Plant disease resistance is augmented in uzu barley lines modified in the brassinosteroid receptor BRI1.

BACKGROUND: Brassinosteroid hormones regulate many aspects of plant growth and development. The membrane receptor BRI1 is a central player in the brassinosteroid signaling cascade. Semi-dwarf 'uzu' barley carries a mutation in a conserved domain of the kinase tail of BRI1 and this mutant allele is recognised for its positive contribution to both yield and lodging resistance. RESULTS: Here we show that uzu barley exhibits enhanced resistance to a range of pathogens. It was due to a combination of preformed, inducible and constitutive defence responses, as determined by a combination of transcriptomic and biochemical studies. Gene expression studies were used to determine that the uzu derivatives are attenuated in downstream brassinosteroid signaling. The reduction of BRI1 RNA levels via virus-induced gene silencing compromised uzu disease resistance. CONCLUSIONS: The pathogen resistance of uzu derivatives may be due to pleiotropic effects of BRI1 or the cascade effects of their repressed BR signaling.

Brassinosteroid enhances resistance to fusarium diseases of barley.

Fusarium pathogens are among the most damaging pathogens of cereals. These pathogens have the ability to attack the roots, seedlings, and flowering heads of barley and wheat plants with disease, resulting in yield loss and head blight disease and also resulting in the contamination of grain with mycotoxins harmful to human and animal health. There is increasing evidence that brassinosteroid (BR) hormones play an important role in plant defense against both biotic and abiotic stress agents and this study set out to determine if and how BR might affect Fusarium diseases of barley. Application of the epibrassinolide (epiBL) to heads of 'Lux' barley reduced the severity of Fusarium head blight (FHB) caused by Fusarium culmorum by 86% and reduced the FHB-associated loss in grain weight by 33%. Growth of plants in soil amended with epiBL resulted in a 28 and 35% reduction in Fusarium seedling blight (FSB) symptoms on the Lux and 'Akashinriki' barley, respectively. Microarray analysis was used to determine whether growth in epiBL-amended soil changed the transcriptional profile in stem base tissue during the early stages of FSB development. At 24 and 48 h post F. culmorum inoculation, there were 146 epiBL-responsive transcripts, the majority being from the 48-h time point (n = 118). Real-time reverse-transcription polymerase chain reaction analysis validated the results for eight transcripts, including five defense genes. The results of gene expression studies show that chromatin remodeling, hormonal signaling, photosynthesis, and pathogenesis-related genes are activated in plants as a result of growth in epiBL.

A single-chain antibody fragment against human thyroglobulin: construction and evaluation of immunoreactivity.

Smaller recombinant antibody fragments are at the forefront of in vivo diagnosis and therapy. These units possess better distribution and faster clearance than larger molecules. Among these, single chain antibody fragments (scFv) are emerging as credible alternatives. These proteins are shown to have same specificities and affinities for their antigens as the parental monoclonal antibody (MAb). We have attempted to produce scFv against human thyroglobulin (H-Tg) using anti-Tg secreting hybridoma cells and PCR-based cloning approach. Hybridoma secreting anti-Tg MAb B10IV was established. cDNA was prepared from hybridoma cells. The V(H) and V(L) genes were amplified and cloned. The gene sequences were submitted to Genebank database (accession nos. AJ508533 and AM072962, respectively.) V(L) and V(H) genes were then linked together with a linker peptide and successfully cloned in pET28a and expressed as His-tag fusion protein in expression host BL21 (DE3). The scFv protein from IPTG-induced cells was purified under native conditions by immobilized metal affinity chromatography on a Ni-NTA agarose column. The yield expressed in Escherichia coli was approximately 8 mg/L. ScFv could be labeled with (125)I and its immunoreactivity evaluated in radioassays. Although scFv demonstrated specific binding to H-Tg, the immunoreactivity was low (10.3%) compared to the parental MAb B10IV, which showed immunoreactivity of 37.27%. Inhibition radioassays exhibited that scFv and MAb interact with the same epitope on the target antigen, indicating its specificity.

Production of hepatitis B surface antigen in recombinant plant systems: an update.

There is a growing interest to develop oral vaccines for infectious diseases, as it is the most convenient and effective way to attain mucosal immunity. Hepatitis B continues to be a major infectious disease in many developing countries despite the availability of recombinant vaccine. On a global scenario, Hepatitis B Virus infection is probably the single most prevalent cause of persistent viraemia in humans. There are about 350 million chronic carriers of HBV, which is about 5% of the total world population. It is estimated that 75-100 million of them will die of liver cirrhosis and/or hepatocellular carcinoma. Progress in plant genetic engineering has enabled the transfer of useful genes for desirable traits. The recent trend is to use this technique to exploit plants as biofactories for the production of therapeutic proteins including vaccines. Rapid progress has been made in this area to develop plant-based vaccines for hepatitis B. This review describes the expression, characterization, and immunogenicity studies of hepatitis B vaccines produced in recombinant plant systems and their implications for developing a plant-based vaccine.

Secretion of hepatitis B surface antigen in transformed tobacco cell suspension cultures.

Six different expression cassettes of hepatitis B surface antigen (HBsAg) were used to transform tobacco cell suspension cultures. The transgenic nature of the cells was confirmed by PCR. The secreted HBsAg was assayed by ELISA and analyzed by Western blotting. A maximum of 31 microg antigen/l was obtained in the spent medium from the transformed cells. The use of an ethylene-forming enzyme promoter and incorporation of C-terminal endoplasmic-reticulum-retention signal enhanced the secretion of HBsAg. Salicylic or jasmonic acid at 10 microM: increased secretion of HBsAg by six fold.

Expression of hepatitis B surface antigen in transgenic banana plants.

Embryogenic cells of bananan cv. Rasthali (AAB) have been transformed with the 's' gene of hepatitis B surface antigen (HBsAg) using Agrobacterium mediated transformation. Four different expression cassettes (pHBS, pHER, pEFEHBS and pEFEHER) were utilized to optimize the expression of HBsAg in banana. The transgenic nature of the plants and expression of the antigen was confirmed by PCR, Southern hybridization and reverse transcription (RT)-PCR. The expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by ELISA for all the four constructs. Maximum expression level of 38 ng/g F.W. of leaves was noted in plants transformed with pEFEHBS grown under in vitro conditions, whereas pHER transformed plants grown in the green house showed the maximum expression level of 19.92 ng/g F.W. of leaves. Higher monoclonal antibody binding of 67.87% of the antigen was observed when it was expressed with a C-terminal ER retention signal. The buoyant density in CsCl of HBsAg derived from transgenic banana leaves was determined and found to be 1.146 g/ml. HBsAg obtained from transgenic banana plants is similar to human serum derived one in buoyant density properties. The transgenic plants were grown up to maturity in the green house and the expression of HBsAg in the fruits was confirmed by RT-PCR. These transgenic plants were multiplied under in vitro using floral apex cultures. Attempts were also made to enhance the expression of HBsAg in the leaves of transgenic banana plants by wounding and/or treatment with plant growth regulators. This is the first report on the expression of HBsAg in transgenic banana fruits.