MicroRNAs in rice innate immunity

MicroRNAs (miRNAs) are short regulatory non-coding RNAs that guide gene silencing in most eukaryotes. They regulate gene expression by triggering sequence-specific cleavage or translational repression of target transcripts. Plant miRNAs are known to play important roles in a wide range of developmental processes. Increasing evidence also supports that the modulation of miRNA levels plays an important role in reprogramming plant responses to abiotic stress (drought, cold, salinity and nutrient deficiency) and biotic stress (antibacterial resistance). Most of these studies were carried out in the model plant Arabidopsis thaliana. During the last years, the adoption of high-throughput sequencing technologies has significantly contributed to uncover multiple miRNAs while allowing miRNA profiling in plants. However, although a plethora of rice miRNAs have been shown to be regulated by pathogen infection, the biological function remains largely unknown for most of them. In this review, we summarize our current understanding on the contribution of miRNAs to rice immunity and discuss their potential applications in rice biotechnology. A better understanding of the miRNA species controlling rice immunity may lead to practical biotechnological applications leading to the development of appropriate strategies for rice protection

OsDCL1a activation impairs phytoalexin biosynthesis and compromises disease resistance in rice

MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression via sequence-specific cleavage or translational repression of target transcripts. They are transcribed as long single-stranded RNA precursors with unique stem-loop structures that are processed by a DICER-Like (DCL) ribonuclease, typically DCL1, to produce mature miRNAs. Although a plethora of miRNAs have been found to be regulated by pathogen infection in plants, the biological function of most miRNAs remains largely unknown. Here, the contribution of OsDCL1 to rice immunity was investigated. OsDCL1a activation enhances susceptibility to infection by fungal pathogens in rice. Activation of OsDCL1a represses the pathogen-inducible host defence response and negatively regulates diterpenoid phytoalexin production. These findings provide a basis to understand the molecular mechanisms through which OsDCL1a mediates rice immunity

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors

[Background] Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis.[Results] Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species.[Conclusion] Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants.P. Baldrich is a recipient of a Ph.D grant from the “Ministerio de Ciencia e Innovación, Formación de Personal Investigador-FPI, ref. BES-2010-032879). This work was supported by grants BIO2009-08719 and BIO2012-32838 to BSS, AGL2010-14949 to JJLM, and BFU2008-04251 to JLR, from the Spanish Ministry of Economy and Competitiveness (MINECO), and grant 2010–0520193 to JLR from the National Science Foundation (NSF). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors

Background: Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis. - Results: Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species. - Conclusion: Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants

Role of microRNAs in plant innate immunity

Los pequeños ARNs son ARNs cortos no codificantes que regulan la expresión génica en la mayoría de eucariotas. Las plantas tienen dos clases de pequeños ARNs, los microARNs (miRNAs) y los pequeños ARNs de interferencia (siRNAs), que se diferencian por su biogénesis y su modo de acción. Actualmente se estima que las pérdidas debidas a patógenos y plagas alcanzan el 50-80%, siendo uno de los factores limitantes de la producción y causando graves pérdidas económicas. Tenemos pues la necesidad de mejorar el conocimiento de los mecanismos de defensa y desarrollar nuevas estrategias para la protección de los cultivos. Con el fin de ampliar los conocimiento en este sector, se han llevado a cabo estudios con Arabidopsis y arroz, los dos sistemas modelo usados en genómica funcional en plantas dicotiledóneas y monocotiledóneas. En el primer capítulo, se analizaron las alteraciones en la acumulación de pequeños ARNs, incluyendo miRNAs, en respuesta al tratamiento con elicitores en plantas de Arabidopsis. Entre los miRNAs regulados por elicitores se encuentra miR168, que regula ARGONAUTE1, el componente principal del complejo RISC (RNA-induced silencing complex) de la maquinaria de funcionamiento de miRNAs. Los microarrays permitieron, además de analizar miRNAs conocidos, la identificación de un pequeño ARN incorrectamente anotado como miRNA en el registro miRBase. Se demostró que este pequeño ARN es un pequeño ARN heterocromático (hc-siRNA) denominado siRNA415. En el segundo capítulo, se usaron librerías de secuenciación masiva de pequeños ARNs para la identificación global de miRNAs de arroz regulados por elicitores fúngicos. Esto permitió también describir 9 miRNAs no caracterizados previamente. Combinando dichas librerías con el análisis de nuestros degradomas, se identificaron sus respectivos targets y se observó la existencia de redes reguladoras enriquecidas en miRNAs regulados por elicitores. Específicamente, se identificó un número importante de miRNAs y sus genes diana, implicados en las rutas de biosíntesis de pequeños ARNs, incluyendo miRNAs, hc-siRNAs y "trans-acting siRNAs" (ta-siRNAs). Así mismo se presentan evidencias de miRNAs y sus genes diana implicados en la señalización mediada por hormonas así como en la interacción entre rutas hormonales, demostrando así un gran potencial en la regulación de la inmunidad del arroz. Asimismo se describe la regulación de genes de arroz que contienen "Conserved-Peptide upstream Open Reading Frame" (CPuORF) mediada por miRNAs. Esto sugiere la existencia de una red reguladora nueva que integra la función de miRNAs y CPuORF en plantas. El conocimiento adquirido en este estudio ayudará al la comprensión de los mecanismos de defensa mediados por miRNAs, así como a desarrollar estrategias apropiadas a la protección del arroz. En el tercer capítulo, se uso la combinación de herramientas bioinformáticas y experimentales para el análisis de miRNAs policistrónicos en arroz, observando la existencia de 23 loci con la habilidad de formar la típica estructura de horquilla de los precursores de miRNAs. Se presentan evidencias experimentales de 7 miRNAs policistrónicos que contienen tanto miRNAs homólogos como miRNAs no homólogos. Se demostró también un patrón de conservación en diferentes especies de arroz (Oryza sativa) cuyo genoma es AA, no presente en especies primitivas de arroz. Conjuntamente, los resultados obtenidos en este trabajo apoyan la idea que los miRNAs pueden ser considerados como componentes de la respuesta de las plantas a la infección por patógenos, posiblemente actuando como nodos de regulación de diferentes procesos fisiológicos durante la adaptación de las plantas a la infección.Small RNAs (sRNAs) are short non-coding RNAs that guide gene silencing in most eukaryotes. Plants have two main classes of sRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), which are distinguished by their mode of biogenesis and mechanisms of action. In this day and age, crop losses due to pathogens and pests are estimated from 50% to 80%, factors limiting crop production and causing important economical losses. There is then an imperative need to improve our knowledge in defense mechanisms and to develop novel strategies for crop protection. To improve the understanding in this field, we carried out studies in Arabidopsis and rice plants, the two model systems used for functional genomic studies in dicot and monocot plant species. In the first chapter, we analyzed alterations on the accumulation of smRNAs in response to elicitor treatment, including miRNAs, in Arabidopsis plants. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA in the miRBase registry. We demonstrated that this smRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. In the second chapter, we used deep sequencing of small RNA libraries for global identification of rice miRNAs that are regulated by fungal elicitors. We also describe 9 previously uncharacterized miRNAs. Combined small RNA and degradome analyses revealed regulatory networks enriched in elicitor-regulated miRNAs supported by the identification of their corresponding target genes. Specifically, we identified an important number of miRNA/target gene pairs involved in small RNA pathways, including miRNA, heterochromatic and trans-acting siRNA pathways. We present evidence for miRNA/target gene pairs implicated in hormone signaling and cross-talk among hormone pathways having great potential in regulating rice immunity. Furthermore, we describe miRNA-mediated regulation of Conserved-Peptide upstream Open Reading Frame (CPuORF)-containing genes in rice, which suggests the existence of a novel regulatory network that integrates miRNA and CPuORF functions in plants. The knowledge gained in this study will help in understanding the underlying regulatory mechanisms of miRNAs in rice immunity and develop appropriate strategies for rice protection. In the third chapter, we used a combination of bioinformatic tools and experimental analyses for the discovery of new polycistronic miRNAs in rice, revealing 23 loci with the ability to form the typical hairpin structure of miRNA precursors in which two or more mature miRNAs mapped along the same structure. Evidence is presented on the polycistronic nature of 7 miRNA precursors containing homologous or non-homologous miRNA species. We also demonstrated a pattern of conservation in the genome of rice (Oryza sativa) species that have an AA genome, but not in primitive rice species. Collectivelly, results obtained in this work support the notion that miRNAs might be considered as components of the plant response to pathogen infection, possible acting as regulatory nodes of different physiological processes during plant adaptation to infection conditions

