Plant Immunity Is Regulated by Biological, Genetic, and Epigenetic Factors

Abstract

An immune system is a protective mechanism that shields plants from environmental stresses. This primary function is to maintain optimal circumstances for the growth and development of plant tissues while avoiding harm from biotic and abiotic stress factors. Plants subjected to various stressors initiate stress signaling cascades that affect multiple gene expressions and induce adaptation. These signaling pathways are coordinated by transcription factors, non-coding RNAs, RNA-binding proteins, and protein–protein interaction networks. Several studies have focused on various immune systems, but no study has collected all of them together to illustrate them efficiently. According to this review, stress-responsive genes encode ion and water transporters, enzymes, and transcription factors, making plants more resistant to biological and abiotic challenges. Plants have also evolved anti-pathogen defense systems such as regulatory hormone pathways, reactive oxygen species generation, gene expression, programmed cell death, and cell survival. Plants produce short RNAs in response to a viral attack, which silences the offensive genome and creates complex epigenetic regulatory mechanisms such as histone changes, chromatin remodeling, and DNA methylation to protect plants from pathogens. This review provides an in-depth description of proteins, effectors, and pathways included in plant resistance against environmental stresses and offers details on future trends, such as metabolic pathways and genetic engineering, to improve the protection of plants against stress-induced responses.

1. Introduction

Undoubtedly, all living organisms’ survival depends on plants. Most food, sanitary, and industrial products are derived directly or indirectly from plants. Given the high conservation of different organisms in biological systems, plant discoveries also have important implications for human health. According to the abovementioned, the importance of plants’ survival and development becomes more apparent. Plant immunity is the innate or induced capacity of plants to eliminate pathogens such as bacteria, fungi, viruses, nematodes, and insects. Plants have also developed immune mechanisms to avoid herbivores [1]. Plant defense against abiotic or biotic perturbations is critical for plant adaptability and survival under suboptimal growth or adverse conditions.

Despite the many similarities between plant and animal immunities, there are also notable differences. For instance, plants lack antibodies, T cells, and circulating immune cells [2]. Plant immune responses can be categorized into functionally different classes: (1) basal responses, including the transcriptional regulation of genes in response to pathogen-associated molecular patterns (PAMPs), (2) hypersensitive responses, which involve the apoptosis of cells at the site of infection, (3) systemic acquired protection, which renders the entire plant resistant to infection, (4) jasmonic acid (JA)/ethylene (ET) pathway responses, which protect the whole plant and neighboring plants from herbivores, and (5) non-host immunity [3,4,5,6].

Environmental changes are usually unfavorable, posing severe threats to plant growth and development [7]. Abiotic stresses under adverse ecological conditions include drought, high or low temperatures, lack of nutrients, excess salt, and heavy metals in the soil. Besides biotic stresses, abiotic stresses significantly affect crop yield and wild plant species distribution. Drought, salinity, and extreme temperatures are the abiotic stresses affecting the geographic distribution and limiting crop yield. The harmful effects of biological and abiotic stresses on plants are worsening because extreme weather is occurring more often [8]. In a natural environment, the ability of plants to cope with abiotic stresses is influenced by microbial communities composed of bacteria, fungi, and oomycetes that inhabit the host plant without causing disease. Upon recognizing environmental stresses via specific sensing mechanisms, plants adapt their growth, metabolism, and development. Hence, like almost all living organisms, plants have developed signaling pathways to sense changes in their environment and adjust their metabolism and biological functions to prevent stress-related damage [7]. Osmotic stress caused by drought has been recognized as the most common abiotic stress, followed by salinity stress [9], causing ion toxicity [7]. The secondary effects of drought and salinity stress are incredibly complex. They include oxidative stress, metabolic dysfunction, and damage to various cell components, such as membrane lipids, proteins, and nucleic acids [10]. Although primary stress signals can induce immune responses in plants, secondary stress signals are the main triggers of plant immune responses [11]. Extensive crosstalk occurs between the signal transduction pathways induced by drought and salt stress. For instance, the hyperosmotic signals induced by drought and salt stress promote the accumulation of the plant hormone abscisic acid (ABA), which is responsible for numerous plant biological responses [12]. This review summarizes the essential principles of immunity in biological defenses such as PAMP and effector-triggered immunity (ETI) stimulators, systematic immunity propagation in plants, host innate immunity, and signal transduction. The importance of epigenetics, such as histone modifications, chromatin remodeling, DNA methylation, and non-coding RNAs, has been discussed. This review discusses recent research on how stress affects gene expression, metabolism, physiology, and plant growth.

2. Biological Defenses Mediate Plant Immunity

The crosstalk among hormone signal transduction pathways in response to invading pathogens can be antagonistic or synergistic. Salicylic acid (SA) and JA are the predominant hormonal pathways in plant defense [13]. Plant defense mechanisms are versatile and tailored according to the pathogens’ characteristics. The SA pathway is induced primarily in response to biotrophic pathogens, whereas the JA/ET pathway is involved in the defense against herbivore or necrotrophic pathogen infections [14]. Typically, only one of these pathways is activated to preserve energy and resources [15]. Although JA, ET, and SA are the predominant hormones in plant defense, additional hormones play a supporting role. These hormones include ABA, auxins, gibberellins, cytokinins, and brassinosteroids. SA has a vital role in the fight against biotrophic pathogens, whereas JA and ET are critical defense hormones in response to herbivores and necrotrophic pathogens. While the JA/ET and SA pathways have been the most antagonistic, studies in mutant plants have suggested a synergistic effect under certain conditions [16]. In pattern-triggered immunity (PTI), PAMPs such as bacterial flagellin and fungal chitin are detected by transmembrane pattern recognition receptors (PRRs), which initiate PTI on the cell surface. Upon activation, PRRs initiate complex defense signaling cascades [17].

Plant immunity relies on innate immune receptors, which are expressed in every cell and recognize invasion signals, inducing PTI or ETI [18]. To prevent pathogen infection, plants use receptor barriers for innate defense. Microbial or altered host molecules are recognized by PRRs in the plasma membrane, activating PTI [19,20]. Damaged endogenous molecules, microbe-associated molecular patterns (MAMPs), and danger-associated molecular patterns are recognized and bound to by PRRs. Activation of PRR by cytosolic Ca²⁺ and apoplastic reactive oxygen species (ROS) responses promotes phosphorylation and activates the NADPH oxidase respiratory burst oxidase D (RBOHD). RBOHD activation results in the rapid production of ROS in a calcium-dependent or calcium-independent manner. Increases in ROS and cytosolic Ca²⁺ cause the activation of Ca²⁺-dependent protein kinases, mitogen-activated protein kinases (MAPKs), and defense hormone networks, as well as a lot of transcriptional, translational, and metabolic reprogramming [21] (Figure 1).

MicroRNAs (miRNAs) are central players in these defense cascades, modulating gene expression, metabolism, and plant development [22]. PTI results in distinct physiological phenomena, including stomata closure to prevent pathogen penetration; ROS and nitric oxide (NO) production; translocation of nutrients from the cytosol to the apoplast; deposition of callose; and production of various antimicrobial metabolites and defense hormones [23,24]. Signaling cascades involved in ETI are more robust than PTI-related cascades. ETI is initiated upon plant recognition of pathogen virulence factors known as effector molecules by resistance proteins (R proteins). R proteins can be extracellular or intracellular and recognize pathogen virulence factors directly or indirectly. Most R proteins contain nucleotide-binding sites and leucine-rich repeat domains. The gene family containing these domains is conserved across plant species and includes highly polymorphic genes often found in clusters [25]. Thus, PTI and ETI result in extensive reprogramming of plant gene expression via various receptor proteins, signal transduction cascades, kinases, ROS, hormones, heat shock proteins, and transcription factors, protecting against invading pathogens [26] (Figure 1).

The overlap between the physiological responses involved in PTI and ETI partially brings together plant immune signaling pathways in response to biological stresses. The transcriptional regulation of downstream signaling cascades involved in ETI and PTI is controlled by molecules such as small RNAs, transcription factors, chromatin remodelers, histone modifiers (like methylase and acetylase), and transcriptional regulatory complexes [27,28] (Figure 1).

2.1. PAMP (PTI) and Effector (ETI)-Stimulated Immunity in Plants

The plant immune system is a complex network of active and passive systems that withstand pathogen invasion of a host [29]. Most plant surface barriers, such as wax coatings, rigid cell walls, cuticular lipids [30], antimicrobial enzymes [31], or secondary metabolites [7], stop pathogens from getting into the plant and causing disease symptoms to appear.

PAMPs/apoplastic effectors, intracellular effectors, or changed effector targets are actively detected by receptors in the plasma membrane or R proteins in the cytoplasm, activating PTI and ETI. Helper proteins and guard proteins/decoys play a role in pathogen-derived component co-perception [32]. Pathogens use host proteins (S-proteins) expressed by plant sensitivity genes (S-genes) to help enter and multiply, which leads to host adaptation [33]. To get past these protective layers, pathogens use microbial (or pathogen)-associated molecular patterns (MAMP/PAMP)-stimulated immunity (MTI/PTI) and enhanced T-cell immunity (ETI) [30] (Figure 2).

2.2. Systemic Propagation of Immunity in Plants

Plants react to their surroundings in a stimulus-specific manner. These reactions frequently converge on multiple phytohormone pathways interacting with one another and presumably fine tune plants’ pathway responses to optimize fitness [34,35]. PTI and ETI are both SA-dependent and elicit a systemic, SA-dependent defense response known as systemic acquired resistance (SAR) [2]. SAR is a type of long-term resistance to a wide range of (hemi-) biotrophic infections [36]. Azelaic acid and glycerol-3-phosphate are also considered as additional signals in SAR [37]. Another common type of induced (pathogen) resistance is induced systemic resistance (ISR), which is triggered by commensal bacteria in the plants rhizosphere [38]. ISR appears to work against a broader spectrum of pathogens than SAR, including both (hemi-) biotrophic and necrotrophic infections. Systemic immunity similar to SAR has been seen in monocots such as maize, barley, wheat, and banana [39,40]. Wheat systemic resistance against Xanthomonas translucens is not related to SA. However, it may be linked to JA/ABAPip (pipecolic acid) and/or WRKY and ETHYLENE RESPONSE FACTORS, which build up in barley petiole exudates after an infection that makes the plant resistant [40] (Figure 2). Systemic immunity against Blumeria graminis f.sp. hordei may depend on SA, whereas Pip induces systemic resistance in barley against both infections [41]. Pip treatment induces NO buildup and primes ROS generation [41], indicating that the Pip pathway of SAR may be conserved between monocotyledonous and dicotyledonous plants (Figure 3). It is important to understand the similarities and differences between induced resistance responses in monocots and dicots. Before using SAR signaling components like those from Arabidopsis to protect cereal crops, it will be important to study SAR in monocots such as barley and wheat.

3. Genetic Effects on Plant Immunity

3.1. Signaling Pathways Host Innate Immunity in Plants

Phytopathogens such as biotrophs, hemibiotrophs, and necrotrophs stress the development of their host plants, reducing yields. Plants are constantly in contact with such phytopathogens, making them vulnerable to their attack. Plants must evolve immunity to these attacks to defend themselves. As a result, plants have evolved ways of recognizing and combating disease via PTI and ETI [42].

3.1.1. Pattern-Triggered Immunity

The first step in the immune response is PTI. PAMPs or MAMPs are identified by PRRs and conserved molecular patterns such as lipopolysaccharides, peptidoglycans, chitin, flagellin, EF-Tu, DNA, and ergosterol. The production of ROS, the activation of MAP kinase, and the stimulation of pathogen-responsive gene transcription are some of the well-known MAMP evaluations included in the activation of signaling pathways and hydrolysis [43,44].

3.1.2. Effector-Triggered Immunity

Plants, including R proteins, are able to recognize many effectors released by pathogens to activate their defense systems. ETI is detected by nucleotide-binding oligomerization-like receptors (NOD), which target microorganism-produced effector molecules [45]. The complex network of cell walls and their key protein constituents is one of the most critical aspects of the plant’s immune system. Significant advances in research on how plant viruses affect cell wall remodeling in both susceptible and resistant plants show that cell wall metabolism components can change virus transmission and turn on apoplast- and symplast-based defense mechanisms [46].

3.2. Signal Transduction

3.2.1. ABA Signaling Pathway

ABA is a plant defense-related phytohormone that positively or negatively regulates the immune system, playing a central role in response to abiotic and biotic stresses. The ABA signaling pathway modulates plant growth and development, seed dormancy and maturation, vegetative growth, and stomatal closure in response to various abiotic stimuli. The ABA signaling pathways have been described from hormone receptors to multiple downstream components as a major system that mediates different abiotic stress responses to drought, osmotic, and salt challenges [47,48]. ABA can be synthesized from farnesyl diphosphate (direct C15 pathway) or 9-cis-violaxanthin (indirect C40 pathway). Plants have developed various defense mechanisms to adapt to environmental changes [49]. Under stress, plants accumulate ABA, which serves as a communication molecule among plant cells and initiates various defense mechanisms [50]. Most ABA is synthesized as 9-cis-violaxanthin. In plastids, carotenoids are catabolized into zeaxanthin, which is converted to lutein by zeaxanthin epoxidase. Lutein is converted to all-trans-violaxanthin, which is transformed into 9-cis-violaxanthin or all-trans-neoxanthin; 9-cis-violaxanthin is derived from all-trans-neoxanthin [51]. Under aerobic conditions, 9-cis-epoxycarotenoid dioxygenase converts 9-cis-violaxanthin into 9-cis-neoxanthin and xanthin, which are transferred from the plastid into the cytoplasm [52]. Oxyxanthin is converted into xanthic acid, which serves as a substrate for short-chain alcohol dehydrogenase to generate ABA and abscisic acid. Abscisic aldehyde is further converted into ABA by aldehyde dehydrogenase [53]. Thus, ABA’s binding to its receptor initiates various signaling cascades to modulate different cellular processes, including gene expression. In plants, abiotic stimuli induce activation of MAPKs and SNF1-related protein kinase 2 (SnRK2) family members, which play a central role in osmotic stress responses. In Arabidopsis, osmotic stress and ABA activate all SnRK2 family members except SnRK2.9 [54]. The activities of SnRK2 family members, osmotic stress-activated protein kinase, and SAPK1 in Nicotiana tabacum were inhibited by phosphatase treatment, suggesting the importance of phosphorylation in activating SnRK2 proteins [55]. Numerous phosphorylation sites in SnRK2 have been identified. The serine at position 175 in SnRK2.6 has been extensively characterized and proven essential for SnRK2.6 activation [56,57]. Furthermore, ABA treatment increased in vivo phosphorylation levels [58]. NO-induced SnRK2.6 modification inhibits the activity of SnRK2.6. It has been suggested that NO induction by ABA inactivates SnRK2.6 in guard cells, serving as a negative feedback loop that regulates SnRK2.6 activity in response to ABA pathway activation [59]. Phosphatidic acid is a signaling lipid involved in various stress-induced pathways. McLoughlin et al. [60] found that SnRK2.4 and SnRK2.10 accumulated on the cell membrane by binding to phosphatidic acid, promoting salt resistance and root growth and morphogenesis under salt stress. SnRK2s have been shown to bind to ABA-responsive element-binding factors (ABFs), which, upon phosphorylation, regulate the expression of numerous ABA-induced genes [61]. In wheat, PKABA1 is the phosphorylation substrate of TaABF, a seed-specific ABF [55]. In rice, the serine at position 102 of the ABF TRAB1 is crucial for SAPK8/9/10 activation, regulating downstream targets of the ABA signal transduction pathway in response to osmotic stress [62]. In Arabidopsis, ABF1, AREB1/ABF2, AREB2/ABF4, and ABF3 are the most important transcription factors downstream of SnRK2.2/2.3/2.6 in the ABA pathway during the osmotic stress response [61]. These findings highlight the crucial role of SnRK2s in regulating ABA response gene expression by phosphorylating ABFs. SnRK2s also activate the slow anion channel-associated 1 (SLAC1) protein, which regulates ion balance and controls stomatal closure in response to ABA accumulation [63].

