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Evolution of Disease Defense Genes and Their Regulators in Plants

Biotic stresses do damage to the growth and development of plants, and yield losses for some crops. Confronted with microbial infections, plants have evolved multiple defense mechanisms, which play important roles in the never-ending molecular arms race of plant–pathogen interactions. The complicated defense systems include pathogen-associated molecular patterns (PAMP) triggered immunity (PTI), effector triggered immunity (ETI), and the exosome-mediated cross-kingdom RNA interference (CKRI) system. Furthermore, plants have evolved a classical regulation system mediated by miRNAs to regulate these defense genes. Most of the genes/small RNAs or their regulators that involve in the defense pathways can have very rapid evolutionary rates in the longitudinal and horizontal co-evolution with pathogens. According to these internal defense mechanisms, some strategies such as molecular switch for the disease resistance genes, host-induced gene silencing (HIGS), and the new generation of RNA-based fungicides, have been developed to control multiple plant diseases. These broadly applicable new strategies by transgene or spraying ds/sRNA may lead to reduced application of pesticides and improved crop yield.


Introduction
The arms race of plants and host-pathogens seems never to stop, and sometimes the race is very intense. During the evolutionary process, plants have had to evolve multiple immunity mechanisms to survive danger signals in extracellular and intracellular milieus. Plants are able to enhance disease resistance and increase the food security, as well as to balance the resource allocation between growth and development. The prevalent defense mechanisms are categorized into three defense layers: the preliminary defense, pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) [1], the secondary defense, effector-triggered immunity (ETI) [2], and the additional defense, the exosome-mediated cross-kingdom RNA interference (CKRI) system [3].
It is well-known that PTI functions in basal defense. Using the cell surface-localized pattern recognition receptors (PRR), plants can detect the infection of invaders by recognizing the conserved microbe-associated or pathogen-associated molecular patterns (MAMPs or PAMPs) [1]. Plant PRRs are cell surface localized, and always are receptor-like kinases (RLKs) and receptor like proteins (RLPs). RLKs are comprised of extracellular domains, transmembrane domains, and intracellular kinase domains, which are required for transmitting the signals to the downstream defense responses, whereas RLPs are only comprised of the basic conformation without intracellular kinase domain. PTI with broad-spectrum defense is not sufficient to prevent most pathogens, and if plants have defect in PRRs, they often become more susceptible to microbes [4][5][6][7]. In turn, pathogens employ kinds of virulence effectors to overcome PTI and establish successful infection, termed effector-triggered immunity. Thus, ETI functions in the second defense of elicitor mediated defenses.
Most of the genes involved in ETI pathway contain intracellular nucleotide-binding site and leucine-rich repeat domains (NBS-LRRs or NLRs), which are typically cytoplasmic receptor proteins. NBS-LRR genes can detect or recognize the polymorphic, strain-specific pathogen-secreted virulence effectors, and then transfer the signals to the downstream of defense genes. Thus, ETI-pathways belong to the species-specific disease resistance, and rapidly co-evolve with their pathogens. Plant species in eudicots and dicots have lots of NB-LRR genes. According to the N-terminal features and functions, the NB-LRR proteins in plants can be termed into two classes with the terminal Toll/interleukin-1receptor (TIR) or coiled-coil (CC)/resistance to powdery mildew8 (RPW8) domains [8][9][10]. The TIR, CC or RPW8 domains are crucial in signaling transmit in cellular targets for effector action or downstream signaling components [11]. Although the NB-LRR genes were demonstrated as the ancient and conserved genes in plants, their comparative genomic analyses have shown great structural diversity. For example, the CC domains are prevalent in eudicots and monocots, while the TIR domains are nearly absent in monocots [12]. Cross-kingdom RNA interference (CKRI) functions in the third layer, which protects plants by extracellular vesicles transport small RNAs or microRNAs (miRNAs) to microbial pathogens and then silence the virulence genes [3].
As one kind of typically small non-coding RNAs, miRNAs function in post-transcriptional gene regulation. Small miRNAs play big roles in a variety of biological processes, such as development, hormone responses and stress adaptations [13][14][15][16]. In PTI and ETI pathways, microRNAs as the classical regulators in post-transcript or translation level regulate defense/defense-associated genes [17,18], which can balance the benefits and costs of their targets. Plants employ miRNAs as shields against the pathogen attacks. MiRNAs respond to virus, bacteria and fungi by negatively regulating of mRNAs, which mainly function in both PTI and ETI. Until now, totally 153 disease resistance genes from PRGdb database [19], which involved in the plant immunity to biotic stresses, were validated by experiments in wet labs. Of them, 62.09% (95 from 153) genes, 17.65% (27 from 153) genes, 20.26% (31 from 153) genes were classified as NBS-LRR families, RLP/RLK, and other kinds of genes, respectively ( Figure 1).
(RLPs). RLKs are comprised of extracellular domains, transmembrane domains, and intracellular kinase domains, which are required for transmitting the signals to the downstream defense responses, whereas RLPs are only comprised of the basic conformation without intracellular kinase domain. PTI with broad-spectrum defense is not sufficient to prevent most pathogens, and if plants have defect in PRRs, they often become more susceptible to microbes [4][5][6][7]. In turn, pathogens employ kinds of virulence effectors to overcome PTI and establish successful infection, termed effector-triggered immunity. Thus, ETI functions in the second defense of elicitor mediated defenses.
Most of the genes involved in ETI pathway contain intracellular nucleotide-binding site and leucine-rich repeat domains (NBS-LRRs or NLRs), which are typically cytoplasmic receptor proteins. NBS-LRR genes can detect or recognize the polymorphic, strain-specific pathogen-secreted virulence effectors, and then transfer the signals to the downstream of defense genes. Thus, ETI-pathways belong to the species-specific disease resistance, and rapidly co-evolve with their pathogens. Plant species in eudicots and dicots have lots of NB-LRR genes. According to the N-terminal features and functions, the NB-LRR proteins in plants can be termed into two classes with the terminal Toll/interleukin-1receptor (TIR) or coiled-coil (CC)/resistance to powdery mildew8 (RPW8) domains [8][9][10]. The TIR, CC or RPW8 domains are crucial in signaling transmit in cellular targets for effector action or downstream signaling components [11]. Although the NB-LRR genes were demonstrated as the ancient and conserved genes in plants, their comparative genomic analyses have shown great structural diversity. For example, the CC domains are prevalent in eudicots and monocots, while the TIR domains are nearly absent in monocots [12]. Cross-kingdom RNA interference (CKRI) functions in the third layer, which protects plants by extracellular vesicles transport small RNAs or microRNAs (miRNAs) to microbial pathogens and then silence the virulence genes [3].
As one kind of typically small non-coding RNAs, miRNAs function in post-transcriptional gene regulation. Small miRNAs play big roles in a variety of biological processes, such as development, hormone responses and stress adaptations [13][14][15][16]. In PTI and ETI pathways, microRNAs as the classical regulators in post-transcript or translation level regulate defense/defense-associated genes [17,18], which can balance the benefits and costs of their targets. Plants employ miRNAs as shields against the pathogen attacks. MiRNAs respond to virus, bacteria and fungi by negatively regulating of mRNAs, which mainly function in both PTI and ETI. Until now, totally 153 disease resistance genes from PRGdb database [19], which involved in the plant immunity to biotic stresses, were validated by experiments in wet labs. Of them, 62.09% (95 from 153) genes, 17.65% (27 from 153) genes, 20.26% (31 from 153) genes were classified as NBS-LRR families, RLP/RLK, and other kinds of genes, respectively ( Figure 1).

