Thomas M. Hammond

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. Transformation of two Aspergilli (A. flavus and A. parasiticus) and a Fusarium (F. graminearum) with inverted repeat transgenes (IRT) containing sequences of mycotoxin-specific regulatory genes suppressed mycotoxin production in all three plant-pathogenic fungi. This atoxigenic phenotype was stable during infection on corn and wheat, and importantly, F. graminearum IRT strains were less virulent on wheat than were wild type. The IRT did not alter physiological characteristics of the fungi, such as spore production and growth rate on solid media. These results indicate that RNA silencing exists in Aspergillus and Fusarium plant pathogens and suggest that RNA silencing technology may be a useful tool for eliminating mycotoxin contamination of agricultural products.

Aspergillus mycoviruses are targets and suppressors of RNA silencing.

ABSTRACT RNA silencing can function as a virus defense mechanism in a diverse range of eukaryotes, and many viruses are capable of suppressing the silencing machinery targeting them. However, the extent to which this occurs between fungal RNA silencing and mycoviruses is unclear. Here, three Aspergillus dsRNA mycoviruses were partially characterized, and their relationship to RNA silencing was investigated. Aspergillus virus 1816 is related to Agaricus bisporus white button mushroom virus 1 and suppresses RNA silencing through a mechanism that alters the level of small interfering RNA. Aspergillus virus 178 is related to RNA virus L1 of Gremmeniella abietina and does not appear to affect RNA silencing. The third virus investigated, Aspergillus virus 341, is distantly related to Sphaeropsis sapinea RNA virus 2. Detection of mycovirus-derived siRNA from this mycovirus demonstrates that it is targeted for degradation by the Aspergillus RNA silencing machinery. Thus, our results indicate that Aspergillus mycoviruses are both targets and suppressors of RNA silencing. In addition, they suggest that the morphological and physiological changes associated with some mycoviruses could be a result of their antagonistic relationship with RNA silencing.

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. The two mechanisms require common RNAi proteins, such as RNA-directed RNA polymerases, Dicers, and Argonaute slicers. We have previously demonstrated that, while Quelling depends on the redundant dicer activity of DCL-1 and DCL-2, only DCL-1 is required for MSUD. Here, we show that QDE-2-interacting protein (QIP), an exonuclease that is important for the production of single-stranded siRNA during Quelling, is also required for MSUD. QIP is crucial for sexual development and is shown to colocalize with other MSUD proteins in the perinuclear region.

RNA Silencing Gene Truncation in the Filamentous Fungus Aspergillus nidulans

ABSTRACT The genus Aspergillus is ideally suited for the investigation of RNA silencing evolution because it includes species that have experienced a variety of RNA silencing gene changes. Our work on this subject begins here with the model species Aspergillus nidulans. Filamentous ascomycete fungi generally each encode two of the core RNA silencing proteins, Dicer and Argonaute, but A. nidulans appears to have lost one of each to gene truncation events. Although a role in growth, development, or RNA silencing was not detected for the truncated genes, they do produce spliced and poly(A)-tailed transcripts, suggesting that they may have an undetermined biological function. Population analysis demonstrates that the truncated genes are fixed at the species level and that their full-length orthologs in a closely related species are also unstable. With these gene truncation events, A. nidulans encodes only a single intact Dicer and Argonaute. Their deletion results in morphologically and reproductively normal strains that are incapable of experimental RNA silencing. Thus, our results suggest that the remaining A. nidulans RNA silencing genes have a “nonhousekeeping” function, such as defense against viruses and transposons.

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. One transformant underwent homologous recombination with deletion of the endogenous TRI6 gene, whereas, in another transformant, the TRI6 IRT was not integrated into the genome. The TRI6 IRT did not alter the physiological characteristics, such as spore production, pigmentation or growth rate, on solid media. In most transformants, a high TRI6 amplification signal was detected by quantitative reverse transcription-polymerase chain reaction, corresponding to a TRI6-hybridizing smear of degraded fragments by Northern analysis, whereas TRI5 expression decreased compared with the respective nontransformed strain. Four transformants showed increased TRI5 expression, which was correlated with a dramatic (up to 28-fold) augmentation of deoxynivalenol production. Pathogenicity assays on durum wheat seedlings confirmed that impairment of deoxynivalenol production in the TRI6 IRT transformants correlated with a loss of virulence, with decreased disease indices ranging from 40% to 80% in nine silenced strains, whereas the overproducing transformants displayed higher virulence compared with the wild-type.

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.

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. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species.

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. Nevertheless, at least a subset of MSUD proteins must be present inside the nucleus, as unpaired DNA recognition undoubtedly takes place there. In this study, we identified and characterized two new proteins required for MSUD, namely SAD-4 and SAD-5. Both are previously uncharacterized proteins specific to Ascomycetes, with SAD-4 having a range that spans several fungal classes and SAD-5 seemingly restricted to a single order. Both genes appear to be predominantly expressed in the sexual phase, as molecular study combined with analysis of publicly available mRNA-seq datasets failed to detect significant expression of them in the vegetative tissue. SAD-4, like all known MSUD proteins, localizes in the perinuclear region of the meiotic cell. SAD-5, on the other hand, is found in the nucleus (as the first of its kind). Both proteins are unique compared to previously identified MSUD proteins in that neither is required for sexual sporulation. This homozygous-fertile phenotype uncouples MSUD from sexual development and allows us to demonstrate that both SAD-4 and SAD-5 are important for the production of masiRNAs, which are the small RNA molecules associated with meiotic silencing.

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.

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 (SkS) 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-2INS1) and part of a >220-kb inversion (Sk-2INV1). 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.