RESUMEN
Complete nucleotide sequences of three strains (I, III, and IV) of Barley yellow mosaic virus (BaYMV) isolated in Japan were determined. The length of the genome was the same among the three strains; RNA1 was 7,642 nt and RNA2 was 3,585 nt. The molecular phylogenetic analysis showed that strain I was most closely related to the Chinese isolate, and these two strains formed one cluster with European isolates. Strains II, III, and IV, and the Korean isolate formed another cluster. Amino acid sequences of each viral gene product were compared among strains. The sequences of the VPg protein showed less identity among almost strains (less than 92%) than the sequences of other proteins (more than 93%). VPg is thought to be involved in interactions with host factors, especially initiation factor 4E (eIF4E) or eIF(iso)4E, and infection. Therefore, the relationship between amino acid substitutions and infection of host plants is analyzed.
Asunto(s)
Filogenia , Potyviridae/clasificación , Potyviridae/genética , Hordeum/virología , Datos de Secuencia Molecular , Enfermedades de las Plantas/virología , Potyviridae/aislamiento & purificación , Proteínas Virales/genéticaRESUMEN
Singlet oxygen was detected from the reaction of lophine hydroperoxides 2 in the presence of 1,3-diphenyl-isobenzofuran as a singlet oxygen detector. In order to examine the substituent effect on the formation of (1)O(2), 2-(p-substituted phenyl) lophine peroxides 2a-c were tested. It was found that an electron-attracting group contributed to the efficient formation of (1)O(2) (80% for a NO(2) group), while an electron-donating group enhanced the efficiency of chemiluminescence.
Asunto(s)
Benzofuranos/química , Imidazoles/química , Peróxidos/química , Oxígeno Singlete/análisis , Oxígeno Singlete/química , Cromatografía Líquida de Alta Presión , Estructura MolecularRESUMEN
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.
Asunto(s)
Acholeplasmataceae/genética , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Genes Bacterianos , Proteínas de Transporte de Membrana/genética , Acholeplasmataceae/metabolismo , Acholeplasmataceae/patogenicidad , Adenosina Trifosfatasas/inmunología , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Asteraceae/microbiología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Clonación Molecular , ADN Bacteriano/genética , Expresión Génica , Inmunohistoquímica , Proteínas de Transporte de Membrana/inmunología , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Mapeo Físico de Cromosoma , Conejos , Canales de Translocación SEC , Proteína SecA , Homología de Secuencia de AminoácidoRESUMEN
The genomes of most prokaryotic and eukaryotic single-stranded (ss) DNA viruses, and some prokaryotic plasmids such as pLS1, commonly replicate via a rolling circle replication (RCR) strategy, and thus the viruses are hypothesized to have evolved from the plasmids, although evidence for this view is sparse. We have sequenced a circular plasmid of 3933 nt, pOYW, obtained from onion yellows phytoplasma (OY-W), a cell-wall-less, unculturable prokaryote that inhabits the cytoplasm of both plant and insect cells. pOYW contains five open reading frames (ORFs) on the same strand and apparently replicates by an RCR mechanism. Its rep gene (ORF5) encodes a unique protein, pOYW-Rep, with an unprecedented structure. The N-terminal region of pOYW-Rep has similarities to the RCR initiator protein (Rep) of pLS1 family plasmids but, unlike the Rep of other plasmids, its C-terminal region was unexpectedly similar to the helicase domain of the replication-associated proteins (Rap) of eukaryotic viruses, especially circoviruses (ssDNA viruses of vertebrates). The pOYW-Rep was specifically detected in OY-W-infected plant phloem cells, suggesting that it is a functional protein. We suggest that an ancestral phytoplasma plasmid pOYW may have acquired a helicase domain from host phytoplasmal DNA, entered the surrounding eukaryotic cytoplasm, and subsequently evolved into an ancestral eukaryotic ssDNA virus. Alternatively, a pOYW ancestor could have obtained the helicase domain by recombination with a virus: this would be the first example of recombination between plasmids and viruses.
Asunto(s)
ADN Bacteriano , ADN Viral , Mycoplasma/genética , Plásmidos , Secuencia de Aminoácidos , Northern Blotting/métodos , Southern Blotting/métodos , Clonación Molecular , Genes Virales , Datos de Secuencia Molecular , Cebollas/microbiología , Análisis de Secuencia de ADNRESUMEN
ABSTRACT Two lines of onion yellows phytoplasma producing milder symptoms were isolated from the original line (OY-W). One has an additional characteristic, non-insect-transmissibility (OY-NIM), compared with the other (OY-M). OY-M was established after maintaining OY-W for 11 years on a plant host (Chrysanthemum coronarium) with an insect vector (Macrosteles striifrons), and OY-NIM was isolated after subsequent maintenance of OY-M in plants by periodic grafting. Polymerase chain analysis suggested that OY-NIM cannot traverse the gut or survive in the hemolymph of the leafhopper. OY-W results in witches'-broom formation and stunted growth in the host plant. In contrast, OY-M and OY-NIM do not cause stunting in the host plant, although they result in witches'-broom. Histopathological analysis of these lines revealed that the hyperplastic phloem tissue and severe phloem necrosis seen in OY-W did not exist in OY-M and OY-NIM. This was attributed to a reduction in the population of phytoplasma in tissues in both OY-M- and OY-NIM-infected plants. The results suggest that the cause of stunting and phloem hyperplasia may be genetically different from the cause of witches'-broom. Pulsed field gel electrophoresis analysis showed that OY-M had a smaller genome size ( approximately 870 kbp) than OY-W ( approximately 1,000 kbp). Thus, some of the OY-W genes responsible for pathogenicity may not be present in OY-M.