Juan Huang , et al.
Citation: Huang J, Zhou W, Zhang X, Li Y (2023) Roles of long non-coding RNAs in plant immunity. PLoS Pathog 19(5):
Editor: Bjorn F. C. Kafsack, Joan and Sanford I Weill Medical College of Cornell University, UNITED STATES
Published: May 11, 2023
Copyright: © 2023 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by the National Natural Science Foundation of China (NSFC 32090012, YL), Program of CAS (ZDBS-LY-SM027, XZ), the Beijing Municipal Natural Science Foundation (5202017, XZ), Hainan Yazhou Bay Seed Lab (B21HJ0104, XZ), and Strategic Priority Research program of the CAS (XDPB16, XZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Throughout their life cycle, plants face challenges of severe environmental conditions, including diverse abiotic and biotic stresses. To overcome these challenges, plants have developed complicated immune systems to recognize stress factors and generate appropriate signal to regulate growth and development, and thus adapting to adversity [1,2]. In response to biotic stress, plants are equipped with cell-surface immune receptors and intracellular immune receptors to sense microbial signals and activate early immune responses, including calcium influx, reactive oxygen species (ROS) burst, and mitogen-activated protein kinase (MAPK) activation [3,4]. These early immune responses in turn regulate downstream transcriptional reprogramming of defense-related genes, including transcription factors and genes involved in hormone synthesis, to form late immune responses.
Long non-coding RNAs (lncRNAs) are defined as a class of endogenous single-stranded non-protein-coding transcripts with a sequence length greater than 200 nucleotides that do not belong to other well-defined non-coding RNA (ncRNA) types . Advances in the past few decades have broadened our understanding of plant signal perception, activation of defense genes, and expression of resistance genes . However, due to their characteristics of non-protein-coding, poor conservation among different species, stage- and cell-type specificity, and low abundance, lncRNAs failed to attract the attention of researchers in the early days. Technical innovations in genome sequencing and the development of bioinformatic tools have greatly improved our understanding of genes at the transcriptional and posttranscriptional levels , especially driving the discovery of ncRNAs, including lncRNAs and small RNAs (sRNAs), and furthered the exploration of their roles in regulating biological processes in animals and plants. Studies in past decades have demonstrated the important and unique roles of lncRNAs in animal growth, development, and immunity [7–9]. In comparison, studies on the function of lncRNAs in plant immunity are lagging behind. However, we should never underestimate the profound potential of lncRNAs in plant immunity. As reported by many articles, a large number of lncRNAs react to pathogen infections or insect infestations [10,11]. Therefore, biological stress could be a good system to study the actions of lncRNAs and expand our knowledge of the RNA world.
Indeed, our understanding of the roles of lncRNAs in plant immunity has improved in recent decades. LncRNAs have been shown to play critical roles in plant responses to various stresses through diverse actions. In this review, we mainly focus on the roles of lncRNAs in plant immunity, aiming to characterize the biogenesis, biological functions, and mechanisms of action of lncRNAs in different immunity stages and distinct plant–microbe/insect interactions. Overall, this review will provide novel insights into plant immunity studies and will help researchers better understand lncRNAs at multiple levels in but not limited to plant immune responses.
1. Biogenesis and modes of action of plant lncRNAs
LncRNAs are ubiquitously present in almost all forms of life ranging among animals, plants, fungi, and prokaryotes, and even including viruses. With the development of genome sequencing and bioinformatic analysis tools, enormous lncRNA candidates have been identified in different plants, including Arabidopsis [12–14], rice [15–21], maize [22–24], cotton [25,26], Medicago , etc. . These lncRNA candidates can be found in many plant databases, including the general plant databases, such as TAIR and Araport, and specific non-coding RNA databases, such as PLncDB, Green Non-coding Database (GREENC), NONCODE, CANTATAdb, PNRD, and PlantNATsDB .
As the most abundant class of ncRNAs, lncRNAs are key regulators of gene expression in various biological processes . According to the positional relationship between an lncRNA and its neighboring protein-coding genes on chromosomes, lncRNAs can be divided into 5 groups (Fig 1): (1) sense lncRNA: located on the same strand as its associated protein-coding gene, and partially or completely, overlapping with the coding region; (2) antisense lncRNA: located on the opposite strand of its associated gene, and partially or completely, overlapping with the coding region; (3) intronic lncRNAs: located within an intron of the associated protein-coding gene; (4) bidirectional lncRNA: located on the opposite strand of the associated protein-coding gene at a distance less than 1 kb from the promoter of the protein-coding gene; and (5) long intergenic non-coding RNA (lincRNA): transcribed from the intergenic region between 2 protein-coding genes [31,32]. Most of well-characterized lncRNAs in plants are antisense lncRNAs and lincRNAs, while rare bidirectional lncRNAs have been characterized in plants (Fig 1).
Fig 1. Classification of lncRNAs based on their genomic location to protein-coding genes.
LncRNAs were classified into 5 categories. Representatives of each category were listed in the figure, such as COLDWRAP  and LDMAR  in sense lncRNAs, COOLAIR , Enod40 , MAS , nalncFL7 , SABC1 , SEAIRa , Sho , SVALKA , TWISTED LEAF , and αsHSFB2α  in antisense lncRNAs, COLDAIR  in intronic lncRNAs, and APOLO , At5NC056820 , DAN1 , ELENA1 , and IPS1  in intergenic lncRNAs. Blue square and arrow represent Exons of gene A; yellow square and arrow represent Exons of gene B; brown square represents lncRNA. The direction of the gene A, gene B, and lncRNA are showed by the direction of the arrow. lncRNA, long non-coding RNA.
