The roles of rice microRNAs in rice-Magnaporthe oryzae interaction
Phytopathology Research volume 1, Article number: 33 (2019)
MicroRNAs (miRNAs) are a class of small (20–24 nucleotides (nt) long) non-coding RNAs. One mature miRNA can be transcribed from one or more gene loci known as miRNA genes (MIRs). The transcript of a MIR forms a stem-loop structure that is processed into a 20–24-nt miRNA-5p/−3p duplex by RNase III family endoribonucleases such as Dicer-like1 (DCL1). In turn, the overhang ends of the duplex are methylated by HUA ENHANCER 1 (HEN1), generating stabilized mature miRNAs. The mature miRNAs are loaded onto ARGONAUTE (AGO) proteins, forming a miRNA-induced gene silencing complex (miRISC). Then, the miRISC binds to target sites with sequences complementary to the miRNAs, leading to either cleavage or translational inhibition of the target mRNAs, or methylation of the target sequences, resulting in post-transcriptional and transcriptional gene silencing, respectively. In the past decade, more than 700 miRNAs have been identified in rice, a subset of which have been found to be responsive to the rice blast fungus, Magnaporthe oryzae, or its elicitors. Moreover, members of 10 miRNA families have been found to positively or negatively regulate rice defense against M. oryzae, namely miR160, miR164, miR166, miR167, miR169, miR319, miR396, miR398, miR444 and miR7695. This review summarizes the identification and functional characterization of the miRNAs, which respond to M. oryzae or its elicitors and describes the current understanding of the complicated but well-organized network in the context of rice-M. oryzae interaction.
Rice is a staple food for half of the world population and thus is one of the most important grains to secure global food supply. Rice blast is one of the most devastating diseases of rice, and hence threatens food security. Exploitation of blast-resistant cultivars is rated to be the most appropriate strategy to control this disease. Rice mounts an innate immune system consisting of two perception layers to defend itself against invasion by the blast fungus, Magnaporthe oryzae. The first layer of immunity, known as pathogen-associated molecular pattern- (PAMP-) triggered immunity (PTI), is activated following recognition of the fungal pattern molecule chitin, by four lysin motif- (LysM-) containing protein (LYP) receptors, i.e., chitin elicitor binding protein (CEBiP), chitin elicitor receptor kinase (CERK1), LYP4 and LYP6 (Shimizu et al. 2010; Liu et al. 2012). Virulent rice blast strains employ effectors, such as secreted LysM protein1 (Slp1), or avirulence genes, such as AVR-Pizt, to subvert PTI, leading to effector-triggered susceptibility (Mentlak et al. 2012; Park et al. 2016; Wang et al. 2017). In turn, rice resistance (R) genes recognize avirulence effectors to activate the second layer of immunity, i.e., effector-triggered immunity (ETI), to contain the M. oryzae infection (Bialas et al. 2018). To date, more than 30 R genes, most of which encode nucleotide-binding site leucine-rich repeat (NLR) family receptors, have been functionally characterized from rice, and nine associated cognate avirulence effectors from M. oryzae have been identified (Wang et al. 2017; Xie et al. 2019). Some of these R genes have been widely exploited in rice production and have made major contributions to the control of rice blast. However, activation of immunity often results in yield penalties in rice, known as fitness costs. Some recent reports have demonstrated that both PTI and ETI can be deliberately regulated by immunity-associated regulators that could reduce or abolish the fitness cost (Deng et al. 2017; Wang et al. 2018b; Chandran et al. 2019). MicroRNAs (miRNAs) are among such regulators capable of fine-tuning growth, development and immunity.
miRNAs are a class of small non-coding RNAs transcribed from miRNA genes (MIRs). To date, more than 500 MIR genes have been identified in the rice genome, generating more than 700 mature miRNAs (Tang and Chu 2017). MIR genes are transcribed into primary miRNAs (pri-miRNAs) that are spliced to form stem-loop precursor miRNAs (pre-miRNAs). The pre-miRNAs are processed into miRNA-5p/miRNA-3p (previously miRNA/miRNA*) duplex by Dicer-like1s (DCL1s) or DCL3 (Rogers and Chen 2013). While DCL3 produces mainly 24-nt miRNAs, DCL1s produce 21-nt miRNAs (Liu et al. 2005; Wu et al. 2010; Wei et al. 2014). The overhang ends of the miRNA-5p/miRNA-3p duplex are methylated by HUA ENHANCER 1 (HEN1) to stabilize the duplex (Abe et al. 2010). Then, the 21-nt and 24-nt miRNAs are loaded to ARGONAUTE 1 s (AGO1s) and AGO4, respectively, forming miRNA-induced gene silencing complex (miRISC) (Seo et al. 2013). Whereas the miRNA-AGO1 miRISC mediates sequence-complementary transcript cleavage or translational inhibition, the miRNA-AGO4 miRISC mediates target site methylation (Wu et al. 2009; Wu et al. 2010). Thus, miRNAs negatively regulate the expression of their target genes at either the transcriptional or post-transcriptional level (Fig. 1).
