Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 https://doi.org/10.1186/s12870-019-2156-5 RESEARCH ARTICLE Open Access Osa-miR7695 enhances transcriptional priming in defense responses against the rice blast fungus Ferran Sánchez-Sanuy1, Cristina Peris-Peris1, Shiho Tomiyama2, Kazunori Okada2, Yue-Ie Hsing3, Blanca San Segundo1,4 and Sonia Campo1* Abstract Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post- transcriptional level in eukaryotes. In rice, MIR7695 expression is regulated by infection with the rice blast fungus Magnaporthe oryzae with subsequent down-regulation of an alternatively spliced transcript of natural resistance- associated macrophage protein 6 (OsNramp6). NRAMP6 functions as an iron transporter in rice. Results: Rice plants grown under high iron supply showed blast resistance, which supports that iron is a factor in controlling blast resistance. During pathogen infection, iron accumulated in the vicinity of M. oryzae appressoria, the sites of pathogen entry, and in cells surrounding infected regions of the rice leaf. Activation-tagged MIR7695 rice plants (MIR7695-Ac) exhibited enhanced iron accumulation and resistance to M. oryzae infection. RNA-seq analysis revealed that blast resistance in MIR7695-Ac plants was associated with strong induction of defense-related genes, including pathogenesis-related and diterpenoid biosynthetic genes. Levels of phytoalexins during pathogen infection were higher in MIR7695-Ac than wild-type plants. Early phytoalexin biosynthetic genes, OsCPS2 and OsCPS4, were also highly upregulated in wild-type rice plants grown under high iron supply. Conclusions: Our data support a positive role of miR7695 in regulating rice immunity that further underpin links between defense and iron signaling in rice. These findings provides a basis to better understand regulatory mechanisms involved in rice immunity in which miR7695 participates which has a great potential for the development of strategies to improve blast resistance in rice. Keywords: Blast, Defense, Iron, Magnaporthe oryzae, microRNA, miR7695, Oryza sativa, Phytoalexins, Rice, Transcriptomics Background the activation of protein phosphorylation/dephosphoryla- Plants have a sophisticated innate immune system for pro- tion cascades, and the production of pathogenesis-related tection against pathogen infection [1, 2]. The activation of (PR) proteins, among others [3, 4]. Successful pathogens, plant defense responses against pathogens occurs via the however, have developed countermeasures to suppress this recognition of conserved pathogen-associated molecular basal defense in certain plant species and promote disease patterns (PAMPs; previously known as elicitors) by host by delivering effectors into the host. Plants have also pattern-recognition receptors (PRR) which, in turn, triggers evolved Resistance (R) genes that recognize microbial ef- a signaling cascade leading to the activation of defense- fectors to activate a much stronger immune response, the related responses. Pathogen-induced defense responses so called effector-triggered immunity [5]. PTI and ETI have include the production of reactive oxygen species (ROS), long been considered protein-based mechanisms. However, increasing evidence supports that microRNAs (miRNAs) * Correspondence: sonia.campo@cragenomica.es 1 are also important players in both PTI and ETI [6–11]. Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus Universitat Autònoma de Barcelona (UAB), Bellaterra (Cerdanyola del MiRNAs are small noncoding RNAs that modulate Vallés), Barcelona, Spain gene expression in eukaryotes by triggering sequence- Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 2 of 16 specific cleavage or translational repression of target genes 166 h) and negative regulators (miR164a, miR169 and [12]. Plant miRNAs play a crucial role in the control of de- miR319) of defense responses against the rice blast fungus velopmental processes and adaptation to environmental M. oryzae [41, 42, 44–47]. Hence, to obtain a comprehen- stresses, both abiotic and biotic stresses [13–16]. Although sive understanding of the regulatory functions of miRNAs numerous miRNAs have been reported to be regulated in the rice response to M. oryzae infection, intense experi- during pathogen infection, the biological role of most of mental validation of miRNA functioning is mandatory. them remains unknown. Furthermore, these studies have We previously reported that the rice miR7695 is in- been conducted mainly in the model dicotyledonous plant volved in blast resistance [41]. This particular miRNA tar- Arabidopsis thaliana during interaction with the bacterial gets an alternatively spliced transcript of OsNramp6 pathogen Pseudomonas syringae. Further experimental (natural resistance-associated macrophage pathogen 6), in validation is required to better understand the regulatory particular the shortest transcript variant (OsNramp6.8), roles of miRNAs in plant immunity. encoding an Fe and manganese transporter, the NRAMP6 In the past few years, studies have demonstrated that protein [48]. Here we show that Fe accumulates at the miRNAs act as regulators of nutrient homeostasis in plants sites of pathogen penetration (appressoria) and cells sur- by modulating the expression of genes involved in nutrient rounding the infection sites in M.oryzae-infected rice homeostasis [17]. It has been shown that miR399 and leaves. MIR7695 activation results in blast resistance miR395 play a fundamental role in phosphate and sulfur which was associated with local iron accumulation at the homeostasis in plants [18, 19]. Plant miRNAs controlling infection sites and superinduction of PR and diterpenoid nutrient homeostasis may also be important factors in con- phytoalexin biosynthesis genes. Consequently, MIR7695 trolling disease resistance. Unfortunately, miRNA-mediated activation plants accumulated major rice phytoalexins in mechanisms involved in disease resistance and nutrient their leaves. These results provide new insights into the homeostasis have been studied separately. role of miR7695 in regulating immune responses and Fe Iron (Fe) is an essential microelement for plant growth signaling pathways in the rice–M. oryzae interaction. required for essential redox reactions in metabolism. Fe is also required for photosynthesis and maintenance of Results chloroplast function [20]. However, excess Fe generates M. oryzae infection alters Fe distribution in rice leaves reactive oxygen species (ROS), which might cause oxida- In this work, we investigated the cellular distribution of tive damage to macromolecules (e.g. nucleic acids, lipids, Fe during M. oryzae infection by using the Perls staining. proteins) and cellular structures [21–25]. During patho- Perls reagent (potassium ferrocyanide) reacts with Fe3+ gen infection, Fe homeostasis must be carefully regu- to form an insoluble pigment, or Prussian blue. Without lated as the host and pathogen compete for the available pathogen infection, Perls staining revealed that Fe pref- Fe. The pathogen must acquire this vital element from erentially accumulated at stomata (Fig. 1a upper left host tissues, whereas the host plant can deprive the panel). Upon pathogen challenge, iron staining showed a invader of Fe as a defensive strategy. Mechanisms for less uniform, but more widespread distribution in the maintaining Fe homeostasis need to be highly dynamic stomatal areas pointing to a possible pathogen-induced in the host plant to allow normal plant growth. Al- iron mobilization (Fig. 1a upper right panel). Of interest, though distinct miRNAs have been shown to be respon- Perls staining revealed iron accumulation forming halo sive to Fe stress [26–30], how such alterations will affect areas around the infection sites (Fig. 1a, lower panels). Fe homeostasis and disease resistance remain to be To increase the sensitivity and definition of Fe detec- determined. tion in rice leaves, we used intensified Perls staining with Rice is one of the most important cereal crops in the DAB/H2O2. This method takes advantage of the redox world and the model plant for genomics research of mono- activity of the Prussian blue reagent. As previously ob- cotyledonous [31, 32]. Rice production is severely affected served by Perls staining, after Perls/DAB staining, strong by blast disease caused by the fungal pathogen Magna- black precipitates appeared at stomatal areas in mock- porthe oryzae [33]. miRNAs controlling traits of agronomic inoculated leaves which showed a diffuse staining upon importance (e.g., tiller growth, early flowering, grain pro- M. oryzae infection (Fig. 1b, upper panels). In those duction) [34–36] and tolerance to abiotic stress (drought, regions, Fe-stained granules were often visible (Fig. 1b, salinity and cold stress) [37–39] have been described in middle left panel). As well, in these regions, Fe accumu- rice. Evidence also supports variations in the accumulation lated with different intensities, with strong black precipi- of rice miRNAs during M. oryzae infection or treatment tates in the center, surrounded by weaker and unevenly with M. oryzae elicitors [40–43], but the biological function distributed halos of black precipitate (Fig. 1b, middle right of only a few of these pathogen-regulated miRNAs has panel). Higher magnification of these regions showed ger- been demonstrated. They include both positive regulators minating spores and germ tubes forming appressoria, the (miR7695, miR160, miR398, and polycistronic miR166k- sites where pathogen entry occurs (Fig. 1b, lower panel). Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 3 of 16 in close proximity to the infection site, supporting that Fe a distribution might be important for blast resistance. Mock M. oryzae Resistance to infection by the rice blast fungus M. oryzae in mutant plants with MIR7695 activation st st We searched publicly available rice mutant collections for mutants with affected MIR7695 expression. Because of the small size of MIR genes, identifying mutant alleles for miRNAs in insertional mutant collections is unlikely. A T-DNA tagged line (M0107013) was identified in the M. oryzae M. oryzae ls Taiwan Rice Insertion Mutants (TRIM) Database [49]; http://trim.sinica.edu.tw) in which the T-DNA was inserted upstream of the MIR7695 locus (Additional file 1: st Figure S1a, left panel). TRIM was designed for gene ls knockout and activation tagging in the Tainung67 (ja- ponica) background. Thus, the presence of an octamer of the cauliflower mosaic virus 35S (CaMV35) transcrip- tional enhancer next to the left border of the T-DNA b can activate the expression of genes located up to 30 Kb Mock M. oryzae from the integration site [50, 51] (Additional file 1: st Figure S1a, left panel). Homozygous and azygous plants were identified by PCR genotyping (Additional file 1: ls Figure S1a, right panel, primers are in Additional file 2: Table S1). Quantitative PCR (qPCR) revealed that MIR7695-Ac plants had a single copy of T-DNA inserted in its genome (Additional file 3: Table S2) Importantly, M. oryzae M. oryzae the accumulation of miR7695 precursor and mature sequences was higher in homozygous mutant plants with MIR7695 activation tagging (hereafter MIR7695-Ac) than wild-type azygous (WT-Az) plants as revealed by RT-qPCR and small-RNA northern blot analyses, respect- ively (Fig. 2a, left panel). Consistent with upregulated MIR7695, the accumulation of miR7695 target transcripts (OsNramp6.8) was decreased in leaves with MIR7695-Ac M. oryzae (Fig. 2a, right panel). gt ap We previously reported that the recognition site of miR7695 locates in the 3′ UTR region of OsNramp6.8 transcripts. In this study, we further investigated whether OsNramp6.8 gene is a real target gene for miR7695 by per- sp forming RNA ligase-mediated 5′ RACE (5′-RLM-RACE). Sequencing of the 5′ -RACE PCR products identified Fig. 1 Histochemical detection of iron (Fe) in M. oryzae-infected rice cleavage fragments at the expected site of OsNramp6.8 (O. sativa cv. Nipponbare, japonica) leaves. Wild-type plants at the transcripts, thus, supporting that OsNramp6.8 transcripts three-leaf stage were sprayed with a M. oryzae spore suspension or are cleaved by miR7695 (Fig. 2b). These observations dem- mock inoculated. At 24–48 h post-inoculation (hpi), the third leaf of onstrated that M0107013 is an activation mutant for each plant was stained with Perls (a) or Perls/DAB (b) (blue and black precipitates, respectively). Scale bar: 100 μm. ap, appressorium; MIR7695 (MIR7695-Ac plants) and that miR7695 cleaves gt, germ tube; ls, lesion; st, stomata; sp, spore OsNramp6.8 transcripts. MIR7695-Ac plants were slightly shorter and contained less chlorophyll than did WT-Az plants, but these differences were not statistically signifi- cant (Additional file 1: Figure S1b and c). Fe was weakly stained further away from the penetration Infection experiments were performed to assess the ef- site. Hence, histochemical analysis of Fe accumulation fect of MIR7695 activation on disease resistance. WT-Az established that Fe accumulates at the sites of attempted and MIR7695-Ac plants were spray-inoculated with M. penetration by the fungus (appressoria) as well as in cells oryzae spores. On visual inspection, MIR7695-Ac plants Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 4 of 16 Fig. 2 Resistance of MIR7695-Ac mutant plants to M. oryzae infection. a MIR7695 a RT-qPCR analysis of MIR7695 precursor transcripts (left panel) and 0.2 precursor 3.2 Nramp6.8 Relative expression miR7695 target (Nramp6.8, Os01g0503400.8) in homozygous mutant * (MIR7695-Ac) and WT (segregated azygous, WT-Az) plants. Data are mean ± SE (n = 3) (Student t test, *p < 0.05). Lower panel: northern 0.1 1.6 blot analysis of mature miR7695 using the miR7695.3-3p sequence * as the hybridization probe (Additional file 2: Table S1). As a loading control, the RNA blot was stained with ethidium bromide (EtBr) (b) Experimental validation of miR7695-mediated cleavage of 0.0 0.0 OsNramp6.8 transcripts by 5′-RLM-RACE. Schematic representation of miR7695 WT-Az the OsNramp6.8 (upper panel), showing the coding sequence (blue), EtBr MIR7695-Ac 5’UTR (green), and 3’UTR (pink). Boxes, exons; lines, introns. Gene- specific primers were used for 5′-RACE and the resulting PCR b Nramp6.8 products were sequenced. The identified cleavage site is indicated by an arrow and the number above indicate the detected cleavage 29/29 site of independent clones. c Leaves of 3-week-old plants were Nramp6.8 1128 5’-CUACGUGGCAUGCCAUGUGGACGAUG-3’1151 sprayed with a M. oryzae spore suspension. The second leaf was ::::::::.. ::.::::: .: photographed at 7 days post-inoculation. d Percentage of leaf area miR7695 3’-UGCACCGUGUAGUGCACCUAUU-5’ affected by blast lesions (upper panel). Relative fungal biomass (lower panel) was determined by qPCR as the ratio of M. oryzae 28S c WT-Az MIR7695-Ac d 0.2 Lesion Area (%) Fungal Biomass ribosomal DNA to the rice Ubiquitin1 gene (primers in Additional file 2: Table S1). Data are mean ± SE (n = 7) from 1 experiment (Student t 0.1 * test, *p < 0.05). Four independent infection assays were performed with similar results. e RT-qPCR analysis of OsPR1a transcripts at 0 different times after inoculation with M. oryzae spores. Blast infection 60 was carried out as in (c). Data are mean ± SE (n = 3, each biological replicate is a pool of 3 individual leaves) (Student t test, **p < 0.01 * ***p < 0.001; infected vs non-infected). Mock inoculated (control) 30 plants; +, M. oryzae-infected plants. 0 WT-Az MIR7695-Ac oryzae infection in miR7695-overexpressing lines and e 5.0 OsPR1a Relative Expression ** Osnramp6 mutant plants [41, 48]. *** As previously mentioned, without pathogen infection, 2.5 iron accumulated in the stomata of leaves from wild-type rice plants whereas M. oryzae infection induced iron mobilization to the infection sites in wild-type plants (see 0.0 Fig. 1). In this work, we determined the accumulation of + + + + + + - - - - - - 24h 48h 72h 24h 48h 72h iron at different time points after inoculation with M. ory- WT-Az MIR7695-Ac zae spores in wild-type and MIR7695-Ac plants. As it was observed in wild-type plants, iron was detected in stomata of MIR7695-Ac leaves in non-infected plants (Fig. 3a). were more resistant to M. oryzae infection than were WT This analysis also revealed a stronger iron accumulation at plants (Fig. 2c). Blast resistance was confirmed by quanti- the infection sites in the MIR7695-Ac plants compared to fying the lesion area and the relative amount of fungal the WT-Az at 24 hpi (Fig. 3b, upper panels). Moreover, a DNA in infected leaves (Fig. 2d). Resistance of MIR7695- general decrease on the iron content occurred at later Ac plants to M. oryzae infection was also observed by time points (48 hpi, 72 hpi) in both wild-type and local inoculation of detached rice leaves (Additional file 4: MIR7695-Ac plants. Figure S2). The induction of PR1 expression is a widely used indica- Transcript profiling of MIR7695-ac mutant plants tor of defense activation in response to pathogen infection To investigate the molecular mechanisms underlying in plants, including infection by M. oryzae in rice [52]. As blast resistance in MIR7695 plants, we used RNA-seq expected, PR1a was induced in WT-Az plants during M. analysis. Initially, we examined the impact of MIR7695 oryzae infection (Fig. 2e). However, PR1a was induced at a activation on the rice transcriptome by comparing the much higher level in fungal-infected MIR7695-Ac than transcript profiles of mock-inoculated MIR7695-Ac and WT-Az plants (Fig. 2e), which is consistent with the WT-Az plants. We identified 281 differentially expressed phenotype of blast resistance observed in MIR7695-Ac genes (DEGs; 153 upregulated and 128 downregulated) plants. The observed phenotype of blast resistance in (Fig. 4a; Additional file 5: Figure S3a). Additional file 6: MIR7695-Ac plants also agreed with resistance to M. Table S3 lists the DEGs in MIR7695-Ac plants. Singular Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 5 of 16 The expression of a vast array of transcription factors a WT-Az MIR7695-Ac (TFs) belonging to different TF families was regulated in Mock Mock mock-inoculated MIR7695-Ac plants (most of them being 48hpi 48hpi upregulated) (Fig. 4c; Additional file 8: Table S5). They in- cluded TFs with a demonstrated role in the rice defense response to blast infection), such as OsWRKY45 and st st OsNAC4 [53]. These TFs function as positive regulators of the rice response to M. oryzae infection [54, 55] and are both upregulated in MIR7695-Ac plants (Fig. 4c). Other b WT-Az MIR7695-Ac TFs that are activated in MIR7695 plants are known to M. oryzae M. oryzae mediate defense hormone signaling, such as ethylene 24hpi 24hpi response factor 5 (OsEREBP5), APETALA2/ethylene-re- sponsive element binding protein (AP2/EREBP), several jasmonate ZIM-domain (JAZ) TFs, and RERJ1 (a jasmonic acid-dependent stress inductive bHLH transcription fac- tor) [56] (Fig. 4c). Genes encoding several wall-associated kinase (WAK) receptors and disease resistance (R) pro- M. oryzae M. oryzae teins were also upregulated in MIR7695-Ac (Fig. 4c). 