Eva C. Thuenemann

miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii

MicroRNAs (miRNAs) in eukaryotes guide post-transcriptional regulation by means of targeted RNA degradation and translational arrest. They are released by a Dicer nuclease as a 21–24-nucleotide RNA duplex from a precursor in which an imperfectly matched inverted repeat forms a partly double-stranded region. One of the two strands is then recruited by an Argonaute nuclease that is the effector protein of the silencing mechanism. Short interfering RNAs (siRNAs), which are similar to miRNAs, are also produced by Dicer but the precursors are perfectly double-stranded RNA. These siRNAs guide post-transcriptional regulation, as with miRNAs, and epigenetic genome modification. Diverse eukaryotes including fungi, plants, protozoans and metazoans produce siRNAs but, until now, miRNAs have not been described in unicellular organisms and it has been suggested that they evolved together with multicellularity in separate plant and animal lineages. Here we show that the unicellular alga Chlamydomonas reinhardtii contains miRNAs, putative evolutionary precursors of miRNAs and species of siRNAs resembling those in higher plants. The common features of miRNAs and siRNAs in an alga and in higher plants indicate that complex RNA-silencing systems evolved before multicellularity and were a feature of primitive eukaryotic cells.

Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii, like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae.

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.

Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.

Recent advances of Cowpea mosaic virus-based particle technology

Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as a means of presenting peptides for vaccine purposes. However, the existing technology has limitations in regard to the size and nature of the peptides which can be presented and has problems regarding bio-containment. Recent developments suggest ways by which these problems can be overcome, increasing the range of potential applications of CPMV-based particle technology.

Identification of an E-box motif responsible for the expression of jasmonic acid-induced chitinase gene OsChia4a in rice.

The plant hormone jasmonic acid (JA) is known to be involved in multiple defence responses against pathogens, which include the production of pathogenesis-related (PR) proteins. In order to investigate the induction mechanism of the rice defence responses by JA, we performed transcriptome analyses and focused on a chitinase gene, OsChia4a, which was identified to be one of the highest JA-inductive genes. The recombinant protein of His-tagged OsChia4a exhibited an inhibitory effect against the spore germination and hyphal growth of Magnaporthe oryzae. The promoter analysis of OsChia4a revealed that the region from -515 bp to -265 bp upstream of the ATG translation initiation site was required for the responsiveness to JA. A subsequent mutation analysis indicated that an E-box (CANNTG) in this region act as a JA-responsive cis element. These results imply that a basic helix-loop-helix transcription factor is likely to be involved in the regulation of the OsChia4a expression in a JA-dependent manner.

The Use of Transient Expression Systems for the Rapid Production of Virus-like Particles in Plants

Advances in transient expression technologies have allowed the production of milligram quantities of proteins within a matter of days using only small amounts (tens of grams) of plant tissue. Among the proteins that have been produced using this approach are the structural proteins of viruses which are capable of forming virus-like particles (VLPs). As such particulate structures are potent stimulators of the immune system, they are excellent vaccine candidates both in their own right and as carriers of additional immunogenic sequences. VLPs of varying complexity derived from a variety of animal viruses have been successfully transiently expressed in plants and their immunological properties assessed. Generally, the plant-produced VLPs were found to have the expected antigenicity and immunogenicity. In several cases, including an M2e-based influenza vaccine candidate, the plant-expressed VLPs have been shown to be capable of stimulating protective immunity. These findings raise the prospect that low-cost plant-produced vaccines could be developed for both veterinary and human use.

Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery

Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification.

Dynamic Gastric Model (DGM)

The Dynamic Gastric Model (DGM) was developed at the Institute of Food Research (Norwich, UK) to address the need for an in vitro model which could simulate both the biochemical and mechanical aspects of gastric digestion in a realistic time-dependent manner. As in the human stomach, masticated material is processed in functionally distinct zones: Within the fundus/main body of the DGM, gastric acid and enzyme secretions are introduced around the outside of the food bolus which is subjected to gentle, rhythmic massaging. Secretion rates adapt dynamically to the changing conditions within this compartment (acidification, fill state). Portions of gastric contents are then moved into the DGM antrum where they are subjected to physiological shear and grinding forces before ejection from the machine (and subsequent separate duodenal processing).

Dynamic Digestion Models: General Introduction

The first section of this chapter has focused on static digestion models and their specific applications. Whilst these static models have many advantages, they mainly function to mimic the biochemical processes in the gastrointestinal (GI) tract and usually use a single set of initial conditions (pH, concentration of enzymes, bile salts, etc.) for each part of the GI tract. However, this simplistic approach is often not a realistic simulation of the more complex in vivo conditions, where the biochemical environment encountered is constantly changing and physical parameters such as shear and grinding forces can have a large impact on the breakdown of larger food particles and the release of nutrients. Several dynamic digestion models have been developed in recent years to address these complex aspects of digestions, and four of these dynamic models will be presented in more detail in the following subchapters. This introduction will provide a brief overview of how the aspects of geometry, biochemistry and physical forces have been addressed in these and other dynamic digestion models.