Collateral damage: spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene in Neurospora crassa

Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G:C to A:T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of theerg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of theerg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1-0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterialhph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation

Collateral Damage: Spread of Repeat-induced Point Mutation from a Duplicated DNA Sequence into an Adjoining Single-copy Gene in Neurospora Crassa

Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of the erg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1⋅5 or 2 kb) and located at defined distances (0, 0⋅5, 1 or 2 kb) upstream or downstream of the erg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0⋅1-0⋅8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterial hph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation. [Vyas M and Kasbekar D P 2005 Collateral damage: Spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene in Neurospora crassa; J. Biosci. 30 [15][16][17][18][19][20

Genetic analysis of wild-isolated Neurospora crassa strains identified as dominant suppressors of repeat-induced point mutation

Repeat-induced point mutation (RIP) in Neurospora results in inactivation of duplicated DNA sequences. RIP is thought to provide protection against foreign elements such as retrotransposons, only one of which has been found in N. crassa. To examine the role of RIP in nature, we have examined seven N. crassa strains, identified among 446 wild isolates scored for dominant suppression of RIP. The test system involved a small duplication that targets RIP to the easily scorable gene erg-3. We previously showed that RIP in a small duplication is suppressed if another, larger duplication is present in the cross, as expected if the large duplication competes for the RIP machinery. In two of the strains, RIP suppression was associated with a barren phenotype-a characteristic of Neurospora duplications that is thought to result in part from a gene-silencing process called meiotic silencing by unpaired DNA (MSUD). A suppressor of MSUD (Sad-1) was shown not to prevent known large duplications from impairing RIP. Single-gene duplications also can be barren but are too short to suppress RIP. RIP suppression in strains that were not barren showed inheritance that was either simple Mendelian or complex. Adding copies of the LINE-like retrotransposon Tad did not affect RIP efficiency

A Dominant Mutation in mediator of paramutation2, One of Three Second-Largest Subunits of a Plant-Specific RNA Polymerase, Disrupts Multiple siRNA Silencing Processes

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes

Chromosome Segment Duplications in Neurospora crassa and Their Effects on Repeat-Induced Point Mutation and Meiotic Silencing by Unpaired DNA

The size and extent of four Neurospora crassa duplications, Dp(AR17), Dp(IBj5), Dp(OY329), and Dp(B362i), was determined by testing the coverage of RFLP markers. The first three duplications were all >∼350 kb and have been shown in earlier studies to act as dominant suppressors of repeat-induced point mutation (RIP) in gene-sized duplications, possibly via titration of the RIP machinery. Dp(B362i), which is only ∼117 kb long, failed to suppress RIP. RIP suppression in gene-sized duplications by large duplications was demonstrated using another test gene, dow, and supposedly applies generally. Crosses homozygous for Dp(AR17) or Dp(IBj5) were as barren as heterozygous crosses. Barrenness of the heterozygous but not the homozygous crosses was suppressible by Sad-1, a semidominant suppressor of RNAi-dependent meiotic silencing by unpaired DNA. A model is proposed in which large duplications recessively suppress semidominant Sad-1 mutations. The wild-isolated Sugartown strain is hypothesized to contain a duplication that confers not only dominant suppression of RIP but also a barren phenotype, which is linked (9%) to supercontig 7.118 in LG VII

Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs

<div><p>Control of the whitefly <i>Bemisia tabaci</i> (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly ‘gut’ genes were selected from an <i>in silico</i>-annotated transcriptome library constructed from the whitefly alimentary canal or ‘gut’ of the B biotype of <i>B</i>. <i>tabaci</i>, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: <i>Acetylcholine receptor subunit α</i>, <i>Alpha glucosidase 1</i>, <i>Aquaporin 1</i>, <i>Heat shock protein 70</i>, <i>Trehalase1</i>, and <i>Trehalose transporter1</i>. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced <i>Acetylcholine receptor subunit α</i> and <i>Heat shock protein 70</i> genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.</p></div

Percent whitefly mortality for <i>Tre1</i> and <i>Tret1</i> knockdowns.

<p>Percent whitefly mortality for <i>Tre1</i> and <i>Tret1</i> knockdowns.</p

Percent mortality post-<i>AChRα</i> dsRNA knock down, and exposure to <i>I MaxxPro</i>^®^&.

<p>Percent mortality post-<i>AChRα</i> dsRNA knock down, and exposure to <i>I MaxxPro</i>^®<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168921#t004fn002" target="_blank">^&</a>.</p

Primers used for PCR and RT-PCR amplification and <i>in vitro</i> transcription.

<p>The T7 promoter sequence is underlined.</p