RdDM pathway components differentially modulate Tobamovirus symptom development

Small RNAs (sRNAs) are important molecules for gene regulation in plants and play an essential role in plantpathogen interactions. Researchers have evaluated the relationship between viral infections as well as the endogenous accumulation of sRNAs and the transcriptional changes associated with the production of symptoms, but little is known about a possible direct role of epigenetics, mediated by 24-nt sRNAs, in the induction of these symptoms. Using diferent RNA directed DNA methylation (RdDM) pathway mutants and a triple demethylase mutant; here we demonstrate that the disruption of RdDM pathway during viral infection produce alterations in the plant transcriptome and in consequence changes in plant symptoms. This study represents the initial step in exposing that DNA methylation directed by endogenous sRNAs has an important role, uncoupled to defense, in the production of symptoms associated with plant-virus interactions.Fil: Leone, Melisa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina. Ministerio de Ciencia. Tecnología e Innovación Productiva. Agencia Nacional de Promoción Científica y Tecnológica; ArgentinaFil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Venturuzzi, Andrea Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thalianaviral infection

BACKGROUND: Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. RESULTS: This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. CONCLUSIONS: Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.Fil: Rodriguez, Maria Cecilia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Manacorda, Carlos A.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

TMV induces RNA decay pathways to modulate gene silencing and disease symptoms

RNA decay pathways comprise a combination of RNA degradation mechanisms that are implicated in gene expression, development and defense responses in eukaryotes. These mechanisms are known as the RNA Quality Control or RQC pathways. In plants, another important RNA degradation mechanism is the posttranscriptional gene silencing (PTGS) mediated by small RNAs (siRNAs). Notably, the RQC pathway antagonizes PTGS by preventing the entry of dysfunctional mRNAs into the silencing pathway to avoid global degradation of mRNA by siRNAs. Viral transcripts must evade RNA degrading mechanisms, thus viruses encode PTGS suppressor proteins to counteract viral RNA silencing. Here, we demonstrate that tobacco plants infected with TMV and transgenic lines expressing TMV MP and CP (coat protein) proteins (which are not linked to the suppression of silencing) display increased transcriptional levels of RNA decay genes. These plants also showed accumulation of cytoplasmic RNA granules with altered structure, increased rates of RNA decay for transgenes and defective transgene PTGS amplification. Furthermore, knockdown of RRP41 or RRP43 RNA exosome components led to lower levels of TMV accumulation with milder symptoms after infection, several developmental defects and miRNA deregulation. Thus, we propose that TMV proteins induce RNA decay pathways (in particular exosome components) to impair antiviral PTGS and this defensive mechanism would constitute an additional counter-defense strategy that lead to disease symptoms.Inst. de BiotecnologíaFil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Venturuzzi, Andrea Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodriguez, Maria Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Crespi, Martin. Institute of Plant Sciences Paris-Saclay; FranciaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Immune receptor genes and pericentromeric transposons as targets of common epigenetic regulatory elements

Pattern recognition receptors (PRR) and nucleotide-binding leucine-rich repeat proteins (NLR) are major components of the plant immune system responsible for pathogen detection. To date, the transcriptional regulation of PRR/NLR genes is poorly understood. Some PRR/NLR genes are affected by epigenetic changes of neighboring transposable elements (TEs) (cis regulation). We analyzed whether these genes can also respond to changes in the epigenetic marks of distal pericentromeric TEs (trans regulation). We found that Arabidopsis tissues infected with Pseudomonas syringae pv. tomato (Pst) initially induced the expression of pericentromeric TEs, and then repressed it by RNA-directed DNA methylation (RdDM). The latter response was accompanied by the accumulation of small RNAs (sRNAs) mapping to the TEs. Curiously these sRNAs also mapped to distal PRR/NLR genes, which were controlled by RdDM but remained induced in the infected tissues. Then, we used non-infected mom1 (Morpheus’ molecule 1) mutants that expressed pericentromeric TEs to test if they lose repression of PRR/NLR genes. mom1 plants activated several PRR/NLR genes that were unlinked to MOM1-targeted TEs, and showed enhanced resistance to Pst. Remarkably, the increased defenses of mom1 were abolished when MOM1/RdDM-mediated pericentromeric TEs silencing was re-established. Therefore, common sRNAs could control PRR/NLR genes and distal pericentromeric TEs and preferentially silence TEs when they are activated.Fil: Cambiagno, Damián Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Nota, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rius, Sebastian Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; ArgentinaFil: Casati, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alvarez, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Grapevine virus L: a novel vitivirus in grapevine

