Treatment with High Fe Enhances Resistance to M. oryzae Infection in Rice Plants
In this work, we investigated the effect of treatment with high Fe on resistance to infection by M. oryzae in rice plants. Rice (O. sativa cv. Nipponbare) plants were grown in soil under sufficient Fe supply (50 µM Fe) for 16 days. Then, half of the plants were supplied with 1 mM Fe while the other half of the plants continued growth under 50 µM Fe condition (henceforth High-Fe and Control Fe plants, respectively). Treatment with high Fe was carried out for 5 days (Fig. 1A). No phenotypic differences were observed between Control and High-Fe plants after 5 days of Fe treatment (Fig. 1B), these plants showing similar shoot biomass, root biomass and chlorophyll content (Fig. 1C). High-Fe plants had a higher content of Fe in root, stem and leaf tissues compared with Control Fe plants (Fig. 1D). Under the same experimental conditions, cupper, manganese or calcium levels were not significantly altered in High-Fe plants compared with Control-Fe plants (Additional file 1: Fig. S1A). When using a longer period of Fe treatment (e.g. 19 days of treatment with 1 mM Fe), plants developed symptoms of Fe toxicity in their leaves, these plants also showing a significant reduction in root and shoot biomass and chlorophyll content (Additional file 1: Fig. S1B, C). To avoid that toxic effects caused by Fe accumulation could confound our results on disease resistance, in this study we used a short-term Fe treatment (5 days of treatment with 1 mM Fe), followed by a M. oryzae infection for 7 days (Fig. 1A).
Most importantly, upon infection with M. oryzae, High-Fe plants consistently exhibited resistance to infection as revealed by visual inspection of disease symptoms, quantification of diseased leaf area and measurement of fungal biomass in leaves of pathogen-infected plants (Fig. 1E). These results were substantially similar to those previous reported in hydroponically-grown rice plants (Peris-Peris et al. 2017) supporting that treatment with high Fe increases resistance to infection by the rice blast fungus in rice.
Transcriptional Responses of Rice Leaves to Treatment with High Fe
Transcriptional changes occurring in rice leaves in response to treatment with high Fe were examined. For this, RNA-Seq analysis was carried out in leaves from High-Fe and Control plants. Differentially expressed genes (DEGs) were identified based on significance level (FDR ≤ 0.05) and log2 fold change (FC) with a threshold of FC ≥ + 0.5 and FC ≤ − 0.5 for up-regulated and down-regulated genes, respectively.
When comparing leaf transcriptomes of High-Fe and Control plants, only 239 genes were found to be regulated by treatment with high Fe (48 genes upregulated; 191 genes down-regulated) (Fig. 2A, Additional file 2: Table S1a). Genes whose expression is known to be induced by Fe deficiency where found to be repressed in leaves of High-Fe plants relative to Control plants (Fig. 2B, Additional file 2: Table S1b) which is consistent with an increase in Fe content in these plants (see Fig. 1C). Down-regulated genes in High-Fe plants included: Iron deficiency-inducible peptide-IRON MAN (OsIMA1, OsIMA2), Iron-related transcription factors (OsIRO2, OsIRO3) and Iron-binding Haemerythrin RING ubiquitin ligases (OsHRZ1, OsHRZ2). Other genes down-regulated in leaves of High-Fe rice plants were those encoding plasma membrane iron transporters, such as Iron-deficiency-regulated oligopeptide transporter 7 (OsOPT7), Natural Resistance-Associated Macrophage Protein 1 (OsNRAMP1), Vacuolar Membrane Ferric-Chelate Reductase (OsFRO1), and Vacuolar mugineic acid transporter (OsVMT) (Fig. 2B, Additional file 2: Table S1a). OsFER2 (FERRITIN 2) was up-regulated in leaves of High-Fe plants (Fig. 2B). As previously mentioned, Ferritins bind Fe (mainly in the form of Fe3+), thus, contributing to protection of plants against Fe-mediated oxidative stress.
