Cauliflower mosaic virus (CaMV) gene VI encodes a multifunctional protein (P6) involved in the translation of viral RNA, the formation of inclusion bodies, and the determination of host range. Arabidopsis thaliana ecotype Tsu-0 prevents the systemic spread of most CaMV isolates, including CM1841. However, CaMV isolate W260 overcomes this resistance. In this paper, the N-terminal 110 amino acids of P6 (termed D1) were identified as the resistance-breaking region. D1 also bound full-length P6. Furthermore, binding of W260 D1 to P6 induced higher beta-galactosidase activity and better leucine-independent growth in the yeast two-hybrid system than its CM1841 counterpart. Thus, W260 may evade Tsu-0 resistance by mediating P6 self-association in a manner different from that of CM1841. Because Tsu-0 resistance prevents virus movement, interaction of P6 with P1 (CaMV movement protein) was investigated. Both yeast two-hybrid analyses and maltose-binding protein pull-down experiments show that P6 interacts with P1. Although neither half of P1 interacts with P6, the N-terminus of P6 binds P1. Interestingly, D1 by itself does not interact with P1, indicating that different portions of the P6 N-terminus are involved in different activities. The P1-P6 interactions suggest a role for P6 in virus transport, possibly by regulating P1 tubule formation or the assembly of movement complexes.