RNA-mediated gene silencing is a widely conserved mechanism for regulating eukaryotic gene expression during development, in response to environmental changes and in response to pathogen infection. In plants, RNA-mediated gene silencing relies on 21to 24 nucleotide (nt)-long small regulatory RNAs (sRNAs), including small interfering RNAs (siRNAs), microRNAs (miRNAs) and trans-acting small interfering RNAs (ta-siRNAs). These sRNAs direct silencing effector proteins to complementary sequences of DNA or RNA, resulting in down-regulation of the targeted locus through either transcriptional gene silencing (TGS) or post-transcriptional gene silencing (PTGS). In plants, the movement of gene silencing signals from cell-to-cell and/or over long distances is important for normal development and plant defence. Previous studies have used micrografting of transgenic Arabidopsis as a model to study movement of silencing signals from rootstocks to scions, and vice versa. These earlier studies showed that components of both the TGS and PTGS pathways, including RNA polymerase IV (NRPD1A), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), ARGONAUTE4 (AGO4), DICER-LIKE 3 (DCL3) and RDR6 are required for reception of PTGS signals in grafted scions. In addition, NRPD1A and DCL3 have been shown to be required for transmitting TGS from scions to roots. However, the mechanisms controlling silencing signal production and transmission from rootstocks to scions have not been defined.
In this study, we developed a transgenic Green Fluorescent Protein (GFP)-based silencing-reporter system to study the phenomenon of root-to-shoot spreading of PTGS in Arabidopsis thaliana. This system is comprised of a constitutively expressed 35S:GFP transgene linked to a root tip-specific transgene expressing double-stranded RNA homologous to nucleotides nine to 400 of GFP. These components are encoded on a single T-DNA vector called pUQC10027. When pUQC10027 is transformed into Arabidopsis, PTGS of GFP is initiated in the root tips and travels through the hypocotyl to the leaves. When grafted as a rootstock under GFP expressing scions, this line transmits GFP PTGS to the scions. Using the 10027-3 reporter line as the genetic background of rootstocks in grafting experiments, we showed that AGO1, RDR6, SUPPRESSOR OF GENE SILENCING 3 (SGS3) and DCL3 are required for transmission of PTGS from rootstocks to scions. AGO1, RDR6 and SGS3 are components of the trans-acting siRNA (ta-siRNA) biogenesis pathway, but we showed that another component of this pathway, DCL4, is not required for transmission of silencing from rootstocks. DCL3 is required for producing 24nt small interfering RNAs (siRNAs) that are normally associated with RNA-directed DNA methylation. Surprisingly, we showed that 24nt siRNAs produced by DCL3 from RDR6-derived double-stranded RNA appear to be necessary for root-to-shoot graft-transmissible PTGS in Arabidopsis.
We also used the 10027-3 transgenic line in a forward genetic screen to identify novel genes required for root-to-shoot transmission of PTGS. Over 40 independent root-to-shoot transmission of PTGS (rtp) mutants were identified. One new mutant, called rtp1-1, showed delayed root-to-shoot transmission of PTGS, as well multiple developmental defects. We mapped rtp1-1 to a nonsense mutation in the coding sequence of PLEIOTROPIC REGULATORY LOCUS 1 (PRL1). Complementation testing confirmed that PRL1 is required for both normal timing of the onset of PTGS in 10027-3 plants, as well as normal development. The literature shows that PRL1 interacts with an importin alpha called MOS6, and is also a component of a widely conserved complex that is associated with mRNA processing. This combined evidence suggests that PRL1 is involved in intracellular processing and/or trafficking of small regulatory RNAs that are required for root-to-shoot transmission of PTGS during embryogenesis and early seedling development.
We also tested candidate genes for their role in sRNA-associated pathways, including root-to-shoot transmission of PTGS. Two of our candidates were the DEFECTIVE EMBRYO AND MERISTEMS (DEM) genes DEM1 and DEM2, which are redundantly required together for gametogenesis in Arabidopsis. DEM1 interacts with Arabidopsis Ras-related nuclear protein (RAN), which is required for nucleocytoplasmic transport processes, including HASTY (HST)-mediated export of small regulatory RNAs from the nucleus. We used microarrays and sRNA Northern blots to show that dem1, but not dem2 mutants have lower levels of miRNAs and endogenous siRNAs compared to WT. We found DNA methylation pattern changes in T-DNA insertion mutants of dem1 and dem2 mutants compared to their wild-type parents, however, these methylation pattern changes may have resulted from the Agrobacterium-mediated transformation process rater than loss of function of DEM1 or DEM2. We also used the 10027 GFP reporter to show that dem1, dem2 and hst mutants are defective in root-to-shoot transmission of PTGS.