Transcriptome analysis also indicated that treatment with high Fe results in alterations in the expression of genes involved in ROS metabolism, in particular peroxidase (prx) genes (Fig. 2B, Additional file 2: Table S1a). Compared with Control plants, the expression of prx genes was either up-regulated (OsPrx62, OsPOX22.3) or down-regulated (OsPrx11, OsPrx15, OsPrx16, OsPrx38, OsPrx81, OsPrx83, OsPrx89) in High-Fe plants. Peroxidases participate in a broad range of processes, such as cross-linking of cell wall components, synthesis of phytoalexins, and ROS production in plant defense responses. They catalyze oxidation of various substrates concomitant with the decomposition of H2O2. Among peroxidases, Class III peroxidases can either produce H2O2 in oxidase cycle or scavenge H2O2 to H2O and O2 through peroxidase activity. POX22.3 was previously identified among proteins accumulating in M. oryzae-infected rice leaves (Sun et al. 2004). A Metallothionein-like protein (OsMT3a), and a glycolipid transfer protein involved in ceramide transport (OsGLTP) were also up-regulated in High-Fe plants compared with Control plants (Fig. 2B). Metallothioneins are metal-binding proteins implicated in metal detoxification and scavenging of ROS (Hassinen et al. 2011), while ceramides regulate the Arabidopsis defense response by promoting H2O2 generation (Li et al. 2022).
Genes related to responses to biotic stress were also regulated by Fe treatment under non-infection conditions, including certain Pathogenesis-Related (PR) genes. Their expression was up-regulated (OsPR1b) or down-regulated (OsCht11 and OsChib3a encoding rice chitinases; Jasmonate-Inducible PR10, or JIOsPR10; and Germin-like Protein, or OsGLP genes) in High-Fe plants (Fig. 2B).
Results obtained by RNA-Seq analysis were validated by RT-qPCR for genes showing either down-regulation (OsIRO2, OsIRO3, OsNRAMP1, OsChib3a, OsJIPR10) or up-regulation (OsFER2, OsMT3a, OsPR1b) by treatment with high Fe (Fig. 2C).
Together, these findings indicated that treatment with Fe provokes transcriptional alterations in genes involved in iron homeostasis and defense responses in the absence of pathogen infection. Down regulation of defense-related genes by treatment with high Fe (e.g. PR genes) is in apparent contradiction with the phenotype of blast resistance that is observed in High-Fe plants. We then reasoned that blast resistance in rice plants that have been treated with high Fe might rely more on the stronger induction of defense responses upon pathogen infection rather than on constitutive expression of defense genes (see below).
Transcriptional Reprogramming in Leaves of High-Fe Rice Plants During M. oryzae Infection
To explore the possibility that treatment of rice plants with high Fe has an effect on the induction of defense genes during pathogen infection, we compared the leaf transcriptomes of Control and High-Fe plants, inoculated with M. oryzae spores or mock-inoculated (at 48 h post-inoculation, hpi). The same criteria described above were used to identify pathogen-regulated genes in each Fe condition (log2 FC ≥ 0.5 or ≤ − 0.5 for up-regulated and down-regulated genes, respectively; P ≤ 0.05). Pair-wise comparisons of DEGs (M. oryzae-inoculated vs mock-inoculated) revealed 5996 genes regulated by M. oryzae infection in Control Fe plants (2879 up-regulated; 3117 down-regulated) (Fig. 3A, Additional file 3: Table S2a). In High-Fe plants, 6470 genes were regulated by M. oryzae infection (3293 up-regulated; 3177 down-regulated) (Fig. 3A, Additional file 3: Table S2b). Most of the genes regulated by pathogen infection, either up-regulated or down-regulated genes, were shared between Control and High-Fe plants (Additional file 1: Fig. S2).
