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.


Introduction
RNA-directed DNA methylation (RdDM) is an epigenetic modification mechanism driven by noncoding small interfering RNAs (siRNAs) [1,2]. siRNAs are present in most eukaryotic organisms, are highly developed in plants and regulate gene expression at the transcriptional and posttranscriptional level in a sequence-specific manner. In contrast to microRNAs (miRNAs) that are derived from the transcripts of miRNA genes generated by RNA Polymerase II, production of RdDM-associated siRNAs requires RNA Polymerase IV (Pol IV) complex activity which includes, among other constituents, the largest and second largest subunits, NRPD1 and NRPD2, respectively [3][4][5]. Upon the action of Pol IV, the resulting single-stranded RNAs are used as templates for RNA-dependent RNA polymerase 2 (RDR2) generating double-stranded RNAs, which are processed by DICER-LIKE 3 (DCL3) [6,7]. Subsequently, RNA methyltransferase HUA ENHANCER-1 (HEN1) generates functional siRNAs that are recruited by ARGONAUTE4 (AGO4) to form the AGO4-RISC multiprotein complex guided to siRNA-complementary genome sequences [8][9][10]. AGO4-siRNA complexes interact with the RNA Polymerase V (Pol V) complex, which includes the largest and second largest subunits, NRPE1 and NRPD2, respectively. Pol V is somehow required to recruit DRM2 methyltransferase as well as histone-modifying complexes to finally establish the methylation pattern in the siRNAcomplementary genome sequences; however, the details of this recruitment are unknown. This process results in the methylation of certain genome repeat regions and their subsequent transcriptional silencing [2]. Among the different classes of siRNA, the 24 nt in lenght hetrocromatic siRNAs (hc-siRNAs) and repeatassociated siRNAs (ra-siRNAs), primarily derived from transposons, repeated elements and heterochromatin regions, are those functioning in the RdDM pathway by mediating DNA methylation and/or histone modification at the target sites [2].
Small RNAs regulate a multitude of biological processes in plants, including sustaining genome integrity, development, metabolism and responses to changing environmental conditions and abiotic stress [11]. Increasing evidences also indicate that plant endogenous small RNAs, including miRNAs and siRNAs, are integral regulatory components of plant defense machinery against microbial pathogens [12]. The Arabidopsis miR393 imparts basal resistance to the bacterial pathogen Pseudomonas syringae DC3000 by targeting the auxin receptors TIR1, ABF2 and ABF3 [13]. Besides miR393, two other miRNA families, miR160 and miR167, are upregulated following PsDC3000 inoculation and target members of auxin-response factors (ARF) [14]. Thus, in response to bacterial infection, plants suppress multiple components of the auxin signaling pathway. In turn, bacteria have developed type III secretion effectors that repress transcription of miRNA genes, the host RNA silencing machinery is suppressed and therefore disease susceptibility increase [15]. Similarly, Lu et al. [16] identified a series of 10 miRNAs families in loblolly pine whose expression were suppressed, and the transcript levels of their target genes increased, upon infection with the rust fungus Cromartium quercuum f. sp. fusiforme. Likewise, upon infection of Brasica rapa with Turnip mosaic virus (TuMV) the miR1885 is upregulated, and its target is predicted to be a member of the TIR-NBS-LRR class of disease-resistance proteins [17]. Thus, it appears that following detection of pathogenassociated molecules, plant cells undergo changes in miRNA global profiles that mediate the establishment of a specific defense response [12,18].
Although plants contain only several hundred miRNAs, they contain huge numbers of endogenous siRNAs but only in a few cases the involvement of siRNAs in plant immunity has been described. In Arabidopsis, the natural antisense transcript (NAT)derived nat-siRNAAATGB2 and the long siRNA lsiRNA-1, which specifically targets the mitochondrial pentatricopeptide protein(PPR)-like gene PPRL and the RNA-binding protein gene AtRAP, respectively, are induced by the bacterial pathogen PsDC3000(avrRpt2) and contribute to plant antibacterial immunity [19,20]. The endogenous siRNAs generated at disease resistance RPP4 locus, which impart resistance to both the bacterial Ps pv. maculicola and the oomycetes Hyaloperonospora arabidopsidis, constitute a third example for siRNA-mediated resistance responses [21]. However, it remains unclear how RdDM participates in this type of processes.