Generally, the biogenesis of lncRNAs is similar to that of mRNAs. LncRNAs are usually transcribed by RNA polymerase (Pol) II from intergenic, exonic, or the distal protein-coding regions of the genome [5,33,34]. After transcription, they undergo 5′-end capping, 3′-end polyadenylation, and sometimes alternative splicing . Interestingly, some non-polyadenylated lncRNAs have been identified and appear to be more specific to the stress response . In addition to Pol II, Pol III, Pol IV, and Pol V can transcribe plant lncRNAs [36,37]. Pol III usually produces relatively short, high-quantity and stable RNAs, such as 5S rRNA and tRNA. Interestingly, lncRNAs AtR8 and AtR18 were efficiently transcribed by Pol III in vitro in tobacco nuclear extracts, with AtR8 being shown to be a functional lncRNA conserved in Brassicaceae and acting in responses to different stress treatments . The lncRNAs transcribed by Pol IV and Pol V have structural differences compared to those transcribed by Pol II, such as lacking a poly (A) tails [36,39]. LncRNAs that transcribed by Pol IV usually serve as RDR2 templates for the synthesis of 24-nt sRNAs, whereas lncRNAs produced by Pol V function as a scaffolds to recruit 24-nt sRNAs to their complementary target loci in the genome [40,41]. Compared with lncRNAs transcribed by Pol II, lncRNAs transcribed by RNA Pol IV or Pol V are poorly characterized. Their low expression and high instability make them more difficult to identify and characterize.
LncRNAs modulate the expression of their target genes in cis, in trans, or through other actions. Cis-acting lncRNAs usually regulate the transcription of genes in close genomic proximity by recruiting or displacing transcription factors at the promoters of neighboring genes . The three-dimensional organization of genomes plays key roles in the transcriptional regulation of genes. Some cis-acting lncRNAs interact with chromatin remodeling complexes and modulate the three-dimensional organization of genomes, such as forming chromatin loops with target genes, to affect histone modifications and transcriptions of target genes [42–46]. Trans-acting lncRNAs, however, usually target genes far from the site of the primary locus of transcription, acting as a scaffold of protein complexes to recruit transcriptional or chromatin-modifying factors, or as a platform to assemble protein complexes [47–52]. In addition, lncRNAs can interact with proteins to modulate their activity, stability, or subcellular localization [52,53]. Moreover, lncRNAs could act as precursors of some sRNAs to modulate the expression of mature sRNAs or functions as decoys of sRNA to interfere RNA silencing to regulate gene expression [54–57]. For lncRNAs that exhibit a coordinated expression profile with their neighboring genes (cis-acting lncRNAs), it is essential to distinguish the function of lncRNAs from that of their neighboring genes. Therefore, generating proper lncRNA mutants without directly affecting the function of neighboring genes is most important. For trans-acting lncRNAs, it is essential to find the primary targets.
2. Regulatory roles of lncRNAs in plant immunity
The enormous lncRNAs in plants form regulatory networks with protein-coding genes, and/or other non-coding RNAs to mediate growth, development, stress responses, and other biological processes. Coupled with their important roles, the expression of lncRNAs is stage- and cell-type specific and tightly regulated in response to abiotic or biotic stimuli, which subsequently facilitates plants to cope with these stimuli. Abiotic stimuli that result in the differential expression of lncRNAs have been reported in Arabidopsis [10,38,58–67], wheat , barley [69,70], rice [71–74], maize [75–80], etc. [81–84]. For example, in response to cold, the lncRNAs COLDWARP, COOLAIR, and COLDAIR are induced and regulate vernalization by transcriptional silencing of FLOWERING LOCUS C (FLC) [43,44,85]. The level of the lncRNA SVALKA was found to gradually increase during the early responses to cold temperatures and to promote cold acclimation by fine-tuning the expression of Crepeat/dehydration-responsive element Binding Factor 1 (CBF1) . Similarly, the expression patterns of lncRNAs also react in a genome-wide manner to biotic stimuli, which in turn modulates the resistance of plants to different pathogens [87–91]. However, due to the specific function and action of each lncRNA, the mechanism by which lncRNAs regulate plant immunity remains scant. Here, we summarize the current knowledge about the roles of lncRNAs in different stages of plant immunity (Fig 2).
Fig 2. LncRNAs in plant immunity.
Pathogens and insects activate plant PRRs or produce effectors to activate NLRs and further trigger different signaling events and immune defense mechanisms. Pathogen infection changes the expression of lncRNAs, and the differentially expressed lncRNAs regulate various aspects of plant immunity, including ROS accumulation, calcium influx, MAPK activation, hormone pathway activity, and defense-related gene expression. Created with Biorender.com. lncRNA, long non-coding RNA; MAPK, mitogen-activated protein kinase; PRR, pattern recognition receptor; NLR, nucleotide-binding domain, leucine-rich–repeat-containing receptors; ROS, reactive oxygen species.
2.1. Roles of lncRNAs in immune perception processes
To properly activate immune defense, plants have developed cell-surface receptors and intracellular receptors to perceive signals from pathogens. Generally, plant cell-surface pattern recognition receptors (PRRs) perceive immunogenic signals from microbes/insects or host-derived molecular patterns, whereas canonical plant intracellular nucleotide-binding domain, leucine-rich–repeat-containing receptors (NLRs) sense the presence of a pathogen effector by directly interacting with effectors that are secreted into plant cells, or recognize changes of guard host proteins, the replication of viruses/pathogens, integrated diverse cues, etc. [92–97]. However, there are a few cell-surface receptors that detect highly specific effector signatures, such as tomato Cf9, which recognizes AvrCf9 [98,99], while a few NLRs recognize other signatures, in addition to pathogen effectors (e.g., a canonical NLR, N, recognizes the replicase protein of Tobacco mosaic virus, p50) .
Many immunogenic signals recognized by PRRs have been identified, among which the most commonly studied are bacterial flagellin, bacterial elongation factor-Tu, and fungal chitin [92–94,101]. In addition, the damage to plant tissues, particularly the plant cell wall, caused by enzymes or toxins of pathogens, as well as the immunogenic peptides produced by plants, is recognized by PRRs [102–105]. Studies have shown that the expression levels of some lncRNAs are significantly altered after treatment with immunogenic signals (Fig 2). For example, in Arabidopsis, the accumulation of the lncRNA At5NC056820 was found to be increased by 22-fold after the treatment with elf18 (Elongation factor-Tu, EF-Tu) . Likewise, in response to the treatment with flg22 or Pseudomonas fluorescens 55, many lncRNAs in tomato were shown to be up- or down-regulated, and the number of differentially expressed lncRNAs was dramatically increased at 6 h post inoculation .
