Papers by Shin-ichi Miyata
Citrus greening (huanglongbing) is the most destructive disease of citrus worldwide. It is spread... more Citrus greening (huanglongbing) is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with phloem-limited bacteria of three species of a-Proteobacteria, namely, ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. americanus’, and ‘Ca. L. africanus’. Recent findings suggested that some Japanese strains lack the bacteriophage-type DNA polymerase region (DNA pol), in contrast to the Floridian psy62 strain. The whole genome sequence of the pol-negative ‘Ca. L. asiaticus ’ Japanese isolate Ishi-1 was determined by metagenomic analysis of DNA extracted from ‘Ca. L. asiaticus’-infected psyllids and leaf midribs. The 1.19-Mb genome has an average 36.32 % GC content. Annotation revealed 13 operons encoding rRNA and 44 tRNA genes, but no typical bacterial pathogenesis-related genes were located within the genome, similar to the Floridian psy62 and Chinese gxpsy. In contrast to other ‘Ca. L. asiaticus ’ strains, the genome of the Japanese Ishi-1 strain...
Phytopathology®, 2009
The aim of this study was to investigate the genetic diversity and relationships among ‘Candidatu... more The aim of this study was to investigate the genetic diversity and relationships among ‘Candidatus Liberibacter asiaticus’ isolates from different hosts and distinct geographical areas in Southeast Asia. Genetic diversity among ‘Ca. Liberibacter asiaticus’ was estimated by sequencing four well-characterized DNA fragments: the 16S ribosomal DNA (rDNA) and 16S/23S intergenic spacer regions; the outer membrane protein (omp) gene region; the trmU-tufB-secE-nusG-rplKAJL-rpoB region (gene cluster region); and the bacteriophage-type DNA polymerase region. The sequences of the 16S rDNA and 16S/23S intergenic spacer regions were identical among all ‘Ca. Liberibacter asiaticus’ isolates. In contrast, nucleotide substitutions were observed in both the omp gene and the gene cluster regions. However, extended bacteriophage-type DNA polymerase sequences acquired by thermal asymmetric interlaced polymerase chain reaction provided the most sequence diversity among isolates. Phylogenetic analysis of...
Journal of General Plant Pathology, 2010
In Japan and Southeast Asia, 'Candidatus Liberibacter asiaticus' (Las) is the dominant causal age... more In Japan and Southeast Asia, 'Candidatus Liberibacter asiaticus' (Las) is the dominant causal agent of citrus greening (huanglongbing) disease. Using PCR techniques, we determined the 11168-nucleotide sequence of the wserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster and the flanking regions for 51 Japanese, four Taiwanese, four Indonesian, and three Vietnamese isolates of Las. The sequence is identical in 62 isolates collected from Japan, Taiwan, Indonesia, and Vietnam, except for nucleotide substitutions at 11 positions. Some Las isolates from Sakishima Islands near Taiwan had unique nucleotide mutations, but all Las isolates around Okinawa Main Island were homologous. On the basis of the pattern of single nucleotide polymorphisms (SNPs) of the 11 nucleotide substitutions, the 62 Las isolates from Japan, Taiwan, Indonesia, and Vietnam could be divided into 12 pattern groups, and the 51 Japanese isolates consisted of six patterns. The results suggested that one unique genetic group is dominant around Okinawa Main Island, whereas several different are commonly distributed around islands near Taiwan.
Journal of General Plant Pathology, 2002
The phytoplasmas are a group of plant pathogenic bacteria that cause devastating damage to over 7... more 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 (760 kbp). We also cloned and sequenced all three of its extrachromosomal (EC) DNAs (7, 6 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 hos! metabolites and affecting normal cellular functiqns. This work is the first genome analysis of a plant pathogen that inhabits plant phloem cells.
