Two Korean isolates of turnip mosaic virus (TuMV), KIH1 and HJY1, show severe and mild symptoms, respectively, in Nicotiana benthamiana. Infectious clones under control of both 35S and T7 promoters allow infection by agroinfiltration or by in vitro transcripts. Analysis of chimeric constructs between KIH1 and HJY1 localized the major determinant of symptom severity in N. benthamiana to the N-terminal 3.9 kb of KIH1; amino acid sequence identities of P1, HC-Pro, P3, 6K1, and the CI N-terminal domain were 90.06%, 98.91%, 93.80%, 100%, and 100% respectively. Expression of either P1 or P3 from a potato virus X (PVX) vector yielded symptom differences only between P3 of KIH1 and HJY1, implicating P3 in symptom severity in N. benthamiana. However, over-expression of P3 from PVX failed to induce systemic necrosis. As an additional coding region, PIPO, lying within the sequence of the P3 gene is expressed by polymerase slippage resulting in a frameshift into the +2 reading-frame, we also examined the differences in the predicted P3N-PIPO sequences of isolates KIH1 and HJY1. Differences in the cell wall localization of GFP:P3, dependent on co-expression of either a P3N::PIPO fusion or PIPO of KIH1 and HJY1 were observed in agroinfiltrated N benthamiana. There were only three residue differences in the P3N portion, and three substitutions in the PIPO segment - (D(168)N, G(215)S, and I(217)V, that are possibly responsible for the differential symptoms in N. benthamiana.