The understanding of the overall contribution and requirement of the different components that conform the RdDM pathway, and how important they are in the regulation of the RdDMmediated processes, particularly in relation to plant immunity, is an issue that still remains to be fully understood. Previously we described a genetic screen in Arabidopsis design to identify mutants (ocp mutants) with altered immune responses [22]. This allowed identifying AGO4, through the characterization of its mutant allele ago4-2/ocp11, as an important component of the RdDM pathway in mediating plant immune responses towards PsDC3000 [23]. Towards characterizing the contribution of other components of the RdDM pathway in plant immunity, we report here on the isolation and characterization of ocp1, a recessive mutant allele of NRPD2. Our results support that RdDM, through the action of RNA Pol V, is pivotal in modulating immune responses towards pathogens.

Characterization of ocp1 Plants
The Arabidopsis ocp mutants were identified previously in a genetic screen [22] designed to isolate negative regulators of pathogen-induced defense responses. The H 2 O 2 -responsive and defense-related Ep5C gene promoter fused to GUS was used as reporter [24]. Here we described the characterization of the ocp1 mutant. Figure 1A shows the constitutive Ep5C::GUS expression in rosette leaves from ocp1 plants compared with its parental Col-0 line (line 5.2). ocp1 plants exhibited similar plant architecture and growth habit to the wild-type plants ( Figure 1B). F 1 hybrids from a backcross between parental and ocp1 plants showed the absence of GUS activity, and GUS activity segregated in the F 2 progeny as a single recessive Mendelian locus [OCP1:ocp1, 111:33 (P,0.05, x 2 test)].
We hypothesize that the constitutive expression of Ep5C::GUS observed in ocp1 plants might be accompanied by an altered disease resistance response to pathogens as previously revealed in ocp3 and ocp11 plants [22,23,25,26]. Therefore, we inoculated ocp1 plants with the virulent necrotrophic fungal pathogen Botrytis cinerea and monitored the disease response in leaves in comparison with the parental line. Disease was scored by recording the extent of necrosis. Wild-type plants exhibited normal susceptibility to B. cinerea ( Figure 1C), with inoculated leaves showing necrosis accompanied by extensive proliferation of the fungal mycelia. In contrast, ocp1 plants showed increased susceptibility to B. cinerea distinguished by moderate but statistical significant enlargement of necrotic areas at inoculation sites ( Figure 1C).
Susceptibility of ocp1 plants to pathogens was also investigated with the bacterial pathogen PsDC3000. The npr1 mutant, which is compromised in resistance towards this pathogen [27] was used as a control. Resulting bacterial growth in inoculated leaves is shown in Figure 1D and indicates the wild-type and ocp1 mutant susceptibility was unchanged towards virulent PsDC3000. In addition, plants were inoculated with an avirulent strain of PsDC3000 carrying the avrRpm1 gene that triggers a hypersensitive cell death response in the plant that stops bacterial growth. The rpm1 mutant, compromised in the hypersensitive response and consequently hypersusceptible to the pathogen, was used as a control. Results showed the growth of PsDC3000 (avrRpm1) in ocp1 plants was not significantly different to that observed in wild-type plants ( Figure 1E). These results were consistent with normal accumulation of transcripts of the salicylic acid (SA)-responsive gene PR-1 at 48 h following inoculation with PsDC3000 ( Figure 1F), however induction occurs earlier in ocp1 plants. Interestingly, induction of the jasmonic acid (JA)-responsive gene PDF1.2a, a characteristic molecular response of plants to fungal attack, was compromised in ocp1 plants following inoculation with B. cinerea ( Figure 1G). This later observation is congruent with the observation that ocp1 plants showed enhanced disease susceptibility to this pathogen ( Figure 1C).

Author Summary
The influence of epigenetic regulation in controlling the adaptive responses of living organisms to changes in the environment is becoming a common theme in biology. RNA-directed DNA methylation (RdDM) is an epigenetic control mechanism driven by a subset of noncoding small interfering RNAs (siRNAs) that influence gene function without changing DNA sequence by inducing de novo methylation of cytosines, or by modification of histones, at their target genomic regions. The implication and roles of the RdDM mechanism in the orchestration of plant immune responses still remains to be characterized. A recent study in the model plant Arabidopsis showed that ARGONAUTE4, one of the characteristic components of the RdDM pathway, was required for plant immunity against bacterial pathogens. Here, in a genetic screen aiming to identify cellular factors integral in regulating immunity in Arabidopsis, we further identified that the RNA polymerases V, another crucial component of the RdDM pathway, is pivotal for plant immunity against fungal pathogens. Similarly, we identified that additional components of the RdDM pathway, but surprisingly not RNA polymerase IV, are similarly required for plant immunity. Based on genetic evidence, our results highlight the importance of RdDM as an additional layer of complexity in the regulation of plant immune responses.
