The Experts below are selected from a list of 96 Experts worldwide ranked by ideXlab platform
Shigetou Namba - One of the best experts on this subject based on the ideXlab platform.
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spatiotemporal dynamics and quantitative analysis of phytoplasmas in insect vectors
Scientific Reports, 2020Co-Authors: Hiroaki Koinuma, Kensaku Maejima, Ryosuke Tokuda, Kohei Kumita, Akio Miyazaki, Shigetou Namba, Yugo Kitazawa, Takamichi Nijo, Yasuyuki YamajiAbstract:Phytoplasmas are transmitted by insect vectors in a persistent propagative manner; however, detailed movements and multiplication patterns of phytoplasmas within vectors remain elusive. In this study, spatiotemporal dynamics of Onion Yellows (OY) phytoplasma in its vector Macrosteles striifrons were investigated by immunohistochemistry-based 3D imaging, whole-mount fluorescence staining, and real-time quantitative PCR. The results indicated that OY phytoplasmas entered the anterior midgut epithelium by seven days after acquisition start (daas), then moved to visceral muscles surrounding the midgut and to the hemocoel at 14–21 daas; finally, OY phytoplasmas entered into type III cells of salivary glands at 21–28 daas. The anterior midgut of the alimentary canal and type III cells of salivary glands were identified as the major sites of OY phytoplasma infection. Fluorescence staining further revealed that OY phytoplasmas spread along the actin-based muscle fibers of visceral muscles and accumulated on the surfaces of salivary gland cells. This accumulation would be important for phytoplasma invasion into salivary glands, and thus for successful insect transmission. This study demonstrates the spatiotemporal dynamics of phytoplasmas in insect vectors. The findings from this study will aid in understanding of the underlying mechanism of insect-borne plant pathogen transmission.
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unique morphological changes in plant pathogenic phytoplasma infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ specific manner
Plant Journal, 2011Co-Authors: Misako Himeno, Yoshiko Ishii, Shigeyuki Kakizawa, Yasuyuki Yamaji, Kenro Oshima, Yutaro Neriya, Nami Minato, Chihiro Miura, Kyoko Sugawara, Shigetou NambaAbstract:Summary Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, Onion Yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.
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In the non-insect-transmissible line of Onion Yellows phytoplasma (OY-NIM), the plasmid-encoded transmembrane protein ORF3 lacks the major promoter region.
Microbiology (Reading England), 2009Co-Authors: Yoshiko Ishii, Ayaka Hoshi, Shigeyuki Kakizawa, Kensaku Maejima, Yasuyuki Yamaji, Kenro Oshima, Satoshi Kagiwada, Shigetou NambaAbstract:'Candidatus Phytoplasma asteris', Onion Yellows strain (OY), a mildly pathogenic line (OY-M), is a phytopathogenic bacterium transmitted by Macrosteles striifrons leafhoppers. OY-M contains two types of plasmids (EcOYM and pOYM), each of which possesses a gene encoding the putative transmembrane protein, ORF3. A non-insect-transmissible line of this phytoplasma (OY-NIM) has the corresponding plasmids (EcOYNIM and pOYNIM), but pOYNIM lacks orf3. Here we show that in OY-M, orf3 is transcribed from two putative promoters and that on EcOYNIM, one of the promoter sequences is mutated and the other deleted. We also show by immunohistochemical analysis that ORF3 is not expressed in OY-NIM-infected plants. Moreover, ORF3 protein seems to be preferentially expressed in OY-M-infected insects rather than in plants. We speculate that ORF3 may play a role in the interactions of OY with its insect host.
