PLANT VIRUS

Author: AGUILAR AGUILAR JUAN MANUEL.
Year: 2004.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: This thesis aims to study general aspects related to the identification and the use of genetic resistance, natural or transgenic, as a strategy for controlling CYSDV, a virus transmitted by whitefly by affecting crops protected from the cucurbits southeast Peninsular español.Inicialmente, was necessary to carry out the molecular characterization of CYSDV. Thus, in this thesis it has been determined the complete nucleotide sequence of the genome of an isolate Spanish-CYSDV from which has been deducted possible organization genómica.Los analysis has shown characteristics that are not shared by other members of the genus and also that the mechanisms involved in gene expression is the generation of messengers subgenómicos. The estratgias proximity to control CYSDV provide results restringidos.Así which has conducted a search for genetic resistance to CYSDV in 300 entries pepino.En this analysis were identificadoo two entries in the natural conditions showed no infection symptoms typical caused by CYSDV, but under controlled conditions if they showed symptoms, but the development and severity of these were minor when compared with those of a grown susceptible.Además, quantitative analysis showed a smaller accumulation of RNA CYSDV over the testifo susceptible.Por Furthermore, the results obtained from analysis of favoring the whitefly revealed that a entrad seems to show resistance to the fly the type antixenoisis. Given the existence of a limited number of potential donors natural resistance to CYSDV, the goal was to generate resistance to the virus through the transformation of plants with genes capable of confeir this carácter.La strategy is based on the genetic transformation of plants with melon a building that bears a fragment with an inverted repeat of the RNA-dependent RNA polymerase (RdRp) CYSDV.Se have identified the lines of melon with a partial resistance to CYSDV.Dicha resistance is characterized by a lower accumulation of viral RNA and less severe symptoms when compared with their respective controles.Los analysis performed with a line of plants suggest that the mechanism responsible for the resistance gene silencing is the level of RNA desecadenado by the transgene.

Author: ROMÁN PERUYERO MARTHA PATRICIA.
Year: 2004.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS.
Place of preparation: UNIVERSIDAD POLITÉCNICA DE VALENCIA.

Summary: The tristeza virus (CTV) citrus in the world has caused the death of more than one hundred million plants grafted on bitter orange since the decade of 1930. In this work we have studied causes of the syndrome of decay by sadness in sweet orange grafted on bitter orange, and tested methods of greenhouse and in vitro to reproduce the observed decay in the field. It also has studied the movement of the virus in different combinations variety / pattern bitter and have been characterized in isolated CTV current natural dispersion field. In parallel, we studied the syndrome decay of trees affected by the Sudden Death of Citrus (MSC), a new disease sweet orange trees that have ca used in Brazil, from 199, the deaths of more than two million plants. All this is enhanced through anatomical studies and estimates of viral load by serological methods. There has been, for the first time, able to reproduce in vitro greenhouse syndrome decay and death by sadness plant sweet orange grafted on bitter orange. There were three kinds of syndromes: A-Death by collapse. B-Death by slow decay. C-severe symptoms of chlorosis and dwarfism. The anatomical study of the bark of the sweet orange and orange bitters, as well as field trees grown in the greenhouse and in vitro, infected with CTV, it was observed early aging or degeneration of phloem. Such degeneration (hypertrophy cells parenquimáticas, obliterating, collapse and necrosis of the tubes cribosos resulted in a noticeable reduction in the foema functional. Cultivated plants in the greenhouse was noted that the survival time of the plant was depending on the resilience of phloem. plants that lived longer (slow decay) phloem degenerated was renewed by the steady production of new phloem. plants acute symptoms only in the variety, the phloem of bitter orange was not affected. In plants cultivated in vitro were also observed large anatomical alterations in the root system. observed changes in the bark of trees affected by sadness in Spain were similar to those affected by MSCS in Brazil. intensity of the symptoms of sadness and MSC has been linked to increased anatomical changes, especially the reduction in the phloem functional. trees affected by sadness there is a positive relationship between viral load and symptoms. viral load CTV trees affected by MSC is not associated with symptoms. uniformity and speed distribution of CTV, depends on the viral isolation and the variety grafted on bitter orange. combinations clementino and Mexican lime on bitter orange, gave a very regular distribution and mobilization of the virus to the roots. In sweet orange grafted on bitter orange, but the distribution was not as regularly for all isolates studied, the rapid distribution of the virus was higher than in other combinations. isolated to be distributed more quickly was the T-300, in any combination variety / bitter orange, it may be related to the speed with which affects the phloem, causing the collapse of the plants. characterization biological and serological isolated captured in the field, revealed variability between them. All the isolates contained epítopos 3DF1 and 3CA5 and some of them were MCA 13 positive and increased aggressiveness that usually found so far in the region of L'Horta Nord.

