Understanding the role of iron homeostasis in rice immunity and novel applications of miRNAs for crop protection

Les plantes s’enfronten constantment a una àmplia gamma d’estressos ambientals que provoquen importants pèrdues en els cultius. Aquests estressos es classifiquen, segons la seva naturalesa, en estressos biòtics o abiòtics. L’estrès biòtic és causat per organismes vius com fongs, oomicets, bacteris, virus, nematodes, insectes; o bé per altres plantes. Els estressos abiòtics inclouen fenòmens diversos com salinitat, sequera, temperatures extremes, inundacions, radiació; així com estrès nutricional o toxicitat per metalls pesants, entre d’altres. A causa del seu estil de vida sèssil, les plantes no poden escapar d’aquests estressos i han desenvolupat un conjunt de mecanismes per superar-los. Per fer front a l’estrès ambiental, les plantes poden percebre els estímuls i activar vies de transducció de senyals que condueixen a la reprogramació transcripcional de l’expressió gènica. Les evidències confirmen que les plantes responen a estressos múltiples de manera diferent a com ho fan a estressos individuals, activant un programa específic d’expressió gènica segons el tipus d’estrès trobat. Per exemple, la presència d’un estrès abiòtic pot reduir o augmentar la susceptibilitat a un patogen, o viceversa. No obstant això, la majoria dels estudis sobre les respostes de les plantes a l’estrès biòtic i abiòtic s’han realitzat en plantes sotmeses a un estrès individual, però en la naturalesa és més probable que hagin d’enfrontar-se a estressos múltiples. En les plantes, els microARN (miARN) són reguladors clau de l’expressió gènica en diversos processos de desenvolupament i adaptació a l’estrès ambiental. S’ha demostrat que diferents miARNs participen en la resposta de la planta a la infecció per patògens o l’homeòstasi de nutrients. Encara que des de fa molt temps en l’agronomia s’ha reconegut una connexió entre el ferro i la immunitat de les plantes, no es comprèn bé el paper exacte de l’homeòstasi del ferro durant la resposta de defensa de les plantes a la infecció per patògens. En aquesta tesi doctoral, he investigat la regulació de processos associats a l’homeòstasi del ferro (Fe) regulada per miARN i la immunitat innata en plantes d’arròs durant la infecció amb el fong de la piriculariosi de l’arròs Magnaporthe oryzae. D’altra banda, se sap que els petits ARN es poden moure entre organismes que interactuen per a la regulació de l’expressió gènica entre regnes mitjançant la interferència d’ARN (ARNi). L’evidència recent també dona suport a la comunicació bidireccional entre regnes de petits ARN entre plantes hospedants i patògens fúngics adaptats que determinen el resultat de la infecció. La majoria d’aquests estudis es centren en el moviment d’ARNs bicatenaris llargs (dsRNAs) o petits ARN interferents (siARNs). A més, l’aplicació de dsARNs o siARNs ha demostrat ser eficaç per al control de malalties fúngiques. En l’actualitat, es disposa de poca informació sobre el moviment de miARNs entre organismes. Com a primer pas per avaluar si els miARNs poden ser eines útils per a la protecció de cultius, en aquesta tesi he explorat la viabilitat d’utilizar miARNs fets a mida per reprimir l’expressió de gens fúngics durant la infecció de plantes d’arròs amb M. oryzae.Las plantas se enfrentan constantemente a una amplia gama de estreses ambientales que provocan importantes pérdidas en los cultivos. Estos estreses se clasifican, según su naturaleza, en estreses bióticos o abióticos. El estrés biótico es causado por organismos vivos como hongos, oomicetos, bacterias, virus, nematodos, insectos; o bien por otras plantas. Los estreses abióticos incluyen fenómenos diversos como salinidad, sequía, temperaturas extremas, inundaciones, radiación; así como estrés nutricional o toxicidad por metales pesados, entre otros. Debido a su estilo de vida sésil, las plantas no pueden escapar de estos estreses y han desarrollado un conjunto de mecanismos para superarlos. Para hacer frente al estrés ambiental, las plantas pueden percibir los estímulos y activar vías de transducción de señales que conducen a la reprogramación transcripcional de la expresión génica. Las evidencias respaldan que las plantas responden a estreses múltiples de manera diferente a como lo hacen a estreses individuales, activando un programa específico de expresión génica según el tipo de estrés encontrado. Por ejemplo, la presencia de un estrés abiótico puede reducir o aumentar la susceptibilidad a un patógeno, o viceversa. Sin embargo, la mayoría de los estudios sobre las respuestas de las plantas al estrés biótico y abiótico se han realizado en plantas sometidas a un estrés individual, pero en la naturaleza es más probable que deban enfrentarse a estreses múltiples. En las