Hisashi Nishigawa

Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma.

The minimal gene set essential for life has long been sought. We report the 860-kb genome of the obligate intracellular plant pathogen phytoplasma (Candidatus Phytoplasma asteris, OY strain). The phytoplasma genome encodes even fewer metabolic functions than do mycoplasma genomes. It lacks the pentose phosphate cycle and, more unexpectedly, ATP-synthase subunits, which are thought to be essential for life. This may be the result of reductive evolution as a consequence of life as an intracellular parasite in a nutrient-rich environment.

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 Dynamic Analysis of Onion Yellows Phytoplasma Using Localized Inoculation by Insect Transmission

ABSTRACT Due to the lack of a means to inoculate plants mechanically, the histological dynamics and in planta spread of phytoplasmas have been studied very little. We analyzed the dynamics of plant infection by phytoplasmas, using a technique to infect a limited area of a leaf, nested polymerase chain reaction (PCR), real-time PCR, and immunohistochemical visualization. Following localized inoculation of a leaf of garland chrysanthemum (Chrysanthemum coronarium) by the vector leafhopper Macrosteles striifrons, the onion yellows (OY) phytoplasma spread within the plant from the inoculated leaf to the main stem (1 day postinoculation [dpi]), to the roots and the top leaf (2 dpi), and to other leaves from top to bottom (from 7 to 21 dpi). The populations of the OY phytoplasmas in inoculated leaves and roots increased approximately sixfold each week from 14 to 28 dpi. At 14 dpi, the OY phytoplasmas colonized limited regions of the phloem tissue in both the root and stem and then spread throughout the phloem by 21 dpi. This information should form the basis for elucidating the mechanisms of phytoplasma multiplication and migration within a plant host.

Positive Selection Acting on a Surface Membrane Protein of the Plant-Pathogenic Phytoplasmas

Phytoplasmas are plant-pathogenic bacteria that cause numerous diseases. This study shows a strong positive selection on the phytoplasma antigenic membrane protein (Amp). The ratio of nonsynonymous to synonymous substitutions was >1 with all the methods we tested. The clear positive selections imply an important biological role for Amp in host-bacterium interactions.

A plasmid from a non-insect-transmissible line of a phytoplasma lacks two open reading frames that exist in the plasmid from the wild-type line

Two novel rolling circle replication (RCR) plasmids, pOYM (3932 nt) and pOYNIM (3062 nt), were isolated from a mildly pathogenic variant line (OY-M) and a mildly pathogenic plus non-insect-transmissible line (OY-NIM), respectively, of onion yellows (OY) phytoplasma, a plant and insect endocellular mollicute. OY-M was isolated from an original wild-type line (OY-W) after regular maintenance using alternate plant/insect infections, while OY-NIM was further isolated from OY-M after maintenance by plant grafting without insect vectors. The RCR-initiator proteins (Rep) of both plasmids, which have a characteristic structure with both plasmid- and virus-like domains, were highly homologous to that of a previously described OY-W plasmid, pOYW (3933 nt), and were expressed in OY-M- and OY-NIM-infected plants, indicating that this replicon is stably maintained in the phytoplasma. Interestingly, pOYNIM lacked two ORFs that exist in both pOYW and pOYM, which encode a single-stranded DNA binding protein (SSB) and an uncharacterized putative membrane protein, indicating that these two proteins are not necessary for the phytoplasma to live in plant cells. These are the first candidates as phytoplasma proteins possibly related to host specificity.

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.

Evidence of intermolecular recombination between extrachromosomal DNAs in phytoplasma: a trigger for the biological diversity of phytoplasma?

Recombination among bacterial extrachromosomal DNAs (EC-DNAs) plays a major evolutionary role by creating genetic diversity, and provides the potential for rapid adaptation to new environmental conditions. Previously, a 7 kbp EC-DNA, EcOYW1, with a geminivirus-like rolling-circle-replication protein (Rep) gene was isolated and characterized from an original wild-type line (OY-W) of onion yellows (OY) phytoplasma, an endocellular cell-wall-less prokaryote that inhabits the cytoplasm of both plant and insect cells. EcOYW1, found in OY-W, was not present in a mild-symptom line (OY-M) derived from OY-W. A 4 kbp EC-DNA, pOYW, was also isolated and characterized from OY-W, and its pLS1-plasmid-like rep gene was expressed. This paper describes the isolation and sequencing of an EC-DNA of 5560 nt, EcOYW2, from OY-W, and its counterpart EC-DNA of 5025 nt, EcOYM, from OY-M. EcOYW2 and EcOYM contained seven and six ORFs, respectively. They both encoded a geminivirus-like Rep and a putative single-stranded-DNA-binding protein (SSB). Southern blot analysis indicated that no more EC-DNAs with a rep gene exist in either OY-W or OY-M, which means that the complete set of EC-DNAs has been cloned from the OY-W and OY-M lines of OY phytoplasmas. Sequence analysis revealed that both EcOYW2 and EcOYM have chimeric structures of previously characterized EcOYW1 and pOYW, suggesting that they have a recombinational origin. This is the first evidence of intermolecular recombination between EC-DNAs in phytoplasma. The possible implications of these findings in increasing the biological diversity of phytoplasma are discussed.

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.

An Antibody Against the SecA Membrane Protein of One Phytoplasma Reacts with Those of Phylogenetically Different Phytoplasmas.

ABSTRACT Antisera raised against phloem-limited phytoplasmas generally react only with the phytoplasma strain used to produce the antigen. There is a need for an antiserum that reacts with a variety of phytoplasmas. Here, we show that an antiserum raised against the SecA membrane protein of onion yellows phytoplasma, which belongs to the aster yellows 16S-group, detected eight phytoplasma strains from four distinct 16S-groups (aster yellows, western X, rice yellow dwarf, and elm yellows). In immunoblots, approximately 96-kDa SecA protein was detected in plants infected with each of the eight phytoplasmas. Immunohistochemical staining of thin sections prepared from infected plants was localized in phloem tissues. This antiserum should be useful in the detection and histopathological analysis of a wide range of phytoplasmas.

Complete set of extrachromosomal DNAs from three pathogenic lines of onion yellows phytoplasma and use of PCR to differentiate each line

Abstract Two lines of onion yellows phytoplasma with reduced pathogenicity have been isolated from the original wild-type line (OY-W). One is a line with mild symptoms (OY-M) and the other is a non-insect-transmissible line, also with mild symptoms (OY-NIM). We previously reported heterogeneity in extrachromosomal DNA (EC-DNA) species in these lines. In this report, another EC-DNA, EcOYNIM, from OY-NIM was cloned and sequenced, providing a complete set of EC-DNAs from the three OY lines. To monitor each phytoplasma in synergism or cross-protection experiments, a pair of polymerase chain reaction (PCR) primers that universally amplify a portion of the EC-DNAs that are characteristic of each line was designed. Using this primer set, a line-specific fragment was amplified from the total DNA of each plant inoculated with one or more phytoplasma lines. The PCR product sizes differ for each phytoplasma line, so the lines can be distinguished even in plants infected with multiple lines. Because EC-DNAs are more abundant than chromosomal genes in phytoplasma cells, this primer set will be valuable for detecting and discriminating these phytoplasma lines and for analyzing their interaction.