Enhanced Abiotic Stress Tolerance of Vicia faba L. Plants Heterologously Expressing the PR10a Gene from Potato.

Pathogenesis-related (PR) proteins are known to play relevant roles in plant defense against biotic and abiotic stresses. In the present study, we characterize the response of transgenic faba bean (Vicia faba L.) plants encoding a PR10a gene from potato (Solanum tuberosum L.) to salinity and drought. The transgene was under the mannopine synthetase (pMAS) promoter. PR10a-overexpressing faba bean plants showed better growth than the wild-type plants after 14 days of drought stress and 30 days of salt stress under hydroponic growth conditions. After removing the stress, the PR10a-plants returned to a normal state, while the wild-type plants could not be restored. Most importantly, there was no phenotypic difference between transgenic and non-transgenic faba bean plants under well-watered conditions. Evaluation of physiological parameters during salt stress showed lower Na+-content in the leaves of the transgenic plants, which would reduce the toxic effect. In addition, PR10a-plants were able to maintain vegetative growth and experienced fewer photosystem changes under both stresses and a lower level of osmotic stress injury under salt stress compared to wild-type plants. Taken together, our findings suggest that the PR10a gene from potato plays an important role in abiotic stress tolerance, probably by activation of stress-related physiological processes.

Lack of efficacy of transgenic pea (Pisum sativum L.) stably expressing antifungal genes against Fusarium spp. in three years of confined field trials.

Fusarium root rot is a major pea disease in Canada and only partial tolerance exists in germplasm. Transgenic technologies may hold promise but the economic benefits of genetically modified (GM) pea will need to surpass the regulatory costs, time and labor involved in bringing a GM crop to market. European pea (Pisum sativum L.) cultivars expressing four antifungal genes, 1-3 ß glucanase (G), endochitinase (C) (belonging to PR proteins family), polygalacturonase inhibiting proteins (PGIPs) (P) and stilbene synthase (V) have been transformed for disease tolerance and showed disease tolerance under laboratory conditions. Transgenic lines with four antifungal genes inserted either individually or stacked through crossing were tested for their efficacy against Fusarium root rot (Fusarium avenaceum) in confined trials over three years (2013 to 2015) in comparison with two parental German lines and three Canadian lines. Superior emergence, higher fresh weight or lower disease ratings above and below ground, of transgenic lines in presence of disease inoculum were not observed consistently in the three years of field experiments when compared to the parental and Canadian lines in the presence of disease inoculum. No indication of an advantage of stacked genes over single genes was observed. Most transgenic lines had lower relative gene expression in the roots than in the leaves in greenhouse trials suggesting a possible explanation for poor tolerance to Fusarium root rot. Field trials are necessary to verify the agronomic performance and ecological relevance of the promising effects detected under laboratory conditions.

Antifungal genes expressed in transgenic pea (Pisum sativum L.) do not affect root colonization of arbuscular mycorrhizae fungi.

Genetically modified crops have raised concerns about unintended consequences on non-target organisms including beneficial soil associates. Pea transformed with four antifungal genes 1-3 ß glucanase, endochitinase, polygalacturonase-inhibiting proteins, and stilbene synthase is currently under field-testing for efficacy against fungal diseases in Canada. Transgenes had lower expression in the roots than leaves in greenhouse experiment. To determine the impact of disease-tolerant pea or gene products on colonization by non-target arbuscular mycorrhizae and nodulation by rhizobium, a field trial was established. Transgene insertion, as single gene or stacked genes, did not alter root colonization by arbuscular mycorrhiza fungus (AMF) or root nodulation by rhizobium inoculation in the field. We found no effect of transgenes on the plant growth and performance although, having a dual inoculant with both AMF and rhizobium yielded higher fresh weight shoot-to-root ratio in all the lines tested. This initial risk assessment of transgenic peas expressing antifungal genes showed no deleterious effect on non-target organisms.

Metabolic engineering of apple by overexpression of the MdMyb10 gene.