Gene ontology (GO) enrichment analysis was used to identify GO terms enriched in the set of pathogen-regulated genes in High-Fe or Control plants. Redundant GO terms were removed using the REVIGO tool (Supek et al. 2011; http://revigo.irb.hr). In Biological Processes, the most abundant subcategories were “Diterpene Phytoalexin Metabolism” followed by “Secondary Metabolism” and “Response to Biotic Stimulus/Defense Responses”, which were enriched in both High-Fe and Control-Fe plants (Fig. 3A, Additional file 4: Table S3a, b). The GO category of “Phosphorus Metabolism” was also enriched in the set of genes that are up-regulated by pathogen infection (Fig. 3A). Indeed, an enrichment of GO terms for “ATP binding” and “Protein kinase activity” annotations were found in the Molecular Function category, both in High-Fe and Control plants (Fig. 3B). Genes involved in “Iron binding” and “Signaling receptor activity” were also over-represented in the set of up-regulated genes by pathogen infection in both High-Fe and Control plants, while genes with “Chitinase activity” were over-represented in High-Fe plants, but not in Control plants (Fig. 3B). In this respect, transgenic expression of chitinase genes has long been recognized to confer resistance to fungal diseases in different plant species, including rice blast disease (Ali et al. 2018). As for genes that are down-regulated by M. oryzae infection, in Biological Processes, they categorized in “Photosynthesis” and “Pigment biosynthetic processes” in both High-Fe and Control plants (Additional file 1: Fig. S3A). In Molecular Function, GO terms for “Iron-sulfur cluster binding” and “Zinc ion binding” were represented in the group of down-regulated genes in Control and High-Fe plants (Additional file 1: Fig. S3B).
Taken together, GO enrichment analysis of genes regulated by M. oryzae infection indicated similar GO terms in High-Fe and Control rice plants, indicating that similar biological processes and mechanisms appear to be activated by pathogen infection in both conditions. Then, quantitative rather than qualitative differences in defense gene expression would explain the phenotype of blast resistance that is observed in High-Fe plants. Indeed, hierarchical clustering of DEGs expression levels in Control and High-Fe plants (infected and mock) revealed that the transcriptional response to M. oryzae infection in High-Fe plants was not qualitatively different from that in Control plants, but quantitatively different (Additional file 1: Fig. S4).
To further investigate differences in the response of Control and High-Fe plants to M. oryzae infection, we examined co-expression patterns. Genes up-regulated by M. oryzae infection classified into 6 hierarchical clusters (Fig. 4). GO terms in co-expressing genes identified terms related to plant defense in clusters I, II, IV, and V, whereas genes playing a role in protein phosphorylation grouped in cluster VI (Fig. 4). In particular, Cluster V contained the GO terms of “Diterpene phytoalexin biosynthetic process” and “Response to fungus/bacterium/oomycetes”. This analysis also revealed that High-Fe plants exhibited stronger induction in the expression of pathogen-responsive genes compared with Control plants (Fig. 4).
Enhanced Defense Responses of Iron-Treated Rice Plants Against the Blast Fungus
The induction of PR genes is regarded as a ubiquitous response to pathogen infection in plants. Presently, PR proteins are grouped into 17 families based on their protein sequence similarities and function (Ali et al. 2018). As expected, our RNASeq analysis revealed that M. oryzae infection activated the expression of an important number of PR genes (Fig. 5A, Additional file 5: Table S4). They included genes belonging to different PR families, such as PR1, PR2 (β-1,3 glucanases), PR3, PR4 and PR8 (chitinases), PR5 (thaumatin-like proteins, TLPs), PR6 (proteinase inhibitors), PR9 (peroxidases), PR10 (ribonuclease-like proteins), PR14 (lipid transfer proteins, LTPs) and PR15 (germin-like proteins) genes. Of them, OsPR1 and OsPBZ1 (PR10 family) are routinely used as marker genes for the induction of the rice response to M. oryzae infection (Midoh and Iwata 1996; Agrawal et al. 2001). Although PR gene expression was induced by M. oryzae infection in both High-Fe and Control plants, their expression was induced to a greater extent in High-Fe plants compared with Control plants (Fig. 5A, Additional file 5: Table S4). Here it is worth mentioning that transgenic expression of distinct PR genes, such as chitinase or PR10 genes, has proven to enhance resistance to M. oryzae infection (Wu et al. 2016; Ali et al. 2018).
