Rapid Communication - Agroinoculation as a Simple Way to Deliver a Tobacco Mosaic Virus-Based Expression Vector

As an expression vector with characteristics of relatively high stability and high-level production of heterologous proteins, the tobacco mosaic virus (TMV)-based 30B vector has been available for the experimental use worldwide (Yusibov et al, 1997; Shivprasad et al, 1999; Nemchinov et al, 2000). In a conventional procedure, however, a step of in vitro transcription is required to produce infectious viral RNA prior to inoculation of host plants. This troublesome and expensive process has hindered the use of the 30B vector to some extent. To conquer such disadvantage, we have adopted a technique called agroinoculation in which the cDNA copy of a viral genome, herein 30B, is inserted into the T-DNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter in such a way that it can be easily delivered into plant cells and initiate an infection. In order to test the agroinoculation method conveniently, the gfp gene for the jellyfish green fluorescent protein (GFP) was used as a reporter gene and inserted into the cloning sites of the viral vector to construct plasmid p35S-30B:: GFP. The agrobacteria harboring the modified vector were simply pressure-infiltrated into a leaf using a needle-less syringe. In this paper, we report high-level expression of GFP in systemic leaves up to 5.2% of total soluble leaf protein, demonstrating that agroinoculation can be used as an efficient and simple way to express foreign proteins in plants from the 30B vector.