Small RNA and Epigenetic Control of Plant Immunity

Abstract

Plants have evolved a complex, multilayered immune system that integrates molecular recognition, signaling pathways, epigenetic regulation, and small RNA-mediated control. Recent studies have shown that DNA-level regulatory mechanisms, such as RNA-directed DNA methylation (RdDM), histone modifications, and chromatin remodeling, are critical for modulating immune gene expression, allowing for rapid and accurate pathogen-defense responses. The epigenetic landscape not only maintains immunological homeostasis but also promotes stress-responsive transcription via stable chromatin modifications. These changes contribute to immunological priming, a process in which earlier exposure to pathogens or abiotic stress causes a heightened state of preparedness for future encounters. Small RNAs, including siRNAs, miRNAs, and phasiRNAs, are essential for gene silencing before and after transcription, fine-tuning immune responses, and inhibiting negative regulators. These RNA molecules interact closely with chromatin features, influencing histone acetylation/methylation (e.g., H3K4me3, H3K27me3) and guiding DNA methylation patterns. Epigenetically encoded immune memory can be stable across multiple generations, resulting in the transgenerational inheritance of stress resilience. Such memory effects have been observed in rice, tomato, maize, and Arabidopsis. This review summarizes new findings on short RNA biology, chromatin-level immunological control, and epigenetic memory in plant defense. Emerging technologies, such as ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing), ChIP-seq (Chromatin Immunoprecipitation followed by Sequencing), bisulfite sequencing, and CRISPR/dCas9-based epigenome editing, are helping researchers comprehend these pathways. These developments hold an opportunity for establishing epigenetic breeding strategies that target the production of non-GMO, stress-resistant crops for sustainable agriculture.

1. Introduction

Plants, unlike animals, are stationary organisms that are constitutionally vulnerable to infection by disease. They lack specialized immune organs and cells, yet each cell is able to initiate immunological responses on its own. Consequently, they have developed a robust and multilayered immune system with two interconnected levels: Pattern-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI). PTI occurs when pattern recognition receptors (PRRs) on the cell surface recognize conserved microbial molecules known as pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin or fungal chitin [1]. The recognition triggers a cascade of defense responses, such as reactive oxygen species (ROS), calcium influx, MAPK activation, and transcriptional reprogramming of immune-related genes [2]. However, successful pathogens use effector proteins that suppress the plant’s innate immunity, which is part of PTI. Plants respond with ETI, mediated by intra-cellular nucleotide-binding leucine-rich repeat (NLR) receptors that detect these effectors and trigger a stronger, typically hypersensitive, immune response in the form of localized cell death to restrict pathogen growth [3]. More recent findings have implicated PTI and ETI as functioning in a synergistic way, converging on shared signaling pathways and transcriptional outputs to constitute an integrated immune response [4].

Beyond the initial immune activation, gene expression regulation plays a critical role in fine-tuning the timing, magnitude, and specificity of responses. The RNA-directed DNA methylation (RdDM) pathway utilizes small RNAs to target specific genomic loci for DNA methylation, such as defense genes and transposable elements, for strict regulation during stress conditions [5]. Chromatin remodeling, such as H3K27me3 and H3K9ac, also regulates defense gene expression during immune activation and priming [6]. One emerging area is epigenetic memory, where previous exposure to pathogens “primes” the plant to mount faster and more robust responses upon subsequent attacks. This immune memory is mediated by stable changes in chromatin, including methylation of histones (e.g., H3K4me3) and dynamic changes in DNA methylation at defense gene loci, some of which are transmitted to future generations. Notably, priming with Pseudomonas syringae has been shown to induce hypomethylation at WRKY and PR gene promoters, sustaining enhanced gene responsiveness in Arabidopsis progeny [7]. Recent studies in rice and tomato suggest that such memory is not limited to model plants; cross-priming induced by abiotic stress (e.g., drought or heat) can also modulate chromatin states that boost biotic stress responses, indicating epigenetic crosstalk across signaling pathways [8,9]. In maize, prolonged JA exposure results in histone acetylation changes that persist beyond the stress period, suggesting a potential mechanism for long-term transcriptional “bookmarking” [10,11] (Figure 1).

Recent progress has demonstrated that these DNA-level regulatory networks extend far beyond classical transcriptional control, encompassing a layered architecture that involves small RNA pathways, chromatin state transitions, and targeted DNA methylation. These processes not only influence the accessibility of immune gene promoters but also modulate the thresholds for defense activation, particularly through interactions between RdDM and chromatin remodelers such as SWI/SNF complexes [12,13]. Increasing evidence highlights the importance of DNA-level regulatory mechanisms, such as RdDM, chromatin remodeling, and histone modifications [14]. Among them, small RNAs, particularly small interfering RNAs (siRNAs) and microRNAs (miRNAs), are outstanding as essential regulatory elements. These molecules guide both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) [15]. Importantly, these mechanisms underpin the establishment of long-term “immunological memory,” also referred to as defense priming, which enables plants to mount faster responses without constantly activating energy-intensive defense systems. This primed state can be environmentally induced or inherited, offering a non-genetically modified (non-GM) route for engineering durable disease resistance in crops [16,17]. Here, we update the current understanding of DNA-level regulation of plant immunity, with a particular focus on small RNA-directed gene silencing, chromatin remodeling, and the role of epigenetic memory. We also highlight new tools such as Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq), Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq), bisulfite sequencing, and Clustered Regularly Interspaced Short Palindromic Repeats/dead Cas9 (CRISPR/dCas9)-based epigenome editing that are revolutionizing our understanding of how chromatin dynamics and non-coding RNAs regulate plant–pathogen interactions. In this review, we dissect the molecular interplay between chromatin remodeling, small RNAs, and epigenetic memory, highlighting their roles in plant immunity and prospects for crop improvement.