RNA-Seq analysis also revealed regulation of an important number of peroxidase (Prx) genes during M. oryzae infection (PR9 family), these genes exhibiting stronger induction in High-Fe plants compared with Control plants (Fig. 5A, Additional file 5: Table S4). It is tempting to hypothesize that M. oryzae-regulated peroxidases might contribute to control the spatial and temporal accumulation of H2O2 in rice plants.
Among the genes regulated by both Fe treatment and M. oryzae infection there were several lipoxygenases (Fig. 5B). Lipoxygenases (LOXs) are crucial for lipid peroxidation processes during plant defense responses to pathogen infection. For instance, LOX are involved in the conversion of polyunsaturated fatty acids into oxylipins and jasmonates (Viswanath et al. 2020). Lipoxygenases have been also implicated in the initiation and execution of ferroptotic cell death in plants (Conrad et al. 2018).
Defensive functions of phenylpropanoid compounds have long been recognized, these metabolites conferring broad-spectrum disease resistance in different plant species (Yadav et al. 2020). To note, genes involved in the general phenylpropanoid pathway were found to be regulated by both iron treatment and M. oryzae infection, most of these genes showing a stronger response in High-Fe plants than in Control plants (Fig. 5B, Additional file 5: Table S4). A schematic representation of these genes in the rice phenylpropanoid pathway is presented in Additional file 1: Fig. S5. They included genes that participate in the general phenylpropanoid pathway (phenylalanine ammonia lyase, PAL; cinnamic acid 4-hydroxylase, C4H; 4-coumarate:CoA ligase, 4CL), as well as genes in branches leading to flavonols and anthocyanins (chalcone synthase, CHS; chalcone isomerase, CHI). The phenylpropanoid pathway also leads to the production of naringenin, the precursor of flavonols. Naringenin is also the precursor molecule for sakuranetin, the only flavonoid phytoalexin so far identified in rice (Hasegawa et al. 2014). Sakuranetin has been shown to accumulate in rice leaves during M. oryzae infection and to exhibit antifungal activity against M. oryzae (Hasegawa et al. 2014). We noticed that OsNOMT1 (Naringenin 7-O-methyltransferase), responsible of methylation of naringenin to sakuranetin (Shimizu et al. 2012) reached a higher expression in High-Fe compared with Control plants (Fig. 5B). RT-qPCR confirmed higher expression levels of defense-related genes in response to pathogen infection in High-Fe plants compared with infected Control plants (OsPR1a, OsPR1b, OsPBZ1, PR10, JIOsPR10, ROsPR10, OsLOX, OsLOX6, OsLOX8) (Fig. 5C).
Altogether, these results indicated that treatment with high Fe is associated with a stronger induction of defense gene expression during M. oryzae infection, which correlates well with resistance to M. oryzae in High-Fe plants.
Treatment with High Fe Promotes the Accumulation of Phytoalexins in Rice Leaves
Phytoalexins are low-molecular-weight antimicrobial compounds that are produced by plants in response to pathogen infection (Ahuja et al. 2012). Major phytoalexins in rice accumulating during M. oryzae infection are diterpene phytoalexins and the flavonoid phytoalexin sakuranetin (Okada et al. 2007; Hasegawa et al. 2010, 2014).
Diterpenoid phytoalexins are synthesized from geranylgeranyl diphosphate (GGDP), the end product of the methylerythritol phosphate (MEP) pathway (Fig. 6A). Rice produces a variety of diterpene phytoalexins, namely momilactones (A, B), phytocassanes (A to E), and oryzalexins (A to F, S). Our RNA-Seq analysis revealed that the expression of genes implicated in the MEP and diterpene phytoalexin biosynthesis pathways was induced to a greater extend in High-Fe plants than in Control plants (Fig. 6B, Additional file 6: Table S5). They included genes in the biosynthetic pathway leading to the production of oryzalexins (A–F), phytocassanes (A–E) and momilactones (A and B) whose expression was strongly induced by pathogen infection in High-Fe plants compared with Control plants (Fig. 6B, Additional file 6: Table S5). Results obtained by RNA-Seq analysis were confirmed by RT-qPCR analysis for selected genes (Additional file 1: Fig. S6). As previously mentioned, RNA-Seq analysis also revealed stronger induction of OsNOMT1 involved in sakuranetin (phenolic phytoalexin) biosynthesis in the response of High-Fe plants to M. oryzae infection compared with Control plants (Fig. 6, Additional file 6: Table S5).
Next, we investigated whether superinduction of phytoalexin biosynthesis genes in High-Fe plants during infection has an effect on phytoalexin content. As shown in Fig. 6C, M. oryzae infection is accompanied by the accumulation of diterpene phytoalexins (Momilactone A and B, Phytocassane B, C and E) in leaves of Control and High-Fe plants. Most importantly, their accumulation was found to be significantly higher in High-Fe plants than in Control plants (Fig. 6C). Equally, the phenolic phytoalexin Sakuranetin accumulated at a higher level during infection of High-Fe plants compared with Control plants (Fig. 6C). These results are in agreement with those obtained on the expression of phytoalexin biosynthetic genes indicating that treatment with high-Fe is associated to a higher accumulation of phytoalexins in rice plants, both diterpene and phenolic phytoalexins.
Phytoalexin content was also measured in rice plants grown under Fe limiting conditions. For this, the rice plants were grown for 16 days under control Fe conditions followed by 5 days under low Fe (Low-Fe) or Control Fe condition (Ctrl-Fe), and then inoculated with M. oryzae spores (Additional file 1: Fig. S7A, left panel). As expected, treatment with high Fe results in a decrease in total Fe content in rice tissues (Additional file 1: Fig. S7A, right panels) Under these experimental conditions, there were no statistically significant differences in phytoalexin content (momilactone A, momilactone B, phytocassane C, phytocassane B, phytocassane E sakuranetin) in M. oryzae-infected Low Fe plants compared with M. oryzae-infected Control plants (Additional file 1: Fig. S7B).
Knowing that rice phytoalexins have been described to exhibit antifungal activity against M. oryzae (Umemura et al. 2003; Hasegawa et al. 2010, 2014), it is tempting to hypothesize that blast resistance in High-Fe plants might result, as least in part, from the superactivation of phytoalexin biosynthesis genes, and subsequent accumulation of phytoalexins in these plants.
M. oryzae Infection Modulates Iron Accumulation in Rice Leaves
Comparative transcriptome analysis of Control and High-Fe plants revealed that, in addition to defense-related genes, M. oryzae infection has an impact on the expression of genes that function in iron homeostasis in leaves. Among them, there were genes that are down-regulated by Fe treatment (e.g. in the absence of pathogen infection) that are also down-regulated by pathogen infection in Control plants. This is the case of the transcription factors OsIRO2, OsIRO3, OsIMA1, OsIMA2, OsHRZ1, OsHRZ2, the OsOPT7 Oligopeptide transporter, and Ferric Reductase Oxidase 2 (OsFRO2) (Fig. 7A, Additional file 7: Table S6).
As chelators of Fe, rice plants produce phytosiderophores of the mugineic acid (MA) family, DMA (2-deoxymugineic acid) being the sole MA produced in rice. DMA is synthesized from S-adenosyl-methionine (SAM). To note, OsSAMS1, OsSAMS2 (encoding SAM synthases 1 and 2) responsible for the conversion of L-methionine to SAM were found to be strongly up-regulated by pathogen infection in High-Fe plants (Fig. 7A). A priori, the up-regulation of these genes would ensure a sufficient supply of SAM for DMA conversion. Other genes in the biosynthesis of DMA that are regulated by M. oryzae infection were OsNAAT1 (nicotianamine aminotransferase 1) and OsDMSA1 (deoxymugineic acid synthase 1). Phytosiderophore efflux transporter genes (OsTOM2, OsTOM3) were also up-regulated by infection, these genes being implicated in the transport of DMA out of the cell (Fig. 7A).
