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1.  Characterization of Resistance Gene Analogues (RGAs) in Apple (Malus × domestica Borkh.) and Their Evolutionary History of the Rosaceae Family 
PLoS ONE  2014;9(2):e83844.
The family of resistance gene analogues (RGAs) with a nucleotide-binding site (NBS) domain accounts for the largest number of disease resistance genes and is one of the largest gene families in plants. We have identified 868 RGAs in the genome of the apple (Malus × domestica Borkh.) cultivar ‘Golden Delicious’. This represents 1.51% of the total number of predicted genes for this cultivar. Several evolutionary features are pronounced in M. domestica, including a high fraction (80%) of RGAs occurring in clusters. This suggests frequent tandem duplication and ectopic translocation events. Of the identified RGAs, 56% are located preferentially on six chromosomes (Chr 2, 7, 8, 10, 11, and 15), and 25% are located on Chr 2. TIR-NBS and non-TIR-NBS classes of RGAs are primarily exclusive of different chromosomes, and 99% of non-TIR-NBS RGAs are located on Chr 11. A phylogenetic reconstruction was conducted to study the evolution of RGAs in the Rosaceae family. More than 1400 RGAs were identified in six species based on their NBS domain, and a neighbor-joining analysis was used to reconstruct the phylogenetic relationships among the protein sequences. Specific phylogenetic clades were found for RGAs of Malus, Fragaria, and Rosa, indicating genus-specific evolution of resistance genes. However, strikingly similar RGAs were shared in Malus, Pyrus, and Prunus, indicating high conservation of specific RGAs and suggesting a monophyletic origin of these three genera.
doi:10.1371/journal.pone.0083844
PMCID: PMC3914791  PMID: 24505246
2.  Terpenoid Variations within and among Half-Sibling Avocado Trees, Persea americana Mill. (Lauraceae) 
PLoS ONE  2013;8(9):e73601.
Chemical analyses were conducted to determine the qualitative and quantitative differences in monoterpenes and sesquiterpenes in plant material from avocado trees, Persea americana Mill. (Lauraceae). The initial study analyzed plant material sampled from the trunk to the leaves through different branch diameters to quantify proximo-distal spatial differences within a tree. All trees were seedlings initiated from a single maternal tree. Two-way analysis of variance was conducted on 34 chemicals that comprised at least 3% of the total chemical content of at least one tree and/or location within a tree. There were significant interactions between genotype and location sampled for most chemicals. Parentage analysis using microsatellite molecular markers (SSR's) determined that the four trees had three fathers and that they represented two full-siblings and two half-sibling trees. Descriptive discriminant analysis found that both genotype and location within a tree could be separated based on chemical content, and that the chemical content from full-siblings tended to be more similar than chemical content from half-siblings. To further explore the relationship between genetic background and chemical content, samples were analyzed from leaf material from 20 trees that included two sets of full-sibling seedling trees, the maternal tree and the surviving paternal tree. Descriptive discriminant analysis found good separation between the two full-sibling groups, and that the separation was associated with chemistry of the parental trees. Six groups of chemicals were identified that explained the variation among the trees. We discuss the results in relation to the discrimination process used by wood-boring insects for site-selection on host trees, for tree selection among potential host trees, and the potential use of terpenoid chemical content in chemotaxonomy of avocado trees.
doi:10.1371/journal.pone.0073601
PMCID: PMC3767776  PMID: 24039994
3.  A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update 
In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system.
doi:10.3389/fmicb.2013.00165
PMCID: PMC3698417  PMID: 23847598
CTV; infectious cDNA clones; agroinoculation; Agrobacterium tumefaciens; oncogenic strains; graft transmission; N. benthamiana protoplasts; RNA silencing suppressor
4.  Identification and Functional Characterization of the Soybean GmaPPO12 Promoter Conferring Phytophthora sojae Induced Expression 
PLoS ONE  2013;8(6):e67670.
Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthorasojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotianabenthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5’ and 3’ deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters.
doi:10.1371/journal.pone.0067670
PMCID: PMC3695865  PMID: 23840763
5.  phiC31 Integrase-Mediated Site-Specific Recombination in Barley 
PLoS ONE  2012;7(9):e45353.
The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.
doi:10.1371/journal.pone.0045353
PMCID: PMC3443236  PMID: 23024817
6.  Overexpression of ORCA3 and G10H in Catharanthus roseus Plants Regulated Alkaloid Biosynthesis and Metabolism Revealed by NMR-Metabolomics 
PLoS ONE  2012;7(8):e43038.
In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of 1H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus.
doi:10.1371/journal.pone.0043038
PMCID: PMC3423439  PMID: 22916202
7.  Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics 
BMC Biotechnology  2012;12:41.