Evidence from Arabidopsis has demonstrated the fine-tuning roles of miRNAs in both PTI and ETI. The conserved flagellin peptide flg22, a PAMP molecule, induces the expression of a subset of miRNAs, including miR160 and miR393 (Li et al. 2010a). miR393 is the first miRNA to be identified to act in both PTI and ETI against Pseudomonas syringe pv. tomato (Pst) DC3000 via targeting of the auxin receptor genes TIR1, AFB2 and AFB3 to block auxin signaling. On the one hand, the expression of miR393 is induced by flg22 to promote basal defense against bacterial strains (Navarro et al. 2006). The expression of miR393 can be suppressed by AvrPto and AvrPtoB, two bacterial effectors that can subvert PTI (Navarro et al. 2008). On the other hand, both miR393-5p and miR393-3p can be up-regulated during ETI as a result of the recognition of the avirulent effector AvrRpt2 by the R receptor RPS2 (Zhang et al. 2011). Whereas miR393-5p is loaded onto AGO1, miR393-3p is loaded onto AGO2 and mediates suppression of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) gene, MEMB12, to promote immunity against this bacterial pathogen (Zhang et al. 2011). Therefore, a miRNA may be involved in both PTI and ETI, and thus be involved in host-pathogen “arms race”. In addition, several miRNAs directly target resistance genes encoding NLR family proteins that mediate ETI, such as miR2109 in Medicago (Zhai et al. 2011), miR482 and miR2118 in tomato (Shivaprasad et al. 2012) and miR472 in Arabidopsis (Boccara et al. 2014). miR472 targets NLRs, such as RPS5 that recognizes the effector AvrPphB to activate ETI against P. syringae in Arabidopsis (Boccara et al. 2014). In rice, miRNAs are well-documented in fine-tuning many biological processes to control many important agronomic traits, such as tiller development, flowering time, panicle establishment, grain formation and yield production, as well as responses to biotic and abiotic stresses (Wu et al. 2015; Bakhshi et al. 2016; Tang and Chu 2017; Wu et al. 2017; Tripathi et al. 2018; Yao et al. 2019). Increasing numbers of reports have shown that miRNAs also play important roles in rice-blast fungus interactions. Because detailed reviews on the biogenesis and functions of miRNAs have been published (Xie et al. 2015; Baldrich and San 2016; Huang et al. 2016b; Tang and Chu 2017; Song et al. 2019), we focus in the current review on rice miRNAs that are responsive to M. oryzae or its elicitors, and summarize our understanding of their roles in rice-M. oryzae interaction.
Core components of the miRNA signaling pathway involved in rice-M. oryzae interaction
DCLs and AGOs are two key groups of core component proteins in the miRNA signaling pathway, encoded separately by distinct groups of genes. The rice genome contains eight DCL and 19 AGO genes (Kapoor et al. 2008). Evidence is emerging that both DCLs and AGOs play important regulatory roles in rice immunity against M. oryzae. For example, AGO4 has been demonstrated to regulate the expression of the blast-susceptibility NLR receptor gene PigmS, which, in turn, balances the tradeoff between yield formation and activation of immunity against M. oryzae mediated by the broad-spectrum blast resistance gene PigmR (Deng et al. 2017), indicating that AGO4 positively regulates PigmR-mediated ETI. However, the roles of the other AGOs in rice immunity against M. oryzae remain to be elusive. Among the eight DCLs, OsDCL1a, OsDCL1b, OsDCL2a/b, OsDCL3a and OsDCL4 have been shown to be responsive to M. oryzae (Zhang et al. 2015; Salvador-Guirao et al. 2019), implying an involvement in rice-M. oryzae interaction.