Vitiviruses are ssRNA(+) viruses in the family Betaflexiviridae (subfamily Trivirinae). There are currently 10 ICTV recognized virus species in the genus; nevertheless, the extended use of NGS technologies is rapidly expanding their diversity and official recognition of six more have been proposed recently. Here, we present the characterization of a novel virus from grapevine, which fits the genomic architecture and evolutionary constraints to be classified within the Vitivirus genus. The detected virus sequence is 7607 nt long, including a typical genome organization of ORFs encoding a replicase (RP), a 22 kDa protein, a movement protein, a coat protein (CP) and a nucleic acid binding protein. Phylogenetic analyses based on the predicted RP and CP proteins unequivocally place the new virus within the Vitivirus genus. Multiple independent RNAseq data confirmed the presence of the detected virus in berries at diverse developmental stages. Additionally, we detected, confirmed, and assembled virus sequences from grapevine samples of distinct cultivars from America, Europe, Asia and Oceania, sharing 74.4%–97.8% nt identity, suggesting that the identified virus is widely distributed and diverse. We propose the name grapevine virus L (GVL) to the detected Vitivirus.Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Soltero Brisbane, Reid. Foundation Plant Services; Estados UnidosFil: Voncina, Darko. University of Zagreb; CroaciaFil: Almeida, Rodrigo P.. University of California at Berkeley; Estados UnidosFil: Blouin, Arnaud G.. No especifíca;Fil: Al Rwahnih, Maher. University of California at Berkeley; Estados UnidosFil: Gómez Talquenca, Sebastián. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

Rooting in Km selective media as efficient in vitro selection method for sunflower genetic transformation

Despite of numerous publications in sunflower genetic transformation, there is no efficient or reproducible protocol with low number of escapes. The latter would indicate that the selection method is not effective. In this work we used Km as selective agent, Agrobacterium tumefaciens EHA105 strain and a vector with the nptII gene under the nos promoter and uidA gene under 35S promoter. The response of agroinfected (A) and control (C) explants during the in vitro culture was studied and in both cases in presence or absence of Km in order to assign a differential morphologic response between transformed and non-transformed plants. The characteristics analyzed were: height, colour/aspect of the plantlets, in vitro rooting and in vitro bud-flower development. Selection was applied from the third regeneration media. Among the A plantlets two were capable of rooting, being positive by PCR, whereas the C were unable to root in presence of Km. One of them gave 6 seeds and in these plants, it was determined the presence of the transgene by PCR and GUS staining. This work shows that in Km selection, colour/aspect of shoots is not useful as selection criteria whereas rooting is an effective selection method in which no escapes were obtained

Rooting in Km selective media as efficient in vitro selection method for sunflower genetic transformation

Despite of numerous publications in sunflower genetic transformation, there is no efficient or reproducible protocol with low number of escapes. The latter would indicate that the selection method is not effective. In this work we used Km as selective agent, Agrobacterium tumefaciens EHA105 strain and a vector with the nptII gene under the nos promoter and uidA gene under 35S promoter. The response of agroinfected (A) and control (C) explants during the in vitro culture was studied and in both cases in presence or absence of Km in order to assign a differential morphologic response between transformed and non-transformed plants. The characteristics analyzed were: height, colour/aspect of the plantlets, in vitro rooting and in vitro bud-flower development. Selection was applied from the third regeneration media. Among the A plantlets two were capable of rooting, being positive by PCR, whereas the C were unable to root in presence of Km. One of them gave 6 seeds and in these plants, it was determined the presence of the transgene by PCR and GUS staining. This work shows that in Km selection, colour/aspect of shoots is not useful as selection criteria whereas rooting is an effective selection method in which no escapes were obtained

Aislamiento y caracterización de regiones promotoras de girasol para dirigir la expresión tejido específica de genes foráneos a semilla