Flavonoids are low-molecular-weight phenolic compounds that are widely distributed in the plant kingdom. They have different roles in plant resistance to biotic and abiotic stresses. The transcription factor gene MdMyb10 (Gene Bank: DQ267896) was introduced into two apple (Malus domestica Borkh.) cultivars i.e. 'Holsteiner Cox (HC)' and 'Gala' via Agrobacterium-mediated transformation. The regenerated shoots were selected on kanamycin containing media. The presence of additional MdMyb10 gene in putative shoots was confirmed by PCR, RT-PCR and Southern blotting. Expression level of introduced MdMyb10 gene was analyzed by quantitative real time PCR. The results confirmed a dramatic increase in overexpression of MdMyb10 in the transgenic plants, up to 1261 and 847-folds for cultivars Holsteiner Cox and Gala, respectively compared to non-transformed negative control plants. HPLC-MS was used to determine the levels of different flavonoid compounds in both non-transgenic and transgenic plants. In MdMyb10 'HC' transgenic plants, some of the polyphenols analyzed were enhanced while others were reduced in comparison to their levels in the non-transgenic plants. On the other hand, all of the analyzed polyphenol classes were induced in MdMyb10 'Gala' transgenic plants in comparison to their levels in the non-transgenic plants. In the present study, the flavonoid pathway was successfully modified in apple by overexpressing the MdMyb10 transcription factor to validate the hypothesis of increased effect on plant disease resistance.

Molecular and functional characterization of cry1Ac transgenic pea lines.

Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

Enhanced tolerance to drought and salt stresses in transgenic faba bean (Vicia faba L.) plants by heterologous expression of the PR10a gene from potato.

KEY MESSAGE: We report for the first time that expression of potato PR10a gene in faba bean causes enhanced tolerance to drought and salinity. Grain legumes such as soybean (Glycine max L. Merrill), pea (Pisum sativum L.) and faba bean (Vicia faba L.) are staple sources of protein for human and animal nutrition. Among grain legumes, faba bean is particularly sensitive to abiotic stress (in particular osmotic stress due to lack of water or enhanced soil salinity) and often suffers from severe yield losses. Many stress responsive genes have been reported with an effect on improving stress tolerance in model plants. Pathogenesis-related proteins are expressed by all plants in response to pathogen infection and, in many cases, in response to abiotic stresses as well. The PR10a gene isolated from the potato cultivar Desiree was selected for this study due to its role in enhancing salt and/or drought tolerance in potato, and transferred into faba bean cultivar Tattoo by Agrobacterium tumefaciens-mediated transformation system based upon direct shoot regeneration after transformation of meristematic cells derived from embryo axes. The transgene was under the control of the constitutive mannopine synthase promoter (p-MAS) in a dicistronic binary vector, which also contained luciferase (Luc) gene as scorable marker linked by internal ribosome entry site elements. Fertile transgenic faba bean plants were recovered. Inheritance and expression of the foreign genes were demonstrated by PCR, RT-PCR, Southern blot and monitoring of Luciferase activity. Under drought condition, after withholding water for 3 weeks, the leaves of transgenic plants were still green, while non-transgenic plants (WT) wilted and turned brown. Twenty-four hours after re-watering, the leaves of transgenic plants remained green, while WT plants did not recover. Moreover, the transgenic lines displayed higher tolerance to NaCl stress. Our results suggested that introducing a novel PR10a gene into faba bean could be a promising approach to improve its drought and salt tolerance ability, and that MAS promoter is not only constitutive, but also wound-, auxin/cytokinin- as well as stress-inducible.

Effect of antifungal genes expressed in transgenic pea (Pisum sativum L.) on root colonization with Glomus intraradices.

Pathogenic fungi have always been a major problem in agriculture. One of the effective methods for controlling pathogen fungi to date is the introduction of resistance genes into the genome of crops. It is interesting to find out whether the induced resistance in crops will have a negative effect on non-target organisms such as root colonization with the AM fungi.   The objective of the present research was to study the influence of producing antifungal molecules by four transgenic pea (Pisum sativum L.) lines expressing PGIP gene from raspberry, VST-stilbene synthase from vine, a hybrid of PGIP/VST and bacterial Chitinase gene (Chit30) from Streptomyces olivaceoviridis respectively on the colonization potential of Glomus intraradices. Four different experiments were done in greenhouse and climate chamber, colonization was observed in all replications. The following parameters were used for evaluation: frequency of mycorrhization, the intensity of mycorrhization, the average presence of arbuscules within the colonized areas and the presence of arbuscules in the whole root system which showed insignificant difference between transgenic and non-transgenic plants. The root/shoot ratio exhibited different values according to the experiment condition. Compared with negative non-transgenic control all transgenic lines showed the ability to establish symbiosis and the different growth parameters had insignificant effect due to mycorrhization. The results of the present study proved that the introduced pathogen resistance genes did not affect the mycorrhization allocations in pea.