3.2.2. Gibberellic Acid (GA) Signaling Pathway

GA is a tetracyclic diterpenoid belonging to the gibberellin family. It is involved in plant growth, metabolism, and response to abiotic stress [52]. The biosynthetic pathways of GA have been extensively investigated [64]. Ent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, ent-kaurenoic acid oxidase, GA13-oxidase, GA20-oxidase (GA20ox), GA3-oxidase (GA3ox), and GA2-oxidase are enzymes essential for GA biosynthesis [65]. Geranylgeranyl diphosphate is converted into the endogen ent-kaurene by ent-copalyl diphosphate synthase and ent-kaurene synthase in plastids. In the endoplasmic reticulum, GA12-aldehyde is formed by ent-kaurene oxidation.

In the cytoplasmic matrix, GA12 and GA53 are oxidized by GA20ox and GA3ox to form different GAs [66] (Figure 4). GA-insensitive and topless-related members of the DELLA (aspartic acid–glutamic acid–leucine–leucine–alanine) protein family and negative regulators of GA signal transduction bind to the promoter of GA20ox after recruitment by the transcription factor GAF1, inhibiting GA biosynthesis. Wu et al. [67] reported the interaction between the zinc finger protein OsLOL1 and the transcription factor OsbZIP58, promoting GA biosynthesis. Serine/threonine phosphorylation has been shown to play an essential role in GA synthesis, as dephosphorylation of the repressor of GA1 induced the transcription of GA20ox and GA3ox [68] (Figure 4).

Plants sense extracellular GA via the soluble GA receptor GID1. The binding of GA to GID1 is concentration-dependent. At low extracellular GA concentrations, GA does not bind to GID1, and the N-terminal extension domain of GID1 binds to GA-responsive genes, suppressing their transcription. When the concentration of extracellular GA is high, GID1 binds to GA via its C-terminal domain, inducing the transcription of GA-responsive genes. GA binding to GID1 leads to a conformational change in GID1, and the N-terminal extension domain of GID1 forms a hydrophobic surface that binds to DELLA proteins. GA–GID1 complexes bind to DELLA proteins, forming trimers, which bind to the ubiquitin E3 linker complex (SCFSLY1/GID2), leading to DELLA protein degradation by the 26S proteasome [69]. In the absence of GA, GID1 binds to DELLA proteins, activating alternative pathways [70]. Therefore, plants can sense extracellular GA via both GA-dependent and GA-independent pathways. Ca²⁺, cyclic guanosine monophosphate (cGMP), and NO are critical second messengers in GA signal transduction [71]. These second messengers transform extracellular cues into intracellular signals, triggering various physiological responses. Ca²⁺ and cGMP are positive regulators of GA signal transduction, whereas NO is a negative regulator of the GA signal pathway. In rice, Ca²⁺ binds to calmodulin to form Ca²⁺/calmodulin, which regulates the expression of Ca²⁺/ATPase-dependent GA-responsive genes [72]. In Arabidopsis, cGMP and the GA response element play essential roles in the GA signaling pathway, modulating the expression of negative regulators of the GA pathway, SPINDLY, and GID1. Lozano-Juste and Leon [71] confirmed that NO reduced the expression levels of SLY (SLEEPY) and promoted the accumulation of DELLA proteins, thereby inhibiting the expression.

3.2.3. ET Signaling Pathway

ET biosynthesis is induced in PTI and ETI, and ET induction is enhanced by SA [39]. ET is involved in response to various abiotic stresses in plants. For instance, low-temperature stress changes the endogenous level of ET. In Arabidopsis seedlings, ET treatment decreased resistance to frost stress, whereas treatment with ET synthesis inhibitors increased resistance to frost stress. This suggests that ET negatively regulates tolerance to low temperatures [73].

In contrast, ET increased the frost resistance in Arabidopsis seedlings grown in soil [74]. The effect of ET on frost resistance may vary depending on the growth conditions [75]. Under salt stress, ET negatively regulated salt tolerance in Arabidopsis. Overexpression of the wheat TaACO1 (aminocyclopropane-1-carboxylate oxidase) gene reduced two crucial transcription factors, DREB1B/CBF1 and DREB1A/CBF3, suggesting the decrease in stress tolerance in Arabidopsis was due to increased salt sensitivity [76]. Although ET promotes stomatal opening by inhibiting ABA-induced stomatal closure, it also promotes stomatal closure by inducing ROS production in guard cells [77]. Methionine-derived 1-aminocyclopropane-carboxylic acid is the primary ET precursor. During the Yang cycle, methionine is converted to S-adenosylmethionine by methionine adenosyltransferase. The ET precursor 1-aminocyclopropane-1-carboxylic acid is formed by 1-aminocyclopropane-1-carboxylic acid synthetase, and 1-aminocyclopropane-carboxylic acid is converted to ET by 1-aminocyclopropane-1-carboxylic acid oxidase. Methionine is replenished by 5-methylthioadenosine, produced simultaneously as 1-aminocyclopropane-carboxylic acid [78] (Figure 5). The ET signaling pathway has been extensively evaluated in Arabidopsis. The ET receptor (ETR) is located on the surface of the endoplasmic reticulum. In the absence of ET, ETR inhibits activation of the ET signaling pathway by binding to CTR. However, ET binds to ETR, releasing the CTR protein in the presence of ET in the cell. Subsequently, CTR activates EIN2 by cleavage; the carboxyl end of EIN2 enters the nucleus, activating the transcription of EIN3, and the resulting EIN3 protein induces ERF expression [79] (Figure 5).

EIN3 is a nuclear protein acting as a plant-specific transcription factor. It plays a critical role in the ET signaling pathway by directly regulating the expression of ET-responsive genes. Arabidopsis has five EIN3 homologous genes (EIL1–5), and EIN3/EIL1 controls the expression of most ET-responsive genes, as reflected by the finding that EIN3/EIL1 defects result in ET insensitivity. Although ET treatment does not alter EIN3 mRNA levels, it causes the accumulation of the EIN3 protein [80]. In the absence of ET, EIN3 binds to the F-box protein EBF, which results in its recruitment to the Cullin protein complex for ubiquitination. Therefore, the level of EIN3 protein in plants is deficient under physiological growth conditions. However, in the presence of ET, a portion of EIN2 translocates into the nucleus, promoting EBF degradation and subsequent EIN3 stabilization [7,81]. Numerous studies have shown that the EIN3/EIL complex promotes salt stress resistance [65] and low-temperature stress [73]. EIN3/EIL1 directly regulates the expression of the ERF transcription factor in plants, and ERF regulates the amount of ET-responsive genes produced in response to abiotic stresses [82,83] (Figure 5).

3.2.4. SA and JA Signaling Pathways

Important roles in plant defense are played by the phytohormones SA and JA. Various studies have shown that SA- and JA-mediated signals interact among themselves (SA-JA crosstalk) to regulate plant innate immunity against pathogens. SA and JA, which are antagonistic defensive hormones involved in defense against biotrophs and necrotrophs, accumulate at high levels during ETI [84]. The plant hormones SA and JA function as critical secondary signaling molecules in plant immunity [39]. In response to plant–pathogen interactions, primary signaling pathways influence the levels and activities of SA and JA [34,85]. Notably, the treatment of cucumber seedlings with SA synthesis inhibitors blocked the accumulation of endogenous SA and rendered plants more susceptible to the damage caused by low temperatures. Inhibition of SA synthesis under low-temperature stress led to the accumulation of hydrogen peroxide, highlighting the importance of SA in response to low-temperature stress by regulating the expression of low-temperature-responsive genes and intracellular hydrogen peroxide levels [86]. JA is involved in tolerance to low temperatures and freezing [75]. Notably, exogenous JA increased frost resistance in Arabidopsis, while mutations in JA biosynthesis genes increased sensitivity to freezing [87]. In addition, JA has also been implicated in salt tolerance. Specifically, the branch of the JA synthesis pathway catalyzed by allene oxide cyclase via MYC2-dependent and ABA-independent pathways is responsible for the JA-mediated increased salt tolerance in plants [88].

3.2.5. Crosstalk in Stress Response Signaling Pathways

Abiotic stresses, such as drought, salinity, and low and high temperatures, initiate systematic responses in plants. Biological crosstalk refers to instances in which one or more components of one signal transduction pathway affect another. This crosstalk can be achieved through various mechanisms, including the interactions among different signaling cascade protein components. In plants, however, stress response pathways are not initiated independently, and apart from local responses, local stress also triggers systemic responses. For example, local salt stress at the root tip can alter calcium and ROS levels in the whole plant, affecting salt tolerance systemically [89]. The zinc finger protein ZAT12 is involved in DNA repair and replication, essential in ensuring cell survival by acclimation to different environmental stress conditions via RBOHD-mediated ROS accumulation [90].

Activating the intracellular Ca²⁺ signal enhances ROS production and the expression of defense genes, initiating systemic plant defense responses [91]. The guard cell hydrogen-peroxide-resistant 1 (GHR1) protein is a plasma membrane receptor kinase, expressed in guard cells, and regulates the activation of plasma membrane calcium channels [92]. It has been reported that JA responses positively relate to SA [93] and ET [94] responses because of synergistic or feedback regulation. In Arabidopsis, EIN3/EIL1 is regulated by EBF1/2. EIN3/EIL1 regulates the transcriptional repressor jasmonate ZIM domain (JAZ) and causes it to activate ET-responsive gene expression.

Additionally, JAZ inhibits the transcriptional activity of EIN3/EIL1, suppressing the expression of the Erf1 gene. MeJA, a vital component of the JA pathway, increased ET levels in plants [95]. Although JA and ET both positively regulate plant defense responses, they may also have antagonistic effects [96]. Studies have shown that EIN3/EIL1 levels are also regulated by transcription factors activated by other hormones, such as GA. DELLA proteins, transcriptional repressors of the GA signaling pathway, bind to regulatory sequences in Ein3/Eil1 genes, suppressing their transcription [97]. However, in the absence of GA, DELLA and MYC2 bind to JAZs, and the binding of MYC2 to G-box motifs in JA-responsive genes activates their expression. When the GA content increases, DELLA proteins are degraded, and JAZs are released, inhibiting the JA signaling pathway [98]. Yaish et al. [99] demonstrated that OsAP2-39 overexpression induced the upregulation of 9-cis-epoxycarotenoid dioxygenase I, a key enzyme in ABA biosynthesis. OsAP2-39 also plays an essential role in maintaining the balance between ABA and GA in plants by regulating the expression of critical enzymes involved in ABA and GA biosynthesis. In response to abiotic stress in plants, DELLA proteins participate in the dynamic balance between ABA and GA by enhancing the expression of the ubiquitin E3 ligase Xerico, reducing GA levels, and increasing ABA levels [100].

4. Epigenetics Mediate Immunity in Plants

4.1. Histone Modifications

Histone modifications are covalent post-translational modifications in histones, including methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation. These post-translational modifications regulate gene expression by altering chromatin structure or recruiting transcriptional regulators, recognizing specific histone modifications. Histones are crucial for the packaging of DNA into chromosomes. It has become evident that histone modifications regulate diverse biological processes, including transcriptional activation and inactivation, chromosome packaging, and DNA damage repair [101]. The contrasting effects of histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate the acetylation levels of lysine residues within the N-terminal tail histones. Acetylation is a feature of actively transcribed genes, and some HATs have been shown to function as transcriptional co-activators [78]. In plants, HDACs and HATs control the expression of genes for stress response and development [102]. Thus, HDACs are essential for defense against plant pathogens. According to Chen et al. [103], HDA19 is the predominant HDAC involved in the defense against plant pathogens. HDA19 is a nuclear RPD3/HDA1 family member with HDAC catalytic activity, and its absence has been shown to increase histone acetylation levels by 10-fold [104]. Significantly, increased AtHDAC19 expression levels have enhanced fungal resistance via activation of the ethylene-responsive factor 1, whereas AtHDAC19 silencing has increased susceptibility to fungal infections [105]. It has been suggested that HDA19 and ERF factors may regulate the expression of JA-responsive genes [106]. Additionally, HDA19 was shown to protect against Pseudomonas syringae infection by suppressing SA-responsive gene expression [107], highlighting the extensive crosstalk between the pathways initiated by different plant hormones. Notably, mutations in HDA19 enhanced the expression of SA-responsive genes and provided resistance to biotrophic pathogens [107].

HDAC6 is another HDAC that activates JA-dependent defense mechanisms in plants [108]. It has been suggested that NO suppresses histone deacetylation, ultimately leading to hyperacetylation and subsequent transcription of defense genes [109].

Histone methylation involves transferring one, two, or three methyl groups from S-adenosyl-L-methionine to lysine or arginine residues in histones by histone methyltransferases. Histone methylation regulates gene expression via chromatin-dependent transcriptional repression or activation. In plants, methylation of lysine residues in histone H3 has been well-documented [110]. Notably, H3K27me3 [111], H3K9me2 [112], and H3K4me1/2/3 [113] have been identified as epigenetic modifications in histone H3 that affect DNA packaging and gene expression. For example, in Arabidopsis, H3K27me3 suppressed the expression of over 4000 genes [114]. Interestingly, H3K4me2 and H3K4me3 appear to be mutually exclusive [113].

The WRKY superfamily comprises approximately 100 transcription factors involved in the defense against pathogens and stress [115]. WRKY70 [116], the most extensively investigated WRKY member, regulates plant immunity. H3K4 methylation in the WRKY70 gene has promoted SA-dependent defense responses in plants [117]. In Arabidopsis, WRKY70 links SA- and JA-mediated responses. In wild-type plant cells, P. syringae induced the expression of WRKY70. WRKY70-deficient mutant plants exhibited greater sensitivity to necrotrophic and biotrophic pathogens, including the bacterial pathogens Erwinia carotovora and Erysiphe cichoracearum, as well as the fungi Botrytis cinerea [118]. Similarly, bacterial (Streptomyces) infection in tomato plants increased WRKY70 expression [119]. Protein ubiquitination involves the addition of ubiquitin, a 76-residue peptide, to different proteins. Ubiquitination is mediated by ubiquitin ligases E1, E2, and E3 [120]. Interestingly, among histones, ubiquitination has only been observed in H2B or H2A. Typically, ubiquitination positively regulates gene expression by opening up chromatin. For example, the Arabidopsis genes HUB1 and HUB2 encode monoubiquitination ligase E3 and are essential for plant defense against necrotrophic pathogens [121]. HUB1 and HUB2 were also required for biotrophic pathogen resistance [122] and cuticle alterations [123]. Cotton plants overexpressed with HUB2 were better able to withstand drought, while those whose expression was silenced were more susceptible [124].

4.2. Chromatin Remodeling

Chromatin remodelers are multi-protein complexes that modify histone–DNA interactions via ATP-dependent transfer or modulation of nucleosomes. Pardal et al. [125] reported that chromatin remodeling ATPases control the plant’s immune response mechanisms. Kang et al. [126] described how a novel regulator, CHR19, affects the transcription of genes, influencing plant resistance to various pathogens by acting at the chromatin level. Additionally, it has been shown that VdDpb4 and VdIsw2 play a role in preserving chromatin structure for nucleosome positioning and transcription regulation, which includes genes responsible for DNA repair in response to ROS stress throughout improvement and plant infectious disease [127]. These protein complexes include catalytic subunits of the sucrose non-fermenting 2 (SNF2) family of DNA helicases/ATPases and are recruited to particular promoters via interaction with transcription factors or accessory proteins. SNF2 proteins are evolutionarily conserved and are categorized into subfamilies according to archetypal members [128,129]. Recently, a tomato SNF2 protein was identified, and its potential role in response to diverse stimuli, including ABA, SA, cold, and salt, has been suggested [130].

4.2.1. Photoperiod-Independent Early Flowering 1 (PIE1)

PIE1 is an SWR1-like protein belonging to the SWR1 subfamily of SNF2 proteins. In this way, PIE1 regulates gene expression by replacing the canonical H2A histone with the H2A.Z variant in a replication-independent manner [131,132]. PIE1 is the Arabidopsis SWR1-like complex catalytic component containing various other proteins, including serrated leaves and early flowering (SEF), actin-related protein 6, and H2A.Z [89,133]. PIE1 cooperates with the H2A.Z histone variant encoded by the genes HTA8 (At2g38810), HTA9 (At1g52740), and HTA11 (At3g54560). In Arabidopsis, the SWR1-like complex regulates SA-dependent defense pathways. H2A.Z residues are often targeted for acetylation [134] or methylation [135], modulating the accessibility of chromatin to regulatory proteins [136]. In Arabidopsis, H2A.Z has been implicated in gene silencing regulation in response to DNA methylation [137]. SEF and PIE1 may directly incorporate H2A.Z at the promoters of genes encoding SA pathway inhibitors.

4.2.2. BRAHMA (BRM) and SPLAYED (SYD)

BRM and SYD belong to the SNF2 protein superfamily [129,138] and regulate the development of reproductive and vegetative structures [138]. SYD regulates the expression of the JA-responsive gene PDF1.2a and is essential for initiating JA-mediated defense mechanisms against the necrotrophic pathogen B. cinerea [139]. Specific SYD mutants (syd-4) exhibited enhanced resistance to the bacterial pathogen P. syringae pv. maculicola ES4326 [140]. In plants, the chromatin remodeler BRM was shown to control ABA-responsive gene expression in response to abiotic stress [141]. Importantly, BRM101-mutant plants exhibited dysregulated expression in some defense-related genes, suggesting that BRM plays a vital role in plant defense pathways by modulating the crosstalk between SA and ABA signaling pathways [142].

4.2.3. Decrease in DNA Methylation 1 (DDM1)

DDM1 is a helicase belonging to the LSH subfamily involved in the methylation of genomic DNA. ddm1-mutant Arabidopsis plants exhibited decreased cytosine methylation, TE pathway activation, and histone modification alterations [143]. Furthermore, DDM1 has been shown to regulate the SA signaling pathway, among other plant defense mechanisms [95]. The variations of DDM1 gene expression were discovered in an infection-resistant Arabidopsis strain [144] that controls other chromatin alterations such as histone modifications [145]. Notably, ddm1-mutant plants exhibited remarkable resistance to Hyaloperonospora arabidopsidis [146], indicating the importance of DDM1 in plant defense mechanisms.

4.2.4. CHR5 (Chromatin Remodeling Factor 5)

The chromatin remodeler CHR5 positively regulates gene expression by relaxing the chromatin structure at Snc1, among other genes. Chr5-mutant plants exhibited deficient SNC1 levels and increased susceptibility to avirulent and virulent strains of P. syringae [145] due to enhanced activation of the SA signaling pathway [147].

4.3. DNA Methylation

DNA methylation is an epigenetic mechanism regulating gene expression by adding methyl groups to DNA. In many eukaryotes, methylation of the fifth carbon of cytosine is a critical mechanism for modulating chromatin structure, thereby regulating DNA recombination, gene expression, and genomic imprinting [148]. High-resolution DNA methylation profiling in Arabidopsis has shown that DNA methylation is widespread in repetitive DNA sequences and pericentromeric transposons [149]. In plants, DNA methylation is mediated by plant DNA methyltransferases, such as DDM1 [150]. An HDA6-dependent, RNA-directed DNA methylation mechanism has been identified [108]. In vegetative structures, the repressor of silencing 1 (ROS1) has been shown to play a critical role in DNA demethylation [151]. Although most plant viruses have RNA genomes, the genomes of Geminiviridae family members are single-stranded DNA. Virus infection in plants is fought partly by a defense mechanism involving DNA methylation of the geminivirus viral genome in plant guard cells [152]. Viral DNA methylation has also been detected in tomato plants. Some infectious viral strains carry the beta satellite encoding βC1, a repressor of viral genome silencing [153]. Resistant strains of tomato plants have developed the ability to polyubiquitinate βC1, targeting βC1 for proteasome-mediated degradation. Apart from βC1, various viral effectors have been shown to repress transcriptional silencing [154]. Plants initiate alterations in DNA methylation throughout the pathogen attack [155]. However, mutant plants defective in DNA methylation may protect against viral infections by upregulating SA-responsive genes [146].

4.4. Non-Coding RNAs (ncRNA)

Non-coding RNAs, either as long ncRNAs (lncRNAs) or processed into small non-coding RNAs (sncRNA), are a group of RNAs that do not encode functional proteins and were initially thought to regulate gene expression only at the post-transcriptional level. However, many recent studies have shown that the most common regulatory RNAs are miRNAs, endogenous small interfering RNA (siRNAs), piRNAs, and lncRNAs. There is increasing evidence that regulatory ncRNAs play an important role in epigenetic control [156]. Recently, it has been discovered that various ncRNAs participate directly or indirectly in various epigenetic phenomena that control different phenotypes within cloned cells and the specific determination of various physiological processes. ncRNAs can regulate the production of defense markers, hormone biosynthesis, signal transduction, or RNA-mediated silencing (Figure 6 and

Table 1. ncRNAs involved in plant immune reactions.

Action	ncRNAs	Target	Plant Type	Plant Defense Response	Reference
ROS accumulation	miR400	PRR1/PRR2	Arabidopsis	Disease resistance genes	[157]
	miR825	At5g38850/At3g04220	Arabidopsis	Resistance to Botrytis cinerea	[158]
	miRNA21 miRNA22	NB-LRR/LRR	Tabacco, Tomato, Potato	Tobacco mosaic virus (TMV)	[159]
	bra-miR1885	TIR-NBS-LRR	Brassica rapa	Turnip mosaic virus (TuMV) infection	[160]
	miR528	AO	Rice	Antiviral response in rice	[161]
	lncRNA16397	SIGRX21/SIGRX22	Tomato	Resistance to Phytophthora infestans	[162]
	lncRNA33732	RBOH	Tomato	Resistance to Phytophthora infestans	[163]
PR gene expression	miR393	MEMB12	Arabidopsis	Silencing of a Golgi-localized SNARE Gene	[164]
	miR163	PXMT1/FAMT	Arabidopsis	Defense against Pseudomonas syringae	[165]
	miR163	Dicer-like 1 (DCL1)	Arabidopsis	Role of RNase III enzymes such as Drosha and Dicer in miRNA biogenesis in animals	[166]
	miR7695	Nramp6	Rice	Pathogen resistance	[167]
	miR7695	Nramp6	Rice	Blast fungus Magnaporthe oryzae	[168]
	ELENA1	PR1	Arabidopsis	Innate immune response genes	[169]
	lncRNA39026	miR168a	Tomato	Resistance to Phytophthora infestans	[170]
	miR 13609	TARs	Arabidopsis	Responsive to Fusarium oxysporum infection	[171]
	circR5g05160	IR25	Rice	Resistance Magnaporthe oryzae	[172]
Callose deposition	miR166k-166h	EIN2	Arabidopsis	Immunity via post-transcriptional control of EIN2 expression	[173]
	miR160a	ARF16	Rice	Positively regulate PAMP-induced callose deposition, negatively regulate PAMP-induced callose deposition, and disease resistance to bacteria	[174]
	miR398b and miR773	COX5b.1, CSD1, and CSD2	Arabidopsis	Negatively regulate PAMP-induced callose deposition and disease resistance to bacteria	[174]
Hormone	miR393	TIR1/AFB2/AFB3	Arabidopsis	Repression of auxin signaling restricts P. syringae growth	[175]
	miR319	TCP21	Rice	Suppression of Jasmonic acid-mediated defense by viral-inducible MicroRNA319 facilitates virus infection	[176]
	miR477	CPB60A	Cotton	Plant defense against Verticillium dahlia	[177,178]
	ALEX1		Rice	Disease resistance	[178]
	GhlncNAT-ANX2GhlncNAT-RLP7	LOX1/LOX2	Cotton	Resistance to Verticillium dahliae, a fungal disease	[179]
miRNA biosynthesis pathway	miR863-3P	SERRATE	Arabidopsis	Bacterial infection	[180]
	RPS2/RPS2	avrRpt2	Arabidopsis	Phytopathogenic Pseudomonas	[181]
	miR168	AGO1	Rice	Virus resistance	[67]
	miR444	MADS23/27a/57	Rice	Antiviral RNA silencing pathway	[182]
	miR403	AGO2	Arabidopsis	Turnip crinkle virus (TCV) and cucumber mosaic virus (CMV) infection	[183]
	miR403a	AGO2	Tobacco (Nicotiana benthamiana)	Antiviral defenses against TMV infection	[184]
	ARGONAUTE	AGO1/AGO10	Arabidopsis	Turnip Mosaic virus infection	[185]

).

Moreover, the joint function among ncRNAs, including lncRNAs, siRNAs, miRNAs, and circular RNAs (circRNAs), plays an essential role in plant immunity. Recent studies have shown that various ncRNAs change their expression levels under abiotic and biotic stresses. Interaction of these ncRNAs usually results in the co-regulation of the target gene expressions to modulate plant immunity (Figure 6). Through the complicated interactions that occur between lncRNAs, siRNAs, miRNAs, and circRNAs, a variety of immune responses may be coordinated either synergistically or antagonistically [186].

4.4.1. MicroRNAs

MicroRNAs are produced from precursors called primary microRNAs (pri-miRNAs). DICER-LIKE 1 (DCL1) sequentially cleaves most pri-miRNAs, stabilized in nuclear foci known as dicing bodies. miRNAs mediate PTGS (post-transcriptional gene silencing) through translational inhibition or mRNA degradation [187]. The mutation of DCL1 in different plants results in pleiotropic developmental effects or lethal embryos, mainly because of the significant reduction in the miRNA levels [188]. These findings indicate the importance of miRNAs in the developmental processes of plants. MiRNAs also seem to regulate the epigenomic mechanism because some of these molecules regulate genes involved in chromatin rearrangement. For example, miR773 targets DNA methyltransferase 2 (MET2) transcripts, and after infection with pathogens, the miRNA levels are reduced, leading to the increased accumulation of MET2 required for a proper immune response. The overexpression of miR773 suppresses MET2 and weakens PAMP-triggered immunity [174]. After the miRNAs are processed and exported to the cytoplasm, these transcripts are loaded into one of the several ARGONAUTE (AGO) complexes (see section Plant Argonautes) present in the plant cell (10 paralogs in Arabidopsis) [189]. Various miRNAs have been found to guide the R gene cleavage, thereby strictly controlling the expression of the R gene to regulate the immune response in Solanaceae, Leguminosae, Arabidopsis, etc. Most of these miRNAs trigger the production of phased secondary small interfering RNAs (phasiRNAs) from their nucleotide-binding leucine-rich repeat (NLR) targets [186]. Some miRNAs suppress NLR genes through mRNA cleavage rather than by initiating phasiRNA production to inhibit further NLR genes [162]. These facts indicate that various plant species may utilize a widely conserved immunity mechanism in which miRNAs directly target NLR genes to fine tune the immune response. Some siRNAs and miRNAs directly target signal transduction regulators to modulate immunity. Moreover, many siRNAs and miRNAs target transcription factors to modulate the transcription of genes involved in defense mechanisms. After pathogen infection, the ncRNAs mediate transcriptional reprogramming via direct targeting of transcription factors to regulate various immune responses [186].

4.4.2. Small Interfering RNAs

Small RNAs are non-protein-coding RNAs that are 18–30 nucleotides long and regulate gene expression [22]. Small RNAs are an epigenetic mechanism modulating plant immunity according to various biological stresses. Plants have developed mechanisms to silence foreign gene expressions as defense mechanisms against viral attacks [190].

The vast majority of plant viruses are RNA viruses. A double-stranded RNA (dsRNA) molecule is produced from the viral genome by a viral replicase that catalyzes the synthesis of a complementary RNA molecule using an RNA template. Viral dsRNA can be modified by plant dicer-like proteins, triggering the assembly of small RNA complexes that promote viral genome silencing via degradation of dsRNA replication intermediates and viral transcripts or inhibition of their translation.

Similarly, single-stranded RNA viral genomes initiate silencing mechanisms after being converted to dsRNA molecules by plant RNA-dependent RNA polymerases (RDR). In plant viruses with a single-stranded DNA genome (e.g., Geminivirus), the viral transcripts are converted to dsRNA by the plant RDR2, generating viral small RNAs resembling heterochromatic small RNAs and thereby silencing the viral genome at the transcriptional level [191]. Upon recognizing viruses and other plant pathogens, defense mechanisms initiate ROS production along with small RNAs to regulate the levels of plant hormones, such as SA and JA [34].

SiRNA precursors are long dsRNAs that can be produced by various mechanisms, such as inverted sequence folding, partial complementarity between unrelated transcripts, hybridization of antisense and sense sequences, a lncRNA, and the activity of RDRs. The processing of siRNAs mainly occurs through the actions of DCL2, DCL3, and DCL4, resulting in various types of siRNAs that can be classified into subgroups [189].

4.4.3. Long Non-Coding RNAs

LncRNAs, which are longer than 200 nt, represent another type of non-coding regulatory RNA in the cytoplasm or nucleus. Unlike miRNAs and siRNAs, lncRNAs can function without being processed and cleaved by DCR proteins. Among their multiple functions, lncRNAs have been reported to be involved in modulating mRNA stability and translation, miRNAs and protein hijack, and chromatin modification at different levels [192]. Sometimes, miRNAs can be hijacked by “target mimicry”. This process, also known as miRNA kidnapping, was first described in plants. It occurs through partial complementary sequences when lncRNAs act as decoys for miRNAs by blocking the interaction between a miRNA and its target. Moreover, in animals, lncRNAs involved in the sequestration of miRNAs, known as competing for endogenous RNAs (ceRNAs), have been reported. This mechanism in different organisms indicates its significance in regulating various genetic networks in the eukaryotic cell. For instance, regulating miR399 through miRNA kidnapping is a significant regulator of plant phosphate homeostasis. MiR399 guides the degradation of the PHO2 mRNA and promotes the expression of two phosphate transporters in roots, enhancing phosphate uptake [189].

