The use of transgenic plants to express orally immunogenic protein antigens is an emerging strategy for vaccine biomanufacturing and delivery. This concept has particular suitability for developing countries. One factor that has limited the development of this technology is the relatively modest levels of accumulation of some antigenic proteins in plant tissues. We used fusion protein design to improve expression of the hepatitis B surface antigen (HBsAg) by attempting to mimic the process of HBsAg targeting to the endoplasmic reticulum of human liver cells during hepatitis B virus infection. We created a gene encoding a recombinant HBsAg modified to contain a plant signal peptide fused to its amino terminus. The signal peptide from soybean vegetative storage protein vspA (VSP␣S) directed endoplasmic reticulum targeting of HBsAg in plant cells, but was not cleaved and resulted in enhanced VSP␣S-HBsAg fusion accumulation. This product was more stable and presented the protective ''a'' antigenic determinant to significantly higher levels than unmodified native HBsAg expressed in plant cells. It also showed a greater extent of intermolecular disulfide bond formation and formation of virus-like particles. Moreover, VSP␣S-HBsAg stimulated higher levels of serum IgG than native HBsAg when injected into mice. We conclude that HBsAg tolerates a polypeptide fusion at the amino terminus and that VSP␣S-HBsAg is an improved antigen for plant-based expression of a subunit vaccine for hepatitis B virus.transgenic plants ͉ tobacco NT-1 cell ͉ vaccine H epatitis B virus (HBV) infection is an important global health problem, and vaccination is a proven strategy to control HBV infection. Current vaccines use yeast-derived recombinant hepatitis B surface antigen (rHBsAg) delivered by intramuscular injection, requiring trained medical practitioners and refrigerated storage, and are thus expensive to use. We have evaluated a new strategy for biomanufacturing the antigen to create a plant-derived oral vaccine, which could provide lower capital costs of production, mucosal immunization, and needlefree delivery of the final product. These advantages are particularly important for immunization programs in developing countries. Plant-derived HBsAg assembles into virus-like particles (VLPs) (1, 2) as in human (3), Chinese hamster ovary (4), and yeast (5) cells. Potato-derived HBsAg is orally immunogenic in mice, but high doses are needed (2, 6), creating a need for enhanced accumulation of the antigen in plant tissues if acceptable amounts of the plant material are to be delivered orally.HBsAg is a transmembrane protein with uncleaved internal signal sequences that facilitate cotranslational translocation and integration of HBsAg into the endoplasmic reticulum (ER) membrane (7). Subsequently, HBsAg dimers are rapidly formed via intermolecular disulfide bonds, catalyzed by protein disulfide isomerase (PDI). The protein-membrane complexes then assemble and extrude into the ER lumen (8) and are transported to the post-ER-pre-Golgi intermedia...

MicroRNAs (miRNAs) are a newly discovered class of noncoding endogenous small RNAs involved in plant growth and development as well as response to environmental stresses. miRNAs have been extensively studied in various plant species, however, only few information are available in cassava, which serves as one of the staple food crops, a biofuel crop, animal feed and industrial raw materials. In this study, the 169 potential cassava miRNAs belonging to 34 miRNA families were identified by computational approach. Interestingly, mes-miR319b was represented as the first putative mirtron demonstrated in cassava. A total of 15 miRNA clusters involving 7 miRNA families, and 12 pairs of sense and antisense strand cassava miRNAs belonging to six different miRNA families were discovered. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. The cis-regulatory elements relevant to drought stress and plant hormone response were identified in the promoter regions of those miRNA genes. The results provided a foundation for further investigation of the functional role of known transcription factors in the regulation of cassava miRNAs. The better understandings of the complexity of miRNA-mediated genes network in cassava would unravel cassava complex biology in storage root development and in coping with environmental stresses, thus providing more insights for future exploitation in cassava improvement.

The efficient analysis of small-molecule mixtures underlies many endeavors in chemical biology. The sensitivity of mass spectrometry (MS) has resulted in its widespread adoption for such analyses, and today rapid automated LC-MS analyses are widely used. Several recent studies have demonstrated the feasibility of NMR spectroscopic analyses of complex smallmolecule mixtures, including the use of diffusion-ordered spectroscopy (DOSY) [1] or principal component analysis (PCA) in metabolomics, [2] as well as the characterization of crude unfractionated natural product extracts using routine two-dimensional NMR spectra.[3] Compared to MS analyses, 2D NMR spectroscopic investigations of small-molecule mixtures offer the benefit of more detailed structural information, which is of particular relevance for the detection of novel chemotypes. However, the complexity of 2D spectra typically obtained for small-molecule mixtures has limited a broader implementation of NMR spectroscopy for their characterization. Herein, we describe a simple procedure for the differential analysis of arrays of 2D NMR spectra and demonstrate its utility for the detection of new natural products from a small library of fungal extracts.Fungi are prolific producers of natural products derived from terpene, [4] polyketide, [5] and nonribosomal peptide pathways.[6] Several lines of evidence indicate that only a fraction of the biosynthetic capabilities of fungi (and other cultured organisms) are discovered in traditional screening operations, as most secondary metabolite pathways are not expressed under the culture conditions used. Various approaches are being pursued to increase the accessible fraction of fungal metabolomes, and anecdotal evidence suggests that fungi respond to even small variations in their culturing protocol by starting (or stopping) the biosynthesis of specific natural products. [7][8][9][10][11] Clearly, a more systematic exploration of factors modulating secondary metabolite biosynthesis in fungi (and, by the same token, in bacteria) would be highly desirable. In a pilot study, we used differential analyses of 2D NMR spectra for the characterization of a small library of fungal extracts derived from a Tolypocladium cylindrosporum strain, cultured with a variety of protocols, which quickly revealed two new terpenoid indole alkaloids.T. cylindrosporum strain TC705 was selected from a group of insect-pathogenic fungi [12] because it has a number of nonribosomal peptide and polyketide biosynthetic genes that suggest a high metabolic potential for the production of secondary metabolites.[13] For our studies, TC705 cultures were grown using seven different protocols, based on four different media (YM, SDY, mEM, and diEM; see Supporting Information for full details). Three protocols (YM-SDY, YMmEM, and YM-diEM) included growing cultures in a twostep fermentation procedure, whereby each culture is initiated using a nutrient-rich medium and then transferred to a minimal or partially nutrient-deficient medium.[14] For subsequent NMR spe...

Development of storage roots is a process associated with a phase change from cell division and elongation to radial growth and accumulation of massive amounts of reserve substances such as starch. Knowledge of the regulation of cassava storage root formation has accumulated over time; however, gene regulation during the initiation and early stage of storage root development is still poorly understood. In this study, transcription profiling of fibrous, intermediate and storage roots at eight weeks old were investigated using a 60-mer-oligo microarray. Transcription and gene expression were found to be the key regulating processes during the transition stage from fibrous to intermediate roots, while homeostasis and signal transduction influenced regulation from intermediate roots to storage roots. Clustering analysis of significant genes and transcription factors (TF) indicated that a number of phytohormone-related TF were differentially expressed; therefore, phytohormone-related genes were assembled into a network of correlative nodes. We propose a model showing the relationship between KNOX1 and phytohormones during storage root initiation. Exogeneous treatment of phytohormones N (6) -benzylaminopurine and 1-Naphthaleneacetic acid were used to induce the storage root initiation stage and to investigate expression patterns of the genes involved in storage root initiation. The results support the hypothesis that phytohormones are acting in concert to regulate the onset of cassava storage root development. Moreover, MeAGL20 is a factor that might play an important role at the onset of storage root initiation when the root tip becomes swollen.