OCP1 Is At3g23780 and Encodes NRPD2, the Second Largest Subunit of the RNA Pol IV and Pol V The genetic lesion carried by ocp1 plants was identified by positional cloning ( Figure S1). A single nucleotide deletion was detected on locus At3g23780, particularly in the third exon of the transcribed gene encoding NRPD2, the second largest subunit of the RNA Pol IV and Pol V protein complexes (Figure 2A and Figure S1C). The loss of a nucleotide residue created a change in the NRPD2 open reading frame that leads to a frame shift starting at residue 595 ( Figure 2A) followed by an incorrect 22 amino acid C-terminal tail sequence before an in-frame stop codon ( Figure  S2). The mutation renders a protein of 616 amino acid residues, instead of the 1172 contained in NRPD2, that thus has lost almost half of the protein sequence, including the amino acids that contribute to the active site of RNA polymerases [28].
The result obtained in our mapping strategy was corroborated with a test of allelism between ocp1 plants and plants carrying a null allele of NRPD2, in particular with nrpd2-2 plants which carry a T-DNA insertion (SALK_046208) [3]. Analysis of GUS expression driven by the Ep5C gene promoter in 20 F1 plants derived from a cross between homozygous ocp1 plants with homozygous nrpd2-2 plants or, alternatively, from a reversed cross between nrpd2-2 plants with ocp1 plants, revealed that all F1 plants showed constitutive GUS expression ( Figure S3). Conversely, control crosses between the parental Col-0 plants carrying the Ep5C::GUS gene construct (line 5.2) with either ocp1 plants or nprd2-2 plants revealed no GUS expression in any of the F1 22 plants analyzed ( Figure S3). The result indicates that the ocp1 and nrpd2-2 are mutant alleles of the same NRPD2 gene and supported the conclusion that the ocp1 mutation represents a loss of function allele. Hence, the ocp1 mutation will be referred also as ocp1/nrpd2-53.
From the type of mutation found, we cannot exclude the possibility that ocp1 plants are still able to produce a truncated version of the NRPD2 protein with a residual ability to interact with other components of the RNA polymerase complexes. Since Pol IV and Pol V complexes are comprised of a variety of interacting subunits, some being polymerase-specific while other subunits shared (i.e., NRPD2) [5,29,30], and with some cross-talk described for some of their subunits (i.e., between NRPD2 and NRPE1; [4]), we can not discard the possibility that the relationships between the different components of the two RNA polymerase complexes may become differentially altered in the ocp1 mutant. In this respect, the availability of the ocp1 allele may represent a valuable experimental tool to approach the biochemical regulation of the RdDM mechanism.
Interestingly, RT-PCR analyses of NRPD2 transcript levels in ocp1 plants revealed the absence of notable changes in gene expression compared with Col-0 plants ( Figure 2B). This is in marked contrast with the expression observed in nrpd2-2 null mutant plants where no transcript amplification products can be obtained ( Figure 2B). A comparison of the disease resistance response between ocp1 and nrpd2-2 plants revealed that while the ocp1 plants showed a moderate increase in susceptibility to B. cinerea, the nrpd2-2 null mutant responded to B. cinerea infection with a remarkable enhancement in susceptibility ( Figure 2C). The enhanced susceptibility phenotype of nrpd2-2 plants was further corroborated by recording the susceptibility towards Plectosphaerella cucumerina, a different fungal necrotroph ( Figure 2D). Consistent with the observed increase in disease susceptibility to P. cucumerina, RT-qPCR experiments revealed that induction of the JAresponsive PDF1.2a gene was disabled in nrpd2-2 plants compared to Col-0 ( Figure 2E). These results mirror what occurs in ocp1 plants following B. cinerea infection ( Figure 1F). Of importance for understanding the immune-related phenotype of nrpd2-2 plants is the observation that expression of the SA-responsive PR-1 gene was clearly enhanced following fungal inoculation in the mutant when compared to wild-type plants ( Figure 2F). Since nrpd2-2 plants show an enhanced disease susceptibility of bigger magnitude than that observed in ocp1/nrpd2-53 plants, subsequently, the experiments related to disease resistance/susceptibility will be carried out employing the nrpd2-2 allele.