Regarding vacuolar iron transporters, we found that the Vacuolar Iron Transporter 1 (OsVIT1) involved in Fe sequestration into vacuoles, was repressed during infection in both Control and High-Fe plants. As for the Vacuolar Mugineic acid Transporter (OsVMT) responsible for sequestration of DMA into the vacuoles, its expression was down-regulated by infection only in Control plants (Fig. 7A). To note, M. oryzae infection strongly induced FERRITIN2 (FER2) in High-Fe plants (Fig. 7A). As previously mentioned, Ferritin is considered to be the major Fe-storage protein in plants.
Other Fe-related genes that are also regulated by pathogen infection were those involved in Fe transport, such as members of the NRAMP (Natural Resistance-Associated Macrophage Protein) family (OsNRAMP1, OsNRAMP3, OsNRAMP5, OsNRAMP6, OsNRAMP7), and the ZIP (Zn-regulated transporter, IRT-like protein) family (OsZIP2, OsZIP4, OsZIP8) (Fig. 7A). Additionally, up to 7 genes encoding Yellow Stripe-Like (YSL) transporters functioning in the import of Fe3+-DMA complexes into the plant cell were found to be regulated during M. oryzae infection in rice leaves. They were: OsYSL5, OsYSL12, OsYSL13, OsYSL16 (up-regulated), OsYSL6, OsYSL14 (down-regulated), and OsYSL9 (up-regulated in Control plants but down-regulated in High-Fe plants) (Fig. 7A). Changes in the expression of selected iron homeostasis-related genes in response to M. oryzae infection in High-Fe and Control plants were confirmed by RT-qPCR analysis (Additional file 1: Fig. S8).
Altogether, this study revealed that M. oryzae infection triggers alterations in the expression of genes with different functions in maintenance of iron homeostasis, including transcriptional regulators of Fe-responsive genes, Fe transporters, and Fe chelators. Presumably, these genes would be of importance in maintaining proper spatio-temporal accumulation of Fe in the host tissue during pathogen infection.
Having established that pathogen infection regulates the expression of genes involved in Fe homeostasis, we determined whether M. oryzae infection provokes alterations in Fe content in rice leaves. Total Fe content was determined by inductively coupled plasma-mass spectrometry (ICP-MS) in leaves of Control and High-Fe plants, that have been inoculated with M. oryzae spores or mock-inoculated. As expected, treatment with high Fe results in an increase in total Fe content (Fig. 7B, grey bars). Upon pathogen challenge, there was a significant increase in Fe content in leaves of Control plants, but its level was not significantly altered in leaves of High-Fe plants (a tendency to a lower content in response to M. oryzae infection could be, however, observed in High-Fe plants) (Fig. 7B).
Next, it was of interest to investigate whether M. oryzae infection has an effect on the accumulation of each Fe ion in rice leaves, Fe3+ and Fe2+. Accordingly, the ferrozine assay was used to assess Fe3+ and Fe2+ accumulation in leaves of Control and High-Fe plants, mock-inoculated and M. oryzae-inoculated. Treatment with high Fe increased both Fe3+ and Fe2+ content in mock-inoculated plants (Fig. 7C, grey bars). Upon pathogen infection, Fe3+ content increased in Control plants, while its level decreased in High-Fe plants (infected vs non-infected plants) (Fig. 7C, left panel, pink bars), a pattern that resembled that of total Fe content (see Fig. 7B). From these results, it appears that Fe3+ content in M. oryzae-infected High-Fe plants reached a level similar to that in non-infected Control plants. As for Fe2+, its level was not significantly altered by pathogen infection either in Control or High-Fe plants (Fig. 7C, right panel).