48hpi 48hpi Upregulated genes in MIR7695-Ac plants also included several heavy metal transporter/metal detoxification (HMTD) protein genes and siroheme uroporphyrinogen methyltransferase1 (SUM1), encoding enzymes respon- sible for the synthesis of the Fe-containing cofactor of enzymes (Fig. 4c). M. oryzae M. oryzae Genes that were downregulated in mock-inoculated 72hpi 72hpi MIR7695-Ac plants included those involved in the syn- thesis of nicotinamine (NA), a chelator of metals and the precursor of phytosiderophores (components for Fe ac- quisition) [57]: OsNAS1 and OsNAS2, encoding nicotina- mine synthases (Fig. 4c). Other downregulated genes are involved in oxidation-reduction processes, such as lac- Fig. 3 Histochemical detection of iron (Fe) in wild-type (O. sativa cv. cases (OsLAC4, OsLAC19, OsLAC24) and peroxidases Tainung 67, japonica) and MIR7695-Ac mutant plants during M. (Prx81, Prx125) (Fig. 4c). oryzae infection. Wild-type and MIR7695-Ac mutant plants at the three-leaf stage were (a) mock-inoculated or (b) inoculated with M. RT-qPCR was used to validate RNA-seq findings. RT- oryzae spores. At 24, 48, 72 h post-inoculation (hpi), the third leaf of qPCR results obtained for selected genes were highly con- each plant was stained with Perls. Iron is detected as blue cordant with RNA-seq results for both upregulated genes precipitates. Representative images of one experiment are shown (OsWRKY45, OsWRKY71, OsNAC4, OsDREB1G, OsDRE (n = 4). Three independent infection assays were performed with B1E, OsRERJ1) and downregulated genes (OsLAC19 and similar results. Scale bar: 100 μm. st, stomata OsNAS1) (Additional file 9: Figure S4). Together, these observations suggest that without patho- enrichment analysis (SEA) of molecular function by gen infection, MIR7695 activation led to altered expression using AgriGO revealed gene ontology (GO) annotations of genes involved in 1) transcriptional regulation, 2) disease in the “binding” and “catalytic activity” categories, which resistance, 3) metal binding and transport, and 4) oxidation- were over-represented for both upregulated and down- reduction mechanisms. Transcriptional changes caused by regulated DEGs (Fig. 4a; Additional file 7: Table S4). MIR7695 activation might well contribute to the resistance Genes in the categories “transcription regulator activity” response of these plants to pathogen infection. and “transporter activity” were specifically enriched in the upregulated DEGs, whereas genes in the “electron Enhanced defense responses to M. oryzae infection in carrier activity” category were enriched in downregulated MIR7695-ac plants DEGs (Fig. 4a). The binding category comprised genes re- Pathogen-induced alterations in the transcriptome of lated to “calcium ion binding” and “zinc ion binding” (up- MIR7695-Ac plants were identified and compared to those regulated only in MIR7695-Ac plants) and “iron ion of fungal-infected WT-Az plants. The number of genes binding” genes (downregulated only in MIR7695-Ac with expression affected by M. oryzae infection at 48 h plants) (Fig. 4b). post-infection (hpi) was 4.5 times higher in MIR7695-Ac Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 6 of 16 a WT-Az vs MIR7695-Ac c Expression Log2 FC 0 1 2 −4 0 4 200 100 0 100 200 of DEGs MIR7695-Ac Number WT-Az MIR7695-Ac vs WT-Az 153 128 WRKY7 WRKY14 WRKY21 UP DOWN WRKY24 WRKY44 WRKY WRKY45 WRKY69 WRKY70 WRKY71 NAC4 SNAC1 ONAC59 NAC GO terms ONAC022 Enriched OsNAC14 AP2/EREBP EREBP5 DREB1E ERF DREB1G DREB1H MYB2 TF Transcription Regulator activity MYB2P−1 MYB Binding Transporter activity JAZ9 Catalytic activity JAZ10 JAZ Electron carrier activity JAZ12 bHLH1 bHLH148 bHLH17 bHLH b bHLH RERJ1 Catalytic GATA8 GATA activity OsRING−1 Zinc finger protein Oxidoreductase Binding ZOS4−01 ZPT ZOS2−19 Transferase Calcium Ion CIGR1 GRAS Hydrolase Metal Ion HSFA4D HSF Zinc Ion Iron ion Protein Kinase WAK4 DNA WAK36 WAK ATP WAK50 20 R-1 R-2 Percent of genes R-3 10 R-4 Resistance R-5 R-6 proteins 0 R-7 R-8 R-9 10 HMTD1 HMTD2 20 HMTD3 UP DOWN HMTD4 Metal SUM1 Id3 binding NAS1 NAS2 LAC4 LAC19 LAC24 Oxidation/ Prx125 Prx81 Reduction POX Fig. 4 Differentially expressed genes (DEGs) in MIR7695-Ac mutant plants relative to WT-Az plants by RNA-seq analysis. Leaves of three-week-old plants were used (a) Number of DEGs and Gene Ontology (GO) analysis of DEG function. Up-regulated genes (log2 fold change [FC] ≥ 1; purple) and down-regulated genes (log2FC ≤ − 1; green) genes (p < 0.05, false discovery rate [FDR] < 0.05, n = 2). Pie charts represent the five general GO terms enriched in up- and downregulated DEGs. b Enriched terms in the “binding” and “catalytic activity” categories. c Heatmaps showing RNAseq expression level (left panel; log10 [FPKM+ 1]) and FC (right panel; log2FC) of DEGs. Gene expression is represented from pale yellow (less expressed) to blue (more expressed). Upregulated (log2FC ≥ 1; purple) and downregulated (log2FC ≤ − 1; green) DEGs. Data are means (n = 2). The full gene names and ID list are in Additional file 8: Table S5 than WT-Az plants (531 and 116, respectively) (Fig. 5a; plants. DEGs for WT-Az and MIR7695-Ac plants are listed Additional file 5: Figure S3bc). This observation already in- in Additional files 10 and 11 (Tables S6 and S7), respect- dicated stronger transcriptional regulation in the mutant ively. Of note, genes typically associated with the plant Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 7 of 16 represented in upregulated genes were clustered in the cat- egories “response to stress” (biotic and oxidative stress), “response to stimulus”, and “secondary metabolism” (phe- nylpropanoids and terpenoids) (Fig. 6a, upper panel). Genes induced in MIR7695-Ac plants in the bifactorial analysis included defense-related genes, such as PR genes (PR1, PR2, PR5 and PR10 family members) and oxidative stress-related enzymes (e.g., several peroxidases) (Fig. 6b and Additional file 14: Table S10). Phenylpropanoid biosynthetic genes were highly repre- sented in the bifactorial analysis of DEGs. They included genes involved in the production of flavonoids (CHS; F3H, FLS, DFR) and monolignols, the building blocks of lignin (CCR, CAD, COMT, PMT, LAC, PRX) (Fig. 6b and Additional file 14: Table S10; additional information on phenylpropanoid and lignin biosynthesis genes that were differentially regulated in infected MIR7695-Ac plants is in Additional file 15: Figure S5a). The expression of flavonoid biosynthesis genes is known to be induced by pathogen infection, and certain plant flavonoids exhibited antifungal activity [59]. The accumulation of lignin in sec- ondary cell walls provides a physical barrier against patho- gen invasion [60]. The expression of several peroxidases was upregulated in MIR7695-Ac versus WT-Az plants and Fig. 5 Comparison of DEGs in MIR7695-Ac and WT plants after also with pathogen infection. Peroxidases are key enzymes challenge with M. oryzae. Leaves of 3-week-old rice plants (WT-Az and MIR7695-Ac) were mock-inoculated or sprayed with a in the biosynthesis of lignin during resistance reactions via suspension of M. oryzae spores, and collected at 48 hpi for RNA cross-linking of lignin monomers. A stronger induction of extraction and RNA-seq. Upregulated (log2FC ≥ 1) and genes involved in flavonoid and lignin biosynthesis might downregulated (log2FC ≤ 1) genes by M. oryzae infection (p < 0.05, play a role in protecting the MIR7695-Ac plants against FDR < 0.05). a Venn diagram of the overlap between fungal- M. oryzae infection. In addition, a important number of responsive genes of each genotype. b Comparison of the expression pattern of defense-related genes with M. oryzae infection. Up- genes involved in the production of diterpenoid phyto- (purple) and downregulated (green) DEGs. For a full list of gene IDs, alexins were among the top induced genes in MIR7695- see Additional file 12: Table S8 Ac plants with infection (bifactorial DEGs) (Fig. 6b; Additional file 14: Table S10) as described below. The GO term most represented in downregulated genes response to pathogen attack, such as PR genes, were in- on bifactorial analysis of DEGs was “iron ion transport” duced in MIR7695-Ac but not in WT-Az plants at 48hpi (Fig. 6a, lower panel). This included genes related to Fe (Fig. 5b; Additional file 12: Table S8). They included PR1, homeostasis, such OsFRO2 (a Fe3+ reductase), OsNAS2 (a β-1,3-glucanase (PR2), chitinase (PR3, PR4, PR8), thauma- nicotianamine synthase), OsIRO2 TF, OsHRZ1, OsHRZ2 tin (PR5), peroxidase (PR9), PBZ1 and other Bet v1 homo- ubiquitin ligases, OsNRAMP1 (Fe2+ transporter), OsMIR logues (PR10), and lipid transfer protein (LTP; PR14). The (mitochondrial Fe-regulated gene), and OsIROPT (an antimicrobial activity of many of these PR proteins has been oligopeptide transporter) (Fig. 6b; Additional file 14: Table demonstrated (e.g., PR1, chitinases, β-1,3-glucanases, PR4, S10). thaumatin, LTPs) [58]. A more detailed expression analysis was performed for To further establish differences in the transcriptional re- genes identified by bifactorial analysis, and their expres- sponse to pathogen infection between MIR7695-Ac and sion was examined by RT-qPCR at different times after WT-Az plants, we used a two-factor analysis (genotype inoculation with M. oryzae spores (24, 48 and 72 hpi). and treatment) of the full dataset of DEGs in each geno- This analysis confirmed stronger induction of PR genes type. A total of 153 and 100 genes were identified as upreg- (OsPR1b, OsPBZ, OsPR10b) and lignin biosynthesis ulated and downregulated, respectively, in MIR7695-Ac genes (OsCAD2, OsCOMT1) in MIR7695-Ac than WT- versus WT plants (Fig. 6a and Additional file 16: Figure Az plants during M. oryzae infection (Fig. 7). Induction S3d; Additional file 13: Table S9). AgriGO was used for of OsPBZ1 and other OsPR10 family members is known GO enrichment analysis of DEGs in the bifactorial analysis, to occur during M. oryzae infection and, when overex- with clustering by Revigo (Fig. 6a). The GO terms over- pressed, the genes confer pathogen resistance [61–63]. Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 8 of 16 a b Expression Log2 FC UP-REGULATED 153 DEG (log2FC ≥ 1) 1 2 3 −4 0 4 phenylpropanoid MIR7695-Ac Secondary metabolism I.Ho vs Ho metabolism I.Az vs Az WT(Az) Ho vs Az Bifactorial -4 Terpenoid (Ho) metabolism C I C I oxidation PR1a PR proteins reduction PR1b PR2 Response to stress 0 PR5 response OSM1 PR10 to stimulus PR10b PBZ1 response JIOSPR10 − 5 to oxidative stress cellular modified Prx13 Oxidative 4 Prx20 stress aa metabolism −15 Prx22 to biotic −25 Prx38 CPX1 stimulus log10 Prx81 to stress pval Prx125 -4 0 4 C4H CHS1 DOWN-REGULATED 100 DEG (log2FC ≤ -1) Phenylpropanoid F3H 0.5 FLS DFR_a Secondary metabolism DFR_b CCR CAD2 COMT1 COMT2 PMT LAC4 iron ion transport LAC19 LAC24 DXS3 Terpenoid 1.0 MAS1 CPS2 CYP701A8 CYP71Z7 0 CYP76M6 CYP76M8 −1.0 FRO2 homeostasis −2.0 NAS2 IRO2 Iron −3.0 HRZ1 log10 HRZ2 pval NRAMP1 1.5 MIR -0.4 0.0 0.4 IROPT1 Fig. 6 Biological processes altered in MIR7695-Ac mutant plants relative to WT-Az plants with M. oryzae infection. Same plant material as in Fig. 4. RNA-seq data underwent bifactorial analysis (upregulated, log2FC ≥ 1; downregulated, log2FC ≤ 1; p < 0.05, FDR < 0.05). a GO analysis of DEG function in MIR7695-Ac plants with blast infection (upper and lower panels show upregulated and downregulated DEGs, respectively). The top GO terms enriched in MIR7695-Ac vs WT-Az DEGs were represented by using REVIGO after reducing redundancy (http://revigo.irb.hr/). Circles represent GO terms and those clustered closer to each other represented similar GO terms. Disc colors (blue to yellow) represent de degree of GO enrichment (p-value) and disc size is proportional to the frequency of the GO term in the GO database (larger and smaller discs represent more general and more specific terms, respectively). b Heatmap showing distribution of RNAseq expression level (log10[FPKM+ 1], pale yellow to blue from less to more expressed) for DEGs belonging to the top enriched GO categories in M. oryzae-infected MIR7695-Ac plants (left panel). Heatmaps show upregulated (purple) and downregulated (green) DEGs (bifactorial analysis, middle panel; Monofactorial analysis for the given comparisons, right panel) Biological processes are indicated to the left. Data are means (n = 2). The full gene ID list is shown in Additional file 14: Table S10. Altogether, comparative transcriptome analysis (bifac- Regulation of Fe homeostasis-related genes in rice leaves torial analysis) revealed stronger induction of defense- during M. oryzae infection related genes in MIR7695-Ac (e.g., PR, oxidative stress- In plant roots, two different mechanisms have been de- related, phenylpropanoid and diterpenoid phytoalexin scribed for Fe uptake from the rhizosphere, the reducing biosynthesis genes), whereas genes that function in Fe and chelating strategies (strategies I and II, respectively) homeostasis appear to be downregulated in MIR7695-Ac [64, 65]. Rice is unique in that as it uses both strategies. plants during M. oryzae infection. Besides strategy I and II genes, other genes contribute to Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 9 of 16 strong downregulation at 48 and 72 hpi (Additional file 16: a WT-Az MIR7695-Ac b WT-Az MIR7695-Ac Figure S6; MIR7695-Ac, infected vs mock). Therefore, 1.2 OsPR1b 0.4 OsCAD2 downregulation of Fe homeostasis genes occurs earlier in MIR7695-Ac than WT-Az plants. relative expression level 0.6 0.2 Phytoalexins accumulate in MIR7695-ac plants during M. oryzae infection relative expression level 0.0 0.0 3.0 1.4 OsPBZ OsCOMT1 Phytoalexins are low-molecular-weight antimicrobial com- pounds that accumulate in plant tissues during pathogen 1.0 0.7 infection [66]. Major phytoalexins accumulating in rice leaves in response to M. oryzae infection are the diterpene 0.0 0.0 1.2 OsPR10b 24 48 72 24 48 72 phytoalexins momilactones, phytocasssenes and oryzalexins hours [67]. As previously mentioned, the expression of genes c WT-Az MIR7695-Ac involved in the biosynthesis of diterpenoid phytoalexins, 0.6 24 48 72 24 48 72 PR1b oryzalexins, phytocassenes and momilactones was induced PR10b 0.0 PBZ1 to a higher level in MIR7695-Ac than WT-Az plants (at 48 24 48 72 24 48 72 CAD2 hpi with M. oryzae) (see Fig. 6b). For details on genes in- hours COMT1 log2FC volved in diterpene phytoalexin biosynthesis with overex- Control Infected 4 2 0 -2 -4 pression in MIR7695 plants, see Additional file 15: Figure Fig. 7 Expression of PR and lignin biosynthesis genes in WT-Az and S5b. RT-qPCR analysis of diterpene phytoalexin biosyn- MIR7695-Ac plants during blast infection. Plants were sprayed with a thesis genes at different times after inoculation with M. ory- M. oryzae spore suspension. Leaves were collected at the indicated zae spores (24, 48, 72 hpi) confirmed earlier and stronger times (24, 48, 72 hpi). a-b Expression analysis of (a) PR (OsPR1b, induction of these genes in MIR7695-Ac than WT-Az OsPBZ, OsPR10b) and (b) lignin (OsCAD2, OsCOMT1) genes determined by RT-qPCR. Data are mean ± SE (n = 3; each sample plants (Fig. 8a, b). Differences in pathogen-induced expres- consisted of a pool of 3 individual leaves). Mock-inoculated (control, sion of these genes were more evident at 48 and 72 h after grey) and M. oryzae-infected (red) plants. Time point used for blast inoculation, as revealed by the higher fold change of RNAseq analysis (48 h) is labeled with a thick line in the x-axis. c gene expression (Fig. 8c). Heatmap showing log2 FC for each transcript and each time To investigate whether superinduction of diterpenoid (infected vs. control) as determined from RT-qPCR values (a-b). Upregulated (purple) and downregulated (green). Dots indicate phytoalexin biosynthesis genes affects phytoalexin accu- significant differences (infected vs control) (Student t test, p < 0.05) mulation, we measured phytocassane and momilactone levels in leaves of MIR7695-Ac and WT-Az plants with- out and with infection. As expected, the expression of Fe transport and/or mobilization through the plant. Al- phytocassanes (B, C and E) and momilactones (A and B) though great progress has been made during the last was barely detected in non-infected rice leaves (Fig. 8d). years to identify mechanisms governing Fe uptake in Upon pathogen challenge, the accumulation of phytocas- roots, the regulation of Fe homeostasis genes in leaves is sanes and momilactones increased in both WT-Az and less understood. MIR7695-Ac plants but was significantly higher in We investigated the expression profile of Fe homeostasis- MIR7695-Ac than WT plants (Fig. 8d). These findings related genes in leaves of wild-type plants during M. oryzae revealed that in response to pathogen infection, diter- infection. Genes examined were: OsFRO2, OsIRO2, OsHRZ1, penoid phytoalexin biosynthesis genes are induced earl- OsNRAMP1 and OsIROPT1 (genes strongly downregulated ier in MIR7695-Ac than WT-Az plants. MIR7695-Ac in MIR7695-Ac plants). These genes were strongly upregu- plants also accumulated higher levels of phytoalexins lated early during infection (24–48 hpi) but downregu- during pathogen infection. Knowing that diterpene phyto- lated at a later stage of the infection process (72 hpi) alexins have antifungal activity against M. oryzae [67–69], (Additional file 16: Figrue S6; WT-Az, infected vs a higher M. oryzae-induced accumulation of phytoalexins mock). Upregulation of these genes early during infec- in leaves of MIR7695-Ac plants might contribute to dis- tion of WT plants correlates with Fe accumulation at ease resistance in these plants. the sites of fungal penetration and infection sites, as re- Finally, we investigated whether Fe supply affects the ex- vealed by histochemical analysis of M. oryzae-infected pression of genes involved in the biosynthesis of diterpen- rice leaves (Fig. 1). oid phytoalexins in rice. We analyzed the effect of Fe For a comparison, we examined the expression profile of supply (low, sufficient and high) on the expression of Fe homeostasis genes in MIR7695-Ac plants. Four of the OsCPS2 and OsCPS4, which function first cyclization steps five genes examined were induced early during infection in the phytoalexin biosynthetic pathway (Additional file 15: (24 hpi), as it was observed in WT plants, followed by a Figure S5b). The expression of these genes was barely Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 10 of 16 Fig. 8 Expression of diterpenoid phytoalexin genes in WT-Az and MIR7695-Ac plants during blast infection. Plant material was treated as in Fig. 7. a-b RT-qPCR analysis of expression of (a) Phytocassane (OsCPS2, OsCYP71Z7, OsCYP76M6) and (b) momilactone (OsCPS4, OsMAS1) biosynthesis genes in rice leaves infected with M. oryzae. Data are mean ± SE (n = 3; each sample consisted of a pool of 3 individual leaves). Mock-inoculated (control, grey) and M. oryzae- infected (red) plants. c Heatmap showing log2 FC for each transcript and each time point (infected vs control) as determined from RT- qPCR values (a-b). Upregulated (purple) and downregulated (green). Dots indicate significant differences (infected vs control) (Student t test, p < 0.05). d Accumulation of diterpenoid phytoalexins, phytocassane E (PE), B (PB) and C (PC) (upper panels) and momilactone A (MA) and B (MB) (lower panels), in leaves of mock- and M. oryzae-infected plants. Each dot represents