2. Epigenetic Regulation of Plant Immunity

Plants use epigenetic control through chromatin structural changes and DNA methylation to control the activation or suppression of their immune-related genes. By these changes, plants can mount an instant and efficient response when they are attacked by pathogens, e.g., bacteria, fungi, or viruses. One of the essential aspects of this regulation is chromatin remodeling, where nucleosomes (the fundamental chromatin units) are moved or altered in place by highly dedicated protein machines to prevent or permit entry to specific genes. Chromatin remodeling is regulated by multi-subunit complexes like SWR1, CHD3, and INO80, which move nucleosome positions to trigger or suppress immune-responsive genes [18]. For example, in Sorghum bicolor, infection by Colletotrichum sublineola triggers the reorganization of chromatin loops at resistance QTLs, enhancing access to promoters of defense genes [19]. In Arabidopsis thaliana, activation of immune responses via SARD1 and other defense-related transcription factors is accompanied by increased chromatin accessibility at target loci, indicating that chromatin remodeling facilitates rapid gene activation during pathogen attack [20]. Histone modifications are the central factors here. Histone marks such as H3K4me3 and H3K9ac are associated with transcriptionally active chromatin, whereas the repressive mark H3K27me3 is typically deposited at defense-related genes that are not immediately required [21]. Exposure of Medicago truncatula to Fusarium oxysporum induces flavonoid biosynthesis genes, corresponding to high antimicrobial metabolite production [22]. Similarly, in rice (Oryza sativa), JMJ705 histone demethylase is known to remove H3K27me3 marks from defense-gene-related genes, like those representing the WRKY transcription factor family, and hence their activation during early stages of pathogen infection [23]. DNA methylation plays a role in repression and gene expression adjustment, particularly through methylation at Cytosine methylation in CG sequence context (CG), Cytosine methylation in CHG sequence context (CHG), and Cytosine methylation in CHH sequence context (CHH) sites. MET1, CMT3, and DRM2 regulate methylation deposition, while ROS1 and members of the DML family catalyze active demethylation. Infection of tomato by Phytophthora infestans results in demethylation of JA-responsive loci, rendering them more accessible. Noncanonical RdDM, which employs 21–22 nt siRNAs in place of the typical 24 nt class, has been discovered in maize reproductive organs during systemic viral stress [24]. Emerging evidence underscores the role of histone variants in stress priming. In Brachypodium distachyon, the deposition of the histone variant H2A.Z at defense gene loci has been associated with enhanced transcriptional responsiveness upon repeated stress exposure. This chromatin feature may act as a molecular bookmark, preserving gene responsiveness and facilitating rapid reactivation of immunity-related genes during subsequent pathogen attacks [25]. Furthermore, some of these epigenetic modifications are robust after the initial exposure to stress and are inherited by the next generation, being susceptible to transgenerational immune memory. In monocots like wheat and barley, inheritance has been established through epigenetic means involving DNA methylation marks and small RNAs, wherein primed defense genes are maintained in hypomethylated, open chromatin conformations in subsequent generations, emphasizing the potential of epigenetic priming for durable disease resistance [26,27] (Figure 1).

2.1. DNA Methylation and Plant Defense

Histone proteins also receive diverse post-translational modifications, including methylation, acetylation, phosphorylation, and ubiquitination, which control chromatin structure and gene expression. Repressive histone marks like H3K9me2 and H3K27me3 are generally in agreement with compacted states of chromatin and dominate defense-associated loci during non-stress conditions. These changes are engaged in transcriptional repression by introducing chromatin-binding factors and limiting RNA polymerase access and hence maintaining genes suppressed until activation is required [28]. Active histone marks, such as H3K9ac, H3K27ac, and H3K4me3, are dynamically deposited at immune gene promoters during pathogen infection, enabling an open chromatin structure that is conducive to transcription. For instance, in barley (Hordeum vulgare), Blumeria graminis f. sp. hordei leads to H3K27ac enrichment at receptor kinase and secondary metabolite enzyme gene loci corresponding to their increased expression. Similarly, in soybean (Glycine max), H3K4me3 accumulation at some WRKY transcription factor genes has been linked with increased expression during infection by Phakopsora pachyrhizi [29]. Further studies have established the role of histone marks in other systems. In Setaria italica, a model C4 crop plant in development, biotic stress induces to redistribution of H3K36me3 marks at metabolic enzyme genes associated with immunity [30]. In cucumber, chromatin immunoprecipitation analyses revealed increased levels of H3K9ac at defense-associated PAL and PR1 gene promoters upon Pseudoperonospora cubensis infection, which were associated with an SA accumulation burst [31]. In Sorghum bicolor, exposure to Cercospora sublineola elicited the expression of a gene homologous to the chromatin remodeler BRM (BRAHMA chromatin remodele), which is consistent with the induction of defense-related genes [32]. Histone acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), which maintain the level of chromatin compaction and relaxation in equilibrium. A. thaliana HDAC HDA19 is a widely characterized HDAC that represses SA-mediated defense signaling and pathogenesis-related (PR) gene expression. Loss of function of HDA19 causes increased resistance to bacterial pathogens such as P. syringae [33]. Suppression of the CaHDA1 gene in Capsicum annuum has been associated with enhanced defense gene expression and increased tolerance to Xanthomonas campestris pv. Vesicatoria bacterial spot disease [34]. Remodeling of chromatin is also achieved by SWI/SNF complex families, utilizing ATP to move nucleosomes in order to expose or protect regulatory DNA elements. SWI3C, part of the SWI/SNF complex, in tomato, has been suggested to regulate JA-responsive expression of genes after necrotrophic fungal infections, based on functional insights accumulated through A. thaliana [35]. In Brassica rapa, silencing of an SNF2-type chromatin remodeler by RNAi reduced the ex-pression of callose synthase genes and resulted in increased susceptibility to powdery mil-dew [36]. Together, these epigenetic features, including histone modifications, chromatin remodeling complexes, and dynamic chromatin accessibility, make up a flexible regulation system enabling rapid transcriptional reprogramming upon pathogen invasion in different plant species.

2.2. Histone Modifications in Defense Signaling

DNA methylation in plants occurs predominantly at cytosine residues in CG, CHG, and CHH contexts and serves as a crucial epigenetic mechanism for transcriptional regulation. It is orchestrated by DNA methyltransferases such as MET1 (maintains CG methylation), CMT3 (maintains CHG methylation), and DRM2 (catalyzes CHH methylation through the RNA-directed DNA methylation, RdDM, pathway). These methylation marks play essential roles in silencing transposable elements (TEs), provide genome stability, and preventing the ectopic activation of adjacent genes, including those involved in immune responses [37]. In addition to genome defense, DNA methylation controls immunity by maintaining repressive marks at defense gene loci under normal conditions. Upon pathogen exposure, selective demethylation at immune gene promoters facilitates rapid transcriptional activation (Figure 1). For instance, in Arabidopsis, ROS1 is upregulated in response to bacterial challenge and removes methylation marks at WRKY70 and PR1 promoters, enabling their expression [38]. Similarly, in cucumber, exposure to P. cubensis leads to site-specific hypomethylation near PAL genes involved in salicylic acid biosynthesis [39]. In tomato, the demethylase SlDML2 has been shown to mediate global DNA hypomethylation during infection by F. oxysporum, promoting resistance-related transcriptional programs [40]. In rice, infection by Xanthomonas oryzae results in dynamic loss of CHH methylation at JA/SA pathway genes, highlighting the interplay between methylation and hormone signaling [41]. DNA demethylation is catalyzed by a family of bifunctional DNA glycosylases, including ROS1, DME (DEMETER), and DML2/3. These enzymes excise methylated cytosines and initiate base excision repair, replacing them with unmethylated cytosines [42]. In Brassica napus, DME homologs are associated with demethylation of R gene promoters under fungal stress, suggesting a conserved role in activating immune genes [43]. Such dynamic and reversible changes in DNA methylation are not only critical for immediate defense responses but also contribute to immune priming and the formation of memory. Transient stress cues can leave persistent epigenetic imprints, establishing an adaptive ‘memory’ that enhances responsiveness to future attacks. This has been observed in maize, where the first exposure to pathogens leads to long-term hypomethylation of immune loci in later generations [44]. Together, the processes of methylation and demethylation constitute a finely balanced regulatory circuit that enables plants to react efficiently to biotic challenge with minimally inappropriate immunity activation.