SUPERMAN, 5S Genes, and the AtSN1 Retroelement Are Hypomethylated in ocp1 Plants To further substantiate the molecular phenotype of ocp1 plants in relation to RdDM, we checked if the methylation status of different RdDM target sequences could be similarly affected in ocp1 and nrpd2-2 plants. We analyzed the methylation status in ocp1 plants of the RdDM pathway DNA target sequences SUPERMAN, ribosomal 5S genes and the retrotransposon AtSN1 [31]. We used methylation tests employing the methylation-sensitive restriction endonuclease HaeIII (where HaeIII will not cut DNA if methylated), with subsequent amplification by PCR [32]. Initial experiments revealed that ocp1, as well as ago4-2/ocp11 plants used as controls, exhibit a higher degree of hypomethylation in SUPERMAN gene compared to Col-0 plants ( Figure 3A). Analyses were extended to the ribosomal 5S genes and the AtSN1 retrotransposon and we incorporated nrpd2-2, nrpd1-3 and nrpe1-1 mutants for comparison. Figure 3B shows mutants demonstrated higher degrees of hypomethylation in the sequences analyzed. DNA samples derived from ocp1 plants exhibited decreased amplification for the 5S and AtNS1 loci, confirming a clear DNA methylation deficiency in this mutant. The ABI5 gene, whose sequence contains no restriction sites for HaeIII, was used as a control. Methylation tests were also used to ascertain whether or not the enhanced induction observed for the PR-1 gene, or the repression of PDF1.2a, in the nrpd2 mutant following fungal infection correlated with defects in the DNA methylation of their promoter regions. Since both genes contain a large number of recognition sites for the methylation-sensitive restriction enzymes FspEI, MspJI and AvaII (168 target sequences in the PR-1 gene and 298 targets in the PDF1.2a gene), and where FspEI and MspJI sites must be methylated for the enzymes to cleave the DNA, we used restriction analysis with these enzymes with subsequent amplification by PCR to check the methylation status of the PR-1 and PDF1.2a genes. The results shown in Figure S4 and Figure S5 revealed that none of the promoters appear methylated, not even in Col-0 plants. Conversely, the sensitivity of the methylated 5S ribosomal DNA ( Figure S4) to the aforementioned enzymes revealed the appropriateness of the method used to identify methylation of cytosine residues. The lack of a methylation footprint in the DNA of the defense-related PR-1 and PDF1.2a genes might suggest that the abnormal expression patterns concurring in nrpd2 mutant plants must obey not to a direct modification of cytosine residues but to other type of chromatin modification or mechanism similarly controlled either directly or indirectly by the RdDM pathway.
The Pol V Complex, But Not Pol IV, Is Required for the Correct Immune Response against B. cinerea and P. cucumerina As for NRPD2, we addressed if other RdDM pathway components are similarly engaged in plant immunity. A comparative analysis of the disease resistance response of nrpd1, nrpe1, and ago4 mutant plants due to inoculation by B. cinerea was performed in relationship to nrpd2. Figure 4A shows an increase in nrpe1 disease susceptibility to B. cinerea; the susceptibility being of a magnitude similar to that attained in nrpd2 plants. This enhancement in susceptibility was comparatively greater than that observed in ocp1 plants but less than in ago4-2/ocp11 plants. Conversely, nrpd1 plants did not exhibit a significant deviation from the normal disease response observed in Col-0 plants. This differential behavior was further corroborated in the Pol IV and Pol V defective mutants by challenging with P. cucumerina ( Figure 4B). The nrpd1 nrpe1 double mutant that would be defective in both Pol IV and Pol V activities was incorporated in this experiment for comparison. nrpd1 nrpe1 plants showed an enhanced disease susceptibility of a magnitude similar to that attained in nrpe1 or nrpd2 plants. Furthermore, fungal biomass determination in leaves inoculated with P. cucumerina, as an alternative method for recording disease resistance, also revealed that the single nrpd2 and nrpe1 mutants, as well as the double nrpd1 nrpe1 mutant support significantly more fungal growth than Col-0 and the nrpd1 mutant ( Figure S6). Therefore, the Pol V complex participates in the regulation of the immune response to necrotrophs while the Pol IV complex appears at least partially dispensable. This is sustained also by the observation that expression patterns of the PDF1.  