Detection of exogenous double-stranded RNA movement in \u3ci\u3ein vitro\u3c/i\u3e peanut plants

New technologies are needed to eliminate mycotoxins and/or fungal pathogens from agricultural products. RNA interference (RNAi) has shown potential to control fungi associated with crops. In RNAi, double-stranded RNA (dsRNA) targets homologous mRNA for cleavage, and can reach the mRNA of pathogens in contact with the plant. The key element in this process is the movement of RNA signals cell-to-cell and over long distances within the plant, and between host plants and parasites. • In this study, we selected a regulatory gene in the aflatoxin biosynthesis pathway, aflS/ aflR, necessary for the production of aflatoxins in Aspergillus spp. We designed a Dicersubstrate RNA (DsiRNA) to study the movement and stability of the duplex over time in in vitro peanut plants using stem-loop primers and RT-PCR for DsiRNA detection. • The preliminary results demonstrated that DsiRNA was absorbed and moved away from the point of application, spread systemically and was transported rapidly, most likely through the phloem of the shoot, to the sink tissues, such as new auxiliary shoots, flowers and newly formed pegs. The DsiRNA remained detectable for at least 30 days after treatment. •This is the first time that movement of exogenous DsiRNA in in vitro peanut plants has been described. Since DsiRNA was detectable in the pegs 15 days after treatment, aflatoxin reduction may be possible if the duplexes containing part of the aflatoxin biosynthesis pathogen gene induce silencing in the peanut seeds colonised by Aspergillus spp. The application of small RNAs could be a non-transformative option for mycotoxin contamination control

Detection of exogenous double-stranded RNA movement in \u3ci\u3ein vitro\u3c/i\u3e peanut plants

• New technologies are needed to eliminate mycotoxins and/or fungal pathogens from agricultural products. RNA interference (RNAi) has shown potential to control fungi associated with crops. In RNAi, double-stranded RNA (dsRNA) targets homologous mRNA for cleavage, and can reach the mRNA of pathogens in contact with the plant. The key element in this process is the movement of RNA signals cell-to-cell and over long distances within the plant, and between host plants and parasites. • In this study, we selected a regulatory gene in the aflatoxin biosynthesis pathway, aflS/aflR, necessary for the production of aflatoxins in Aspergillus spp. We designed a Dicersubstrate RNA (DsiRNA) to study the movement and stability of the duplex over time in in vitro peanut plants using stem-loop primers and RT-PCR for DsiRNA detection. • The preliminary results demonstrated that DsiRNA was absorbed and moved away from the point of application, spread systemically and was transported rapidly, most likely through the phloem of the shoot, to the sink tissues, such as new auxiliary shoots, flowers and newly formed pegs. The DsiRNA remained detectable for at least 30 days after treatment. • This is the first time that movement of exogenous DsiRNA in in vitro peanut plants has been described. Since DsiRNA was detectable in the pegs 15 days after treatment, aflatoxin reduction may be possible if the duplexes containing part of the aflatoxin biosynthesis pathogen gene induce silencing in the peanut seeds colonised by Aspergillus spp. The application of small RNAs could be a non-transformative option for mycotoxin contamination control

Marcadores SSR y EST-SSR aplicados al análisis del genoma de especies silvestres del genero Arachis, y el anfiploide sintético [(A. Correntina x A. Cardenasii) x A. Batizocoi] 4x

Ponencia presentada en XXVIII Jornada Nacional del Maní. General Cabrera, Córdoba, Argentina, 19 de septiembre de 2013Numerosas especies vegetales cultivadas son, desde el punto de vista genético, poliploides naturales. Esta condición presenta tanto ventajas como desventajas, entre éstas, la ocurrencia del aislamiento reproductivo con respecto a los progenitores lo cual, sumado al proceso de domesticación y selección de genotipos superiores de interés productivo, repercute sobre la variabilidad genética. Tal es el caso de los cultivares de maní (Arachis hypogaea L.), que presentan entre otros problemas, alta susceptibilidad a enfermedades y plagas que afectan al rendimiento del cultivo. La sección Arachis, una de las nueve dentro del género Arachis, incluye al 40% de las especies silvestres, y constituyen un reservorio de genes de resistencia. En esta sección, las especies diploides silvestres (2n=20, x=10 y 2n=18, x=9), presentan genomas diferentes denominados A, B, D, F y K, en tanto que, A. hypogaea y A. montícola son las únicas tetraploides, cultivada y silvestre respectivamente. Esta diferencia en los niveles de ploidía e incompatibilidad genómica, dificulta la transferencia de genes de resistencia al maní cultivado, siendo una estrategia posible, la obtención de un anfiploide sintético con 40 cromosomas, a partir de un híbrido diploide. Mediante trabajos de hibridación y retrocruzas, junto a técnicas biotecnológicas, se pueden obtener variedades de maní con atributos deseables, en menor tiempo y costo. Los marcadores moleculares de tipo microsatélites genómicos ─SSRs─ y de secuencias expresadas ─EST-SSRs─, entre otros, permiten asistir la tarea del mejorador, mediante la caracterización genómica de los materiales sintéticos con respecto a sus progenitores, incrementando así la eficiencia y predictibilidad de los resultados. A partir de librerías genómicas tanto de A. hypogaea (genoma AA-BB) como de otras pertenecientes a tribus o secciones relacionadas a Arachis, se han desarrollado cientos de marcadores basados en la reacción en cadena de la polimerasa (PCR) que permiten identificar los genomas propuestos para las especies del género. El objetivo del trabajo fue analizar mediante marcadores SSR y EST-SSR, el genoma de especies silvestres de maní y su permanencia en el híbrido [(A. correntina x A. cardenasii) x A. batizocoi] y en el anfiploide derivado [(A. correntina x A. cardenasii) x A. batizocoi]4x.Fil: Torres, Laura Ester. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: Costero, Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: Teich, Ingrid. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias. Cátedra de Estadística y Biometría; Argentina.Fil: Teich, Ingrid. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.Fil: Taborda, Ricardo Jorge. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: Cisneros, M. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: Franceschini, L. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: De Blas, F. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: Soave, S. J. Criadero El Carmen; Argentina.Fil: Buteler, M. I. Criadero El Carmen; Argentina.Fil: Faustinelli, P. C. Universidad Católica de Córdoba; Argentina.Fil: Faustinelli, P. C. Criadero El Carmen; Argentina