Figure 1.
Categories of the genes/regulators in the three defense layers in plants. The data was downloaded from PRGdb database and the recent publications . PTI: pathogen-associated molecular patterns (PAMP) triggered immunity; ETI: effector-triggered immunity; CRKI: cross-kingdom RNA interference.
In regard to defense genes, studies have shown a number of genes/small RNAs linked to anti-pathogen immunity. Here, we mainly summarize the current knowledge of the defense genes  . PTI: pathogen-associated molecular patterns (PAMP) triggered immunity; ETI: effector-triggered immunity; CRKI: cross-kingdom RNA interference.
In regard to defense genes, studies have shown a number of genes/small RNAs linked to anti-pathogen immunity. Here, we mainly summarize the current knowledge of the defense genes and their evolution paths regulated by miRNAs in plants, and then discuss their potential applications in crop improvements in the last section.

Three Layers of Defense Mechanisms to Biotic Stresses in Plants
2.1. The First Layer of Defense: Defense Genes in PTI As one of the most important sensory protein groups, RLKs and RLPs in plants play crucial roles both in cell-cell and the plant-environment communications such as plant-pathogen interaction. In addition, RLKs and RLPs play fundamental roles in plant growth and development. Plants deploy a wide assay of RLKs and RLPs as the first layer of inducible defense to detect microbe-and hostderived molecular patterns ( Figure 2A, the first layer) [63]. Numbers of RLKs/RLPs have been cloned in plants [64]. The best classical example is FLAGELLIN-SENSITIVE2 (FLS2), belonging to RLK family, which have been verified to response to Flagellin fragment flg22 of bacteria in Arabidopsis [65], grapevine [66], tobacco [67], rice [68] and tomato [69]. As a "molecular glue", flg22 induces the activity of the heterodimerization complex FLS2-BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE). In different plant species, FLS2 receptors display different affinities for the conserved part of flagellin from different bacteria, which possibly reflect the coevolution with specific-pathogens [66]. Except FLS2, EF-TU RECEPTOR (EFR), PEP 1 RECEPTOR (PEPR1), PEPR2, RLP23, RLP30 [70], the endogenous AtPep1 [71], NLPs [72], and SCFE1 [73], can also recognize bacterial EF-Tu, respectively. All of them are associated with the regulatory BAK1 that acts as a co-receptor for flg22/EF-Tu/AtPep1/nlp30/SCFE1 of pathogens and are crucial for signaling activation [74].
Long chitin oligomers as bivalent ligands, lead to the homodimerization of CHITIN ELICITOR RECEPTOR KINASE 1 (AtCERK1) and generate an active receptor complex in Arabidopsis, which directly trigger chitin-induced immune signaling [75]. The chitin perception system in rice is significantly different from the one in Arabidopsis. OsCERK1 dimmer does not bind chitin since the single LysM domain, while the dimer elicitor-binding LysM-RLP (OsCEBiP) can bind the chitin by ligand. The OsCERK1-chitin-OsCEBiP then forms a sandwich-type receptor dimerization for chitin oligomers [76].
There are a number of RLKs/RLPs involved in plant immunity, which have been well summarized by Tang et al [63]. After plant sensing of pathogen/microbe-associated molecular patterns, these pattern recognition receptors instantly trigger a number of downstream responses, such as the activation of mitogen-activated protein kinases (MAPKs) ( Figure 2A, the first layer), which is one of the earliest signaling events [77]. By phosphorylation to transmit response signals, MAPKKK actives MKK, and then MKK actives MPK [78]. MAPK cascades is involved in multiple signaling defense responses, including the biosynthesis/signaling of plant stress/defense hormones, reactive oxygen species generation, stomatal closure, defense gene activation, phytoalexin biosynthesis, cell wall strengthening, and hypersensitive response (HR) cell death ( Figure 2A, the first layer) [77]. The activation of MAPK cascades is essential for plant immunity.
In addition, some transcription factors were found to regulate the defense-related genes that involved in signal transduction in rice. For example, a bZIP gene OsBBI1 in rice, is a major transcription factor to regulate the resistance spectrum for diverse groups of M. oryzae by altering the first level of innate immunity in host plants [79]. WRKY13 as another major regulatory factor was identified to transfer signals from WRKY45 to downstream WRKY42 as functioning WRKY-type transcription factors (TFs) [80]. Following the SA-pathway-dependent disease response mechanism, WRKY13 shows correlation of the defense to M. oryzae and Xoo [81]. By activation of NPR1 protein, the SA pathway plays a crucial role in the systemic acquired resistance response mechanism ( Figure 2A, the first layer) [82]. As a result, kinds of genes comprised of cellulase surface disease resistance genes and intracellular transcript factors could function in the complex PTI.

The Second Layer of Defense: The Defense Genes in ETI
In ETI pathway, plants have developed NBS-LRR proteins to recognize effectors and trigger the ETI response [2], which can cause programmed cell death together with the downstream of WRKY and lead to hypersensitive response (HR) ( Figure 2A, the second layer) [97]. NBS-LRRs as an interesting class of disease resistance genes own a larger member in plants. In Table 1

The Second Layer of Defense: The Defense Genes in ETI
In ETI pathway, plants have developed NBS-LRR proteins to recognize effectors and trigger the ETI response [2], which can cause programmed cell death together with the downstream of WRKY and lead to hypersensitive response (HR) ( Figure 2A, the second layer) [97]. NBS-LRRs as an interesting class of disease resistance genes own a larger member in plants. In Table 1, about 1.19-3.48% of total coding genes were defined as NBS-LRR genes. Although NB-LRR genes are abundant in plants, only 93 genes are validated to play important roles in the innate immunity of plants up to now. Of the validated NBS-LRR genes, 65.59% (61 from 93) genes contain the CC domains, while only 19.35% (18 from 93) genes contain the TIR domains, and the others contain only one domain of either NBS, LRR, TIR, CC, or RPW8 ( Figure 1). The verified disease resistance genes with CNL or TNL domains are listed in Table 2. For example, seven CNLs and seven TNLs in Arabidopsis thaliana, eleven CNLs in Oryza sativa, five CNLs and one TNL in Solanum lycopersicium, seven CNLs in Triticum aestivum, three CNLs in Hodeum vulgare had been exemplified by experiments. These defense genes in plants can confer the resistance to fungi, oomycetes, bacteria, viruses, nematodes, and insects. 1 the percentage of the R-genes from the total coding genes; 2 percentage of the miRNA target genes from the R-genes.
The disease resistance genes were abundant in the wild resource. In Triticeae for example, the defense genes Sr31 and Sr50 [133] from cereal rye (Secale cereale), can confer the resistance to stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt). Sr35 gene from Triticum monococcum confers the resistance to Ug99 Stem Rust Race Group [134]. In addition, some non-NBS-LRR genes can also provide the defense to pathogens. For example, Stb6 in wheat can directly interacted with the effector AvrStb6 that produced by wheat pathogen Zymoseptoria tritici [135]. The X10 gene, which has four potential transmembrane helices in rice, can be induced by transcription activator-like (TAL) effector AvrXa10. The gene can confer disease resistance to rice bacterial blight by inducing programmed cell death in rice [136,137].
By introgression or transgene strategy, these defense genes confer the disease resistance in plants. For example, by overexpressing Pm3a/c/d/f/g in wheat, all tested transgenic lines showed the significantly more resistance than their respective non-transformed sister lines in field experiments [138]. The T0 and T1 transgenic lines with the Sr50 gene were resistant to Puccinia graminis f. sp. tritici (Pgt), while lines without the transgene were susceptible [133].