To date, of the eight rice DCL genes, only OsDCL1a has been deeply investigated, being found to negatively regulate PTI against M. oryzae. Knock-down of OsDCL1a led to enhanced resistance to the blast disease (Zhang et al. 2015). This resistant phenotype could be due to constitutive activation of defense-related genes, such as 13 Pathogenesis-related genes (PRs) and the PTI-related genes OsKS4 and OsNAC4, as well as higher and faster production of H2O2 in the knock-down mutant than in the control (Zhang et al. 2015). By contrast, up-regulation of OsDCL1a led to increased susceptibility to both M. oryzae and the bakanae disease fungus Fusarium fujikuroi in an activation-tagged mutant (Salvador-Guirao et al. 2019). Moreover, such activation mutation also resulted in reprogramming of both the transcriptome and the miRNAome, leading to suppression of defense-related genes, phytoalexin biosynthesis and ROS detoxification (Salvador-Guirao et al. 2019). These reports indicate that the functions of DCL1 in rice differ from those in Arabidopsis, in which DCL1 is required for PTI against bacterial pathogens (Navarro et al. 2008).
Consistent with the function of OsDCL1 in the biogenesis of miRNAs, the up-regulation of this gene remarkably influenced the expression of more than 90 miRNAs from 61 miRNA families, including up-regulation and down-regulation of a subset of miRNAs, which, in turn, resulted in altered expression of 216 genes in the categories of “biotic stress,” “signaling,” and “metabolism” (Salvador-Guirao et al. 2019).
Therefore, OsDCL1s seem to act as a key node in the miRNA signaling pathway, mediating crosstalk between the miRNA network and rice-M. oryzae interaction (Fig. 1).
Identification of rice miRNAs responsive to M. oryzae or its elicitors
To investigate the roles of miRNAs in rice-M. oryzae interaction, it is vital to first identify those miRNAs that are responsive to M. oryzae or its elicitors. A number of research groups have independently identified such miRNAs, as a result of using one of three methods described below. In the first approach, rice tissues were treated with M. oryzae mycelial extracts containing the fungal elicitors. Samples from mock- and elicitor-treated rice tissues were collected for miRNA analysis. Candidate miRNAs were those showing altered accumulation between elicitor- and mock-treated samples. Using this method, some conserved miRNAs/miRNA families and novel miRNAs were found to be responsive to the blast fungal elicitors, suggesting their possible involvement in rice-M. oryzae interaction (Baldrich et al. 2015). These miRNAs have been shown to target a wide range of genes involved in a number of pathways, via degradomics analysis (Baldrich et al. 2015). In the second strategy, rice seedlings were inoculated with a virulent M. oryzae strain, following which samples were collected for miRNA analysis. Candidate miRNAs were those showing altered abundance following inoculation with M. oryzae. Using this method, the expression levels of a number of miRNAs were found to be significantly altered in the rice accession Nipponbare (NPB) or its mutant following infection with M. oryzae (Zhang et al. 2015; Wang et al. 2018a; Zhang et al. 2018). In the third approach, rice seedlings from one susceptible and one resistant accession were inoculated with a M. oryzae strain, with samples subsequently collected for miRNA analysis. Candidate miRNAs were those showing altered abundance between the susceptible and resistant accessions in response to M. oryzae infection. Using this method, a subset of miRNAs was found to be differentially expressed in the susceptible and resistant accessions, suggesting their possible roles in rice-M. oryzae interaction (Li et al. 2014; Li et al. 2016b; Dong et al. 2018).
To date, more than 70 miRNAs from 61 MIR families have been identified to be responsive to M. oryzae or its elicitors (Table 1). These miRNAs have been predicted to regulate various signaling pathways by targeting a wide range of genes, including those involved in the biogenesis of small RNAs, auxin signaling, salicylic acid (SA)/jasmonic acid (JA)/ethylene (ET) signaling, and ROS signaling, as well as genes containing upstream open reading frames (Baldrich et al. 2015; Wang et al. 2018a; Zhang et al. 2018; Li et al. 2019a). Intriguingly, candidate miRNAs responsive to M. oryzae included members from the miR162 and miR168 families (Campo et al. 2013; Li et al. 2014). It is known that miR162 regulates DCL1 and miR168 controls AGO1 (Wu et al. 2015; Zhang et al. 2015), suggesting that the miRNA signaling pathway plays important roles in rice immunity against M. oryzae. The identification of these miRNAs opens up a door to explore the fine-tuning mechanisms underlying rice-M. oryzae interaction.
The microRNAs that play positive roles in rice immunity against M. oryzae
To date, members of four miRNA families have been reported to act as positive regulators of rice immunity against M. oryzae, namely miR160, miR166, miR398 and miR7695.