To gain more information about effect of pathogen infection on Fe distribution in rice leaves, we measured apoplastic Fe levels. Malate dehydrogenase (MDH) activity, an indicator of cytosolic contamination, was not detected in apoplast samples, thus, excluding cytosolic contamination in apoplastic samples (Additional file 1: Fig. S9). This study revealed that treatment with high-Fe was accompanied by a significant increase in apoplastic Fe concentrations (Fig. 7D, grey bars). Upon M. oryzae infection, apoplastic Fe decreased in both Control and High-Fe plants (Fig. 7D, pink bars). In this way, M. oryzae infection decreased Fe accumulation in the leaf apoplast of High-Fe plants to a level similar to that in non-infected Control plants.
Collectively, transcriptome analysis in combination with Fe determinations revealed that M. oryzae infection provokes alterations in the expression of genes involved in Fe homeostasis leading to alterations in Fe content, total Fe and apoplastic Fe, in rice leaves. Different responses to pathogen infection are, however, observed depending on the Fe supply condition. In High-Fe plants, there is a reduction on total Fe, Fe3+ and apoplastic Fe during pathogen infection, whereas pathogen infection increases total and Fe3+ content, but not apoplastic Fe, in control plants. Clearly, a tight control of Fe homeostatic mechanisms occurs in rice plants at the intracellular and extracellular levels during infection by M. oryzae, which is also dependent on the Fe status of the rice plant.
ROS and Fe Accumulation in High-Fe Plants During Pathogen Infection
The production of ROS is a generalized plant defense response against pathogen attack (Torres 2010). Among ROS, H2O2 is considered to be an important signaling molecule in regulating plant immunity. It is also known that pathogen infection triggers ROS accumulation and localized cell death around the site of infection, thus, limiting the spread of the pathogen. Additionally, Fe-mediated ROS production (Fenton reaction) is a central executioner of ferroptosis, an iron-dependent form of cell death which is also associated with increased lipid peroxidation (Distéfano et al. 2017; Conrad et al. 2018). The observation that M. oryzae infection was accompanied by alterations in Fe accumulation in rice leaves prompted us to investigate Fe and ROS accumulation in leaves of High-Fe plants during M. oryzae infection at the histochemical level.
Histochemical detection of ROS was carried out using the fluorescent probe H2DCFDA (2′, 7′ dichlorofluorescein diacetate) which mainly detects H2O2 accumulation (Fichman et al. 2019). In the absence of M. oryzae infection, ROS was not detected in leaves of either Control or High-Fe plants (Fig. 8A, mock). Upon pathogen challenge, however, discrete regions accumulating ROS could be observed in both Control and High-Fe plants, but the H2DCFDA-fluorescent signals were more intense and abundant in High-Fe plants relative to Control plants (Fig. 8A, infected). Quantification of H2DCFDA fluorescence using ImageJ software confirmed higher pathogen-induced accumulation of ROS in High-Fe plants compared with Control plants (Fig. 8A, right panel). Regions accumulating ROS, most probably, correspond to the M. oryzae infection sites.
To further investigate Fe accumulation during infection of High-Fe plants we used an histochemical procedure based on 3, 3′-diaminobenzidine (DAB) staining followed by Perls staining. In mock-inoculated leaves from Control and High Fe plants, Fe3+ ions were detected in stomata (Fig. 8B, blue color). Upon M. oryzae infection of Control-Fe plants, ROS was found to accumulate at the invaded leaf cells (Fig. 8B, brown color; Ctrl. Fe infected). Interestingly, in M. oryzae-infected High Fe plants, ROS (brown) and Fe3+ ions (blue) accumulated at the infection sites with the surrounding cells also accumulating Fe3+ ions (Fig. 8B, High Fe infected).
