Quantifying Vg-Portiera Interaction via IC-PCR

The interaction between Vg and Portiera was examined using an IC-PCR assay by previously described protocols (29 (link)). PCR tubes were coated with 25 μL antibody against Vg (1:1,000 diluted in coating buffer), for 1.5 h at 37°C, and then washed five times for 5 min each time with 50 μL washing buffer. Homogenates of bacteriocytes and heads that were collected from 8 to 10 whiteflies in 5 μL PBS were incubated for 18 h at 4°C in the coated PCR tubes. The tubes were washed five times, 5 min each time, with 25 μL washing buffer and dried. PCR amplification of Portiera bound to the Vg protein, which was itself bound to the antibody-coated tubes, was performed with Portiera-specific 16S rRNA gene fragment primers (Table S1). The control no-antibody-coated tubes were incubated with a homogenate of the bacteriocytes and heads of whiteflies, and antibody-coated tubes incubated with a homogenate of whitefly heads not containing bacteriocytes served as controls.