Author: MORENO LOZANO ARANZAZU.
Year: 2004.
University: POLITÉCNICA DE MADRID [www.upm.es].
Place of defense: ETSI AGRONOMOS.
Place of preparation: CENTRO INVESTIGACIONES AGRARIAS, CCMA, CSIC.

Summary: During the thesis has been made on the one hand, the identification, surveys indicencia and prevalence of the major viruses affecting crops of lettuce and brásicas and in the adventitia flora associated with them. Further analysis has been done of the evolution of space mosaic virus lettuce in lettuce crops in the central region. The results show as the mosaic virus (LMV) lettuce and cucumber mosaic virus (CMV) in the central region, and the tan tomato virus (TSWV) and Virus western yellow beet (BWYV ) in Murcia, were the main viruses found in lettuce. In cultured brásicas was Virus cauliflower mosaic (CaMV), which appeared more frequently. Major viruses found in crops also appeared in the wild flora associated. The presence of multiple infections was common sobret particularly in wild flora associated. The results obtained by analyzing space of LMV show how the dispersal of the virus in the test conditions is typically policíclica. On the other hand, we studied the relationship of the main virus vector virus found in crops. We evaluated the efficacy of transmission LMV by different species of aphids, being M yzus persicae and Macrosiphum euphorbiae species that best conveyed the virus. Nasonovia ribisnigri could not transmit the virus to any of the plants tested. It was also nearing techniques IC-RT-PCR and IC-RT-Nested-PCR comparing its sensitivity and specificity. The technique IC-RT-Nested-PCR was used for the detection of the virus in aphids, remain possible in detecting viral vector species not as vector. In studies of the behavior of aphid associated with the inoculation of CaMV was seen as the virus is inoculated by salivation during the first intracellular penetration, differences in the pesistencia virus by the two species studied, M.persicae and Brevycorine brassicae. Further studies with several variants mutadas virus show as the amino acid 6 (Gln) of the protein P2 of CaMV is specifically involved in the recognition between this protein and receiver cuticular in vector.

Author: VARGAS CONCHA YOLANDA MARISOL.
Year: 2005.
University: POLITÉCNICA DE MADRID [www.upm.es].
Place of defense: ESCUELA TECNICA SUPERIOR DE INGENIEROS AGRONOMOS.
Place of preparation: CENTRO DE INVESTIGACIONES BIOLOGICAS CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS (CSIC).

