<p>Soybean mosaic virus-N (SMV-N) and clover yellow vein virus (ClYVV-No.30) are two recognized potyviruses with distinct host ranges and host specificities. Both infect systemically wild soybean (<i>Glycine soja</i>) whereas SMV-N, but not ClYVV-No.30, infects systemically cultivated soybean (<i>G. max</i>) as well. In contrast, ClYVV-No.30, but not SMV-N, infects systemically broad bean (<i>Vicia faba</i>). To investigate whether P3, P3N-PIPO, and 6K1 from SMV-N play role in host specificity for systemic infection, each was replaced precisely with those from ClYVV-No.30 including the corresponding NIaPro protease recognition sequences at the junctions between P3-6K1 or 6K1-CI cistrons. A second set of SMVN/ClYVV-No.30 chimeras were also synthesized with the P3, P3N-PIPO, and 6K1 from ClYVV-No.30 but with NIaPro protease recognition site at P3-6K1 or 6K1-CI junctions from SMV-N. Regardless of the origin of NIaPro recognition sites, none of the SMV-N-derived chimeras gained the ability to infect systemically broad bean. On the contrary, unlike parental SMV-N, all SMV-N-derived chimeras lost the ability to infect systemically cultivated and wild soybeans. Site-directed mutagenesis of SMV-N NIaPro recognition site at P3-6K1 junction showed replacement of one of the amino acids with the corresponding residue from ClYVV-No.30 at proximal C-terminus of P3 abolished systemic infection of the SMV-N-derived mutant in both cultivated and wild soybeans. Our data suggest that in addition to processing of the NIaPro recognition site at the P3-6K1 junction, P3N-PIPO and 6K1 are also involved in systemic infection of cultivated and wild soybeans by SMV-N.</p>