During the coevolution of plants and pathogens, pathogens have evolved diverse effectors to facilitate pathogens to overcome the basic immune response of plants [107,108]. Plant NLRs recognize effectors either through direct physical interaction or sensing of host protein modifications caused by effectors and subsequently activate immune responses [102,109–113]. In healthy plants, NLRs are suppressed to balance plant growth and immunity . Conserved regions of NLR genes are widely targeted by microRNAs (miRNAs) and phasiRNAs, especially 22-nt microRNAs, to repress plant immunity under normal conditions [115–121]. Upon pathogen infection, the accumulation of these 22-nt microRNAs decreases, which releases the accumulation of miRNA-targeted NLR genes and thus increases plant immunity [115,120]. LncRNAs have also been shown to be differentially expressed corresponding to the activation of NLRs (Fig 2). Genomic analysis revealed 145 up- and 118 down-regulated lncRNAs in response to AvrPto and AvrPtoB, 2 well-studied Pseudomonas syringae pv. tomato (Pst) DC3000 effectors that could interfere with PRR signaling . Some of these lncRNAs modulate the expression of NLR genes through their interactions with miRNAs that target NLR genes. For example, miR482 targets the coiled-coil domains of the N terminal of NLR genes in Solanum species . Tomato lncRNA23468, which contains conserved endogenous target mimic sites for miR482b, was shown to suppress miR482b expression to up-regulate the expression of NLRs, and thereby enhancing tomato resistance to Phytophthora infestans . On the other hand, the overexpression of tomato lncRNA15492 and lncRNA08489 resulted in increased the expression of NLRs, corresponding with decreased expression of miR482a and miR482e-3p, respectively, and subsequently enhanced plant resistance to P. infestans [123,124]. LncRNAs appear to regulate plant immunity by acting as decoys of sRNAs or sRNA precursors to mediate the expression of NBS-LRR resistance genes.
2.2. Roles of lncRNAs in immune responses
Immune responses triggered by cell-surface immunogenic signals and intracellular pathogen effectors have obvious differences in their mechanisms of action, but they also have mutual relations, and have developed into an interconnected mode of action in the coevolution of plants and pathogens [125,126]. The activation of PRRs phosphorylates immediate downstream receptor-like cytoplasmic kinases (RLCKs) and leads to a subsequent series of downstream signaling events, including ROS accumulation, calcium influx, MAPK phosphorylation cascades, defense gene expression, stomata closure, callose deposition, and biosynthesis of defense hormones [4,127–130]. The physiological responses mediated by NLRs overlap with those induced by PRRs, such as increased ROS production, activation of MAP kinases, but are delayed, stronger and prolonged, which usually leads to programmed cell death, known as the hypersensitive response (HR) . These immune responses are fine-tuned not only by protein-coding genes but also by lncRNAs (Fig 2).
2.2.1. Roles of lncRNAs in the accumulation of ROS.
Studies have found that lncRNAs alter the accumulation of ROS by regulating the expression of genes in close genomic proximity [132,133]. Tomato lncRNA16397 reduces ROS accumulation, alleviates cell membrane injury, and subsequently enhances plant resistance to P. infestans, probably by inducing the expression of its neighboring gene SlGRX . Meanwhile, tomato lncRNA33732 was reported to induce the expression of respiratory burst oxidase (RBOH) to increase the accumulation of H2O2 during early defense against P. infestans attack .
2.2.2. Roles of lncRNAs in calcium influx.
Transient and rapid calcium influx upon infection is important for early cellular responses in plant immunity and essential for triggering downstream signaling . Currently, no lncRNA has been identified to directly regulate calcium influx, but some lncRNAs have been found to act downstream of calcium influx. MuLnc1 in mulberry forms a mulmiR3954-MuLnc1-siRNAs-mRNAs network to enhance resistance to Botrytis cinerea and Pst DC3000 . When cleavaged by mulmiR3954, MuLnc1 was found to produces si161579, a siRNA that cleavages the transcript of the calmodulin-like protein gene CML27. CML27 belongs to the CML family whose members are important Ca2+ sensors. Therefore, the lncRNA MuLnc1 may act downstream of calcium influx via CML27. ROS and calcium influx also contributes to the down-regulation of the lncRNA salicylic acid biogenesis controller 1 (SABC1) upon pathogen treatment .
2.2.3. Roles of lncRNAs in the activation of MAPK cascades.
The activation of MAPK cascades is a major early signaling event downstream of PAMP perception and response for the transduction of extracellular stimuli into intracellular responses [135,136]. The activation of MAPK cascades triggers multiple defense responses, including regulating the transcription of defense-related genes, immune signaling proteins, and biosynthetic enzymes of defense hormones, ROS generation, cell wall strengthening, and HR cell death . The activity of MAPK is regulated by the dephosphorylation of protein serine/threonine phosphatases (PSPs) . Effectors employed by pathogens could suppress the activation of MAPK to attenuate resistance [139–141]. Recently, a nalncFL7–FL7-HAI1-MAPK3/6 cascade was reported to regulate MAPK cascade immunity responses . The cis-natural antisense lncRNA of FL7 (nalncFL7) is protected by BPL3, a conserved negative regulator of plant immunity, and suppresses the accumulation of FL7 transcripts. In response to pathogens, the transcript levels of BPL3 decrease, resulting in the degradation of nalncFL7 and thus releasing its suppression on FL7. FL7 interacts with HIGHLY ABA-INDUCED PP2C1 (HAI1), a kind of PSPs, and inhibits the phosphatase activity of HAI1. By decreasing the phosphatase activity of HAI1, FL7 increases the phosphorylation levels of MPK3 and MPK6, which enhances immunity responses.
2.2.4. Roles of lncRNAs in altering the defense-related gene expression.
PRRs and NLRs triggered immune responses involve the activation of a series of overlapping downstream defense responses . Many of these reactions transmit signals from the cell membrane to the nucleus, where these signals modulate the transcriptional level of some defense related genes, pathogenesis related (PR), lipoxygenase (LOX), phenylalanine ammonia-lyase (PAL), catalase (CAT), GDSL lipase, antimicrobial peptides (AMPs), etc. [143–146]. Considering the low accumulation of lncRNAs, transcriptional reprogramming of genes is one of the profound functions of lncRNAs to manifest the actions of lncRNAs. LncRNAs target or interact with transcription factors, splicing factors, epigenetic regulators, and some other key proteins to modulate their activities and regulate the expression of genes in downstream signaling pathways.