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movement of Onion Yellows phytoplasma and cryptotaenia japonica witches broom phytoplasma in the nonvector insect nephotettix cincticeps
Japanese Journal of Phytopathology, 2009Co-Authors: S. Nakajima, Shigetou Namba, Norio Nishimura, I. Fujisawa, Young H J Jung, S Kakizawa, Tsuneo TsuchizakiAbstract:Two strains of ‘Candidatus Phytoplasma asteris’ (Onion Yellows phytoplasma: OYP and Cryptotaenia japonica witches’ broom phytoplasma: CJWP) were detected by PCR from the nonvector leafhopper (Nephotettix cincticeps) that had fed on phytoplasma-infected plants or had been injected with one of the phytoplasmas. In all experiments, each phytoplasma was detected from the mid-gut, abdomen, thorax, head and salivary glands of insects after acquisition feeding or injection. However, neither phytoplasma was transmitted after inoculation feeding. This report is the first that phytoplasmas can pass through the mid-gut wall and reach the salivary glands of a nonvector leafhopper that had fed on phytoplasma-infected plants.
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in planta dynamic analysis of Onion Yellows phytoplasma using localized inoculation by insect transmission
Phytopathology, 2004Co-Authors: Wei Wei, Shigeyuki Kakizawa, Kenro Oshima, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Masashi Ugaki, Shiho Suzuki, Tadaaki Hibi, Shigetou NambaAbstract:Wei, W., Kakizawa, S., Suzuki, S., Jung, H.-Y., Nishigawa, H., Miyata, S., Oshima, K., Ugaki, M., Hibi, T., and Namba, S. 2004. In planta dynamic analysis of Onion Yellows phytoplasma using localized inoculation by insect transmission. Phytopathology 94:244-250. 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.
Kenro Oshima - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of phyllogen a phyllody inducing effector protein of phytoplasma
Biochemical and Biophysical Research Communications, 2019Co-Authors: Nozomu Iwabuchi, Kensaku Maejima, Ryosuke Tokuda, Akio Miyazaki, Yugo Kitazawa, Hiroaki Koinuma, Takamichi Nijo, Hideyuki Miyatake, Masanobu Nishikawa, Kenro OshimaAbstract:Abstract Phytoplasmas are plant pathogenic bacteria that often induce unique phyllody symptoms in which the floral organs are transformed into leaf-like structures. Recently, a novel family of bacterial effector genes, called phyllo dy-inducing gen e s (phyllogens), was identified as being involved in the induction of phyllody by degrading floral M ADS-domain t ranscription f actor s (MTFs). However, the structural characteristics of phyllogens are unknown. In this study, we elucidated the crystal structure of PHYL1OY, a phyllogen of ‘Candidatus Phytoplasma asteris’ Onion Yellows strain, at a resolution of 2.4 A. The structure of PHYL1 consisted of two α-helices connected by a random loop in a coiled-coil manner. In both α-helices, the distributions of hydrophobic residues were conserved among phyllogens. Amino acid insertion mutations into either α-helix resulted in the loss of phyllody-inducing activity and the ability of the phyllogen to degrade floral MTF. In contrast, the same insertion in the loop region did not affect either activity, indicating that both conserved α-helices are important for the function of phyllogens. This is the first report on the crystal structure of an effector protein of phytoplasmas.
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unique morphological changes in plant pathogenic phytoplasma infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ specific manner
Plant Journal, 2011Co-Authors: Misako Himeno, Yoshiko Ishii, Shigeyuki Kakizawa, Yasuyuki Yamaji, Kenro Oshima, Yutaro Neriya, Nami Minato, Chihiro Miura, Kyoko Sugawara, Shigetou NambaAbstract:Summary Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, Onion Yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.
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In the non-insect-transmissible line of Onion Yellows phytoplasma (OY-NIM), the plasmid-encoded transmembrane protein ORF3 lacks the major promoter region.