Figure S7B). Notorious is the higher JA-triggered PDF1.2 gene induction in nrpd1 plants in comparison to Col-0 ( Figure S7B). Conversely, this JA-triggered PDF1.2a gene induction is notably repressed in the Pol V defective mutants ( Figure 4C and Figure  S7B). This is in marked contrast with the altered expression pattern observed in nrpd2 plants where induction of PR-1 gene expression showed enhancement following inoculation with P. cucumerina ( Figure 4D) or upon external application of SA ( Figure  S7A). Importantly, this pattern of gene expression was reproduced in nrpe1 plants ( Figure 4D and Figure S7A). Moreover, the transcription factors WRKY6 and WRKY53 that bind W-box and transcriptionally regulate gene expression of SA-related genes, including PR-1 [33], and are themselves induced by pathogen infection [34], show similar enhanced level of induction following SA application in nrpd2 and nrpe1 plants when compared to Col-0 or nrpd1 plants ( Figure S7C and S7D). drd1, rdr2, and drm1drm2 Mutants Show Compromised Immune Responses to P. cucumerina To further extent the implication of RdDM mechanism in plant immunity we inoculated the drm1drm2 double mutant plants, which is compromised in de novo DNA methylation [35], with P. cucumerina and recorded the disease response. Results in Figure 4E reveal that loss of the functional RDM methyltransferase compromise disease resistance and results in plants showing enhanced susceptibility to P. cucumerina to levels even higher than in nrpd2 plants. This reinforces the proposal that RdDM is pivotal for plant immunity. Likewise, we observed that the chromatinremodeling factor DRD1, which is required for the association of NRPE1 with chromatin [36], is also pivotal for plant immunity. Figure 4E shows that drd1-6 plants phenocopy Pol V defective mutants. The RNA-dependent RNA polymerase 2 (RDR2), which functions early in the RdDM pathway by generating dsRNA from the ssRNA transcripts thought to emanate presumably from the the Pol IV complex was also entertained in these experiments. Intriguingly, rdr2 plants also show a strikingly enhancement in susceptibility to fungal infection ( Figure 4E), achieving highest levels of susceptibility to P. cucumerina. This observation strongly argues in favor of RDR2 as required for plant immunity. Then, how can a mechanism explain the exclusion of RNA Pol IV in mediating plant immunity while the rest of downstream components of the RdDM pathway are engaged in this biological process? There are previous evidences where Pol V has been described to operate independently of Pol IV, such as in the mechanism for maintaining the methylation status of target sequences [37], and thus for some processes Pol IV and Pol V act not in concert [38]. A hypothesis that could help explain the paradoxical observations indicating that Pol V, RDR2, AGO4, DRD1 and DRM2, but not Pol IV, are required for plant immunity to fungal pathogens could be one where RDR2 can accept RNA transcripts derived from the action of RNA Pol V, and not necessarily only from RNA Pol IV. These putative transcripts thought to be acted upon by RDR2, which generates dsRNA, will be processed into siRNAs and feed into the RdDM pathway. In support for the existence of Pol V-dependent transcripts required for DNA methylation and silencing is the recent identification of low-abundance intergenic non-coding (IGN) transcripts [36]. It could be that a similar situation is on the basis to explain the requirement of RdDM for plant immune responses. This possibility merits future research approaches.

SA-Mediated Defense Genes Are Poised for Enhanced Activation by Histone Modifications in Pol V Defective Mutants
The previous results suggest that in Pol V defective mutants SArelated defense genes are poised for enhanced activation following perception of pathogenic cues and concurrently JA-related defenses appear impeded for induction. This will be congruent with a notion where Pol V may regulate a priming phenomenon for SA-mediated defense responses that ultimately would modulate the speed and extent of gene activation. However, the lack of a methylation footprint in the DNA of the defense-related PR-1 and PDF1.2a genes ( Figure S5 and Figure S6) suggest that the observed abnormal gene expression patterns concurring in the Pol V defective mutants is not to be due to an altered DNA methylation pattern resulting from a defective RdDM pathway. However, one could still entertained the possibility that changes in chromatin structure such as those obeying to covalent modification of histones, which are also under the control of the RdDM pathway, may be on the basis for the enhanced expression observed for PR-1 and, therefore, for the altered resistance phenotypes in the mutant plants. This would be congruent with the recent identification of a mechanism linking chromatin modification in wild type plants, through the differential modification of histones in several genes encoding WRKY transcription factors (i.e. WRKY6, WRKY29 or WRKY53), with priming of a defense response following pharmacological treatment with the SA analogue acidobenzolar S-methyl (BTH) which functions as a priming agent in plants [39]. Thus, we hypothesized that in Pol V defective mutants PR-1 could be poised for enhanced activation of gene expression by a differential modification of histones.