2.3. Chromatin Remodeling in Plant Immunity

Chromatin remodeling is an essential process by which plants dynamically control gene expression upon pathogen attack. Chromatin remodeling involves structural alterations to chromatin, influencing the accessibility of the transcription machinery to DNA, thereby facilitating fine modulation of immune responses [45]. Unlike rigid epigenetic marks such as DNA methylation, chromatin remodeling is extremely dynamic, with the capacity to provide plants with versatility for instant activation or repression of defense genes. Plant immunity chromatin remodeling facilitates the rapid reprogramming of transcriptional networks in basal and induced immune responses and therefore is central to both local and systemic resistance. Chromatin structure consists of nucleosomal DNA wrapped around histone octamers, which are modified by either ATP-dependent chromatin remodeling complexes or histone modifications. These modifications alter nucleosome position or composition and therefore gene accessibility [20]. In plant–pathogen interactions, this remodeling enables recruitment of defense genes to be rapidly activated upon recognition of the pathogen. In addition, chromatin remodelers have functions in the establishment of transcriptional memory, allowing the plants to respond more efficiently to subsequent infections.

ATP-dependent chromatin remodeling complexes harness energy from hydrolysis of ATP to move nucleosomes by sliding along the DNA, evicting them, or altering histone–DNA interactions. Members of this family are implicated in the transcriptional activation of defense-linked genes. BRM, for instance, is involved in the activation of pathogenesis-related (PR) genes during systemic acquired resistance (SAR) [46]. The ISWI family, through its role in nucleosome spacing and chromatin compaction, is also implicated in immune responses, though little is known about its role in plant immunity. Current research suggests that ISWI complexes may suppress some defense genes under non-stress conditions in a bid to provide energy efficiency and growth, releasing this suppression upon the detection of a pathogen [36]. The CHD family (chromodomain–helicase–DNA-binding proteins) includes remodelers like PICKLE (PKL), which has been associated with repression of embryonic developmental gene expression but is increasingly recognized as being involved in defense and stress responses [47]. The INO80 complex, known for its roles in DNA replication and repair, also participates in chromatin remodeling during plant immunity. Its involvement in plant defense is supported by the observed upregulation of INO80 subunits following pathogen infection [48]. Together, these complexes orchestrate a nuanced regulatory network that fine-tunes gene expression to ensure an optimal balance between growth and defense.

Priming and Epigenetic Memory Mechanisms

Priming is a phenomenon where a plant that has experienced a stress event exhibits a more rapid and robust response when re-exposed to the same or similar stress. Epigenetic memory facilitates a priming effect, enabling plants to ‘remember’ previous pathogen interactions through stable changes in chromatin structure that affect gene expression. One of the best-documented priming cases is the SAR pathway, in which systemic signals generated at the infection site lead to the activation of PR genes in the entire plant. Histone marks like H3K4me3 (histone H3 lysine 4 trimethylation) and H3K9ac (histone H3 lysine 9 acetylation) normally occur with the primed state of defense genes. These marks maintain chromatin in a more open configuration, allowing for rapid transcription upon re-stimulation [49]. The role of histone methyltransferases (e.g., ATX1) and demethylases (e.g., IBM1) in modulating H3K4me3 levels at PR gene loci during priming events has been well-studied. ATX1 facilitates H3K4me3 deposition, whereas IBM1 removes repressive H3K9me2 marks, both contributing to a transcriptionally permissive chromatin state [50]. Interestingly, this primed state remains stable for extended periods and, in some cases, even persists transgenerationally. For instance, pre-exposure of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 has been shown to induce amplified resistance in the progeny, mediated through heritable epigenetic modifications such as histone acetylation and DNA hypomethylation at defense-related loci [51]. DNA methylation changes, particularly at transposable elements near defense genes, have also been implicated in this transgenerational memory. RNA-directed DNA methylation (RdDM) may also play a role in establishing or maintaining priming. siRNAs generated in response to pathogens could guide DNA methylation to specific loci, reinforcing chromatin-based memory mechanisms. Furthermore, the chromatin remodelers BRM and SYD are not only necessary for transient defense gene expression but also for maintaining chromatin states that facilitate memory establishment. Those plants that do not have these remodelers cannot start or sustain priming, confirming their important role in epigenetic memory [52,53]. Chromatin remodeling typically provides a dynamic and flexible structure for immunity, enabling a quick response to pathogen assault, but also incorporating a memory of past exposure. Such a dual capacity of plants to be economical with resources allows them to maintain growth and defense in equilibrium as conditions around them change.

3. Small RNA-Mediated Gene Silencing in Plant Immunity

Small RNA-mediated gene silencing is a central process of gene expression regulation in plant development, stress response, and particularly immunity. The small RNAs are typically 20–24 nucleotides in length and are produced from double-stranded RNA (dsRNA) precursors by the DCL enzymes [54]. The sRNAs guide the AGO (Argonaute proteins) to complementary target sequences in RNA or DNA, guiding either transcriptional gene silencing (TGS) or post-transcriptional gene silencing (PTGS). At the plant immunity level, small RNAs are gene expression fine-tuners, acting on negative regulators or enhancing immune-responsive cascades to maintain homeostasis and prevent fitness costs. Notably, numerous miRNAs have emerged as crucial regulators of immune gene networks. For example, miR393 controls auxin receptors such as TIR1, suppressing auxin signaling to trigger antibacterial defense, particularly against P. syringae infection [55]. Likewise, miR398 is repressed during oxidative stress or pathogen infection, which derepresses CSD1/2 and CCS, thereby promoting ROS detoxification as part of the immune response [56]. In Solanaceae plants, miR482/2118 plays a central role in regulating the expression of NLR genes. These miRNAs induce phased secondary siRNA (phasiRNA) production, which modulates resistance gene levels so that the plant is able to break autoimmunity but remain sensitive to pathogens [57,58]. New evidence in tomato and cotton shows that dynamic expression of family members of miR482 in pathogen and tissue contexts during infection with fungi and viruses drives resistance or susceptibility [59,60]. Besides endogenous regulation, pathogens themselves possess the ability to hijack host small RNA pathways. The necrotrophic fungus Botrytis cinerea also possesses the ability to deliver small RNA effectors into host cells to suppress host immunity. RNAs of B. cinereas (Bc-sRNAs) are delivered into host plants and hijack the plant RNAi machinery to induce cross-kingdom RNAi of host immune-responsive genes. This discovery has opened new perspectives on pathogen-derived small RNAs as virulence factors, prompting efforts to identify and block these sRNAs to restore plant immunity [61]. Furthermore, new classes of small RNAs, including natural antisense transcript-derived siRNAs (nat-siRNAs) and siRNAs derived from long non-coding RNA (lncRNA), have emerged as context-specific regulators under pathogen attack. For example, Arabidopsis produces nat-siRNAs in response to P. syringae that suppress negative regulators of immunity, such as PPRL [62,63]. It has been demonstrated that during Magnaporthe oryzae infection, rice’s salicylic acid and jasmonic acid signaling nodes are modulated by infection-triggered lncRNA-derived siRNAs. New lncRNA-derived siRNAs with functional roles in plant–pathogen interactions are continually being discovered thanks to developments in sRNA-seq and degradome profiling (

