Ana J Distéfano

Centro Nacional de Investigaciones Agropecuarias, Buenos Aires, Buenos Aires F.D., Argentina

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Publications (20)39.07 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: Plants employ RNA silencing pathway as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that is induced by sense or inverted repeat transgene in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection.
    Virus Research 12/2013; · 2.75 Impact Factor
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    ABSTRACT: Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate-resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K-means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from glyphosate-resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target-site resistance mechanism.
    Ecology and Evolution 09/2013; 3(10):3388-400. · 1.18 Impact Factor
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    ABSTRACT: Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNA were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse genetic approach.
    Virus Research 04/2013; · 2.75 Impact Factor
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    ABSTRACT: In a large cropping area of northern Argentina, Sorghum halepense (Johnsongrass) has evolved towards glyphosate resistance. This study aimed to determine the molecular and biochemical basis conferring glyphosate resistance in this species. Experiments were conducted to assess target EPSPS gene sequences and (14)C-glyphosate leaf absorption and translocation to meristematic tissues. Individuals of all resistant (R) accessions exhibited significantly less glyphosate translocation to root (11% versus 29%) and stem (9% versus 26%) meristems when compared with susceptible (S) plants. A notably higher proportion of the applied glyphosate remained in the treated leaves of R plants (63%) than in the treated leaves of S plants (27%). In addition, individuals of S. halepense accession R(2) consistently showed lower glyphosate absorption rates in both adaxial (10-20%) and abaxial (20-25%) leaf surfaces compared with S plants. No glyphosate resistance endowing mutations in the EPSPS gene at Pro-101-106 residues were found in any of the evaluated R accessions. The results of the present investigation indicate that reduced glyphosate translocation to meristems is the primary mechanism endowing glyphosate resistance in S. halepense from cropping fields in Argentina. To a lesser extent, reduced glyphosate leaf uptake has also been shown to be involved in glyphosate-resistant S. halepense.
    Pest Management Science 09/2011; 68(3):430-6. · 2.59 Impact Factor
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    Ana J Distéfano, Ivan Bonacic Kresic, H Esteban Hopp
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    ABSTRACT: Cotton blue disease is the most important virus disease of cotton in the southern part of America. The complete nucleotide sequence of the ssRNA genome of the cotton blue disease-associated virus was determined for the first time. It comprised 5,866 nucleotides, and the deduced genomic organization resembled that of members of the genus Polerovirus. Sequence homology comparison and phylogenetic analysis confirm that this virus (previous proposed name cotton leafroll dwarf virus) is a member of a new species within the genus Polerovirus.
    Archives of Virology 11/2010; 155(11):1849-54. · 2.03 Impact Factor
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    ABSTRACT: Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins.
    Virus Research 09/2010; 152(1-2):96-103. · 2.75 Impact Factor
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    ABSTRACT: Mal de Río Cuarto virus (MRCV) is a newly described species of the genus Fijivirus, family Reoviridae. Compared with other plant-infecting genus of the family, the function and localization of MRCV and other Fijivirus proteins are poorly understood. Through analysis of viral particle purifications, we positively identified five structural proteins of approximately 170, 140, 130, 66, and 62 kDa. The protein encoded by MRCV S3 genomic segment was expressed as a fusion protein in Escherichia coli, purified and used for rabbit immunization. The resulting antiserum reacted with the 140 kDa structural protein and with incomplete virus particles in immunoelectron microscopy assays, suggesting that MRCV S3 codes for the major core capsid protein.
    Virus Genes 04/2009; 38(3):455-60. · 1.77 Impact Factor