In plants, disease resistance is usually conferred by a diverse intracellular system of NLR innate immune receptors, which detect pathogen proteins and their effects on the host [193]. These receptors represent valuable agronomic traits that plant breeders rely on to maximize yields in the face of devastating pathogens. PRRs and NLRs are regulated at different transcription levels: post-transcription and post-translation. Transcription factors and kinases are among the downstream signaling components of these receptors.

• Circular RNAs

CircRNA, a new type of endogenous ncRNA, has been studied and characterized in various species. These ncRNAs are commonly generated by thousands of genes and have important functions in regulating gene expression at multiple levels in eukaryotic cells [172]. Based on the spatial- and/or tissue-specific expression patterns, plant circRNAs have been found to function during flowering, fiber development, and fruit coloration [194,195]. The expression of plant circRNAs is also affected by biotic and abiotic stimuli, including pathogen invasion [196,197]. For instance, circRNA45 and circRNA47 in tomato function as positive immunity regulators against Phytophthora infestans by regulating the expression of SpRLK and miR477-3P [198]. CircRNAs are the elaborate regulators of immune signaling that modulate plant immunity [186].

4.5. RNA-Binding Proteins (RBPs)

To modify transcription and translation, RNA-binding proteins (RBPs) frequently phase separate RNAs into condensates [199]. From synthesis to decomposition, RBPs are essential regulators of RNA fate. Therefore, changes in the RBPome coordinate RNA metabolism and gene expression reprogramming during plants’ responses to several signals, including pathogen attacks [200]. The number of RBPs characterized in plants is limited, and their contribution to plant immunity is even less evaluated. Plant RBPs containing RNA-binding domains have putative roles in RNA processing and are also involved in plant immune responses (

Table 2. Several RBPs are involved in plant immunity.

RNA-Binding Protein	Conserved RNA-Binding Motif	Other Properties	Possible Functions	Plant Origin	Reference
PRP-BP		Binds U-rich region	PvPRP1 mRNA binding protein may function in the elicitor-induced destabilization of PvPRP7 mRNA	Bean	[202]
RPP13			ETI and hypersensitive response, and the overlap between NHR-induced genes and a gene for host resistant	Wheat	[203]
tcI14	SR-rich repeat region RNP1 and RNP2		Alternative splicing	Tobacco	[204]
GaPR10		K-A-X-E-X-Y-L domain and a P-loop	Ribonuclease activity	Cotton	[205]
GRP7	RRM, glycine-rich motif RNP-binding domain		mRNA stability and control, pathogen defense	Arabidopsis	[206]
CPR5			RNA splicing activator NineTeen Complex and RNA polyadenylation factor CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR coordinately function downstream of CPR5	Arabidopsis	[207]
eIF4E		Eukaryotic initiation factor 4E homolog domain	Components of 5 cap-binding complex (CBC), response to the virus, translational initiation	Arabidopsis	[208]
MOS2	G-patch and KOW motif		RNA binding	Arabidopsis	[134]
MOS11		Homologous to human RNA binding protein CIP29	mRNA export	Arabidopsis	[209]
DCL2	Double-stranded RNA-binding motif, double-stranded RNA-binding domain	Helicase superfamily c-terminal domain, RIBOc. Ribonuclease III C-terminal domain, PAZ domain	Defense response to a virus, maintenance of DNA methylation, production of ta-siRNAs involved in RNA interference	Arabidopsis	[210]
DCL4	Double-stranded RNA binding motif, double-stranded RNA-binding domain	Helicase superfamily c-terminal domain, RIBOc. Ribonuclease III C-terminal domain, PAZ domain	Maintenance of DNA methylation, production of lsiRNA, siRNA, and ta-siRNAs involved in RNA interference, virus-induced gene silencing	Arabidopsis	[210]
ALFIN-LIKE 6 (AL6)			ChEP-P is well suited to gain holistic insights into chromatin-related processes in plants	Arabidopsis	[211]
AGO1	PIWI domain, PAZ domain	DUF1785 domain, PLN03202 domain, Glycine-rich region of argonaut	RNA interference, gene silencing by miRNA, innate immune response, leaf morphogenesis, virus-induced gene silencing siRNA binding, defense response to the virus, RISC components	Arabidopsis	[164]
AGO2	PIWI domain, PAZ domain	DUF1785 domain, PLN03202 domain		Arabidopsis	[164,210]
AGO7	PIWI domain, PAZ domain	DUF1785 domain, PLN03202 domain	Nucleic binding, RISC components, gene silencing by miRNA, production of lsiRNA and ta-siRNAs involved in RNA interference	Arabidopsis	[164]
MAC5A/5B	CCCH-type zinc-finger domain, RNA recognition motif		Component of MAC, associated with spliceosome, defense response to bacterium	Arabidopsis	[212]
AtRBP-DR1	Three RNA recognition motifs		Positive regulation of salicylic-acid-mediated signaling pathway	Arabidopsis	[213]
FgRbp1	FgRbp1 binds to the motif CAAGR in its target mRNAs		Leading to enhanced recruitment of FgU2AF23 to the target mRNAs	Fusarium graminearum	[214]

). Many RBPs have been identified through genomic analysis in the model plant Arabidopsis thaliana, among which 50% are specific to plants, most with unknown functions. Studying plant RBPs related to pathogen infection response can extend our understanding of similar RBPs in other taxa. Because of the instability of their mRNA targets, RBPs have traditionally been challenging to study. Next-generation sequencing (NGS) methods such as RNA-seq will significantly enhance our ability to analyze both RBPs and RBP knockout mutants [201].

4.6. Plant ARGONAUTES

AGOs are the effector proteins in eukaryotic small RNA-based gene silencing pathways controlling gene expression and transposon activity. Eukaryotes produce many small RNAs, such as siRNA, miRNA, scanRNAs, piRNAs, and 21U-RNAs, that each group associates with different AGO family members, such as AGO PIWI, etc. Small RNA-guided AGO proteins regulate gene expression at various levels, including deletion of internal genomic DNA sequences (in ciliates), inhibition of translation (animals), and RNA cleavage (all eukaryotes), which is sometimes followed by chromatin remodeling and DNA methylation. As the model plant species, Arabidopsis contains 10 AGO proteins belonging to 3 phylogenetic groups [215]. AGOs in plants regulate vital biological processes such as genome structure and integrity, development, response to stress, and pathogen defense. Typical functions of plant AGO–sRNA complexes comprise the translational inhibition or endonucleolytic cleavage of target RNAs and the methylation of target DNAs [216] (

Table 3. The biological pattern of plant ARGONAUTEs.