Summary: The silencing gene postranscripcional is a mechanism of degradation of RNA-dependent sequence homology, which acts as a natural defense system in plants compared to viral infections. We know also that RNA bicatenario (dsRNA) is the inducer of this adaptive response of defense and highly conserved in evolution. In this thesis work has been dealt with two different strategies that although both are based on the use of molecules dsRNAs derived sequence for interfering with viral infection of the virus. The first strategy is based on the expression of dsRNA by a stable transgene encoding the transcription of viral sequences arranged in inverted repeat (IR). In this regard, there are transgenic lines of Nicotiana benthamiana in which it has introduced a transgene containing an inverted repeat of the region 54k of replicasa of mottle virus soft pepper (PMMoV). The expression of this construction resulted in the accumulation stable in the floor of a transcribed in length corresponding to the sequence transgenic. In trials of protection against PMMoV, nearly 70% of these lines R0 reclaimed showed resistance to the virus. By contrast, buildings arranged in repeating direct encoding the same region of the virus but without the ability to hibridar intramolecularmente showed resistance to the virus in just a 6-20% of plants R0 reclaimed. Resistance to PMMoV was associated with the accumulation of small RNAs of 21 and 25 nts characteristic of PTGS in a stage prior to the viral inoculation. It has been characterized a line R2 in homocigosis, IR4 # 8, which expresses the NYT bicatenario. This line has been subject to increasing degrees of viral inoculum in the road and a high inoculum pressure and continuing via vascular bundles, using grafts. In all cases, the transgenic plants had high resistance (immunity) against the virus until the end of the life cycle. Moreover, when this line of transgenic plants were inoculated with tobamovirus with low nucleotide similarity with PMMoV-S as TMV or ToMV, they showed a susceptibility phenotype, however, when they were inoculated with PMMoV-I a tobamovirus high nucleotide similarity with PMMoV, plants showed complete resistance phenotype, indicating the high specificity of this mechanism that promotes resistance in these plantas.La second strategy is presented as an alternative to the genetic transformation of plants and is based on production dsRNAs derived viral sequences in bacteria. Previously it had been shown that inoculation joint viruses and viral dsRNA sequences derivative produced in vitro specifically prevents viral infection in plants. In order to have large amounts of dsRNA that could be used in studies of PTGS, as well as for potential use in the control of viral diseases in plants, has developed a system of inducible expression of these molecules in bacteria. The dsRNAs well expressed relate to various genomic sequences derived from PMMoV, virus the sharka (PPV) and the potato virus Y (PVY). The dsRNAs purified from bacteria and co-inoculados with the virus counterpart in plants N. Benthamiana and Capsicum annuum filed a specific interference with the viral infection. Moreover, when crude extracts of dsRNAs obtained by the lysate of the bacterium by the French press, without any additional purification step, were co-inoculados with the virus or pulverized counterpart on the leaf surface of plants, were observed to be equally effective the viral protection, even when spraying was made several days before the inoculation of the virus. There was obtained protection of pl 8 doors po 3ea r this method when the virus inoculation was conducted by the vector Myzus persicae. However, when the molecules dsRNAs were transiently expressed in plants, it received a total interference with the transmission of the virus vector, resulting plants protected from viral infection.

Author: CASTRO DE SOUSA JOAO PAULO.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: ESCUELA DE POSTGRADO.

Author: GARCIA ANDRES SUSANA.
Year: 2005.
University: CÓRDOBA [www.uco.es].
Place of defense: ETSIAM.
Place of preparation: ETSIAM.

Summary: The bengomovirus (Family Geminiviridade) associated with the disease curly yellow tomato (TYLCD) causing major damage in tomato (Lycopersicon esculentum Mill.) In many tropical and subtropical regions of the world, also are involved in very serious epidemic episodes in Spain and Italy since the late delos 80's. However, despite its economic importance, are very few studies on the genetic diversity and structure of their populations, which are essential to understanding the factors affecting its development and develop control strategies durable. Therefore, the objectives of this work has focused on the analysis of the structure and genetic diversity of populations bengomovirus causing TYLCD, studying different causes that may alter the structure of these populations and their evolution, as well as mechanisms generators diversity is as recombination. The structure of the populations of these begomovirus in Spain and Italy proved to be very complex, finding differences host, geographic area or time. It identified five different viral variants in Spain and three in Italy. It has also highlighted the importance played by wildlife as Solanumnigrum in the evolution of these viruses, as they act as reservoirs of genetic diversity. In fact, it has detected the presence at this sort of a new variant of begomovirus of recombinant nature with a new phenotype pathogenic, which could have unpredictable consequences epidemiological. It has been demonstrated in this paper that recombination is a common phenomenon among begomovirus which provides a wide variety of viral variants in the population that allows these viruses rapid adaptation to environmental change. It has also been revealed that the genetic structure of populations begomovirus in Spain has been disrupted by the movement of a species viral another appeared to be more resistant to the new tomato varieties introduced into crops with genes for resistance to TYLCD. This paper gives an insight into the developments that have followed the begomovirus present in the Mediterranean basin since its detection.