Microbiology (Reading England), 2009Co-Authors: Yoshiko Ishii, Ayaka Hoshi, Shigeyuki Kakizawa, Kensaku Maejima, Yasuyuki Yamaji, Kenro Oshima, Satoshi Kagiwada, Shigetou NambaAbstract:'Candidatus Phytoplasma asteris', Onion Yellows strain (OY), a mildly pathogenic line (OY-M), is a phytopathogenic bacterium transmitted by Macrosteles striifrons leafhoppers. OY-M contains two types of plasmids (EcOYM and pOYM), each of which possesses a gene encoding the putative transmembrane protein, ORF3. A non-insect-transmissible line of this phytoplasma (OY-NIM) has the corresponding plasmids (EcOYNIM and pOYNIM), but pOYNIM lacks orf3. Here we show that in OY-M, orf3 is transcribed from two putative promoters and that on EcOYNIM, one of the promoter sequences is mutated and the other deleted. We also show by immunohistochemical analysis that ORF3 is not expressed in OY-NIM-infected plants. Moreover, ORF3 protein seems to be preferentially expressed in OY-M-infected insects rather than in plants. We speculate that ORF3 may play a role in the interactions of OY with its insect host.
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in planta dynamic analysis of Onion Yellows phytoplasma using localized inoculation by insect transmission
Phytopathology, 2004Co-Authors: Wei Wei, Shigeyuki Kakizawa, Kenro Oshima, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Masashi Ugaki, Shiho Suzuki, Tadaaki Hibi, Shigetou NambaAbstract:Wei, W., Kakizawa, S., Suzuki, S., Jung, H.-Y., Nishigawa, H., Miyata, S., Oshima, K., Ugaki, M., Hibi, T., and Namba, S. 2004. In planta dynamic analysis of Onion Yellows phytoplasma using localized inoculation by insect transmission. Phytopathology 94:244-250. 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.
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secretion of immunodominant membrane protein from Onion Yellows phytoplasma through the sec protein translocation system in escherichia coli
Microbiology, 2004Co-Authors: Shigeyuki Kakizawa, Kenro Oshima, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Masashi Ugaki, Shiho Suzuki, Minoru Tanaka, Shigetou NambaAbstract:A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the Onion Yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster Yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.
Tsuneo Tsuchizaki - One of the best experts on this subject based on the ideXlab platform.
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movement of Onion Yellows phytoplasma and cryptotaenia japonica witches broom phytoplasma in the nonvector insect nephotettix cincticeps
Japanese Journal of Phytopathology, 2009Co-Authors: S. Nakajima, Shigetou Namba, Norio Nishimura, I. Fujisawa, Young H J Jung, S Kakizawa, Tsuneo TsuchizakiAbstract:Two strains of ‘Candidatus Phytoplasma asteris’ (Onion Yellows phytoplasma: OYP and Cryptotaenia japonica witches’ broom phytoplasma: CJWP) were detected by PCR from the nonvector leafhopper (Nephotettix cincticeps) that had fed on phytoplasma-infected plants or had been injected with one of the phytoplasmas. In all experiments, each phytoplasma was detected from the mid-gut, abdomen, thorax, head and salivary glands of insects after acquisition feeding or injection. However, neither phytoplasma was transmitted after inoculation feeding. This report is the first that phytoplasmas can pass through the mid-gut wall and reach the salivary glands of a nonvector leafhopper that had fed on phytoplasma-infected plants.
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Transmission of Cryptotaenia japonica Witches' Broom and Onion Yellows by Hishimonoides sellatiformis
Japanese Journal of Phytopathology, 2004Co-Authors: Norio Nishimura, S. Nakajima, H. Kawakita, Mamoru Sato, I. Fujisawa, Tsuneo TsuchizakiAbstract:Cryptotaenia japonica witches' broom phytoplasma (CJWP) and Onion Yellows phytoplasma (OYP) were transmitted by the leafhopper H. sellatiformis. CJWP and OYP were also detected by PCR in H. sellatiformis fed on infected plants. Latent periods in H. sellatiformis of CJWP and OYP were 13-17 days. The phytoplasmas were detected by PCR in leafhoppers at about 7 days after the start of acquisition feeding. This is the first report of the transmission of CJWP and OYP by H. sellatiformis.
