Juan Jose Contreras Garcia - Academia.edu (original) (raw)
Papers by Juan Jose Contreras Garcia
Spanish Journal of Psychology, 2011
In 1999, Wilkinson and the Task Force on Statistical Inference published "Statistical Methods and... more In 1999, Wilkinson and the Task Force on Statistical Inference published "Statistical Methods and Psychology: Guidelines and Explanation." The authors made several recommendations about how to improve the quality of Psychology research papers. One of these was to report some effect-size index in the results of the research. In 2001, the fifth edition of the Publication Manual of the American Psychological Association included this recommendation. In Spain, in 2003, scientific journals like Psicothema or the International Journal of Clinical and Health Psychology (IJCHP) published editorials and papers expressing the need to calculate the effect size in the research papers. The aim of this study is to determine whether the papers published from 2003 to 2008 in the four Spanish journals indexed in the Journal Citation Reports have reported some effect-size index of their results. The findings indicate that, in general, the followup of the norm has been scanty, though the evolution over the analyzed period is different depending on the journal.
Spanish Journal of Psychology, 2012
Entrepreneurship research is receiving increasing attention in our context, as entrepreneurs are ... more Entrepreneurship research is receiving increasing attention in our context, as entrepreneurs are key social agents involved in economic development. We compare the success of the dichotomic logistic regression model and the Bayes simple classifier to predict entrepreneurship, after manipulating the percentage of missing data and the level of categorization in predictors. A sample of undergraduate university students (= 1230) completed five scales (motivation, attitude towards business creation, obstacles, deficiencies, and training needs) and we found that each of them predicted different aspects of the tendency to business creation. Additionally, our results show that the receiver operating characteristic (ROC) curve is affected by the rate of missing data in both techniques, but logistic regression seems to be more vulnerable when faced with missing data, whereas Bayes nets underperform slightly when categorization has been manipulated. Our study sheds light on the potential entrepreneur profile and we propose to use Bayesian networks as an additional alternative to overcome the weaknesses of logistic regression when missing data are present in applied research.
PloS one, 2014
Plum pox virus (PPV) infects Prunus trees around the globe, posing serious fruit production probl... more Plum pox virus (PPV) infects Prunus trees around the globe, posing serious fruit production problems and causing severe economic losses. One variety of Prunus domestica, named…
PloS one, 2014
The aim of this study was to compare the composition of two deep-sea viral communities obtained f... more The aim of this study was to compare the composition of two deep-sea viral communities obtained from the Romanche Fracture Zone in the Atlantic Ocean (collected at 5200 m depth) and the southwest Mediterranean Sea (from 2400 m depth) using a pyro-sequencing approach. The results are based on 18.7% and 6.9% of the sequences obtained from the Atlantic Ocean and the Mediterranean Sea, respectively, with hits to genomes in the non-redundant viral RefSeq database. The identifiable richness and relative abundance in both viromes were dominated by archaeal and bacterial viruses accounting for 92.3% of the relative abundance in the Atlantic Ocean and for 83.6% in the Mediterranean Sea. Despite characteristic differences in hydrographic features between the sampling sites in the Atlantic Ocean and the Mediterranean Sea, 440 virus genomes were found in both viromes. An additional 431 virus genomes were identified in the Atlantic Ocean and 75 virus genomes were only found in the Mediterranean ...
Plant methods, 2014
Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they a... more Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent protein genes are among the most commonly used reporters in transient RNA silencing and heterologous protein expression assays. Fluorescence intensity is used to quantify fluorescent protein accumulation by image analysis or spectroscopy of protein extracts; however, these methods might not be suitable for medium- to large-scale comparisons. We report that laser scanners, used routinely in proteomic studies, are suitable for quantitative imaging of plant leaves that express different fluorescent protein pairs. We developed a microtiter plate fluorescence spectroscopy method for direct quantitative comparison of fluorescent protein accumulation in intact leaf discs. We used this t...
Molecular plant-microbe interactions : MPMI, 2014
Research performed on model herbaceous hosts has been useful to unravel the molecular mechanisms ... more Research performed on model herbaceous hosts has been useful to unravel the molecular mechanisms that control viral infections. The most common Plum pox virus (PPV) strains are able to infect Nicotiana species as well as Chenopodium and Arabidopsis species. However, isolates belonging to strain C (PPV-C) that have been adapted to Nicotiana spp. are not infectious either in Chenopodium foetidum or in Arabidopsis thaliana. In order to determine the mechanism underlying this interesting host-specific behavior, we have constructed chimerical clones derived from Nicotiana-adapted PPV isolates from the D and C strains, which differ in their capacity to infect A. thaliana and C. foetidum. With this approach, we have identified the nuclear inclusion a protein (VPg+Pro) as the major pathogenicity determinant that conditions resistance in the presence of additional secondary determinants, different for each host. Genome-linked viral protein (VPg) mutations similar to those involved in the bre...
