Expression and purification of an anti-Foot-and-mouth disease virus single chain variable antibody fragment in tobacco plants.

Low-cost recombinant antibodies could provide a new strategy to control Foot-and-mouth disease virus (FMDV) outbreaks by passive immunization of susceptible animals. In this study, a single chain variable antibody fragment (scFv) recognizing FMDV coat protein VP1 was expressed in transgenic tobacco plants. To enhance the accumulation of scFv protein, the codon-usage of a murine hybridoma-derived scFv gene was adjusted to mimic highly expressed tobacco genes and fused to an elastin-like polypeptide (ELP) tag. This scFv-ELP fusion accumulated up to 0.8% of total soluble leaf protein in transgenic tobacco. To recover scFv-ELP protein from the leaf extract, a simple and scalable purification strategy was established. Purified scFv-ELP fusion was cleaved to separate the scFv portion. Finally, it was shown that the purified scFv proteins retained their capacity to bind the FMDV in the absence or presence of ELP fusion.

Transgenic plants for animal health: plant-made vaccine antigens for animal infectious disease control.

A variety of plant species have been genetically modified to accumulate vaccine antigens for human and animal health and the first vaccine candidates are approaching the market. The regulatory burden for animal vaccines is less than that for human use and this has attracted the attention of researchers and companies, and investment in plant-made vaccines for animal infectious disease control is increasing. The dosage cost of vaccines for animal infectious diseases must be kept to a minimum, especially for non-lethal diseases that diminish animal welfare and growth, so efficient and economic production, storage and delivery are critical for commercialization. It has become clear that transgenic plants are an economic and efficient alternative to fermentation for large-scale production of vaccine antigens. The oral delivery of plant-made vaccines is particularly attractive since the expensive purification step can be avoided further reducing the cost per dose. This review covers the current status of plant-produced vaccines for the prevention of disease in animals and focuses on barriers to the development of such products and methods to overcome them.

Steviol glycoside biosynthesis.

Steviol glycosides are found in high concentrations in the leaves of the Paraguayan perennial herb Stevia rebaudiana and their intense sweetness, as well as high concentration in Stevia leaf tissue, has made them the subject of research interest for over 100 years. Steviol glycosides are diterpenoids whose biosynthetic pathways share four steps in common with gibberellic acid formation. The convergence of genomics and plant biochemistry has led to the rapid elucidation of the genes coding for the various enzymes in the biosynthetic pathway. Functional characterization of the enzymes coded for by those genes is on-going. The first committed step in the pathway is the synthesis of the aglycone steviol and the various glycosides found in the leaf tissue result from the elaboration of steviol by a number of glucosyltransferases.

Evaluation of Host Resistance and Soil Fumigation for the Management of Black Root Rot of Tobacco in Ontario.

Black root rot of tobacco, caused by the soilborne fungus Thielaviopsis basicola, is a serious problem in many tobacco (Nicotiana tabacum L.)-growing regions of the world. In Ontario, the disease is favored by cool, wet soil conditions and heavy textured or poorly drained soils. Yield loss can be severe under these conditions and fumigants containing chloropicrin are used extensively for controlling the disease. Usually, fumigants control the disease reasonably well, but they are costly and could cause a negative environmental impact. A 2-year study was conducted to evaluate the performance of resistant (AC Gayed) and moderately susceptible (Delgold) tobacco cultivars and soil fumigation to black root rot. T. basicola reduced yield of the susceptible Delgold cultivar. The interaction between genotype and fumigation was significant for most traits examined, indicating that the two genotypes responded differently. Orthogonal comparisons indicate that yield from nonfumigated AC Gayed was higher than that of nonfumigated Delgold. Yield of nonfumigated AC Gayed was also not significantly different from the yield of AC Gayed treated either with Vorlex Plus (1,3-dichloropropene+methyl isothiocyanate) or with Vorlex Plus CP (1,3-dichloropropene+methyl isothiocyanate+chloropicrin). In contrast, the yield of nonfumigated Delgold was lower than Delgold treated with Vorlex Plus CP.

Expressed sequence tags from the Yukon ecotype of Thellungiella reveal that gene expression in response to cold, drought and salinity shows little overlap.

Thellungiella salsuginea (also known as T. halophila) is a close relative of Arabidopsis that is very tolerant of drought, freezing, and salinity and may be an appropriate model to identify the molecular mechanisms underlying abiotic stress tolerance in plants. We produced 6578 ESTs, which represented 3628 unique genes (unigenes), from cDNA libraries of cold-, drought-, and salinity-stressed plants from the Yukon ecotype of Thellungiella. Among the unigenes, 94.1% encoded products that were most similar in amino acid sequence to Arabidopsis and 1.5% had no match with a member of the family Brassicaceae. Unigenes from the cold library were more similar to Arabidopsis sequences than either drought- or salinity-induced sequences, indicating that latter responses may be more divergent between Thellungiella and Arabidopsis. Analysis of gene ontology using the best matched Arabidopsis locus showed that the Thellungiella unigenes represented all biological processes and all cellular components, with the highest number of sequences attributed to the chloroplast and mitochondria. Only 140 of the unigenes were found in all three abiotic stress cDNA libraries. Of these common unigenes, 70% have no known function, which demonstrates that Thellungiella can be a rich resource of genetic information about environmental responses. Some of the ESTs in this collection have low sequence similarity with those in Genbank suggesting that they may encode functions that may contribute to Thellungiella's high degree of stress tolerance when compared with Arabidopsis. Moreover, Thellungiella is a closer relative of agriculturally important Brassica spp. than Arabidopsis, which may prove valuable in transferring information to crop improvement programs.