The miR160 family is a highly conserved miRNA family, consisting of six loci in the rice genome and being capable of generating three mature isoforms (Table 1). The abundance of each of the three mature isoforms was altered in response to infection by M. oryzae or treatment with its elicitors (Campo et al. 2013; Li et al. 2014). The mature miR160a/b/c/d isoform seems to be transcribed mainly from OsMIR160a and OsMIR160b and accumulates to greater concentrations than miR160e and miR160f in leaves (Li et al. 2014; Huang et al. 2016a). miR160 targets five Auxin Response Factors (ARFs) transcription factor genes, namely ARF8 (LOC_Os02g41800), ARF10 (LOC_Os04g43910), ARF13 (LOC_Os04g59430), ARF18 (LOC_Os06g47150) and ARF22 (LOC_Os10g33940) (Wu et al. 2009; Li et al. 2010b). Transgenic lines overexpressing miR160a showed reduced susceptibility to M. oryzae, a phenotype which was associated with suppression of the expression of target genes such as ARF8, ARF10 and ARF13, indicating that miR160 positively regulates rice blast disease resistance via ARFs that may involve auxin signaling; this finding, is also, consistent with the opinion that auxin signaling antagonizes immunity. However, the mechanism by which suppression of ARFs leads to increased resistance remains to be elucidated.
The miR166 family has 13 members in the rice genome, forming six mature isoforms (Table 1). The function of miR166 has been reported in plant development, targeting transcription factor genes of the class III homeodomain-leucine zipper family, such as PHABULOSA and PHABOLUTA, to specify the fate of the shoot apical meristem (Zhu et al. 2011; Li et al. 2019b). Its function in plant immunity has been reported only in rice. The difference in expression between resistant and susceptible accessions or in response to M. oryzae or its elicitors varied between individual miR166 members. For example, miR166m was down-regulated following treatment with M. oryzae elicitors (Campo et al. 2013). The expression of miR166k−/l-3p was constitutively higher in the susceptible accession Lijiangxin Tuan Hegu (LTH) than in the resistant accession IRBLkm-Ts, but was up-regulated in both accessions in response to infection by M. oryzae (Li et al. 2014). In the intermediate susceptible/resistant accession NPB, expression of both miR166k-3p and miR166j-3b was up-regulated following M. oryzae infection (Zhang et al. 2018). In a knock-down dcl1 mutant, expression of miR166j-5 was down-regulated, whereas, expression of miR166a/b/c/d-3p/f was up-regulated following M. oryzae infection (Zhang et al. 2015). The expression patterns suggest that the functions may differ among different members of the miR166 family. However, in-depth functional investigation has been carried out only with respect to the miR166k-166 h polycistron, that co-expresses miR166k and miR166h (Baldrich et al. 2016). Up-regulation of miR166k and miR166h from an activation mutant miR166k-166 h-Ac led to increased resistance to rice blast disease, a phenotype which was associated with activation of the ET-signaling pathway and high marked up-regulation of defense-related genes, although miR166k-5p suppresses the expression of two OsEIN2 genes (Salvador-Guirao et al. 2018). This finding was consistent with an involvement of ET signaling in rice immunity against M. oryzae (Yang et al. 2017), although it was unclear how suppression of OsEIN2s led to activation of the ET-signaling pathway.
The miR398 family has two members in rice genome, generating two mature isoforms (Table 1). This family is highly conserved in both monocotyledons and dicotyledons, including rice, wheat, barley, switchgrass, Arabidopsis, Brassica rapa and Populus trichocarpa. miR398 targets four members of the 15 superoxidase dismutase (SOD) gene family in rice, i.e., CSD1, CSD2, SODX and CCSD (Li et al. 2019a). Overexpression of miR398b led to increased resistance to the rice blast disease, in association with the down-regulation of all four target genes, up-regulation of defense-related genes and increased accumulation of H2O2 (Li et al. 2014). By contrast, blocking miR398b by overexpressing a target gene mimic resulted in increased susceptibility, which was associated with up-regulation of all four target genes, delayed induction of defense-related genes and reduced H2O2 accumulation (Li et al. 2019a). Detailed analysis indicated that CSD1, CSD2 and SODX negatively regulated H2O2 accumulation, whereas CCSD positively regulated it; the loss-of-function mutants csd1, csd2 and sodx exhibited increased susceptibility. CSD1, CSD2 and SODX appear to determine the production of H2O2 by coordinately regulating the expression of the other SOD family members and hence the total SOD enzyme activity. In contrast, CCSD is probably required for CSD enzyme activity because the ccsd mutant exhibited little CSD activity (Li et al. 2019a). Therefore, miR398 fine-tunes ROS signaling through these four SOD members to regulate rice immunity against M. oryzae. Nevertheless, the function of miR398 in rice immunity obviously differs from those in Arabidopsis and barley, where miR398 negatively regulates immunity to the bacterial pathogen Pst DC3000 and the powdery mildew pathogen Blumeria graminis f. sp. hordei, respectively (Li et al. 2010a; Xu et al. 2014). Therefore, the detailed functions of miR398 need to be further investigated in different phytopathological systems.
