Daijiro Hosokawa

Immunocytochemical localization of the 130K and 180K proteins (putative replicase components) of tobacco mosaic virus

We have revealed the cellular localization of the putative replicase components of tobacco mosaic virus (TMV), 130K and 180K proteins, in TMV-infected tobacco leaves by the immunogold technique with antisera which specifically react with these two proteins. When sections of TMV-infected tobacco leaves were treated with anti-130K protein antiserum and then with protein A-gold complex, most of the gold label was strongly localized on granular inclusion bodies which were found specifically in the cytoplasm of TMV-infected cells. Very small amounts of label present in other regions, including the nuclei, chloroplasts, and mitochondria, seemed to be nonspecific. Gold-labeled 180K protein was also dispersed over the granular inclusion bodies. The granular inclusion bodies appeared to be oval-shaped structures with various diameters ranging from 0.2 to 2.8 microm. TMV particles were usually observed near the granular inclusion bodies as aggregates but not inside them. Considering the involvement of the 130K and 180K proteins in replication, the granular inclusion bodies may be the site for replication of TMV RNA.

Defective Tobamovirus Movement Protein Lacking Wild-Type Phosphorylation Sites Can Be Complemented by Substitutions Found in Revertants

ABSTRACT We reported previously that the movement protein (MP) of tomato mosaic tobamovirus is phosphorylated, and we proposed that MP phosphorylation is important for viral pathogenesis. Experimental data indicated that phosphorylation enhances the stability of MP in vivo and enables the protein to assume the correct intracellular location to perform its function. A mutant virus designated 37A238A was constructed; this virus lacked two serine residues within the MP, which prevented its phosphorylation. In the present study, we inoculated plants with the 37A238A mutant, and as expected, it was unable to produce local lesions on the leaves. However, after an extended period, we found that lesions did occur, which were due to revertant viruses. Several revertants were isolated, and the genetic changes in their MPs were examined together with any changes in their in vivo characteristics. We found that reversion to virulence was associated first with increased MP stability in infected cells and second with a shift in MP intracellular localization over time. In one case, the revertant MP was not phosphorylated in vivo, but it was functional.

Reinvestigation of intracellular localization of the 30K protein in tobacco protoplasts infected with tobacco mosaic virus RNA.

It has been shown that the 30K protein of tobacco mosaic virus (TMV) is responsible for the cell-to-cell movement function of the virus. It is still obscure how the protein is involved in this function at the molecular level. We formerly found that the 30K protein is localized to the plasmodesmata of TMV-infected plants. We also reported that the 30K protein was detected in a nuclei-rich fraction of TMV-infected protoplasts after biochemical fractionation. To clarify the inconsistency, the 30K protein was immunocytologically localized in TMV-infected protoplasts using a newly prepared antibody against the 30K protein. On some sections, the 30K protein was found near the nucleus but not in or on the nucleus. At later stages of infection a novel electron-transparent structure was detected in the cytoplasm where the 30K proteins were localized. This structure might reflect an intermediate form between its synthesis in the cytoplasm and its targeting to the plasmodesmata in whole plants.

Sequence-specific binding of protein factors to two independent promoter regions of the acidic tobacco pathogenesis-related-1 protein gene (PR-1)

Gel shift mobility analysis, using the proximal 0.3 kb fragment of the tobacco pathogenesis-related protein la gene (PR-1a) and nuclear extracts from healthy Samsun NN tobacco leaves, which do not produce PR-1 proteins, showed a broad shifted signal with low mobility. This signal was not detected with nuclear proteins from the interspecific hybrid of Nicotiana glutinosa x Nicotiana debneyi, which constitutively produces the PR-1a protein. Similar shifted signals were detected with both proximal and distal regions of the 0.3 kb fragment using nuclear proteins from healthy Samsun NN tobacco, but not with proteins from the interspecific hybrid. Further experiments, performed using 5′ or 3′ truncated fragments of the 0.3 kb fragment, identified two independent binding sites: a distal site between −179 and −168 by from the transcription start site, and a proximal site between −61 and −37 bp. Footprint analysis revealed two protected sequences, a distal region between −184 and −172 bp, and a proximal region between −68 and −51 bp. These results indicate the presence of regulatory factor(s) for expression of the acidic PR-1a gene. The possibility of negative regulation of the gene is discussed.