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detection of mulberry dwarf and Onion Yellows phytoplasmas by pcr from vector insects and nonvector insects
Japanese Journal of Phytopathology, 2002Co-Authors: S. Nakajima, Toshiki Shiomi, Shigetou Namba, Norio Nishimura, Izumi Matsuda, Tsuneo TsuchizakiAbstract:Phytoplasmas were detected by PCR in vector leafhoppers Hishimonus sellatus and Macrosteles striifrons of Onion Yellows phytoplasma (OYP), and in a vector leafhopper H. sellatus of mulberry dwarf phytoplasma (MDP), respectively, after they fed on infected plants or were injected with the extract from infective insects. MDP was not detected by PCR in nonvector M. striifrons fed on infected plants, but was detected in preparations from whole bodies, headless bodies and heads of M. striifrons injected with the extract from infective insects. M. striifrons injected with infective MDP failed to transmit MDP to test plants.
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in planta expression of a protein encoded by the extrachromosomal dna of a phytoplasma and related to geminivirus replication proteins
Microbiology, 2001Co-Authors: Hisashi Nishigawa, Kenro Oshima, Shin-ichi Miyata, Tsutomu Kuboyama, Toshimi Sawayanagi, Akihiro Komoto, I Matsuda, Tsuneo Tsuchizaki, Shigetou NambaAbstract: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.
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a plasmid isolated from phytopathogenic Onion Yellows phytoplasma and its heterogeneity in the pathogenic phytoplasma mutant
Molecular Plant-microbe Interactions, 1998Co-Authors: Tsutomu Kuboyama, Toshimi Sawayanagi, Tsuneo Tsuchizaki, Chiehchen Huang, Tokiko Kanazawa, Takashi Kagami, Izumi Matsuda, Shigetou NambaAbstract: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 (...
Hisashi Nishigawa - One of the best experts on this subject based on the ideXlab platform.
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in planta dynamic analysis of Onion Yellows phytoplasma using localized inoculation by insect transmission
Phytopathology, 2004Co-Authors: Wei Wei, Shigeyuki Kakizawa, Kenro Oshima, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Masashi Ugaki, Shiho Suzuki, Tadaaki Hibi, Shigetou NambaAbstract:Wei, W., Kakizawa, S., Suzuki, S., Jung, H.-Y., Nishigawa, H., Miyata, S., Oshima, K., Ugaki, M., Hibi, T., and Namba, S. 2004. In planta dynamic analysis of Onion Yellows phytoplasma using localized inoculation by insect transmission. Phytopathology 94:244-250. 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.
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secretion of immunodominant membrane protein from Onion Yellows phytoplasma through the sec protein translocation system in escherichia coli
Microbiology, 2004Co-Authors: Shigeyuki Kakizawa, Kenro Oshima, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Masashi Ugaki, Shiho Suzuki, Minoru Tanaka, Shigetou NambaAbstract:A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the Onion Yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster Yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.
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Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the Onion Yellows phytoplasma genome.
Microbiology, 2003Co-Authors: Shin-ichi Miyata, Shigeyuki Kakizawa, Kenro Oshima, Hisashi Nishigawa, Hee-young Jung, Tsutomu Kuboyama, Masashi Ugaki, Shigetou NambaAbstract:Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. Two homologues of bacterial thymidylate kinase genes were identified in a genomic library of the Onion Yellows (OY) phytoplasma, a plant pathogen that inhabits both plant phloem and the organs of insects. Southern blotting analysis suggested that the OY genome contained one copy of the tmk-b gene and multiple copies of the tmk-a gene. Sequencing of PCR products generated by amplification of tmk-a enabled identification of three other copies of tmk-a, although the ORF in each of these was interrupted by point mutations. The proteins, TMK-a and TMK-b, encoded by the two intact genes contained conserved motifs for catalytic activity. Both proteins were overexpressed as fusion proteins with a polyhistidine tag in Escherichia coli and purified, and TMK-b was shown to have thymidylate kinase activity. This is believed to be the first report of the catalytic activity of a phytoplasmal protein, and the OY phytoplasma is the first bacterial species to be found to have two intact homologues of tmk in its genome.