plantas, los microARNs (miARNs) son reguladores clave de la expresión génica en diversos procesos de desarrollo y adaptación al estrés ambiental. Se ha demostrado que distintos miARNs participan en la respuesta de la planta a la infección por patógenos o la homeostasis de nutrientes. Aunque desde hace mucho tiempo en la agronomía se ha reconocido una conexión entre el hierro y la inmunidad de las plantas, no se comprende bien el papel exacto de la homeostasis del hierro durante la respuesta de defensa de las plantas a la infección por patógenos. En esta tesis doctoral, he investigado la regulación mediada por miARNs en procesos asociados a la homeostasis del hierro (Fe) y la inmunidad innata en plantas de arroz durante la infección con el hongo del añublo del arroz Magnaporthe oryzae. Por otro lado, es bien sabido que los pequeños ARN pueden moverse entre organismos que interactúan para la regulación de la expresión génica entre reinos a través de la interferencia de ARN (ARNi). La evidencia reciente también respalda la comunicación bidireccional entre reinos de pequeños ARNs entre plantas hospedantes y patógenos fúngicos adaptados que determinan el resultado de la infección. La mayoría de estos estudios se centran en el movimiento de ARN bicatenarios largos (dsARNs) o pequeños ARN interferentes (siARNs). Además, la aplicación de dsARNs o siARNs ha demostrado ser eficaz para el control de enfermedades fúngicas. En la actualidad, se dispone de poca información sobre el movimiento de miARNs entre organismos. Como primer paso para evaluar si los miARNs pueden ser herramientas útiles para la protección de cultivos, en esta tesis he explorado la viabilidad de usar miARNs hechos a medida para reprimir la expresión de genes fúngicos durante la infección de plantas de arroz con M. oryzae.Plants are constantly challenged with a wide range of environmental stresses that cause major losses in crops. These stresses are categorized into biotic or abiotic stresses depending on their nature. Biotic stresses are caused by living organisms like fungi, oomycetes, bacteria, viruses, nematodes, insects and weeds. Abiotic stresses include salinity, drought, extreme temperatures, flooding, radiation, as well as nutrient stress or heavy metals toxicity, among others. Due to their sessile lifestyle, plants cannot escape from stresses and have evolved a set of mechanisms to overcome them. To cope with environmental stress, plants are able to sense the stimuli for the activation of signal transduction pathways leading to transcriptional reprogramming of gene expression. Evidence supports that plants respond to multiple stresses differently from how they do to individual stresses, by activating a specific programme of gene expression depending on the type of stress encountered. For instance, the presence of an abiotic stress can have the effect of reducing or enhancing susceptibility to a pathogen, or vice versa. However, most studies on plant responses to biotic and abiotic stresses have been approached on plants subjected to an individual stress, but in nature they are more likely to occur simultaneously. In plants, microRNAs (miRNAs) are key regulators of gene expression in diverse developmental processes and adaptation to environmental stress. Distinct miRNAs have been shown to be involved in the plant response to pathogen infection or nutrient homeostasis. Although a connection between iron and plant immunity has long been recognized in agronomy, the exact role of iron homeostasis during the plant defense response to pathogen infection is not well understood. In this Ph. D. Thesis, I investigated miRNA-mediated regulation in processes associated to iron (Fe) homeostasis and innate immunity in rice plants during infection with the rice blast fungus Magnaporthe oryzae. On the other hand, it is well recognized that small RNAs are able to move between interacting organisms for cross-kingdom regulation of gene expression through RNA interference (RNAi). Recent evidence also supports bidirectional cross‐kingdom communication of small RNAs between host plants and adapted fungal pathogens that determine the outcome of infection. Most of these studies focused on the movement of long double stranded RNAs (dsRNAs) or small interfering RNAs (siRNAs). Moreover, the application of dsRNAs or siRNAs has proven to be effective for the control of fungal diseases. At present, little information is available about movement of miRNAs between organisms. As a first step to evaluate whether miRNAs can be useful tools for crop protection, in this PhD, I explored the feasibility of using tailor-made miRNAs to repress the expression of fungal genes during infection of rice plants with M. oryzae.Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia i Biotecnologia Vegeta