Effects of single and double infections with Potato virus X and Tobacco mosaic virus on disease development, plant growth, and virus accumulation in tomato

The tomato cv. Fukuju no. 2 was used for studying the effect of single and double infections with Potato virus X (PVX) and Tobacco mosaic virus (TMV). Mixed infection resulted in a synergistic increase of disease severity, where more growth reduction was seen with simultaneous inoculations than with sequential inoculations at four-day intervals. At five and 12 days post-inoculation, the increased severity of the disease coincided with enhancement of virus accumulation in the rapidly expanding upper leaves. The PVX concentration in leaves no 5 to 7 of doubly infected plants was three to six fold that of singly infected ones, as determined by DAS-ELISA. Mixed infection with the L strain led to higher enhancement of PVX than with the TMV-L11A strain. The concentration of TMV-L was lower in double infection and significantly higher than TMV-L11A in both singly and doubly infected plants. Analyses of the PVX ORF2 by Western blot and Northern hybridization revealed the pattern of accumulation of the 25 kDa protein and the RNAs, respectively, following those of the virion and coat protein. The strain TMV-L11A overcame the resistance gene in cv. GCR 237 (Tm-1). In the upper leaf no. 8, the concentration of PVX was three times higher in plants with mixed infection than with L11A. The concentrations of the L and OM (TMV strains) in both singly and doubly infected plants were at very low levels, and the synergistic effect on PVX concentration and disease severity was not observed.

Possible Role of Concanavalin A-Binding Glycoprotein(s) Secreted from Germinating Conidia of Magnaporthe Grisea

Many plant species contain carbohydrate-binding proteins, which are commonly referred to as lectins or agglutinins. But their physiological role in plants has not yet been elucidated. One of the possible roles is the idea that lectins play a role in the plant’s defense mechanism as does the immunoglobulin in the animal owing to their binding specificity.

Studies on multiplication and distribution of viruses in plants by use of fluorescent antibody technique. (VI). Progressive distribution of tobacco mosaic virus and necrotic local lesion formation in Nicotiana tabacum cv. Samsun NN leaves.

蛍光抗体法を用いて,タバコモザイクウイルス(TMV)のNicotiana tabacum cv. Samsun NNの接種葉組織内における増殖・分布と局部病斑形成との関係をTMVが全身感染するN. tabacum cv. Samsunの場合とも比較しながら検討した。Samsun NNにおいてはウイルス抗原は接種24時間後に初めて認められた。この時期にはウイルス抗原が表皮,柵状組織とこれに接した少数の海綿状組織の細胞に分布した。その後ウイルス抗原は葉の水平方向と垂直方向に拡がり,接種32時間後には下側表皮にまで達した。またこの間にウイルスの増殖部位の篩部細胞でもウイルスの増殖が認められ,篩部を経由するウイルスの遠隔部への移行が局部病斑が形成される前にすでに始まりつつあるように思われた。接種36時間後になるとウイルスの増殖した部位の中央部附近の柵状組織に接した海綿状組織の少数の細胞にえ死が起こり,その後細胞のえ死は海綿状組織の他の細胞に急速に拡がり,続いて柵状組織の細胞から表皮の細胞へと及んだ。接種44∼48時間後にはウイルスの増殖した部位のほとんどの細胞がえ死を起こし,局部病斑が形成された。しかし局部病斑が形成されたのちにおいても,その周縁には特異蛍光が認められてもえ死を起こしていない細胞が少数存在し,ウイルスはこれらの細胞からさらに周囲の細胞へ移行,増殖を続け,これらの細胞もやがて内側からえ死を起こすため局部病斑はゆっくり拡大した。接種60時間後にはこの局部病斑周縁部の特異蛍光は認められなくなり,病斑の周縁には強い紫色の自家蛍光を発する物質が集積した。一方,Samsunの葉組織内におけるTMV抗原の分布経過はSamsun NNで細胞え死が始まる接種36時間後までは,Samsun NNのそれと同様であった。しかし,Samsunではその後もウイルスは組織内にさらに広く分布し,細胞内の特異蛍光の輝度も高まった。