miRNA/phasiRNA mediated regulation of plant defense response against P. syringae

Gene silencing is a mechanism of regulation of gene expression where the small RNAs (sRNAs) are key components for giving specificity to the system. In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs). DCL proteins acting on large RNA precursors produce the mature forms of sRNAs (20-24nt) that can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have validated this regulation, and have also established that the corresponding pri-miRNA is down-regulated upon PAMPs or bacteria perception. Using GUS reporters, we have characterized the expression pattern of both pri-miRNA and its best target R genes. We demonstrate that plants with altered levels of miRNA* (knockdown or overexpression lines) exhibit altered PTI-associated phenotypes, supporting a role for this miRNA* in the defence response against this bacterial pathogen. Finally, we identify phasiRNAs that arise from the transcript of one of the R target genes in a miRNA*-RDR6-DCL4-dependent manner.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Evaluation of agronomic performance of grafted tomato plants in greenhouse cultivation

El trabajo tuvo como objetivo evaluar el crecimiento, rendimiento y comportamiento fenológico de plantas de tomate injertadas cultivadas bajo cubierta. Se condujeron tres ensayos en un invernadero parabólico ubicado en La Plata, Buenos Aires, Argentina (34°58’ S; 57°54’ W). Los tratamientos fueron plantas del híbrido Elpida (Enza Zaden®) injertadas sobre pie Efialto (Enza Zaden®) y Elpida sin injertar, usado como testigo. Las plantas testigo se condujeron a una rama (2 plantas.m-2) y las injertadas a dos ramas (1 planta.m-2). Se registró la altura de planta, el rendimiento total y por categorías comerciales y la fecha de floración y fructificación por racimo, calculando la cantidad de días transcurridos entre transplante y cada una de las fases. Se utilizó un diseño en bloques completos aleatorizados con 4 repeticiones. La cantidad de días entre fases se evaluó con la prueba no paramétrica de Kruskal Wallis (p ≤ 0,05). Los datos de rendimiento y altura de planta se sometieron a análisis de la varianza (p ≤ 0,05). Las plantas injertadas presentaron un incremento estadísticamente significativo en la cantidad de días requeridos para alcanzar la floración y fructificación del primer, tercer y quinto racimo solo en el primer ensayo. La altura, el rendimiento total y por categorías comerciales no presentaron diferencias entre tratamientos. El injerto del cv. Elpida sobre el pie Efialto produjo plantas más vigorosas que pueden conducirse a dos tallos reduciendo la cantidad de plantas necesarias y manteniendo niveles de producción equivalentes al híbrido sin injertar.This study aimed to evaluate plant height, total and commercial category yields and date of flowering and fruiting of each truss, computing the number of days elapsed from transplanting and each phase of grafted plants growing under greenhouse conditions. Three trials were carried out in a parabolic greenhouse in La Plata, Buenos Aires, Argentina (34°58’ S; 57°54’ W). Treatments were hybrid tomato plants cv. Elpida (Enza Zaden®) grafted on Efialto (Enza Zaden®) and ungrafted Elpida as control. Ungrafted plants were conducted to one branch (2 plants.m-2) and grafted plants were conducted to 2 branches (1 plant. m-2). Experimental design was a randomized complete block with 4 replications. The number of days between phases was evaluated by nonparametric Kruskal Wallis (p ≤ 0,05). Yield data and plant height were submitted to variance analysis (p ≤ 0,05). Grafted plants showed a statistically significant increase in the number of days required to reach the flowering and fruiting of the first, third and fifth cluster only in the first trial. Treatments did not differ in either the height or total yield and commercial categories. Grafting cv. Elpida on Efialto resulted in more vigorous plants that can be conducted to two stems reducing the number of plants needed for growing, maintaining production levels equivalents to the ungrafted hybrid led to a stem.Facultad de Ciencias Agrarias y Forestale

Evaluation of agronomic performance of grafted tomato plants in greenhouse cultivation

