Chloroplast GFP expression in tobacco plants agroinfiltrated with tobacco mosaic virus based vectors

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Viruses are submicroscopic infectious particles comprising of nucleic acid and protein which can replicate only within living host cells. TMV is a positive single-stranded RNA virus which produces a typical mosaic symptom in infected leaves. TMV has an interesting association with the chloroplast of a plant cell. we have shown that GFP expressed from the TMV vector TMV- 30B-GFP is also found in chloroplasts and we are using TMV-30B-GFP to understand how GFP is expressed inside the chloroplast. Two putative Shine-Dalgarno motifs were found immediately upstream of GFP. To find out the exact role of these motifs, we introduced point mutations in each of them. When the mutation was introduced in both the motifs, GFP expression in the chloroplast was even lesser. Most importantly, the results obtained with p35SGFP show that GFP is somehow able to gain entry to chloroplasts without the aid of any TMV sequences. My work is important because it shows that GFP is an imperfect negative control for the analysis of chloroplast transit sequences.