By using chromatin immunoprecipitation (ChIP) we analyzed trimethylation of histone H3 Lys4 (H3K4me3) and acetylation of histone H3 Lys9 (H3K9ac) on the promoter of the PR-1 gene. For comparison, the promoter of the JA-inducible PDF1.2a gene, that of the constitutively expressed Actin2 gene and also those of the WRKY6 and WRKY53 genes were similarly studied. The specificity of the ChIP reaction was evaluated in advance by measuring histone modifications on these genes in Col-0 plants treated with BTH ( Figure S8A and S8B). On the PR-1 promoter H3K4me3 and H3K9ac marks increased after BTH application while these marks did not change in the promoters of Actin2 or PDF1.2a ( Figure S8A and S8B). As for PR-1, these chromatin marks were similarly increased in the promoters of WRKY6 and WRKY53 upon treatment with BTH ( Figure S8C). Thus chromatin marks normally associated with active genes [39,40] are set in the promoters of SA-related defense genes by the priming stimulus of BTH. Interestingly, determination of H3K4me3 ( Figure 5A) and H3K9ac ( Figure 5B) chromatin marks in the PR-1 promoter in ChIP samples derived from nrpd2 and nrpe1 plants, revealed that these marks are already set in these two mutants, although PR-1 gene activation does not take place. Thus, Pol V defective mutants mimic Col-0 plants treated with the priming agent BTH. This reconciles with the idea that the PR-1 gene is switch on for priming in the Pol V defective mutant and explains why this gene shows enhanced induction upon pathogenic attack in the same mutants ( Figure 4D). In the nrpd1 mutant only a moderate increase in the setting of these chromatin marks in the promoter of PR-1 was detected ( Figure 5A and 5B). No variation in similar activation marks was observed in the promoters of the Actin2 and PDF1.2a genes (Figure 5A and 5B). Other histone marks, such as H3K9me2 and H3K27me3, both of which repressive marks normally associated with heterochromatin and established through the RdDM pathway [41], appear notably reduced in the PR-1 promoter in ChIP samples derived from nrpd2 and nrpe1 plants, and much less reduced in nrpd1 plants, when compared to Col-0 plants ( Figure S9A and S9B). Moreover, Col-0 plants respond to P.cucumerina infection with reduction in the setting of these two repressive histone marks in the PR-1 gene promoter but not in the promoters of the PDF1.2a or Actin2 genes ( Figure S9C). The dismantling of histone repressive marks in infected plants, along with the concurring increase in histone activation marks and decrease in repressive marks in the promoter of the PR-1 gene, as observed in nrpd2 and nrpe1 plants, gives further support to the implication of Pol V in regulating defense gene activation.
As for PR-1, H3K4me3 activation marks are also constitutively set in the promoters of the WRKY6 and WRKY53 genes in healthy nrpd2 and nrpe1 plants ( Figure S8C), again mirroring the effect carried out by BTH on Col-0 for these promoters ( Figure S8C). Further analysis demonstrated that Col-0 plants respond to P. cucumerina infection with a drastic increase in the setting of H3K4me3 and H3K9ac activation marks in the promoters of PR-1 ( Figure 5C and 5D). In nprd2 plants, in which these chromatin marks are already set in PR-1, P. cucumerina inoculation further increase H3K4me3 marks on the PR-1 promoter to levels that are even higher than those attained in Col-0 ( Figure 5C). However, for H3K9ac marks no further increase was observed in nrpd2 plants, suggesting that this type of mark is completely set in the mutant. In contrast, no variation in the setting of these chromatin marks was detected in the PDF1.2a promoter upon fungal infection ( Figure 5C and 5D). For WRKY6 and WRKY53 gene promoters, Col-0 plants respond to P. cucumerina infection by similarly increasing H3K4me3 mark setting in both promoters ( Figure S10). Compared to Col-0, nrpd2 plants constitutively carry increased H3K4me3 mark setting in WRKY6 and WRKY53 gene promoters and do not show further increases upon inoculation, but instead slightly decrease ( Figure S9). Together, these data imply that Pol V, either directly or indirectly, regulates the extent of chromatin modifications on SA defense-related gene promoters, and may be the underlying mechanism controlling priming marks facilitating the more rapid activation of gene expression observed upon perception of pathogenic cues. As reported for other genes, the observed covalent modifications in chromatin might provoke increases in the accessibility of DNA or perhaps in the provision of docking sites for gene activators [42,43].

nrpd2 and nrpe1 Plants Show Enhanced Resistance to PsDC3000
Enhanced activation of SA-mediated defenses is characteristic of plants resistant to biotrophic pathogens, like PsDC3000, and is on the basis for a systemic type of immunity known as systemic acquired resistance (SAR) [44]. Our results on a priming effect for enhanced expression of SA defense-related genes in nrpd2 and nrpe1 plants suggest these mutants may be altered in the resistance to PsDC3000. Consequently, we addressed Pol IV and Pol V defective mutants in search for defects in the immune response to PsDC3000. We used ago4-2/ocp11 and npr1 plants as controls, both exhibiting heightened PsDC3000 disease susceptibility [23,27]. Interestingly, a significant enhanced disease resistance to PsDC3000 was observed in nrpd2, nrpe1, and in nrpd1 nrpe1 plants, when compared to Col-0 plants ( Figure 6). In contrast, statistically significant effects were not observed in nrpd1 plants relative to Col-0 in response to PsDC3000, giving further support to the idea that RNA Pol IV seems not engaged in plant immunity. The observed heightened resistance towards PsDC3000 in nrpd2 and nrpe1 plants indicated that in wild-type plants Pol V is required for susceptibility to this pathogen. However, in ago4-2/ocp11 plants resistance to PsDC3000 is severely compromised. Although there is no obvious explanation for this contrasting effect, as previously stated [23] one can speculate that AGO4 can serve a novel function, and while required for an effective defense response it may operate independently of the RdDM pathway.
