The RNA Silencing Enzyme RNA Polymerase V Is Required for Plant Immunity

Abstract

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.

The Arabidopsis Elongator Complex Subunit2 Epigenetically Regulates Plant Immune Responses

“…Mutations in several components of the RNA-directed DNA methylation pathway have been shown to alter immune responses to Pst DC3000. While mutations in AGRONAUTE4 (AGO4) compromise resistance to Pst DC3000 (Agorio and Vera, 2007), mutations in NRPE1 and NRPD2, which encode the largest subunit of the RNAP V complex and second largest subunit of the RNAP IV and V complexes, respectively (Law and Jacobsen, 2010), enhance resistance to this pathogen (López et al, 2011). The DNA methylation mutants met1-3 and ddc (drm1-2 drm2-2 cmt3-11), which are deficient in CG maintenance methylation and non-CG maintenance/de novo methylation, respectively (Saze et al, 2003;Chan et al, 2006;Penterman et al, 2007a), are highly resistant to Pst DC3000 (Dowen et al, 2012;Luna et al, 2012).…”

“…The DNA methylation mutants met1-3 and ddc (drm1-2 drm2-2 cmt3-11), which are deficient in CG maintenance methylation and non-CG maintenance/de novo methylation, respectively (Saze et al, 2003;Chan et al, 2006;Penterman et al, 2007a), are highly resistant to Pst DC3000 (Dowen et al, 2012;Luna et al, 2012). As AGO4 might play some unknown functions independent of the RNA-directed DNA methylation pathway (López et al, 2011), it has been proposed that DNA methylation represses immune responses to Pst DC3000 in the absence of the pathogen (Dowen et al, 2012). However, we found that the DNA demethylase triple mutant rdd (ros1-3 dml2-1 dml3-1), which contains genome-wide DNA hypermethylation (Penterman et al, 2007b;Lister et al, 2008), also exhibits constitutively elevated resistance to bacterial pathogens (see Supplemental Figure 8 online).…”

Epigenetic Changes in Hybrids

“…The expression of a number of pathogen-responsive genes is up-regulated in mutants defective in DNA methylation (met1 and ddc1 [a drm1, drm2 and cmt3 triple mutant] mutants; Dowen et al, 2012). López et al (2011) reported that mutants defective in RNA POLYMERASE V, which is involved in DNA methylation through the RdDM pathway, show an up-regulation of SA-responsive genes and enhanced resistance toward Pseudomonas syringae. In rice, hypomethylation by treatment with the demethylating agent 5-azadeoxycytidine causes increased resistance to virulent strains of the bacterium Xanthomonas spp.…”

Diverse Gene-Silencing Mechanisms with Distinct Requirements for RNA Polymerase Subunits in Zea mays

“…There is also evidence in Arabidopsis that Pol IV and Pol V activity can be uncoupled (Wierzbicki et al 2008(Wierzbicki et al , 2012Pontes et al 2009;Lopez et al 2011) and that Pol II-and Pol V-occupied sites are frequently found in close proximity to one another (Wierzbicki et al 2012). Therefore, it is feasible that, in the absence of Pol V, Pol II might be positioned to compensate for this activity.…”