Plant 4/1 protein: potential player in intracellular, cell-to-cell and long-distance signaling

Originally isolated as a result of its ability to interact with the movement protein of Tomato spotted wilt virus in a yeast two-hybrid system, the 4/1 protein is proving to be an excellent tool for studying intracellular protein trafficking and intercellular communication. Expression of 4/1 in vivo is tightly regulated, first appearing in the veins of the cotyledon and later in the vasculature of the leaf and stem in association with the xylem parenchyma and phloem parenchyma. Structural studies indicate that 4/1 proteins contain as many as five coiled–coil (CC) domains; indeed, the highest level of sequence identity among 4/1 proteins involves their C-terminal CC domains, suggesting that protein–protein interaction is important for biological function. Recent data predict that the tertiary structure of this C-terminal CC domain is strikingly similar to that of yeast protein She2p; furthermore, like She2p, 4/1 protein exhibits RNA-binding activity, and mutational analysis has shown that the C-terminal CC domain is responsible for RNA binding. The 4/1 protein contains a nuclear export signal. Additional microscopy studies involving leptomycin and computer prediction suggest the presence of a nuclear localization signal as well

DataSheet_1_Effect of the At-CDC27a gene on Nicotiana benthamiana phenotype and accumulation of recombinant proteins.pdf

In this study the anaphase promoting complex subunit CDC27a from Arabidopsis thaliana was introduced in the genome of Nicotiana benthamiana by Agrobacterium tumefaciens. The presence of the At-CDC27a gene facilitates plant biomass production. Compared to wild type N. benthamiana the leaf mass fraction of the best performing transgenic line At-CDC27a-29 was increased up to 154%. The positive effect of the At-CDC27a expression on leaf biomass accumulation was accompanied by an enlarged total leaf area. Furthermore, the ectopic expression of the At-CDC27a also affected cellular conditions for the production of foreign proteins delivered by the TRBO vector. In comparison to the non-transgenic control, the protein accumulation in the At-CDC27a-29 plant host increased up to 146% for GFP and up to 181% for scFv-TM43-E10. Collectively, the modified N. benthamiana plants developed in this study might be useful to improve the yield of recombinant proteins per biomass unit in closed facilities.</p

Alternatives to antibiotic resistance marker genes for in vitro selection of genetically modified plants: scientific developments, current use, operational access and biosafety considerations

Genes conferring resistance to antibiotics have been widely used as markers for the selection of transformed cells in the development of genetically modified (GM) plants. Their presence in GM plants released in the environment or used as food or feed has raised concerns over the past years regarding possible risks for human health and the environment. Although these concerns have not been supported so far by scientific evidence, the implementation of selection approaches avoiding the presence of antibiotic resistance marker genes (ARMGs) in the final GM plant is increasingly considered by GM plant developers, not only to alleviate the above-mentioned concerns, but also to circumvent technical limitations associated with the use of ARMGs. In the current paper, we present the results of a three-step analysis of selectable markers and reporter genes as well as methods aiming at developing marker-free GM plants. First, based on a comprehensive review of the scientific literature, technical developments in this domain are presented. Second, a state-of-the-art of the current use of selection approaches is provided based on publicly available information on GM plants tested in the field or authorized for commercialization. Third, in order to get more insight in the underlying practical, scientific and/or regulatory arguments supporting the choice of selection approaches, we present the results of a survey directed at relevant developers and users of GM plants. The applicability, efficiency, operational access and biosafety of the various selection approaches is discussed and considered in light of their current use, and in perspective to the long history of use of ARMGs in plant biotechnology

Improvement of conditional Cre-lox system through application of the regulatory sequences from Cowpea mosaic virus

To study the impact of regulatory sequences from Cowpea mosaic virus (CPMV) on Cre-mediated recombination rates, the cre gene was flanked by the 5′ non-translated and 3′ non-translated regions of CPMV. This cre configuration was tested by simultaneous excision of nptII selectable marker gene and heat-inducible cre recombinase gene in potato. Fusion of the cre recombinase sequence with modified regulatory sequences of CPMV increased both the excision efficiencies in primary regenerants and transmission frequencies of recombined loci to vegetative progeny as was confirmed by molecular analysis. These data might have practical implication with regard to selection of putative recombinants in vegetative progeny of potato and other clonally propagated plants as well.Instituto de Patología VegetalFil: Kopertekh, Lilya. Institute for Biosafety in Plant Biotechnology. Julius Kuehn Institute, Federal Research Centre for Cultivated Plants; AlemaniaFil: Krebs, Erika. Institute for Biosafety in Plant Biotechnology. Julius Kuehn Institute, Federal Research Centre for Cultivated Plants; AlemaniaFil: Guzman, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentin

Transient plant production of Salmonella Typhimurium diagnostic antibodies

Salmonella Typhimurium is one of the most important zoonotic pathogens worldwide and a major cause of economic losses in the pig production chain. The emergence of multi-drug resistant strains over the past years has led to considerations about an enhanced surveillance of bacterial food contamination. Currently, ELISA is the method of choice for high throughput identification of S. Typhimurium. The sensitivity and specificity of this assay might be improved by application of new diagnostic antibodies. We focused on plant-based expression of candidate diagnostic TM43-E10 antibodies discovered using as antigen the S. Typhimurium OmpD protein. The scFv-TM43-E10 and scFv-Fc-TM43-E10 antibody derivatives have been successfully produced in N. benthamiana using a deconstructed movement-deficient PVX vector supplemented with the γb silencing suppressor from Poa semilatent virus. The plant-made antibodies showed the same antigen-binding specificity as that of the microbial/mammalian cell-produced counterparts and could recognize the OmpD antigen in S. Typhimurium infected plant samples. Keywords: Molecular farming, Diagnostic antibody, Transient expressio

Tuber-specific cphA expression to enhance cyanophycin production in potatoes

Hühns M, Neumann K, Hausmann T, et al. Tuber-specific cphA expression to enhance cyanophycin production in potatoes. Plant Biotechnology Journal. 2009;7(9):883-898.The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphATe) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphATe) as well as tuber-specific plastidic (pB33-PsbY-cphATe) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphATe, both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphATe tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphATe tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed