RNA polymerase V-dependent small RNAs in Arabidopsis originate from small,intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.     

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small,intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.     

Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation

Argonaute (AGO) family proteins are conserved key components of small RNA‐induced silencing pathways. In the RNA‐directed DNA methylation (RdDM) pathway in Arabidopsis, AGO6 is generally considered to be redundant with AGO4. In this report, our comprehensive, genomewide analyses of AGO4‐ and AGO6‐dependent DNA methylation revealed that redundancy is unexpectedly negligible in the genetic interactions between AGO4 and AGO6. Immunofluorescence revealed that AGO4 and AGO6 differ in their subnuclear co‐localization with RNA polymerases required for RdDM. Pol II and AGO6 are absent from perinucleolar foci, where Pol V and AGO4 are co‐localized. In the nucleoplasm, AGO4 displays a strong co‐localization with Pol II, whereas AGO6 co‐localizes with Pol V. These patterns suggest that RdDM is mediated by distinct, spatially regulated combinations of AGO proteins and RNA polymerases. Consistently, Pol II physically interacts with AGO4 but not AGO6, and the levels of Pol V‐dependent scaffold RNAs and Pol V chromatin occupancy are strongly correlated with AGO6 but not AGO4. Our results suggest that AGO4 and AGO6 mainly act sequentially in mediating small RNA‐directed DNA methylation.     

The RNA silencing enzyme RNA polymerase v is required for plant immunity

RNA-directed DNA methylation (RdDM) is an epigenetic control mechanism driven by small interfering RNAs (siRNAs) that influence gene function. In plants, little is known of the involvement of the RdDM pathway in regulating traits related to immune responses. In a genetic screen designed to reveal factors regulating immunity in Arabidopsis thaliana, we identified NRPD2 as the OVEREXPRESSOR OF CATIONIC PEROXIDASE 1 (OCP1). NRPD2 encodes the second largest subunit of the plant-specific RNA Polymerases IV and V (Pol IV and Pol V), which are crucial for the RdDM pathway. The ocp1 and nrpd2 mutants showed increases in disease susceptibility when confronted with the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina. Studies were extended to other mutants affected in different steps of the RdDM pathway, such as nrpd1, nrpe1, ago4, drd1, rdr2, and drm1drm2 mutants. Our results indicate that all the mutants studied, with the exception of nrpd1, phenocopy the nrpd2 mutants; and they suggest that, while Pol V complex is required for plant immunity, Pol IV appears dispensable. Moreover, Pol V defective mutants, but not Pol IV mutants, show enhanced disease resistance towards the bacterial pathogen Pseudomonas syringae DC3000. Interestingly, salicylic acid (SA)-mediated defenses effective against PsDC3000 are enhanced in Pol V defective mutants, whereas jasmonic acid (JA)-mediated defenses that protect against fungi are reduced. Chromatin immunoprecipitation analysis revealed that, through differential histone modifications, SA-related defense genes are poised for enhanced activation in Pol V defective mutants and provide clues for understanding the regulation of gene priming during defense. Our results highlight the importance of epigenetic control as an additional layer of complexity in the regulation of plant immunity and point towards multiple components of the RdDM pathway being involved in plant immunity based on genetic evidence, but whether this is a direct or indirect effect on disease-related genes is unclear.