The Third Layer of Defense: Cross-Kingdom/Organism RNA Interference
It had been demonstrated that plasmodesmata sRNAs can presumably move from cell to cell, and they systemically travel through vasculature [139]. Remarkably, sRNAs also move and induce their target gene silencing between interacted organisms and hosts. The phenomenon was defined as cross-kingdom/organism RNA interference (CKRI) [20,93,[140][141][142]. Pathogens can deliver sRNAs into plants. It was recently discovered as a novel class of pathogen effectors ( Figure 2A, the third layer). Botrytis cinerea can deliver small RNAs (Bc-sRNAs) to plant cells to silence host immunity genes [140]. Such small RNA effectors in B. cinerea are mostly produced by Dicer-like protein 1/2 (Bc-DCL1/2). In reverse, over-expressing sRNAs that target Bc-DCL1 and Bc-DCL2 in tomato and Arabidopsis, would silence Bc-DCL genes and inhibit fungal growth and pathogenicity. It exemplified bidirectional CKRI and sRNA trafficking between plants and fungi [93]. The easy traveling phenomenon suggests naturally occurring small RNAs might exchange each other across cross-kingdom/organism.
Conversely, hosts also can transfer naturally occurring small RNAs into pests or pathogens to attenuate their virulence ( Figure 2A, the third layer). Recently, two reports have demonstrated that naturally occurring plant small RNAs might be delivered into pathogens to silence their target genes. In response to the infection of Verticillium dahliae, cotton plants increase the dose of miR159 and miR166 in expression level and then export both to the fungal hyphae for specific silencing. Two genes encoding an isotrichodermin C-15 hydroxylase and a Ca 2+ -dependent cysteine protease, were targeted by miR159 and miR166, respectively. Both of the target genes are essential for fungal virulence [20]. Another example is that host Arabidopsis cells by secreting exosome-like extracellular vesicles can also transfer small RNAs into fungal pathogen Botrytis cinerea. At the infection sites, these sRNA-containing vesicles accumulate and then are taken up by the fungal cells. Delivered host small RNAs induce the silence of fungal genes that is critical for pathogenicity. TAS1c-siR483 target two genes BC1G_10728 and BC1G_10508 from B. cinerea, and TAS2-siR453 targets BC1T_08464. All of the three genes involving in vesicle trafficking pathways are critical for pathogenicity [3]. Of them, BC1G_10728 encodes a vacuolar protein sorting 51 and plays a crucial role in Candida albicans virulence [21]. Thus, Arabidopsis has adapted exosome-mediated CKRI mechanism as part of its immune responses during the evolutionary arms race with the pathogens [3].
Based on the above description, since only two miRNAs and two small RNAs in plants were identified to function in CKRI, data are inefficient to deduce their evolution among species. Thus, in the next section, we only discussed the evolution of disease resistance genes and their regulator miRNAs in PTI and ETI.