Due to the profound roles of transcription factors in transcriptional reprogramming and hormone activation, lncRNAs targeting nearby transcription factors to efficiently exert their actions have been well characterized, such as the lncRNAs COOLAIR, COLDAIR, and COLDRAP to FLC in Arabidopsis , and the lncRNAs TWISTED LEAF to R2R3-MYB in rice . A recent study identified 15 defense-related transcription factors in Arabidopsis that may be targeted by adjacent lncRNAs . Among these lncRNAs, the lncRNA SABC1 represses the transcriptional level of its neighboring gene NAC3, a NAC transcription factor, to repress plant immunity in healthy plants. Upon pathogen infection, calcium influx and ROS burst decrease the accumulation of SABC1, release the expression of NAC3 to activate transcriptional reprogramming and hormone activation, thus tilting the balance from plant growth to plant immunity. In addition to modulating the expression of adjacent genes, lncRNAs can act in trans to regulate the activity of transcription factors. In addition to modulating adjacent genes, the Arabidopsis lncRNA ELF18-INDUCED LONG-NONCODING RNA1 (ELENA1) was shown to increase plant resistance against Pst DC3000 by directly interacting with the mediator subunit 19a (MED19a), a positive regulator, to enrich MED19a on the PR1 promoter, then inducing PR1 expression [148,149]. Furthermore, ELENA1 also interacts with FIB2 (MED36a), a transcriptional repressor, to release MED19 from the FIB2/MED19a complex, and the dissociation of FIB2 from MED19 results in the full activation of PR1 expression by MED19 . Moreover, a genome-wide analysis of lncRNA and miRNA networks in tomatoes upon P. infestans infection identified lncRNAs that were predicted to decoy miRNAs and modulate the transcription of target genes, including transcription factors . LncRNA42705/lncRNA08711, lncRNA39896, and lncRNA11265/lncRNA15816 were predicted to decoy miR159, miR166b, and miR164a-5p, respectively, and to modulate the transcriptional level of MYB, HD-Zip, and NAC transcription factors, respectively. These transcription factors further regulated the expression of defense-related genes and altered the plant response to pathogens.
In addition to targeting or regulating transcription factors, lncRNAs interact with splicing components to fine-tune the plant transcriptional response to pathogens. In response to flagellin, the Arabidopsis lncRNA ALTERNATIVE SPLICING COMPETITOR (ASCO) interacts with the spliceosome-core components PRP8a and SmD1b, alters SmD1b/PRP8a-dependent transcriptome diversity, differentially alternatively splices flg22-response regulatory genes, and subsequently attenuates root growth sensitivity to flg22 . The lncRNA ASCO was also found to hijack the alternative splicing (AS) regulators NUCLEAR SPECKLE RNA-BINDING PROTEINS (NSRs) to modulate the AS of NSR targets and alter the plant response to auxin . Interestingly, ASCO presented different regulatory mechanisms in response to flagellin, a peptide released by bacteria and acting as a triggering PAMP, and auxin, a hormone that balances plant growth and immunity. Further study suggests that other lncRNAs than ASCO may also interact with NSRs to modulate AS . The participation of lncRNAs in plant development and immunity may be far more complicated than current model.
LncRNAs can also regulate gene transcription by interacting with chromatin regulatory proteins, including CURLY LEAF (CLF), LIKE HETEROCHROMATIC PROTEIN 1 (LHP1), etc., to regulate the chromatin topology on a genome-wide scale. The modified chromatin topology recruits regulatory protein/lncRNA complexes to specific sites on DNA and performs chromatin modification [153–155]. The repression of lncRNA COLDAIR, COLDWRAP, COOLAIR, and AG-incRNA4 on FLC was performed by lncRNAs interacting with CLF, a key component of polycomb repressive complex 2 (PRC2), catalyzing histone H3 lysine 27 trimethylation (H3K27me3) of FLC, and repressing its transcription [43,44,85]. Among them, COLDAIR and COLDWRAP cooperatively formed chromatin loops between the promoter and the 3′ end of the first intron of FLC to maintain the polycomb-mediated silencing of FLC. The lncRNA AUXIN REGULATED PROMOTER LOOP (APOLO) associates with LIKE HETEROCHROMATIC PROTEIN 1 (LHP1), the key component of PRC1, forming a chromatin loop to encompass the intergenic region between the APOLO loci and its neighboring gene PINOID, and thus regulating the expression of PINOID [156,157]. The lncRNA SABC1, which is down-regulated in response to Pst (avrRpt2) inoculation and Turnip mosaic virus (TuMV) infection, represses the transcription of NAC3 by associating with CLF and recruiting CLF/PRC2 complexes to increase the H3K27me3 of NAC3, which subsequently decreases the association of Pol II to NAC3 promoter . Although COLDAIR/COLDWRAP, APOLO, and SABAC1 all form a repressive chromatin loop to associate with target genes, lncRNAs are required for the formation of the chromatin loop of COLDAIR/COLDWRAP-FLC and APOLO-PINOID, but this is not the case for the SABC1-NAC3 loop. The chromatin loops of COLDAIR/COLDWRAP-FLC and APOLO-PINOID are unstable during vernation and auxin treatment, respectively, while the loop at the SABC1-NAC3 locus is stable upon pathogen infection [42,43,156,157]. The general roles of lncRNAs in the formation of chromatin loops need to be further determined. In addition, other chromatin regulatory proteins were revealed to interact with lncRNAs and induce chromatin modification of target genes. The lncRNA MAS interacts with WDR5a, a core component of COMPASS-like complexes, and recruits WDR5a to MAF4 to enhance H3K4me3, thus activating MAF4 , while the intragenic lncRNA SEAIRa interacts with PUB25/26 and RUB1 and induces H3K27me3 and H2A monoubiquitination (H2Aub) deposition on its neighboring target SE to cause transcriptional and epigenetic repression of SE .