Latitudinal Variation in the Toxicity and Sexual Compatibility of Alexandrium catenella Strains from Southern Chile

The bloom-forming toxic dinoflagellate Alexandrium catenella was first detected in southern Chile (39.5–55° S) 50 years ago and is responsible for most of the area’s cases of paralytic shellfish poisoning (PSP). Given the complex life history of A. catenella, which includes benthic sexual cysts, in this study, we examined the potential link between latitude, toxicity, and sexual compatibility. Nine clones isolated from Chilean Patagonia were used in self- and out-crosses in all possible combinations (n = 45). The effect of latitude on toxicity, reproductive success indexes, and cyst production was also determined. Using the toxin profiles for all strains, consisting of C1, C2, GTX4, GTX1, GTX3, and NeoSTX, a latitudinal gradient was determined for their proportions (%) and content per cell (pg cell−1), with the more toxic strains occurring in the north (−40.6° S). Reproductive success also showed a latitudinal tendency and was lower in the north. None of the self-crosses yielded resting cysts. Rather, the production of resting cysts was highest in pairings of clones separated by distances of 1000–1650 km. Our results contribute to a better understanding of PSP outbreaks in the region and demonstrate the importance of resting cysts in fueling new toxic events. They also provide additional evidence that the introduction of strains from neighboring regions is a cause for concern.En prens

Sensitization to isothiazolinones in the Spanish Contact Dermatitis Registry (REIDAC): 2019–2021 epidemiological situation

Background: Current frequency and risk factors for sensitization to methylisothiazolinone (MI), methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), benzisothiazolinone (BIT) and octylisothiazolinone (OIT) in Spain are not well known. Objectives: To study the frequency of sensitization, risk factors and simultaneous sensitization between the four isothiazolinones. Materials and Methods: We analysed all 2019-2021 consecutive patients patch-tested with MI (0.2% aq.), MCI/MI (0.02% aq.), BIT (0.1% pet.) and OIT (0.1% pet) within the Spanish Contact Dermatitis Registry (REIDAC). Results: A total of 2511 patients were analysed. Frequencies of sensitization were: any isothiazolinone 15.7%, MI 6.8%, MCI/MI 4.8%, BIT 3.5% and OIT 0.5%. MI and MCI/MI sensitization was associated with being occupationally active, hand dermatitis, detergents and age over 40. BIT sensitization was associated with leg dermatitis and age over 40. About one in nine MI-positive patients were positive to BIT, whereas one in five BIT-positive patients were positive to MI. Conclusions: Sensitization to MI, MCI/MI and BIT is still common in Spain, while sensitization to OIT is rare. Currently, sensitization to MI and MCI/MI seems to be occupationally related. Although its origin is unknown, sensitization to BIT is more frequent in patients aged over 40 years. Simultaneous sensitization between MI and BIT is uncommon.The Spanish Registry of Contact Dermatitis (REIDAC) is promoted by the Fundación Piel Sana (Academia Española de Dermatología y Venereología), which has received financial support from the Spanish Medicines and Health Products Agency (Agencia Española de Medicamentos y Productos Sanitarios. https://www.boe.es/boe/dias/2022/04/11/pdfs/BOE-A-2022-5975.pdf) and Sanofi. The funders were not involved in the design and conduct of the study, collection, management, analysis and interpretation of data, preparation, review, approval of the manuscript, or decision to submit the manuscript for publication