Japan Agricultural Research Quarterly: JARQ, 2010
The Asian type "Candidatus Liberibacter asiaticus" (Las, citrus greening organism) is severely da... more The Asian type "Candidatus Liberibacter asiaticus" (Las, citrus greening organism) is severely damaging citrus production in Asia including Japan. Our previous study suggested that the bacteriophagetype DNA polymerase region (DNA pol) would be useful for molecular differentiation in different Southeast Asian Las isolates. Moreover, Las isolates originated from most of the Southeast Asian regions harbor the DNA pol gene, whereas Japanese ones lack this region. These preliminary findings lead us to a hypothesis that all Japanese isolates lack DNA pol. To try this hypothesis, we collected citrus leaf samples infected with Las throughout the Ryukyu Islands, and examined them by a duplex PCR that could simultaneously amplify two DNA fragments of DNA pol and nusG-rplK operon of Las. The duplex PCR was applied to the collection of 65 Las isolates. Both DNA pol and nusG-rplK operon were successfully amplified from nine isolates, whereas only nusG-rplK operon was amplified with the other 56 isolates. These nine isolates with DNA pol originated from the Hateruma, Irabu, Kohama, Miyako, Tarama, and Yonaguni Islands, which are geographically close to Taiwan. The nucleotide sequence of DNA pol of these nine isolates was identical, and was also the same as four Taiwanese isolates reported previously. These results suggest that Japanese Las isolates comprise at least two distinct genotypes, and the genotype that had DNA pol is highly homogeneous.
The Japanese Journal of Genetics, 1995
Gene, 2002
Two novel rolling circle replication (RCR) plasmids, pOYM (3932 nt) and pOYNIM (3062 nt), were is... more 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-NIMinfected 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.
DNA and Cell Biology, 2002
An 11.4-kbp region of genomic DNA containing the complete S10-spc operon was constructed by an in... more An 11.4-kbp region of genomic DNA containing the complete S10-spc operon was constructed by an integrative mapping technique with eight plasmid vectors carrying ribosomal protein sequences from onion yellows phytoplasma. Southern hybridization analysis indicated that phytoplasmal S10-spc is a single-copy operon. This is the first complete S10-spc operon of a phytoplasma to be reported, although only a part of six serial genes of the S10 operon is reported previously. The operon has a context of 59-rps10,
DNA and Cell Biology, 2003
Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that ... more Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects. Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced. Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma. Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons. However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly. Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional. Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas. The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute. Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis. This data should shed light on the evolutionary relationships and phylogeny of the mollicutes.
Current Genetics, 1998
Previous investigations located nine of the genes for rice tRNAs on plastid (pt)-derived sequence... more Previous investigations located nine of the genes for rice tRNAs on plastid (pt)-derived sequences in mitochondrial DNA. In the present study, we examined whether these genes were also transcribed in rice mitochondria. Northern-blot hybridization revealed that seven of these genes (trnC, trnF, trnH, trnM, trnN, trnS and trnW) are transcribed and are precisely processed to mature tRNAs. One of the other two genes (trnP) is transcribed but cannot be processed efficiently, while the other (trnR), which has 100% identity to the original plastid tRNAArg gene, is not transcribed in rice mitochondria. These results suggest that seven of the nine pt-derived tRNAs may be utilized for the biosynthesis of protein in plant mitochondria.
Biochemical and Biophysical Research Communications, 2008
Several lines of evidence suggest different allocations of the physiological roles of aminopropyl... more Several lines of evidence suggest different allocations of the physiological roles of aminopropyl transferase genes, SPMS and ACL5 in plants. To get deeper insights into the physiological role of apple ACL5 (MdACL5), we performed yeast two-hybrid (Y2H) assay to identify proteins which interact with MdACL5. After intense screening processes, including the swapping of the bait and prey vectors and in vitro coimmunoprecipitation, we identified three MdACL5-interacting proteins: putative translation elongation factor 1A (eEF-1A), putative S-adenosyl-L-methionine synthetase (SAMS) and an unknown protein. Results from Y2H and RNA gel blot analysis suggested the involvement of MdACL5 and eEF-1A or SAMS complexes in the plant growth and development of the organized tissues and/or organs.