miR7695 was the first miRNA to be identified as being responsive to the elicitors of M. oryzae, and is expressed in leaves throughout the rice vegetative growth stage (Campo et al. 2013). Its abundance is reduced in the dcl4, but not in dcl1 mutant, indicating that MIR7695 is a recently evolved MIR locus, the primary transcript of which is processed by DCL4, producing multiple miRNAs (Campo et al. 2013). Indeed, five mature miR7695s were detected by northern blotting analysis (Campo et al. 2013). Mature miR7695.3-3p targets one transcript of NRAMP6 (Natural resistance-associated macrophage protein 6), a locus that generates eight transcripts as a result of alternative splicing (Campo et al. 2013). miR7695 seems to occur specifically in rice, particularly in the japonica subspecies and in some indica accessions. Its expression is higher in blast-resistant accessions than in blast-susceptible ones, and is further up-regulated in response to infection by M. oryzae (Quoc et al. 2019). Overexpression of its precursor leads to increased resistance to M. oryzae. Therefore, miR7695 plays positive roles in rice immunity against M. oryzae. Consistent with this role of miR7695, loss-of-function mutants of its target gene NRAMP6 exhibited reduced plant growth but increased resistance to M. oryzae (Peris-Peris et al. 2017). NRAMP6 encodes a metal transporter that has iron and manganese transport activity. Therefore, it is presumed that NRAMP6 acts via metal homeostasis, linking immunity to M. oryzae to rice plant growth, a process which is fine-tuned by miR7695.
Taken together, some miRNAs played positive roles in regulating rice immunity against M. oryzae via regulating a number of signaling pathways, such as auxin signaling, ethylene signaling, ROS signaling and metal homeostasis.
The microRNAs that facilitate infection by M. oryzae in rice
Currently, members of six miRNA families have been reported to facilitate the infection by M. oryzae, namely miR164, miR167, miR169, miR319, miR396 and miR444.
The miR164 family is a highly conserved miRNA family containing six loci, which generate four mature isoforms, targeting six genes encoding NAC (NAM, ATAF1/2 and CUC2) transcription factors in rice (Fang et al. 2014). The miR164-NAC regulatory module functions in ET-signaling to control leaf senescence in Arabidopsis and drought tolerance in rice (Li et al. 2013; Fang et al. 2014). Recently, evidence has emerged of its roles in rice-M. oryzae interactions. The abundance of miR164a/b/f was altered in response to infection by M. oryzae or treatment with the blast fungus elicitors (Campo et al. 2013; Li et al. 2014; Wang et al. 2018a). Overexpressing miR164a led to increased susceptibility to M. oryzae, failing to up-regulate the expression of defense-related genes and H2O2 production following infection by M. oryzae (Wang et al. 2018a). Consistently, mutation in one target gene, OsNAC60, resulted in increased susceptibility to M. oryzae. In contrast, transient overexpression of OsNAC60 activated cell death that was associated with H2O2 production in Nicotiana benthamiana (Wang et al. 2018a). Therefore, miR164 negatively regulated rice immunity to M. oryzae via at least one of its targeted NAC transcription factor genes coupled to ET signaling. However, it is unclear whether the other target genes were involved in miR164-mediated regulation of rice-M. oryzae interaction.
In rice, the miR167 family has 10 members, forming two mature isoforms that target four Auxin Responsive Factors and three other genes, including one encoding a NLR family protein (Zhao et al. 2019). Over-expressing miR167d resulted in increased susceptibility to M. oryzae in a resistant accession genetic background, a phenomenon that was associated with failure to induce defense-related genes and consequent reduced H2O2 production (Zhao et al. 2019). In contrast, blocking miR167d by expressing a target mimic led to increased resistance to M. oryzae, associated with high expression of defense-related genes and increased H2O2 production. The miR167d-mediated regulation of immunity seemed to be associated with neither ARF25 (LOC_Os12g41950) nor the gene encoding the NLR protein (LOC_Os07g29820), because knocking-out ARF25 or overexpressing or knocking-out of LOC_Os07g29820 did not generate any obvious change in the rice blast disease phenotype (Zhao et al. 2019). In contrast, knocking-out of ARF12 led to increased susceptibility to M. oryzae (Zhao et al. 2019), implying that miR167d mediates its functions via ARF12. In addition, the miR167d-mediated regulation of rice-M. oryzae interaction may be associated with both IAA- and JA-signaling pathways, because JA concentration decreased in transgenic lines overexpressing miR167d, but increased in those overexpressing the target mimicry (Zhao et al. 2019). Therefore, it would be interesting to investigate the context of the connection between ARF12 and IAA/JA signaling in terms of rice immunity against M. oryzae.
