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PubMed1995

Structure and induction pattern of a novel proteinase inhibitor class II gene of tobacco.

A cDNA and a corresponding genomic clone encoding a protein with partial identity to type II proteinase inhibitors from potato, tomato and Nicotiana alata, were isolated from tobacco libraries. The protein of 197 amino acids contains a putative signal peptide of 24 residues and three homologous domains, each with a different reactive site. The tobacco PI-II gene is not expressed in leaves of healthy plants, but is locally induced in leaves subjected to different types of stress (TMV infection, wounding, UV irradiation) and upon ethephon treatment. As opposed to the analogous PI-II genes of potato and tomato, the tobacco gene is not systemically induced by wounding or pathogenic infection. A far-upstream region in the PI-II promoter, containing various direct and indirect repeats, shares considerable sequence similarity to a similar region in the stress-inducible Cu/Zn-superoxide dismutase gene of N. plumbaginifolia.

Amino Acid Sequence↗
PubMed1995

Cloning and properties of a rice gene encoding phenylalanine ammonia-lyase.

Phenylalanine ammonia-lyase (PAL) genomic sequences were isolated from a rice (Oryza sativa L.) genomic library using a PCR-amplified rice PAL DNA fragment as a probe. There is a small family of PAL genes in the rice genome. The nucleotide sequence of one PAL gene, ZB8, was determined. The ZB8 gene is 4660 bp in length and consists of two exons and one intron. It encodes a polypeptide of 710 amino acids. The transcription start site was 137 bp upstream from the translation initiation site. Rice PAL transcripts accumulated to a high level in stems, with lower levels in roots and leaves. Wounding of leaf tissues induced ZB8 PAL transcripts to a high level. In rice suspension-cultured cells treated with fungal cell wall elicitors, the ZB8 PAL transcript increased within 30 min and reached maximum levels in 1-2 h. The transcription of the ZB8 gene was investigated by fusing its promoter to the reporter gene beta-glucuronidase (GUS) and transforming the construct into rice and tobacco plants, as well as rice suspension-cultured cells. High levels of GUS activity were observed in stems, moderate levels in roots and low levels in leaves of transgenic rice and tobacco plants. Histochemical analysis indicated that in transgenic rice the promoter was active in root apical tips, lateral root initiation sites, and vascular and epidermal tissues of stems and roots. In rice flowers, high GUS activity was observed in floral shoots, receptacles, anthers and filaments, occasionally GUS activity was also detected in lemma and awn tissues. In tobacco flowers, high GUS activity was detected in the pink part of petals. Consistent with the activity of endogenous PAL transcripts, wounding of rice and tobacco leaf tissues induced GUS activity from low basal levels. Tobacco mosaic virus (TMV) infection of tobacco leaves induced GUS activity to a high level. Fungal cell wall elicitors strongly induced GUS activity and GUS transcripts to high levels in transgenic rice suspension-cultured cells. We demonstrated that the promoter of ZB8 gene is both developmentally regulated and stress-inducible.

Amino Acid Sequence↗
PubMed1993

Transient gene expression of foreign genes in preheated protoplasts: stimulation of expression of transfected genes lacking heat shock elements.

Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at 42 degrees C also increased expression in protoplasts transfected with a plasmid bearing the tobacco mosaic virus (TMV) translational enhancer, omega. Northern blot analysis revealed that heat treatment of protoplasts before the transfection event greatly increased the amount of the newly synthesized transcripts. Preheating of protoplasts did not affect the transfection efficiency, namely the number of transfected cells in the population, nor the amount of DNA in transfected nuclei, as was inferred from histochemical staining and Southern blot analysis, respectively. The possible mechanism by which heat treatment stimulates transient gene expression of genes lacking obvious heat shock elements is offered. The relevance of the present findings to transient gene expression in plants in general and to viral gene expression in particular is discussed.

Cell Division↗
PubMed1990

Evidence for a third structural class of beta-1,3-glucanase in tobacco.

Glucan endo-1,3-beta-glucosidases (beta-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of beta-1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a beta-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized beta-1,3-glucanase. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q'. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of beta-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.