Background
The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes β-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees.
Results
The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability.
Conclusions
Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.
doi:10.1186/1472-6750-12-41
PMCID: PMC3462728  PMID: 22794278
Citrus; Transgenic trees; Selectable marker genes; uidA; nptII; Substantial equivalence; Pleiotropic effects; Long-term transgene stability; Phenotypic assessment; Field performance.
8.  The Effects of Seed Size on Hybrids Formed between Oilseed Rape (Brassica napus) and Wild Brown Mustard (B. juncea) 
PLoS ONE  2012;7(6):e39705.
Background
Seed size has significant implications in ecology, because of its effects on plant fitness. The hybrid seeds that result from crosses between crops and their wild relatives are often small, and the consequences of this have been poorly investigated. Here we report on plant performance of hybrid and its parental transgenic oilseed rape (Brassica napus) and wild B. juncea, all grown from seeds sorted into three seed-size categories.
Methodology/Principal Findings
Three seed-size categories were sorted by seed diameter for transgenic B. napus, wild B. juncea and their transgenic and non-transgenic hybrids. The seeds were sown in a field at various plant densities. Globally, small-seeded plants had delayed flowering, lower biomass, fewer flowers and seeds, and a lower thousand-seed weight. The seed-size effect varied among plant types but was not affected by plant density. There was no negative effect of seed size in hybrids, but it was correlated with reduced growth for both parents.
Conclusions
Our results imply that the risk of further gene flow would probably not be mitigated by the small size of transgenic hybrid seeds. No fitness cost was detected to be associated with the Bt-transgene in this study.
doi:10.1371/journal.pone.0039705
PMCID: PMC3382164  PMID: 22745814
9.  The monoterpene limonene in orange peels attracts pests and microorganisms 
Plant Signaling & Behavior  2011;6(11):1820-1823.
Plant volatiles include terpenoids, which are generally involved in plant defense, repelling pests and pathogens and attracting insects for herbivore control, pollination and seed dispersal. Orange fruits accumulate the monoterpene limonene at high levels in the oil glands of their fruit peels. When limonene production was downregulated in orange fruits by the transgenic expression of a limonene synthase (CitMTSE1) in the antisense configuration, these fruits were resistant to the fungus Penicillium digitatum (Pers.) Sacc. and the bacterium Xanthomonas citri subsp. citri and were less attractive to the medfly pest Ceratitis capitata. These responses were reversed when the antisense transgenic orange fruits were treated with limonene. To gain more insight into the role of the limonene concentration in fruit responses to pests and pathogens, we attempted to overexpress CitMTSE1 in the sense configuration in transgenic orange fruits. Only slight increases in the amount of limonene were found in sense transgenic fruits, maybe due to the detrimental effect that excessive limonene accumulation would have on plant development. Collectively, these results suggest that when limonene reaches peak levels as the fruit develops, it becomes a signal for pest and pathogen attraction, which facilitate access to the fruit for pulp consumers and seed dispersers.
doi:10.4161/psb.6.11.16980
PMCID: PMC3329358  PMID: 22212123
co-evolution; defense; repellency; secondary metabolism; seed dispersers; trophic interaction; volatiles
10.  Pollen Competition as a Reproductive Isolation Barrier Represses Transgene Flow between Compatible and Co-Flowering Citrus Genotypes 
PLoS ONE  2011;6(10):e25810.
Background/Objective
Despite potential benefits granted by genetically modified (GM) fruit trees, their release and commercialization raises concerns about their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. Information compiled from field trials on GM trees is essential to propose measures to minimize the transgene dispersal. We have conducted a field trial of seven consecutive years to investigate the maximum frequency of pollen-mediated crop-to-crop transgene flow in a citrus orchard, and its relation to the genetic, phenological and environmental factors involved.
Methodology/Principal Findings
Three different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM self-incompatible contiguous citrus genotype (recipient) were used in conditions allowing natural entomophilous pollination to occur. The examination of 603 to 2990 seeds per year showed unexpectedly low frequencies (0.17–2.86%) of transgene flow. Paternity analyses of the progeny of subsets of recipient plants using 10 microsatellite (SSR) loci demonstrated a higher mating competence of trees from another non-GM pollen source population that greatly limited the mating chance of the contiguous cross-compatible and flowering-synchronized transgenic pollen source. This mating superiority could be explained by a much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations.
Conclusions/Significance
Pollen competition strongly contributed to transgene confinement. Based on this finding, suitable isolation measures are proposed for the first time to prevent transgene outflow between contiguous plantings of citrus types that may be extendible to other entomophilous transgenic fruit tree species.
doi:10.1371/journal.pone.0025810
PMCID: PMC3185051  PMID: 21991359

Results 1-10 (10)