Role of microRNAs in plant innate immunity

Los pequeños ARNs son ARNs cortos no codificantes que regulan la expresión génica en la mayoría de eucariotas. Las plantas tienen dos clases de pequeños ARNs, los microARNs (miRNAs) y los pequeños ARNs de interferencia (siRNAs), que se diferencian por su biogénesis y su modo de acción. Actualmente se estima que las pérdidas debidas a patógenos y plagas alcanzan el 50-80%, siendo uno de los factores limitantes de la producción y causando graves pérdidas económicas. Tenemos pues la necesidad de mejorar el conocimiento de los mecanismos de defensa y desarrollar nuevas estrategias para la protección de los cultivos. Con el fin de ampliar los conocimiento en este sector, se han llevado a cabo estudios con Arabidopsis y arroz, los dos sistemas modelo usados en genómica funcional en plantas dicotiledóneas y monocotiledóneas. En el primer capítulo, se analizaron las alteraciones en la acumulación de pequeños ARNs, incluyendo miRNAs, en respuesta al tratamiento con elicitores en plantas de Arabidopsis. Entre los miRNAs regulados por elicitores se encuentra miR168, que regula ARGONAUTE1, el componente principal del complejo RISC (RNA-induced silencing complex) de la maquinaria de funcionamiento de miRNAs. Los microarrays permitieron, además de analizar miRNAs conocidos, la identificación de un pequeño ARN incorrectamente anotado como miRNA en el registro miRBase. Se demostró que este pequeño ARN es un pequeño ARN heterocromático (hc-siRNA) denominado siRNA415. En el segundo capítulo, se usaron librerías de secuenciación masiva de pequeños ARNs para la identificación global de miRNAs de arroz regulados por elicitores fúngicos. Esto permitió también describir 9 miRNAs no caracterizados previamente. Combinando dichas librerías con el análisis de nuestros degradomas, se identificaron sus respectivos targets y se observó la existencia de redes reguladoras enriquecidas en miRNAs regulados por elicitores. Específicamente, se identificó un número importante de miRNAs y sus genes diana, implicados en las rutas de biosíntesis de pequeños ARNs, incluyendo miRNAs, hc-siRNAs y “trans-acting siRNAs” (ta-siRNAs). Así mismo se presentan evidencias de miRNAs y sus genes diana implicados en la señalización mediada por hormonas así como en la interacción entre rutas hormonales, demostrando así un gran potencial en la regulación de la inmunidad del arroz. Asimismo se describe la regulación de genes de arroz que contienen “Conserved-Peptide upstream Open Reading Frame” (CPuORF) mediada por miRNAs. Esto sugiere la existencia de una red reguladora nueva que integra la función de miRNAs y CPuORF en plantas. El conocimiento adquirido en este estudio ayudará al la comprensión de los mecanismos de defensa mediados por miRNAs, así como a desarrollar estrategias apropiadas a la protección del arroz. En el tercer capítulo, se uso la combinación de herramientas bioinformáticas y experimentales para el análisis de miRNAs policistrónicos en arroz, observando la existencia de 23 loci con la habilidad de formar la típica estructura de horquilla de los precursores de miRNAs. Se presentan evidencias experimentales de 7 miRNAs policistrónicos que contienen tanto miRNAs homólogos como miRNAs no homólogos. Se demostró también un patrón de conservación en diferentes especies de arroz (Oryza sativa) cuyo genoma es AA, no presente en especies primitivas de arroz. Conjuntamente, los resultados obtenidos en este trabajo apoyan la idea que los miRNAs pueden ser considerados como componentes de la respuesta de las plantas a la infección por patógenos, posiblemente actuando como nodos de regulación de diferentes procesos fisiológicos durante la adaptación de las plantas a la infección.Small RNAs (sRNAs) are short non-coding RNAs that guide gene silencing in most eukaryotes. Plants have two main classes of sRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), which are distinguished by their mode of biogenesis and mechanisms of action. In this day and age, crop losses due to pathogens and pests are estimated from 50% to 80%, factors limiting crop production and causing important economical losses. There is then an imperative need to improve our knowledge in defense mechanisms and to develop novel strategies for crop protection. To improve the understanding in this field, we carried out studies in Arabidopsis and rice plants, the two model systems used for functional genomic studies in dicot and monocot plant species. In the first chapter, we analyzed alterations on the accumulation of smRNAs in response to elicitor treatment, including miRNAs, in Arabidopsis plants. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA in the miRBase registry. We demonstrated that this smRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. In the second chapter, we used deep sequencing of small RNA libraries for global identification of rice miRNAs that are regulated by fungal elicitors. We also describe 9 previously uncharacterized miRNAs. Combined small RNA and degradome analyses revealed regulatory networks enriched in elicitor-regulated miRNAs supported by the identification of their corresponding target genes. Specifically, we identified an important number of miRNA/target gene pairs involved in small RNA pathways, including miRNA, heterochromatic and trans-acting siRNA pathways. We present evidence for miRNA/target gene pairs implicated in hormone signaling and cross-talk among hormone pathways having great potential in regulating rice immunity. Furthermore, we describe miRNA-mediated regulation of Conserved-Peptide upstream Open Reading Frame (CPuORF)-containing genes in rice, which suggests the existence of a novel regulatory network that integrates miRNA and CPuORF functions in plants. The knowledge gained in this study will help in understanding the underlying regulatory mechanisms of miRNAs in rice immunity and develop appropriate strategies for rice protection. In the third chapter, we used a combination of bioinformatic tools and experimental analyses for the discovery of new polycistronic miRNAs in rice, revealing 23 loci with the ability to form the typical hairpin structure of miRNA precursors in which two or more mature miRNAs mapped along the same structure. Evidence is presented on the polycistronic nature of 7 miRNA precursors containing homologous or non-homologous miRNA species. We also demonstrated a pattern of conservation in the genome of rice (Oryza sativa) species that have an AA genome, but not in primitive rice species. Collectivelly, results obtained in this work support the notion that miRNAs might be considered as components of the plant response to pathogen infection, possible acting as regulatory nodes of different physiological processes during plant adaptation to infection conditions