Table 1. Examples of cross-kingdom small RNAs in plant–Pathogen interactions.

sRNA Name/Type	Origin	Class and Biogenesis	Molecular Target(s)	Defense Role	Reference
miR393	Arabidopsis	miRNA	TIR1 (auxin receptor)	Enhances PTI via auxin suppression	[64]
miR398	Arabidopsis	miRNA	CSD1, CSD2, CCS	Enhances ROS detox during stress	[65]
miR160	Arabidopsis	miRNA	ARF10/16/17	Regulates auxin–immune crosstalk	[66]
miR482	Tomato	miRNA/phasiRNA	NBS-LRR	Controls immune receptor expression	[67]
miR2118	Glycine max	miRNA/phasiRNA	R-genes	Triggers phasiRNA production	[68]
miR167	Arabidopsis	miRNA	ARF6/8	Regulates auxin in stress responses	[69]
miR399	Arabidopsis	miRNA	PHO2	Controls phosphate signaling and defense	[70]
miR156	Rice	miRNA	SPL transcription factors	Modulates development and immunity	[71]
TAS1-derived siRNAs	Arabidopsis	ta-siRNA	F-box proteins	Controls auxin signaling	[72]
nat-siRNA (PPRL)	Arabidopsis	nat-siRNA	PPR-like proteins	Suppresses immunity repressors	[72]
miR528	Maize	miRNA	L-ascorbate oxidase	Regulates oxidative stress and defense	[73]
miR159	Wheat	miRNA	MYB33/MYB65	Involved in Fusarium resistance	[74]
miR444	Rice	miRNA	MADS-box TFs	Implicated in defense against pathogens	[75]
phasiRNA (NLRs)	Soybean	phasiRNA	NLRs	Controls immune gene bursts	[76]
Bc-siR37	B. cinerea	sRNA	MAPKs, WRKYs	Silences host defense genes	[61,77]
Bc-sRNA3	B. cinerea	sRNA	Host AGO1 pathway	Hijacks RNA silencing	[78]
Vd-sRNA1	V. dahliae	sRNA mimic	PR1, defense genes	Suppresses host immunity	[79]
Vd-sRNA2	V. dahliae	sRNA mimic	ETI-related genes	Promotes virulence	[79]
gma-miR1508	Phytophthora sojae	miRNA mimic	NBS-LRR class of R gen	ETI	[80]
5′AlaCGC tRF	Piper nigrum	tRF (tRNA-derived fragment)	NPR1	Mediates cleavage of NPR1 mRNA, downregulating the SAR pathway	[81]
5′MetCAT tRF	P. nigrum	tRF	E3 ubiquitin ligase	Targets the mRNA of ubiquitin ligase, affecting PTI/ETI signaling	[81]
miR162	Arabidopsis/Nicotiana benthamiana	miRNA	DCL1 mRNA	p19 VSR binds miR162, upregulates DCL1, and alters miRNA biogenesis to aid early infection	[82]
miR168	Arabidopsis/N. benthamiana	miRNA	AGO1 mRNA	Weak p19 binding; feedback on AGO1; minor role in early infection	[82]
miR403	Arabidopsis/N. benthamiana	miRNA	AGO2 mRNA	Strong p19/2b binding; blocks AGO2 defense early in infection.	[82]

tRF = tRNA-derived fragment; phasiRNA = phased small interfering RNA; PR1 = pathogenesis-related protein 1; PTI = Pattern-Triggered Immunity; ETI = Effector-Triggered Immunity; SAR = Systemic Acquired Resistance; NBS-LRR = Nucleotide-Binding Site Leucine–Rich Repeat; TF = Transcription Factor; AGO = Argonaute protein; DCL = Dicer-like.

).

3.1. Small RNAs in Plant Immunity

Small RNAs (sRNAs) are key regulators of plant immunity, which are generated through specific biogenesis pathways and function through transcriptional and post-transcriptional gene silencing. They are classified into different categories, like microRNAs (miRNAs), small interfering RNAs (siRNAs), natural antisense siRNAs (nat-siRNAs), and long non-coding RNA (lncRNA)-derived siRNAs [83]. These RNA molecules control gene expression in response to environmental stimuli, as well as pathogen infection, and are thus crucial components in plant defense. These miRNAs comprise miR393, miR160, and miR167, which play a crucial role in immune signaling by regulating genes involved in auxin, such as TIR1, ARF10/16/17, and ARF6/8, respectively [55,66,69]. miR398 regulates CSD1, CSD2, and CCS for the control of oxidative stress tolerance [65], and miR399 regulates phosphate signaling and immunity by downregulating PHO2 [70]. miR156 regulates SPL transcription factors to balance growth and immunity in rice [71], and miR528 targets L-ascorbate oxidase in maize, affecting ROS-related defenses [73]. Phased siRNAs (phasiRNAs) and trans-acting siRNAs (ta-siRNAs) form another layer of sRNA regulation. miR482 and miR2118 trigger phasiRNA production from NBS-LRR transcripts in tomato and soybean, fine-tuning immune receptor levels [68,84]. Similarly, TAS1-derived siRNAs target F-box proteins, modulating auxin signaling [72]. nat-siRNAs, such as those derived from the PPRL locus, are induced during P. syringae infection to silence negative regulators of immunity [72]. These findings highlight the role of sRNAs in balancing defense and growth. Pathogen-derived sRNAs also exploit the host silencing machinery. B. cinerea produces Bc-siR37 and Bc-sRNA3, which silence host MAPKs, WRKYs, and AGO1-dependent defenses [61,78]. V. dahliae secretes Vd-sRNA1 and Vd-sRNA2, targeting PR1 and ETI-related genes to suppress plant immunity [42]. P. sojae delivers gma-miR1508, a miRNA mimic that represses NBS-LRR genes [80]. In Piper nigrum, tRNA-derived fragments (5′AlaCGC tRF, 5′MetCAT tRF) target NPR1 and E3 ubiquitin ligase genes, interfering with Systemic Acquired Resistance (SAR) and PTI/ETI pathways [81]. Viral suppressors of RNA silencing (VSRs) such as p19 and 2b proteins manipulate host RNA pathways. miR162 is sequestered by p19, leading to DCL1 upregulation. miR168 and miR403, which regulate AGO1 and AGO2, are differentially targeted by p19 and 2b to disrupt early antiviral responses [82]. Mutant analysis of OsRDR1 and OsRDR6 revealed disrupted immune synergy against viral, fungal, and bacterial pathogens, further emphasizing the central role of small RNAs in cross-kingdom plant defense [85]. Collectively, sRNAs from both host and pathogens orchestrate a multilayered regulatory network in plant immunity. Advances in strand-specific RNA-seq and degradome analysis continue to uncover new roles for lncRNA-derived siRNAs [86,87,88,89], emphasizing the potential of RNA-based interventions for crop improvement (Table 1).

3.2. Role in Transcriptional Gene Silencing

Transcriptional silencing by small RNAs is predominantly mediated through the RdDM pathway, a plant-specific epigenetic mechanism crucial for both genome stability and adaptive immune regulation. In this process, 24-nt siRNAs are generated from double-stranded RNAs transcribed by RNA Polymerase IV and processed by DCL3 [90,91]. These siRNAs are then loaded onto AGO4, which directs the silencing complex to scaffold RNAs produced by RNA Polymerase V [92]. The recruitment of Domains Rearranged Methyltransferase 2 (DRM2) facilitates de novo cytosine methylation in symmetric (CG, CHG) and asymmetric (CHH) contexts. This pathway plays a dual role: (1) It suppresses transcription from transposable elements (TEs) and repetitive DNA, preserving genome integrity; and (2) it regulates immune-related genes, preventing inappropriate activation in the absence of pathogens During infection, selective demethylation or targeted reprogramming of RdDM marks enables activation of key defense genes [93]. In rice, RdDM activity involving OsDRM2 is essential for silencing endogenous viral elements and retrotransposons, highlighting its importance in antiviral immunity [94].

Similarly, Arabidopsis mutants defective in RdDM components, such as NRPD1 and RDR2, display heightened susceptibility to pathogens, suggesting that RdDM helps maintain a primed, but non-costly, immune state, Recent studies also indicate a connection between RdDM and defense priming. For instance, sustained methylation at certain defense loci has been correlated with enhanced responsiveness during secondary pathogen exposure [95]. Moreover, crosstalk between RdDM and histone modifications, such as H3K9me2 and H3K27me3, provides an additional epigenetic layer to regulate transcriptional responsiveness [96]. In tomato, methylome reprogramming through RdDM has been shown to influence salicylic acid-responsive genes following P. syringae infection, supporting the role of RdDM in adaptive transcriptional immunity [45]. As advances in single-base resolution bisulfite sequencing and epigenome editing tools emerge, RdDM continues to be a promising target for engineering stable, heritable, and pathogen-responsive transcriptional control in plants [97] (Table 1).

3.3. Role in Post-Transcriptional Gene Silencing

PTGS operates through the sequence-specific degradation or translational repression of target RNAs. This mechanism is primarily mediated by 21–25 nucleotide siRNAs or miRNAs loaded into AGO or related effector proteins. In the context of plant immunity, PTGS plays a vital role in buffering immune activation by fine-tuning the expression of resistance (R) genes, particularly NBS-LRR receptors [98,99,100]. A well-characterized example includes the miR482/2118 superfamily, which targets NBS-LRR transcripts for cleavage. This cleavage triggers the production of phasiRNAs, which further amplifies the silencing signal and reinforces the downregulation of immune receptors. This layered silencing ensures that immune receptor levels are dynamically regulated in response to pathogenic stimuli while minimizing autoimmune damage [101]. In tomato, miR6027 has also been shown to regulate TIR-NBS-LRR genes and affect susceptibility to viral infections, demonstrating crop-specific PTGS networks [84]. Pathogens, in turn, have evolved sophisticated mechanisms to disrupt host programmed cell death and programmed cell death signaling (PTGS). Viral suppressors of RNA silencing (VSRs) are commonly deployed to neutralize this defense [102]. For instance, Tombusvirus P19 protein binds siRNAs with high affinity, effectively sequestering them and preventing their incorporation into AGO complexes [103]. Similarly, the HC-Pro protein from potyviruses interferes with both miRNA and siRNA accumulation, destabilizing endogenous silencing pathways [104]. In addition to viral suppressors, bacterial and fungal pathogens produce effectors that inhibit components of the PTGS machinery. The P. syringae effector HopT1-1 impairs AGO1 activity [105], while Verticillium dahliae delivers Vd-sRNA1/2, which mimic endogenous plant miRNAs to repress host immune genes [106]. RDR6, a key enzyme in secondary siRNA biogenesis, contributes to PTGS and resistance against viruses, fungi, and bacteria in rice [107]. These examples illustrate an intricate molecular arms race, where PTGS represents a key battleground in plant–pathogen interactions. Emerging studies using degradome sequencing and transient silencing assays are uncovering new sRNA–target relationships that fine-tune the immune response. As tools such as artificial miRNAs and mobile sRNA delivery evolve, PTGS mechanisms offer powerful biotechnological avenues for engineering precision resistance in crops [108]. These insights into PTGS highlight the central role of small RNAs in controlling immune gene expression post-transcriptionally. Notably, recent discoveries suggest that small RNAs also contribute to gene regulation at the chromatin level, pointing to a dynamic interplay between RNA silencing pathways and epigenetic modifications.