An important observation derived from the results presented is the co-existence of an enhanced disease resistance to a biotrophic bacteria, like PsDC3000, with an enhanced susceptibility to necrotrophic fungi in Pol V defective mutants. This reveals an underlying complexity in the control of disease resistance by RdDM. The SA and JA signal pathways are under an antagonistic equilibrium that occasionally culminates with the partial inhibition of one pathway when the other is facilitated. Consequently the interaction between pathways serves to optimize responses to a specific type of pathogenic insult [45]. Our results demonstrated that nrpd2 and nrpe1 plants are poised for enhanced activation of SA defense-related genes and respond to pathogen attack with a marked enhancement in the induced expression of marker genes, which suggests these plants are more prone to mobilize the defense arsenal controlled by SA. A simpler explanation for these observations is that in wild type plants Pol V negatively regulates a priming mechanism for SA-mediated disease resistance while keeping intact a JA-mediated disease resistance. Defects in Pol V function, such as those observed in nrpd2 and nrpe1 mutants, derepress the priming mechanism for SA-mediated resistance through pertinent chromatin modifications, and renders enhanced resistance to PsDC3000. As a tradeoff, presumably mediated through endogenous antagonistic cross talk mechanisms, misregulation of the JA-mediated disease resistance occurs. This thus explaining the repressed expression of JA-marker gene and the heightened susceptibility of nrpd2 and nrpe1 plants to fungal pathogens. However, although this mechanism seems very likely, we still cannot disregard the possibility that RdDM may be similarly required for normal expression of one or more unknown genes involved in JA signaling. Disruption of RdDM could thus lead to a disruption of JA signaling which would in turn result in hyper-activation of SA signaling. In fact, mutant plants with JAmediated signaling pathway defects and hypersensitivity to fungal necrotrophs concurrently present a less repressed SA-mediated signaling pathway, resulting in a more efficient defense response when challenged with biotrophic pathogens [45,46]. Experiments directed towards identification of an epigenetic footprint associated to the JA pathway merits future reach and will help clarify the complexity of the antagonistic cross-talk mechanism between the SA and the JA signal transduction pathways.
A deeper understanding on how the RdDM and associated chromatin modification acts as a mechanism controlling gene priming and induced immune responses in plants, and how pathogens may counteract this epigenetic regulation for their own benefit will open new avenues for the a better knowledge on how plant immunity is orchestrated.

GUS Staining
Plant leaves were incubated overnight at 37uC in GUS staining buffer as previously described [22].
The ocp1 mutant was backcrossed twice to the P Ep5C :GUS line to confirm its recessive inheritance. ocp1 plants were crossed to Ler, and F 1 plants were allowed to self. F 2 plants were scored for cosegregation of high constitutive GUS activity with simple sequence length polymorphisms (SSLP) [40]. Molecular markers were derived from the polymorphism database between the Ler and Col-0 ecotypes (http://www.arabidopsis.org).

PCR-Based Methylation Assays
Methylation tests using the methylation-sensitive endonuclease HaeIII, FspEI, AvaII and MspJI were performed as described [32]. The relative DNA fragment amounts corresponding to SUPER-MAN, 5S and AtSN1 were obtained after 30, 25 and 35 respective PCR cycles. For ABI5, 30 (A) or 26 (B) PCR cycles were used. PR-1 and PDF1.2a methylation assays are provided in a supplemental file.

Expression Analysis
Gene expression analysis, by either RT-PCR or RT-qPCR was performed as described previously [23]. The primers used to amplify the different genes and DNA regions, and the PCR conditions employed for genotyping T-DNA insertions, and RT-PC and qRT-PC experiments are provided in the supporting information file Text S1.

Bacterial and Fungal Bioassays
Bacterial strains were grown overnight and used to infect 5week-old Arabidopsis leaves by infiltration and bacterial growth determined as described [23]. Twelve samples were used for each data point and represented as the mean 6 SEM of log c.f.u./cm 2 . B. cinerea and P. cucumerina bioassays were performed as previously described [24]. Fungal disease symptoms were evaluated by determining the lesion diameter (in mm) of a minimum of 30 lesions. All experiments were repeated at least three times with similar results.