The First Layer of Defense Regulation: miRNAs Involved in the PTI Pathway
During pathogen infection, plant small RNAs play key roles in gene regulation level. According to the targets of miRNAs that how to respond to the pathogen infection, miRNAs were divided into active and repressed regulation in basal resistance ( Figure 1A, Table 3). In the positive regulation, overexpression of miRNAs conferred the resistance to defense diseases in plants. For example, miR393 in Arabidopsis, was discovered to contribute to the antibacterial resistance by negatively targeting the transcripts of the F-box auxin receptors TIR1 [22]. Repressing auxin signaling through miR393 overexpression increases bacterial resistance; conversely, augmenting auxin signaling through over-expressing a TIR enhances susceptibility to virulent Pto DC3000. miR444/OsMADS directly monitors OsRDR1 transcription, and involves in the rice antiviral response [23]. Overexpression of miR444 enhanced rice resistance against rice stripe virus (RSV) infection by diminishes the repressive roles of OsMADS23, OsMADS27a, and OsMADS57 and concomitant by the up-regulation of OsRDR1 expression. Thus, miR444 can indirectly activate the OsRDR1-dependent antiviral RNA-silencing pathway. Over-expression of osa-miR171b conferred less susceptibility to rice stripe virus infection by regulating the target OsSCL6. OsSCL6-IIa/b/c was down-regulated or up-regulated in plants, where osa-miR171b was over-expressed or interfered [24]. In the negative regulation, overexpression their target genes could confer the resistance to pathogens in plants. miR169 suppresses the expression of NFYA in immunity against the infection of bacterial wilt Ralstonia solanacearum [25] and the blast fungus Magnaporthe oryzae in Arabidopsis and rice, respectively [26]. The transgenic lines of over-expressing miR169a, became hyper-susceptible to pathogens. MiR156 and miR395 regulate apple resistance to Leaf Spot Disease [27]. In apple, Md-miR156ab and Md-miR395 suppress MdWRKYN1 and MdWRKY26 expression, which decreases the expression of some pathogenesis-related genes, and results in susceptibility to Alternaria alternaria f. sp. mali. In Arabidopsis, miR396/GRF module mediates innate immunity against P. cucumerina infection without growth costs. Reduced activity of miR396 (MIM396 plants) was found to improve broad resistance to necrotrophic and hemibiotrophic fungal pathogens [28]. MiR319/TCP module involves in the rice blast disease. Increasing expression level of rice miR319 or decreasing expression level of its target TCP21, LIPOXYGENASE2 (LOX2) and LOX5 can facilitate rice ragged stunt virus (RRSV) infection [29], which caused the decreased endogenous jasmonic acid (JA) [30]. Inhibiting ath-miR773 activity accompanied with up-regulation of its target gene METHYLTRANSFERASE 2 increased resistance to hemibiotrophic (Fusarium oxysporum, Colletototrichum higginianum) and necrotrophic (Plectosphaerrella cucumerina) fungal pathogens in Arabidopsis [31]. By regulating the transcription of GhMKK6 gene in cotton, ghr-miR5272a involved in the immune response. Over-expressing ghr-miR5272a increased sensitivity to Fusarium oxysporum by decreasing the expression of GhMKK6 and the followed disease-resistance genes, which lead a similar phenotype to GhMKK6-silenced cotton [32]. In addition, miRNAs could also be involved in the resistance to nematode invasion. For example, miR827 in Arabidopsis down-regulated the expression of NITROGEN LIMITATION ADAPTATION (NLA) gene. It suppressed the basal defense pathway by enhancing susceptibility to the cyst nematode Heterodera schachtii [33].
Except these miRNAs indirectly regulation the PTI pathway, a few of miRNAs were predicted to directly regulate the receptor-like genes. For example, when osa-miR159a.1 was repressed, the expression of OsLRR-RLK2 was induced, which is responded to Xanthomonas oryzae pv. Oryzae in rice [31]. In future, some miRNAs regulation of pattern recognition receptors (PRR) genes may be validated by experiments.