An integrated approach to oilseed rape cultivar selection using phenotypic stability.

Various methods of evaluating phenotypic stability have been proposed; however, no single method can adequately describe cultivar performance. The objectives of this study were to integrate a number of methods of evaluating stability and to use this approach for cultivar selection. These objectives were considered in the context of the broad-based oilseed rape cultivar (Brassica napus spp. oleifera) evaluation system currently used in western Canada. Regression analysis was used to assess cultivar response to environments. Cluster analysis was used to assemble cultivars into groups with similar regression coefficients (b i ) and mean yield. Three parametric stability parameters, years within locations mean square (MS; Y/L), Shukla's stability variance (σ i (2) ), and Francis and Kannenberg's coefficient of variability (CV i ), were compared to determine which method would be most suitable for selection of oilseed rape cultivars from within clustered groups. Yield data from three cultivars and six breeding lines that had been tested for 2 years at 26 locations in the Western Canola Cooperative Test 'A' were used for all calculations. The cluster analysis was successful in identifying commercially acceptable breeding lines. The parameter MS i Y/L was considered to be more appropriate than either CV i or σ i (2) , because it measured only the unpredictable portion of the genotype x environment interaction and was independent of the other cultivars in the test. The use of cluster analysis to group entries with similar b i values and mean yields, followed by selection for stability within groups, was advocated.

Field performance and heavy metal concentrations of transgenic flue-cured tobacco expressing a mammalian metallothionein-beta-glucuronidase gene fusion.

Cadmium (Cd) is a nonessential heavy metal that can cause acute and chronic illness in humans. Some plant species such as tobacco (Nicotiana tabacum L.) tend to accumulate high levels of Cd in leaf tissue, the consumed portion of the plant. Tissue-specific expression of mammalian metallothionein has been suggested as a means of partitioning Cd in nonconsumed portions of transgenic plants. The purpose of the experiment reported here was to evaluate Cd concentration and agronomic performance of four field-grown transgenic tobacco lines harbouring a metallothionein-beta-glucuronidase (MG) gene fusion driven by the constitutive 35S promoter of cauliflower mosaic virus. The trial was grown in a region of Canada known to have high background levels of Cd. The agronomic evaluation showed that some of the transgenic lines were equal to, while others performed more poorly than, the untransformed control for yield, days to flower, and leaf number. Gene expression measured by beta-glucuronidase activity showed that all of the transgenic lines expressed the MG gene in the upper portion of the plant. One line did not express the MG gene in the roots. Cd levels in the leaf tissue of transformed lines were not significantly different from the untransformed control.

Diterpene synthesis in Stevia rebaudiana: recruitment and up-regulation of key enzymes from the gibberellin biosynthetic pathway.

Stevia rebaudiana Bertoni leaves accumulate a mixture of at least eight different glycosides derived from the tetracyclic diterpene steviol. These natural products taste intensely sweet and have similar biosynthetic origins to those of gibberellic acid (GA). The initial steps leading to the formation of GA result from the two-step cyclization of geranylgeranyl diphosphate (GGDP) to (-)-kaurene via the action of two terpene cyclases (-)-copalyl diphosphate synthase (CPS) and (-)-kaurene synthase (KS). Steviol biosynthesis probably uses the same mechanism although the genes and enzymes from S. rebaudiana that are involved in the cyclization of GGDP have not been characterized. We have isolated both the CPS and KS genes from S. rebaudiana and found that recombinant CPS and KS were catalytically active, suggesting that the CPS and KS genes participate in steviol biosynthesis. The genes coding for CPS and KS are usually present in single copies in most plant species and their expression is normally low and limited to rapidly growing tissues. The KS gene has been duplicated in the S. rebaudiana genome and both the KS and CPS genes are highly expressed in mature leaves, a pattern opposite to that found with GA biosynthesis. This pattern may, at least in part, lead to temporal and spatial separation of GA and steviol biosynthesis and probably helps to prevent over-expression from interfering with normal GA metabolism. Our results show that CPS and KS are part of the steviol glycoside biosynthetic pathway and that Stevia rebaudiana has recruited two genes to secondary metabolism from a highly regulated pathway involved in hormone biosynthesis.

Leaf Ests from Stevia rebaudiana: a resource for gene discovery in diterpene synthesis.

Expressed sequence tags (ESTs) are providing a new approach to gene discovery in plant secondary metabolism. Stevia rebaudiana Bert. leaves produce high concentrations of diterpene steviol glycosides and should be a rich source of transcripts involved in diterpene synthesis. In order to create a resource for gene discovery and increase our understanding of steviol glycoside biosynthesis, we sequenced 5,548 ESTs from a S. rebaudiana leaf cDNA library. The EST collection was fully annotated based on database search results. ESTs involved in diterpene synthesis were identified using published sequences as electronic probes, by keyword searches of search results, and by differential representation. A significant portion of the ESTs were specific for standard leaf metabolic pathways; energy and primary metabolism represented 17.6% and 13.1% of total transcripts respectively. Diterpene metabolism in S. rebaudiana represented 1.1% of total transcripts. This study identified candidate genes for 70% of the known steps in the steviol glycoside pathway. One candidate, kaurene oxidase, was the 8th most abundant EST in the collection. Identification of many candidate genes specific to the I -deoxyxylulose 5-phosphate pathway suggests that the primary source of isopentenyl diphosphate, a precursor of geranylgeranyl diphosphate, is via the non-mevalonic acid pathway. The use of ESTs has greatly facilitated the identification of candidate genes and increased our understanding of diterpene metabolism.