a biological replicate. FW, fresh weight. e RT-qPCR of expression of upstream diterpenoid biosynthetic genes (OsCPS2 and OsCPS4) in mock- and M. oryzae-infected leaves of rice plants treated under three different Fe supply conditions (low, sufficient, high). Data are mean ± SE (n = 3), each sample consisting of a pool of 4 individual leaves) Discussion Although an increasing number of miRNAs have been shown to be differentially expressed in response to pathogen infection or nutrient stress, most of this re- search involved plants exposed to one or another type of stress separately. Furthermore, few studies aimed to understand the regulation of Fe homeostasis in rice dur- ing M. oryzae infection. Here, we present evidence of a miR7695-guided cleavage of OsNramp6.8 transcripts en- coding the NRAMP6 iron transporter from rice. More- over, we investigated the role of miR7695 in the rice response to infection by M. oryzae. Upon challenge with M. oryzae, Fe accumulated near M. oryzae appressoria and in cells surrounding infected regions of rice leaf. Very recently, Dangol et al. reported that incompatible rice/M.oryzae interactions trigger iron- and ROS- dependent ferroptotic cell death in leaf sheaths of rice plants where iron accumulated at sites of infection to mediate the oxidative burst [22].. Activation-tagged MIR7695 rice plants showed enhanced resistance and a stronger accumulation of iron at the sites of infection. On RNA-seq analysis, defense-related genes, including PR and diterpenoid biosynthetic genes were strongly in- duced along with blast resistance in MIR7695-Ac plants. detected in plants without infection (Fig. 8e). Upon patho- Levels of phytoalexins during pathogen infection were gen challenge, the highest expression of phytoalexin genes higher in MIR7695-Ac than WT azygous plants and occurred in plants grown under high Fe supply as com- genes in the phytoalexin biosynthetic pathway were pared with low or sufficient Fe (Fig. 8e), which supports highly induced in rice plants grown under high Fe sup- that Fe supply affects phytoalexin biosynthesis. Presum- ply. This piece of evidence support that miR7695 posi- ably, a localized accumulation of Fe at the sites of patho- tively regulates immune responses and establish links gen penetration and/or invasion would activate the between defense signaling and Fe homeostasis in rice. expression of phytoalexin biosynthetic genes for phyto- However, the exact mechanisms by which Fe signaling alexin accumulation, thus arresting fungal colonization in regulates the expression of defense-related genes re- infected leaves of MIR7695-Ac plants. mains to be determined. Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 11 of 16 Being a foliar pathogen, M. oryzae has an absolute typical of graminaceous species) [74]. Our results indi- requirement for Fe from host tissues, so rice plants cate that during M. oryzae infection, genes that function might capitalize on the toxicity or the essentiality of Fe in Fe uptake via strategy I (e.g. OsFRO2) or strategy II to arrest M. oryzae invasion. Different scenarios can be (e.g. OsIRO2, OsNAS2) in roots are downregulated in considered. On the one hand, mechanisms that exploit leaves in both WT-Az and MIR7695-Ac plants. Other Fe Fe toxicity might be used by the host plant against M. homeostasis genes such as NRAMP1 (a Fe transporter), oryzae. On the other, the host plant might develop with- OsHRZ1 and OsIROPT1 are also downregulated during holding strategies to restrict Fe availability to the invad- infection. In line with this, the Fe homeostasis genes ing pathogen, a process that in humans and animals has TmFER1 and TmNAS1 (marker genes for monitoring been called “nutritional immunity” [70]. An examination intracellular Fe status in wheat) were found downregu- of Fe distribution in M. oryzae-infected WT rice leaves lated in B. graminis-infected wheat leaves [72]. Further- revealed Fe accumulation in close vicinity of appressoria more, downregulation of Fe homeostasis genes was and in cells surrounding the infection sites, thus, reinfor- accompanied by cytosolic Fe depletion and induction of cing the notion that rice plants use strategies to locally PR genes. A better understanding of the mechanisms in- increase Fe levels to prevent penetration and spread of volved in Fe homeostasis in rice leaf tissues is needed to the pathogen into the leaf tissue. Local accumulation of know whether M. oryzae also provokes intracellular Fe Fe would avoid Fe poisoning caused by a generalized depletion in rice leaves. accumulation of Fe in rice leaf while providing a signal MIR7695-Ac plants exhibited resistance to M. oryzae in- for the activation of host immune responses. If so, this fection, which is consistent with the phenotype of disease localized accumulation at the sites of pathogen penetra- resistance observed in loss-of-function OsNramp6 plants tion and invasion might mediate a localized oxidative [48]. Disease resistance in MIR7695-Ac plants is associated burst that can be toxic to the invading pathogen. Local with a basal expression of resistance genes and defense accumulation of H2O2 would also serve for cell-wall regulatory genes (e.g., OsWRKY45, OsNAC4) without reinforcement (lignification, oxidative cross-linking of pathogen infection and a superinduction of defense-related cell wall components) and induction of defense-related genes with infection. Thus, MIR7695-Ac plants mount a genes (e.g., PR genes). stronger defense response to pathogen infection, a response Without infection, Fe preferentially accumulated in that is reminiscent of defense priming [75]. Whether leaf stomata. In this respect, Fe has been shown to be defense responses are activated earlier in MIR7695-Ac than important in regulating aperture of stomata [71]. During WT plants is unknown. Furthermore, proteins encoded by M. oryzae infection, a re-distribution of Fe appears to defense-related genes that are strongly induced during occur in the rice leaf, Fe moving around stomata and to- infection in MIR7695-Ac plants are known to possess anti- ward the sites of pathogen penetration and colonization. microbial activity (e.g., chitinases, β-1,3-glucanases, PR10 In support of this notion, a localized accumulation of Fe and LTP proteins), and their overexpression in plants con- in cell wall appositions and subsequent defensive H2O2 fers pathogen resistance, including blast resistance [63, 76]. production was previously linked to basal defense in Stronger expression and induction of peroxidases is also a wheat leaves after infection with Blumeria graminis f. sp. feature of MIR7695-Ac plants, these genes being typically tritici [72]. Also, altered Fe distribution in Arabidopsis induced in host plant tissues upon pathogen infection. plants infected with the bacterial pathogen Dickeya Peroxidases are important for generating highly toxic envi- dadantii was reported [73]. Although not proven, the ronments by producing ROS species during resistance reac- activation of toxic oxidative bursts caused by localized tions [77] and for lignin biosynthesis (cross-linking of lignin accumulation of Fe in rice leaves might be important to monomers). A miR7695-mediated regulation of peroxidases restrict M. oryzae growth while maintaining normal might then function to generate an oxidative burst at the plant development. Iron accumulation at the sites of sites where Fe accumulates, thus helping to limit pathogen pathogen infection was observed in both wild-type and spread on the rice leaf. Also, an important number of genes MIR7695-Ac plants, the later ones accumulating more involved in the flavonoid and lignin branches of the general iron at the infection sites than wild-type plants. phenylpropanoid pathway were upregulated in MIR7695- During M. oryzae infection, genes involved in Fe Ac versus WT plants (bifactorial analysis). The antifungal homeostasis were strongly downregulated in leaves of activity of phenylpropanoid compounds against phytopath- MIR7695-Ac plants compared with WT plants. As previ- ogens has been reported [78–80]. The superactivation of ously mentioned, the rice plant uses a combined strategy these various defense genes might be responsible for the for Fe uptake from the rhizosphere that has features of blast resistance phenotype observed in MIR7695-Ac plants. both strategy I (reduction of Fe3+ to Fe2+, a system that Notably, upon pathogen challenge, diterpenoid phyto- operates in roots of most non-graminaceous species) alexin biosynthesis genes were highly upregulated in and strategy II (release of phytosiderophores by the root, MIR7695-Ac plants (bifactorial analysis), accompanied Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 12 of 16 by increased accumulation of major rice phytoalexins. For For Fe treatment, 10 rice seeds were grown in 0.35-L some of these phytoalexins, antifungal activity against M. pots containing soil (turface: vermiculite:quartz sand [2:1: oryzae has been described [67–69]. Other studies pro- 3]) for 14 days and then watered with a half-strength Hoag- posed that rapid biosynthesis of diterpene phytoalexins land solution (5 mM KNO3, 5 mM Ca (NO3)2·4H2O, 2 contributes to resistance to M. oryzae, whereas delayed in- mM MgSO4·7H2O, 1 mM NH4NO3, 0.5 mM KH2PO4 (pH duction of these genes results in enhanced susceptibility to 6.0), 46.3 μM H3BO3, 9.1 μM MnCl2·4H2O, 0.76 μM to blast infection [67]. The accumulation of phytoalexins ZnSO4·7H2O, 0.2 μM CuSO4·5H2O, 0.28 μM would enhance the ability to cope with pathogen infection Na2MoO4·2H2O, 51.7 μM Fe-EDDHA). To assess the ef- in MIR7695-Ac plants. fect of Fe supply, the same nutrient solution was used but with a lower or higher Fe concentration (0.1 μM or 1 mM Conclusions Fe-EDDHA). After 5 days of Fe treatment, plants were in- Overall, this study highlights the relevance of miR7695 in fected with M. oryzae spores (see below for inoculation blast resistance via regulation of rice immune responses. method). Because miR7695 regulates OsNramp6 encoding a Fe transporter from rice, these results support the existence Perls staining and DAB/H2O2 intensification of links between miR7695/OsNramp6 functioning to con- Rice leaves (mock- and blast-inoculated, 48 h post- trol Fe signaling and defense signaling in rice. At the cellu- infection [hpi]) were stained with Prussian blue dye ac- lar level, M. oryzae infection altered Fe distribution in rice cording to [83] with some modifications. Briefly, rice leaves, a process probably involving miR7695. Because leaves were vacuum-infiltrated in a fixing solution (chloro- miRNAs function as fine-tuners of gene expression in- form:methanol:glacial acetic acid; 6:3:1, v/v) for 1 h and in- stead of turning-on or turning-off target gene expression, cubated overnight at room temperature. After washing miR7695 would be well suited to maintain appropriate Fe with distilled water (three times), samples were vacuum- levels in host cells during pathogen infection. If so, infiltrated with a pre-warmed (37 °C) staining solution (4% miR7695 might well be involved in modulation of iron ac- HCl and 4% K-ferrocyanide at equal volumes) for 1 h, in- cumulation in tissues of the rice leaf which, in turn, would cubated 1 h more at 37 °C in the same solution without affect the expression of Fe homeostasis genes. The current vaccuum and washed three times with distilled water challenge of basic and applied plant research is to under- (Perls staining). For DAB intensification reaction, samples stand interconnected regulations between miR7695- were incubated in a methanol solution (0.01 M NaN, 0.3% mediated mechanisms involved in Fe homeostasis and dis- [v/v] H2O) for 1 h, washed with 0.1 M phosphate buffer ease resistance in plants. Deciphering the mechanisms pH 7.2, then incubated with the intensification solution involved in Fe distribution and remobilization during M. (0.025% [w/v] DAB [Sigma], 0.005% [v/v] H2O in 0.1 M oryzae infection with the participation of miR7695 will phosphate buffer, pH 7.2) for 15 min. The reaction was help in designing innovative strategies for blast disease stopped by washing with distilled water. Leaves were control. Knowing how plants integrate immune responses mounted in glycerol 50% in glass slides and observed and Fe signaling pathways is an issue of great importance under a microscope (AixoPhot DP70 under with light). in both basic and applied plant research. Chlorophyll content Methods The mean of 10 readings from the chlorophyll meter Plant material, growth conditions and genotyping (SPAD 502 Plus Chlorophyll Meter, Spectrum Tech- Rice plants were grown at 28 °C with a 14 h/10 h light/ nologies) was obtained from the third leaf of rice plants dark cycle. The T-DNA insertion line for MIR7695 (O. grown in different Fe concentrations. The measurement sativa cv. Tainung67, japonica) was obtained from the was taken at the same position in all leaves. Taiwan Rice Insertion Mutant (TRIM) collection from the Academia Sinica of Taiwan ([49]; http://trim.sinica. Blast resistance assays edu.tw), and propagated under controlled conditions The fungus M. oryzae (strain Guy-11, courtesy of Ane (CRAG greenhouse Service). For genotyping, genomic Sema) was grown in Complete Media Agar (CMA, 9 cm DNA was extracted as described [81] but with mixed plates, containing 30 mg/L chloramphenicol) for 15 days alkyltri-methylammoniumbromide (MATAB) used as at 28 °C under a 16 h/8 h light/dark photoperiod condi- the extraction buffer (0.1 M Tris–HCl pH 8.0, 1.4 M tion. M. oryzae spores were prepared as previously de- NaCl, 20 mM EDTA, 2% MATAB, 1% PEG 6000, 0.5% scribed [41]. Soil-grown plants (3–4 leaf stage) were sodium sulphite). PCR genotyping (100 ng DNA/PCR re- infected by two different methods, 1) whole-plant spray action) involved specific primers (P1 and P3) and T- inoculation assays [84], and 2) drop inoculation on de- DNA–specific primers (P2) (Additional file 2: Table S1). tached leaves [85]. Briefly, the spray inoculation method T-DNA copy number was estimated as described [82]. consisted of spraying whole rice plants with a M. oryzae Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 13 of 16 spore suspension (105 spores/ml; 0.2 ml/plant) by using the whole-plant infection method (105 spores/ml, 0.2 ml/ an aerograph at 2 atm of pressure. Plants were main- plant, at 48 hpi) with the Maxwell 16 LEV Plant RNA tained overnight in the dark under high humidity. For Kit (Promega). Raw reads were checked for quality by the drop inoculation method, the second detached leaf using FastQC v0.11.3 (www.bioinformatics.babraham.ac. was placed into square plate dishes (12 leaves/plate) with uk/projects/fastqc/) was used to check quality of raw 1% (w/v) water agar containing kinetin (2 mg/l). Then, reads; adapters were trimmed and removed with Trim- Whatman filter paper discs saturated with a M. oryzae momatic v0.33 [87] (minimum quality score 35, mini- spore suspension (104–106 spores/ml) were placed onto mum length 25). Reads obtained were mapped to the the upper face of the leaf for 60 h. The percentage of leaf reference rice genome (MSU 7.0) provided with the ref- area affected by blast lesions was determined at 4 days erence gene annotation file (RGSP 7.0) by using STAR (drop-inoculated leaves) or 7 days (spray-inoculated leaves) (v2.4.0j) [88]). Reads with mapping quality (MAPQ) < 30 post-inoculation with M. oryzae spores by using the APS were removed. FeatureCounts (v1.4.5-p1) [89] was used Assess 2.0 program [86]. to perform read summarization at the gene level, with the strand-specific option “reversely stranded”. Statistical Expression analysis analysis of read counts was performed with R, with the Total RNA was extracted from plant tissues by using TRi- HTSFilter package [90] to remove low-expressed genes zol reagent (Invitrogen). For northern blot analysis of rice and the edge R package [91] for differential expression miRNAs, RNAs were fractionated in a 17.5% denaturing analysis. To identify genes with significant difference in polyacrylamide gel containing 8 M urea, transferred to expression, a FDR cutoff < 0.05 and log2FC 1 ≤ or ≥ 1 nylon membranes and probed with a γ 32P-ATP end- was applied. Gene Ontology (GO) enrichment of differ- labeled miR7695.3-3p oligonucleotide (Additional file 2: entially expressed genes involved Singular Enrichement Table S1). Blots were pre-hybridized and hybridized in analysis (SEA) using the AgriGO webtool (p < 0.01 Fish- Perfect-Hyb Plus buffer (Sigma) at 42 °C. Hybridization sig- er’s test, TIGR genemodel) (http://bioinfo.cau.edu.cn/ nals were detected by using STORM Phosphorimager (GE agriGO/) [92]. Enriched GO terms were grouped, sum- Healthcare). marized and 2D-plotted by semantic clustering with the For quantitative RT-PCR (RT-qPCR), the first comple- online analysis tool ReviGO (http://revigo.irb.hr/) [93]. mentary DNA was synthesized from DNase-treated total RNA (1 μg) with High Capacity cDNA Reverse Transcrip- tion (Life technology, Applied Biosystems). Amplification Quantification of Rice Diterpene Phytoalexins involved 2 μl cDNA (5 ng/μl) in optical 96-well plates Leaf segments were collected from mock and M. oryzae-in- (Roche Light Cycler 480; Roche Diagnostics, Mannheim, fected plants. Three biological replicates with two technical Germany) with SYBR Green I dye and gene-specific replicates each were performed. Approximately 200–300 primers (Additional file 2: Table S1). The Ubiquitin1 gene mg of fresh plant material was soaked in 40 vol of 70% (Os06g0681400) was used to normalize transcript levels. methanol and incubated at 4 °C overnight with constant ro- tation. A 1 ml aliquot was centrifuged at maximum speed 5′-RLM-race to remove cell debris. Phytoalexins were quantified using 5′ RNA ligase-mediated rapid amplification of cDNA 5 μl of the extract by LC-MS/MS as described [94]. Signifi- ends (5′ -RLM-RACE) was done using a GeneRacer™ kit cant differences in phytoalexin accumulation were evalu- according to the manufacturer’s instructions (Invitrogen, ated with ANOVA. CA) but omitting the dephosphorylation and decapping steps. Briefly, 3 μg of DNAse-treated total RNA was li- gated to a GeneRacer Oligo RNA Adapter. First-strand Supplementary information Supplementary information accompanies this paper at https://doi.org/10. cDNA was synthesized using oligo-dT. Specific primers 1186/s12870-019-2156-5. were used to amplify 5′ ends by nested PCR from cDNA (Additional file 2: Table S1). The nested PCR products Additional file 1: Figure S1. Characterization and phenotype of were separated on a 2% agarose gel, gel purified, ligated MIR7695-Ac and wild-type azygous (WT-Az) plants. to a Zero Blunt TOPO vector (Invitrogen, CA) trans- Additional file 2: Table S1. Oligonucleotides used in this study formed into Topo 10 cells and sequenced to determine Additional file 3: Table S2. qPCR of T-DNA copy number in MIR7695- Ac mutant plants with the sucrose phosphate synthase (SPS) gene as an the cleavage site in target genes. Specific control were endogenous reference. done using the specific primers. Additional file 4: Figure S2.Resistance of MIR7695-Ac mutant plants to M. oryzae infection. RNA-seq library sample preparation and sequencing Additional file 5: Figure S3. Differentially expressed genes (DEGs) in Total RNA was extracted from rice leaves that had been leaves of MIR7695-Ac mutant plants relative to WT-Az plants, under non- infection or infection. treated or not with a M. oryzae spore solution following Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 14 of 16 Additional file 6: Table S3. Differentially expressed genes (DEGs) in Author details 1 MIR7695-Ac plants relative to wild-type azygous (WT-Az) plants (MIR7695- Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Ac vs WT-Az). Campus Universitat Autònoma de Barcelona (UAB), Bellaterra (Cerdanyola del Vallés), Barcelona, Spain. 2Biotechnology Research Center, The University of Additional file 7: Table S4. Gene Ontology terms enriched in MIR7695- Tokyo, Tokyo, Japan. 3Institute of Plant and Microrbial Biology, Academia Ac versus WT-Az plants according to molecular function. Sinica, Taipei, Taiwan. 4Consejo Superior de Investigaciones Científicas (CSIC), Additional file 8: Table S5. DEGs in MIR7695-Ac versus WT-Az plants. Barcelona, Spain. Additional file 9: Figure S4. Validation of RNAseq data by qRT-PCR. Received: 26 February 2019 Accepted: 21 November 2019 Additional file 10: Table S6. DEGs in WT plants after infection with M. oryzae (48 h post-infection [hpi]) (INF-WT-Az vs. WT-Az). Additional file 11: Table S7. DEGs in MIR7695-Ac plants after infection with M. oryzae (48 hpi) (INF-MIR7695-Ac vs. MIR7695-Ac). References 1. Jones JDG, Dangl JL. The plant immune system. Nature. 2006;444:323–9. Additional file 12: Table S8. Pathogenesis-related (PR) gene expression 2. Doughari J. An overview of plant immunity. J Plant Pathol Microbiol. in WT-Az and MIR7695-Ac plants during M. oryzae infection at 48 hpi. 2015;6:1–11. Additional file 13: Table S9. DEGs in bifactorial analysis. 3. Boller T, Felix G. A renaissance of elicitors: perception of microbe-associated Additional file 14: Table S10. DEGs in the top GO categories that are molecular patterns and danger signals by pattern-recognition receptors. overrepresented in MIR7695-Ac with M. oryzae infection (bifactorial Annu Rev Plant Biol. 2009;60:379–406. analysis). 4. Macho AP, Zipfel C. Plant PRRs and the activation of innate immune signaling. Mol Cell. 2014;54:263–72. Additional file 15: Figure S5. Pathways for the biosynthesis of 5. Cui H, Tsuda K, Parker JE. Effector-triggered immunity: from pathogen phenylpropanoids and diterpenoid phytoalexins in rice. perception to robust defense. Annu Rev Plant Biol. 2015;66:487–511. Additional file 16: Figure S6. RT-qPCR analysis of expression pattern of 6. Li Y, Zhang Q, Zhang J, Wu L, Qi Y, Zhou J-M. Identification of MicroRNAs Fe homeostasis genes in WT-Az and MIR7695-Ac plants with M. oryzae involved in pathogen-associated molecular pattern-triggered plant innate infection. immunity. Plant Physiol. 2010;152:2222–31. 7. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, et al. A plant miRNA contributes to antibacterial resistance by repressing Auxin signaling. Abbreviations Science. 2006;312:436–9. DEG: Differentially Expressed Gene; Fe: Iron; GO: Gene Ontology; LTP: Lipid 8. Shivaprasad PV, Chen H-M, Patel K, Bond DM, Santos BACM, Baulcombe DC. Transfer protein; miR: MicroRNA; Nramp6: Natural Resistance-Associated A MicroRNA superfamily regulates nucleotide binding site-Leucine-rich Macrophage Pathogen 6; PR: Pathogenesis-Related; R: Resistance; repeats and other mRNAs. Plant Cell. 2012;24:859–74. ROS: Reactive Oxygen Species; TF: Transcription Factor 9. Boccara M, Sarazin A, Thiébeauld O, Jay F, Voinnet O, Navarro L, et al. The Arabidopsis miR472-RDR6 silencing pathway modulates PAMP- and Acknowledgements effector-triggered immunity through the post-transcriptional control of Not applicable disease resistance genes. PLoS Pathog. 2014;10:e1003883. 10. Liu J, Cheng X, Liu D, Xu W, Wise R, Shen Q-H. The miR9863 family Authors’ contributions regulates distinct Mla alleles in barley to attenuate NLR receptor-triggered Conceived and designed experiments: SC, BSS, FSS, KO, YIH. Performed disease resistance and cell-death signaling. PLoS Genet. 2014;10:e1004755. experiments: FSS, CPP, ST, SC; Analysed data: SC, FSS, BSS, KO, YIH. Wrote and 11. Li F, Pignatta D, Bendix C, Brunkard JO, Cohn MM, Tung J, et al. MicroRNA revised manuscript: SC, BSS, KO, YIH. All authors read and approved the final regulation of plant innate immune receptors. Proc Natl Acad Sci U S A. manuscript. 2012;109:1790–5. 12. Jones-Rhoades M, Bartel D, Bartel B. MicroRNAs and their regulatory roles in Funding plants. Annu Rev Plant Biol. 2006;57:19–53. This work was supported by the Spanish Ministry of Economy and 13. de Lima JC, Loss-Morais G, Margis R. MicroRNAs play critical roles during Competitiveness (MINECO, BIO2015–67212-R) to BSS, the CSIC/NSC (Spanish plant development and in response to abiotic stresses. Genet Mol Biol. Research Council/National Science Council of Taiwan)-Formosa Program 2012;35:1069–77. (2009TW0041 and NSC 99–2923-B-001-002-MY3), the Academia Sinica 14. Li C, Zhang B. MicroRNAs in control of plant development. J Cell Physiol. Summit Project to YICH, and the Japan Society for the Promotion of Science 2016;231:303–13. (JSPS KAKENHI, 17H03811 to KO). The funders have no role in the design of 15. Baldrich P, San SB. MicroRNAs in Rice innate immunity. Rice. 2016;9:1–6. the study; collection, analysis and interpretation of data; and in writing the 16. Chen X. A MicroRNA as a translational repressor of APETALA2 in Arabidopsis manuscript. We acknowledge support from the CERCA Programme flower development. Science. 2004;303:2022–5. (“Generalitat de Catalunya”) and the “Severo Ochoa Programme for Centres 17. Paul S, Datta SK, Datta K. miRNA regulation of nutrient homeostasis in of Excellence in R&D” (MINECO, 2016–2019, SEV-2015-0533). plants. Front Plant Sci. 2015;6:232. 18. Chiou T-J, Aung K, Lin S-I, Wu C-C, Chiang S-F, Su C-L. Regulation of phosphate homeostasis by MicroRNA in Arabidopsis. Plant Cell. 2006; Availability of data and materials 18:412–21. The RNA sequence datasets generated during the current study will be 19. Liang G, Yang F, Yu D. MicroRNA395 mediates regulation of sulfate available after May 2020 at the National Center for Biotechnology accumulation and allocation in Arabidopsis thaliana. Plant J. 2010;62:1046–57. Information (NCBI) Gene Expression Omnibus (GEO) with the GSE122258 20. Abadía J. Leaf responses to Fe deficiency: a review. J Plant Nutr. 1992;15: accession number (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= 1699–713. GSE122258). Until this date, the datasets are available from the 21. Naranjo-Arcos MA, Bauer P. Iron Nutrition, Oxidative Stress, and Pathogen corresponding author on reasonable request. Defense. In: Erkekoglu P, Kocer-Gumusel B, editors. Nutritional Deficiency. Rijeka: InTechOpen; 2016. p. 63–98. Ethics approval and consent to participate 22. Dangol S, Chen Y, Hwang BK, Jwa N-S. Iron- and reactive oxygen species- Not applicable dependent Ferroptotic cell death in Rice-Magnaporthe oryzae interactions. Plant Cell. 2019;31:189–209. Consent for publication 23. Torres MA. ROS in biotic interactions. Physiol Plant. 2010;138:414–29. Not applicable 24. Huang H, Ullah F, Zhou D-X, Yi M, Zhao Y. Mechanisms of ROS regulation of plant development and stress responses. Front Plant Sci. 2019;10:800. Competing interests 25. Marcec MJ, Gilroy S, Poovaiah BW, Tanaka K. Mutual interplay of Ca2+ and The authors declare that they have no competing interests. ROS signaling in plant immune response. Plant Sci. 2019;283:343–54. Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 15 of 16 26. Waters BM, McInturf SA, Stein RJ. Rosette iron deficiency transcript and 51. Lo S-F, Fan M-J, Hsing Y-I, Chen L-J, Chen S, Wen I-C, et al. Genetic microRNA profiling reveals links between copper and iron homeostasis in resources offer efficient tools for rice functional genomics research. Plant Arabidopsis thaliana. J Exp Bot. 2012;63:5903–18. Cell Environ. 2016;39:998–1013. 27. Kong WW, Yang ZM. Identification of iron-deficiency responsive microRNA 52. Agrawal GK, Rakwal R, Jwa NS, Agrawal VP. Signalling molecules and blast genes and cis-elements in Arabidopsis. Plant Physiol Biochem. 2010;48:153–9. pathogen attack activates rice OsPR1a and OsPR1b genes: a model 28. Valdés-López O, Yang SS, Aparicio-Fabre R, Graham PH, Reyes JL, Vance CP, illustrating components participating during defence/stress response. Plant et al. MicroRNA expression profile in common bean (Phaseolus vulgaris) Physiol Biochem. 2001;39:1095–103. under nutrient deficiency stresses and manganese toxicity. New Phytol. 53. Wei T, Ou B, Li J, Zhao Y, Guo D, Zhu Y, et al. Transcriptional profiling of 2010;187:805–18. Rice early response to Magnaporthe oryzae identified OsWRKYs as important 29. Agarwal S, Mangrauthia SK, Sarla N. Expression profiling of iron deficiency regulators in Rice blast resistance. PLoS One. 2013;8:e59720. responsive microRNAs and gene targets in rice seedlings of Madhukar x 54. Shimono M, Koga H, Akagi A, Hayashi N, Goto S, Sawada M, et al. Rice Swarna recombinant inbred lines with contrasting levels of iron in seeds. WRKY45 plays important roles in fungal and bacterial disease resistance. Mol Plant Soil. 2015;396:137–50. Plant Pathol. 2012;13:83–94. 30. Paul S, Gayen D, Datta SK, Datta K. Analysis of high iron rice lines reveals new 55. Kaneda T, Taga Y, Takai R, Iwano M, Matsui H, Takayama S, et al. The miRNAs that target iron transporters in roots. J Exp Bot. 2016;67:5811–24. transcription factor OsNAC4 is a key positive regulator of plant 31. Yu J, Hu S, Wang J, Wong GK-S, Li S, Liu B, et al. A draft sequence of the hypersensitive cell death. EMBO J. 2009;28:926–36. Rice genome (Oryza sativa L. ssp. indica). Science. 2002;296:79–92. 56. Miyamoto K, Shimizu T, Mochizuki S, Nishizawa Y, Minami E, Nojiri H, et al. 32. Goff SA, Ricke D, Lan T-H, Presting G, Wang R, Dunn M, et al. A draft sequence Stress-induced expression of the transcription factor RERJ1 is tightly of the Rice genome (Oryza sativa L. ssp. japonica). Science. 2002;296:92–100. regulated in response to jasmonic acid accumulation in rice. Protoplasma. 33. Wilson RA, Talbot NJ. Under pressure: investigating the biology of plant 2013;250:241–9. infection by Magnaporthe oryzae. Nat Rev Microbiol. 2009;7:185–95. 57. Inoue H, Higuchi K, Takahashi M, Nakanishi H, Mori S, Nishizawa NK. Three 34. Miura K, Ikeda M, Matsubara A, Song X-J, Ito M, Asano K, et al. OsSPL14 rice nicotianamine synthase genes, OsNAS1, OsNAS2, and OsNAS3 are promotes panicle branching and higher grain productivity in rice. Nat expressed in cells involved in long-distance transport of iron and Genet. 2010;42:545–9. differentially regulated by iron. Plant J. 2003;36:366–81. 35. Wang S, Wu K, Yuan Q, Liu X, Liu Z, Lin X, et al. Control of grain size, shape 58. Ali S, Ganai BA, Kamili AN, Bhat AA, Mir ZA, Bhat JA, et al. Pathogenesis- and quality by OsSPL16 in rice. Nat Genet. 2012;44:950–4. related proteins and peptides as promising tools for engineering plants 36. Zhang Y-C, Yu Y, Wang C-Y, Li Z-Y, Liu Q, Xu J, et al. Overexpression of with multiple stress tolerance. Microbiol Res. 2018;212–213:29–37. microRNA OsmiR397 improves rice yield by increasing grain size and 59. Galeotti F, Barile E, Curir P, Dolci M, Lanzotti V. Flavonoids from carnation promoting panicle branching. Nat Biotechnol. 2013;31:848–52. (Dianthus caryophyllus) and their antifungal activity. Phytochem Lett. 2008;1:44–8. 37. Fang Y, Xie K, Xiong L. Conserved miR164-targeted NAC genes negatively 60. Miedes E, Vanholme R, Boerjan W, Molina A. The role of the secondary cell regulate drought resistance in rice. J Exp Bot. 2014;65:2119–35. wall in plant resistance to pathogens. Front Plant Sci. 2014;5:358. 38. Yang W, Fan T, Hu X, Cheng T, Zhang M. Overexpressing Osa-miR171c 61. Kawahara Y, Oono Y, Kanamori H, Matsumoto T, Itoh T, Minami E, et al. decreases salt stress tolerance in rice. J Plant Biol. 2017;60:485–92. Simultaneous RNA-Seq analysis of a mixed Transcriptome of Rice and blast 39. Wang S, Sun X, Hoshino Y, Yu Y, Jia B, Sun Z, et al. MicroRNA319 positively fungus interaction. PLoS One. 2012;7:e49423. regulates cold tolerance by targeting OsPCF6 and OsTCP21 in Rice (Oryza 62. Wang Y, Kwon SJ, Wu J, Choi J, Lee Y-H, Agrawal GK, et al. Transcriptome sativa L.). PLoS One. 2014;9:e91357. analysis of early responsive genes in Rice during Magnaporthe oryzae 40. Baldrich P, Campo S, Wu M-T, Liu T-T, Hsing Y-IC, Segundo BS. MicroRNA- infection. Plant Pathol J. 2014;30:343–54. mediated regulation of gene expression in the response of rice plants to 63. Huang L-F, Lin K-H, He S-L, Chen J-L, Jiang J-Z, Chen B-H, et al. Multiple fungal elicitors. RNA Biol. 2015;12:847–63. patterns of regulation and overexpression of a Ribonuclease-like 41. Campo S, Peris-Peris C, Siré C, Moreno AB, Donaire L, Zytnicki M, et al. pathogenesis-related protein gene, OsPR10a, Conferring Disease Resistance Identification of a novel microRNA (miRNA) from rice that targets an in Rice and Arabidopsis. PLoS One. 2016;11:e0156414. alternatively spliced transcript of the Nramp6 (Natural resistance-associated 64. Pereira MP, Santos C, Gomes A, Vasconcelos MW. Cultivar variability of iron macrophage protein 6) gene involved in pathogen resistance. New Phytol. uptake mechanisms in rice (Oryza sativa L.). Plant Physiol Biochem. 2014;85:21–30. 2013;199:212–27. 65. Kobayashi T, Nishizawa NK. Iron uptake, translocation, and regulation in 42. Li Y, Lu Y-G, Shi Y, Wu L, Xu Y-J, Huang F, et al. Multiple rice microRNAs are higher plants. Annu Rev Plant Biol. 2012;63:131–52. involved in immunity against the blast fungus Magnaporthe oryzae. Plant 66. Ahuja I, Kissen R, Bones AM. Phytoalexins in defense against pathogens. Physiol. 2014;164:1077–92. Trends Plant Sci. 2012;17:73–90. 43. Li Z-Y, Xia J, Chen Z, Yu Y, Li Q-F, Zhang Y-C, et al. Large-scale rewiring of 67. Hasegawa M, Mitsuhara I, Seo S, Imai T, Koga J, Okada K, et al. Phytoalexin innate immunity circuitry and microRNA regulation during initial rice blast accumulation in the interaction between Rice and the blast fungus. Mol infection. Sci Rep. 2016;6:25493. Plant-Microbe Interact. 2010;23:1000–11. 44. Zhang X, Bao Y, Shan D, Wang Z, Song X, Wang Z, et al. Magnaporthe 68. Umemura K, Ogawa N, Shimura M, Koga J, Usami H, Kono T. Possible role of oryzae induces the expression of a MicroRNA to suppress the immune Phytocassane, Rice Phytoalexin, in disease resistance of Rice against the blast response in Rice. Plant Physiol. 2018;177:352–68. fungus Magnaporthe grisea. Biosci Biotechnol Biochem. 2003;67:899–902. 45. Salvador-Guirao R, Hsing Y-I, San SB. The Polycistronic miR166k-166h 69. Dillon VM, Overton J, Grayer RJ, Harborne JB. Differences in phytoalexin positively regulates Rice immunity via post-transcriptional control of EIN2. response among rice cultivars of different resistance to blast. Front Plant Sci. 2018;9:337. Phytochemistry. 1997;44:599–603. 46. Li Y, Zhao S-L, Li J-L, Hu X-H, Wang H, Cao X-L, et al. Osa-miR169 negatively 70. Hood MI, Skaar EP. Nutritional immunity: transition metals at the pathogen- regulates Rice immunity against the blast fungus Magnaporthe oryzae. Front host interface. Nat Rev Microbiol. 2012;10:525–37. Plant Sci. 2017;8:2. 71. Pereira EG, Oliva MA, Rosado-Souza L, Mendes GC, Colares DS, Stopato CH, 47. Wang Z, Xia Y, Lin S, Wang Y, Guo B, Song X, et al. Osa-miR164a targets et al. Iron excess affects rice photosynthesis through stomatal and non- OsNAC60 and negatively regulates rice immunity against the blast fungus stomatal limitations. Plant Sci. 2013;201–202:81–92. Magnaporthe oryzae. Plant J. 2018;95:584–97. 72. Liu G, Greenshields DL, Sammynaiken R, Hirji RN, Selvaraj G, Wei Y. Targeted 48. Peris-Peris C, Serra-Cardona A, Campo S, Ariño J. Two NRAMP6 isoforms alterations in iron homeostasis underlie plant defense responses. J Cell Sci. function as iron and manganese transporters and contribute to disease 2007;120:596. resistance in rice. Mol Plant-Microbe Interact. 2017;30:385–98. 73. Aznar A, Patrit O, Berger A, Dellagi A. Alterations of iron distribution in 49. Hsing YI, Chern CG, Fan MJ, Lu PC, Chen KT, Lo SF, et al. A rice gene Arabidopsis tissues infected by Dickeya dadantii. Mol Plant Pathol. 2015; activation/knockout mutant resource for high throughput functional 16:521–8. genomics. Plant Mol Biol. 2007;63:351–64. 74. Ricachenevsky FK, Sperotto RA. There and back again, or always there? The 50. Wu H-P, Wei F-J, Wu C-C, Lo S-F, Chen L-J, Fan M-J, et al. Large-scale evolution of rice combined strategy for Fe uptake. Front Plant Sci. 2014;5:189. phenomics analysis of a T-DNA tagged mutant population. Gigascience. 75. Conrath U, Beckers GJM, Langenbach CJG, Jaskiewicz MR. Priming for 2017;6:1–7. enhanced defense. Annu Rev Phytopathol. 2015;53:97–119. Sánchez-Sanuy et al. BMC Plant Biology (2019) 19:563 Page 16 of 16 76. Selitrennikoff CP. Antifungal proteins. Appl Environ Microbiol. 2001;67:2883–94. 77. Almagro L, Gómez Ros LV, Belchi-Navarro S, Bru R, Ros Barceló A, Pedreño MA. Class III peroxidases in plant defence reactions. J Exp Bot. 2009;60:377–90. 78. Cho M-H, Lee S-W. Phenolic Phytoalexins in Rice: biological functions and biosynthesis. Int J Mol Sci. 2015;16:29120. 79. Dixon RA, Achnine L, Kota P, Liu C-J, Reddy MSS, Wang L. The phenylpropanoid pathway and plant defence-a genomics perspective. Mol Plant Pathol. 2002;3:371–90. 80. Camargo-Ramírez R, Val-Torregrosa B, San SB. MiR858-mediated regulation of flavonoid-specific MYB transcription factor genes controls resistance to pathogen infection in Arabidopsis. Plant Cell Physiol. 2018;59:190–204. 81. Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980;8:4321–6. 82. Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, et al. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep. 2005;23:759–63. 83. Roschzttardtz H, Conejero G, Curie C, Mari S. Identification of the endodermal vacuole as the Iron storage compartment in the Arabidopsis embryo. Plant Physiol. 2009;151:1329–38. 84. Sesma A, Osbourn AE. The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi. Nature. 2004;431:582. 85. Coca M, Bortolotti C, Rufat M, Peñas G, Eritja R, Tharreau D, et al. Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast gungus Magnaporthe grisea. Plant Mol Biol. 2004;54:245–59. 86. Lamari L. Assess 2.0: image analysis software for plant disease quantification. St. Paul, MN: APS Press; 2008. 87. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20. 88. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. 89. Liao Y, Smyth GK, Shi W. FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30. 90. Rau A, Gallopin M, Celeux G, Jaffrézic F. Data-based filtering for replicated high-throughput transcriptome sequencing experiments. Bioinformatics. 2013;29:2146–52. 91. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009;26:139–40. 92. Du Z, Zhou X, Ling Y, Zhang ZH, Su Z. AgriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res. 2010;38:W64–70. 93. Supek F, Bosnjak M, Skunca N, Smuc T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One. 2011;6:1–9. 94. Miyamoto K, Fujita M, Shenton MR, Akashi S, Sugawara C, Sakai A, et al. Evolutionary trajectory of phytoalexin biosynthetic gene clusters in rice. Plant J. 2016;87:293–304. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.