Understanding the role of iron homeostasis in rice immunity and novel applications of miRNAs for crop protection

Les plantes s'enfronten constantment a una àmplia gamma d'estressos ambientals que provoquen importants pèrdues en els cultius. Aquests estressos es classifiquen, segons la seva naturalesa, en estressos biòtics o abiòtics. L'estrès biòtic és causat per organismes vius com fongs, oomicets, bacteris, virus, nematodes, insectes; o bé per altres plantes. Els estressos abiòtics inclouen fenòmens diversos com salinitat, sequera, temperatures extremes, inundacions, radiació; així com estrès nutricional o toxicitat per metalls pesants, entre d'altres. A causa del seu estil de vida sèssil, les plantes no poden escapar d'aquests estressos i han desenvolupat un conjunt de mecanismes per superar-los. Per fer front a l'estrès ambiental, les plantes poden percebre els estímuls i activar vies de transducció de senyals que condueixen a la reprogramació transcripcional de l'expressió gènica. Les evidències confirmen que les plantes responen a estressos múltiples de manera diferent a com ho fan a estressos individuals, activant un programa específic d'expressió gènica segons el tipus d'estrès trobat. Per exemple, la presència d'un estrès abiòtic pot reduir o augmentar la susceptibilitat a un patogen, o viceversa. No obstant això, la majoria dels estudis sobre les respostes de les plantes a l'estrès biòtic i abiòtic s'han realitzat en plantes sotmeses a un estrès individual, però en la naturalesa és més probable que hagin d'enfrontar-se a estressos múltiples. En les plantes, els microARN (miARN) són reguladors clau de l'expressió gènica en diversos processos de desenvolupament i adaptació a l'estrès ambiental. S'ha demostrat que diferents miARNs participen en la resposta de la planta a la infecció per patògens o l'homeòstasi de nutrients. Encara que des de fa molt temps en l'agronomia s'ha reconegut una connexió entre el ferro i la immunitat de les plantes, no es comprèn bé el paper exacte de l'homeòstasi del ferro durant la resposta de defensa de les plantes a la infecció per patògens. En aquesta tesi doctoral, he investigat la regulació de processos associats a l'homeòstasi del ferro (Fe) regulada per miARN i la immunitat innata en plantes d'arròs durant la infecció amb el fong de la piriculariosi de l'arròs Magnaporthe oryzae. D'altra banda, se sap que els petits ARN es poden moure entre organismes que interactuen per a la regulació de l'expressió gènica entre regnes mitjançant la interferència d'ARN (ARNi). L'evidència recent també dona suport a la comunicació bidireccional entre regnes de petits ARN entre plantes hospedants i patògens fúngics adaptats que determinen el resultat de la infecció. La majoria d'aquests estudis es centren en el moviment d'ARNs bicatenaris llargs (dsRNAs) o petits ARN interferents (siARNs). A més, l'aplicació de dsARNs o siARNs ha demostrat ser eficaç per al control de malalties fúngiques. En l'actualitat, es disposa de poca informació sobre el moviment de miARNs entre organismes. Com a primer pas per avaluar si els miARNs poden ser eines útils per a la protecció de cultius, en aquesta tesi he explorat la viabilitat d'utilizar miARNs fets a mida per reprimir l'expressió de gens fúngics durant la infecció de plantes d'arròs amb M. oryzae.Las plantas se enfrentan constantemente a una amplia gama de estreses ambientales que provocan importantes pérdidas en los cultivos. Estos estreses se clasifican, según su naturaleza, en estreses bióticos o abióticos. El estrés biótico es causado por organismos vivos como hongos, oomicetos, bacterias, virus, nematodos, insectos; o bien por otras plantas. Los estreses abióticos incluyen fenómenos diversos como salinidad, sequía, temperaturas extremas, inundaciones, radiación; así como estrés nutricional o toxicidad por metales pesados, entre otros. Debido a su estilo de vida sésil, las plantas no pueden escapar de estos estreses y han desarrollado un conjunto de mecanismos para superarlos. Para hacer frente al estrés ambiental, las plantas pueden percibir los estímulos y activar vías de transducción de señales que conducen a la reprogramación transcripcional de la expresión génica. Las evidencias respaldan que las plantas responden a estreses múltiples de manera diferente a como lo hacen a estreses individuales, activando un programa específico de expresión génica según el tipo de estrés encontrado. Por ejemplo, la presencia de un estrés abiótico puede reducir o aumentar la susceptibilidad a un patógeno, o viceversa. Sin embargo, la mayoría de los estudios sobre las respuestas de las plantas al estrés biótico y abiótico se han realizado en plantas sometidas a un estrés individual, pero en la naturaleza es más probable que deban