Secretion of immunodominant membrane protein from onion yellows phytoplasma through the Sec protein-translocation system in Escherichia coli

1 Division of Integrated Biosciences, The University of Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Chiba 277-8562, Japan 2 Division of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 3 National Agricultural Research Center, National Agricultural Research Organization, 3-1-1 Kannondai, Tsukuba-shi, Ibaraki 305-8666, Japan Correspondence Shigetou Namba snamba{at}ims.u-tokyo.ac.jp A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the onion yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli , purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli . A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp. Abbreviations: Amp, antigenic membrane protein; AP, apple proliferation phytoplasma; AY, aster yellows phytoplasma; CP, clover phyllody phytoplasma; ESFY, European stone fruit yellows phytoplasma; E/H/T, export leader, hydrophilic and transmembrane domains, respectively, in the putative Amp; OY, onion yellows phytoplasma; PD, pear decline phytoplasma; PYLR, peach yellow leaf roll phytoplasma; SPWB, sweet potato witches' broom phytoplasma; WX, western X-disease phytoplasma The GenBank accession numbers for the secE - and amp -containing DNA fragmen