3.4. Crosstalk Between Small RNAs and Chromatin Remodeling

An emerging hallmark of plant immune regulation is the intricate coordination between small RNA pathways and chromatin dynamics. These interactions link TGS to chromatin state transitions, enabling plants to mount precise and sustainable immune responses. A key example is the RdDM pathway, where 24-nt siRNAs generated by RNA Polymerase IV are processed by DCL3 and loaded onto AGO4. These complexes are guided to target loci via scaffold RNAs transcribed by RNA Polymerase V. Upon binding, DRM2 catalyzes DNA methylation in CHH contexts. This process is often accompanied by deposition of histone repressive marks such as H3K9me2, indicating a functional link between small RNAs and chromatin-level silencing [109]. Functional disruption of RdDM machinery alters both DNA and histone methylation patterns, often resulting in compromised immunity. For instance, in Setaria viridis, knocking down an RdDM component led to susceptibility to necrotrophic fungi and reduced H3K27me3 deposition at defense gene loci [46]. Similarly, Lotus japonicus mutants lacking RDR2 exhibited diminished methylation at NBS-LRR clusters, accompanied by lowered basal resistance [110]. Beyond canonical RdDM, miRNAs and phasiRNAs modulate chromatin regulators. In M. truncatula, miR482 family members suppress transcripts encoding SET-domain histone methyltransferases, altering chromatin accessibility at pathogenesis-related gene loci [111]. In Solanum lycopersicum (tomato), miR6024 acts as a negative regulator of NLR genes, which are key components of the plant immune system. Overexpression of miR6024 leads to repression of NLRs and increased susceptibility to Alternaria solani, indicating that miR6024 contributes to immune suppression potentially through chromatin-associated pathways [112]. In A. thaliana, the chromatin remodeler CHR11 has been shown to recruit the RNA-binding protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) to chromatin, facilitating the production of small interfering RNAs (siRNAs) from endogenous protein-coding genes. This interaction is critical for initiating PTGS, particularly under biotic stress such as viral infections. SGS3 also binds to RDR6, forming a functional complex essential for the conversion of aberrant RNAs into dsRNAs, a key step in siRNA biosynthesis [113]. Furthermore, SGS3 is indispensable for producing ta-siRNAs and the establishment of PTGS [114,115]. Notably, SGS3 also contributes to epigenetic regulation, with evidence suggesting its involvement in DNA methylation of promoter regions, linking RNA silencing to transcriptional repression. In rice, the OsSGS3b paralog participates in the biogenesis of small RNAs derived from retrotransposons [116], further expanding its functional repertoire in genome stability and immunity [117,118]. Together, these studies highlight a conserved yet adaptable regulatory framework in which small RNAs guide or influence chromatin modifications. This crosstalk not only ensures tight immune gene control but also facilitates epigenetic memory and defense priming.

4. Epigenetic Memory and Transgenerational Immunity

Plants possess the remarkable ability to establish and retain a form of “immunological memory” following exposure to biotic stress, a phenomenon known as defense priming. Unlike animals, which rely on specialized adaptive immune cells, plants encode immune experiences through epigenetic mechanisms, including DNA methylation, histone modifications, and small RNA activity, that do not alter the underlying DNA sequence. This epigenetically encoded memory allows plants to mount faster and stronger defense responses upon subsequent encounters with pathogens [119]. Epigenetic memory in plants is maintained through persistent changes in DNA methylation, histone modifications, and sustained small RNA activity. For instance, in A. thaliana, infection by P. syringae leads to hypomethylation at promoter regions of defense genes such as PR1 and WRKY6, with these methylation states persisting long after pathogen clearance, contributing to a primed defense state [120]. Similarly, in B. napus, biotic stress results in the durable enrichment of H3K4me3 and depletion of H3K27me3 at salicylic acid-responsive loci, facilitating the long-term transcriptional activation of defense-related genes [121]. Notably, small RNAs also play a pivotal role in this memory. In O. sativa, prolonged expression of pathogen-responsive siRNAs has been documented after X. oryzae exposure, correlating with sustained silencing of negative regulators of immunity [122]. Similar long-lasting phasiRNA responses have been observed in Solanum tuberosum under viral pressure, suggesting a mechanism of RNA-mediated defense imprinting [123]. Epigenetic priming not only enhances immunity within the exposed plant but may also affect progeny, leading to transgenerational immune priming. In A. thaliana, the progeny of plants infected with P. syringae exhibits hypomethylation at the promoter regions of defense genes, such as PR1 and WRKY6, resulting in increased basal expression of these markers and contributing to enhanced resistance in subsequent generations [51]. Similarly, in S. tuberosum, progeny of plants primed with β-aminobutyric acid (BABA) show reduced DNA methylation on the promoter of the R3a NLR gene, correlating with higher transcription levels and increased resistance to Phytophthora infectants, mechanisms underlying this heritable immune memory involve stable chromatin configurations, preservation of non-coding RNA profiles, and intergenerational maintenance of DNA methylation marks [124]. While the extent and universality of this epigenetic memory are still under active investigation, it represents a promising non-GMO strategy for enhancing disease resistance in crop breeding programs. Collectively, these findings emphasize that epigenetic memory forms a crucial layer of plant immunity, encoding environmental stress experiences into long-term and inheritable defense strategies. Understanding this process could open new avenues for sustainable agriculture and pathogen-resilient crop development.

4.1. Priming and Immune Memory

Priming refers to a physiological state in which a plant, upon exposure to a mild or avirulent pathogen, beneficial microorganism, or abiotic cue, becomes sensitized for enhanced defense upon subsequent stress. Unlike constitutive defense activation, priming allows plants to maintain normal metabolic function while remaining poised for faster and stronger immune responses. This heightened responsiveness includes more rapid transcriptional activation of defense genes, accelerated signaling via MAPKs and phytohormones, and quicker accumulation of antimicrobial compounds [125]. A well-studied case of priming is Systemic Acquired Resistance (SAR), where localized infection by a necrotizing pathogen results in systemic immune activation. SAR involves the accumulation of salicylic acid, elevated expression of pathogenesis-related (PR) genes, and chromatin remodeling that maintains genes in a transcriptionally permissive state [126,127]. Alongside SA, pipecolic acid, azelaic acid, and N-hydroxypipecolic acid (NHP) function as mobile signals in SAR, enabling long-distance immune communication. Beneficial microbes can also trigger priming. For instance, colonization by Trichoderma harzianum in cucumber roots enhances defense against P. cubensis by promoting the deposition of H3K4me3 at defense gene promoters [128]. Similarly, Bacillus subtilis primes defense in C. annuum via increased JA biosynthesis and persistent chromatin activation at WRKY genes [129]. Chemical-induced priming is another effective strategy. BABA is a well-known priming agent that induces long-term resistance to multiple pathogens through epigenetic modifications, including H3K9ac enrichment and DNA hypomethylation, without imposing significant fitness costs [130]. In Vitis vinifera, BABA primes the transcriptional activation of stilbene biosynthesis genes against B. cinerea [131]. Cross-priming, where abiotic stress enhances defense against biotic challenges, has also been observed. In tomato, prior drought stress enhances resistance to P. syringae, likely due to persistent histone modifications at immune regulatory genes [132]. In Arabidopsis, heat stress primes the JA pathway, leading to faster induction of JA-responsive defenses under fungal attack. Moreover, in rice, UV-B radiation induces broad-spectrum resistance to X. oryzae via small RNA-dependent activation of epigenetically silenced R genes [133]. In maize, sulfur deficiency primes the expression of oxidative stress genes and improves resistance to Fusarium verticillioides, demonstrating nutrient-based priming potential [134]. In barley, cold pre-treatment has been shown to enhance basal resistance to Blumeria graminis by remodeling chromatin accessibility at genes involved in ROS signaling [135]. Collectively, these findings demonstrate that priming and immune memory represent critical adaptive strategies in plants, integrating environmental signals into durable epigenetic landscapes that support future resilience to diverse stresses and pathogens (Figure 2).

4.2. Epigenetic Memory and Transgenerational Priming in Plant Immunity

Stable epigenetic modifications at specific gene loci underpin the long-term nature of immune memory. These include covalent histone modifications such as H3K4me3 (associated with active chromatin) and H3K27ac3 (linked to repression), as well as DNA methylation and changes in nucleosome positioning [136]. These marks act as epigenetic bookmarks that facilitate faster reactivation of defense genes. DNA demethylation at promoter regions of transcription factors like WRKY and defense genes such as PR1 leads to transcriptional de-repression during or after priming events [137]. Enzymes such as ROS1, DML2, and DME actively remove methylation marks and are upregulated in response to stress [138]. In addition to DNA- and histone-based mechanisms, small RNAs, including siRNAs and phasiRNAs, regulate the chromatin environment by reinforcing silencing of negative regulators or tuning expression of NLR genes [139]. Some of these modifications are retained in progeny, giving rise to transgenerational immune memory. For instance, progeny of primed Arabidopsis plants exhibits enhanced resistance to P. syringae and Hyaloperonospora arabidopsidis due to inherited epigenetic marks. This phenomenon suggests a form of Lamarckian inheritance mediated through epigenetics, though the exact mechanism of reprogramming and resetting in subsequent generations remains under active investigation [140]; examples: SAR, fungal priming, and others. Other examples of priming-induced immune memory include responses to necrotrophic fungi such as B. cinerea and hemibiotrophic pathogens like Colletotrichum higginsianum. In Arabidopsis, exposure to sub-lethal doses of Botrytis enhances H3K4me3 enrichment at defense gene promoters and accelerates gene induction during secondary infection. Similarly, priming by beneficial microbes such as Trichoderma spp. or Piriformospora indica not only enhances local immunity but also induces systemic resistance (ISR) through modifications in chromatin and hormone pathways [141,142]. Priming induced by root-colonizing beneficial microbes often relies on jasmonic acid (JA) and ethylene signaling and is associated with reduced DNA methylation at defense loci. For example, inoculation with Pseudomonas simiae WCS417 primes the JA-responsive gene PDF1.2 via chromatin modification, enhancing resistance to leaf-infecting pathogens [143,144]. Expression levels of OsRDR6 correlate with virus resistance across rice cultivars, suggesting its role in priming-like transcriptional readiness [145]. The study of epigenetic memory and transgenerational immune priming offers a promising approach to develop sustainable crop protection strategies. Rather than relying solely on conventional R gene-based resistance, harnessing plant memory through epigenetic breeding or priming agents may provide broad-spectrum and durable resistance.

4.3. Implications for Epigenetic Breeding and Crop Improvement

The understanding of epigenetic memory has opened novel strategies for crop protection beyond conventional breeding. Unlike traditional resistance breeding that focuses on major R genes often prone to pathogen breakdown, epigenetic priming enables plants to acquire broad-spectrum and potentially durable resistance through reversible, heritable changes in gene expression. Priming agents such as BABA, chitosan, and beneficial microbes like Pseudomonas simiae or Trichoderma spp. are already being explored as epigenetic elicitors. These treatments enhance the transcriptional responsiveness of key immune genes via chromatin remodeling or DNA methylation changes. BABA is a non-protein amino acid known to prime plant defenses by inducing epigenetic modifications, such as changes in DNA methylation and histone acetylation, thereby enhancing resistance to biotic and abiotic stresses. Chitosan acts as a natural elicitor, triggering plant defense responses through the activation of signaling pathways and epigenetic modifications, including histone acetylation and DNA methylation changes. Pseudomonas simiae WCS417: This beneficial rhizobacterium is known to induce systemic resistance in plants by modulating the expression of defense-related genes through epigenetic mechanisms, such as chromatin remodeling and histone modifications. Trichoderma spp. These fungi can prime plant immunity by inducing epigenetic changes, including DNA hypomethylation and histone modifications, leading to enhanced resistance against a range of pathogens. In crops like potato, maize, and tomato, these primed states can be stabilized through clonal propagation or passed to progeny, forming a basis for “epibreeding” [146,147]. Epigenetic variants (epialleles) may also be identified using methylome profiling or ChIP-seq datasets, expanding the breeder’s toolkit without altering the DNA sequence. Recent advances in CRISPR/dCas9-based epigenome editing tools such as dCas9 fused to TET demethylases, histone acetyltransferases (e.g., p300), or histone methyltransferases have enabled precise, locus-specific manipulation of chromatin states in plants. These epigenetic effectors, guided by sequence-specific sgRNAs, can modulate DNA methylation or histone modifications without inducing double-stranded breaks, offering a highly controlled and reversible strategy to regulate gene expression [148]. Epigenetic regulation also integrates abiotic cues, as seen in cotton where overlapping pathways mediate resilience to both heat and pathogen stress [149]. Such tools hold significant promise for the targeted induction of primed immune states in elite cultivars. For instance, targeted acetylation of defense gene promoters may activate basal resistance pathways without compromising yield. By enabling non-genetic, epigenetic activation of immunity, these technologies open new avenues for GMO-free, climate-resilient crop improvement under both biotic and abiotic stress conditions.

4.4. Natural and Engineered Epigenetic Mechanisms in Plant Immune Regulation

Recent advancements in high-throughput sequencing and bioinformatics have greatly expanded our understanding of the epigenetic control of plant immunity. Genome-wide studies using ChIP-seq, bisulfite sequencing, and small RNA profiling have revealed how chromatin marks and small RNA populations shift in response to pathogen attack [150].

