Global analysis of RNA-dependent RNA Polymerase-dependent small RNAs reveals new substrates and functions for these proteins and SGS3 in Arabidopsis

In plants, smRNAs are often derived from long double-stranded RNA (dsRNA) molecules synthesized by one of the six genomically-encoded RNA-dependent RNA Polymerase (RDR) proteins. However, the full complement of the RDR-dependent smRNAs and functions that these proteins and their RNA-binding co-factors play in plant RNA silencing has not been fully uncovered. To address this gap, we performed a global genomic analysis of all six RDRs and two of their co-factors to find new substrates for RDRs and targets of the resulting RDR-derived siRNAs to uncover new functions for these proteins in plants. Based on these analyses, we identified substrates for the three RDR clade proteins (RDR3 - 5), which had not been well-characterized previously. We also identified new substrates for the other three RDRs (RDR1, 2, and 6) as well as the RDR2 co-factor RNA-DIRECTED DNA METHYLATION 12 (RDM12) and the RDR6 co-factor SUPRESSOR OF GENE SILENCING 3 (SGS3). These findings revealed that the target substrates of SGS3 are not limited to those solely utilized by RDR6, but that this protein seems to be a more general co-factor for the RDR family proteins. Additionally, we found that RDR6 and SGS3 are involved in the production of smRNAs that target transcripts related to abiotic stresses including water deprivation, salt stress, and ABA response, and as expected the levels of these mRNAs are increased in rdr6 and sgs3 mutant plants. Correspondingly, plants that lack these proteins (rdr6 and sgs3 mutants) are hypersensitive to ABA treatment, tolerant to high levels of PEG8000, and have higher survival rate under salt treatment in comparison to wild-type (Col-0). In total, our analyses have provided an extremely data-rich resource for uncovering new functions of RDR-dependent RNA silencing in plants, while also revealing a previously unexplored link between the RDR6/SGS3-dependent pathway and plant abiotic stress responses. Small RNA sequencing (15 bp-50 bp) and toal RNA sequencing in T-DNA knockout mutants of six RDRs, SGS3, and RDM12 in Arabidopsis thanliana 30 days unopened flower buds