Enhancing transgenic pea (Pisum sativum L.) resistance against fungal diseases through stacking of two antifungal genes (chitinase and glucanase).

One way of enhancing and broadening resistance of plants to different biotic and abiotic stresses is to combine transgenes expressing several genes into a single line. This can be done using different strategies such as crossing, single vector with multiple genes, co-transformation, sequential transformation and IRES elements. In the present study conventional crossing method was used. Parental transgenic lines transformed via Agrobacterium tumefasciens-mediated gene transformation with pGreenII binary vector harbouring a bar gene as selectable marker in combination with the family 19 chitinase gene from Streptomyces olivaceoviridis for one line and 1,3-ß-glucanase from barley (Hordeum vulgare) for the other line were used for crossing. Both chitinase and glucanase genes were cloned into pGreenII vector under the control of the constitutive double 35S-promoter from cauliflower mosaic virus. Progenies expressing the two genes were characterised at the molecular level using PCR, RT-PCR and Southern blot analysis, as well as segregation and stability studies of the respective expression levels. Leaf paint assay was used as functional test for herbicide resistant gene. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated. The synergistic effect of crossed plants was tested using in vitro assay which shows higher inhibition of spore germination.

Over-expression of PR-10a leads to increased salt and osmotic tolerance in potato cell cultures.

The PR-10a protein (formerly STH-2) is known to be induced by biotic stress in potato. The present study demonstrates that transgenic suspension cells of the potato cultivar Desiree over-expressing the PR-10a protein exhibit significantly increased salt and osmotic tolerance compared to the respective wild type cells. A comparison of the proteome pattern of Solanum tuberosum suspension cultures cv. Desiree before and after the treatment with NaCl or sorbitol under equiosmolar conditions (740mOs/kg) revealed the pathogenesis related protein PR-10a to be one of the predominant differentially expressed proteins in potato cell cultures. The pr-10a mRNA was confirmed to be present by RT-PCR from salt challenged suspension cells and was transcribed into cDNA. For PR-10a over-expression Agrobacterium tumefaciens mediated transformation of the potato cells and a dicistronic vector harboring the cDNA of the pr-10a gene linked to a luciferase gene by an IRES (Internal Ribosome Binding Site) was used. The IRES mediated translation leads to co-expression of PR-10a and luciferase in a fixed ratio. By non-invasive luciferase assay homologous PR-10a over-expressing callus was identified after selection on phosphinothricin supplemented medium. This callus was used for the setup of a transgenic suspension culture. Along with increased salt and osmotic tolerance the transformed culture showed changed proline and glutathione levels under abiotic stress conditions in comparison to the wild type.

Development of a simple and effective protocol for Agrobacterium tumefaciens mediated leaf disc transformation of commercial tomato cultivars.

The transformation of tomato (Solanum lycopersicum) through Agrobacterium tumefaciens is still far from being routine, particularly when it comes to commercial varieties. In the present paper, we present an efficient and simple protocol for leaf disc transformation of three Vietnamese tomato cultivars (DM8, MTS, FM372C) by comparing shoot regeneration media for expanding leaves and examining different parameters of inoculation, co-culture and selection conditions. The present transformation method requires neither feeder layers of cell suspension cultures nor pre-culture. The data clearly show that appropriate cytokinin- and auxin combinations and concentrations provide competent tissues for transformation. Supplementing of 8 µM trans-zeatin and 5 µM indoleacetic acid (IAA) into pre-treatment, inoculation and co-culture media resulted in higher frequency of transformation and stronger GUS-expression than that of media supplemented with 4 µM trans-zeatin and 2 µM IAA. The experiments also exhibited that tomato leaf tissues were more sensitive to glufosinate after inoculation with Agrobacteria compared to the untreated controls, so a more sophisticated scheme for the glufosinate selection had to be established.

