Aphid gene of bacterial origin encodes a protein transported to an obligate endosymbiont

Supplemental Experimental Procedures Aphids Strain ISO, a parthenogenetic clone of the pea aphid Acyrthosiphon pisum was used in this study. The insects were reared on Vicia faba at 15°C in a long-day regime of 16 h light and 8 h dark. Parthenogenetic apterous adults (12 to 15 days old) were used for the experiments. The antibody against A. pisum RlpA4 was raised in rabbits using recombinant protein as described previously [S1]. Complementary DNA was prepared from isolated bacteriocytes as described [S2]. The cDNA sequence encoding the mature RlpA4 protein was amplified by PCR using PrimeSTAR GXL DNA Polymerase (TaKaRa) and the primer set RlpA4_X_1F (5-CACCGAATGCAAGCGTCTCGGAAATAATTC-3) and RlpA4_X_579R (5-TTATTTCGGTAAGCAAAACCCTTTATCTG-3). The PCR product was cloned into the pET100/D-TOPO expression vector (Life Technologies), and the 6x-His fusion polypeptide was expressed in Escherichia coli Rosetta (DE3) cells, purified using nickel–nitrilotriacetic acid agarose resin (HisTrap HP; GE Healthcare), and separated electrophoretically. Polyacrylamide gel slices were homogenized and injected into rabbits to stimulate antibody production. The antibody was affinity-purified from the antisera using recombinant proteins coupled to a HiTrap NHS-activated HP column (GE Healthcare).