Browsing by Author "Arce-Johnson, P"

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    Comparative analysis of TMV-Cg and TMV-U1 detection methods in infected Arabidopsis thaliana
    (2000) Pereda, S; Ehrenfeld, N; Medina, C; Delgado, J; Arce-Johnson, P
    The common strain of the tobacco mosaic virus (TMV-U1), and the crucifer-infecting tobacco mosaic virus (TMV-Cg), both members of Tobamovirus genus, infect efficiently the solanaceous plants such as tomato and tobacco. The crucifer-infecting tobacco mosaic virus (TMV-Cg) also infects Arabidopsis thaliana plant, spreading systemically without causing severe symptoms. In contrast, Arabidopsis is a poor host for TMV-U1 infection. Within the past 10 years, Arabidopsis has developed into a powerful model system for studying plant-pathogen interaction. However, a detailed analysis comparing the accuracy of various viral detection methods has not been reported previously. Four detection methods were evaluated in A. thaliana (ecotype Po-1), infected with TMV-U1 or TMV-Cg. Western blots, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ RNA hybridization methods were used to determine Viral spread at Various days post inoculation (dpi) in inoculated and apical non-inoculated leaves. The detection of viral spread of TMV-U1 and TMV-Cg in Arabidopsis, using these four detection methods, supports previous studies, which demonstrate that the systemic spreads of these two viruses differ in Arabidopsis. Western blotting and ELISA detected TMV-Cg at 5dpi, and TMV-U1 at 12 dpi in systemic tissues. Viral spread was detected earlier when using RNA detection methods. Reverse transcriptase-polymerase chain reaction (RT-PCR) was very sensitive for detecting TMV-CS in A. thaliana, but less sensitive for TMV-U1 detection. In situ RNA hybridization showed differential distribution of TMV-Cg and TMV-U1 in the inoculated leaf and systemic tissues. (C) 2000 Elsevier Science B.V. All rights reserved.
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    Expression of the chicken lysozyme gene in potato enhances resistance to infection by Erwinia carotovora subsp, Atroseptica
    (2000) Serrano, C; Arce-Johnson, P; Torres, H; Gebauer, M; Gutierrez, M; Moreno, M; Jordana, X; Venegas, A; Kalazich, J; Holuigue, L
    Infection of potato plants and tubers with the bacterium Erwinia carotovora subsp, atroseptica produces blackleg and soft rot diseases, which cause significant losses to crops and stored potatoes. In order to obtain resistance against this bacterium, the gene chly encoding the enzyme lysozyme from chicken was introduced into potato plants (cv. Desiree) via Agrobacterium-mediated transformation. Sixty-three and 69 transgenic potato clones were evaluated in the greenhouse for resistance to blackleg and soft rot diseases, respectively. Results reported in this paper indicate that 21%-29% of the potato clones showed increased resistance to infection by the bacterium E. c, subsp, atroseptica T7, as revealed by a reduced severity of blackleg or soft rot symptoms. Nine clones showing different levels of resistance were selected for further molecular analysis. The number of copies of the transgene integrated in the plant genome of these clones was estimated by Southern blot analysis. The level of transgene expression, detected by Northern blot analysis, correlated with the level of resistance detected in these clones.
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    Tobamovirus coat protein CPCg induces an HR-like response in sensitive tobacco plants
    (2005) Ehrenfeld, N; Cañón, P; Stange, C; Medina, C; Arce-Johnson, P
    When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg),is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg -that carries CPCg in a TMV-U1 context- are characterized by cell death and by the presence of autoflorescent phenolic compounds and H2O2, just like the HR lesions. In addition, defense-related genes and detoxifying genes are induced in tobacco Xanthi-nn plants after TMV-Cg and U1-CPCg inoculation. We postulate that in our system, CPCg is recognized by sensitive tobacco plants that mount an incomplete defense response. We call this an HR-like since it is not enough to induce plant resistance.
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    Identification and characterization of a novel tobacco mosaic virus resistance N gene homologue in Nicotiana tabacum plants
    (2004) Stange, C; Matus, JT; Elorza, A; Arce-Johnson, P
    Nicotiana tabacum cv. Xanthi nn plants are susceptible to infection by most tobamoviruses (TMV). However, such plants display a partial hypersensitive resistance response (HR-like response) to TMV-Cg. The genetic mechanism of the HR-like response has yet not been determined, but it may involve a gene with a function similar to that of a resistance gene, responsible for HR in resistant plants. We have cloned a gene homologous to the resistance N gene, named NH, from Nicotiana Xanthi nn plants. The coding region of NH is 5.028 base pairs ( bp) long and has 82.6% nucleotide identity with the N gene. In contrast to the N gene, the NH gene lacks intron 4 and does not have sites for alternative splicing of intron 3. Analysis of its sequence revealed that NH belongs to the TIR/NSB/LRR gene class. We were able to detect stable levels of NH-transcript in Nicotiana Xanthi nn plants from 0 to 18 h post-inoculation (hpi) with TMV-Cg. Transcript levels increased slightly at 24 hpi and dropped below basal values at 48 hpi. The NH transcript was also detected in a range of resistant Nicotiana plants ( N. tabacum Xanthi NN,N. glutinosa, N. glauca and N. rustica) suggesting that NH is a homologue of the N gene, rather than an allele. We have cloned and characterised the NH gene ( GenBank acc. no. bankit598573 AY535010) from nn susceptible plants and postulate that this gene might be involved in the HR-like response seen in these plants.