Action	Plant	AGO Involved	ncRNAs	Reference
Antibacterial immunity	Arabidopsis thaliana	AGO2	MEMB12 miR393b	[164]
	Arabidopsis thaliana	AGO4	OCP11	[217]
Antiviral defense	Arabidopsis thaliana	AGO1	PAZ/PIWI	[218]
	Arabidopsis thaliana	AGO1, AGO2, and AGO7	HC-Pro	[185]
	Arabidopsis thaliana	AGO1	cpr5-2	[219]
	Arabidopsis thaliana	AGO2, AGO5	miR163-LL/miR163-UL	[220]
	Arabidopsis thaliana Arabidopsis thaliana Nicotiana benthamiana	AGO1, AGO2, and AGO7	-	[221]
	Arabidopsis thaliana	AGO1 AGO2	RDR6	[222]
	Oryza sativa	Argonaute18 (AGO18) AGO1	microRNA168 (miR168)	[67]
Cell specification	Arabidopsis thaliana	ARGONAUTE 9 (AGO9)	SUPPRESSOR OF GENE SILENCING 3 RNA-DEPENDENT RNA POLYMERASE 6	[223]
Gamete Production	Zea mays	AGO9	-	[224]
Chromosome segregation	Zea mays	AGO9	-	[224]
Development	Arabidopsis thaliana	AGO1	-	[225]
	Oryza sativa	AGO4b	DCL3	[226]
DNA repair	Arabidopsis thaliana	AGO2	DSB	[226]
	Arabidopsis thaliana	AGO9	-	[227]
Germ cell development	Zea mays	AGO18b	-	[228]
Leaf development	Arabidopsis thaliana	AGO1	microRNA165/166	[229]
	Arabidopsis thaliana	AGO7	TAS3 ta-siRNA	[230]
	Oryza sativa	AGO10	OsPNH1	[231]
	Zea mays	AGO7	miR166	[232]
Meiosis	Arabidopsis thaliana	AGO4	-	[233]
	Oryza sativa	AGO5c	-	[234]
Mega gametogenesis	Arabidopsis thaliana	AGO5	P1/Hc-Pro	[235]
Phase transition	Arabidopsis thaliana	AGO7	miR390-ARGONAUTE7	[236]
	Arabidopsis thaliana	AGO7, AGO2, and AGO1	-	[221]
	Arabidopsis thaliana	AGO7	-	[237]
SAM development	Oryza sativa	AGO1 AGO7	miR166	[238]
	Arabidopsis thaliana	AGO10	miR165/166	[239]
	Arabidopsis thaliana	AGO10 AGO7	miR165/166	[240]
SAM maintenance	Oryza sativa	AGO10	OsPNH1	[231]
Small RNA biogenesis
miRNA	Arabidopsis thaliana	AGO1	miRNA161, miRNA173	[241]
siRNA	Arabidopsis thaliana	AGO4	DCL3	[242]
tasiRNA	Arabidopsis thaliana	AGO1	mirR173	[243]
	Arabidopsis thaliana	AGO7	miR390-ARGONAUTE7	[236]
	Oryza sativa	AGO7	miR166	[238]
	Zea mays	AGO7	miR166 3a (arf3a)	[232]
Stress response	Arabidopsis thaliana	AGO1 AGO10	miR166/165	[244]
	Arabidopsis thaliana	AGO1	F-box FBW2	[245]
	Arabidopsis thaliana	AGO10 AGO7	miR165/166	[240]
Tapetum development	Zea mays	AGO18b	-	[228]

).

4.7. RNAi-Based Antiviral Innate Immunity in Plants

According to numerous studies, RNAi is found to be evolutionarily conserved in eukaryotes and to govern all elements of biological events [246]. Non-coding small RNA (ncRNA) is activated by self-complementary or dsRNA and serves as the signal and specificity determinant of gene silencing. Pathogen RNA-derived siRNAs of various sizes are created during pathogen infection to induce RNAi-based antimicrobial immunity and confer host resistance [158,247]. Plant researchers discovered the first RNAi-based antiviral defense agent [12,248] to play an essential role in antiviral immunity in invertebrates [12] and mammals [18,29]. The function of DCLs, AGOs, and RDRs in antiviral immunity was based on discoveries in transgenic silencing and endogenous gene silencing. It is now known that RNAi-based antiviral innate immunity is developed in virtually all eukaryotes to resist invasion by various RNA or DNA viruses. The function of DCLs, AGOs, and RDRs in antiviral immunity was based on discoveries in transgenic silencing and endogenous gene silencing. It is now understood that RNAi-based antiviral innate immunity will be triggered in practically all eukaryotes to resist the aggression of all types of RNA or DNA viruses (Figure 7). Many viruses, particularly pathogenic viruses, evolve viral suppressors of RNAi (VSRs) to attack distinct steps of the RNAi-based antiviral pathway (Figure 7). The predominance of VSR contributes to viral epidemics and blinds us to the importance of RNAi-based antiviral innate immunity. Furthermore, VSR hampered the use of traditional genetic screens to uncover new regulators in the antiviral pathway for decades until recently, when an effective genetic technique to overcome the barrier was devised [249,250].

5. Conclusions

Plants respond to biotic and abiotic stressors by modifying gene expression, protein structure, and metabolic circuits. These physiological responses depend on the perception and transmission of numerous stress cues, which cause multiple defensive mechanisms to be activated. Given the importance of protein phosphorylation and dephosphorylation in signal transduction, protein kinases and phosphatases are essential in plant defense pathways. In general, abiotic stressors cause large-scale alterations in gene expression, which correspond to fundamental plant physiological functions such as photosynthesis, respiration, and metabolic pathways.

However, the processes that control GA biosynthesis are still unknown. Because of GA’s importance in the agricultural economy, the molecular processes controlling plant hormone interactions, particularly between DELLA proteins and other vital members of hormone signaling pathways, have been intensively studied. JA and SA are also required for stress signal transduction in plants. Plant stimulation with JA or SA stimulates various defensive systems via the stearic acid pathway, protecting plants from abiotic stressors. Despite this, little is known about JA and SA’s roles in plant responses to biotic stress. Future research must uncover the processes that allow JA-and SA-mediated signals to be transmitted across plant cells.

ET is another plant hormone that is induced by biotic and abiotic stressors. Despite extensive research into ET signal transduction in plants, little is known about the function of ET-responsive genes. Plant adaptation to abiotic stress requires ET pathway components such as EIN2 and EIN3/EIl1. However, the roles of ET pathway components in stress reactions are unknown. The discovery of the molecular processes underlying ET-mediated stress adaptation is predicted to improve crop yield and have a high agricultural economic value. In conclusion, ET signaling is critical in plant responses to drought and salinity challenges, but its role in other abiotic stresses is unknown.

The ABA signal transduction pathway is critical for plants’ responses to abiotic stresses, and SnRK2 is a vital component of the ABA signal transduction pathway, relating metabolism to stress responses under extreme abiotic stresses. SnRK2 regulates stress signal transduction in response to drought, salt, and osmotic stress. Although the function of SnRK2 in the ABA pathway is well established, its role in ABA-independent responses remains unknown. Furthermore, despite phosphorylation activating SnRK2, the effect of other post-translational modifications on SnRK2 activation and function is unknown. The role of SnRK2 in osmotic stress adaptation is also unknown. Future research must elucidate the mechanisms underlying SnRK2 activation and function in response to various stress signals.

Significant progress has been made in understanding the role of GA metabolism in plants in recent years. The role of GA metabolism in plant growth and development regulation is now relatively well understood. Unexpectedly, the function of GA metabolism in responding to abiotic stress is still largely unknown.

In the promoter regions of genes, many regulatory sequences are typically found. These regulatory sequences make it possible to fine tune the expression of genes in response to a variety of stimuli from the environment. The increased adaptability of plants is largely due to the crosstalk between the various plant hormones. Plants have developed sophisticated mechanisms for responding to stress, which allow them to mount a strong defense and successfully adapt without compromising their ability to continue growing. This system is able to detect stressful conditions such as low temperatures, high temperatures, drought, toxicity from heavy metals, and oxidative stress.

Plant epigenetic regulation of immunity, including histone modifications, DNA methylation, chromatin remodeling, and RNA silencing, has been extensively explored in plants, particularly Arabidopsis. The primary defense mechanism against viruses is mediated by small RNAs, triggering viral genome degradation. However, how plants differentiate new viruses from previously exposed ones is unclear. Future studies are required to assess the role of histone ubiquitination-mediated regulation of gene expression in plant adaptation to stress. Identification of the mechanisms involved in the propagation of epigenetic modifications regulating plant immunity merits further investigation.