Peanut stunt virus 2b cistron plays a role in viral local and systemic accumulation and virulence in Nicotiana benthamiana.

To analyze the role of the 2b protein (2bP) of Peanut stunt virus (PSV) in the viral infection cycle, we constructed PSV mutants that express either no 2bP or N-terminal-truncated 2bP. The accumulation of wild-type and mutant viruses in tobacco protoplasts indicated that the 2b cistron is not essential for viral replication. Viral accumulation in Nicotiana benthamiana plants suggested that the 2b cistron is responsible for viral accumulation in inoculated and upper leaves and has a role in virulence. The involvement of eight N-terminal amino acids of 2bP in these functions is discussed.

A single amino acid in the RNA-dependent RNA polymerase of Plantago asiatica mosaic virus contributes to systemic necrosis.

From a lily isolate of Plantago asiatica mosaic virus (PlAMV-Li), two sub-isolates (Li1 and Li6) were obtained. Although the nucleotide sequences of Li1 and Li6 were highly conserved, they showed different pathogenicity in Nicotiana benthamiana. Li1 caused necrosis, whereas Li6 infected the plant asymptomatically. Inoculation tests with chimeric and point-mutated viruses revealed that amino acid 1154 of the RNA-dependent RNA polymerase (RdRp) contributes to the necrotic symptoms. The accumulation of the mutant viruses, in which amino acid 1154 of the RdRp was exchanged to the wild-type codon in Li1 and Li6, was almost equal.

Antisense expression of a rice sucrose transporter OsSUT1 in rice (Oryza sativa L.).

We analyzed the function of a rice sucrose transporter, OsSUT1, by using antisense rice. There was no difference between antisense and wild-type plants in carbohydrate content and photosynthetic ability of the flag leaves in the vegetative growth stage, suggesting that OsSUT1 may not play an important role in carbon metabolism, at least in these materials.

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.

A plasmid of phytoplasma encodes a unique replication protein having both plasmid- and virus-like domains: clue to viral ancestry or result of virus/plasmid recombination?

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.

Random mutagenesis in a plant viral genome using a DNA repair-deficient mutator Escherichia coli strain.

Random mutagenesis in a plant viral genome is valuable for generating attenuated strains or for analyzing viral gene function at the molecular level. A DNA repair-deficient mutator Escherichia coli strain was used for random mutagenesis of a plant viral genome. A full-length infectious cDNA clone of Citrus tatter leaf virus (genus Capillovirus) L strain (CTLV-L) genomic RNA under the T7 promoter sequence (pITCL) was introduced into the mutator E. coli strain XL1-Red and mutagenized overnight. To fix mutations, the mixture of plasmid DNA isolated from colonies of the mutator bacteria was introduced into another E. coli strain, JM109, which has normal DNA repair function. Infectious viral genomic RNA was transcribed in vitro from each mutagenized pITCL clone and inoculated on host plants. Phenotypic mutants were selected for altered pathogenicity in the inoculated plants. Nucleotide sequence analysis of each mutant revealed that mutations were introduced randomly into the CTLV-L genome regardless of the function of the viral gene. The nucleotide substitutions were biased towards single point mutations, which consisted of more transitions than transversions or single-base frameshifts. These mutations were preserved stably in plants subject to sequential mechanical inoculation. The strategy presented below is a simple and very efficient way to generate virus mutants for analyzing the functions of viral genes.

Isolation and Characterization of Derivative Lines of the Onion Yellows Phytoplasma that Do Not Cause Stunting or Phloem Hyperplasia.

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.

Complete nucleotide sequence of Tulip virus X (TVX-J): the border between species and strains within the genus Potexvirus.

The complete nucleotide sequence of the genomic RNA of Tulip virus X Japanese isolate (TVX-J) has been determined. The sequence is 6056 nucleotides in length, excluding the poly(A) tail at the 3' terminus, and contains five open reading frames (ORFs) coding for proteins of Mr 153, 25, 12, 10, and 22 kDa (ORFs 1 through 5, respectively). The genome organization of TVX-J is similar to that of potexviruses, and the encoded proteins share a high degree of homology to the corresponding proteins of other potexviruses. Phylogenetic analyses based on the RNA-dependent RNA polymerase (RdRp) protein (the methyltransferase, helicase, and polymerase domains) encoded by ORF1 and the capsid protein (CP) encoded by ORF5, revealed a close relationship of TVX-J to Plantago asiatica mosaic virus (PlAMV). Pairwise comparison analyses revealed that the relationship between TVX and PlAMV is intermediate between that of strains and species, though previously they have not been considered related. Due to the relatively distant relationships of their replication apparatus and triple gene blocks, we conclude that TVX and PlAMV should be classified as distinct viruses. In addition, the borderline between species and strains of potexviruses is discussed.

Ser162-Dependent inactivation of overproduced sucrose-phosphate synthase protein of maize leaf in transgenic rice plants.

To investigate the role of Ser162 in phosphorylation-dependent regulation of maize sucrose-phosphate synthase (SPS) activities in rice, transgenic rice plants expressing wild-type or mutagenized maize SPS were produced. Our results indicate that Ser162 was responsible for overproduction-induced inactivation of SPS protein and for light/ dark modulation of this protein in vivo.

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus.

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene.

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase.

Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R(0)), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines.

Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants.

