Identification,purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).     

Purification and characterization of 8 of the pathogenesis-related proteins in tobacco leaves reacting hypersensitively to tobacco mosaic virus

Summary Leaves of tobacco plants (Nicotiana tabacum cv. Samsun NN) which are reacting hypersensitively to infection with tobacco mosaic virus contain 10 major pathogenesis-related (PR) proteins which are absent, or present in small amounts in uninfected leaves. We describe here a preparative procedure of purification of the tobacco PR-proteins which involves a combination of conventional and high-performance liquid chromatography. The separation and isolation of the proteins were based on differences in net charge at different pH values, in isoelectric point and in apparent molecular weight. This procedure led to the purification to homogeneity of 8 PR-proteins, as shown by polyacrylamide slab gel electrophoresis (PAGE) of the purified proteins under denaturing and non-denaturing conditions. These were the 3 well-known proteins PR-1a,-1b and-1c, and 5 other major PR-proteins, called PR-2,-N,-O,-P and-Q, according to the nomenclature of Van Loon (39). None of the purified PR-proteins gave a positive Schiff reaction for carbohydrate content. Molecular weight determinations from gel permeation chromatography and from sodium dodecyl sulphate (SDS)-PAGE indicated that all 8 PR-proteins were monomers and that three groups could be distinguished among them. The first group is the PR-1 group containing PR-1a,-1b and-1c (12000 MW), the second consists of PR-P and PR-Q (14000 MW) and the third of PR-2, PR-N and PR-O (25000 MW). In the PR-1 group, PR-1a can be distinguished clearly from the two other members on denaturing slab gels containing both SDS and urea.     

Changes in key enzymes of viral-RNA biosynthesis in chloroplasts from PVY and TMV infected tobacco plants

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.This study was supported by grant No. 522/02/0708 of the Grant Agency of the Czech Republic.     

Identification and characterization of maize pathogenesis-related proteins. Four maize PR proteins are chitinases

Eight pathogenesis-related proteins extractable at pH 2.8 were found to accumulate in maize leaves after mercuric chloride treatment or brome mosaic virus infection. These proteins were called PRm (pathogenesis-related maize) proteins. Seven PRm proteins were purified to homogeneity by preparative polyacrylamide gel electrophoresis and their amino acid compositions determined. Estimated molecular weights in SDS-containing gels were: PRm 1 14.2 kDa; Prm 2 16.5 kDa; PRm 3 and PRm 4 25 kDa; PRm 6b 30.5 kDa; PRm 6a 32 kDa; PRm 7 34.5 kDa. Antisera raised against either PRm 3 or PRm 4 reacted specifically each with PRm 3 or PRm 4. Antisera raised against PRm 6b reacted with PRm 6b as well as with PRm 6a and antisera against PRm 7 reacted with PRm 7 and PRm 5. Tobacco anti-PR 1b antisera reacted with maize PRm 2.Chitinase (poly[1,4-(N-acetyl--D-glucosamide)]glycanhydrolase, EC 3.2.1.14) activity was found for PRm 3, PRm 4, PRm 5, and PRm 7.     

The detection of PR (b) protein and TMV by ELISA in systemic and localised virus infections of tobacco

An antiserum was prepared to the b₁ protein purified from TMV infectedN. tabacum cv. Xanthi-nc leaves and used to study PR proteins. The Xanthi-nc proteins b₂ and b₃ were shown to be serologically closely related to b₁. Antisera to b₁ protein and TMV were used in a F(ab′)₂ enzyme linked immunosorbent assay to monitor PR protein and TMV concentrations, respectively, during the first 6 days of a systemic TMV infection (cv. Xanthi) and a localised TMV infection (cv. Xanthi-nc).     

Alleviation of plant virus infection by humic acids

K-humates, obtained from oxihumolites, alleviate infection of tobacco with tobacco mosaic virus both in mixture with virus inoculum and by spraying of leaves before inoculation. However, applications of K-humates after inoculation did not influence the virus infectivity.     

Changes with time in the distribution of virus and PR protein around single local lesions of TMV infected tobacco

Summary ELISA was used to determine PR la protein and TMV accumulation in local necrotic lesions produced on salicylic acid and water sprayed Nicotiana tabacum cv Xanthi-nc leaves. The amount of PR la protein produced is the result of an interaction between the salicylic acid treatment and lesion growth. The implication of these observations for experiments investigating the relationship between PR proteins and resistance are discussed.The distribution of TMV and PR la protein in and around single local necrotic lesions up to 14 days after inoculation was measured by ELISA. The highest concentration of TMV was in the centre of the lesion and decreased rapidly with distance from the centre. In contrast there was very little PR la protein in the centre of the lesion, the largest amounts were just outside the centre, and the concentration then decreased with distance from the centre. This is the distribution that might be expected for a substance closely associated with the restriction of virus spread.     

