Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin

Three genes encoding anthocyanidin reductase (ANR) in apple (Malus×domestica Borkh.), designated MdANR1, MdANR2a, and MdANR2b, have been cloned and characterized. MdANR1 shows 91% identity in coding DNA sequences with MdANR2a and MdANR2b, while MdANR2a and MdANR2b are allelic and share 99% nucleotide sequence identity in the coding region. MdANR1 and MdANR2 genes are located on linkage groups 10 and 5, respectively. Expression levels of both MdANR1 and MdANR2 genes are generally higher in yellow-skinned cv. Golden Delicious than in red-skinned cv. Red Delicious. Transcript accumulation of MdANR1 and MdANR2 genes in fruits gradually decreased throughout fruit development. Ectopic expression of apple MdANR genes in tobacco positively and negatively regulates the biosynthesis of proanthocyanidins (PAs) and anthocyanin, respectively, resulting in white, pale pink-coloured, and white/red variegated flowers. The accumulation of anthocyanin is significantly reduced in all tobacco transgenic flowers, while catechin and epicatechin contents in transgenic flowers are significantly higher than those in flowers of wild-type plants. The inhibition of anthocyanin synthesis in tobacco transgenic flowers overexpressing MdANR genes is probably attributed to down-regulation of CHALCONE ISOMERASE (CHI) and DIHYDROFLAVONOL-4-REDUCTASE (DFR) genes involved in the anthocyanin pathway. Interestingly, several transgenic lines show no detectable transcripts of the gene encoding leucoanthocyanidin reductase (LAR) in flowers, but accumulate higher levels of catechin in flowers of transgenic plants than those of wild-type plants. This finding suggests that the ANR gene may be capable of generating catechin via an alternative route, although this mechanism is yet to be further elucidated

Expression profiles of differentially regulated genes during the early stages of apple flower infection with Erwinia amylovora

To identify genes involved in the response to the fire blight pathogen Erwinia amylovora in apple (Malus×domestica), expression profiles were investigated using an apple oligo (70-mer) array representing 40, 000 genes. Blossoms of a fire blight-susceptible apple cultivar Gala were collected from trees growing in the orchard, placed on a tray in the laboratory, and spray-inoculated with a suspension of E. amylovora at a concentration of 108 cfu ml−1. Uninoculated detached flowers served as controls at each time point. Expression profiles were captured at three different time points post-inoculation at 2, 8, and 24 h, together with those at 0 h (uninoculated). A total of about 3500 genes were found to be significantly modulated in response to at least one of the three time points. Among those, a total of 770, 855, and 1002 genes were up-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively; while, 748, 1024, and 1455 genes were down-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively. Over the three time points post-inoculation, 365 genes were commonly up-regulated and 374 genes were commonly down-regulated. Both sets of genes were classified based on their functional categories. The majority of up-regulated genes were involved in metabolism, signal transduction, signalling, transport, and stress response. A number of transcripts encoding proteins/enzymes known to be up-regulated under particular biotic and abiotic stress were also up-regulated following E. amylovora treatment. Those up- or down-regulated genes encode transcription factors, signaling components, defense-related, transporter, and metabolism, all of which have been associated with disease responses in Arabidopsis and rice, suggesting similar response pathways are involved in apple blossoms

Expresión en tomate de un polipéptido antigénico con epítopos de 3 exotoxinas bacterianas (DPT) codificado por un gen sintético.

Tesis (Doctorado en Ciencias en Biología Molecular)."Una prioridad actual en el campo de vacunas es el desarrollo de vacunas de subunidades y multicomponentes que no produzcan reacciones adversas y protejan contra varios patógenos. Los problemas de salud causados por las exotoxinas de Corynebacterium diphteriae, Bordetella pertussis y Clostridium tetani van desde síntomas leves hasta la muerte. El presente trabajo se enfoca al desarrollo de una vacuna comestible contra la difteria, tos ferina y tétanos en plantas. Se transformó genéticamente a plantas de tomate por medio de Agrobacterium tumefaciens portador de un gen sintético optimizado para plantas el cual codifica para un polipéptido novedoso que contiene dos adyuvantes y seis epítopos inmunoprotectores de las toxinas DPT, unidos por péptidos conectores. Se verificó la integración del gen sintético DPT (sDPT) por PCR en las plantas transgénicas de tomate y se confirmó por Southern blot observándose de 1-2 copias del gen en diversas líneas. Asi mismo se detectó un transcrito específico del tamaño esperado (570 pb) por RT-PCR. El polipéptido codificado por el gen sDPT fue detectado por inmunoensayos in vitro con anticuerpos específicos, con valores equivalentes a 1.9-5.8 μg/g de toxoide diftérico, 0.0003-0.15 μg/g de toxoide pertúsico y 4.5-16.8 μg/g de toxoide tetánico en hojas de tomates transgénicos. Además, se observó que la proteína recombinante expresada en las plantas de tomate fue capaz de inducir anticuerpos específicos en animales.Ratones Balb/c fueron inmunizados por vía oral con el tejido vegetal en tres dosis semanales. En el suero analizado se encontró respuesta del tipo IgG a las exotoxinas diftérica, pertúsica y tetánica inducida por el polipéptido sintético. En el fluído traqueopulmonar se observó una modesta respuesta del tipo IgG. Una respuesta alta del tipo IgA hacia el epítope tetánico fue evidente en los lavados intestinales. Los niveles de respuesta en estos grupos fueron más altos que en el grupo de ratones alimentado con tomate no transformado. Estos resultados sugieren que la proteína recombinante DPT expresada en plantas de tomate puede inducir anticuerpos específicos in vivo y faltaría retar con las toxinas a ratones preinmunizados con tomates que contenienen el gen sDPT para ver si existe protección. Este es el primer reporte de la expresión de un polipéptido codificado por un gen no presente es la naturaleza, con secuencias antigénicas de las exotoxinas diftérica, pertúsica y tetánica, en un sistema vegetal y podria ser utilizado como una vacuna comestible práctica contra la difteria, tos ferina y tétanos. Este trabajo prueba además, que es posible diseñar genes que codifiquen multiepítopos que induzcan inmunidad protectora en ratones.""A current priority of vaccinology is the development of subunit and multicomponent vaccines which protect and avoid secondary effects. The health problems caused by the exotoxins of Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani range from light symptoms to death. We have attempted to develop an edible vaccine against diphtheria, pertussis and tetanus in plants. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimized synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. We detected the integration of the synthetic DPT (sDPT) gene by PCR in transformed tomato plants and confirmed it by Southern blot observing 1-2 copies of the gene in diverse plants. A specific transcript of the expected molecular size (570 bp) was detected by RT-PCR. In addition we detected a polypeptide encoded by the sDPT gene by immunoassay in vitro, with specific antibodies with average values of 1.9-5.8 μg/g of diphtheria toxoid, 0.0003-0.15 μg/g of pertussis toxoid and 4.5-16.8 μg/g of tetanus toxoid in leaves of transgenic tomatoes. We also assayed if the recombinant protein expressed in tomato plants could induce specific antibodies in animals. Balb/c mice were immunized by the route with the plant material in three weekly doses. Sera tested for IgG antibody to pertussis, tetanus and diphtheria toxin showed responses to the synthetic polypeptide. In the tracheopulmonary fluid a modest IgG response was observed. High IgA response against tetanus toxin was evident in gut washes. The levels of response in these groups were higher than those of mice that had been fed with wild-type tomatoes. Our results suggest that the recombinant DPT protein expressed in tomato plants induces specific antibodies in vivo. Challenge with toxins in mice previously immunized with the tomatoes containing sDPT wood be needed to test for protection. This is the first report of the expression of a polypeptide encoded by a gene, which does not exist in nature,l containing diphteria, pertussis and tetanus exotoxin antigen sequences in plants, and may be used as a practical edible vaccine against diphtheria, pertussis and tetanus. Our work also demonstrates that it is possible to design genes encoding multiepitopes polipeptides that induce protective immunity in mice.

Transgenic plants expressing a novel polypeptide which is encoded by a synthetic gene that contains immunoprotective sequences from corinebacterium diphteriae, bordetella pertussis and clostridium tetani exotoxins

"La presente invención se refiere a una vacuna producida en plantas contra difteria tos ferina y tétanos conocida como vacuna triple DPT. La vacuna incluye un complejo inmunogénico a partir de la transformación de jitomate con un gen quimérico optimizado para expresarse en plantas, que codifica un nuevo polipéptido recombinante el cual no existe en la naturaleza y que contiene los principales epítopos inmunoprotectores de difteria, tos ferina y tétanos. También se describen los métodos para construir el vector de transformación genética y los métodos de regeneración mediante cultivo de tejidos vegetales. Asimismo se presentan los resultados de la producción de anticuerpos en pruebas realizadas en ratones los cuales pueden ser útiles para la inmunización de mamíferos.""Provided is a plant produced vaccine acting against diphtheria, pertussis and tetanus, which is best known as DPT vaccine. The vaccine includes an immunogenic complex resulting from the transformation of tomatoe with a chimeric gene optimised to be expressed in plants, which encodes a novel recombinant polypeptide that is not available in nature and which contains the main immunoprotective epitopes of diphtheria, pertussis and tetanus. In addition, methods for building the genetic transformation vector are described, as well as regeneration methods based on the culture of vegetable tissues. The results of the antibody production in tests performed to mice are also shown, which may be useful for the mammals immunisation.

Ectopic Expression of Apple F3′H Genes Contributes to Anthocyanin Accumulation in the Arabidopsis tt7 Mutant Grown Under Nitrogen Stress1[C][W][OA]

Three genes encoding flavonoid 3′-hydroxylase (F3′H) in apple (Malus × domestica), designated MdF3′HI, MdF3′HIIa, and MdF3′HIIb, have been identified. MdF3′HIIa and MdF3′HIIb are almost identical in amino acid sequences, and they are allelic, whereas MdF3′HI has 91% nucleotide sequence identity in the coding region to both MdF3′HIIa and MdF3′HIIb. MdF3′HI and MdF3′HII genes are mapped onto linkage groups 14 and 6, respectively, of the apple genome. Throughout the development of apple fruit, transcriptional levels of MdF3′H genes along with other anthocyanin biosynthesis genes are higher in the red-skinned cv Red Delicious than that in the yellow-skinned cv Golden Delicious. Moreover, patterns of MdF3′H gene expression correspond to accumulation patterns of flavonoids in apple fruit. These findings suggest that MdF3′H genes are coordinately expressed with other genes in the anthocyanin biosynthetic pathway in apple. The functionality of these apple F3′H genes has been demonstrated via their ectopic expression in both the Arabidopsis (Arabidopsis thaliana) transparent testa7-1 (tt7) mutant and tobacco (Nicotiana tabacum). When grown under nitrogen-deficient conditions, transgenic Arabidopsis tt7 seedlings expressing apple F3′H regained red color pigmentation and significantly accumulated both 4′-hydrylated pelargonidin and 3′,4′-hydrylated cyanidin. When compared with wild-type plants, flowers of transgenic tobacco lines overexpressing apple F3′H genes exhibited enhanced red color pigmentation. This suggests that the F3′H enzyme may coordinately interact with other flavonoid enzymes in the anthocyanin biosynthesis pathway