Double-stranded RNA interferes with gene function at the single-cell level in cereals

175

137

Cell death regulation is linked to pathogen defense in plants and animals. Execution of apoptosis as one type of programmed cell death in animals is irreversibly triggered by cytochrome c release from mitochondria via pores formed by BAX proteins. This type of programmed cell death can be prevented by expression of BAX inhibitor 1 (BI-1), a membrane protein that protects cells from the effects of BAX by an unknown mechanism. In barley, a homologue of the mammalian BI-1 is expressed in response to inoculation with the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). We found differential expression of BI-1 in response to Bgh in susceptible and resistant plants. Chemical induction of resistance to Bgh by soil drench treatment with 2,6-dichloroisonicotinic acid led to down-regulation of the expression level of BI-1. Importantly, single-cell transient overexpression of BI-1 in epidermal leaf tissue of susceptible barley cultivar Ingrid led to enhanced accessibility, resulting in a higher penetration efficiency of Bgh on BI-1-transformed cells. In Bgh-resistant mlo5 genotypes, which do not express the negative regulator of defense and cell death MLO, overexpression of BI-1 almost completely reconstituted susceptibility to fungal penetration. We suggest that BI-1 is a regulator of cellular defense in barley sufficient to substitute for MLO function in accessibility to fungal parasites.

Section: Resultssupporting

confidence: 66%

“…Resulting pBI-1 and antisense pasBI-1 were sequenced to confirm that the original ORF was unchanged. To induce posttranscriptional gene silencing via RNA interference, particles were coated with dsRNA as described (18,22).…”

Section: Transient Transformation and Evaluation Of Penetration Efficmentioning

confidence: 99%

Overexpression of barley BAX inhibitor 1 induces breakdown of mlo -mediated penetration resistance to Blumeria graminis

Hückelhoven

Dechert²,

Kögel³

2003

175

137

“…Although it had been established that high molecular weight dsRNAs can trigger silencing confined to bombarded cereal cells (32) and systemic silencing in C. elegans (33), it had not previously been shown that ca. 21-nt siRNAs as well as high molecular weight singlestranded RNAs of both polarities can trigger silencing able to spread from cell to cell and systemically.…”

Section: Discussionmentioning

confidence: 99%

High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants

Klahre

Crété

Leuenberger

et al. 2002

168

132

The underlying mechanisms are thought to be highly conserved in evolution (2, 4). RNAi in animals is initiated by doublestranded RNAs (dsRNAs) similar in sequence to the transcribed region of target genes. These dsRNAs undergo endonucleolytic cleavage to generate 21-to 23-nt-long small interfering RNAs (siRNAs), which then promote RNA degradation (5-7) Remarkably, the silent state in transgenic plants and in C. elegans can spread from cell to cell and even systemically throughout the organism, implying the existence of mobile silencing signals (2, 8). Little is known about the chemical nature of these signals, but it seems likely that the sequence-specific component is an RNA (8-11). The finding that siRNA and dsRNA accumulate in silent tissues, together with studies of informative stable transformants and PTGS induced by RNA viruses, supports the view that dsRNAs and siRNAs have key roles in plant PTGS (12-14). Nevertheless, direct evidence that these or other RNAs can induce systemic PTGS or comprise silencing signals in plants is lacking.In the present study, we used a positive marker system and real-time monitoring of green fluorescent protein (GFP) expression to show that double-stranded siRNAs, large sense, antisense, and double-stranded RNAs delivered biolistically into plant cells trigger PTGS capable of spreading locally and systemically. The introduced siRNAs trigger the production of siRNAs derived from sequences both 3Ј and 5Ј of the inducing siRNAs. Our findings support the hypothesis that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and are generated in a self-amplifying fashion. Materials and Methods

“…Recently it was shown that siRNAs of 21 nt in size, an intermediate of the RNA-interference pathway, are effective in suppression of gene expression in animal systems including mammals. Gene silencing induced by long dsRNA constructs has been demonstrated in transgenic plants showing resistance to virus infection (9): transient interference with endogenous gene expression (28), with virus infection in plants (29), and with reporter gene expression in plant cells (30,31). The siRNA technology offers animal virologists a new way to control viruses because the use of long dsRNA triggers the IFN response, which leads to apoptosis in most mammalian cells (32).…”

mentioning

confidence: 99%

Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells

Ramachandran

Padmanabhan

Fauquet

2003

120

Gene silencing mediated by double-stranded RNA is a sequencespecific RNA degradation mechanism highly conserved in eukaryotes that serves as an antiviral defense pathway in both plants and Drosophila. Short interfering RNAs (siRNAs), the 21-to 23-nt double-stranded intermediates of this natural defense mechanism, are becoming powerful tools for reducing gene expression and countering viral infection in a variety of mammalian cells. Here we report the use of siRNAs to target reporter gene expression and viral DNA accumulation in cultured plant cells. Transient expression of reporter genes encoding either GFP or red fluorescent protein from Discosoma was specifically reduced by 58% and 47%, respectively, at 24 h after codelivery of cognate siRNAs in BY2 protoplasts. In contrast to mammalian systems, the siRNA-induced silencing of GFP expression was transitive as indicated by the presence of siRNAs representing parts of the target RNA outside the region homologous to the triggering siRNA. Codelivery of an siRNA designed to target the mRNA encoding the replicationassociated protein (AC1) of the geminivirus African cassava mosaic virus (ACMV) from Cameroon blocked AC1 mRNA accumulation by Ϸ91% and inhibited accumulation of the ACMV genomic DNA by Ϸ66% at 36 and 48 h after transfection. As with siRNA-induced reporter gene silencing, the siRNA targeting ACMV AC1 was specific and did not affect the replication of East African cassava mosaic Cameroon virus. This report demonstrates the occurrence of siRNA-mediated suppression of gene expression in cultured plant cells and that siRNA can interfere with and suppress accumulation of a nuclear-replicated DNA virus.