Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis

Abstract

In plants, silencing of mRNA can be transmitted from cell to cell and also over longer distances from roots to shoots. To investigate the long-distance mechanism, WT and mutant shoots were grafted onto roots silenced for an mRNA. We show that three genes involved in a chromatin silencing pathway, NRPD1a encoding RNA polymerase IVa, RNA-dependent RNA polymerase 2 (RDR2), and DICER-like 3 (DCL3), are required for reception of long-distance mRNA silencing in the shoot. A mutant representing a fourth gene in the pathway, argonaute4 (ago4), was also partially compromised in the reception of silencing. This pathway produces 24-nt siRNAs and resulted in decapped RNA, a known substrate for amplification of dsRNA by RDR6. Activation of silencing in grafted shoots depended on RDR6, but no 24-nt siRNAs were detected in mutant rdr6 shoots, indicating that RDR6 also plays a role in initial signal perception. After amplification of decapped transcripts, DCL4 and DCL2 act hierarchically as they do in antiviral resistance to produce 21- and 22-nt siRNAs, respectively, and these guide mRNA degradation. Several dcl genotypes were also tested for their capacity to transmit the mobile silencing signal from the rootstock. dcl1-8 and a dcl2 dcl3 dcl4 triple mutant are compromised in micro-RNA and siRNA biogenesis, respectively, but were unaffected in signal transmission.

Fig. 1.

Graft-transmissible mRNA silencing in WT Arabidopsis. (a) Schematic representation of seedling grafting and the transgene constructs used to produce the target and silencer (S1, S2, and S3) plant lines. (b) Phenotype of WT target scion grafted onto S2 rootstocks at 18 and 23 days after grafting. (c) Small RNA analysis from silencers (S1 and S2) and from target scions grafted onto S1 and S2 rootstocks, using GF and P DNA probes; identical results were obtained by using sense and antisense riboprobes (data not shown).

Fig. 2.

Genetic requirements for long-distance mRNA silencing. (a) Small RNA analysis of WT S2 and the triple mutant dcl2 dcl3 dcl4 (dcl234) S2 lines. miRNA159 serves as a loading control. (b) Phenotypes of grafts using WT S2 and dcl234 S2 as the rootstock. (c) Phenotypes of dcl3, nrpd1a, rdr2, rdr6, and ago4 target scions grafted onto WT S2 rootstocks. Some ago4 scions, ago4(B), showed delayed silencing. (d) Northern blot analysis of GFP mRNA levels in various grafted and nongrafted plants.

Fig. 3.

Long-distance down-regulation of transcription. (a) Target GFP transgene and flanking 35S:BAR selectable marker. (b) Northern blot analysis of BAR transcript levels in WT GFP-silenced scions grafted onto WT S2 rootstocks. (c) Northern blot analysis of BAR transcript levels in WT and ago4 GFP-silenced scions grafted onto WT S2 rootstocks. (d) Northern blot analysis of GFP and BAR mRNA levels in nrpd1b silenced scions grafted onto WT S2 rootstock.

Fig. 4.

Molecular genetic analysis of graft-transmissible mRNA silencing. (a Upper) Agarose gel electrophoresis of 5′-RACE products from GFP-silenced target scions grafted onto S2 rootstocks. The white bracket indicates the region cloned and sequenced (see SI Fig. 9). (a Lower) 5′-RACE amplification of the miR171-cleaved SCL6-IV transcript. (b) Target GFP transgene showing the regions used as probes for small RNA analysis. (c) Small RNA analysis of mutant scions grafted onto WT S2 rootstocks. (d) Small RNA analysis of WT scions within the first 70 nt of P. (e) Small RNA analysis of dcl4 target scions grafted onto WT S2 rootstocks.

Fig. 5.

Model for long-distance mRNA silencing in Arabidopsis. In the case of silencer S1 and S2, the mobile GF signal is delivered into nuclei in the shoot apex and stimulates Pol IVa–RDR2–DCL3 to produce P-specific 24-nt siRNAs (blue) from either the GFP transgene or mRNA. This process also depends on RDR6. AGO4 uses the 24-nt siRNAs to mediate cleavage of some GFP transcript to produce P-specific polyadenylated RNAs, which become a substrate for RDR6. DCL4 or DCL2 then produce 21- or 22-nt siRNAs, respectively, and these direct mRNA silencing.