The Second Layer of Defense Regulation: The Defense Signal Small RNAs in ETI
In addition to the basal defense, miRNAs are also involved in ETI pathway to directly and indirectly regulate the disease resistance genes (Figure 2A & Table 3). MiR393*, the complementary strand of miR393 within the sRNA duplex, by targeting a protein trafficking gene Membrin 12 promote the secretion of antimicrobial PR proteins, which functions in ETI during infection of Pseudomonas syringae pv. Tomato in Arabidopsis [34]. The miR863-3p is induced by the bacterial pathogen Pseudomonas syringae. During early infection, miR863-3p silences two negative regulators of plant defense, namely atypical receptor-like pseudokinase1 (ARLPK1) and ARLPK2, both of which trigger immunity through mRNA degradation. Later during infection, miR863-3p silences SERRATE, and positively regulates defense. And SERRATE is essential for miR863-3p accumulation by a negative feedback loop. Thus, miR863-3p targets both negative and positive regulators of immunity through two modes of action to fine-tune in the timing and amplitude of defense responses [35].
High expression of plant NBS-LRR defense genes is often lethal to plant cells, which is associated with the fitness costs. Thus, plants develop several mechanisms to regulate the transcript level of NBS-LRR genes. One of the key mechanism is the suppression of regulation network in microRNAs and NBS-LRRs, which may play a crucial role in plant-microbe interactions by sRNA silencing mechanism [18]. NBS-LRR genes confer defense against the pathogen infections in gene dosage dynamic expression level by multiple duplications and diversification, while miRNAs minimized the cost of gene copies by inhibiting their expression [36]. One miRNA can regulate dozens to hundreds of NBS-LRRs by targeting the similar motif sites [37], which make it more economical to balance the benefits and costs of these copies in genome. Until now, a few of miRNAs had been validated to be involved in the regulation of NBS-LRR genes.
The regulation between miRNAs and CC-NB-LRR or TIR-NB-LRR gene classes was mostly characterized in eudicots. In most of the post-transcriptional regulation networks, the miRNA can trigger the 21-nt phased siRNA generation in NB-LRR transcripts, which were processed by RNA-dependent RNA polymerase 6 (RDR6) and DICER-LIKE 4 (DCL4) [38]. For example, in Brassica miR1885 were validated to induce by Turnip Mosaic Virus (TuMV) infection, which cleaved TIR-NB-LRR class genes [39]. In Tobacco, by cleaving TIR-NB-LRR immune receptors, both of nta-miR6020 and nta-miR6019 provide resistance to Tobacco mosaic virus (TMV) [40,41]. In tomato, sl-miR5300 and sl-miR482f controlled NB domain-containing proteins in mRNA stability and translation level, which involved in plant immunity [42]. In Arabidopsis, miR472 modulated the disease resistance genes mediated by RDR6 silencing pathway [43]. In Medicago, miR2109, miR482/miR2118 and miR1507 were found to influence NB-LRR gene family [37]. In legumes, miR482, miR1507, miR1510, and miR2109 suppressed NB-LRR gene class with CC or TIR domains, which were proposed to function in the regulation of defense response or host specificity during rhizobium colonization [38,44]. In addition, miR482/miR2118, miR946, miR950, miR951, miR1311, miR1312, miR3697, miR3701, and miR3709 were also mediated to generate phased siRNAs by targeting NBS-LRR gene class in Norway Spruce [45]. In monocots, miR2009 (also named miR9863 in miRBase) was first predicted in wheat to target the Mla alleles [46]. In barley, the miR9863 family was confirmed to trigger response to the Mla alleles [47]. Table 3. List of regulators involved in the immunity response to pathogens in plants.