Biochemical and Biophysical Research Communications, 2008
Biochemical and Biophysical Research Communications, 2002
Arabidopsis ARR4/ATRR1/IBC7 and ARR8/ATRR3 are homologous genes of prokaryotic response regulator... more Arabidopsis ARR4/ATRR1/IBC7 and ARR8/ATRR3 are homologous genes of prokaryotic response regulators that are involved in the His-Asp phosphorelay signal transduction. We analyzed the function of these genes as response regulators using transgenic plants. Overexpression of ARR4 in cultured stems of the transgenics markedly promoted shoot formation in the presence of cytokinin, while overexpression of ARR8 repressed shoot formation and greening of calli. The expression level of cytokinin-inducible genes, cycD3 and cab increased in the ARR4 overexpressor but decreased in the ARR8 overexpressor. By contrast, two drought stress-inducible genes, rd29A and erd1, were expressed in both overexpressors as that in control plants. These results suggest that ARR4 and ARR8 are involved in cytokinin signal transduction, and that ARR4 functions as a positive-regulator, whereas ARR8 functions as a negative-regulator. Furthermore, microarray analysis showed that several genes were up-regulated in the ARR4 overexpressor. Consistent with these results, ARR4 and ARR8 might play important roles in the sensoring system of cytokinin signal transduction pathway in various developmental and environmental conditions and the regulation of gene expression.
Japan Agricultural Research Quarterly: JARQ, 2020
Citrus tristeza virus (CTV) commonly infects yuzu (Citrus junos) in most parts of the citrus-grow... more Citrus tristeza virus (CTV) commonly infects yuzu (Citrus junos) in most parts of the citrus-growing areas in Japan. This study examined the occurrence of CTV in the northernmost commercial citrusproducing areas of Japan. Colonies of the brown citrus aphid (Toxoptera citricidus) were detected within the areas investigated. However, 23 of the 41 trees that were examined by RT-PCR and both or either subject to stem-pitting observation and immunostrip testing were free of CTV. Several CTVfree trees were apparently more than 60 years old. A field trial revealed that all eight uninoculated seedlings tested negative for CTV at 52 months after transplanting. These results suggest that CTVfree yuzu production is possible in the Rikuzen-Takata and Ofunato areas of Japan.
Japan Agricultural Research Quarterly: JARQ, 2019
Citrus greening disease, huanglongbing (HLB), is one of the most destructive citrus diseases worl... more Citrus greening disease, huanglongbing (HLB), is one of the most destructive citrus diseases worldwide. The causal agent, Candidatus Liberibacter asiaticus (Las), is transmitted by the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). In Okinawa, in the beginning of 2000, HLB was widespread. Local control and planting of HLB-free trees were considered necessary measures to manage HLB. In 2006, 25 HLB-free trees were planted at an experimental field in Okinawa. Then, insecticides were sprayed in the experimental field according to the spray calendar (during the experimental period: imidacloprid 20%, thiamethoxam 10%, dinotefuran 20%, and clothianidin 16%; each application rate was 0.1 L/m 2). In addition, "local control," that is, insecticide spraying [fenitrothion 50% twice a year from 2006 to 2008, mineral oil 97% once, and methidathion 40% (June-July) once after 2008; each application rate was 0.1 L/m 2 ] and removal of HLB-positive trees in the residential area, were continued. Until 2010, psyllids were very few, and none of the 25 planted trees were infected with HLB in the experimental field. Even in 2011, only 3 of the 25 trees were infected, indicating that the HLB infection rate was low when greening-free trees were initially planted and local control (pest control and removal of infected trees in the residential area) was effective at an early stage of cultivation. This result suggests that even in Japan, local control and planting HLB-free trees are effective in managing HLB.
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Papers by Shin-ichi Miyata