Type I IFNs were discovered as the primary antiviral cytokines and are now known to serve critical functions in host defense against bacterial pathogens. Accordingly, established mediators of IFN antiviral activity may mediate previously unrecognized antibacterial functions. RNase-L is the terminal component of an RNA decay pathway that is an important mediator of IFN-induced antiviral activity. Here, we identify a role for RNase-L in the host antibacterial response. RNase-L ؊/؊ mice exhibited a dramatic increase in mortality after challenge with Bacillus anthracis and Escherichia coli; this increased susceptibility was due to a compromised immune response resulting in increased bacterial load. Investigation of the mechanisms of RNase-L antibacterial activity indicated that RNase-L is required for the optimal induction of proinflammatory cytokines that play essential roles in host defense from bacterial pathogens. RNase-L also regulated the expression of the endolysosomal protease, cathepsin-E, and endosome-associated activities, that function to eliminate internalized bacteria and may contribute to RNase-L antimicrobial action. Our results reveal a unique role for RNase-L in the antibacterial response that is mediated through multiple mechanisms. As a regulator of fundamental components of the innate immune response, RNase-L represents a viable therapeutic target to augment host defense against diverse microbial pathogens.cathepsin-E ͉ interferon ͉ 2Ј, 5Ј-oligoadenylate ͉ cytokine ͉ endosome

Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

The cholera toxin B subunit ͑CTB͒ contains five identical polypeptides and targets glycosphingolipid receptors on eukaryotic cell surfaces. Increased expression of CTB in plants is critical for the development of edible vaccines. In this study, the coding sequence of the CTB gene was optimized, based on the modification of codon usage to that of tobacco plant genes and the removal of mRNA-destabilizing sequences. The synthetic CTB gene was cloned into a plant expression vector and expressed in tobacco plants under the control of the CaMV 35S promoter. The recombinant CTB protein constituted approximately 1.5% of the total soluble protein in transgenic tobacco leaves. This level of CTB production was approximately 15-fold higher than that in tobacco plants that were transformed with the bacterial CTB gene. The recombinant CTB produced by tobacco plants demonstrated strong affinity for GM1-ganglioside, which indicates that the sites required for binding and proper folding of the pentameric CTB structure were conserved. This is the first report on the optimization of the CTB-coding sequence to give a dramatic increase in CTB expression in plants.

Herbicide-tolerant Zoysia grass (Zoysia japonica Steud.) has been generated previously through Agrobacterium tumefaciens-mediated transformation. The genetically modified (GM) Zoysia grass survived Basta spraying and grew to maturity normally while the wild-type (WT) grass stopped growing and died. GM Zoysia grass will permit more efficient weed control for various turf grass plantings such as home lawns, golf courses, and parks. We examined the environmental/biodiversity risks of herbicide-tolerant GM Zoysia before applying to regulatory agencies for approval for commercial release. The GM and WT Zoysia grass' substantial trait equivalence, ability to cross-pollinate, and gene flow in confined and unconfined test fields were selectively analyzed for environmental/biodiversity effects. No difference between GM and WT Zoysia grass in substantial traits was found. To assess the potential for cross-pollination and gene flow, a non-selective herbicide, Basta, was used. Results showed that unintended cross-pollination with and gene flow from GM Zoysia grass were not detected in neighboring weed species examined, but were observed in WT Zoysia grass (on average, 6% at proximity, 1.2% at a distance of 0.5 m and 0.12% at a radius of 3 m, and 0% at distances over 3 m). On the basis of these initial studies, we conclude that the GM Zoysia grass generated in our laboratory and tested in the Nam Jeju County field does not appear to pose a significant risk when cultivated outside of test fields.

Escherichia coli heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant for coadministered antigens. We synthesized a gene encoding the B-subunit of LT (LTB) adapted to the coding sequence of tobacco plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its level of expression in plants. The synthetic LTB gene was cloned into a plant expression vector adjacent to the CaMV 35S promoter and was introduced into tobacco by Agrobacterium-mediated transformation. The amount of LTB protein detected in transgenic tobacco leaves was 2.2% of the total soluble plant protein, which is approx 200-fold higher than in previous reports of native LTB gene expression in transgenic plants. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers.