P1P2 fusion proteins are unstable in plants, both in a viral context and in transient expression assays, and are dispensable for infectivity.

<p>(A) Immunodetection of P1P2 fusion proteins in CaMV Cabb B-JI- or Cabb-S-infected turnip plants. Total proteins were analyzed by SDS-PAGE and western blotting was performed with anti-P1 and anti-P2 polyclonal antibodies. (B) Immunodetection of HA- or myc-tagged P1P2(D2) protein upon <i>Agrobacterium tumefaciens</i>-mediated transient expression assay in <i>Nicotiana benthamiana</i> leaves. Total proteins were extracted from agroinfiltrated leaves and the presence of P1P2(D2) fusion protein was assessed by western assays with anti-HA or anti-myc antibodies. Leaves agroinfiltrated with HA- or myc-GFP expression vectors were used as positive controls. Blots in (A) and (B) are overexposed deliberately to maximize chances of detection of P1P2 proteins. (C) Tagged-P1P2(D2) and GFP transcripts were assessed by RT-PCR on total RNA from infiltrated leaves. The recombinant plasmid pP1P2(D2) used for expression of the fusion protein in <i>E</i>. <i>coli</i> and pCK-EGFP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132665#pone.0132665.ref027" target="_blank">27</a>] were used as controls. (D) Immunodetection of viral proteins P2 and P4 in systemic leaves of Cabb B-JI- or P1P2stop-infected turnip plants at 21 dpi.