Plants rely on the innate immune system to defend against various pathogens. Immune responses are triggered by perceiving pathogen-derived molecules by corresponding host immune receptors (Dodds and Rathjen, 2010). Cell surface-localized pattern recognition receptors detect microbe-associated molecule patterns (MAMPs) and activate pattern-triggered immunity (PTI) in plants (Yu et al., 2017). A series of typical defense responses occur on the plasma membrane or in the cytosol, such as Ca2+ influx, reactive oxygen species (ROS) burst, and activation of mitogen-activated protein kinase (MAPK) (Couto and Zipfel, 2016). Immune signaling is transduced into the nucleus to initiate global transcriptional reprogramming (Li et al., 2016). Proper regulation of gene expression is critical for establishing immunity, and the production of defense-related proteins and metabolites plays an essential role in fighting against pathogens. Stimulation by MAMPs puts plants in the defense priming stage, resulting in faster and stronger resistance to subsequent pathogen invasion (Mauch-Mani et al., 2017).

Perception of MAMPs, such as flg22 (a synthetic peptide derived from bacterial flagellin), triggers complex gene transcriptional changes and upregulation of several thousand genes (Hillmer et al., 2017). Many immune-related genes are induced or repressed as early as 30 min after MAMP perception (Navarro et al., 2004; Zipfel et al., 2004). Such changes in gene transcriptional reprogramming may further intensify after infection by a pathogen (Lewis et al., 2015). Upregulated genes are often associated with the immune response, particularly involved in the defense hormone signaling pathway, whereas downregulated genes are related to photosynthesis (Lewis et al., 2015). A network of multiple signaling sectors regulates immune gene transcriptional reprogramming (Kim et al., 2014; Tsuda and Somssich, 2015). Although plant defense hormones, including salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and phytoalexin deficient 4 (PAD4), contribute greatly to flg22-mediated disease resistance, they are not required for MAMP-induced early immune gene expression (Tsuda et al., 2009).

Gene transcription is regulated by multiple cis and trans-factors. Plant transcription factors (TFs), including the WRKY, ERF, and MYC families, exert important functions by activating immune gene expression (Pandey and Somssich, 2009; Moore et al., 2011; Buscaill and Rivas, 2014). Arabidopsis SH4-related 3 (ASR3) and GT2-LIKE 1 belong to the plant-specific trihelix transcription factor family and are phosphorylated by MAPKs. These two TFs negatively or positively regulate immune gene expression (Li et al., 2015; Volz et al., 2018). General transcriptional regulators, such as RNA polymerase II, mediator subunits, and elongation factors, regulate the expression of subgroups of immune genes (Li et al., 2016). The phosphorylation status of the Arabidopsis RNA Pol II carboxy-terminal domain undergoes changes related to regulating immune gene expression after a pathogen infection (Li et al., 2014). Epigenetic histone and DNA modifications play a role in regulating immune gene expression, possibly by modulating open chromatin and transcription complex binding (Ramirez-Prado et al., 2018). The histone methyltransferases set domain group 8 (SDG8) and SDG25 regulate gene induction in multiple layers of immunity (Lee et al., 2016).

DNA methylation is an important epigenetic marker involved in regulating gene expression and diverse biological processes in organisms (Luo et al., 2018). In plants, DNA methylation occurs in CG, CHG, and CHH contexts (H represents A, T, or C), whose patterns are modified by methylation and demethylation reactions. Arabidopsis de novo methyltransferases, methyltransferase 1, and chromomethylase 3 establish and maintain DNA methylation (Zhang et al., 2018). Conversely, active DNA demethylation is catalyzed by DNA glycosylases, including repressor of silencing 1 (ROS1), demeter-like 2 (DML2), and DML3, which are functionally redundant in plant somatic cells. Additionally, demeter functions during embryo development (Zhang et al., 2018; Liu and Lang, 2020). Several upstream regulatory factors have been implicated in the DNA demethylation pathway. Recruitment of ROS1 to its target is facilitated by a protein complex mainly consisting of methyl-CpG-binding domain protein 7 (MBD7) and histone acetyltransferase increased DNA methylation 1 (IDM1), which respectively recognize methylated DNA and acetylated histone H3 (Liu and Lang, 2020). HDP1 and HDP2, a pair of Harbinger transposon-derived proteins (HDPs), act as a functional module to determine the target specificity of the IDM1 complex (Duan et al., 2017). The SWR1 chromatin-remodeling complex has been recently found to recognize histone acetylation marks and histone H2A.Z recruits ROS1 to specific target loci (Nie et al., 2019).

In most cases, promoter DNA methylation inhibits gene expression by interfering with the binding of the transcription activator to the target gene promoter (Zhang et al., 2018). Changes in DNA methylation status affect gene expression in many biological processes. DNA methylation increases during sweet orange ripening and the expression of many genes is negatively correlated with DNA methylation (Huang et al., 2019). DNA methylation levels tend to be low in plant-pathogen interactions. DNA hypomethylation has been detected on specific centromeric repeats and retrotransposons after Pseudomonas syringae (Pst) infection (Pavet et al., 2006). Moreover, the DNA methylation levels in certain specific genomic regions decrease five days after Pst infection when the disease symptoms are well developed (Dowen et al., 2012). Notably, demethylation regulates the expression of a nucleotide-binding leucine rich-repeat receptor RESISTANCE METHYLATED GENE 1 (RMG1) by targeting sequences in the RMG1 promoter (Yu et al., 2013). Nematode-associated molecular pattern and flg22 treatment on day three post-infection trigger a significant loss of methylation in rice and tomato (Atighi et al., 2020). DNA methylation and demethylation have been associated with disease resistance to diverse pathogens (Deleris et al., 2016). Nevertheless, it remains unknown whether and how DNA demethylation actively regulates gene expression at the genome-wide level and functions in transcriptome responses at the early infection stage.

Our previous study revealed that the trihelix transcription factor ASR3 functions as a transcriptional repressor to regulate the expression of a subset of early immune genes (Li et al., 2015). In this study, we further investigated the involvement of the other trihelix family members in immune gene regulation and disease resistance and found that HDP2, another trihelix transcription factor in the SH4 subclade, was involved in flg22-mediated disease resistance. Similar to that previously reported, HDP2 with DNA binding activity is a subunit of the histone acetylation complex IDM1, a key complex for active DNA demethylation in Arabidopsis (Kaplan-Levy et al., 2012; Duan et al., 2017). Core components of DNA demethylation are required for pattern-triggered immunity and defense priming, and flg22 induces rapid changes in DNA methylation and transcriptional reprogramming in an RDD pathway-dependent manner. Integrative analyses of the methylome and transcriptome revealed DNA demethylation regulated the expression of multiple immune-related genes. Our results provide unique insight into the control of active DNA demethylation by RDD proteins and their regulation of immune genes and defense priming.