Changes in endogenous cyanide and 1-aminocyclopropane-1-carboxylic acid levels during the hypersensitive response of tobacco mosaic virus-infected tobacco leaves

Leaves of Nicotiana tabacum L. cv. Xanthi necroticum plants form local necrotic lesions at the site of infection by tobacco mosaic virus. During the first seven days post-inoculation, endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-ACC increased in the lesion area. The time course of ACC accumulation coincided with an increase in the endogenous cyanide level which began within two days after inoculation. Concomitantly, the activity of -cyanoalanine synthase, the main HCN detoxifying enzyme, decreased. Likewise, treatment of leaf discs of uninfected plants with ACC led to cyanide accumulation. Exogenously applied KCN caused necrotic spots on tobacco leaves very similar to the whitish centers of virus-induced local lesions. Possible implications of cyanide in cell death during TMV-induced lesion development are discussed.     

Isolation and characterization of two middle repetitive DNA sequences of nuclear tobacco genome

Summary Two DNA sequences, R8.1 and R8.3, representing two distinct classes of tobacco genomic repeated DNA, were cloned and characterized by Southern blot analysis. Both R8.1 and R8.3 were found to be homologous to the Nicotiana tomentosiformis component of the allotetraploid Nicotiana tabacum genome, and each of them represents about 0.3% of nuclear DNA. The R8.1 and R8.3 differ in the mode of distribution in chromosomes, as revealed by in situ DNA/DNA hybridization.     

Molecular tagging of the tobacco chromosome carrying the TMV-resistance gene (N gene) by Agrobacterium-mediated transformation

Summary The hypersensitive response of tobacco to inoculation with tobacco mosaic virus (TMV) is controlled by a single dominant gene, the N gene. As a first step in localizing and transferring the N gene, we have prepared a line of tobacco plants in which the kanamycin-resistance (Km^r) gene is closely linked to the N gene. Nicotiana tabacum plants heterozygous for the N gene were transformed to Km^r by Agrobacterium carrying pMON200. Eighty-nine independent transformed clones were regenerated and were backcrossed with nontransformed, TMV-sensitive plants. Progeny from these crosses were screened first for Km^r; then the Km^r progeny were inoculated with TMV and scored for the hypersensitive response. Of the initial 89 clones, 68 appeared to have integrated a single functional Km^r gene. Initial tests for TMV resistance indicated possible linkage between Km^r and the N gene in 11 plants. With further testing, linkage has been established for two of these plant lines. In one of these lines, the two genes were 30–40 map units apart, and evidence of somatic instability in the linkage was obtained. However, in the second line, linkage between Km^r and the N gene was tight, and recombination between the genes in this case was only 5%. Southern hybridization revealed that this plant contained only a single copy of the Km^r gene. Linkage between Km^r and the N gene in this plant line has been verified in each of two additional backcross generations.Abbreviations nptII Neomycin phosphotransferase gene - Km^r kanamycin resistant - Km^s kanamycin sensitive - TMV tobacco mosaic virus - TMV-R TMV resistant - TMV-S TMV sensitive     

Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco

Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.     

Selection of an atrazine-resistant tobacco cell line having a mutant psbA gene

Summary A mutant cell line that shows high resistance to the photosynthesis-inhibiting herbicide atrazine was selected from cultured photomixotrophic Nicotiana tabacum cv. Samsun NN cells by repeated exposure to toxic levels of the herbicide. This resistance was confirmed by measurements of Hill reaction activity in isolated thylakoid membranes. Nucleotide sequencing revealed that the resistant cell line had a point mutation in its chloroplast psbA gene. The 264th codon, AGT (serine) was changed to ACT (threonine) in this mutant. This new type of mutation also conferred moderate cross-resistance to diuron and subsequently was stable in the absence of continued selection pressure.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

An extraction method for tobacco mosaic virus movement protein localizing in plasmodesmata

Summary. The intercellular communication by plasmodesmata (PD) is important for the growth and development of plants, and the transport of macromolecules through PD is likely to be regulated by developmental signals. While PD in the apical meristem transport macromolecules such as mRNAs, the branched PD in the mature leaf do not transport large macromolecules freely. The changes in PD during development might be important for sink-to-source changes in leaves, but the molecular mechanism is still unknown. Movement proteins (MPs) of the tobacco mosaic virus localize in the branched PD and increase the size exclusion limit, allowing transport of viral RNA. We developed a method for differential extraction of MP from isolated cell walls of transgenic tobacco leaves expressing MP or MP tagged with green-fluorescent protein. Lithium chloride at a concentration of 8 M removed filamentous structures in branched PD, the possible attachment site of MP. As some endogenous proteins were coeluted with MP by the treatment, this extraction method might be a powerful tool for investigating MP-interacting proteins in branched PD. Correspondence and reprints: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.