Tsuneo Tsuchizaki

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.

The Outer Capsid Protein of Rice Dwarf Virus Is Encoded by Genome Segment S8

The nucleotide sequence of DNA complementary to the eighth largest (S8) of the 12 genome segments of rice dwarf virus was determined. This genome segment is 1424 nucleotides in length and has a single long open reading frame extending 1260 nucleotides from the first AUG triplet (residues 24 to 26). The predicted translational product comprises 420 amino acids and has an Mr of 46,422. The amino acid sequences of several peptide fragments of the major outer capsid protein were found to be contained in the predicted translational product of the above nucleotide sequence. This protein, previously reported to be 43K, is encoded by genome segment S8 and therefore renamed the 46K protein.

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.

Comparison of four serological methods for practical detection of ten viruses of rice in plants and insects

Four serological methods (ELISA, simplified ELISA, latex flocculation test [LF], and passive hemagglutination test [PHA]) were compared for practical detection of 10 viruses of rice in plants and viruliferous insects. All of the viruses were detected by ELISA and simplified ELISA. The detection of the viruses in plants with systemic infections was easier than the detection of phloem-restricted viruses. Some viruses in plants could not be detected in LF and PHA because of inhibition or nonspecific reactions. All of the viruses that are transmitted in a persistent manner were easily detected in individual insect vectors by ELISA and simplified ELISA, but some viruses were unable to detect by LF and PHA (.)

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.

Complete nucleotide sequence of wheat yellow mosaic bymovirus genomic RNAs

SummaryThe complete sequences of wheat yellow mosaic bymovirus (WYMV) RNAff1 and RNAff2 were determined. RNAff1 is 7 636 nucleotides long [excluding the 3′-poly(A)], and codes for a 269 kDa polyprotein of 2 404 amino acids which contains the capsid protein (CP) at the C terminus and seven putative non-structural proteins. RNAff2 is 3 659 nucleotides long and codes for a polyprotein of 904 amino acids which contains a 28 kDa putative proteinase and a 73 kDa polypeptide. These functional proteins are arranged as in RNAff1 and RNAff2 of barley yellow mosaic bymovirus (BaYMV). Comparisons with the sequence reported for the 3′ half of RNAff1 of wheat spindle streak mosaic bymovirus (WSSMV) from Southern France show that WYMV and WSSMV have a similar genetic organization. However, WYMV and WSSMV share only 77% amino acid sequence identity in their deduced CPs in spite of their close serological relationship, and 74% nucleotide sequence identity in their 3′ non-coding regions. Thus, the sequence data indicate that WYMV and WSSMV are not strains of the same virus, which has long been suggested, but are distinct virus species within the genus Bymovirus of the family Potyviridae.

Location of Structural Proteins in Particles of Rice Gall Dwarf Virus

Summary Protein from particles of rice gall dwarf virus was resolved into seven components by polyacrylamide gel electrophoresis. The estimated molecular weights (× 103) were 183, 165, 150, 143, 120, 56 and 45 (K). Cores prepared by treating intact particles with CsCl contained the 183K, 165K, 120K and 56K proteins; the 150K, 143K and 45K proteins were recovered from the top layer of gradients after CsCl equilibrium centrifugation. Antiserum against intact particles reacted mainly with 150K and 45K proteins and antiserum against core particles reacted mainly with 183K and 120K proteins. The results suggest that the 150K and 45K proteins are located on the surface of the outer capsid, that the 183K and 120K proteins are on the surface of the core, that the 165K and 56K proteins are inside the core, and that the 143K protein is either inside the outer capsid or inside the core.

Comparative electron microscopy of cytoplasmic inclusions induced by 9 isolates of soil-borne wheat mosaic virus

Abstract Rye and wheat leaf cells infected with 9 isolates of soil-borne wheat mosaic virus were examined with the electron microscope. Seven isolates induced large masses of cytoplasmic inclusions consisting of tubules or smooth-surfaced endoplasmic reticulum, differing in fine structure with different isolates. One isolate induced large masses of vesicles and membrane-bound bodies, while another isolate induced small masses of vesicles or endoplasmic reticulum on rare occasions. Virus rods were scattered or aggregated within or near inclusions. In all isolates from Japan, some of these aggregates were parallel in a crystalline arrangement. No crystalline aggregates were observed in the cytoplasm in any American isolates examined. Aggregates of rods were also found in the vacuole of leaf cells infected with each isolate; they were often in crystalline arrays. Concentrically arrayed membranous structures appeared in the cytoplasm of older leaf cells infected with each of 4 isolates. The types of inclusions previously classified by light microscopy did not fully coincide with the types of fine structures observed by electron microscopy. Small crystalline bodies were found in the cytoplasm of both infected and healthy wheat leaf cells, but were not found in rye plants.

Electron microscopy of inclusion development in rye leaf cells infected with soil-borne wheat mosaic virus.

Abstract Rye leaves infected with soil-borne wheat mosaic virus isolate US-A at six consecutive developmental stages were examined with the electron microscope. Virus was not detected in leaves shorter than 5 mm, and viral inclusions were first observed in leaves 10–30 mm in length. The inclusions consisted of tangled tubules or smooth-surfaced endoplasmic reticulum and contained virus rods scattered in interstices between the tubules. The inclusions enlarged with the growth of leaves and changed to a looser conformation. In leaves longer than 100 mm, inclusions were mostly disintegrated and aggregates of virus rods appeared in the cytoplasm and vacuole. In such leaves, inclusions consisted of concentrically arrayed membranes with virus rods sandwiched in between. Both types of inclusions were also found in wheat and barley leaves infected with the US-A isolate.

Infection by Rice Gall Dwarf Virus of Cultured Monolayers of Leafhopper Cells

Summary Synchronous multiplication of rice gall dwarf virus (RGDV) and an assay of its infectivity in cell monolayers of its vector, the green rice leafhopper Nephotettix cincticeps, are described. The number of foci of infected cells was linearly related to the concentration of virus. The method was about 103 times more sensitive than enzymelinked immunosorbent assay. All the vector cells in monolayers were infected when inocula were dilutions of 10-3.5 of sap from infected plants, 10-4.5 of extracts of viruliferous insects, 10-5.5 of infected monolayer cells or purified virus with A 1cm 260nm = 10-5. RGDV was first detected in monolayer cells 10 h after inoculation, and multiplied 105-fold in the subsequent 40 h. Vector cell monolayers are thus an excellent experimental system for the study of RGDV.