Author: SAJNANI PÉREZ CARLOTA.
Year: 2005.
University: GRANADA [www.ugr.es].
Place of defense: ESTACIÓN EXPERIMENTAL DEL ZAIDÍN (CSIC).
Place of preparation: ESTACIÓN EXPERIMENTAL DEL ZAIDÍN.

Summary: In previous work the group has shown that the virus of pepper mild mottle (PMMoV) induces a decrease in the levels of various proteins PSII, as well as changes in the expression of genes of various proteins cloroplastídicas both coding nuclear cloroplastídica. Also imaging of fluorescence emitted by the clorofial (Chl EF) proved to be very useful for keeping track of viral infection and the defense mechanisms that developed during chloroplast it. The systems huésped-patógeno used in this study have been Nicotiana benthamiana infected with the strains of Spanish and Italian mottle virus soft pepper (PMMoV-S, PMMoV-I) and Nicotiana tabacum infected with snuff mosaic virus (TMV) . addition, the company has used a line of transgenic work (CrtZ) sobreexpresa the beta-carotínhidroxilasa. For the study of stress by viruses in plants N.benthamiana have spent two biophysical techniques that allow perform (like the EF Chl) measures Sheet: thermoluminescence (TL) and green fluorescence (GDB), also called fluorescence of the Chl induced by UV. The TL revealed changes in band C by leaps and bounds in the band AG. The TL also measured high temperature (HTL) leaf infected with the bands were identified (HTL2), indicative of oxidative stress. But these changes affected in a different way to the young leaf symptomatic and the mature leaf showing no symptoms (asymptomatic sheet) plant infected. GDB measures enabled us to detect an increase in the fluorescence in the face adaxial and especially in the abaxial leaf of the young and mature after two weeks of inoculation. Moreover, these measures showed that the adaxial side of the road infected, compared with the healthy, absorbing compounds accumulate in the UV region. This phenomenon was detected in the young leaf even before the symptoms of the infection were visible. The CHl FLj has been used in this paper for the study of a second system huésped-patógeno, plant Nicotiana tabacum infected with TMV. In this case were analyzed under different regimes lumínicos, healthy and infected plants from the wild-line snuff (WT) and a transgenic line (crtZ), which sobreexpresa enzyme beta-carotínhidroxilasa that lets you produce larger quantities of zeaxantina (Zea ). Images NPQ (non-photochemical quenching), which measures the energy dissipated in the form of heat and not used in photosynthesis, and of PSII (yield cuántido of PSII) suggest that the line of snuff crtZ submitted simultaneously to both stressors ( lighting and viral infection) has no advantages compared to WT. This would be consistent with the data from the profiles of these plants pigment obtained by HPLC, which did not present a significant difference. Lastly, in this Doctoral Thesis we raised an initial approach to proteome chloroplast of N.benthamiana, also comparing the differences in the levels of expression of proteins cloroplastídicas between samples infected with tobamovirus and samples of healthy plant. We have positively identified a total of 55 spots protein, gels 2D displays of chloroplasts, of which 26 were diminishing in sample infected. The proteins affected by the infection described herein belong mainly to the CTE and the Calvin cycle.

Author: NIETO GARCIA CRISTINA.
Year: 2005.
University: POLITÉCNICA DE MADRID [www.upm.es].
Place of defense: ETSI AGRONOMOS.
Place of preparation: ETSI AGRONOMOS.

Summary: The diseases caused by viruses are a growing problem in modern agriculture and intensive. The cycle of viral infection is a complex process that requires interaction at various levels between host factors and factors of the virus. The study of the processes involved in the molecular interactions between viruses and cellular factors can generate new knowledge about the mechanisms of resistance and the development of resistance to the virus more durable and broad spectrum. The virus of necrotic spots of melon (Melon necrotic spot virus, MNSV) is a virus RNA positive sense of endemic crops cucurbitaceas worldwide. The recessive gene nsv melon (Cucumis melo L.) resistant to all isolated MNSV except MNSV-264. This has further isolated the ability to infect guests who do not belong to the family of cucurbits (Nicotiana benthamiana and Gomphrena globosa). A study using the mutant chimeric built between MNSV-264 and MNSV-M 5 (an isolated avirulento) has shown that the determinant of avirulencia of MNSV resides in the region 3'-terminal the viral genome. Previous studies have identified factor in eukaryotic translation initiation of melon, Cm-elF4E as a candidate gene to nsv. One main aim of this thesis has been the characterization of the molecular interaction between MNSV-nsv. To address this study were carried out tests complementation in melon by transient expression of the susceptibility allele of Cm-el F4E in genotypes resistant melon. These tests led to the multiplication of MNSV-M 5, confirming that Cm-el F4E is the product encoded by nsv. This is therefore the first example of a recessive resistance mediated elF4E effective against a virus does not poliadenilado without 5'cap that does not belong to the family Potyviridae. This paper has done a molecular characterization of the region MNSV involved in determining avirulencia. The experiments have shown that the determinant of avirulencia is an RNA molecule itself. At the same time we have identified in the predictions of the secondary structure of 3'UTRs several isolated MNSV possible structural elements that may be involved in overcoming the resistance. It is the first case described in which the determinant of avirulencia that interacts with elF4E, directly or indirectly, is a molecule of RNA. Controlling MNSV is based primarily on the use of varieties that incorporate the gene nsv. This paper has conducted a search for new sources of natural resistance to MNSV by phenotypic screening of 147 entries C.melo and species purposes after mechanical inoculation. The results have shown the existence of 2 entries C.melo and 4 of related species resistant MSNV-M 5. Only one entry, C.zehyeri was not infected by any of the isolates. The usefulness of the entries related species to C.melo depend largely on the development of gene transfer strategies to overcome the barriers of compatibility with C.melo depend largely on the development of strategies for gene transfer in excess barriers compatibility C.melo. Moreover, in this study we have adapted and developed point technique EcoTILLING for melon with the same collector used in the phenotypic scrutiny. This technique has been applied as genetic screening and allowed confirm the correlation between SNP (single nucleotide change) found in genotype resistant melon and resistance to MNSV. Thanks to the implementation of EcoTILLING has analyzed the variability of Cm-elF4E of entries in the collection, there has been a search for new alleles resistance and has analyzed the correlation between haplotypes found and resistance / susceptibility to other viruses for which the collection has been previously characterized. Finally, another objective has been raised molecular characterization dela interaction MNSV-N.benthamiana. The complementation tests carried cab 8 or mostra 4ef rum that the susceptibility allele of Cm-elF4E not supplementing infection MNSV-M5 in this species. This suggests that the mechanisms controlling the resistance in N.benthamiana and melon are different. The homology of the secondary structure prediction of 3'UTR of isolated avirulentos with precursor miRNA sequences raised the possibility of the involvement of silencing mechanisms mediated by small RNAs. Analysis was carried out by Northern-blot that showed the existence of small RNAs encoded by N.benthamiana with homology with 3'UTR genome MNSV- M5.

Author: Díaz Vivancos Pedro.
Year: 2006.
University: POLITÉCNICA DE CARTAGENA [www.upct.es].
Place of defense: E.T.S. de Ingeniería Agronómica.
Place of preparation: Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC).

Summary: The sharka disease caused by Plum pox virus (PPV), is one of the limiting factors in the production of fruit trees of the genus Prunus, yielding significant economic losses. It has studied the effect of the infection on PPV different physiological and biochemical parameters in Prunus species with different susceptibility to PPV [peach "GF305" and apricot Real Fine, "as susceptible and apricot" Stark Early Orange "(" SEO " ) as resistant], subcellular level. These studies have also been undertaken in a variety of pea ( "Alaska") highly susceptible to PPV. In susceptible plants, infection PPV produce changes in some photosynthetic parameters. Both Prunus pea as has been described in the presence of activity APX resistant acid p-hidroximercuro benzóico (pHMB) (APX Class III). In soluble fraction of susceptible plants apricot infection decreased activity APX sensitive pHMB (APX Class I) deshidroascorbato reductase (DHAR) and peroxidase (POX), while in the resistant variety was an increase in the activities of APX Class III, monodeshidroascorbato reductase (MDHAR), glutathione reductase (GR) and superoxide dismutase (SOD). The catalase activity decreased in the soluble fraction of both varieties. The infection resulted in a drop in PPV enzymes cycle ascorbato-glutation (APX, MDHAR, DHAR and GR) only in chloroplasts of the susceptible variety. In pea studies were performed at 3 and 15 days post-inoculation (dpi). After 3 dpi infection PPV resulted in a drop in Class I and APX increased POX in soluble fraction, while in chloroplasts there was a decline of APX and POX. After 15 dpi infection resulted in an increase in enzyme secuestradoras of H2O2 (POX and APXs) and a decrease in the activities glutation-S-transferasa (GST) and catalase in soluble fraction. In chloroplasts there was a decline in activities glutathione peroxidase (GPX), SOD and SW. It also studied systems antioxidant apoplasto in response to PPV in varieties of Prunus with different susceptibility to PPV. Both peach and apricot described in the presence of APX Class I and Class III, SOD, POX, NADH-POX and polifenoloxidasa (PPO). In apoplasto of peach leaf infection resulted in an increase of APX Class I, POX, NADH-POX and PPO. In apoplasto leaves apricot susceptible to infection resulted in a decline in POX and SOD and an increase in PPO. However, the apoplasto leaves apricot PPV resistant to infection resulted in an increase of APX Class I POX and SOD. The infection resulted in a PPV oxidative stress, as indicated by increased levels of lipid peroxidation, protein oxidation, loss of electrolytes and accumulation of H2O2 only in the susceptible varieties studied. The decline in photosynthetic parameters, indicating generation of reactive oxygen species (ROS) in chloroplasts, and the decline in the levels of antioxidant enzymes in the chloroplasts of susceptible varieties could be responsible for the symptoms of chlorosis. The results obtained in pea, which used a strain of genetically modified PPV with GFP showed that the virus appeared in areas of chlorosis and in the same areas was observed by histochemical techniques, an accumulation of H2O2 and O2-in infected leaves. Ultrastructural studies showed that the virus was accumulated in the cytosol of infected cells and the chloroplast was orgánulo hardest hit by infection with PPV. By proteomics studies, we studied the effect of infection PPV in the expression of proteins peach and pea. The identification of proteins showed an induction taumatina and 8 a desc 712 enso of mandelonitrilo liasa in apoplasto of peach leaves infected with PPV. However, most of the polypeptides showed no homology with known proteins. This fact shows that most of the functions apoplasto are still unknown, at least in Prunus. In pea infection PPV mainly produced an alteration of photosynthesis and metabolism of carbohydrates. As a general conclusion that can describe the infection produces a PPV oxidative stress in both leaf varieties of Prunus in pea leaves susceptible to the virus and that the observed imbalance in the metabolism antioxidativo could be related to the progression of infection and symptoms . Moreover, the results suggest that greater antioxidant capacity shown by the cultivar apricot "SEO" could be the result of a systemic response acquired induced penetration PPV in the tissues of the stem at the point of graft. These facts might be related, among other factors, its resistance to PPV.