Virology, Jan 3, 2015
The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serin... more The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serine proteases present in Potyviridae family members. P1a is located at the N-terminal end of the viral polyprotein, and is closely related to potyviral P1 protease. For its proteolytic activity, P1a requires a still unknown host factor; this might be linked to involvement in host specificity. Here we built a series of constructs and chimeric viruses to help elucidate the role of P1a cleavage in host range definition. We demonstrate that host-dependent separation of P1a from the remainder of the polyprotein is essential for suppressing RNA silencing defenses and for efficient viral infection. These findings support the role of viral proteases as important determinants in host adaptation.
Reis, 1995
Uno de los problemas más difíciles de abordar en el estudio psicosocial del «prejuicio» durante e... more Uno de los problemas más difíciles de abordar en el estudio psicosocial del «prejuicio» durante el presente siglo ha sido, sin duda, el de explicar los repentinos cambios que el sujeto moderno ha experimentado en el proceso de «internacionalización» de sus coordenadas espaciales. Tratando esta cuestión, el ejemplo de la Alemania de entreguerras brota inmediatamente en la imaginación del investigador. Pero el problema no debiera ser acotado por sus casos más dramáticos, sino más bien por la generalidad y frecuencia de su aparición en la era de la Modernidad. Pudiera no ser casual que uno de los rasgos caracterizadores de la «época del nacionalismo» (Kohn, 1949) haya sido la tendencia periódica a anunciar su final, como diversos autores han señalado recientemente. En este ensayo quisiera repensar las discutibles premisas que el estudio del nacionalismo ha asumido tradicionalmente para abordar esta cuestión. Frente
RNA, 2011
RNA silencing mediated by siRNAs plays an important role as an anti-viral defense mechanism in pl... more RNA silencing mediated by siRNAs plays an important role as an anti-viral defense mechanism in plants and other eukaryotic organisms, which is usually counteracted by viral RNA silencing suppressors (RSSs). The ipomovirus Cucumber vein yellowing virus (CVYV) lacks the typical RSS of members of the family Potyviridae, HCPro, which is replaced by an unrelated RSS, P1b. CVYV P1b resembles potyviral HCPro in forming complexes with synthetic siRNAs in vitro. Electrophoretic mobility shift assays showed that P1b, like potyviral HCPro, interacts with double-stranded siRNAs, but is not able to bind single-stranded small RNAs or small DNAs. These assays also showed a preference of CVYV P1b for binding to 21-nt siRNAs, a feature also reported for HCPro. However, these two potyvirid RSSs differ in their requirements of 2-nucleotide (nt) 3′ overhangs and 5′ terminal phosphoryl groups for siRNA binding. Copurification assays confirmed in vivo P1b–siRNA interactions. We have demonstrated by deep ...
Molecular Plant-Microbe Interactions®, 2012
Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silen... more Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silencing suppressors (RSS) to counteract antiviral RNA silencing responses. HCPro is the RSS of Potyvirus spp., and its activity is enhanced by the upstream P1 protein. Distinctively, the ipomovirus Cucumber vein yellowing virus (CVYV) lacks HCPro but contains two P1 copies in tandem (P1aP1b), the second of which functions as RSS. Using chimeras based on the potyvirus Plum pox virus (PPV), we found that P1b can functionally replace HCPro in potyviral infections of Nicotiana plants. Interestingly, P1a, the CVYV protein homologous to potyviral P1, disrupted the silencing suppression activity of P1b and reduced the infection efficiency of PPV in Nicotiana benthamiana. Testing the influence of RSS in host specificity, we found that a P1b-expressing chimera poorly infected PPV's natural host, Prunus persica. Conversely, P1b conferred on PPV chimeras the ability to replicate locally in cucumbe...
Molecular Plant-Microbe Interactions®, 2013
Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specif... more Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N terminus of the coat protein (CP) includes host-specific pathogenicity determinants. Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3′ region of PPV-D (including the region coding for the CP) in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and N. benthamiana when specific amino acids were modified or deleted in a short 30-amino-acid region of the N terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus spp. although others impaired systemic infection in this host. We propose a model in which the N terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanis...