Genome-wide analysis of polycistronic microRNAs in cultivated and wild rice

MicroRNAs (miRNAs) are small noncoding RNAs that direct posttranscriptional gene silencing in eukaryotes. They are frequently clustered in the genomes of animals and can be independently transcribed or simultaneously transcribed into single polycistronic transcripts. Only a few miRNA clusters have been described in plants, and most of them are generated from independent transcriptional units. Here, we used a combination of bioinformatic tools and experimental analyses to discover new polycistronic miRNAs in rice. A genome-wide analysis of clustering patterns of MIRNA loci in the rice genome was carried out using a criterion of 3 kb as the maximal distance between two miRNAs. This analysis revealed 28 loci with the ability to form the typical hairpin structure of miRNA precursors in which 2 or more mature miRNAs mapped along the same structure. RT-PCR provided evidence for the polycistronic nature of seven miRNA precursors containing homologous or nonhomologous miRNA species. Polycistronic miRNAs and candidate polycistronic miRNAs are located across different rice chromosomes, except chromosome 12, and resided in both duplicated and nonduplicated chromosomal regions. Finally, most polycistronic and candidate polycistronic miRNAs showed a pattern of conservation in the genome of rice species with an AA genome. The diversity in the organization of MIR genes that are transcribed as polycistrons suggests a versatile mechanism for the control of gene expression in different biological processes and supports additional levels of complexity in miRNA functioning in plants