Chromatin Immunoprecipitation
Chromatin isolation and immunoprecipitation were performed as described [47]. Chip samples, derived from three biological replicates, were amplified in triplicate and measured by quantitative PCR using primers for PR-1, WRKY6, WRKY53 and Actin2 as reported [39]. The rest of primers are described in Text S1. All ChIP experiments were performed in three independent biological replicates. The antibodies used for immunoprecipitation of modified histones from 2 g of leaf material were antiH3K4m3 (#07-473 Millipore), antiH3K4ac (#07-352 Millipore), antiH3K9me2 (ab1772 Abcam) and anti-H3K27me3 (ab6002 Abcam). Figure S1 ocp1 is At3g23780 and Encodes NRPD2. To identify the genetic lesion carried by ocp1 plants, we performed positional cloning of the mutation. To map the position of ocp1 in the genome, we crossed ocp1 plants to Landsberg erecta (Ler) plants, and F 2 plants were scored for co-segregation of high constitutive GUS activity with simple sequence length polymorphisms (SSLP) [48]. An initial analysis of 40 ocp1 individuals allocated the ocp1 mutation in chromosome III, between markers nga162 and AthGAPAB which define an interval of 22.2 cM. Further analysis of 472 plants with 12 new polymorphic markers allowed narrowing the position of ocp1 to an interval of 246.291 pb located between markers CER455355 and CER454777 and comprising 6 BAC clones (A). Four new SSLP markers and one dCAPF (Derived Cleaved Amplified Polymorphic Sequences) marker were analyzed for this mapping interval, and we deduced that the ocp1 lesion was located between markers CER457821 and CER457824, delimiting an interval of 36 kb that comprised a region of 10 ORFs (B). DNA sequencing of this 36 kb interval allowed us to find a guanosine residue deleted in the third exon of the NRPD2 gene (C). (TIF) Figure S2 Comparative amino acid sequences of NRPD2 and OCP1. In blue is indicated the 22 extra amino acid residues preceding the premature stop codon arising due to the nucleotide deletion identified in the ocp1 mutant. In red is indicated the S to T transition due to the change in the open reading frame as a consequence of the deleted nucleotide. (TIF) Figure S3 ocp1 is allelic to nrpd2. The result obtained in our cloning strategy was corroborated with a test of allelism between ocp1 plants and plants carrying the nrpd2-2 allele. Analysis of GUS expression driven by the Ep5C gene promoter in 20 F1 plants derived from a cross between homozygous ocp1 plants with homozygous nrpd2-2 plants or, alternatively, from a reversed cross between nrpd2-2 plants with ocp1 plants, revealed that all F1 plants showed constitutive GUS expression. Conversely, control crosses between the parental Col-0 plants carrying the Ep5C::GUS gene construct (line 5.2) with either ocp1 plants or nprd2-2 plants revealed no GUS expression in any of the 22 F1 plants analyzed. These complementation analyses indicate that the ocp1 and nrpd2 are mutant alleles of the same NRPD2 gene. Hence, the ocp1 mutation will be referred also as ocp1/nrpd2-53. (TIF) Figure S4 PR-1 and PDF1.2a genes appear not to be methylated in their DNA sequences. Genomic DNA isolated from Col-0, nrpd2, nrpd1 and nrpe1 plants were digested (+) or not (2) with FspEI, MspJI or AvaII and amplified by PCR using specific primers for the indicated promoter regions. The ribosomal 5S DNA sequences, which are methylated, were used as a control. (TIF) Figure S5 Nucleotide sequence of PR-1 and PDF1.2a 59 promoter regions. Restriction sites for FspEI (green) and MspJI (blue) endonucleases are indicated by color sequences. Red circle marks AvaII restriction site. Arrows denote position of primers used to amplify the respective promoter regions as indicated in supplemental Methods. The ATG translation initiation codon for the transcribed genes is shown in bold. (TIF) Figure S6 Growth of P. cucumerina on leaves from Col-0, nrpd1, nrpe1, nrpd1 nrpe1 and nrpd2 plants quantified by qPCR. Plants were inoculated with P. cucumerina by spraying full expanded leaves with a solution containing 5610 6 spores/ml. Five days after inoculation DNA was extracted from leaves and the amount of the P. cucumerina b-tubulin gene quantified by qPCR. Data are standarized for the presence of the P. cucumerina btubulin gene in Col-0. Data represent the mean 6 SD; n = 3 biological replicates. (TIF) Text S1 Primer sequences. (DOCX)