The epigenetic machinery controlling transgenerational systemic acquired resistance

Abstract

Progeny from diseased Arabidopsis shows enhanced resistance, which is associated with priming of defense genes.¹ This transgenerational systemic acquired resistance (SAR) is effective against biotrophic pathogens, such as the downy mildew pathogen Hyaloperonospora arabidopsidis. In this study, we have examined mutants in RNA-directed DNA methylation (RdDM) for transgenerational SAR. Our analysis suggests that transgenerational SAR is regulated by the RdDM pathway and transmitted by hypomethylation at CpNpG sites.

Priming of defense is an adjustment of the plant’s immune system, which results in a faster and stronger activation of inducible defense mechanisms after exposure to environmental stress.^2-4 A well-characterized form of defense priming takes place during expression of systemic acquired resistance (SAR). This resistance response is effective against biotrophic pathogens, requires regulation by the defense regulatory gene NON EXPRESSOR OF PR GENES (NPR1), and is associated with priming of salicylic acid (SA)-dependent genes.^5,6 Recent evidence suggests that priming of SA-inducible genes involves epigenetic regulatory mechanisms, such as post-translational modifications of histone proteins and the RNA Polymerase V.^7,8

We recently demonstrated that progeny from Pseudomonas syringae pv Tomato (Pst) DC3000-infected Arabidopsis (P₁ progeny) are primed for SA-dependent defense compared with progeny from control-treated healthy plants (C₁ progeny). We named this phenomenon “transgenerational SAR,” since the resistance in P₁ progeny is effective against (hemi)-biotrophic pathogens, it requires NPR1, and it is associated with priming of SA-inducible defense genes.¹ In the same journal issue, two complementary publications demonstrated transgenerational defense priming upon exposure to herbivory and the chemical priming agent β-aminobutyric acid.^9,10 Transgenerational SAR in P₁ progeny from diseased plants was associated with increased levels of acetylated histone 3 at lysine 9 (H3K9) at SA-inducible gene promoters¹, a chromatin mark that is associated with a permissive state of transcription.¹¹ Moreover, the drm1drm2cmt3 (ddc) triple mutant, which is affected in non-CpG DNA methylation,¹² mimicked the transgenerational SAR phenotype.¹ Since infection by PstDC3000 induces DNA hypomethylation in Arabidopsis,¹³ we hypothesized that transgenerational SAR is transmitted through DNA hypomethylation at non-CpG sites.

In this study, we have tested transgenerational SAR phenotypes of various Arabidopsis mutants in the RNA-directed DNA methylation (RdDM) pathway (Figs. 1 and 2). Parental plants were repeatedly inoculated with either mock solution, or a suspension containing PstDC3000 bacteria, and allowed to set seeds. At least three independent C₁ and P₁ progenies of each line were tested for resistance against the downy mildew pathogen Hyaloperonospora arabidopsidis, as described before.¹ The results of these bioassays are summarized in Figure 2.

Figure 1. Model of the RNA-directed DNA methylation (RdDM) pathway, adapted from.^25,26 Multi-subunit DNA-dependent RNA Polymerase IV (Pol IV) generates single stranded RNAs, which are used as a template by RNA-dependent RNA polymerase 2 (RDR2) to generate double-stranded RNAs (dsRNAs). DICER-LIKE 3 (DCL3) processes dsRNAs into 24 nucleotide RNAs that become methylated by the RNA-methyltransferase HEN1 and subsequently recruited by the AGO4-RISC complex to targeted genomic sites via interaction with the DNA-dependent RNA Polymerase V (Pol V). Pol V is required for DNA methyltransferase activity by DRM1, DRM2 and CMT3 and chromatin remodelling enzymes DRD, CLSY and KYP. The RdDM pathway targets cytosine (C) methylation at non-CpG sites. Signaling components affected in the mutants tested for transgenerational SAR are shown in color; green: enzymes in the siRNA machinery; red: chromatin remodelling enzymes; blue: DNA methyltransferase enzymes.

