Involvement of RDR6 in short-range intercellular RNA silencing in Nicotiana benthamiana

In plants, non-cell autonomous RNA silencing spreads between cells and over long distances. Recent work has revealed insight on the genetic and molecular components essential for cell-to-cell movement of RNA silencing in Arabidopsis. Using a local RNA silencing assay, we report on a distinct mechanism that may govern the short-range (6–10 cell) trafficking of virus-induced RNA silencing from epidermal to neighbouring palisade and spongy parenchyma cells in Nicotiana benthamiana. This process involves a previously unrecognised function of the RNA-dependent RNA polymerase 6 (RDR6) gene. Our data suggest that plants may have evolved distinct genetic controls in intercellular RNA silencing among different types of cells

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production

RDR2 Partially Antagonizes the Production of RDR6-Dependent siRNA in Sense Transgene-Mediated PTGS

Background: RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and SUPPRESSOR of GENE SILENCING 3 (SGS3) are required for DNA methylation and post-transcriptional gene silencing (PTGS) mediated by 21-nt siRNAs produced by sense transgenes (S-PTGS). In contrast, RDR2, but not RDR6, is required for DNA methylation and TGS mediated by 24-nt siRNAs, and for cellto-cell spreading of IR-PTGS mediated by 21-nt siRNAs produced by inverted repeat transgenes under the control of a phloem-specific promoter. Principal Findings: In this study, we examined the role of RDR2 and RDR6 in S-PTGS. Unlike RDR6, RDR2 is not required for DNA methylation of transgenes subjected to S-PTGS. RDR6 is essential for the production of siRNAs by transgenes subjected to S-PTGS, but RDR2 also contributes to the production of transgene siRNAs when RDR6 is present because rdr2 mutations reduce transgene siRNA accumulation. However, the siRNAs produced via RDR2 likely are counteractive in wildtype plants because impairement of RDR2 increases S-PTGS efficiency at a transgenic locus that triggers limited silencing, and accelerates S-PTGS at a transgenic locus that triggers efficient silencing. Conclusions/Significance: These results suggest that RDR2 and RDR6 compete for RNA substrates produced by transgenes subjected to S-PTGS. RDR2 partially antagonizes RDR6 because RDR2 action likely results in the production of counteractiv

RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration

Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system

RNA silencing can explain chlorotic infection patterns on plant leaves

<p>Abstract</p> <p>Background</p> <p>RNA silencing has been implicated in virus symptom development in plants. One common infection symptom in plants is the formation of chlorotic tissue in leaves. Chlorotic and healthy tissue co-occur on a single leaf and form patterns. It has been shown that virus levels in chlorotic tissue are high, while they are low in healthy tissue. Additionally, the presence of siRNAs is confined to the chlorotic spots and the boundaries between healthy and infected tissue. These results strongly indicate that the interaction between virus growth and RNA silencing plays a role in the formation of infection patterns on leaves. However, how RNA silencing leads to the intricate patterns is not known.</p> <p>Results</p> <p>Here we elucidate the mechanisms leading to infection patterns and the conditions which lead to the various patterns observed. We present a modeling approach in which we combine intra- and inter-cellular dynamics of RNA silencing and viral growth. We observe that, due to the spread of viruses and the RNA silencing response, parts of the tissue become infected while other parts remain healthy. As is observed in experiments high virus levels coincide with high levels of siRNAs, and siRNAs are also present in the boundaries between infected and healthy tissue. We study how single- and double-stranded cleavage by Dicer and amplification by RNA-dependent RNA polymerase can affect the patterns formed.</p> <p>Conclusion</p> <p>This work shows that RNA silencing and virus growth within a cell, and the local spread of virions and siRNAs between cells can explain the heterogeneous spread of virus in leaf tissue, and therewith the observed infection patterns in plants.</p

RNA silencing is required for Arabidopsis defence against Verticillium wilt disease

RNA silencing is a conserved mechanism in eukaryotes that plays an important role in various biological processes including regulation of gene expression. RNA silencing also plays a role in genome stability and protects plants against invading nucleic acids such as transgenes and viruses. Recently, RNA silencing has been found to play a role in defence against bacterial plant pathogens in Arabidopsis through modulating host defence responses. In this study, it is shown that gene silencing plays a role in plant defence against multicellular microbial pathogens; vascular fungi belonging to the Verticillium genus. Several components of RNA silencing pathways were tested, of which many were found to affect Verticillium defence. Remarkably, no altered defence towards other fungal pathogens that include Alternaria brassicicola, Botrytis cinerea, and Plectosphaerella cucumerina, but also the vascular pathogen Fusarium oxysporum, was recorded. Since the observed differences in Verticillium susceptibility cannot be explained by notable differences in root architecture, it is speculated that the gene silencing mechanisms affect regulation of Verticillium-specific defence responses

Graft-Transmitted siRNA Signal from the Root Induces Visual Manifestation of Endogenous Post-Transcriptional Gene Silencing in the Scion

In plants, post-transcriptional gene silencing (PTGS) spreads systemically, being transmitted from the silenced stock to the scion expressing the corresponding transgene. It has been reported that a graft-transmitted siRNA signal can also induce PTGS of an endogenous gene, but this was done by top-grafting using silenced stock. In the present study involving grafting of Nicotiana benthamiana, we found that PTGS of an endogenous gene, glutamate-1-semialdehyde aminotransferase (GSA), which acts as a visible marker of RNAi via inhibition of chlorophyll synthesis, was manifested along the veins of newly developed leaves in the wild-type scion by the siRNA signal synthesized only in companion cells of the rootstock