El trabajo tuvo como objetivo evaluar el crecimiento, rendimiento y comportamiento fenológico de plantas de tomate injertadas cultivadas bajo cubierta. Se condujeron tres ensayos en un invernadero parabólico ubicado en La Plata, Buenos Aires, Argentina (34°58’ S; 57°54’ W). Los tratamientos fueron plantas del híbrido Elpida (Enza Zaden®) injertadas sobre pie Efialto (Enza Zaden®) y Elpida sin injertar, usado como testigo. Las plantas testigo se condujeron a una rama (2 plantas.m-2) y las injertadas a dos ramas (1 planta.m-2). Se registró la altura de planta, el rendimiento total y por categorías comerciales y la fecha de floración y fructificación por racimo, calculando la cantidad de días transcurridos entre transplante y cada una de las fases. Se utilizó un diseño en bloques completos aleatorizados con 4 repeticiones. La cantidad de días entre fases se evaluó con la prueba no paramétrica de Kruskal Wallis (p ≤ 0,05). Los datos de rendimiento y altura de planta se sometieron a análisis de la varianza (p ≤ 0,05). Las plantas injertadas presentaron un incremento estadísticamente significativo en la cantidad de días requeridos para alcanzar la floración y fructificación del primer, tercer y quinto racimo solo en el primer ensayo. La altura, el rendimiento total y por categorías comerciales no presentaron diferencias entre tratamientos. El injerto del cv. Elpida sobre el pie Efialto produjo plantas más vigorosas que pueden conducirse a dos tallos reduciendo la cantidad de plantas necesarias y manteniendo niveles de producción equivalentes al híbrido sin injertar.This study aimed to evaluate plant height, total and commercial category yields and date of flowering and fruiting of each truss, computing the number of days elapsed from transplanting and each phase of grafted plants growing under greenhouse conditions. Three trials were carried out in a parabolic greenhouse in La Plata, Buenos Aires, Argentina (34°58’ S; 57°54’ W). Treatments were hybrid tomato plants cv. Elpida (Enza Zaden®) grafted on Efialto (Enza Zaden®) and ungrafted Elpida as control. Ungrafted plants were conducted to one branch (2 plants.m-2) and grafted plants were conducted to 2 branches (1 plant. m-2). Experimental design was a randomized complete block with 4 replications. The number of days between phases was evaluated by nonparametric Kruskal Wallis (p ≤ 0,05). Yield data and plant height were submitted to variance analysis (p ≤ 0,05). Grafted plants showed a statistically significant increase in the number of days required to reach the flowering and fruiting of the first, third and fifth cluster only in the first trial. Treatments did not differ in either the height or total yield and commercial categories. Grafting cv. Elpida on Efialto resulted in more vigorous plants that can be conducted to two stems reducing the number of plants needed for growing, maintaining production levels equivalents to the ungrafted hybrid led to a stem.Facultad de Ciencias Agrarias y Forestale

R gene regulation mediated by miRNA/phasiRNA during plant defense response against P. syringae

In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs), differing these in their biogenesis and mode of action, but sharing similar sizes (20-24 nt). In plants, their mature forms are products of the activity of DCL proteins and can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. By bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception. We demonstrate that plants with altered levels of this miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. We have characterized the expression pattern of both primiRNA and its best target R genes. Finally, we identify phasiRNAs that arise from the transcript of this R gen in a miRNA*-RDR6-DCL4-dependent mannerUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Regulación de genes de resistencia de la familia TIR-NBS-LRR mediada por miRNA/phasiRNA durante la interacción con P. syringae