Detection of exogenous double-stranded RNA movement in \u3ci\u3ein vitro\u3c/i\u3e peanut plants

New technologies are needed to eliminate mycotoxins and/or fungal pathogens from agricultural products. RNA interference (RNAi) has shown potential to control fungi associated with crops. In RNAi, double-stranded RNA (dsRNA) targets homologous mRNA for cleavage, and can reach the mRNA of pathogens in contact with the plant. The key element in this process is the movement of RNA signals cell-to-cell and over long distances within the plant, and between host plants and parasites. • In this study, we selected a regulatory gene in the aflatoxin biosynthesis pathway, aflS/ aflR, necessary for the production of aflatoxins in Aspergillus spp. We designed a Dicersubstrate RNA (DsiRNA) to study the movement and stability of the duplex over time in in vitro peanut plants using stem-loop primers and RT-PCR for DsiRNA detection. • The preliminary results demonstrated that DsiRNA was absorbed and moved away from the point of application, spread systemically and was transported rapidly, most likely through the phloem of the shoot, to the sink tissues, such as new auxiliary shoots, flowers and newly formed pegs. The DsiRNA remained detectable for at least 30 days after treatment. •This is the first time that movement of exogenous DsiRNA in in vitro peanut plants has been described. Since DsiRNA was detectable in the pegs 15 days after treatment, aflatoxin reduction may be possible if the duplexes containing part of the aflatoxin biosynthesis pathogen gene induce silencing in the peanut seeds colonised by Aspergillus spp. The application of small RNAs could be a non-transformative option for mycotoxin contamination control

Detection of exogenous double-stranded RNA movement in \u3ci\u3ein vitro\u3c/i\u3e peanut plants

• New technologies are needed to eliminate mycotoxins and/or fungal pathogens from agricultural products. RNA interference (RNAi) has shown potential to control fungi associated with crops. In RNAi, double-stranded RNA (dsRNA) targets homologous mRNA for cleavage, and can reach the mRNA of pathogens in contact with the plant. The key element in this process is the movement of RNA signals cell-to-cell and over long distances within the plant, and between host plants and parasites. • In this study, we selected a regulatory gene in the aflatoxin biosynthesis pathway, aflS/aflR, necessary for the production of aflatoxins in Aspergillus spp. We designed a Dicersubstrate RNA (DsiRNA) to study the movement and stability of the duplex over time in in vitro peanut plants using stem-loop primers and RT-PCR for DsiRNA detection. • The preliminary results demonstrated that DsiRNA was absorbed and moved away from the point of application, spread systemically and was transported rapidly, most likely through the phloem of the shoot, to the sink tissues, such as new auxiliary shoots, flowers and newly formed pegs. The DsiRNA remained detectable for at least 30 days after treatment. • This is the first time that movement of exogenous DsiRNA in in vitro peanut plants has been described. Since DsiRNA was detectable in the pegs 15 days after treatment, aflatoxin reduction may be possible if the duplexes containing part of the aflatoxin biosynthesis pathogen gene induce silencing in the peanut seeds colonised by Aspergillus spp. The application of small RNAs could be a non-transformative option for mycotoxin contamination control

Analysis of small RnA populations generated in peanut leaves after exogenous application of dsRnA and dsDnA targeting aflatoxin synthesis genes

Previously, we have shown that RNA interference (RNAi) can prevent aflatoxin accumulation in transformed peanuts. To explore aflatoxin control by exogenous delivery of double-strand RNA (dsRNA) it is necessary to understand the generation of small RNA (sRNA) populations. We sequenced 12 duplicate sRNA libraries of in-vitro-grown peanut plants, 24 and 48 h after exogenous application of five gene fragments (RNAi-5x) related to aflatoxin biosynthesis in Aspergillus flavus. RNAi-5x was applied either as double-stranded RNA (dsRNA) or RNAi plasmid DNA (dsDNA). Small interfering RNAs (siRNAs) derived from RNAi-5x were significantly more abundant at 48 h than at 24 h, and the majority mapped to the fragment of aflatoxin efflux-pump gene. RNAi-5x-specific siRNAs were significantly, three to fivefold, more abundant in dsDNA than dsRNA treatments. Further examination of known micro RNAs related to disease-resistance, showed significant down-regulation of miR399 and up-regulation of miR482 in leaves treated with dsDNA compared to the control. These results show that sRNA sequencing is useful to compare exogenous RNAi delivery methods on peanut plants, and to analyze the efficacy of molecular constructs to generate siRNAs against specific gene targets. This work lays the foundation for non-transgenic delivery of RNAi in controlling aflatoxins in peanut