Thus, transcriptome analysis in combination with histochemical detection of iron indicated that pathogen infection causes a redistribution of Fe in the rice leaf. Fe preferentially accumulates around the infection sites, most probably, as a consequence of alterations in the expression of genes involved in iron homeostasis-related genes during pathogen infection. The higher content of Fe in leaves of High-Fe plants would contribute to a higher accumulation of Fe (hence, ROS) at the infection sites in High-Fe plants. Alterations in iron distribution have been previously described in Arabidopsis tissues infected by Dickeya dadantii, and in maize plants infected by Curvularia lunata (Aznar et al. 2014; Fu et al., 2022). However, additional investigation needs to be carried out to determine the precise spatio-temporal accumulation of Fe during M. oryzae infection at both tissue and subcellular levels.
Finally, trypan blue staining revealed a pattern of cell death in leaves of M. oryzae-infected High-Fe plants that was absent in leaves of M. oryzae-infected Control plants (Additional file 1: Fig. S10). A closer examination of these regions revealed dead cells clustered in the vicinity of fungal penetration sites (appressoria) in the M. oryzae-infected High-Fe plants (Additional file 1: Fig. S10). These results confirmed that cell death occurs during infection of Fe-treated rice plants.
Treatment with the Ferroptosis Inhibitor Ferrostatin-1 Suppresses Fe Accumulation During M. oryzae Infection
The ferroptosis inhibitor Ferrostatin-1 (Fer-1) was used to examine Fe accumulation during infection of rice plants that have been grown under high, control or low Fe supply. Perls staining of M. oryzae-infected leaves indicated that treatment with Fer-1 suppresses Fe accumulation at the infection sites, hence ferroptosis (Fig. 9). Brown spots observed in non-treated High-Fe plants (-Fer-1) points to a HR reaction in these plants. Treatment with Fer-1 of M. oryzae-infected leaf sheaths gave similar results. Here, Fe accumulation was also suppressed upon infection with M. oryzae (Additional file 1: Fig. S11). These findings confirm ferroptosis mediating blast resistance in High-Fe rice plants. In other studies, the use of ferroptosis inducers was found to prevent invasive growth of M. oryzae in leaf sheaths of an otherwise susceptible indica-type rice cultivar (O. sativa cv CO 39) (Shen et al. 2020).
As noted above, lipid peroxidation is also a hallmark of ferroptosis which is mainly caused by iron-mediated ROS accumulation (Yang et al. 2016; Conrad et al. 2018). In this work, we measured lipid peroxidation as malondialdehyde (MDA) content, MDA being a typical breakdown product of peroxidized fatty acids in plant membranes. Under no infection conditions, the MDA levels were similar in Control and High-Fe plants (Fig. 10A, grey bars). Under infection conditions, however, MDA accumulated at a higher level in High-Fe plants than in Control plants in response to pathogen infection, a response that was more evident at 24 hpi (Fig. 10A, pink bars).
To further investigate lipid peroxidation in High-Fe plants, we used C11-BODIPY581/591 (from now on BODIPY), a fluorescent lipid peroxidation reporter molecule that allows monitoring lipid peroxidation in cell membranes associated to pro-oxidant activity during ferroptosis (upon oxidation, BODIPY shows green fluorescence). As shown in Fig. 10B (left panels), leaves of High Fe plants showed the characteristic green fluorescence of oxidized BODIPY at the infected regions, which was not observed in M. oryzae-infected leaves of either Control plants or Low-Fe plants. Treatment with Fer-1 showed a near-complete loss of accumulation of oxidized BODIPY (Fig. 10B, right panels). These findings further support lipid peroxidation and ferroptosis during M. oryzae infection in leaves of High-Fe plants.
Collectively, results here presented indicated that rice plants that have been exposed to treatment with high Fe develop a ferroptotic cell death during infection with a virulent isolate of M. oryzae, a response that also occurs during infection of rice leaf sheaths with an incompatible M. oryzae isolate (Dangol et al. 2019).