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    Isolation of Pinus radiata genomic DNA suitable for RAPD analysis
    (1998) Stange, C; Prehn, D; Arce-Johnson, P
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    Optimization of in vitro culture conditions for Pinus radiata embryos and histological characterization of regenerated shoots
    (1999) Stange, C; Prehn, D; Gebauer, M; Arce-Johnson, P
    Different in vitro culture conditions were tested on Pinus radiata organogenic embryos. Optimum shoot induction occurred at 26.1 degrees C, whereas the best elongation resulted at 21.4 degrees C. Supplements of 2.5 mg/l or 5 mg/l of BAP added to the induction media produced a similar number of regenerated shoots, which differed statistically from 1.0 mg/l of BAP and 0.025 mg/l TDZ. Addition of 10 mg/l MnSO4 to LP1/2 medium significantly increased the number and quality of in vitro regenerated shoots. The removal the apical region of shoots cultured in LP 2.5 mg/l of BAP increased the number of de novo generated shoots by 23%, compared to a control group with intact shoots. Approximately 70% of the in vitro shoots of P. radiata were of wet phenotype (hyperhydrated appearance); the rest were waxy in appearance. Histological cuts did not produce any differences in phenotypes, but scanning electronic microscopy of needles gave evidence of differences in epicuticular wax deposits. Abbreviations: LP: Quoirin and LePoivre basal medium, without plant growth regulators; LP,: LP medium + 1 mg/l BAP; LP2.5: LP medium+ 2.5 mg/l BAP; LP5 : LP medium + 5 mg/l BAP; LP1/2: LP basal medium at half strength of macroelements, 2%; commercial sugar, ammonium nitrate 100 mg/l, calcium nitrate 564.5 mg/l, hydroxyquinoleine 1.25 mg/l, MS vitamins and without plant growth regulators; LPT0.025: LP medium + 0.025 mg/l TDZ; BAP: N-6 benzylaminopurine; TDZ: Thidiazuron.
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    Protein(s) from the gram-positive bacterium Clavibacter michiganensis subsp. michiganensis induces a hypersensitive response in plants
    (1998) Alarcón, C; Castro, J; Muñoz, F; Arce-Johnson, P; Delgado, J
    The gram-positive tomato pathogen Clavibacter michiganensis subsp. michiganensis induced a local necrotic response on four-o'clock (Mirabilis jalapa) and tobacco (Nicotiana tabacum) plants. This necrosis response was characteristic of the hypersensitive response (HR). The cell-free culture supernatant from strain CMM623 also induced a necrosis that was phenotypically similar to that induced by the bacteria. Inhibitors of plant metabolism suppressed the necrotic reaction of both M. jalapa and tobacco. The HR-inducing activity present in the supernatant was heat stable, sensitive to proteases; and had hn apparent molecular mass in the range of 35 to 50 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The properties observed for the necrosis-inducing activity resembled bacteria.
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    Regeneration of whole plants from apical meristems of Pinus radiata
    (2003) Prehn, D; Serrano, C; Mercado, A; Stange, C; Barrales, L; Arce-Johnson, P
    A methodology to regenerate whole plants of Pinus radiata from apical meristems of 3- and 7-year-old trees was developed. Meristematic domes with two or three leaf primordia were excised from surface-sterilized branch tips of field-grown plants and cultured in LP medium with half strength macronutrients (1/2 LP) and full strength micronutrients. The early growth of meristems required approximately 12 weeks, including a recovery stage during the first 2 weeks. After 8 weeks, some meristems developed abnormal phenotypes and died during the subsequent stages of development. However, healthy meristems elongated and formed shoots when they were transferred to LP medium supplemented with MS vitamins, 30 mg l(-1) casein hydrolysate, and 0.4 g l(-1) agar plus 2.85 g l(-1) Gelrite. Meristems that developed vigorous shoots were used for rooting experiments when they were 2 cm in length. Whole plants were obtained after 5 days of root induction in water-agar medium containing 8.2 muM IBA and 5.4 muM NAA and 1 month culture in LP medium with 10 g l(-1) sucrose. Plants regenerated from meristems were further propagated by rooting of cuttings. Of the rooted cuttings, 10% were morphologically juvenile.
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    Replicase mediated resistance against Potato Leafroll Virus in potato Desiree plants
    (2004) Ehrenfeld, N; Romano, E; Serrano, C; Arce-Johnson, P
    Potato leafroll virus (PLRV) is a major menace for the potato production all Over the world. PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides. Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases. The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicase transgene in potato plants cv. Desiree. Plants were genetically modified to express the complete sense PLRV replicase gene. Two constructions were used. one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes. Transgenic plants were infected with PLRV in vitro, using infested aphids. In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected. although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection. In turn, those plants with the RolA promoter displayed an initial resistance that was later overcome. Different molecular mechanisms are likely to participate in the response to PLRV infection of these two types of transgenic plants.
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    Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens
    (2002) Cerda, F; Aquea, F; Gebauer, M; Medina, C; Arce-Johnson, P
    Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing beta-glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation.