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A Plasmid Isolated from Phytopathogenic Onion Yellows Phytoplasma and Its Heterogeneity in the Pathogenic Phytoplasma Mutant

November 1998 , Volume 11 , Number  11
Pages  1,031 - 1,037

Tsutomu Kuboyama , 1 Chieh-Chen Huang , 1 Xiaoyun Lu , 1 Toshimi Sawayanagi , 1 Tokiko Kanazawa , 1 Takashi Kagami , 1 Izumi Matsuda , 2 Tsuneo Tsuchizaki , 3 and Shigetou Namba 1

1Laboratory of Bioresource Technology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; 2National Agricultural Research Center, Tsukuba, Ibaraki 305-0856, Japan; 3Koibuchi College of Agriculture, Uchihara-machi, Ibaraki 319-0323, Japan

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Accepted 3 August 1998.

A 3.6-kbp DNA fragment was cloned from the extrachromosomal DNA of a pathogenic plant mollicute, onion yellows phytoplasma (OY-W). Sequence analysis of the fragment revealed an open reading frame (ORF) encoding the replication (Rep) protein of rolling-circle replication (RCR)-type plasmids. This result suggests the existence of a plasmid (pOYW1) in OY-W that uses the RCR mechanism. This assumption was confirmed by detecting the single-stranded DNA (ssDNA) of a replication intermediate that is specifically produced by the RCR mechanism. This is the first report on the identification of the replication system of this plasmid and the genes encoded in it. With a DNA fragment including the Rep gene region of pOYW1 used as a probe, Southern and Northern (RNA) blot hybridizations were employed to examine the heterogeneity between the plasmids found in OY-W and a pathogenic mutant (OY-M) isolated from OY-W. Multiple bands were detected in the DNA and RNA extracted from both OY-W and OY-M infected plants, although the banding patterns were different. Moreover, the copy number of plasmids from OY-W was about 4.2 times greater than that from OY-M. These results indicate constructive heterogeneity between OY-W and OY-M plasmids, and the possibility of a relationship between the plasmid-encoded genes and the pathogenicity of the phytoplasma was suggested.

© 1998 The American Phytopathological Society