The RNA-dependent RNA polymerase, QDE-1, is a rate-limiting factor in post-transcriptional gene silencing in Neurospora crassa

Abstract

The RNA-dependent RNA polymerase (RdRP) qde-1 is an essential component of post-transcriptional gene silencing (PTGS), termed 'quelling' in the fungus Neurospora crassa. Here we show that the overexpression of QDE-1 results in a dramatic increase in the efficiency of quelling, with a concomitant net increase in the quantity of al-1 siRNAs. Moreover, in overexpressed strains there is a significant reduction in the number of transgenes required to induce quelling, and an increase in the phenotypic stability despite progressive loss of tandemly repeated transgenes, which normally determines reversion of a silenced phenotype to wild type. These data suggest that the activation and maintenance of silencing in Neurospora appear to rely both on the cellular amount of QDE-1 and the amount of transgenic copies producing RNA molecules that act as a substrate for the RdRP, implicating QDE-1 as a rate-limiting factor in PTGS.

Figure 1

Northern analysis of wild-type strains transformed with the qde-1 overexpression cassette. Overexpression of qde-1, induced by the addition of quinic acid, was revealed in transformants 3 and 6. Both transformants showed the same silencing efficiency (data not shown). Subsequent experiments were carried out with transformant 6 as the qde-1-overexpressing strain OQ1. The shadow seen in the non-induced lanes reflects a low level of QDE-1 expression. N, non-induced; I, induced.

Figure 2

Effect of qde-1 overexpression on the phenotype of al-1 transformants. Phenotype of a non-silenced (orange) and a silenced (albino) strain, irrespective of qde-1 overexpression, and an inducibly silenced strain where silencing (albino phenotype) was a result of qde-1 overexpression induced by quinic acid.

Figure 3

Northern analysis of mRNA and siRNA. (A) Northern analysis of qde-1 and al-1 mRNA in a wild-type (WT) control and two overexpressed strains in both non-induced (N) and induced (I) conditions. al-1 mRNA was silenced in OQ1-S1, whereas for OQ1-IS60 it was only silenced when qde-1 was overexpressed. Expression of the al-2 gene was assessed for normalization purposes. (B) Northern analysis of al-1 siRNAs in the same strains as in (A). As expected, no al-1 siRNAs were detected in the wild type, whereas in OQ1-S1 and OQ1-IS60 an increase of al-1 siRNA was detected under induced conditions. The ribosomal RNA is shown as a control for equal loading.

Figure 4

NS, non-silenced; IS, inducibility silenced; S, constitutely silenced. (A) Southern analysis of al-1 copy number in transformed wild-type and OQ1 strains. Schematic representation of the al-1 endogenous locus and of plasmid pX16 carrying the al-1 transgene. (B) Southern analysis of al-1 copy number in wild-type and OQ1 strains transformed with pX16. Eleven out of a total data set of 52 are shown. Lane 1: wild type (control); lanes 2–6: non-silenced OQ1 strains; lanes 7–12: inducibly- silenced OQ1 strains; lanes 13–15: silenced OQ1 strains. The 3.1-kb band corresponds to the endogenous al-1 gene and the 5.5-kb band corresponds to ectopic al-1 transgenes. The more intense 5.5-kb signal in silenced OQ1 strains denotes tandem arrays of transgenic al-1 sequences, while diverse bands represent randomly integrated transgene copies. Non-silenced OQ1 transformants harbour only one, two or no al-1 transgenes.

Figure 5

Reversion of silencing in OQ1 and wild-type al-1 transformants. (A) Phenotype of the original culture (‘ori’) with respect to nine single colonies of a silenced OQ1 transformant (OQ1-S1), an inducibly silenced OQ1 transformant (OQ1-IS60) and a silenced wild-type al-1 transformant (WT-S3). (B) Southern analysis of al-1 copy number in the original culture (ORI) as compared with revertant R5 of WT-S3, R8 of OQ1-IS60 and R4 of OQ1-S1. Around 30% of the descendants showed a characteristic loss of transgenic copies.