Toshimi Sawayanagi

Cloning and expression analysis of Phytoplasma protein translocation genes.

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.

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

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.

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

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.

Minimal Set of Metabolic Pathways Suggested from the Genome of Onion Yellows Phytoplasma

The phytoplasmas are a group of plant pathogenic bacteria that cause devastating damage to over 700 plant species worldwide. They can propagate intracellularly in both insect and plant hosts. Despite their economic importance and unique biological features, phytoplasmas remain the most poorly characterized plant pathogens, primarily because efforts at in vitro culture, gene delivery, and mutagenesis have been unsuccessful. To shed light on these microorganisms, we constructed a DNA library for the onion yellows (OY) phytoplasma, Candidatus Phytoplasma sp. 16S-group I, AY-subgroup (onion), and obtained a draft sequence of phage clones that cover the whole chromosome, which is estimated to be 1 Mbp at most, and completely sequenced and annotated a significant portion of the chromosome (750 kbp). We also cloned and sequenced all three of its extrachromosomal (EC) DNAs (7, 5 and 4 kbp). The EC DNAs are thought to replicate via a rolling-circle replication mechanism with their own unique replicases. The chromosome encodes genes for basic housekeeping functions, such as DNA replication, transcription and translation, but none for amino acid or fatty acid biosynthesis, the TCA cycle, or oxidative phosphorylation. These genes seem to have been replaced by homolog in the host nuclear genome, since phytoplasmas can import many biological substances from host cells, as is the case with parasitic mycoplasmas. Surprisingly, the phytoplasma genome encodes even fewer genes for metabolic functions than that of mycoplasmas, which are known to possess minimal gene sets; genes for the pentose phosphate cycle, conserved in the genomes of all other reported bacteria, were not found in the OY phytoplasma genome. Phytoplasmas appear to possess the most minimal set of metabolic pathways identified in an organism to date; this minimalism may be related to the fact that phytoplasmas inhabit the nutrient-rich environment of the phloem. More than half of the unknown proteins are predicted to localize in the cell membrane, suggesting that they participate in interactions between the intracellular phytoplasma and the host cell surface or cytoplasm. Although the OY phytoplasma does not possess the typical genes related to pathogenicity found in other phytopathogenic bacteria, some of these unknown genes may be related to pathogenicity via their unique metabolic profile, such as actively importing host metabolites and affecting normal cellular functions. This work is the first genome analysis of a plant pathogen that inhabits plant phloem cells.

Gene Arrangement and Sequence of str Operon of Phytoplasma Resemble Those of Bacillus More Than Those of Mycoplasma

The complete region of a putative streptomycin operon (str operon) of onion yellows (OY) phytoplasma, a phytopathogenic mollicute, was isolated and sequenced. This operon contains four genes, rps12, rps7, fus, and tuf, encoding ribosomal proteins S12 and s7, elongation factor (EF) -G, and EF-Tu, respectively. These four genes constitute the str operon in non-mollicute bacteria, such as Escherichia coli and Bacillus subtilis. In two species of mollicute Mycoplasma, the tuf gene was reported not to be included in this operon, but was located apart, indicating that the gene arrangement of this operon in phytoplasmas resembles that of B. subtilis more than that of Mycoplasma spp. In addition, the deduced amino acid sequence of EF-G of phytoplasmas also resembles that of B. subtilis more than that of Mycoplasma spp. These results suggest that analyses of the gene organization and sequence of the phytoplasma genome will provide valuable insights into evolutionary relationships among the culturable mollicutes, phytoplasmas and other Gram-positive bacteria.