The Evolution of Defense Gene in PTI
In land plants, RLKs expanded extensively and fulfilled these diverse roles including perceive growth hormones, environmental/danger signals derived from pathogens [143]. In Arabidopsis, 44 RLK subgroups were defined, and leucine-rich repeat receptor-like kinases (LRR-RLK) belong to the largest receptor-like kinase family and are focused by researchers [144]. According to characters of unique basic gene structures and protein motif compositions, plant LRR-RLKs constitute 19 subfamilies, most of which were derived from the common ancestors in land plants. The proportions of LRR-RLK genes in Lycophytes and moss genome are 0.30% and 0.36%, respectively, while the proportions of LRR-RLK genes in angiosperms are 0.67-1.39% [145], which indicated the special expansion of defense genes in angiosperm genomes. LRR-RLK involved in the defense/resistance-related genes was less conserved than that involved in development. Defense-associated LRR-RLKs undergone many duplication events, and most of them were massively lineage-specific expansion mainly by tandem duplication [143,144]. These discoveries provide important resources for future functional research for these critical signaling genes in PTI.

The Evolution of Defense Gene in ETI
NBS-LRR genes as a class of ancient and conserved genes have been detected in gymnosperms, angiosperm plants and animals to ensure immunity [12,146,147]. However, comparative genomic analyses have demonstrated that NBS-LRR genes have a great structural diversity in plants and animals. For example, TIR domains were established in the ancestor plants conifers and mosses, and also in animals shared functionality regarding innate immunity [148][149][150]. TIR genes specially expanded in dicot genomes, but are absent or at least rare in monocot genomes [8,147,[151][152][153]. For NBS-LRR genes, tandem duplication in genome is the major expansion mechanism in plants. More than 60% of NBS-LRR genes organized in a general pattern of clusters in plant genomes ( Figure 2B) [98]. During whole genome duplication, biased deletions happened in the duplicated paralogous blocks with NB-LRR genes, and it could be possibly compensated by their local tandem duplication mechanism ( Figure 2B).
The miRNAs typically target highly duplicated NBS-LRRs, and families of heterogeneous NBS-LRRs were rarely targeted by miRNAs in Brassicaceae and Poaceae genomes [18]. miRNAs/NBS-LRR-genes interactions drove functional diploidization of structurally retained NBS-LRR genes duplicates by suppression regulation ( Figure 2B) [98]. Evolutionary shuffling events such as diploidization and tandem duplication, leaded to copy number variations and presence absence variations in the synteny collapse of NBS-LRR genes [154][155][156][157]. In addition, the polymorphisms often exist in a population [158]. A contrasted conservation of NBS-LRR genes was observed with only 23.8% for monocots and 6.6% for dicots. Thus, NBS-LRR genes as one of the most plastic gene family in plants have less conservation such as synteny erosion or alternatively loss in plants compared with the other coding protein genes [98].