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    ABSTRACT: Micro RNAs (miRs) constitute a large group of endogenous small RNAs that have crucial roles in many important plant functions. Virus infection and transgenic expression of viral proteins alter accumulation and activity of miRs and so far, most of the published evidence involves post-transcriptional regulations. Using transgenic plants expressing a reporter gene under the promoter region of a characterized miR (P-miR164a), we monitored the reporter gene expression in different tissues and during Arabidopsis development. Strong expression was detected in both vascular tissues and hydathodes. P-miR164a activity was developmentally regulated in plants with a maximum expression at stages 1.12 to 5.1 (according to Boyes, 2001) along the transition from vegetative to reproductive growth. Upon quantification of P-miR164a-derived GUS activity after Tobacco mosaic virus Cg or Oilseed rape mosaic virus (ORMV) infection and after hormone treatments, we demonstrated that ORMV and gibberellic acid elevated P-miR164a activity. Accordingly, total mature miR164, precursor of miR164a and CUC1 mRNA (a miR164 target) levels increased after virus infection and interestingly the most severe virus (ORMV) produced the strongest promoter induction. This work shows for the first time that the alteration of miR pathways produced by viral infections possesses a transcriptional component. In addition, the degree of miR alteration correlates with virus severity since a more severe virus produces a stronger P-miR164a induction.
    BMC Plant Biology 01/2009; 9:152. · 4.35 Impact Factor
  • BMC Plant Biology 01/2009; · 4.35 Impact Factor
  • el II Congreso Internacional-REDBIO-Argentina “LA BIOTECNOLOGÍA y LOS FUTUROS ESCENARIOS MUNDIALES”; 01/2009
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    ABSTRACT: The aim of this study was to characterize the dehydrin content in mature embryos of two quinoa cultivars, Sajama and Baer La Unión. Cultivar Sajama grows at 3600-4000 m altitude and is adapted to the very arid conditions characteristic of the salty soils of the Bolivian Altiplano, with less than 250 mm of annual rain and a minimum temperature of -1 degrees C. Cultivar Baer La Unión grows at sea-level regions of central Chile and is adapted to more humid conditions (800 to 1500 mm of annual rain), fertile soils, and temperatures above 5 degrees C. Western blot analysis of embryo tissues from plants growing under controlled greenhouse conditions clearly revealed the presence of several dehydrin bands (at molecular masses of approximately 30, 32, 50, and 55 kDa), which were common to both cultivars, although the amount of the 30 and 32 kDa bands differed. Nevertheless, when grains originated from their respective natural environments, three extra bands (at molecular masses of approximately 34, 38, and 40 kDa), which were hardly visible in Sajama, and another weak band (at a molecular mass of approximately 28 kDa) were evident in Baer La Unión. In situ immunolocalization microscopy detected dehydrin-like proteins in all axis and cotyledon tissues. At the subcellular level, dehydrins were detected in the plasma membrane, cytoplasm and nucleus. In the cytoplasm, dehydrins were found associated with mitochondria, rough endoplasmic reticulum cisternae, and proplastid membranes. The presence of dehydrins was also recognized in the matrix of protein bodies. In the nucleus, dehydrins were associated with the euchromatin. Upon examining dehydrin composition and subcellular localization in two quinoa cultivars belonging to highly contrasting environments, we conclude that most dehydrins detected here were constitutive components of the quinoa seed developmental program, but some of them (specially the 34, 38, and 40 kDa bands) may reflect quantitative molecular differences associated with the adaptation of both cultivars to contrasting environmental conditions.
    Protoplasma 01/2008; 233(1-2):149-56. · 2.86 Impact Factor
  • Ref. No: INPI Nº P040100887., Year: 01/2008
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    ABSTRACT: Euterpe edulis Martius, a tropical palm species characterized as highly recalcitrant, accumulated dehydrin proteins in both the endosperm and the embryo of the mature seed, as detected by Western blot analysis and immunogold electron microscopy. Three major bands at molecular masses of approximately 16, 18, and 24 kDa were identified in both samples analysed. Immunogold electron microscopy studies detected the presence of dehydrins in the embryo and endosperm. In both cases, dehydrins were immunolocalized in cytoplasm and chromatin. No labelling associated with either membranes or organelles was detected. It is known that dehydrins are produced as part of the developmental program of orthodox seeds and are also present in some recalcitrant seeds of temperate regions. The constitutive presence of dehydrins in embryos of extremely recalcitrant species of tropical origin has not been previously reported.
    Protoplasma 02/2007; 231(1-2):1-5. · 2.86 Impact Factor
