A DEAD-box RNA helicase mediates meiotic silencing by unpaired DNA.

During the sexual phase of Neurospora crassa, unpaired genes are subject to a silencing mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD targets the transcripts of an unpaired gene and utilizes typical RNA interference factors for its process. Using a reverse genetic screen, we have identified a meiotic silencing gene called sad-9, which encodes a DEAD-box RNA helicase.

Gene drive by Fusarium SKC1 is dependent on its competing allele.

The Fusarium verticillioides SKC1 gene driver is transmitted to offspring in a biased manner through spore killing. The mechanism that allows SKC1 to kill non-SKC1 offspring while sparing others is poorly understood. Here we report that gene drive by SKC1 is dependent on SKC1's competing allele.

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.

An introgressed gene causes meiotic drive in .

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 , the spore killer element is found in many natural populations. In this study, we identify the gene responsible for spore killing in by generating both long- and short-read genomic data and by using these data to perform a genome-wide association test.

An NCBP3-Domain Protein Mediates Meiotic Silencing by Unpaired DNA.

In the filamentous fungus , genes unpaired during meiosis are silenced by a process known as meiotic silencing by unpaired DNA (MSUD). MSUD utilizes common RNA interference (RNAi) proteins, such as Dicer and Argonaute, to target homologous mRNAs for silencing. Previously, we demonstrated that nuclear cap-binding proteins NCBP1 and NCBP2 are involved in MSUD.

Identification of , a Meiotic Driver Undergoing RNA Editing in .

is a meiotic drive element that was discovered in wild populations of fungi over 40 years ago. While early studies quickly determined that transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize , a gene required for -based spore killing.

Suppressors of Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD) is a gene silencing process that occurs within meiotic cells of and other fungi. We have previously developed a high-throughput screen to identify suppressors of this silencing pathway. Here, a list of MSUD suppressor candidates from a single pass of the first 84 plates of the knockout library is provided.

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia.

An RNA Recognition Motif-Containing Protein Functions in Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD) is a biological process that searches pairs of homologous chromosomes (homologs) for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of , a gene that encodes a protein with an RNA recognition motif (RRM).

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 (Sk(K)) that causes nearly all surviving meiotic progeny from an Sk(K) × Spore killer-susceptible (Sk(S)) cross to inherit the Sk(K) allele. Sk(K) has been mapped to chromosome V but the genetic element responsible for meiotic drive has yet to be identified.

A RIP allele for transformation of .

This report describes the construction and characterization of , an allele for high-efficiency targeted integration of transgenes into the genome of the model eukaryote . Two of the strains investigated in this work (RZS27.10 and RZS27.

Complex formation of RNA silencing proteins in the perinuclear region of Neurospora crassa.

In Neurospora, genes not paired during meiosis are targeted by meiotic silencing by unpaired DNA (MSUD). Here, our bimolecular fluorescence complementation (BiFC) study suggests that RNA-directed RNA polymerase, Dicer, Argonaute, and others form a silencing complex in the perinuclear region, with intimate interactions among the majority of them. We have also shown that SAD-2 is likely the anchor for this assembly.

Efficient detection of unpaired DNA requires a member of the rad54-like family of homologous recombination proteins.

Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology.

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.

Novel proteins required for meiotic silencing by unpaired DNA and siRNA generation in Neurospora crassa.

During meiosis in the filamentous fungus Neurospora crassa, unpaired genes are identified and silenced by a process known as meiotic silencing by unpaired DNA (MSUD). Previous work has uncovered six proteins required for MSUD, all of which are also essential for meiotic progression. Additionally, they all localize in the perinuclear region, suggesting that it is a center of MSUD activity.

Identification of small RNAs associated with meiotic silencing by unpaired DNA.

In Neurospora crassa, unpaired genes are silenced by a mechanism called meiotic silencing by unpaired DNA (MSUD). Although some RNA interference proteins are necessary for this process, its requirement of small RNAs has yet to be formally established. Here we report the characterization of small RNAs targeting an unpaired region, using Illumina sequencing.

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.

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery.

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library.

Fine-scale mapping in Neurospora crassa by using genome-wide knockout strains.

Fine-scale genetic mapping is often hindered by the lack of adequate markers surrounding the locus of interest. In the filamentous ascomycete Neurospora crassa, the genome has been sequenced and an effort has been made to generate genome-wide deletion strains for the entire gene set. Accordingly, the hygromycin-resistant marker in each deletion strain can be used as a mapping locus in a classical three-point cross, along with the mapping target and a standard marker.

Altered trichothecene biosynthesis in TRI6-silenced transformants of Fusarium culmorum influences the severity of crown and foot rot on durum wheat seedlings.

An RNA silencing construct was used to alter mycotoxin production in the plant pathogenic fungus Fusarium culmorum, the incitant of crown and foot rot on wheat. The transformation of a wild-type strain and its nitrate reductase-deficient mutant with inverted repeat transgenes (IRTs) containing sequences corresponding to the trichothecene regulatory gene TRI6 was achieved using hygromycin B resistance as a selectable marker. Southern analysis revealed a variety of integration patterns of the TRI6 IRT.

Fluorescent and bimolecular-fluorescent protein tagging of genes at their native loci in Neurospora crassa using specialized double-joint PCR plasmids.

The double-joint polymerase chain reaction (DJ-PCR) is a technique that can be used to construct vectors for targeted genome integration without laborious subcloning steps. Here we report the availability of plasmids that facilitate DJ-PCR-based construction of Neurospora crassa tagging vectors. These plasmids allow the creation of green or red fluorescent protein (GFP or RFP) tagging vectors for protein localization studies, as well as split-yellow fluorescent protein (YFP) tagging vectors for bimolecular fluorescence complementation (BiFC) analyses.

QIP, a protein that converts duplex siRNA into single strands, is required for meiotic silencing by unpaired DNA.

RNA interference (RNAi) depends on the production of small RNA to regulate gene expression in eukaryotes. Two RNAi systems exist to control repetitive selfish elements in Neurospora crassa. Quelling targets transgenes during vegetative growth, whereas meiotic silencing by unpaired DNA (MSUD) silences unpaired genes during meiosis.

DCL-1 colocalizes with other components of the MSUD machinery and is required for silencing.

In Neurospora, a gene present in an abnormal number of copies is usually a red flag for mischief. One way to deal with these potential intruders is by destroying their transcripts. Widely known as RNA interference (RNAi), this mechanism depends on the "dicing" of a double-stranded RNA intermediate into small-interfering RNA, which in turn guide the degradation of mRNA from the target gene.

RNA silencing of mycotoxin production in Aspergillus and Fusarium species.

Mycotoxins are natural fungal products that are defined by their harmful effects on humans and animals. Aflatoxin contamination of maize by Aspergillus species and trichothecene contamination of small grains by Fusarium species are two of the most severe mycotoxin problems in the United States. We are investigating RNA silencing in an effort to identify novel ways to control mycotoxin contamination of crops.