Molecular Plant-Microbe Interactions®, 1999
Plum pox potyvirus (PPV) infection of transgenic Nicotiana benthamiana plants that expressed the ... more Plum pox potyvirus (PPV) infection of transgenic Nicotiana benthamiana plants that expressed the PPV NIb RNA replicase carrying a Gly to Val mutation at the GDD motif (NIbV lines) induced a phenotype of virus resistance and transgene silencing, which was not transmissible to the progeny after self-fertilization (H. S. Guo and J. A. García, Mol. Plant-Microbe Interact. 10:160-170, 1997). Here, we demonstrate that the induced resistance of NIbV plants is mitotically stable after plant propagation by grafting and by in vitro regeneration. Virus replication or residual virus RNA seem not to be required to maintain transgene silencing and virus resistance. Analysis by PCR (polymerase chain reaction) amplification after treatment with methylation-sensitive restriction nucleases indicates that DNA methylation is associated with establishment and maintenance of transgene silencing and virus resistance. Restoration of transgene activity and susceptibility to PPV in sexual progeny correlated ...
Molecular Plant Pathology, 2012
Subisolates segregated from a M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in patho... more Subisolates segregated from a M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the HCPro protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in some subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings, suggested the existence of uncharacterized attenuating factors in these subisolates. In this study we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host.
Molecular Plant Pathology, 2014
Taxonomic relationships: Plum pox virus (PPV) is a member of the genus Potyvirus in the family Po... more Taxonomic relationships: Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae. PPV diversity is structured into at least eight monophyletic strains. Geographical distribution: First discovered in Bulgaria, PPV is nowadays present in most of continental Europe (with an endemic status in many central and southern European countries) and has progressively spread to many countries on other continents. Genomic structure: Typical of potyviruses, the PPV genome is a positive-sense single-stranded RNA (ssRNA), with a protein linked to its 5' end and a 3'-terminal poly A tail. It is encapsidated by a single type of capsid protein (CP) in flexuous rod particles and is translated into a large polyprotein which is proteolytically processed in at least 10 final products: P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIapro, NIb and CP. In addition, P3N-PIPO is predicted to be produced by a translational frameshift. Pathogenicity features: PPV causes sharka, the most damaging viral disease of stone fruit trees. It also infects wild and ornamental Prunus trees and has a large experimental host range in herbaceous species. PPV spreads over long distances by uncontrolled movement of plant material, and many species of aphid transmit the virus locally in a nonpersistent manner. Sources of resistance: A few natural sources of resistance to PPV have been found so far in Prunus species, which are being used in classical breeding programmes. Different genetic engineering approaches are being used to generate resistance to PPV, and a transgenic plum, 'HoneySweet', transformed with the viral CP gene, has demonstrated high resistance to PPV in field tests in several countries and has obtained regulatory approval in the USA.
Journal of Virology, 2011
Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the F... more Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genet...
Journal of General Virology, 2012
HCPro, the RNA-silencing suppressor (RSS) of viruses belonging to the genus Potyvirus in the fami... more HCPro, the RNA-silencing suppressor (RSS) of viruses belonging to the genus Potyvirus in the family Potyviridae, is a multifunctional protein presumably involved in all essential steps of the viral infection cycle. Recent studies have shown that plum pox potyvirus (PPV) HCPro can be replaced successfully by cucumber vein yellowing ipomovirus P1b, a sequence-unrelated RSS from a virus of the same family. In order to gain insight into the requirement of a particular RSS to establish a successful potyviral infection, we tested the ability of different heterologous RSSs from both plant- and animal-infecting viruses to substitute for HCPro. Making use of engineered PPV chimeras, we show that PPV HCPro can be replaced functionally by some, but not all, unrelated RSSs, including the NS1 protein of the mammal-infecting influenza A virus. Interestingly, the capacity of a particular RSS to replace HCPro does not correlate strictly with its RNA silencing-suppression strength. Altogether, our r...
BMC Plant Biology, 2010
Background Plant genomes have been transformed with full-length cDNA copies of viral genomes, giv... more Background Plant genomes have been transformed with full-length cDNA copies of viral genomes, giving rise to what has been called 'amplicon' systems, trying to combine the genetic stability of transgenic plants with the elevated replication rate of plant viruses. However, amplicons' performance has been very variable regardless of the virus on which they are based. This has boosted further interest in understanding the underlying mechanisms that cause this behavior differences, and in developing strategies to control amplicon expression. Results Nicotiana benthamiana plants were transformed with an amplicon consisting of a full-length cDNA of the potyvirus Plum pox virus (PPV) genome modified to include a GFP reporter gene. Amplicon expression exhibited a great variability among different transgenic lines and even among different plants of the same line. Plants of the line 10.6 initially developed without signs of amplicon expression, but at different times some of them ...
