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A conditional silencing suppression system for transient expression.

ABSTRACT: RNA silencing is a powerful tool deployed by plants against viral infection and abnormal gene expression. Plant viruses have evolved a suite of silencing suppressors for counter-defense, which are also widely used to boost transcript and protein accumulation in transient assays. However, only wild type silencing suppressor proteins have been reported to date. Here we demonstrate that P0 of Potato leafroll virus (PLRV), PLP0, can be split into two proteins that only show silencing suppression activity upon co-expression. We cloned each of these proteins in two different constructs and transiently co-infiltrated them in N. benthamiana leaves. We expressed a fluorescent protein from one of the vectors and observed that cells expressing both halves of PLP0 suppressed gene silencing. Further, we showed that Q system of Neurospora crassa, based on co-expression of a transcription activator and inhibitor, is functional in agroinfiltrated leaves of N. benthamiana. Q system combined with the split PLP0 system showed very tight co-expression of Q system's transcriptional activator and inhibitor. Altogether, our experiments demonstrate a functioning conditional silencing suppressor system and its potential as a powerful tool for transient expression in N. benthamiana leaves, as well as the application of the Q system in plants.

SUBMITTER: Reis RS

PROVIDER: S-EPMC6013485 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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A conditional silencing suppression system for transient expression.

Reis Rodrigo Siqueira RS Litholdo Celso G CG Bally Julia J Roberts Thomas H TH Waterhouse Peter M PM

Scientific reports 20180621 1

RNA silencing is a powerful tool deployed by plants against viral infection and abnormal gene expression. Plant viruses have evolved a suite of silencing suppressors for counter-defense, which are also widely used to boost transcript and protein accumulation in transient assays. However, only wild type silencing suppressor proteins have been reported to date. Here we demonstrate that P0 of Potato leafroll virus (PLRV), PLP0, can be split into two proteins that only show silencing suppression act ...[more]

PMID: 29930292

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Project description:Aberrant epigenetic silencing plays a major role in cancer formation by inactivating tumor suppressor genes. While the endpoints of aberrant silencing are known, i.e., promoter region DNA methylation and altered histone modifications, the triggers of silencing are not known. We used the tet-off system to test the hypothesis that a transient reduction in gene expression will sensitize a promoter to undergo epigenetic silencing.The tet responsive promoter (P(TRE)) was used to drive expression of the selectable human HPRT cDNA in independent transfectants of an Hprt deficient mouse cell line. In this system, high basal HPRT expression is greatly reduced when doxycycline (Dox) is added to the culture medium. Exposure of the P(TRE)-HPRT transfectants to Dox induced HPRT deficient clones in a time dependent manner. A molecular analysis demonstrated promoter region DNA methylation, loss of histone modifications associated with expression (i.e., H3 lysine 9 and 14 acetylation and lysine 4 methylation), and acquisition of the repressive histone modification H3 lysine 9 methylation. These changes, which are consistent with aberrant epigenetic silencing, were not present in the Dox-treated cultures, with the exception of reduced H3 lysine 14 acetylation. Silenced alleles readily reactivated spontaneously or after treatment of cells with inhibitors of histone deacetylation and/or DNA methylation, but re-silencing of reactivated alleles did not require a new round of Dox exposure. Inhibition of histone deacetylation inhibited both the induction of silencing and re-silencing, whereas inhibition of DNA methylation had no such effect.This study demonstrates that a transient reduction in gene expression triggers a pathway for aberrant silencing in mammalian cells and identifies histone deacetylation as a critical early step in this process. DNA methylation, in contrast, is a secondary step in the silencing pathway under study. A model to explain these observations is offered.

Dataset Information

A conditional silencing suppression system for transient expression.

Publications

A conditional silencing suppression system for transient expression.

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