miR169 is a relatively large miRNA family containing 17 members, which generate nine mature isoforms in rice, targeting eight of the 11 nuclear transcription factor Y subunit A (NF-YA) genes (Wu et al. 2009; Li et al. 2010b). NF-YA, together with NF-YB and NF-YC, forms a heterotrimer to control the expression of downstream genes. The different isoforms of miR169 show considerable variation in expression patterns and are differentially responsive to M. oryzae or its elicitors (Campo et al. 2013; Li et al. 2014; Li et al. 2017). However, they may play negative roles in rice immunity against M. oryzae, because the total miR169 abundance was significantly up-regulated in the susceptible rice accession in response to M. oryzae, but varied only slightly in the resistant accession in response to infection by M. oryzae (Li et al. 2017). Consistent with this observation, overexpression of miR169a resulted in increased susceptibility to M. oryzae, which was associated with compromised defense responses, such as reduced induction of defense-related genes and decreased H2O2 production (Li et al. 2017). In contrast, blocking miR169 via expressing a target mimicry of miR169a led to increased resistance to M. oryzae, which may be attributed to up-regulation of some or all of the eight target NF-YAs (Li et al. 2017). However, it remains to be determined whether (or which of) the target NF-YAs positively regulates rice immunity against M. oryzae and which signal transduction pathway is involved.
The miR319 family includes two members, which form one mature miRNA targeting teosinte branched/cycloidea/proliferation cell factors21 (TCP21) and the positive regulator of gibberellin (GA) signaling, GAMYB, in rice (Zhang et al. 2018). The expression of miR319 was up-regulated, whereas that of its target genes was down-regulated in response to M. oryzae infection in a susceptible accession (Zhang et al. 2018). Similarly, a miR319-resistant TCP21 mutant exhibited resistance to M. oryzae, whereas knock-down of TCP21 led to increased susceptibility to M. oryzae. The resistant phenotypes were associated with up-regulation of expression of genes involved in JA biosynthesis/signaling, marked up-regulation of defense-related genes and increased accumulation of H2O2. In contrast, the susceptible phenotypes were associated with down-regulation of genes involved in JA biosynthesis/signaling and a failure of H2O2 production (Zhang et al. 2018). Therefore, miR319 probably regulates rice immunity against M. oryzae by TCP21-mediated regulation of JA biosynthesis and signaling.
The miR396 family is a highly conserved and abundant miRNA family. It consists of eight members in the rice genome, forming five mature isoforms, which target 11 Growth Regulating Factor (GRF) transcription factor genes (Duan et al. 2015; Gao et al. 2015; Li et al. 2016a). The miR396-GRF module has been reported to fine-tune inflorescence development controlling panicle and individual grain size (Tang and Chu 2017). Over-expressing miR396a, miR396c, miR396d or miR396h led to increased susceptibility to M. oryzae, which was associated with reduced H2O2 production and a failure to induce defense-related genes (Chandran et al. 2019). In contrast, blocking miR396 by expressing a target mimicry of miR396d or a short tandem target mimicry (STTM) of miR396d-396e led to increased resistance to M. oryzae, which was associated with marked up-regulation of defense-related genes and increased H2O2 production. Consistent with these being the target genes of miR396, overexpression of GRF6, GRF7, GRF8 or GRF9 resulted in increased resistance to M. oryzae, whereas, knock-down of GRF7 via RNA interference (RNAi) led to increased susceptibility (Chandran et al. 2019). Thus, miR396 facilitates the infection by M. oryzae via at least four GRF genes. Because blocking miR396 led to improvements in both rice yield and immunity against M. oryzae, the miR396-GRF module is invaluable in breeding programs aiming to improve both of yield and disease resistance simultaneously.