Biotechnology Journal, 2006
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2011
One of the challenges being faced in the twenty-first century is the biological control of 19 pla... more One of the challenges being faced in the twenty-first century is the biological control of 19 plant viral infections. Among the different strategies to combat virus infections, those 20 based on pathogen-derived resistance (PDR) are probably the most powerful approaches 21 to confer virus resistance in plants. The application of the PDR concept not only 22 revealed the existence of a previously unknown sequence-specific RNA-degradation 23 mechanism in plants, but has also helped to design antiviral strategies to engineer viral 24 resistant plants in the last 25 years. In this article, we review the different platforms 25 related to RNA silencing that have been developed during this time to obtain plants 26 resistant to viruses and illustrate examples of current applications of RNA silencing to 27 protect crop plants against viral diseases of agronomic relevance. 28 29 1. Introduction 30 Plant viruses represent important threats to modern agriculture. Although accurate 31 figures for crop losses due to viruses are not available, it is generally accepted that 32 among the different plant pathogens, the economic relevance of viruses comes second to 33 fungi. Until the emergence of genetic engineering technologies, plant viruses have been 34 partially controlled using conventional cultivation techniques such as crop rotation, 35 early detection and eradication of the diseased plants, cross protection, breeding for 36 resistance, or chemical control of their vectors [1]. In the 1980s, the successful transfer 37 of foreign DNA into the nuclear genome using Agrobacterium as a vector prompted the 38 introduction of genetic engineering for crop improvement and the development of virus-39 resistant plants [2, 3]. Today, different antiviral strategies are being undertaken, either 40 by exploiting natural plant defence mechanisms, or designing new tools, which in most 41 cases are ultimately also based on natural defence mechanisms. 42 Most of the achievements obtained in plant biotechnology in the area of plant virus 43 resistance are based on the principle of pathogen-derived resistance (PDR) [4]. The 44 concept of PDR was proposed by Sanford and Johnston [5] twenty-five years ago using 45 the bacteriophage Qß as a model, and considers that expression of pathogen genetic 46 elements outside the context of infection may lead to resistance. This approach opened 47 an interesting possibility for the practical control of diseases. For plant viruses, the 48 concept of PDR was first validated with its use in tobacco plants transformed with the 49 tobamovirus Tobacco mosaic virus (TMV) coat protein (CP) gene [6]. Soon this 50 observation was validated using other viral CPs and other viral sequences that code for 51 proteins such as replicases, proteinases and movement proteins [for review, see 7-11]. 52 CP is the most successful and widely applied viral protein for PDR. However, the 53 protection conferred by CP-mediated resistance varies significantly from strong 54 interference with virus multiplication to delay or attenuation of symptoms. The PDR 55 based on the expression of viral proteins, with either the wild type or the mutated one, 56 in transgenic plants has several general characteristics: i) it is not very specific, and 57 protects against a broad range of viral strains; ii) it shows a positive correlation between 58 the levels of accumulation of the viral product and the effectiveness in resistance; iii) it 59 is usually overcome by high doses of inoculum. Despite extensive studies, the 60 molecular mechanisms underlying protein-mediated resistance are not fully understood. 61 What appears to be certain is that they are diverse, that they probably affect several 62 steps of the infection process, and that each virus/transgenic plant combination has 63 specific features. Moreover, it soon became apparent that many virus resistances 64 initially envisaged as protein-mediated PDR did not rely on the expression of the 65 corresponding viral proteins and that a majority of PDR phenomena seemed to work 66 through RNA-mediated mechanisms [12]. 67 68 2. RNA silencing and virus resistance 69 In the early nineties, two independent research groups found that the expression of 70 a transgene mRNA with a high sequence similarity to an endogenous mRNA, led to 71 specific degradation of both mRNAs through post-transcriptional gene silencing 72 (PTGS), also known as "cosuppression" [13, 14]. Later, the W. Dougherty research 73 group suggested that a similar mechanism might be involved in the resistance 74 phenomena observed in transgenic plants transformed with viral genes. Some of the 75 transgenic lines showed anomalous phenotypes; unexpectedly and unpredictably the 76 highest level of resistance was observed in the transgenic lines showing very low levels 77 of transgene mRNA accumulation, whereas plant lines expressing the same gene at high 78 levels were fully susceptible. Interestingly, the virus resistant plants had actively 79 transcribed genes but they had low steady-state levels of transgene mRNA. A 80 breakthrough discovery, from transgenic lines included to serve as negative controls, 81 showed that resistance occurred even with non-translatable versions of the viral genes, 82 which demonstrated that the RNA itself was responsible for the virus resistance 83 observed in the transgenic plants [15-17]. All the molecular analysis of these transgenic 84 plants challenged the existing paradigm of genetic regulation and became the first 85
Spanish Journal of Psychology, 2011
In 1999, Wilkinson and the Task Force on Statistical Inference published "Statistical Methods and... more In 1999, Wilkinson and the Task Force on Statistical Inference published "Statistical Methods and Psychology: Guidelines and Explanation." The authors made several recommendations about how to improve the quality of Psychology research papers. One of these was to report some effect-size index in the results of the research. In 2001, the fifth edition of the Publication Manual of the American Psychological Association included this recommendation. In Spain, in 2003, scientific journals like Psicothema or the International Journal of Clinical and Health Psychology (IJCHP) published editorials and papers expressing the need to calculate the effect size in the research papers. The aim of this study is to determine whether the papers published from 2003 to 2008 in the four Spanish journals indexed in the Journal Citation Reports have reported some effect-size index of their results. The findings indicate that, in general, the followup of the norm has been scanty, though the evolution over the analyzed period is different depending on the journal.
