An endogene-resembling transgene is resistant to DNA methylation and systemic silencing

Dadami, Elena; Dalakouras, Athanasios; Zwiebel, Michèle; Krczal, Gabi; Wassenegger, Michael

doi:10.4161/rna.29623

Cited by 41 publications

(42 citation statements)

References 72 publications

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“…Yet, reception of systemic silencing in the sink tissues requires RDR6 processing of the target (Schwach et al, 2005). A growing body of recent evidence has suggested that in contrast to intron-containing genes, intronless genes are much more susceptible to RDR6 processivity and thus transitivity and systemic silencing Dadami et al, 2013Dadami et al, , 2014. Taking these observations into consideration, the chances of systemic RNAi upon exogenous RNA application may significantly increase when then the trigger is 22-nt sRNA and the target gene is intronless (see "Outstanding Questions").…”

Section: Transitivity and Systemic Silencingmentioning

confidence: 99%

Genetically Modified Organism-Free RNA Interference: Exogenous Application of RNA Molecules in Plants

et al. 2019

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Section: Transitivity and Systemic Silencingmentioning

confidence: 99%

Genetically Modified Organism-Free RNA Interference: Exogenous Application of RNA Molecules in Plants

et al. 2019

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“…As was shown previously for other geminiviral proteins (78)(79)(80), the coexpression of a PTGS suppressor such as P19 may enhance protein yield, a prerequisite which was also important for AbMV AC2 in this study. Placing an intron into the gene of interest may additionally reduce PTGS effects (81). A TGS effect of multiple 35S promoters on different plasmid constructs is also conceivable, but we did not observe any such effect in our agroinfiltration experiments.…”

Section: Discussionmentioning

confidence: 61%

Early Function of the Abutilon Mosaic Virus AC2 Gene as a Replication Brake

Krenz

Deuschle

Deigner

et al. 2015

J Virol

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The C2/AC2 genes of monopartite/bipartite geminiviruses of the genera Begomovirus and Curtovirus encode important pathogenicity factors with multiple functions described so far. A novel function of Abutilon mosaic virus (AbMV) AC2 as a replication brake is described, utilizing transgenic plants with dimeric inserts of DNA B or with a reporter construct to express green fluorescent protein (GFP). Their replicational release upon AbMV superinfection or the individual and combined expression of epitope-tagged AbMV AC1, AC2, and AC3 was studied. In addition, the effects were compared in the presence and in the absence of an unrelated tombusvirus suppressor of silencing (P19). The results show that AC2 suppresses replication reproducibly in all assays and that AC3 counteracts this effect. Examination of the topoisomer distribution of supercoiled DNA, which indicates changes in the viral minichromosome structure, did not support any influence of AC2 on transcriptional gene silencing and DNA methylation. The geminiviral AC2 protein has been detected here for the first time in plants. The experiments revealed an extremely low level of AC2, which was slightly increased if constructs with an intron and a hemagglutinin (HA) tag in addition to P19 expression were used. AbMV AC2 properties are discussed with reference to those of other geminiviruses with respect to charge, modification, and size in order to delimit possible reasons for the different behaviors. IMPORTANCEThe (A)C2 genes encode a key pathogenicity factor of begomoviruses and curtoviruses in the plant virus family Geminiviridae. This factor has been implicated in the resistance breaking observed in agricultural cotton production. AC2 is a multifunctional protein involved in transcriptional control, gene silencing, and regulation of basal biosynthesis. Here, a new function of Abutilon mosaic virus AC2 in replication control is added as a feature of this protein in viral multiplication, providing a novel finding on geminiviral molecular biology.

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“…22,32 So far, and at least in the case of transgenes, PTGS is always associated with RdDM of transgene coding regions. 22,[56][57][58][59][60][61][62][63][64] RdDM seems be interwoven with PTGS in a mutual-reinforcing loop, but whether RdDM acts upstream or downstream of PTGS or whether one is a prerequisite of the other is not clear. We were thus interested to investigate whether the failure to post-transcriptionally silence the GFPint98(C)/(¡) transgenes was, nevertheless, accompanied by RdDM.…”

Section: Viroid Infection Efficiently Triggered Rddm Of Gfpint98 Tranmentioning

confidence: 99%

“…Capillary blotting of long RNAs, electro-blotting of small RNAs, and hybridization with radiolabelled probes were performed as previously described. 58 In the cases of GFP and PSTVd probes, fulllength cDNAs were used. For the intron ('-ron') probe, the 410 bp fragment amplified by PCR with the primers 5-TCT AAA GGC ATA TTG AAA TAC-3 and 5-TCA TCA AAG TTT TAA CAC TTG-3was used.…”

Section: Plasmid Constructionmentioning

confidence: 99%

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Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA

et al. 2015

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In plants, Potato spindle tuber viroid (PSTVd) replication triggers post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) of homologous RNA and DNA sequences, respectively. PTGS predominantly occurs in the cytoplasm, but nuclear PTGS has been also reported. In this study, we investigated whether the nuclear replicating PSTVd is able to trigger nuclear PTGS. Transgenic tobacco plants carrying cytoplasmic and nuclear PTGS sensor constructs were PSTVd-infected resulting in the generation of abundant PSTVd-derived small interfering RNAs (vd-siRNAs). Northern blot analysis revealed that, in contrast to the cytoplasmic sensor, the nuclear sensor transcript was not targeted for RNA degradation. Bisulfite sequencing analysis showed that the nuclear PTGS sensor transgene was efficiently targeted for RdDM. Our data suggest that PSTVd fails to trigger nuclear PTGS, and that RdDM and nuclear PTGS are not necessarily coupled.

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An endogene-resembling transgene is resistant to DNA methylation and systemic silencing

Cited by 41 publications

References 72 publications

Genetically Modified Organism-Free RNA Interference: Exogenous Application of RNA Molecules in Plants

Genetically Modified Organism-Free RNA Interference: Exogenous Application of RNA Molecules in Plants

Early Function of the Abutilon Mosaic Virus AC2 Gene as a Replication Brake

Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA

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