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    ABSTRACT: The nucleotide sequences of genomic segments S7 and S9 of Mal de Río Cuarto virus (MRCV, Fijivirus group II) have been determined, thus completing the entire genome sequence of the virus. These segments showed a non-overlapping bicistronic structure, as in other members of the genus. MRCV S7 ORF-1 had a length of 1086 bp and encoded a 41.5 kDa putative polypeptide, whereas MRCV S7 ORF-2 had a length of 930 bp and encoded a 36.8 kDa putative polypeptide. Proteins of 39 and 20.5 kDa were predicted for the 1014 bp long MRCV S9 ORF-1 and the 537 bp long MRCV S9 ORF-2, respectively. The terminal 5' and 3' sequences of both segments were 5'AAGUUUUU3' and 5'CAGCUnnnGUC3', respectively. Specific imperfect inverted repeats of each segment were identified. Comparison of the predicted proteins with those of related virus genome segments counterparts in maize rough dwarf virus (MRDV) and rice black streaked dwarf virus (RBSDV), showed 64.5-44.3% identities. These values are lower than those resulting from comparisons between MRDV and RBSDV. The topology of the trees obtained using the complete nucleotide and amino acid sequences of MRCV S7 and MRCV S9 was consistent with the analysis of the other MRCV segments previously published.
    Archives of Virology 02/2007; 152(3):565-73. · 2.03 Impact Factor
  • A J Distéfano, H E Hopp, M del Vas
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    ABSTRACT: Mal de Rio Cuarto virus (MRCV) was recently described as a new species of the genus Fijivirus, family Reoviridae. The nucleotide sequence of two MRCV genome segments was determined. MRCV S5 and S10 were predicted to encode proteins of 106.9 and 63.5 kDa respectively. The protein coded by MRCV S5 had 62.8% and 35.7% identity to fijiviruses RBSDV S5 and FDV S5 coded proteins, and contained a rarely reported type-1 C-terminal peroxisomal targeting signal. The protein coded by MRCV S10 had identity levels of 72.4% and 21.7% to the major outer capsid proteins of fijiviruses RBSDV S10 and NLRV S8.
    Archives of Virology 07/2005; 150(6):1241-8. · 2.03 Impact Factor
  • VIII Congreso Nacional de Maíz; 01/2005
  • V Encuentro Latinoamericano de Biotecnología Agrícola, REDBIO2004; 01/2004
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    ABSTRACT: Mal de Río Cuarto virus (MRCV) is a newly described species of the genus Fijivirus, family Reoviridae. The nucleotide sequence of four MRCV genome segments was determined. MRCV S1, S2, S3 and S6 were predicted to encode proteins of 168.4, 134.4, 141.7 and 90 kDa, respectively. MRCV S1 encodes a basic protein that contains conserved RNA-dependent RNA polymerase motifs, and is homologous to Rice black streaked dwarf virus (RBSDV), Fiji disease virus (FDV) and Nilaparvata lugens reovirus (NLRV) polymerases as well as to corresponding proteins of members of other genera of the Reoviridae. MRCV S2 codes for a protein with intermediate homology to the ones coded by RBSDV S4 and FDV S3 'B' spike, which is presumably the B-spike protein. MRCV S3 most probably encodes the major core protein and is highly homologous to corresponding proteins of RBSDV S2 and FDV S3. MRCV S6-encoded protein has low homology to the proteins of unknown function coded by RBSDV S6 and FDV S6. The identity levels between all analyzed MRCV coded proteins and their RBSDV counterparts varied between 84.5 and 44.8%. The analysis of the reported sequences allowed a phylogenetic comparison of MRCV with other reovirus and supported its taxonomic status within the genus.
    Virus Research 04/2003; 92(1):113-21. · 2.75 Impact Factor
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    ABSTRACT: This is the first sequence-based characterization of Mal de Río Cuarto virus (MRCV), currently classified as a variant of Maize rough dwarf virus (MRDV) and exclusively found in South America. We sequenced and analyzed genome segments S4 and S8. MRCV S4 coded for a putative 131.67 kDa protein while MRCV S8 coded for a putative 68.26 kDa protein containing an ATP/GTP-binding motif. The 5' and 3' ends of MRCV segments, were 5'AAGUUUUU3' and 5'CAGCUnnnGUC3', respectively. Prediction of secondary structure of both segments coding strands showed that terminal regions were able to form structures that are proposed to be replication and packaging signals. MRCV S4 showed identity to members of Fijivirus as well as to two other genera of the Reoviridae family. MRCV S8 revealed identity with Rice black streaked dwarf virus (RBSDV) S8, MRDV S7, Oat sterile dwarf virus (OSDV) S9 and Nilaparvata lugens reovirus (NLRV) S7. While MRDV and RBSDV segments are highly homologous between each other, MRCV identity levels with them was considerably lower. We discussed the evolutionary relationships of MRCV to other Reoviridae, and based on phylogenetic analysis we proposed that although MRCV is related to MRDV, it could be regarded as a new species of the Fijivirus genus.
    Archives of Virology 10/2002; 147(9):1699-709. · 2.03 Impact Factor
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    ABSTRACT: Genetically modified organisms, GMO, Living modified organisms, LMO, PCR, Transgenic.
    Electronic Journal of Biotechnology (Chile) Num.2 Vol.3.

Publication Stats

121 Citations
187 Downloads
1k Views
39.07 Total Impact Points

Institutions

  • 2010–2013
    • Centro Nacional de Investigaciones Agropecuarias
      Buenos Aires, Buenos Aires F.D., Argentina
  • 2008–2010
    • Instituto Nacional de Tecnología Agropecuaria
      • Instituto de Biotecnología
      Buenos Aires, Buenos Aires F.D., Argentina
  • 2007
    • University of Buenos Aires
      Buenos Aires, Buenos Aires F.D., Argentina