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complete set of extrachromosomal dnas from three pathogenic lines of Onion Yellows phytoplasma and use of pcr to differentiate each line
Journal of General Plant Pathology, 2003Co-Authors: Hisashi Nishigawa, Shin-ichi Miyata, Kenro Oshima, Masashi Ugaki, Shigetou NambaAbstract: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.
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minimal set of metabolic pathways suggested from the genome of Onion Yellows phytoplasma
Journal of General Plant Pathology, 2002Co-Authors: Kenro Oshima, Shigeyuki Kakizawa, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Toshimi Sawayanagi, Shiho Suzuki, Kenichiro Furuki, Masaki Yanazaki, Wei WeiAbstract: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.
Shin-ichi Miyata - One of the best experts on this subject based on the ideXlab platform.
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in planta dynamic analysis of Onion Yellows phytoplasma using localized inoculation by insect transmission
Phytopathology, 2004Co-Authors: Wei Wei, Shigeyuki Kakizawa, Kenro Oshima, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Masashi Ugaki, Shiho Suzuki, Tadaaki Hibi, Shigetou NambaAbstract:Wei, W., Kakizawa, S., Suzuki, S., Jung, H.-Y., Nishigawa, H., Miyata, S., Oshima, K., Ugaki, M., Hibi, T., and Namba, S. 2004. In planta dynamic analysis of Onion Yellows phytoplasma using localized inoculation by insect transmission. Phytopathology 94:244-250. 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.
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secretion of immunodominant membrane protein from Onion Yellows phytoplasma through the sec protein translocation system in escherichia coli
Microbiology, 2004Co-Authors: Shigeyuki Kakizawa, Kenro Oshima, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Masashi Ugaki, Shiho Suzuki, Minoru Tanaka, Shigetou NambaAbstract:A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the Onion Yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster Yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.
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Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the Onion Yellows phytoplasma genome.
Microbiology, 2003Co-Authors: Shin-ichi Miyata, Shigeyuki Kakizawa, Kenro Oshima, Hisashi Nishigawa, Hee-young Jung, Tsutomu Kuboyama, Masashi Ugaki, Shigetou NambaAbstract:Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. Two homologues of bacterial thymidylate kinase genes were identified in a genomic library of the Onion Yellows (OY) phytoplasma, a plant pathogen that inhabits both plant phloem and the organs of insects. Southern blotting analysis suggested that the OY genome contained one copy of the tmk-b gene and multiple copies of the tmk-a gene. Sequencing of PCR products generated by amplification of tmk-a enabled identification of three other copies of tmk-a, although the ORF in each of these was interrupted by point mutations. The proteins, TMK-a and TMK-b, encoded by the two intact genes contained conserved motifs for catalytic activity. Both proteins were overexpressed as fusion proteins with a polyhistidine tag in Escherichia coli and purified, and TMK-b was shown to have thymidylate kinase activity. This is believed to be the first report of the catalytic activity of a phytoplasmal protein, and the OY phytoplasma is the first bacterial species to be found to have two intact homologues of tmk in its genome.
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complete set of extrachromosomal dnas from three pathogenic lines of Onion Yellows phytoplasma and use of pcr to differentiate each line
Journal of General Plant Pathology, 2003Co-Authors: Hisashi Nishigawa, Shin-ichi Miyata, Kenro Oshima, Masashi Ugaki, Shigetou NambaAbstract: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.
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minimal set of metabolic pathways suggested from the genome of Onion Yellows phytoplasma
Journal of General Plant Pathology, 2002Co-Authors: Kenro Oshima, Shigeyuki Kakizawa, Shin-ichi Miyata, Hisashi Nishigawa, Hee-young Jung, Toshimi Sawayanagi, Shiho Suzuki, Kenichiro Furuki, Masaki Yanazaki, Wei WeiAbstract: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.