Spanish Journal of Psychology, 2012
Entrepreneurship research is receiving increasing attention in our context, as entrepreneurs are ... more Entrepreneurship research is receiving increasing attention in our context, as entrepreneurs are key social agents involved in economic development. We compare the success of the dichotomic logistic regression model and the Bayes simple classifier to predict entrepreneurship, after manipulating the percentage of missing data and the level of categorization in predictors. A sample of undergraduate university students (= 1230) completed five scales (motivation, attitude towards business creation, obstacles, deficiencies, and training needs) and we found that each of them predicted different aspects of the tendency to business creation. Additionally, our results show that the receiver operating characteristic (ROC) curve is affected by the rate of missing data in both techniques, but logistic regression seems to be more vulnerable when faced with missing data, whereas Bayes nets underperform slightly when categorization has been manipulated. Our study sheds light on the potential entrepreneur profile and we propose to use Bayesian networks as an additional alternative to overcome the weaknesses of logistic regression when missing data are present in applied research.
PloS one, 2014
Plum pox virus (PPV) infects Prunus trees around the globe, posing serious fruit production probl... more Plum pox virus (PPV) infects Prunus trees around the globe, posing serious fruit production problems and causing severe economic losses. One variety of Prunus domestica, named…
PloS one, 2014
The aim of this study was to compare the composition of two deep-sea viral communities obtained f... more The aim of this study was to compare the composition of two deep-sea viral communities obtained from the Romanche Fracture Zone in the Atlantic Ocean (collected at 5200 m depth) and the southwest Mediterranean Sea (from 2400 m depth) using a pyro-sequencing approach. The results are based on 18.7% and 6.9% of the sequences obtained from the Atlantic Ocean and the Mediterranean Sea, respectively, with hits to genomes in the non-redundant viral RefSeq database. The identifiable richness and relative abundance in both viromes were dominated by archaeal and bacterial viruses accounting for 92.3% of the relative abundance in the Atlantic Ocean and for 83.6% in the Mediterranean Sea. Despite characteristic differences in hydrographic features between the sampling sites in the Atlantic Ocean and the Mediterranean Sea, 440 virus genomes were found in both viromes. An additional 431 virus genomes were identified in the Atlantic Ocean and 75 virus genomes were only found in the Mediterranean ...
Plant methods, 2014
Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they a... more Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent protein genes are among the most commonly used reporters in transient RNA silencing and heterologous protein expression assays. Fluorescence intensity is used to quantify fluorescent protein accumulation by image analysis or spectroscopy of protein extracts; however, these methods might not be suitable for medium- to large-scale comparisons. We report that laser scanners, used routinely in proteomic studies, are suitable for quantitative imaging of plant leaves that express different fluorescent protein pairs. We developed a microtiter plate fluorescence spectroscopy method for direct quantitative comparison of fluorescent protein accumulation in intact leaf discs. We used this t...
Molecular plant-microbe interactions : MPMI, 2014
Research performed on model herbaceous hosts has been useful to unravel the molecular mechanisms ... more Research performed on model herbaceous hosts has been useful to unravel the molecular mechanisms that control viral infections. The most common Plum pox virus (PPV) strains are able to infect Nicotiana species as well as Chenopodium and Arabidopsis species. However, isolates belonging to strain C (PPV-C) that have been adapted to Nicotiana spp. are not infectious either in Chenopodium foetidum or in Arabidopsis thaliana. In order to determine the mechanism underlying this interesting host-specific behavior, we have constructed chimerical clones derived from Nicotiana-adapted PPV isolates from the D and C strains, which differ in their capacity to infect A. thaliana and C. foetidum. With this approach, we have identified the nuclear inclusion a protein (VPg+Pro) as the major pathogenicity determinant that conditions resistance in the presence of additional secondary determinants, different for each host. Genome-linked viral protein (VPg) mutations similar to those involved in the bre...