The miR444 family is a monocot-specific miRNA family with six gene loci in the rice genome, each generating 1–3 mature miRNAs, leading to six mature isoforms (Table 1). miR444 targets four MADS-box transcription factor genes and four other genes (Wu et al. 2009; Li et al. 2010b), and plays roles in root and tiller development, as well as in response to Rice stripe virus (RSV) (Yan et al. 2014; Wang et al. 2016). Overexpression of miR444s resulted in increased resistance to RSV that was associated with up-regulation of OsRDR1 expression via suppression of the miR444-targeted MADS genes (Wang et al. 2016), indicating that miR444a plays a positive role in resistance to the virus. On the other hand, miR444b seemed to play negative roles with respect to rice immunity against M. oryzae. In response to M. oryzae infection, the abundance of miR444b.2 was increased in a blast-susceptible rice accession, but was decreased in a blast-resistant accession (Xiao et al. 2017). In contrast, expression of the target genes of miR444b.2, including MADS27b and MADS57, changed only slightly in the susceptible accession but increased significantly in the resistant accession, indicating that miR444b facilitates the infection by M. oryzae via impact on these target genes. In addition, overexpression of miR444b resulted in increased susceptibility to M. oryzae; whereas blocking miR444b.2 via overexpression of a target mimicry of miR444b.2 resulted in increased rice blast disease resistance (Xiao et al. 2017). However, the identity of the target genes, which contribute to miR444b-mediated facilitation of infection by M. oryzae, has still to be confirmed.
Taken together, the signaling pathways associated with rice defense against the blast disease were also regulated by the miRNAs that facilitate infection of M. oryzae, including auxin signaling, ET signaling, JA signaling and some yet-to-be identified signaling pathways, which may also regulate the trade-off between plant growth and immunity.
Challenges facing functional investigations into the roles of miRNAs in rice-M. oryzae interaction
It is quite challenging to investigate the roles of miRNAs in rice-M. oryzae interaction due to the complicated miRNA regulatory nature. Usually, one mature miRNA can be transcribed from one or more MIR gene loci, whereas one gene locus may differ from another in terms of response to M. oryzae. One mature miRNA may suppress the expression of one or several target genes and such suppression may vary among different target genes. Moreover, one target gene can be regulated by multiple miRNAs. However, the most challenging problem is to identify the authentic target genes in planta that contribute to the miRNA-mediated phenotypes, because functional analysis of the target genes is essential for an investigation of a miRNA-mediated regulatory mechanism. In this section, we briefly summarize methods reported to achieve the identification of target genes.
The strategy for target gene identification is based on how the miRNA regulates the expression of its target genes. Most plant miRNAs regulate target gene expression via mRNA cleavage. In such a situation, the target genes can be relatively and easily predicted and experimentally validated via degradome sequencing and/or RNA ligase-mediated 5′-rapid amplification of cDNA ends (RLM-5′-RACE) (German et al. 2008). Because miRNA-mediated mRNA cleavage generates 3′ decay products that have a 3′ poly(A) tail and a 5′ monophosphate end, the 5′ monophosphate ends can be ligated to an adaptor sequence by an RNA ligase. The ligated products can then be reverse-transcribed into cDNA and subjected to high-throughput sequencing or 5′-RACE analysis (Wu et al. 2009). However, this strategy is invalid for those target genes that are translationally inhibited by miRNAs or where target site-methylation is mediated by miRNAs. In such cases, the target genes can first be predicted based on sequence complementarity, using online programs such as psRNATarget (http://plantgrn.noble.org/psRNATarget/) or psRobot (http://omicslab.genetics.ac.cn/psRobot/) (Wu et al. 2012; Dai et al. 2018). The predicted target genes can be confirmed via a reporter assay, in which the reporter gene is constructed by fusing the predicted target site to a fluorescence protein such as the Yellow Fluorescence Protein (YFP). Then, the reporter gene is transiently expressed or co-expressed with the miRNA. If the predicted target site is authentic, the YFP signal should be weaker or the YFP protein concentration lower as a result of co-expression with miRNA than that from expression of the reporter alone (Li et al. 2017). The reporter assay approach should be valid for targets regulated by miRNA-mediated cleavage and translational inhibition, but may not be valid for identification of target genes regulated by miRNA-mediated methylation. In the latter case, the predicted target sites could be confirmed by examining the DNA methylation status via bisulfite sequencing analysis (Wu et al. 2010). After testing each of the above strategies, it may still be impossible to confirm the predicted target genes. Under such circumstances, a combination of the above strategies may be appropriate, with multiple omics analyses, by comparing the transgenic line overexpressing the miRNA with the transgenic line overexpressing its target mimicry with the authentic target genes being among the predicted target genes showing opposite expression patterns in the two transgenic lines. Finally, even if we identified the authentic target genes, we may still face the problem that the target genes identified did not contribute to the miRNA-mediated phenotypes. Therefore, the process can be very challenging. Fortunately, most of the blast fungus-responsive miRNAs have known target genes, the regulatory roles of which have been confirmed in a variety of biological processes, such as growth and development (Table 1) (Tang and Chu 2017).