Durante un estrés biótico, las plantas modulan la expresión de una batería de genes involucrados en la respuesta de defensa, proceso donde recientemente se ha determinado el papel esencial que desempeña el silenciamiento génico. El silenciamiento génico es un mecanismo de regulación de la expresión génica, donde destacan como principales moléculas efectoras los pequeños RNAs (sRNAs). En plantas, estos sRNAs, son clasificados en pequeños RNAs interferentes (siRNAs) o microRNAs (miRNAs), presentando tamaños similares (20-24 nt) pero difiriendo en su biogénesis y modo de acción. Los miRNAs son pequeños RNAs de cadena sencilla que actúan regulando negativamente la expresión de genes, mediante su unión al complejo RISC (Rna Induced Silencing Complex) y en una forma dependiente de secuencia. En nuestro laboratorio, mediante el análisis de datos transcriptómicos, y el uso de herramientas bioinformáticas, identificamos un miRNA* de 22 nt como potencial regulador de la expresión de genes de resistencia (“R”) del tipo TIR-NBS-LRR. Posteriormente hemos validado dicha regulación y caracterizado los patrones de expresión tanto del Pri-miRNA como de un gen “R” regulado por este, en diferentes tejidos y estadios del desarrollo, así como durante la interacción con P. syringae. Por otro lado, hemos generado plantas transgénicas que presentan niveles alterados del miRNA* (incremento y reducción) y hemos observado que muestran fenotipos alterados de PTI y una mayor/menor colonización de P. syringae. Finalmente hemos identificado la producción de sRNAs (phasiRNAs) a partir del gen de resistencia, en una forma dependiente de miRNA*-RDR6-DCL4, pudiendo estos sRNAs secundarios regular otros transcritos de la misma familia de genes de resistencia.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Ecofisiología de los cultivos protegidos: trabajos de experimentación hacia una horticultura sustentable : Proyecto 11 A/269

En el marco del Proyecto de Investigación se realizaron ensayos con el objetivo de responder diversas inquietudes del sector productivo del Cordón Hortícola Platense (CHP). A continuación se enumeran los trabajos realizados y presentados en Jornadas y Congresos de la especialidad, como así también resultados de diversas Tesis de Grado realizadas por alumnos de la FCAyF, UNLP.Facultad de Ciencias Agrarias y Forestale

Ecofisiología de los cultivos protegidos: trabajos de experimentación hacia una horticultura sustentable : Proyecto 11 A/269

En el marco del Proyecto de Investigación se realizaron ensayos con el objetivo de responder diversas inquietudes del sector productivo del Cordón Hortícola Platense (CHP). A continuación se enumeran los trabajos realizados y presentados en Jornadas y Congresos de la especialidad, como así también resultados de diversas Tesis de Grado realizadas por alumnos de la FCAyF, UNLP.Facultad de Ciencias Agrarias y Forestale

Characterization of a Gelatin/Chitosan/Hyaluronan scaffold-polymer

Gelatin, chitosan and hyaluronic acid are natural components used to prepare polymeric scaffold in tissue engineering. The physical properties of these materials confer an appropriate microenvironment for cells, which can be used as a regeneration system for skin and cartilage. In this work, we prepared and characterized a Gelatin/Chitosan/Hyaluronan lyophilized-polymer. Physical properties of lyophilized-polymer changed slightly with moisture, but when polymer was totally hydrated the elasticity changed significantly. Thermophysical characterisation indicated that temperatures higher than 30ºC could modify irreversibly the polymeric matrix probably due to protein denaturation. Besides, we used the polymer as scaffold to prepare a biosynthetic-skin, reporting biological behaviour and its mechanical properties

Functional Specialization of the Plant miR396 Regulatory Network through Distinct MicroRNA–Target Interactions

MicroRNAs (miRNAs) are ∼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the GROWTH-REGULATING FACTOR (GRF) family of transcription factors, which are known to control cell proliferation in Arabidopsis leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor bHLH74, that is necessary for Arabidopsis normal development. bHLH74 homologs with a miR396 target site could only be detected in the sister families Brassicaceae and Cleomaceae. Still, bHLH74 repression by miR396 is required for margin and vein pattern formation of Arabidopsis leaves. MiR396 contributes to the spatio-temporal regulation of GRF and bHLH74 expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5′ portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in Arabidopsis thaliana revealed that they have an enhanced activity toward GRF transcription factors. The interaction between the GRF target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of GRF2 expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant