Exploring the limits of vector construction based on Citrus tristeza virus

We examined the limits of manipulation of the Citrus tristeza virus (CTV) genome for expressing foreign genes in plants. We previously created a vector with a foreign gene cassette inserted between the major and minor coat protein genes, which is position 6 from the 3′ terminus. Yet, this virus has 10 3′-genes with several other potential locations for expression of foreign genes. Since genes positioned closer to the 3′ terminus tend to be expressed in greater amounts, there were opportunities for producing greater amounts of foreign protein. We found that the virus tolerated insertions of an extra gene in most positions within the 3′ region of the genome with substantially increased levels of gene product produced throughout citrus trees. CTV was amazingly tolerant to manipulation resulting in a suite of stable transient expression vectors, each with advantages for specific uses and sizes of foreign genes in citrus trees.