2.2.5. Roles of lncRNAs in regulating defense-related hormones and hormone pathways.
Plant hormones, including salicylic acid (SA), jasmonic acid (JA), ethylene (ET), gibberellin (GA), and abscisic acid (ABA), regulate plant defense against pathogens, among which SA and JA are major defense hormones. SA plays essential roles in resistance against biotrophic and hemi-biotrophic pathogens and some phloem-feeding herbivores, whereas JA is critical in defense against necrotrophic pathogens, some phloem-feeding herbivores, and chewing herbivores [160,161]. SA and JA often function antagonistically . In basal resistance, SA blocks JA production and JA-mediated gene activities. However, in NLR-induced immunity, the initial activation of JA-responsive genes is dependent on SA and SA receptors. The interplay between SA and JA allows the plant to generate defense against different pathogens [163,164].
In Arabidopsis, the pathogen-induced production of SA requires 3 proteins: isochorismate synthase 1 (ICS1), which converts chorismate into isochorismate in plastids; enhanced disease susceptibility 5 (EDS5), which transports isochorismate from plastids to the cytosol; and AVRPPHB susceptible 3 (PBS3), which conjugates isochorismate with glutamate to form isochorismate-9-glutamate. The degradation of isochorismate-9-glutamate spontaneously produces SA and 2-hydroxy-acryloyl-N-glutamate . Pathogen-induced ICS1 expression and SA biosynthesis are tightly regulated by positive and negative transcription factors [166,167]. The lncRNA SABC1 was found to regulate the biosynthesis of SA to modulate plant immunity [45,167]. Upon pathogen infection, the activation of calcium influx and ROS burst decrease SABC1 accumulation and subsequently activate NAC3. The activation of NAC3 then promotes the biosynthesis of SA by binding to the promoter of ICS1 . The profound roles of SA in the induction of defense-related genes and amplification of immune signaling allow SABC1 to mediate the balance between plant defense and growth [45,167]. Furthermore, the lncRNA AtR8, which can be induced by low-level SA, was also found to participate in SA response-related defense upon P. syringae infection .
JA, a vital plant hormone essential for plant defense responses and developmental processes, exhibits diverse responses to different biotic stresses [169,170]. The JA-mediated defense system enhances host defense against insect herbivores and necrotrophic fungi, such as Alternaria brassicicola, B. cinerea, Plectosphaerella cucumerina, and Fusarium oxysporum [171,172]. LncRNAs have been reported to participate in these regulatory processes. Invasion by V. dahliae was shown to increase the expression of the lncRNA GhlncLOX3 and subsequently improve plant resistance, probably through the repressive effect of GhlncLOX3 on the transcription level of GhLOX3 (lipoxygenase 3, a JA pathway gene) and lipoxygenase 2 (LOX2), and JA content . JA mediates plant defense through the regulation of CORONATINE INSENSITIVE1 (COI1)-JASMONATE-ZIM-DOMAIN (JAZ)-transcription factors signaling cascades [174,175]. Many transcriptional activators and repressors in the JA response pathway have been identified, including MYC2, MYC3, MYC4, basic-helix-loop-helix (bHLH) 3, bHLH13, bHLH14, and bHLH17/JAM1 [176–179]. An lncRNA, An Leaf Expressed and Xoo- induced lncRNA 1 (ALEX1), was identified to be specially induced by Xanthomonas oryzae pv. (Xoo) infection in rice . The expression of ALEX1 significantly up-regulates JA-related genes such as JAZ8, MYC2, PR1a, PR1b, PR10a, and RSOsPR10, and increases the endogenous levels of JA, conferring broad-spectrum resistance to bacterial pathogens. Correspondingly, some other enzymes and transcription factors in the JA biosynthetic signaling pathway are hijacked by pathogens to attenuate plant immunity [181,182]. Two cotton lncRNAs, GhlncNAT-ANX2 and GhlncNAT-RLP7, have been found to be induced by the infection with Verticillium dahliae or B. cinerea, repress the expression of 2 JA pathway genes, lipoxygenase 1 (LOX1) and lipoxygenase 2 (LOX2), and further attenuate plant resistance against fungi . LncRNA39896 in tomatoes, which is induced by P. infestans infection, act as endogenous target mimic of miR166b and negatively regulates tomato resistance through the lncRNA39896–miR166b–HDZs module . In lncRNA39896-knockout mutant, miR166b activity is increased, resulting in increased cleavage of SlHDZ34 and SlHDZ45, and increased JA and ET contents, which was not favorable for P. infestans infection. However, the molecular mechanism underlying the regulation is still unclear.
3. Roles of lncRNAs in various plant–microbe/insect interactions
Plants have evolved sets of defense mechanisms to effectively mitigate different diseases. We next summarized the roles of lncRNAs in various plant–microbe/insect interactions, including viruses, fungi, bacteria, oomycetes, nematodes, and insects (Fig 3). The well-studied lncRNAs that are categorized into different plant–microbe/insect interactions are listed in Table 1.
Fig 3. Roles of lncRNAs during plant–pathogen/insect interactions.