Virology, Jan 3, 2015
The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serin... more The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serine proteases present in Potyviridae family members. P1a is located at the N-terminal end of the viral polyprotein, and is closely related to potyviral P1 protease. For its proteolytic activity, P1a requires a still unknown host factor; this might be linked to involvement in host specificity. Here we built a series of constructs and chimeric viruses to help elucidate the role of P1a cleavage in host range definition. We demonstrate that host-dependent separation of P1a from the remainder of the polyprotein is essential for suppressing RNA silencing defenses and for efficient viral infection. These findings support the role of viral proteases as important determinants in host adaptation.
Reis, 1995
Uno de los problemas más difíciles de abordar en el estudio psicosocial del «prejuicio» durante e... more Uno de los problemas más difíciles de abordar en el estudio psicosocial del «prejuicio» durante el presente siglo ha sido, sin duda, el de explicar los repentinos cambios que el sujeto moderno ha experimentado en el proceso de «internacionalización» de sus coordenadas espaciales. Tratando esta cuestión, el ejemplo de la Alemania de entreguerras brota inmediatamente en la imaginación del investigador. Pero el problema no debiera ser acotado por sus casos más dramáticos, sino más bien por la generalidad y frecuencia de su aparición en la era de la Modernidad. Pudiera no ser casual que uno de los rasgos caracterizadores de la «época del nacionalismo» (Kohn, 1949) haya sido la tendencia periódica a anunciar su final, como diversos autores han señalado recientemente. En este ensayo quisiera repensar las discutibles premisas que el estudio del nacionalismo ha asumido tradicionalmente para abordar esta cuestión. Frente
RNA, 2011
RNA silencing mediated by siRNAs plays an important role as an anti-viral defense mechanism in pl... more RNA silencing mediated by siRNAs plays an important role as an anti-viral defense mechanism in plants and other eukaryotic organisms, which is usually counteracted by viral RNA silencing suppressors (RSSs). The ipomovirus Cucumber vein yellowing virus (CVYV) lacks the typical RSS of members of the family Potyviridae, HCPro, which is replaced by an unrelated RSS, P1b. CVYV P1b resembles potyviral HCPro in forming complexes with synthetic siRNAs in vitro. Electrophoretic mobility shift assays showed that P1b, like potyviral HCPro, interacts with double-stranded siRNAs, but is not able to bind single-stranded small RNAs or small DNAs. These assays also showed a preference of CVYV P1b for binding to 21-nt siRNAs, a feature also reported for HCPro. However, these two potyvirid RSSs differ in their requirements of 2-nucleotide (nt) 3′ overhangs and 5′ terminal phosphoryl groups for siRNA binding. Copurification assays confirmed in vivo P1b–siRNA interactions. We have demonstrated by deep ...
Molecular Plant-Microbe Interactions®, 2012
Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silen... more Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silencing suppressors (RSS) to counteract antiviral RNA silencing responses. HCPro is the RSS of Potyvirus spp., and its activity is enhanced by the upstream P1 protein. Distinctively, the ipomovirus Cucumber vein yellowing virus (CVYV) lacks HCPro but contains two P1 copies in tandem (P1aP1b), the second of which functions as RSS. Using chimeras based on the potyvirus Plum pox virus (PPV), we found that P1b can functionally replace HCPro in potyviral infections of Nicotiana plants. Interestingly, P1a, the CVYV protein homologous to potyviral P1, disrupted the silencing suppression activity of P1b and reduced the infection efficiency of PPV in Nicotiana benthamiana. Testing the influence of RSS in host specificity, we found that a P1b-expressing chimera poorly infected PPV's natural host, Prunus persica. Conversely, P1b conferred on PPV chimeras the ability to replicate locally in cucumbe...
Molecular Plant-Microbe Interactions®, 2013
Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specif... more Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N terminus of the coat protein (CP) includes host-specific pathogenicity determinants. Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3′ region of PPV-D (including the region coding for the CP) in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and N. benthamiana when specific amino acids were modified or deleted in a short 30-amino-acid region of the N terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus spp. although others impaired systemic infection in this host. We propose a model in which the N terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanis...
