Fig. 5
In planta interaction of HopM1 with VLRM1. Co-Immunoprecipitation of HopM1 and its corresponding VLRB in Nicotiana benthamiana. Thirteen and one-half mg of proteins were immunoprecipitated (IP) with α-GFP agarose beads without the use of reducing agents in the buffers. Proteins were detected with either α-GFP (IP) or α-c-Myc (co-IP) antibodies. Interactions between HopM1 and HopM1-specific VLRB were tested with both proteins fused to two different epitope tags. Highlighted in orange are those proteins detected in the Western blot, while in black are those proteins also expressed but not detected. VLRM1 = SP-VLRM1, VLRTLR5 = SP-VLRTLR5, M1–300 = SP-HopM11–300, K1 = HopK1. a Total protein input of YFP-tagged proteins for IP, Western blot used α-GFP antibodies for protein detection. Ponceau S staining of the input PVDF membrane is shown below the Western blot. The asterisk represents the position of YFP cleaved from the fusion protein. b Total protein input of c-Myc-tagged proteins for IP. Western blot used α-c-Myc antibodies for protein detection. Ponceau S staining of the input PVDF membrane is shown below the Western blot. c IP of YFP-tagged proteins with α-GFP agarose beads. Western blot used α-GFP antibodies for protein detection. The asterisk indicates the position of YFP cleaved from the fusion protein. d Co-IP of c-Myc-tagged proteins with α-GFP agarose beads. Western blot used α-c-Myc antibodies for protein detection. VLRM1 only interacted with HopM1. The open circle marks the position of a probable dimer formed between VLRM1 and HopM11–300