Attack by pathogens/insects significantly changes the expression of lncRNAs, and these lncRNAs function in plant immunity through different mechanisms. (A) In response to viral infection, lncRNAs can act as sponges of sRNAs to regulate the expression of host defense mRNAs and further mediate plant immunity. In addition, the lncRNA SABC1, which represses the transcription of its neighboring gene, NAC3, by interacting with CLF and increasing the H3K27me3 of NAC3, is down-regulated during TuMV infection and plays a negative role in plant resistance to virus. The lncRNA AP2, which is up-regulated by TCV, promotes the infection of TCV, probably by regulating its neighboring gene, AP2. As a counter strategy, viruses can produce vsiRNAs to silence host lncRNAs, to attenuate host immunity. Moreover, some non-coding satellite RNAs are considered to be function as lncRNAs. (B) In response to fungal infection, lncRNAs can regulate plant immunity by acting as precursors of miRNAs or sponges of miRNAs to indirectly inhibit the cleavage of miRNA target genes. In addition, the lncRNAs ANX2 and RLP7 in cotton decrease the expression of their neighboring genes, regulate the JA response by affecting the JA pathway genes, LOX1 and LOX, and promote the infection of V. dahliae and B. cinerea. (C) During plant–bacteria interactions, lncRNAs regulate the expression of defense mRNAs to mediate plant defense by acting as precursors of miRNAs or sponges of miRNAs. Moreover, the expression of SABC1 is suppressed in response to Pst DC3000 infection, and its suppression triggers the transcription of NAC3 and biosynthesis of SA, thus activating plant resistance. ELENA1 enhances the resistance of Arabidopsis to Pst DC3000 by interacting with the mediator subunit 19a and FIB2 to promote the gene expression of PR1, while the lncRNA SUNA1 promotes plant defense against Pst DC3000 by interacting with fibrillarin to enhance the pre-rRNA processing and translational efficiency of some defense genes. In addition, the lncRNA ALEX1 enhances rice resistance to Xoo by up-regulating the endogenous levels of JA and expression of JA-responsive genes. (D) LncRNAs mediate plant defense against oocymetes by affecting ROS accumulation, changing the expression of PR genes, or acting as decoys of miRNAs. Many lncRNAs serve as positive regulators of plant immunity in response to oocymetes by acting as decoys of miRNAs, while lncRNA39896 negatively regulates plant defense by inhibiting miR166 activity. (E) In response to nematodes, lncRNAs interact with their corresponding miRNAs, exerting miRNA-related regulatory effects, or may regulate host defense mRNAs through other mechanisms. (F) In response to insect attack, lncRNAs are involved in the regulation of JA accumulation, probably by mediating the gene expression of JAZ genes. On the other hand, the aphid transcripts Yas serves as an lncRNA when being translocated into plants and promotes the fecundity of aphids. RNAs produced by pathogens/insects are shown in brown. JA, jasmonic acid; lncRNA, long non-coding RNA; ROS, reactive oxygen species; sRNA, small RNA.
3.1. Roles of lncRNAs in plant–virus interactions
Viruses are obligate intracellular parasites that seriously threaten plant growth. LncRNAs are involved in the interaction between viruses and their hosts (Fig 3A). This interaction is mutual, with some lncRNAs helping the host to perform antiviral functions, while other lncRNAs are induced by the pathogen or directly encoded by the pathogen and facilitate the replication of virus, weaken the immune system, and even evade immune defenses (Fig 3A). Tomato yellow leaf curl virus (TYLCV) causes leaf curl disease in several crops. Identification of lncRNAs in a resistant tomato cultivar following TYLCV infection has highlighted the role of lncRNAs during viral pathogenesis . A total of 1,565 lncRNAs were predicted to be involved in TYLCV infection, among which the lncRNAs slylnc0049 and slylnc0761 (which were significantly up-regulated by TYLCV infection) were selected for verification. The accumulation of TYLCV CP increased 200- and 6-fold in slylnc0049- and slylnc0761-silenced plants . Another study revealed that silencing of lncRNA0957 resulted in reduced disease severity and viral load of TYLCV in susceptible tomato varieties . In response to Rice black-streaked dwarf virus infection, 17 up-regulated and 5 down-regulated lncRNAs were identified. These lncRNAs are probably associated with viral infection probably by regulating the expression of defense-related mRNAs . In Arabidopsis, the lncRNA SABC1 represses Arabidopsis immune responses to TuMV, and the accumulation of SABC1 decreases upon TuMV infection to promote plant resistance . Meanwhile, the lncRNA AP2, which negatively correlates with the APETALA2 (AP2) gene, is significantly up-regulated by the infection of Turnip crinkle virus and promotes the infection of Turnip crinkle virus .
RNA silencing plays major roles in plant resistance to viruses . In response to viral infection, some lncRNAs are induced and inhibit the function of miRNAs by acting as their target mimics (Fig 3A). The slylnc0195, which is significantly induced by TYLCY inoculation, was shown to dramatically increase the mRNA abundance of the corresponding miR166 targets by competing for the binding of miR166 and attenuated virus accumulation . Meanwhile, slylnc1077 may act as a decoy of miR399 to regulate plant resistance against TYLCV . Moreover, lncRNA39026, which is induced by P. infestans infection, was shown to decrease the expression level of miR168a, and increase the level of the SlAGO1 gene . Since AGO proteins play important roles in virus resistance, lncRNA39026 might play a role in virus resistance. Correspondingly, viruses are able to produce vsiRNAs to silence host lncRNAs to promote viral disease development. The tomato lncRNA SlLNR1 is targeted by TYLCV-derived siRNA with almost perfect complementary match and silenced, thereby attenuating host antiviral immunity . However, studies on other viruses apart from TYLCV are also very limited and restricted to only transcriptomic studies. SEAIRa, an antisense intragenic lncRNA that generated from the 3′ end of SE, represses the expression of SE, a core component of the miRNA biogenesis pathway . However, its roles in plant resistance to viruses and other pathogens have not been determined. Hopefully, more studies on functional characterization of identified lncRNAs will bear interesting results in the future.
Intriguingly, many defective/defective interfering (D/DI) RNAs, satellite RNAs, and even incompletely degraded viral genomic RNAs are considered to be lncRNAs [191–194] (Fig 3A). They have the non-protein-coding features and are involved in the host–virus interactions. For example, citrus tristeza virus (CTV) produces a lncRNA called low molecular weight tristeza 1 (LMT1), which is involved in maintaining the accumulation, movement, and infectivity of the virus by lowering the production of SA and reactive ROS required for antiviral defense . CMV Y- and Q- satRNAs, which are 300 to 400 nt in size and do not encode any functional protein, probably function as lncRNAs . CMV Y-satRNA functions as an siRNA precursor to produce Y-sat siRNAs and targets the host ChlI mRNA to bring in bright yellowing symptoms in tobacco, while CMV Q-satRNA can bind to a bromodomain-containing protein (BRP) and probably plays a role in histone remodeling [192,196]. However, the virulence of CMV Y-satRNA results from sRNAs derived from satRNAs, and the role of CMV Q-satRNA has not been verified, which makes it controversial to group these satRNAs as lncRNAs [197,198]. Plant satellites of other viruses, including Tobacco ring spot virus satRNA, RNA C, D and F of Turnip crinkle virus, and Cymbidium ring spot virus satRNA, all possess features of lncRNAs and generate disease symptoms in infected plants, but have not yet been studied as a lncRNA [199–202]. With the study on further discovering the mechanism of satRNAs, there might be more solid evidence to link satRNAs with lncRNAs.
