Cloning and Expression Analysis of Phytoplasma Protein Translocation Genes

Abstract

Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.