At present, our understanding of the miRNA-mediated regulation of rice-M. oryzae interaction is quite limited and fragmentary. In the context of the rice-M. oryzae interaction, the activation of either PTI or ETI, or both, could lead to either activation or silencing of certain subsets of MIR genes, which, in turn, could recruit networks of downstream genes that may achieve feedback regulation of PTI and/or ETI (Fig. 1). On the one hand, the key component genes of the miRNA signaling pathway, such as DCLs and AGOs, are regulated during PTI and/or ETI, which, in turn, mediate the engagement of the miRNA network in the regulation of rice-M. oryzae interaction. Evidence supporting this conclusion comes from DCL1 and AGO4, in that DCL1 negatively and AGO4 positively regulates rice immunity against M. oryzae (Zhang et al. 2015; Deng et al. 2017; Salvador-Guirao et al. 2019). On the other hand, a subset of MIR genes may be down- or up-regulated during PTI and/or ETI, depending on their roles as negative or positive regulators of PTI and/or ETI, whereas the positive regulators would be up-regulated during PTI and/or ETI, the negative regulators would be down-regulated. Evidence supporting this conclusion comes from functionally characterized miRNAs, including miR160, miR164, miR166, miR167, miR169, miR319, miR396, miR398, miR444 and miR7695 (Campo et al. 2013; Li et al. 2014; Li et al. 2017; Xiao et al. 2017; Wang et al. 2018a; Zhang et al. 2018; Chandran et al. 2019; Quoc et al. 2019; Zhao et al. 2019). However, we still do not know how PTI and/or ETI is coupled with the miRNA signaling pathway or with each of the blast fungus-responsive miRNAs.
Some miRNAs may be involved in either PTI or ETI, or both, according to their response to M. oryzae or its elicitors. For example, miR398b may act in PTI, because its abundance was up-regulated in both the susceptible and resistant accessions following M. oryzae infection, with overexpression of miR398b leading to increased induction of the PTI marker genes OsKS4 and OsNAC4 in response to PAMP treatment (Li et al. 2014). However, current data cannot exclude the involvement of miR398 in ETI. Nevertheless, the miRNA-mediated regulatory nature bestows miRNA with the potential to simultaneously regulate multiple genes. For example, miR169 targets eight of the NF-YA family transcription factor genes and miR396 targets eleven of the GRF genes. In such cases, blocking or overexpressing a single miRNA can achieve up- or down-regulation of multiple genes, facilitating gene manipulation when these target genes have functional redundancy. For example, in tomato, blocking miR482 and miR2118 via STTM resulted in increased resistance to at least two different pathogens (Canto-Pastor et al. 2019). Therefore, it would be worthwhile investigating the roles of miRNAs in rice-M. oryzae interaction and applying them to breeding programs to improve agronomic traits such as disease resistance.
Taken together, progress is clearly being made with respect to unveiling the roles of miRNAs in conferring rice immunity against M. oryzae. The future focus should be on dissecting the functions of key components in the appropriate miRNA signaling pathway and of each blast fungus-responsive miRNA in the rice-M. oryzae interactions. It is highly anticipated that, through MIR genes manipulation we can improve important agronomic traits with lower fitness costs.
Availability of data and materials
Auxin response factor
Copper chaperone for superoxide dismutase
Chitin elicitor binding protein
Chitin elicitor receptor kinase
Growth regulating factor
Hua enhancer 1
Lijiangxin Tuan Hegu
Lysin motif–containing protein
miRNA-induced silencing complex
Nuclear transcription factor Y subunit A
Nucleotide-binding site leucine-rich repeat
- NRAMP 6:
Natural resistance-associated macrophage protein 6
Pathogen-associated molecular pattern
Reactive oxygen species
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We are grateful to Ji-Qun Zhao, Mei Pu and Dr. Yan-Yan Huang for management of the miRNA-related projects in the lab. We apologize to all colleagues whose work could not be cited due to space limitations.
This work was supported by the National Natural Science Foundation of China grants (31430072 and 31672090 to W-MW, and 31471761 to YL).
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Li, Y., Jeyakumar, J.M.J., Feng, Q. et al. The roles of rice microRNAs in rice-Magnaporthe oryzae interaction. Phytopathol Res 1, 33 (2019). https://doi.org/10.1186/s42483-019-0040-8
- Dicer-like 1