3.2. Roles of lncRNAs in plant–fungi interactions
Fungi are eukaryotic pathogens that cause serious diseases to crops. At present, emerging evidence has shown that lncRNAs play important regulatory roles in plant immunity upon the infection of many fungal species (Fig 3B and Table 1). In Arabidopsis, 15 lncNATs and 20 lincRNAs were identified to be differentially expressed in response to infection with Fusarium oxysporum, a soil-borne plant fungal pathogen, and some of these lncRNAs were demonstrated to affect disease development, probably through their associations with neighboring genes . In wheat, lncRNAs participate in plant immunity in the response to powdery mildew and stripe rust infection [89,203]. Seventy-one wheat lncRNAs were identified in response to powdery mildew infection. These lncRNAs displayed tissue-specific expression patterns, and some of them functioned in plant immunity through their feature as miRNA precursors . In Brassica napus, 41 lncRNAs have been identified to respond to Sclerotinia sclerotiorum infection, and they probably function as precursors of miRNAs to produce miRNAs such as miR156 and miR169 . Likewise, a further study identified 254 differentially expressed lncRNAs in response to Blumeria graminis f. sp. tritici stress and 52 lncRNAs in response to Puccinia striiformis f. sp. tritici in Triticum aestivum. Some of these lncRNAs were predicted to be the targets or target mimics of miRNAs and regulate wheat resistance to powdery mildew and stripe rust stress via miRNA regulation  (Fig 3B). The roles of lncRNAs in plant antifungal defense networks were also determined in Vitis vinifera (grapevine) responses to Erysiphe necator (powdery mildew, PM) and Plasmopara viticola (downy mildew, DM), and 71 PM- and 83 DM-responsive V. vinifera lncRNAs were identified . These lncRNAs and their associated protein-coding genes are involved in the modulation of basal and specific defense responses. However, the exact roles of these lncRNAs in plant–fungi interactions and the underlying mechanism are largely unknown. A recent study showed that lncRNAs mediate plant resistance against fungi through their regulation of the JA pathway. GhlncNAT-ANX2 and GhlncNAT-RLP7 (Fig 3B) in cotton promote V. dahliae and B. cinerea infection, probably by decreasing the expression of their neighboring genes ANX2 and RLP7, respectively, exhibiting associations with the decreases in JA pathway genes, LOX1 and LOX2 , while GhlncLOX3 positively regulates plant defense against V. dahlia, exhibiting associations with the increased levels of GhLOX3 expression and JA content  (Table 1).
3.3. Roles of lncRNAs in plant–bacteria interactions
In addition to viruses and fungi, bacteria are another major threat to plants, causing serious yield loss. Studies have demonstrated the involvement of lncRNAs in bacterial disease resistance (Fig 3C and Table 1). Bacterial canker disease of kiwi fruit is caused by the Pseudomonas syringae pv. actinidiae (Psa). The up-regulation of lncRNAs and their interactions with various signaling and defense-related genes have been reported in Psa-infected kiwi fruit . A total of 110 lncRNAs responding to phytoplasma infection have been identified in Paulownia by high-throughput sequencing . When the interaction between tomato and Ralstonia solanacearum was studied, 23 differentially expressed lincRNAs were identified. These lncRNAs were found to respond to bacterial wilt infection, probably by their involvement in JA and ethylene signaling pathways, or by regulating the expression of the AGO protein . Dickeya zeae responsive lncRNAs were also identified in rice (Oryza sativa L.) . Through genomic-wide analysis, 2,518 and 2,191 predicted lncRNAs were found to be up- and down-regulated in response to D. zeae infection, respectively. Several of these lncRNAs are known to participate in rice immune systems as target mimics of miRNAs. In Arabidopsis, 12 lncRNAs react to the infection of Pst D3000 . Among them, the lncRNA SABC1, which plays negative roles in plant defense by inhibiting the transcription of its neighboring gene NAC3 and reducing SA biosynthesis, was suppressed in response to Pst D3000 infection to activate the plant immunity. The lncRNA ELENA1 enhances the resistance of Arabidopsis to Pst DC3000 by interacting with the mediator subunit 19a and FIB2 to promote the gene expression of PR1 [148,149]. The lncRNA SUNA1, the expression of which is triggered by SA, also plays a positive role in Arabidopsis defense against Pst DC3000 . SUNA1 appears to regulate plant defense by interacting with fibrillarin to enhance the pre-rRNA processing and translational efficiency of some defense genes (Table 1). In addition, the accumulation of large amounts of rice lncRNAs was shown to be significantly altered upon the infection with Xoo. The lncRNA ALEX1 enhances Oryza sativa resistance to Xoo by up-regulating the endogenous levels of JA and the expression of JA-responsive genes .
3.4. Roles of lncRNAs in plant–oomycete interactions
Oomycetes are filamentous microbes that represent one of the biggest threats to crops. Among the ubiquitous and highly diverse species of oomycetes, P. infestans is most notorious, as this oomycete causes late blight of tomato and potato and is blamed for the cause of the Irish potato famine . In tomatoes, more than 600 differentially expressed lncRNAs were identified in response to P. infestans infection . Tomato lncRNA16397 and lncRNA33732 were found to regulate plant defense against P. infestans by mediating ROS accumulation [132,133], while lncRNA39026 increased resistance by inducing the expression of PR genes  (Fig 3D and Table 1). Furthermore, many lncRNAs have been reported to modulate the defense response to P. infestans by regulating the function of miRNAs (Fig 3D and Table 1). LncRNA39026, 42705, 08711, 40787, 15492, 23468, and 08489 positively regulate plant resistance against P. infestans by acting as competitive endogenous RNAs of miR168a, miR394, miR159, miR482a, miR482b, and miR482e-3p, respectively, while lncRNA39896 negatively regulates resistance to P. infestans through its action on miR166b [122–124,184,189,211]. In potatoes (Solanum tuberosum L.), 133 differentially expressed lncRNAs were identified in response to P. infestans infection . Among them, StLNC0004 suppresses the growth of P. infestans in Nicotiana benthamiana, probably by regulating the transcriptional level of the EXT gene.