Figure 2. Transgenerational SAR against Hyaloperonospora arabidopsidis in mutants of Arabidopsis that are impaired in RNA-directed DNA methylation: nrpd2a-1, dcl3–1, ago4–3, clsy1–1 and drd1–6 (experiment A), and kyp-6, drm1drm2 (dd) and drm1drm2kyp (ddk) (experiment B). Induction of transgenerational SAR and challenge inoculation with H. arabidopsidis were performed as described.¹ At 6 d after conidiospore inoculation, stained leaves were microscopically examined and assigned to different classes, as described.¹ Asterisks and P values at the bottom of the graphs indicate statistical differences in class distributions between C₁ and P₁ progenies of each genotype (χ² Test).

P₁ progeny from the RdDM pathway mutants ago4-3,¹⁴ clsy1-1,¹⁵ nrpd2a-1,¹⁶ drd1-6¹⁷ and dcl3-1¹⁸ failed to express increased resistance in comparison to their C₁ progenies. Furthermore, C₁ progeny of the ago4-3 mutant showed constitutively enhanced resistance in comparison to C₁ progeny of wild-type plants (χ² = 14.2; p = 0.002). Since mutations in AGO4 cause reduced levels of non-CpG DNA methylation,¹⁹ the phenotype of the ago4-3 mutant supports our hypothesis that hypomethylation at non-CpG sites transmits SAR. Surprisingly, however, C₁ progeny from the other RdDM mutants displayed similar levels of susceptibility as C₁ progeny from wild-type plants. Considering their inability to express transgenerational SAR, these phenotypes suggest positive regulation by the corresponding RdDM components. In a second experiment, we tested additional Arabidopsis mutants impaired in non-CpG DNA methyltransferase activity. As observed for the ago4-3 mutant and ddc triple mutant,¹ C₁ and P₁ progenies from the drm1drm2kyp (ddk) triple mutant¹² showed no difference in resistance to H. arabidopsidis, while the C₁ progeny of this triple mutant expressed elevated levels of basal resistance in comparison to C₁ progeny from wild-type plants (χ² = 17.31; p = 0.001). By contrast, the drm1drm2 (dd) double mutant, which is specifically impaired in asymmetric CpHpH DNA methylation,¹² displayed a wild-type phenotype and was unaffected in transgenerational SAR and basal resistance. Finally, the kyp-6¹² single mutant, which is specifically affected in CpNpG methylation,²⁰ resembled the defense phenotype of clsy1-1, nrpd2a-1, drd1-6, and dcl3-1: there was no significant difference in resistance between C₁ and P₁ progenies, and C₁ progeny of this mutant displayed similar levels of susceptibility as C₁ progeny from wild-type plants.

It is clear from the data presented in this communication that transgenerational SAR in Arabidopsis involves regulation by the RdDM pathway. The exact role of each individual RdDM component remains difficult to decipher on the basis of the presented disease phenotypes. Rasmann et al., (2012) used two RdDM pathway Arabidopsis mutants to assess the contribution of siRNAs in transgenerational priming of jasmonic-acid (JA)-dependent defense against herbivory⁹: the nrpd2a nrpd2b double mutant²¹ and the dcl2dcl3dcl4 triple mutant.²² Progenies from healthy, herbivore-exposed and jasmonic acid-treated plants of these mutants failed to show differences in resistance against the specialist herbivore Pieris rapae, suggesting a critical role for the RdDM pathway in transgenerational priming of JA-dependent defenses. Hence, the RdDM pathway controls trans-generational priming of both JA- and SA-dependent defenses. We therefore propose that the RdDM pathway provides the machinery for different transgenerational defense responses, while the specificity of the response is determined by the 24-nucleotide sequence of the siRNAs.

Our findings that the dd double mutant expressed a wild-type phenotype (Fig. 2), whereas C₁ and P₁ progenies of the ddk and ddc triple mutant display similar levels of constitutively enhanced resistance (Fig. 2;¹), points to a critical role for KYP- and CMT3-dependent DNA methylation. KYP directs CMT3 activity through methylation of the lysine 9 residue of histone H3.²³ Its involvement in transgenerational SAR supports our previous observation that transgenerational SAR is marked by increased acetylation of H3K9 at SA-inducible gene promoters.¹ Since KYP and CMT3 predominantly mediate cytosine methylation at symmetrical CpNpG sites,²⁴ we conclude that transgenerational SAR is transmitted by DNA hypomethylation at CpNpG sites.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.