A series of chimeric promoters for higher-level expression of foreign genes in plants was constructed as fusions of a gene for beta-glucuronidase (GUS) with the terminator of a gene for nopaline synthase (nos) or of the cauliflower mosaic virus (CaMV) 35S transcript, and the strength of these promoters was assayed in transient and stable expression systems in tobacco and rice. As parts of these promoters, the CaMV 35S core promoter, three different 5'-upstream sequences of the 35S promoter, the first intron of a gene for phaseolin, and a 5'-untranslated sequence (omega sequence) of tobacco mosaic virus were used in various combinations. In tobacco and rice protoplasts, all three fragments of the 35S promoter (-419 to -90, -390 to -90 and -290 to -90, relative to the site of initiation of transcription), the intron, and the omega sequence effectively enhanced GUS activity. Some chimeric promoters allowed levels of GUS activity that were 20- to 70-fold higher than those obtained with the 35S promoter in pBI221. In tobacco protoplasts, the two longer fragments of the 35S promoter were more effective than the shortest fragment. In rice cells, by contrast, the shortest fragment was as effective as the two longer ones. The terminator of the 35S transcript was more effective than that of the nos gene for gene expression. In transgenic tobacco plants, a representative powerful promoter, as compared to the 35S promoter, allowed 10- and 50-fold higher levels of expression on average and at most, respectively, with no clear qualitative differences in tissue- and organ-specific patterns of expression. When the representative promoter was introduced into tobacco with a gene for luciferase, the autofluorescence of detached leaves after a supply of luciferin to petioles was great and was easily detectable by the naked eye in a dark room.

[Silicosis associated with autoimmune hemolytic anemia].

A 60-year-old man with pneumoconiosis complained of general fatigue, exertional dyspnea, and anorexia. The patient had severe anemia, and laboratory examination revealed autoimmune hemolytic anemia with positive direct and indirect Coombs tests. After corticosteroid therapy, the anemia resolved markedly, and the antinuclear antibody test became negative. However, the silicotic shadow on chest X-ray film showed no remarkable change. The autoimmune hemolytic anemia in this case was probably due to an immunological disturbance caused by silicosis.

Transgenic plants of Agrostis alba obtained by electroporation-mediated direct gene transfer into protoplasts.

A system for genetic transformation of Agrostis alba plants by electroporation-mediated DNA transfer to protoplasts is described. The npt II gene was used as a selectable marker. Selection with 20 mg/1 G418 (geneticin) yielded a total of over 50 resistant cell colonies from three independent experiments. Overall frequency of resistant colony formation was 1-3 × 10(-6) based on the number of protoplasts plated and 1-2 × 10(-5) based on the number of cell colonies recovered. Subsequent subcultures led to the development of plants with an apparently normal morphology. DNA analysis (PCR and Southern hybridization) and enzymatic analysis showed that the G418 resistant plants carried the transgene and expressed it. This is the first successful genetic transformation of an economically important temperate grass, Agrostis.

The complete nucleotide sequence of cucumber green mottle mosaic virus (SH strain) genomic RNA.

The complete nucleotide sequence of the genomic RNA of cucumber green mottle mosaic virus watermelon strain SH (CGMMV-SH) was determined using cloned cDNA. This sequence is 6421 nucleotides long containing at least four open reading frames, which correspond to 186K, 129K, 29K and 17.3K proteins. The 17.3K protein is the coat protein. Sequence analysis shows that CGMMV-SH is very closely related to another watermelon strain. CGMMV-W, although three amino acid substitutions in the 29K protein were found between these strains. The sequence was also compared to those of other tobamoviruses, tobacco mosaic virus (TMV) vulgare, TMV-L (a tomato strain) and tobacco mild green mosaic virus reported by other groups. It shows 55 to 56% identity with these viruses. The size and location of the open reading frames are very similar to those of TMV but the 129K and 186K proteins are composed of 1142 and 1646 amino acids, being larger than those of TMV by 27 and 31 amino acids, respectively. The deduced amino acid sequences of these proteins are highly homologous to those of TMV, especially in the readthrough downstream region of the 186K protein.

Replication of a geminivirus derived shuttle vector in maize endosperm cells.

A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus. To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11. The p15A origin of replication, functional in E. coli, was introduced into the viral sequences. We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene. The resulting NPT II gene fusion can serve as a selectable marker in both plant and E. coli systems. Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator. By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E. coli in the presence of kanamycin. The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.

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.

Hepatocellular carcinoma combined with hepatic mesenchymal cell tumor and sarcoma.

An autopsy case of hepatocellular carcinoma combined with sarcoma and mesenchymal cell tumor or cholangiole cell carcinoma is presented. The main tumor nodes in the liver consisted of hepatocellular carcinoma and sarcoma. Extrahepatic metastases showed characteristics of monocytic or reticulum cells and hemangioendothelial sarcomas. In this report, some problems concerning hepatocellular carcinoma and sarcomatous representation of the liver tumor are proposed. Differentiation to the monocyte-macrophage-like cells and a reticulum cell pattern were present in the metastatic locations. Thus strongly suggesting that the sarcoma may have arisen from mesenchymal cells, particularly from the Kupffer cells or endothelia consisted of the sinusoid. However, mesenchymal tumor from cholangiole cells might be associated in a part of the sarcomatous cells.