Plastid transformation: Advances and challenges for its implementation in agricultural crops

Traditionally, reactive oxygen intermediates (ROIs) were considered to be toxic by-products of aerobic metabolism, which were disposed of using antioxidants. However, in recent years, it has become apparent that plants actively produce ROIs as signaling molecules to control processes such as programmed cell death, abiotic stress responses, pathogen defense and systemic signaling. Recent advances including microarray studies and the development of mutants with altered ROI-scavenging mechanisms provide new insights into how the steady-state level of ROIs are controlled in cells. In addition, key steps of the signal transduction pathway that senses ROIs in plants have been identified. These raise several intriguing questions about the relationships between ROI signaling, ROI stress and the production and scavenging of ROIs in the different cellular compartments.

Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. Toward this end, we demonstrate that ingestion of double-stranded (ds)RNAs supplied in an artificial diet triggers RNA interference in several coleopteran species, most notably the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte. This may result in larval stunting and mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage in a growth chamber assay, suggesting that the RNAi pathway can be exploited to control insect pests via in planta expression of a dsRNA.

Large proteins are usually expressed in a eukaryotic system while smaller ones are expressed in prokaryotic systems. For proteins that require glycosylation, mammalian cells, fungi or the baculovirus system is chosen. The least expensive, easiest and quickest expression of proteins can be carried out in Escherichia coli. However, this bacterium cannot express very large proteins. Also, for S-S rich proteins, and proteins that require post-translational modifications, E. coli is not the system of choice. The two most utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Yeasts can produce high yields of proteins at low cost, proteins larger than 50 kD can be produced, signal sequences can be removed, and glycosylation can be carried out. The baculoviral system can carry out more complex post-translational modifications of proteins. The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells. Genetically modified animals secrete recombinant proteins in their milk, blood or urine. Similarly, transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.