In planta expression of a protein encoded by the extrachromosomal DNA of a phytoplasma and related to geminivirus replication proteins

Laboratory of Bioresource Technology, Graduate School of Frontier Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 1 Laboratory of Plant Breeding and Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2 National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan 3 Koibuchi College of Agriculture, 5965 Koibuchi, Uchihara-cho, Higashi-ibaraki, Ibaraki 319-0323, Japan 4 Author for correspondence: Shigetou Namba. Tel: +81 424 69 3125. Fax: +81 424 69 8786. e-mail: snamba{at}ims.u-tokyo.ac.jp A new extrachromosomal DNA, EcOYW1, was cloned from the onion yellows phytoplasma (OY-W). Southern blot and PCR analysis showed that EcOYW1 is not present in the OY-M, a mild symptom line derived from OY-W. We determined the complete nucleotide sequence of EcOYW1; it is a circular dsDNA of 7·0 kbp in length, which contains seven ORFs. ORF1 encoded a homologue of the geminivirus Rep protein. Western immunoblot analysis revealed that this Rep homologue is expressed in OY-W infected plants, suggesting that EcOYW1 replicates via a geminivirus-like rolling-circle replication mechanism. EcOYW1 is the first phytoplasmal extrachromosomal DNA shown to express encoded genes. Keywords: phytoplasma, extrachromosomal DNA, Rep protein, geminivirus Abbreviations: AY, aster yellows; OY, onion yellows; OY-M, mild symptom line of onion yellows; OY-W, wild-type onion yellows phytoplasma; SCWL, sugar cane white leaf; TGMV, tomato golden mosaic virus; Vaccinium witches’ broom The GenBank accession number for EcOYW1 is AB010426 .