Tesis para obtener el grado de Doctor en el área de Ciencias Biológicas, de la Universidad de Buenos Aires, en 2011Desde el inicio de la utilización de variedades transgénicas, la superficie agrobiotecnológica ha aumentado exponencialmente, con un incremento del orden de 80 veces en solo 14 años, y su uso se ha expandido a más de veinte especies vegetales. Las ventajas obtenidas con estos cultivos transgénicos de primera generación incluyen: mejor control de insectos y malezas, mayor productividad, y una reducción del uso de agroquímicos. Los avances de la biotecnología en los últimos años permitirán, probablemente en un futuro muy cercano, el desarrollo de cultivos con características de interés para los consumidores, relacionadas con una mejora en la calidad nutritiva.. En este sentido las semillas constituyen un blanco ideal para este tipo de cultivos debido a sus propiedades de dormición, almacenamiento de nutrientes y la capacidad de germinar en condiciones limitantes de nutrientes. En este trabajo, se aislaron las regiones promotoras de tres genes específicos de semillas de girasol (Helianthus annuus L. HA89), que codifican para una proteína de transferencia de lípidos (HaAP10), una oleato desaturasa (HaFAD2-1) y una oleosina (HaOLE), las cuales fueron clonadas y caracterizadas “in silico”. Los fragmentos aislados de 964, 867 y 1838pb respectivamente, contienen en sus secuencias varios motivos específicos de semilla como elementos GCN4, motivos (AACA) y (ACTG), Prolamine-Box, E-Box, G-Box, sitos de unión SEF, Sh1 Box y elementos RY/G repetidos entre otros. A su vez, contienen motivos de respuesta a hormonas ABRE y GARE (ácido abscísico y giberélico) relacionados con desarrollo embrionario. Se realizaron análisis funcionales mediante la fusión de los promotores aislados al gen reportero GUS en plantas transgénicas de Arabidopsis y lechuga. No se observó actividad de la proteína reportera en ningún tejido vegetativo exceptuando los cotiledones de plántulas pequeñas. Al ser analizados a lo largo del desarrollo de la semilla, se observó tinción específica restringida a tejido embrionario. En los ensayos fluorométricos cuantitativos se observó actividad en estadios tardíos del desarrollo, similares a los patrones típicos de genes de reserva, siendo el HaFAD2-1 un promotor con una fuerte actividad especifica de semilla, con niveles similares a los del promotor constitutivo 35S. En este trabajo de tesis se confirmó que las regiones promotoras aisladas dirigen la expresión a semilla en forma específica y fuerte, con diferencias temporales, constituyendo, por lo tanto, importantes herramientas que podrán ser de utilidad en futuras aplicaciones biotecnológicas.The successful engineering of genetically modified (GM) crops has been increasing exponentially, and its use has been widely extended in more than 20 species. The advantages obtained with this first generation of transgenics include: herbicide and pathogen tolerance, increased productivity and reduction of agrochemical use. Second generation crops will soon bring to the consumers products with better nutritious properties. In this regard, seeds are natural storage organs that accumulate high levels of lipids and proteins and constitute ideal targets for expression of recombinant proteins to increase nutritional contents and to develop molecular farming strategies. Here, we report the isolation and functional characterization of three novel sunflower seed-specific promoters which enconded for a sunflower lipid transfer protein HaAP10, an oleate desaturase HaFAD2-1 and an oleosin HaOLE. The 5’ fragments of 964 bp, 867 bp and 1838 bp respectively, were isolated, cloned and sequenced. Bioinformatic analyses of these sequences allowed the identification of several cis-elements involved in seed-specific transacting factors such as GCN4 motif, AACA motif, ACGT element E-Boxes, SEF binding sites and Sh1 Box. Among them, it is worth mentioning that ABRE and GARE elements which are related to embryo or seed development, were also identified. Putative promoter regions of these three genes were cloned into expression vector, directing the expression of the GUS gene. Arabidopsis thaliana and Letucce plants were transformed with these constructions. No GUS activity was displayed in any vegetative tissue. Although, promoter activity at different seed developmental stages showed GUS expression in embryos and mature seeds. Quantitative fluorometric assays showed activity on late seed developmental stage, similar to seed storage protein patterns, being the HaFAD2-1 a strong specific seed promoter with similar expression level to the 35S constitutive promoter. In this thesis we confirmed that the isolated regions are strong specific seed promoters, with distinct temporal expression patterns. These promoters represent potencial biotechnological tools that may be useful for future biotechnological applications.Instituto de BiotecnologíaFil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina

MITE Tracker: an accurate approach to identify miniature inverted-repeat transposable elements in large genomes

Abstract Background Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II transposable elements present in a high number of conserved copies in eukaryote genomes. An accurate identification of these elements can help to shed light on the mechanisms controlling genome evolution and gene regulation. The structure and distribution of these elements are well-defined and therefore computational approaches can be used to identify MITEs sequences. Results Here we describe MITE Tracker, a novel, open source software program that finds and classifies MITEs using an efficient alignment strategy to retrieve nearby inverted-repeat sequences from large genomes. This program groups them into high sequence homology families using a fast clustering algorithm and finally filters only those elements that were likely transposed from different genomic locations because of their low scoring flanking sequence alignment. Conclusions Many programs have been proposed to find MITEs hidden in genomes. However, none of them are able to process large-scale genomes such as that of bread wheat. Furthermore, in many cases the existing methods perform high false-positive rates (or miss rates). The rice genome was used as reference to compare MITE Tracker against known tools. Our method turned out to be the most reliable in our tests. Indeed, it revealed more known elements, presented the lowest false-positive number and was the only program able to run with the bread wheat genome as input. In wheat, MITE Tracker discovered 6013 MITE families and allowed the first structural exploration of MITEs in the complete bread wheat genome