Ricardo J. Ordás

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

Induction of a Virus-Specific Antibody Response to Foot and Mouth Disease Virus Using the Structural Protein VP1 Expressed in Transgenic Potato Plants

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

Agrobacterium tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer

Abstract This study is the first report of a protocol for transfer and expression of foreign chimeric genes into cotyledons excised from Pinus pinea L. embryos. Agrobacterium tumefaciens EHA105 harbouring the plasmid p35SGUSint was more infective than LBA4404 or C58 GV3850, as determined by the percentage of cotyledons showing uidA expression. Factors which significantly affected the T-DNA transfer included: (1) preinduction and concentration of bacteria, (2) days of coculture and (3) the wounding procedure applied. More efficient transfer of the uidA gene was achieved growing the bacteria in YEP medium at pH 7, infecting the cotyledons according to the sonication-assisted Agrobacterium-mediated transformation procedure with a bacterial density of 1 (OD600 nm) for 5 min, and coculture for 72 h. Using this protocol, 49.7% of the cotyledons showed a diffuse blue staining 7 days after infection. However, all were necrotic 30 days after inoculation. Since a decrease in bacterial density to 0.01 allowed the recovery of about 4% of cotyledons forming buds 1 month after inoculation, we conclude that the high mortality associated with the infection may be related to the hypersensitive response of the plant to bacterial infection.

Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus.

Summary. Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV.

Genetic transformation of potato (Solanum tuberosum): An efficient method to obtain transgenic plants

A quick procedure for efficient transformation of potato (cv. Desiree) has been devised. Leaf disc have been inoculated with an Agrobacterium tumefaciens harbouring a Ti plasmid derived binary vector. Transformed shoots carrying the neomycin phosphotransferase gene were regenerated using a feeder layer technique after 4 weeks on selective medium containing kanamycin. Transgenic plants obtained in large numbers appeared phenotypically normal and expressed the NPT II gene. The present work points out that culture conditions are fundamental to obtain the highest transformation efficiency.

Relationships between hormonal contents and the organogenic response in Pinus pinea cotyledons

Isolated cotyledons from Pinus pinea L. germinated embryos cultured in vitro showed shoot organogenesis in response to treatment with 4.4 µM N 6 -benzyladenine (BA). In late germination phases, the extent of the response onset decreased and adventitious buds were redistributed to the basal zone of the cotyledons. To understand these two events, analyses of indole-3-acetic acid (IAA) and several cytokinins have been carried out in apical and basal portions of cotyledons taken at four different culture periods of the embryos on germination medium (0, 2, 4 or 6 d). The highest endogenous content of all hormones, except N 6 -isopentenyladenosine, was found in the most responsive explants, those that were taken from 0-d-old cultured embryos. Apical portions of cotyledons taken from 0 to 4-d-old cultured embryos showed a reduction in the content of IAA and several cytokinins, but basal portions did not show the same pattern. BA dose-response curves were made to explain the loss of caulogenic potential with the age of the explants. The results suggested the combination of two factors, changes in the endogenous hormonal content in original explants and loss of their sensitivity to exogenously applied BA, as the cause for the reduction of the ability of Pinus pinea cotyledons to form adventitious buds.

EuroPineDB: a high-coverage web database for maritime pine transcriptome

BackgroundPinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases.DescriptionEuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided.ConclusionsThe EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

The effect of the promoter on expression of VP60 gene from rabbit hemorrhagic disease virus in potato plants

Abstract We have investigated the use of five different promoter-5′UTR leader combinations to develop a potato plant-based expression system for the production of VP60 protein from rabbit haemorrhagic disease virus (RHDV). The relative efficiency of CaMV 35S, modified CaMV 35S, sunflower polyubiquitin and patatin promoters, as well as the addition of the 5′ leader sequence from φ10 gene of T7 phage, on VP60 gene expression in leaf and tuber tissues of plants, cultivated both in vitro and as potted plants, was investigated. Our results indicated that the B33 promoter with the φ10 leader gave the highest level of expression in tubers. The rabbits immunised with tuber plant extracts containing VP60 elicited specific antibody responses and were protected against challenge with virulent RDHV.

Oral Immunization using Tuber Extracts from Transgenic Potato Plants Expressing Rabbit Hemorrhagic Disease Virus Capsid Protein

Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult animals that is characterized by acute liver damage and disseminated intravascular coagulation. In this study, we report the production of the major structural protein VP60 of rabbit hemorrhagic disease virus in transgenic tubers of potato plants and its use as an oral immunogen in rabbits.

The toxicity of antibiotics and herbicides on in vitro adventitious shoot formation of Pinus pinea L. cotyledons

SummaryThe toxicity of three antibiotics commonly used to eliminate Agrobacterium tumefaciens from plant tissue during transformation were tested to determine their effect on Pinus pinea L. morphogenesis. Cefotaxime and vancomycin at a dose of 250 µg ml−1, as well as ticarcillin at 300 µg ml−1, were essentially nontoxic to the culture and significantly enhanced regeneration and shoot development. Kanamycin, a widely used selection agent for plant transformation, strongly inhibited regeneration even at very low doses. On the contrary, phosphinothricin and its ammonium salt glufosinate in the commercial formulation Finale® proved to be the best selection agents because they were less toxic to stone pine cotyledons. Thus, they could be applied immediately after transformation to avoid escapes. Other schemes for selection and recovery of transgenic stone pine trees are discussed.