Dicistronic binary vector system-A versatile tool for gene expression studies in cell cultures and plants.

Dicistronic binary vector constructs based on pGreenII vectors for Agrobacterium mediated gene transfer alleviate the translational expression monitoring of a target gene in plants. The functionality of the transformation vectors was proven by marker gene constructs containing a mannopine synthase promoter (p-MAS) fused to a beta-glucuronidase (gus) gene followed by an internal ribosome entry site and a firefly luciferase (luc) gene. The cap-dependent translation of a physically independent target protein can be monitored by the cap-independently co-translated luciferase, because both mRNAs are located on the same strand. Among three different IRES elements, the tobamo IRES element showed highest activity in transient expression. As a proof of principle for physiological studies the gus gene was replaced by a sodium antiporter gene (Atnhx1). Comparative studies with Atnhx1 transgenic luc expressing tobacco cell cultures and pea plants (Pisum sativum L.) showed improved salt tolerance in relation to their wild type counterparts grown under corresponding conditions. A coincidence of the luc gene expression and increased sodium chloride tolerance is demonstrated by measurement of luminescence and cell growth.

A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro.

Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. 'Sponsor' were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants.

Unrevealed structural requirements for auxin-like molecules by theoretical and experimental evidences.

An computational-biostatistical approach, supported by ab initio optimizations of auxin-like molecules, was used to find biologically meaningful relationships between quantum chemical variables and fresh bioassay's data. It is proven that the auxin-like recognition requires different molecular assembling states. We suggest that the carboxyl group is not the determining factor in explaining the biological auxin-like conduct. The biological effects depends essentially on the chemical condition of the ring system. The aim to find active molecules (quantum objects) via statistical grouping-analysis of molecular quantum similarity measures was verified by bioactivity assays. Next, this approach led to the discovery of a non-carboxylated active auxin-like molecule (2,6-dibromo-phenol). This is the first publication on structure activity relationship of auxin-like molecules, which relies on highly standardized bioassays of different auxins screened in parallel as well as analysed by multi-dimensional scaling.

Coulomb and overlap self-similarities: a comparative selectivity analysis of structure-function relationships for auxin-like molecules.

Auxins are defined mainly by a set of physiological actions, but the structure-effect relationship still is based on chemical intuition. Currently a well-defined auxin molecular structure is not available. The existence of different auxin binding proteins and mechanisms of auxin action, the wide diversity of the auxin molecules, and the pleiotropic effects of auxin imply a completely different mechanism as described for the animal hormone concept. Here, we present a computational approach dealing with semiempirical optimizations of the auxin molecules themselves, which represent a number of about 250 different chemical structures. Our approach uses molecular quantum similarity measures and additional quantum variables for the analysis of auxin-like molecules. The finding of similarities in molecules by focusing basically on their electron structure results in new insights in the relationship of the different auxin groups. Additional statistical analysis allows the identification of relationships between similarity groups and their biological activity, respectively. It is postulated that the auxin-like molecular recognition depends more on specific molecular assembling states than on a specific ring system or side chain.

Production of a single-chain variable fragment antibody against fumonisin B1.

The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free fumonisin B1 and fumonisin-biotin conjugate. Also fumonisin B2 was bound by the scFv. Modeling of both scFv and fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentially be used for developing a rapid and affordable immunoassay for detection of food contamination and can be applied in immunoaffinity chromatography, usually carried out prior to HPLC analysis of mycotoxin-contaminated food and feed.

The production of a genus-specific recombinant antibody (scFv) using a recombinant potyvirus protease.

A single chain variable fragment antibody (scFv; anti-NIa scFv102) was selected from a synthetic human antibody library by using a NIa protease of Plum pox virus (PPV) as an antigen, which was expressed in bacteria. The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. The NIa protein was detected with anti-NIa scFv102 after expression in Escherichia coli cells as well as from PPV-infected Nicotiana benthamiana plants. Furthermore, the scFv102 has the ability to identify not only PPV from infected plants but also can detect other infections with members of the potyviruses. Nineteen different potyviruses were recognized by the scFv102 in various infected plants tested through dot blot assays. Therefore, the antibody scFv102 has the potential of becoming a general tool to detect potyvirus infections in different plant species.