One prominent pathway is RdDM, wherein 24-nt siRNAs generated by RNA Polymerase IV are processed via DCL3 and loaded onto AGO4 to direct DNA methylation at promoter regions of immune genes such as WRKYs, PR1, and R genes. Studies in A. thaliana and O. sativa have demonstrated that RdDM not only contributes to silencing of transposable elements near immune loci but also maintains immune gene homeostasis [151,152]. Histone modifications also play pivotal roles. For instance, genome-wide H3K4me3 mapping has shown priming of SA-responsive genes during pathogen memory phases. Histone methyltransferases such as ATX1 and demethylases like IBM1 modulate chromatin accessibility at PR1 and WRKY6 loci [50]. Additionally, the INO80 complex has been upregulated in pathogen-infected Arabidopsis, suggesting its role in chromatin remodeling during biotic stress [48]. Small RNA-mediated PTGS is another critical layer. Discovery pipelines such as miRDeep-P and ShortStack have facilitated the annotation of stress-responsive miRNAs and phasiRNAs across species. For example, miR393 regulates auxin signaling through TIR1 receptor suppression, while miR482 and its associated phasiRNAs target NLR genes in S. lycopersicum during Phytophthora infection [153]. Furthermore, epigenetic priming provides long-term transcriptional memory via stable chromatin states. Genome-wide analyses in rice and tomato have reported sustained H3K4me3 enrichment and reduced CHH methylation at defense genes after priming stimuli. Remodelers such as BRM and SYD are crucial here; mutants deficient in these factors show impaired memory establishment [46,52]. Finally, CRISPR/dCas9-based epigenome editing has enabled locus-specific chromatin modifications. For example, targeting PR gene promoters with dCas9-TET (a nuclease-deactivated Cas9 fused to a DNA demethylase) or dCas9-HAT (dCas9 fused to a histone acetyltransferase) fusions has resulted in robust immune activation without DNA sequence changes, offering promise for non-GMO breeding strategies [154,155]. These findings underscore the integrative role of small RNAs and chromatin regulators in fine-tuning plant immunity. A comparative overview of these mechanisms is presented in

Table 2. Epigenetic and small RNA-based regulatory strategies involved in plant immunity.

Approach	Mechanism	Outcome	Example Genes or Targets	No. of Genes Affected	Regulation Status	Reference
RdDM (RNA-directed DNA Methylation)	siRNA-guided DNA methylation at promoter regions	Transcriptional gene silencing of defense regulators	WRKYs, PR1, R genes	10–100	Regulated as non-GMO (varies by region)	[151,152,156,157]
Histone Modifications	Acetylation/methylation (e.g., H3K4me3, H3K27me3)	Chromatin remodeling and immune gene activation	SA/JA pathway genes, TFs	Dozens to hundreds	Not regulated as GMO	[49,158,159]
Small RNA-Mediated PTGS	miRNA/phasiRNA-mediated silencing of target transcripts	Post-transcriptional silencing; immune homeostasis	miR393, miR482, phasiRNA–NLRs	1–50	Generally unregulated (endogenous pathways)	[64,138,160,161]
Epigenetic Priming	Long-term chromatin state changes via stress or immune cues	Faster and stronger immune response upon re-exposure	PR1, WRKY6, DML2 loci	Context dependent	No regulation (natural induction)	[45,162]
Epigenome Editing (CRISPR/dCas9)	Site-specific modulation of DNA methylation or histone marks	Targeted immune gene activation/repression with heritable outcomes	PR promoters, NLR enhancers	1–10	Mixed (regulated or non-GMO by delivery)	[155,163,164]

.

4.5. Technological Advances and Challenges in Epigenomic Immunity

To unravel the complexities of epigenetic regulation in plant immunity, researchers have adopted advanced genomic tools. CRISPR/dCas9-based epigenome editing has emerged as a powerful method to target specific genomic regions for methylation or demethylation, or to modify histone marks without altering the DNA sequence [165]. CRISPR-based genome editing, including viral delivery systems, opens new avenues for targeted immunity enhancement [166]. Fusions of dCas9 with DNA methyltransferases or histone modifiers enable precise functional validation of epigenetic regulation of defense genes [167]. ATAC-seq is used to assess chromatin accessibility at a genome-wide level and has been applied to study rapid changes in chromatin openness during plant–pathogen interactions [168]. Similarly, ChIP-seq (Chromatin Immunoprecipitation sequencing) provides insight into histone modification landscapes and transcription factor binding during immune responses [169]. Bisulfite sequencing remains the gold standard for mapping DNA methylation at single-base resolution and is used to monitor dynamic changes in methylation at defense loci during priming or infection [170]. In recent years, single-cell omics technologies, such as single-cell RNA-seq and ATAC-seq, have opened new avenues to resolve cell-type-specific immune responses and epigenetic states during pathogen attacks. These methods are beneficial for uncovering heterogeneous responses in complex tissues like leaves and roots and for studying mobile defense signals [171]. Despite these technological advancements, several challenges and future opportunities remain. First, our understanding of locus-specific chromatin dynamics during various stages of infection remains limited. Many chromatin and epigenetic changes are tissue- or cell-type-specific, and capturing these events in real time across spatial and temporal scales remains technically demanding. Moreover, the transient and reversible nature of some epigenetic marks poses challenges for interpretation and functional validation. Additionally, integrating multi-omics datasets, including transcriptomics, epigenomics, metabolomics, and proteomics, at single-cell resolution could uncover previously unknown regulatory modules [172]. There is also a need for functional epigenetic annotations in crop genomes, which lag behind model systems like Arabidopsis [173]. Ultimately, leveraging these insights could pave the way for engineering durable disease resistance through epigenetic reprogramming. This might include the use of epigenome editing to stably modify stress-responsive loci or developing chemical agents that prime immunity without growth penalties. With more refined tools and better data integration, plant epigenetics may unlock new strategies in sustainable agriculture.

5. Conclusions

Plant immunity is orchestrated through a sophisticated interplay between transcriptional regulators, chromatin remodelers, DNA methylation pathways, and small RNA-mediated silencing mechanisms. This review underscores how these deoxyribonucleic acid-level regulatory systems not only govern rapid and localized defense responses, but also facilitate long-term, systemic, and even heritable immunity. Small regulatory ribonucleic acids play pivotal roles in guiding both transcriptional gene silencing and post-transcriptional gene silencing, while dynamic changes in chromatin accessibility and deoxyribonucleic acid methylation modulate immune gene expression with temporal and spatial precision. The emerging concept of epigenetic memory, where prior stress exposure primes plants for enhanced future responses, has added a new dimension to plant immune regulation. Such priming, whether triggered by beneficial microorganisms, non-lethal environmental cues, or chemical elicitors, equips plants with heightened resilience without incurring physiological penalties. However, key challenges remain, including deciphering the precise molecular mechanisms that stabilize beneficial epigenetic modifications, understanding the specificity of small regulatory ribonucleic acid and chromatin interactions, and ensuring that epigenetic changes are heritable without negative effects on plant growth or productivity.

Future research must focus on integrating high-resolution chromatin profiling, clustered regularly interspaced short palindromic repeats-based epigenome editing, and transgenerational tracking of chromatin states. Overcoming technical limitations in mapping tissue-specific epigenetic changes and scaling biotechnological applications to real-world agricultural systems will be essential. Furthermore, establishing practical frameworks for epigenetic crop improvement through the selection of stable chromatin modifications and predictive molecular markers offers promising routes to sustainable agriculture. In summary, realizing the full potential of epigenetic regulation in plant defense requires coordinated efforts across molecular biology, genetics, breeding, and biotechnology. Through such multidisciplinary collaboration, we can develop resilient, high-performing crops fortified with durable, heritable immunity suited for the challenges of future agriculture.