Molecular Plant-Microbe Interactions®, 1999
Plum pox potyvirus (PPV) infection of transgenic Nicotiana benthamiana plants that expressed the ... more Plum pox potyvirus (PPV) infection of transgenic Nicotiana benthamiana plants that expressed the PPV NIb RNA replicase carrying a Gly to Val mutation at the GDD motif (NIbV lines) induced a phenotype of virus resistance and transgene silencing, which was not transmissible to the progeny after self-fertilization (H. S. Guo and J. A. García, Mol. Plant-Microbe Interact. 10:160-170, 1997). Here, we demonstrate that the induced resistance of NIbV plants is mitotically stable after plant propagation by grafting and by in vitro regeneration. Virus replication or residual virus RNA seem not to be required to maintain transgene silencing and virus resistance. Analysis by PCR (polymerase chain reaction) amplification after treatment with methylation-sensitive restriction nucleases indicates that DNA methylation is associated with establishment and maintenance of transgene silencing and virus resistance. Restoration of transgene activity and susceptibility to PPV in sexual progeny correlated ...
Molecular Plant Pathology, 2012
Subisolates segregated from a M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in patho... more Subisolates segregated from a M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the HCPro protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in some subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings, suggested the existence of uncharacterized attenuating factors in these subisolates. In this study we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host.
Molecular Plant Pathology, 2014
Taxonomic relationships: Plum pox virus (PPV) is a member of the genus Potyvirus in the family Po... more Taxonomic relationships: Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae. PPV diversity is structured into at least eight monophyletic strains. Geographical distribution: First discovered in Bulgaria, PPV is nowadays present in most of continental Europe (with an endemic status in many central and southern European countries) and has progressively spread to many countries on other continents. Genomic structure: Typical of potyviruses, the PPV genome is a positive-sense single-stranded RNA (ssRNA), with a protein linked to its 5' end and a 3'-terminal poly A tail. It is encapsidated by a single type of capsid protein (CP) in flexuous rod particles and is translated into a large polyprotein which is proteolytically processed in at least 10 final products: P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIapro, NIb and CP. In addition, P3N-PIPO is predicted to be produced by a translational frameshift. Pathogenicity features: PPV causes sharka, the most damaging viral disease of stone fruit trees. It also infects wild and ornamental Prunus trees and has a large experimental host range in herbaceous species. PPV spreads over long distances by uncontrolled movement of plant material, and many species of aphid transmit the virus locally in a nonpersistent manner. Sources of resistance: A few natural sources of resistance to PPV have been found so far in Prunus species, which are being used in classical breeding programmes. Different genetic engineering approaches are being used to generate resistance to PPV, and a transgenic plum, 'HoneySweet', transformed with the viral CP gene, has demonstrated high resistance to PPV in field tests in several countries and has obtained regulatory approval in the USA.
Journal of Virology, 2011
Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the F... more Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genet...
Journal of General Virology, 2012
HCPro, the RNA-silencing suppressor (RSS) of viruses belonging to the genus Potyvirus in the fami... more HCPro, the RNA-silencing suppressor (RSS) of viruses belonging to the genus Potyvirus in the family Potyviridae, is a multifunctional protein presumably involved in all essential steps of the viral infection cycle. Recent studies have shown that plum pox potyvirus (PPV) HCPro can be replaced successfully by cucumber vein yellowing ipomovirus P1b, a sequence-unrelated RSS from a virus of the same family. In order to gain insight into the requirement of a particular RSS to establish a successful potyviral infection, we tested the ability of different heterologous RSSs from both plant- and animal-infecting viruses to substitute for HCPro. Making use of engineered PPV chimeras, we show that PPV HCPro can be replaced functionally by some, but not all, unrelated RSSs, including the NS1 protein of the mammal-infecting influenza A virus. Interestingly, the capacity of a particular RSS to replace HCPro does not correlate strictly with its RNA silencing-suppression strength. Altogether, our r...
BMC Plant Biology, 2010
Background Plant genomes have been transformed with full-length cDNA copies of viral genomes, giv... more Background Plant genomes have been transformed with full-length cDNA copies of viral genomes, giving rise to what has been called 'amplicon' systems, trying to combine the genetic stability of transgenic plants with the elevated replication rate of plant viruses. However, amplicons' performance has been very variable regardless of the virus on which they are based. This has boosted further interest in understanding the underlying mechanisms that cause this behavior differences, and in developing strategies to control amplicon expression. Results Nicotiana benthamiana plants were transformed with an amplicon consisting of a full-length cDNA of the potyvirus Plum pox virus (PPV) genome modified to include a GFP reporter gene. Amplicon expression exhibited a great variability among different transgenic lines and even among different plants of the same line. Plants of the line 10.6 initially developed without signs of amplicon expression, but at different times some of them ...