enfrentarse a estreses múltiples. En las plantas, los microARNs (miARNs) son reguladores clave de la expresión génica en diversos procesos de desarrollo y adaptación al estrés ambiental. Se ha demostrado que distintos miARNs participan en la respuesta de la planta a la infección por patógenos o la homeostasis de nutrientes. Aunque desde hace mucho tiempo en la agronomía se ha reconocido una conexión entre el hierro y la inmunidad de las plantas, no se comprende bien el papel exacto de la homeostasis del hierro durante la respuesta de defensa de las plantas a la infección por patógenos. En esta tesis doctoral, he investigado la regulación mediada por miARNs en procesos asociados a la homeostasis del hierro (Fe) y la inmunidad innata en plantas de arroz durante la infección con el hongo del añublo del arroz Magnaporthe oryzae. Por otro lado, es bien sabido que los pequeños ARN pueden moverse entre organismos que interactúan para la regulación de la expresión génica entre reinos a través de la interferencia de ARN (ARNi). La evidencia reciente también respalda la comunicación bidireccional entre reinos de pequeños ARNs entre plantas hospedantes y patógenos fúngicos adaptados que determinan el resultado de la infección. La mayoría de estos estudios se centran en el movimiento de ARN bicatenarios largos (dsARNs) o pequeños ARN interferentes (siARNs). Además, la aplicación de dsARNs o siARNs ha demostrado ser eficaz para el control de enfermedades fúngicas. En la actualidad, se dispone de poca información sobre el movimiento de miARNs entre organismos. Como primer paso para evaluar si los miARNs pueden ser herramientas útiles para la protección de cultivos, en esta tesis he explorado la viabilidad de usar miARNs hechos a medida para reprimir la expresión de genes fúngicos durante la infección de plantas de arroz con M. oryzae.Plants are constantly challenged with a wide range of environmental stresses that cause major losses in crops. These stresses are categorized into biotic or abiotic stresses depending on their nature. Biotic stresses are caused by living organisms like fungi, oomycetes, bacteria, viruses, nematodes, insects and weeds. Abiotic stresses include salinity, drought, extreme temperatures, flooding, radiation, as well as nutrient stress or heavy metals toxicity, among others. Due to their sessile lifestyle, plants cannot escape from stresses and have evolved a set of mechanisms to overcome them. To cope with environmental stress, plants are able to sense the stimuli for the activation of signal transduction pathways leading to transcriptional reprogramming of gene expression. Evidence supports that plants respond to multiple stresses differently from how they do to individual stresses, by activating a specific programme of gene expression depending on the type of stress encountered. For instance, the presence of an abiotic stress can have the effect of reducing or enhancing susceptibility to a pathogen, or vice versa. However, most studies on plant responses to biotic and abiotic stresses have been approached on plants subjected to an individual stress, but in nature they are more likely to occur simultaneously. In plants, microRNAs (miRNAs) are key regulators of gene expression in diverse developmental processes and adaptation to environmental stress. Distinct miRNAs have been shown to be involved in the plant response to pathogen infection or nutrient homeostasis. Although a connection between iron and plant immunity has long been recognized in agronomy, the exact role of iron homeostasis during the plant defense response to pathogen infection is not well understood. In this Ph. D. Thesis, I investigated miRNA-mediated regulation in processes associated to iron (Fe) homeostasis and innate immunity in rice plants during infection with the rice blast fungus Magnaporthe oryzae. On the other hand, it is well recognized that small RNAs are able to move between interacting organisms for cross-kingdom regulation of gene expression through RNA interference (RNAi). Recent evidence also supports bidirectional cross-kingdom communication of small RNAs between host plants and adapted fungal pathogens that determine the outcome of infection. Most of these studies focused on the movement of long double stranded RNAs (dsRNAs) or small interfering RNAs (siRNAs). Moreover, the application of dsRNAs or siRNAs has proven to be effective for the control of fungal diseases. At present, little information is available about movement of miRNAs between organisms. As a first step to evaluate whether miRNAs can be useful tools for crop protection, in this PhD, I explored the feasibility of using tailor-made miRNAs to repress the expression of fungal genes during infection of rice plants with M. oryzae