The Evolution of miRNAs in PTI
In the PTI pathway, most of miRNAs were very conserved and directly/indirectly involve multiple biological processes in the development and abiotic/biotic stresses. All of the MiR169, miR171, miR393, miR395, and miR396 were ancient miRNAs present in both dicots and monocots [48]. miR444 was specific in monocots [49], whereas miR773 and miR5272 were lineage-specific in Arabidopsis and Medicago. The miRNAs conserved in plants mostly regulate the important transcript factors. These transcript factors tend to involve multiple biological processes. Take miR169 and miR396 for example, miR169/NFYA in Arabidopsis indirectly affected lateral root initiation [50], nitrogen-starvation [51], drought stress [52], and biotic stress [25,26]. In Arabidopsis roots, miR396/GRF regulates the switch between stem cells and transit-amplifying cells [53], which affects rice yield by shaping inflorescence architecture [54], and biotic stresses [28].
Both of the miRNA/target regulation and their function are very conserved in plants. MiR169/NFYA module influences the Ralstonia solanacearum pathogenicity in Arabidopsis [25] and the resistance to M. oryzae strains in rice [26]. In addition, these conserved miRNAs' targets were expanded except for their classical miRNA/target model. For example, miRNA156 regulates of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) family involve in the timing of vegetative and reproductive phase change, which is highly conserved among phylogenetically distinct plant species [55]. miR395 by targeting a high-affinity sulphate transporter and three ATP sulfurylases involved in the sulfate homeostasis, is also conserved in plants [56,57]. Differently, both miR156 and miR395 regulate apple resistance to leaf spot disease by targeting WRKY. Thus, miRNAs involved in PTI pathway, are conserved in PTI defense pathway and in plant development such as miR393 vs TIR in auxin signal pathway [22] and miR319 vs. TCL in JA pathway [29]. Only few of miRNAs were reported to potentially regulating the RLK/RLP by osa-miR159a.1 [58], MiR5638 and miR1315 [59]. Genes involved in the PTI pathway were relatively conserved compared to these genes involved in ETI pathway. Thus, most of their regulator miRNAs were also conserved miRNAs or neofunctionalization of miRNAs in plants.

The Evolution of miRNAs in ETI
Although there are many miRNAs regulated NB-LRR genes, the conservation level of miRNAs is lower than the development associated miRNAs or PTI-associated miRNAs. In the eudicots and monocots, there is no conserved miRNAs targeting the NB-LRR genes. Lineage-or species-specific disease resistance-associated miRNAs were continually present and accompanies the continually varied pathogens. And some miRNAs with similar sequences had obvious functional diversity. miR482/miR2118 in eudicots mostly targeted NB-LRR genes, however, it only initiated the generation of 21-nt phased siRNAs in rice, and most of the target transcripts were noncoding sequences and specifically expressed in the rice stamen and the maize premeiotic and meiotic anther [60][61][62]. It clearly concluded that miR2118 initiated the phased siRNA in male reproductive organs. Therefore, a functional switch occurred in miR482/miR2118 between eudicots and dicots. Their expression level also varies in the lineage-related species. Tae-miR3117 was predicted to target the numbers of NBS-LRRs with higher expression in the tetraploid and hexaploid Triticum seedlings, while it had lower expression levels in Aegilops tauschii (not published data). And in rice, maize, and sorghum, miR3117 also displayed lower expression levels.
Diverse miRNAs, as negative transcriptional regulators, inhibit NBS-LRRs in plants. The highly duplicated NBS-LRRs were typically targeted by miRNAs ( Figure 2B), while families of heterogeneous NBS-LRR genes were rarely regulated by miRNAs such as in Poaceae and Brassicaceae genomes. For example, some miRNAs also have a high duplication rate such as miR482/miR2118 in tandem duplication in genomes [60][61][62], which may enhance the expression dosage.
Newly emerged miRNAs were periodically derived from duplicated/redundant NBS-LRRs from different gene families. And most of these new birth miRNAs target these NBS-LRR gene regions of conserved, encoded protein motif, which follow in the convergent evolution model ( Figure 2B). The miRNAs may drive the rapid diploidization of these NBS-LRR genes in polyploid plants. These NBS-LRR associated miRNAs had a rapid diversity. The nucleotide diversity of the target site region in the wobble position of the codons drives the diversification of miRNAs. These characters of high duplication rate and rapid diversity were similar to their target genes. The co-evolutionary model between NBS-LRRs and miRNAs in plants makes the plants balance the costs and benefits of disease resistance [18].

The First Strategy: Utilize the Disease Resistance Genes by a Molecular Switch
Up to now, a number of genes were exemplified to be involved in plant immunity defense. By over-expressing such defense genes can dramatically enhance disease resistance in plants, while is often associated with significant penalties to fitness and make the resulting products undesirable. Thus, it is difficult in agricultural applications. Recently, it has been developed a strategy to utilize these disease defense genes from the angle of plant genes or their regulators [83]. The strategy is to introduce immunity-inducible promoter and other two pathogen-responsive upstream open reading frames of the TBF1 gene. It is called uORFsTBF1, which is a key immune regulator and its translation is transiently and rapidly induced upon pathogen challenge ( Figure 2C, uORF). It has been demonstrated that inclusion of the uORFsTBF1-mediated translational control over the production of AtNPR1 in rice and an auto-activated immunity receptor snc1-1 in Arabidopsis did not reduce the plant fitness in the laboratory or in the field [83]. This strategy using a molecular switch enables us to engineer more broad-spectrum disease resistance genes with minimal adverse effects on plant growth and development in the agriculture application.

The Second Strategy: Host-Induced Gene Silencing (HIGS)
Transgene-derived artificial sRNAs in plants can induce the target gene silencing in certain interacting insects [84,85], nematodes [86], fungi [87][88][89][90], oomycetes [91,92], and even plants-plants [141]. The phenomenon was called host-induced gene silencing (HIGS). The artificial sRNAs can travel from host plants to pathogens or pests and then function in trans ( Figure 2C, HIGS). It had been well used in many plants in the decades. By plant RNAi suppressing a bollworm P450 monooxygenase gene of cotton impaired larval tolerance of gossypol [85]. In transgenic plants, by RNAi silencing of a conserved and essential root-knot nematode parasitism gene engineered broad root-knot resistance [86]. HIGS of nematode fitness and reproductive genes decreases fecundity of Heterodera glycines Ichinohe. Double-stranded RNA complementary to cytochrome P450 lanosterol C14 alpha-demethylase-encoding genes of Fusarium in Arabidopsis and barley contributes to strong resistance to Fusarium species [90]. HIGS to the MAPKK gene PsFUZ7 in wheat enhance stable resistance to wheat stripe rust [159]. HIGS of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici conferred resistance of wheat to stripe rust [160]. By transgene-mediated cross-kingdome RNAi mechanism, HIGS by transgene is a good and effect strategy to improve the crop disease resistance in a broad spectrum.

The Third Strategy: Spray-Induced Gene Silencing (SIGS)
The pathogens and pests are capable to take up the double RNAs or small RNAs from the plants or the environments [93]. Based on this and according to the mechanism of cross-kingdom/organism RNA interference, researchers have developed a strategy to control crop disease. It is spray-induced gene silencing (SIGS) that spraying dsRNAs and sRNAs on plant surfaces can target pathogen genes to repression pathogen virulence ( Figure 2C, SIGS). For modern crop protection strategies, it is a natural blueprint. Evidences suggest that nematodes [94], insects [84] and fungi [95] could uptake up the environmental dsRNA or sRNAs. Directly spraying the dsRNAs that target the fungal cytochrome P450 lanosterol C-14alpha-demethylases of fungal gene can suppress fungal growth [95]. On barley leaves, spraying CYP51-targeting dsRNA at a concentration range of 1-20 ng/mL, inhibited growth of Fusarium species [3]. It has been demonstrated that spraying naked sRNAs and dsRNA on plants was successful to protect fruits and vegetables against pathogens. However, pesticide effect of the naked sRNAs and dsRNAs can only last 5-8 days. Mitter, et al. developed a method to load dsRNAs on designer, non-toxic, degradable, layered double hydroxide (LDH) clay nanosheets. This LDH made the dsRNA does not be wash off, and can be sustained released for 30 days [96]. This SIGS broadly application of new strategy may contribute to reduced use of chemical pesticides and lightening of selective pressure for resistant pathogens. The new-generation of RNA-based fungicides and pesticides are powerful, eco-friendly, which can be easily adapted to control multiple plant diseases simultaneously.

Conclusions
Plants deployed PTI, ETI, and CKRI innate immune systems to arm race with different pathogen stresses. Pathogens developed more advanced effectors to defeat plant defense immunity. A number of genes have been exemplified to play important role between the host-pathogen interactions in plants. These signaling genes will be helpful to improve plant disease resistance against various pathogens. The sustainable and broaden spectrum resistance genes and their regulators such as miRNAs will be applied in developing crop varieties by introducing the molecular switch. From the cross-kingdom angle, the HIGS can also be used to crop breeding by transgenic approach, which can also confer the broaden spectrum resistance to hosts. The SIGS can also make plants yield the broaden spectrum resistance by spraying the designed dsRNAs/sRNA. Further function studies in plants will dissect more and more defense genes and hopefully unravel the intricate defense regulation network. More and more molecular technologies will be invented and adapted to help develop the eco-friendly disease-resistance cultivars.
Author Contributions: S.Z. and R.Z. conceived and designed the project. F.Z., R.Z., S.Z., S.W., and P.C. downloaded the data and analyzed the data. R.Z., F.Z., and G.L. prepared and drafted the manuscript. S.Z. and P.C. revised the manuscript. All the authors read and approved the final manuscript.