An RNA-Dependent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus

Abstract

Posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and RNA interference in animals. Here, we describe four genetic loci that are required for posttranscriptional gene silencing in Arabidopsis. One of these, SDE1, is a plant homolog of QDE-1 in Neurospora crassa that encodes an RNA-dependent RNA polymerase. The sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. We propose that the role of SDE1 is to synthesize a double-stranded RNA initiator of posttranscriptional gene silencing. According to this idea, when a virus induces posttranscriptional gene silencing, the virus-encoded RNA polymerase would produce the double-stranded RNA and SDE1 would be redundant.

Specific degradation of 3??? regions of GUS mRNA in posttranscriptionally silenced tobacco lines may be related to 5???-3??? spreading of silencing

“…The unexpected observation that virus-induced silencing appeared to spread more readily to transgene sequences downstream of the inducer region than to sequences upstream may point to an entirely different explanation for the predominance of 39 regions as PTGS targets+ Mechanistically spreading of the target region in the 39-59 direction is more easily explained, as a host RdRP (Dalmay et al+, 2000;Mourrain et al+, 2000) could use antisense RNA from the double-stranded inducer region as primer for synthesis of cRNA on a transgene mRNA template (Sijen et al+, 2001b)+ Indeed, experiments in Drosophila embryo extracts have shown that small RNAs can serve as primers to transform target mRNA into dsRNA, which is subsequently degraded into new small RNAs (Lipardi et al+, 2001)+ Similarly, small RNAs corresponding to target mRNA sequences upstream of the dsRNA inducer were produced in C. elegans in a reaction requiring a cellular RdRP (Sijen et al+, 2001a)+ Because RNA synthesis only occurs by 39 addition of nucleotides, the use of small RNAs as primers can only generate new dsRNA from the mRNA region upstream of the initial inducer sequence+ Indeed, only spreading of silencing in the 39-59 direction was observed in the Drosophila and C. elegans experiments (Lipardi et al+, 2001;Sijen et al+, 2001a)+ However, several observations in plants have suggested that PTGS can spread to transgene regions downstream as well as upstream of the primary target+ Jones et al+ (1999) used PVX carrying 59 or 39 regions of the green fluorescent protein (GFP) coding region to initiate silencing in GFP-expressing N. benthamiana plants+ The virus-induced DNA methylation of the entire coding region of the GFP transgene occurred irrespectively of whether 59 or 39 GFP sequences were carried by the virus+ However, a causal connection between DNA methylation and RNA degradation in PTGS has not been demonstrated+ Hamilton et al+ (1998) transformed tobacco with extra copies of the tobacco ACCoxidase gene (ACO1) carrying an inverted repeat of the 59 UTR+ Transformants showed posttranscriptional 1040…”

RNAi: an ever-growing puzzle

“…Similarly, in Neurospora, co-suppression is not active in mutants with lesions in its RdRP homolog, qde-1 [29]. By contrast, virus-induced gene silencing is unaffected in sgs2/sde1 mutants [28]. Viruses encode their own RdRPs that are essential for producing dsRNA during replication, and thus targeting of viral mRNAs might not be dependent on the function of a host RdRP.…”

“…sgs2/sde1 mutant plants are defective in the suppression of transgenes [28]. The SGS2/SDE1 locus encodes a putative RNA-dependent RNA polymerase, which might function either to convert aberrant RNAs into the dsRNA silencing trigger, or to amplify silencing initiated by very small amounts of dsRNA that could originate from the transgene locus.…”

Guest list or black list: heritable small RNAs as immunogenic memories

“…By using the target RNA as a template, RNA-dependent RNA polymerases catalyze the biogenesis of secondary small RNAs [83]. siRNAs and piRNAs trigger such amplification, a process that generates a secondary RNAi response that persists long after the original RNA transcript is degraded [84][85][86][87].…”