Biotechnology Journal, 2006
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2011
One of the challenges being faced in the twenty-first century is the biological control of 19 pla... more One of the challenges being faced in the twenty-first century is the biological control of 19 plant viral infections. Among the different strategies to combat virus infections, those 20 based on pathogen-derived resistance (PDR) are probably the most powerful approaches 21 to confer virus resistance in plants. The application of the PDR concept not only 22 revealed the existence of a previously unknown sequence-specific RNA-degradation 23 mechanism in plants, but has also helped to design antiviral strategies to engineer viral 24 resistant plants in the last 25 years. In this article, we review the different platforms 25 related to RNA silencing that have been developed during this time to obtain plants 26 resistant to viruses and illustrate examples of current applications of RNA silencing to 27 protect crop plants against viral diseases of agronomic relevance. 28 29 1. Introduction 30 Plant viruses represent important threats to modern agriculture. Although accurate 31 figures for crop losses due to viruses are not available, it is generally accepted that 32 among the different plant pathogens, the economic relevance of viruses comes second to 33 fungi. Until the emergence of genetic engineering technologies, plant viruses have been 34 partially controlled using conventional cultivation techniques such as crop rotation, 35 early detection and eradication of the diseased plants, cross protection, breeding for 36 resistance, or chemical control of their vectors [1]. In the 1980s, the successful transfer 37 of foreign DNA into the nuclear genome using Agrobacterium as a vector prompted the 38 introduction of genetic engineering for crop improvement and the development of virus-39 resistant plants [2, 3]. Today, different antiviral strategies are being undertaken, either 40 by exploiting natural plant defence mechanisms, or designing new tools, which in most 41 cases are ultimately also based on natural defence mechanisms. 42 Most of the achievements obtained in plant biotechnology in the area of plant virus 43 resistance are based on the principle of pathogen-derived resistance (PDR) [4]. The 44 concept of PDR was proposed by Sanford and Johnston [5] twenty-five years ago using 45 the bacteriophage Qß as a model, and considers that expression of pathogen genetic 46 elements outside the context of infection may lead to resistance. This approach opened 47 an interesting possibility for the practical control of diseases. For plant viruses, the 48 concept of PDR was first validated with its use in tobacco plants transformed with the 49 tobamovirus Tobacco mosaic virus (TMV) coat protein (CP) gene [6]. Soon this 50 observation was validated using other viral CPs and other viral sequences that code for 51 proteins such as replicases, proteinases and movement proteins [for review, see 7-11]. 52 CP is the most successful and widely applied viral protein for PDR. However, the 53 protection conferred by CP-mediated resistance varies significantly from strong 54 interference with virus multiplication to delay or attenuation of symptoms. The PDR 55 based on the expression of viral proteins, with either the wild type or the mutated one, 56 in transgenic plants has several general characteristics: i) it is not very specific, and 57 protects against a broad range of viral strains; ii) it shows a positive correlation between 58 the levels of accumulation of the viral product and the effectiveness in resistance; iii) it 59 is usually overcome by high doses of inoculum. Despite extensive studies, the 60 molecular mechanisms underlying protein-mediated resistance are not fully understood. 61 What appears to be certain is that they are diverse, that they probably affect several 62 steps of the infection process, and that each virus/transgenic plant combination has 63 specific features. Moreover, it soon became apparent that many virus resistances 64 initially envisaged as protein-mediated PDR did not rely on the expression of the 65 corresponding viral proteins and that a majority of PDR phenomena seemed to work 66 through RNA-mediated mechanisms [12]. 67 68 2. RNA silencing and virus resistance 69 In the early nineties, two independent research groups found that the expression of 70 a transgene mRNA with a high sequence similarity to an endogenous mRNA, led to 71 specific degradation of both mRNAs through post-transcriptional gene silencing 72 (PTGS), also known as "cosuppression" [13, 14]. Later, the W. Dougherty research 73 group suggested that a similar mechanism might be involved in the resistance 74 phenomena observed in transgenic plants transformed with viral genes. Some of the 75 transgenic lines showed anomalous phenotypes; unexpectedly and unpredictably the 76 highest level of resistance was observed in the transgenic lines showing very low levels 77 of transgene mRNA accumulation, whereas plant lines expressing the same gene at high 78 levels were fully susceptible. Interestingly, the virus resistant plants had actively 79 transcribed genes but they had low steady-state levels of transgene mRNA. A 80 breakthrough discovery, from transgenic lines included to serve as negative controls, 81 showed that resistance occurred even with non-translatable versions of the viral genes, 82 which demonstrated that the RNA itself was responsible for the virus resistance 83 observed in the transgenic plants [15-17]. All the molecular analysis of these transgenic 84 plants challenged the existing paradigm of genetic regulation and became the first 85