Transcriptional analysis of iron stressed rice plants under Magnaporthe oryzae infection

Trabajo presentado al Seminario del CRAG (Stress Seminar), celebrado el 25 de febrero de 2020.Peer reviewe

The microRNA miR7695 is involved in the rice immune response to pathogen infection

Resumen del póster presentado al Congreso 'At the Forefront of Plant Research', celebrado en Barcelona (España) del 6 al 8 de mayo de 2019.MicroRNAs (miRNAs) are short regulatory non-coding RNAs that guide gene silencing by triggering sequence-specific cleavage or translational repression of target transcripts. In plants, miRNAs emerged as versatile regulators of gene expression in developmental processes and adaptive responses to environmental stresses, including pathogen infection. Certain plant miRNAs have been shown to function in cross-kingdom regulation of gene expression (i.e. miR159 and miR166 can move from cotton plants to the fungal pathogen Verticillium dahliae for specific silencing of pathogen virulence genes). In rice, the fungus Magnaporthe oryzae is the causal agent of the rice blast disease, one of the most devastating fungal diseases of cultivated rice worldwide. Although a plethora of rice miRNAs have been shown to be regulated during M. oryzae infection, their biological function remains largely unknown. Here, we report that a miRNA, miR7695, contributes to rice immunity. MiR7695 targets an alternatively spliced transcript of the Natural resistance-associated macrophage protein 6 (OsNramp6) gene encoding an iron transporter from rice. Activation-tagged MIR7695 rice plants (MIR7695-Ac) exhibited enhanced resistance to M. oryzae infection. RNA-seq analysis revealed that blast resistance in MIR7695-Ac plants is associated with stronger induction of defense-related genes, including pathogenesis-related and diterpenoid biosynthetic genes. Furthermore, rice plants grown under high iron supply showed blast resistance, indicating that iron is a factor in controlling blast resistance. During pathogen infection, iron accumulates in the vicinity of fungal aspersoria, the sites of pathogen entry, and in cells surrounding infected regions of the rice leaf. This observation supports that rice plants use strategies to locally increase Fe content to prevent penetration and spread of the pathogen into the leaf tissue. A better understanding of the mechanisms that are regulated by miR7695 during rice immunity and crosstalk with iron homeostasis will help in designing novel strategies to control rice blast disease

Two NRAMP6 isoforms function as Iron and manganese transporters and contribute to disease resistance in rice

