Fig. 5.

In vivo coimmunoprecipitation (co-IP) assays confirm the interactions between histone H3 and the Bean dwarf mosaic virus (BDMV) movement proteins (NSP and MP) and reveal a complex composed of histone H3, NSP, MP, and viral DNA. (A) The Mycx4-H3, NSP, MP, and BCMNV HC-Pro suppressor and BDMV DNA-A and DNA-B components were coexpressed in leaves of N. benthamiana via A. tumefaciens-mediated transient expression. Controls were healthy N. benthamiana leaves and leaves expressing Mycx4, NSP, MP, HC-Pro, and BDMV DNA-A and DNA-B. Total proteins were extracted from healthy and infiltrated leaves (3 dpi), and co-IP was performed with the Myc-Tag (9B11) mouse MAb (Sepharose bead conjugate) kit. Input (total) and eluted (co-IP fraction) proteins were separated by SDS-PAGE, and proteins were transferred to PVDF membranes. Western blot analyses were performed with Myc, MP, and NSP antibodies. Asterisks indicate the immunoprecipitated Mycx4-H3 (*), MP (**), and NSP (***) proteins. (B) Detection of BDMV DNA-A and DNA-B components in co-IP fractions by PCR with DNA-A and DNA-B primers. DNA extracts were prepared from co-IP fractions from healthy N. benthamiana leaves and leaves infiltrated with Mycx4, NSP, MP, HC-Pro, and BDMV DNA-A and DNA-B (Mycx4) or with Mycx4-H3, NSP, MP, HC-Pro, and BDMV DNA-A and DNA-B (Mycx4-H3). These extracts were used in the PCR with degenerate primers for the begomovirus DNA-A component (BDMV A) or DNA-B component (BDMV B). Internal controls were DNA extracts prepared from healthy (−) and BDMV-infected (+) N. benthamiana leaves. The expected sizes of the DNA-A (1.1 kb) and DNA-B (0.5 kb) fragments are indicated by arrowheads.