Exploring Citrus tristeza virus-based vector limits for heterologous gene/s expression

Virus vectors are key tools in basic molecular biology research and have great potential for commercial applications in expressing foreign genes. Stability of foreign inserts is a major drawback for long term potential applications of virus vectors for protein expression. Citrus tristeza virus (CTV) has been unique among plant virus vectors in having the ability to accommodate a foreign insert for many years. CTV has ten genes at the 3' half of the genome that are expressed by subgenomic (sg) RNAs. The controller elements for the sgRNA are located upstream of the open reading frames. Previously, a foreign gene was inserted between the major and minor coat protein genes. In this study I tested the vector limits of using CTV to express foreign genes ranging from 806 to 3480 nucleotides in size. The gene cassettes were introduced into the CTV genome as replacements of the p13 gene, as added extra genes at three different locations (p13-p20, p20-p23 and p23-3'non-translated region (NTR)), as fusions to p23 and protease processing, and behind IRES sequences to create bi-cistronic messages. Twenty seven expression vectors were created and tested in Nicotinia benthamiana protoplasts and plants. The most successful strategies were examined in citrus. Remarkably, most of the vector constructs replicated, spread systemically in plants, and expressed the foreign gene. The highest expressing vectors were the "add a gene" constructs with an insertion between the p13 and p20 genes or between the p23 gene and the 3'NTR. Similarly, the vectors with the inserted gene replacing the p13 gene effectively expressed different reporter genes. However, optimal expression of the reporter gene depended both on the size and location of the insertion. Efficient expression of two genes simultaneously from the same vector was accomplished in both N. benthamiana and citrus. This research demonstrates the elasticity (size of inserts) and flexibility (different locations) of the CTV genome to accommodate and express foreign gene/s by different strategies.