In addition to P. infestans, lncRNAs also involve in the resistance to other oomycetes. The differentially expressed pepper lncRNAs in response to P. capsici were found to increase pepper resistance to soil-borne diseases by interacting with their coordinated miRNA-mRNA and regulating the expression of disease-defense–related genes  (Fig 3D and Table 1). Genes encoding zinc finger proteins, pentatricopeptide repeat-containing proteins, and LRR receptor-like serine/threonine-protein kinases are potentially regulated by lncRNAs to regulate plant immune responses to P. capsici . On the other hand, the expression levels of lncRNAs in oomycetes were also altered during their infection of plant. Eighty-five P. sojae lncRNAs were found to exhibit different transcriptional patterns 3 h after inoculation onto susceptible soybean leaves compared to their transcription in other growth stages, including mycelia, zoospores, and germinated cysts . A high proportion of these lncRNAs associated with effector-coding genes.
3.5. Roles of lncRNAs in plant–nematode interactions
The invasion of nematodes may affect the growth and development of plants, leading to plant deformity. Genome-wide identification and functional deciphering has revealed the involvement of lncRNAs in the responses to nematodes in different plants [215–218] (Fig 3E and Table 1). However, the action mechanisms of these lncRNAs are not clear. The mechanism reported most frequently is that lncRNAs interacts with their corresponding miRNAs and exhibit miRNA-related regulatory effects. For example, in soybeans, 384 and 284 potential lncRNAs were identified in response to 2 nematode species, Heterodera glycines and Rotylenchulus reniformis, respectively, and 15 and 6 lncRNAs were predicted to be involved in the regulation of nematode-responsive gene expression by their interactions with miRNAs . In response to root-knot nematode stress, 10 peanut lncRNAs were identified to participate in defense-related processes . These lncRNAs formed a regulatory network with corresponding miRNAs and mRNAs, and engaged in peroxidase activity, the lignin biosynthetic process, and oxidation–reduction processes. In the tomato response to M. incognita, 43 up-regulated and 35 down-regulated lncRNAs were identified, 12 of which were predicted to be sponges of their corresponding miRNAs and to regulate tomato resistance . In rice (Oryza sativa), lncRNAs responsive to Meloidogyne graminicola infection were predicted to regulate the expression of genes involved in phosphotransferase activity and influence DNA methylation levels in cis . These studies revealed the great potential of lncRNAs in plant resistance to nematodes. However, to explore effective plant protection strategies against parasitic nematodes, further studies on specific lncRNAs are needed to confirm the functions of these lncRNAs.
3.6. Roles of lncRNAs in plant–insect interactions
Recent studies have also revealed the involvement of lncRNAs in plant resistance to insects (Fig 3F and Table 1). A large number of tobacco lncRNAs were found to be induced by the phytophagous insects Manduca sexta . Silencing of the lncRNAs JAL1 and JAL3 attenuated plant resistance to M. sexta, probably through their roles in inhibiting the accumulation of JA and JA derivatives. In addition, a total of 238 armyworm (AW)-responsive lncRNAs were identified in monocot rice, and one lncRNA was the antisense transcript of the JA ZIM-domain gene JAZ10 . A total of 606 differentially expressed lncRNAs were identified in cotton upon the infestation of whitefly . Among them, lncA07 and lncD09 potentially increased plant resistance to insect infestation through their regulation of JA content. Intriguingly, during the interaction between plant and insect, RNAs from the insect can translocate into plants and function as virulence factors. Ya transcripts from aphid Myzus persicae translocate into plants during aphid feeding and migrate systemically to distal leaves in several plant species . M. persicae that feed on A. thaliana expressing Ya1 RNA show increased fecundity. Ya1 acts as an aphid lncRNA virulence factor to modulate plant processes.
3.7. Future perspectives
In summary, compared with the adequate databases and well-developed bioinformatic tools for mRNA, sRNA, and protein, plant-related lncRNA databases are relatively small in number, and their annotation is insufficient, which makes it difficult to study lncRNAs systematically. Moreover, the low abundance, high diversity, and the specific function of each lncRNA also increase the difficulty of discovering the functions of lncRNAs and exploring the underlying mechanisms. Therefore, research on the roles and actions of plant lncRNAs in plant immune responses and other biological processes is at a relatively early stage.
On the other hand, genome-wide analysis has identified plant lncRNAs that are induced or repressed upon stress. Emerging studies on lncRNAs have revealed the essential roles of lncRNAs not only in cellular and developmental processes but also in stress responses, hormone signaling, and pathogenesis. LncRNAs have unique characteristics that make them important players in plant immunity responses . The non-protein-coding nature of lncRNAs allows them to evolve more rapidly than protein-coding genes, and this rapid evolution can lead to the emergence of new lncRNAs with specific functions in plant–pathogen/insect arms races. Moreover, lncRNAs can react to stress responses more rapidly than protein-coding genes, which can be important in the early stages of immune responses when rapid action is needed. By linking early and later immune responses, lncRNAs can play a key role in shaping the overall immune response of the plant. The highly cell type-specific expression of lncRNAs can also regulate the expression of immune and growth genes in different cells, providing an elegant balance between growth and immunity as some protein-coding genes . Overall, the unique characteristics of lncRNAs make them important players in plant immunity responses, and studying their functions can provide insights into the complex interplay between growth and immunity in plants.
As stress responses manifest the accumulation and actions of lncRNAs, more studies should focus on the roles of lncRNAs in plant immune responses. In addition to the genome-wide analysis of the accumulation alternations in lncRNAs upon the infection/infestation by different pathogens/insects, we need to pay more attention on the detailed actions and roles of lncRNAs in immune responses. As it is still difficult to determine the targets of trans-acting lncRNAs, future studies may focus on cis-acting lncRNAs in plant immune responses, especially on lncRNAs generated from loci close to key immune response genes. With an increasing number of reports on the functional characterization of plant lncRNAs, we may be able to draw an lncRNA regulatory network with protein-coding genes in plant immune responses. At that time, we will be able to identify the differences and correlations between lncRNAs and protein-coding genes in plant immune responses. In addition to the unique roles of hormones, Ca2+, protein-coding receptors, protein-coding transcription factors, and other well-defined biomolecules, lncRNAs may perform unique roles in plant immune systems. Studies on lncRNAs may uncover many mysterious phenomena and improve our understanding of plant immune systems.