Metal ions are essential elements for all living organisms. However, metals can be toxic when present in excess. In plants, metal homeostasis is partly achieved through the function of metal transporters, including the diverse natural resistance-associated macrophage proteins (NRAMP). Among them, the OsNramp6 gene encodes a previously uncharacterized member of the rice NRAMP family that undergoes alternative splicing to produce different NRAMP6 proteins. In this work, we determined the metal transport activity and biological role of the full-length and the shortest NRAMP6 proteins (l-NRAMP6 and s-NRAMP6, respectively). Both l-NRAMP6 and s-NRAMP6 are plasma membrane-localized proteins that function as iron and manganese transporters. The expression of l-Nramp6 and s-Nramp6 is regulated during infection with the fungal pathogen Magnaporthe oryzae, albeit with different kinetics. Rice plants grown under high iron supply show stronger induction of rice defense genes and enhanced resistance to M. oryzae infection. Also, loss of function of OsNramp6 results in enhanced resistance to M. oryzae, supporting the idea that OsNramp6 negatively regulates rice immunity. Furthermore, nramp6 plants showed reduced biomass, pointing to a role of OsNramp6 in plant growth. A better understanding of OsNramp6-mediated mechanisms underlying disease resistance in rice will help in developing appropriate strategies for crop protection.This project was funded by the Ministry of Economy and Competitiveness (MINECO) and the European Regional Development’s funds (BIO2012-32838 and BIO2015-67212 to B. San Segundo and BFU2014-54591-C2-1-P to J. Ariño). J. Ariño is recipient of a 2014SGR-4 grant from the Generalitat de Catalunya. We acknowledge the support of the MINECO for the “Centro de Excelencia Severo Ochoa 2016-2019” award SEV-2015-0533.Peer reviewe

Osa-miR7695 enhances transcriptional priming in defense responses against the rice blast fungus

Altres ajuts: CERCA Programme/Generalitat de CatalunyaBackground: 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

Iron Induces Resistance Against the Rice Blast Fungus Magnaporthe oryzae Through Potentiation of Immune Responses

Abstract Iron is an essential nutrient required for plant growth and development. The availability of iron might also influence disease resistance in plants. However, the molecular mechanisms involved in the plant response to iron availability and immunity have been investigated separately from each other. In this work, we found that exposure of rice plants to high iron enhances resistance to infection by the fungal pathogen Magnaporthe oryzae, the causal agent of blast disease. RNA-Seq analysis revealed that blast resistance in iron-treated rice plants was associated with superinduction of defense-related genes during pathogen infection, including Pathogenesis-Related genes. The expression level of genes involved in the biosynthesis of phytoalexins, both diterpene phytoalexins and the flavonoid phytoalexin sakuranetin, was also higher in iron-treated plants compared with control plants, which correlated well with increased levels of phytoalexins in these plants during M. oryzae infection. Upon pathogen infection, lipid peroxidation was also higher in iron-treated plants compared with non-treated plants. We also show that M. oryzae infection modulates the expression of genes that play a pivotal role in the maintenance of iron homeostasis. Histochemical analysis of M. oryzae-infected leaves revealed colocalization of iron and reactive oxygen species in cells located in the vicinity of fungal penetration sites (e.g. appressoria) in rice plants that have been exposed to iron. Together these findings support that ferroptosis plays a role in the response of iron-treated rice plants to infection by virulent M. oryzae. Understanding interconnected regulations between iron signaling and immune signaling in rice holds great potential for developing novel strategies to improve blast resistance in rice

Additional file 7 of Iron Induces Resistance Against the Rice Blast Fungus Magnaporthe oryzae Through Potentiation of Immune Responses

1 table.Additional file 7: Table S6. Expression data (FPKM and Log2FC) of genes involved in Fe homeostasis in leaves of Control and High-Fe plants (M. oryzae-infected and mock-inoculated plants).Consejo Superior de Investigaciones Cientificas (CSIC)Peer reviewe

Osa-miR7695 enhances transcriptional priming in defense responses against the rice blast fungus

[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.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, BIO2015–67212-R) to BSS, the CSIC/NSC (Spanish Research Council/National Science Council of Taiwan)-Formosa Program (2009TW0041 and NSC 99–2923-B-001-002-MY3), the Academia Sinica Summit Project to YICH, and the Japan Society for the Promotion of Science (JSPS KAKENHI, 17H03811 to KO). We acknowledge support from the CERCA Programme (“Generalitat de Catalunya”) and the “Severo Ochoa Programme for Centres of Excellence in R&D” (MINECO, 2016–2019, SEV-2015-0533).Peer reviewe

Additional file 2 of Iron Induces Resistance Against the Rice Blast Fungus Magnaporthe oryzae Through Potentiation of Immune Responses

1 table.Additional file 2: Table S1. Differentially expressed genes (DEGs) in leaves of High-Fe plants relative to Control plants identified by RNASeq analysis (no infection conditions).Peer reviewe