 degrees N). Species collected included N. crassa, N. discreta, N. sitophila and N. tetrasperma. The species distribution and spatial dynamics of Neurospora populations showed both similarities and differences when compared between temperate Europe and western North America, both regions of similar latitude, climate and vegetation. For example the predominant species in western North America, N. discreta phylogenetic species 4B, is common but not predominant in Europe, whereas species rare in western North America, N. crassa NcB and N. sitophila, are much more common in Europe. The meiotic drive element Spore killer was also common in European populations of N. sitophila and at a higher proportion than anywhere else in the world. The methods by which organisms spread and adapt to new environments are fundamental ecosystem properties, yet they are little understood. The differences in regional diversity, reported here, can form the basis of testable hypotheses. Questions of phylogeography and adaptations can be addressed specifically by studying Neurospora in nature.

New findings of Neurospora in Europe and comparisons of diversity in temperate climates on continental scales

The life cycles of the conidiating species of Neurospora are adapted to respond to fire, which is reflected in their natural history. Neurospora is found commonly on burned vegetation from the tropic and subtropical regions around the world and through the temperate regions of western North America. In temperate Europe it was unknown whether Neurospora would be as common as it is in North America because it has been reported only occasionally. In 2003 and 2004 a multinational effort surveyed wildfire sites in southern Europe. Neurospora was found commonly from southern Portugal and Spain (37° N) to Switzerland (46° N). Species collected included N. crassa, N. discreta, N. sitophila and N. tetrasperma. The species distribution and spatial dynamics of Neurospora populations showed both similarities and differences when compared between temperate Europe and western North America, both regions of similar latitude, climate and vegetation. For example the predominant species in western North America, N. discreta phylogenetic species 4B, is common but not predominant in Europe, whereas species rare in western North America, N. crassa NcB and N. sitophila, are much more common in Europe. The meiotic drive element Spore killer was also common in European populations of N. sitophila and at a higher proportion than anywhere else in the world. The methods by which organisms spread and adapt to new environments are fundamental ecosystem properties, yet they are little understood. The differences in regional diversity, reported here, can form the basis of testable hypotheses. Questions of phylogeography and adaptations can be addressed specifically by studying Neurospora in nature.

Genes in conflict: the biology of selfish genetic elements

Genes in conflict: the biology of selfish genetic elements

Het up mould unleashes a sporekiller prion.

het-s/het-S incompatibility is one of only a handful of fungal heterokaryon incompatibility systems that have begun to be molecularly analysed (for a review see Saupe 2000). The spore killer phenotype also is one of only a few segregation ratio distortions that has been studied in any detail. Finally the [HET-s] prion is one of only three examples known in non-mammalian systems (for a review see Wickneret al 1999). Clearly thehet-s/het-S story intersects with several exciting areas of research that offer many opportunities for young scientists.

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).