Amino Acid Sequence↗
PubMed1995

Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana.

We compared the organ specificity and the strength of different constitutive (CaMV-35S, CaMV-35Somega, Arabidopsis ubiquitin UBQ1, and barley leaf thionin BTH6 promoter) and one inducible promoter (soybean heat-shock promoter Gmhsp17.3) in stably transformed Arabidopsis thaliana plants. For this purpose we constructed a set of plant expression vectors equipped with the different promoters. Using the uidA reporter gene we could show that the CaMV-35S promoter has the highest expression level which was enhanced two- to threefold by the addition of a translational enhancer (TMV omega element) without altering the organ specificity of the promoter. The barley leaf thionin promoter was almost inactive in the majority of lines whereas the ubiquitin promoter exhibited an intermediate strength. The heat-shock promoter was inducible up to 18-fold but absolute levels were lower than in the case of the ubiquitin promoter. Conclusive quantitative results for different organs and developmental stages were obtained by the analysis of 24 stably transformed lines per promoter construct.

Antimicrobial Cationic Peptides↗
PubMed1981

A proteinless mutant of tobacco mosaic virus: evidence against the role of a viral coat protein for interference.

A coat-protein-free mutant of tobacco mosaic virus as well as mutants with a non-functional coat protein were found to interfere with the establishment and spread of challenging strains of TMV. The results do not support an earlier concept, according to which the genome of a related challenging virus could be captured by the coat protein of the virus introduced in advance. The presence of a viral coat protein is obviously not essential and a competition among the viral genomes for some specific site seems to be a more likely mechanism of cross protection.

Mutation↗
PubMed1992

Accumulation of the 126 kDa protein of tobacco mosaic virus during systemic infection analysed by immunocytochemistry and ELISA.

Systemic infection of tobacco with tobacco mosaic virus (TMV) strain WU1, is accompanied by massive accumulation of the virus-coded non-structural 126 kDa protein in X-bodies. The development of X-bodies and the time course of the increase in 126 kDa protein in systemically infected leaves were analyzed by immunocytochemistry and ELISA, respectively, using an antiserum raised against a fusion protein of beta-galactosidase and part of the 126 kDa protein. The ELISA assay developed enabled routine detection of viral 126 kDa (as well as 183 kDa) protein in samples of less than 5 mg of systemically infected leaves. Plants were inoculated by differential temperature treatment, whereafter the accumulation of 126 kDa protein was related to viral multiplication, the development of X-bodies and the formation of symptoms. Both 126 kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h. By electron microscopical analysis small X-bodies, weakly labelled with antibodies against the 126 kDa protein, were detected as early as 24 h after transfer. At this stage they were not associated with nuclei. Thereafter, however, X-bodies increased in size and 126 kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves that developed mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations are consistent with the hypothesis that association of X-bodies with nuclei may lead to symptom induction, when the leaf is invaded by the virus early in its development.

Cell Compartmentation↗
PubMed1993

On the relationship between X-bodies and symptom development in plants infected with different tobamoviruses.

The relationship between systemic mosaic symptoms and the occurrence of viral 126-kDa protein in X-bodies was studied in tobacco infected with the tobacco mild green mosaic virus (TMGMV) strains U2, U5, and ribgrass mosaic virus (RMV) strain HR, and in other plant species infected with tobacco mosaic virus (TMV) strain W U 1. Strains U2, U5, and HR coded for proteins of 126, 126, and 130 kDa, respectively, but these were not recognized by antisera against the corresponding protein from W U 1. Only the HR 130-kDa protein reacted with an antiserum raised against a peptide of amino acids 849-863 from the sequence of W U 1. Electron microscopic analysis established the presence of virus clusters in the cytoplasm, as well as in chloroplasts, in leaf tissue infected with U 2 or U 5, and adjacent to nuclei and chloroplasts in scattered cells infected with HR. X-bodies were not detected after infection with any of these strains, but were large and adjacent to nuclei in W U 1-infected tomato displaying severe mosaic symptoms. Large X-bodies were detected near nuclei in W U 1-infected tomato displaying severe mosaic symptoms, but none were detected after infection of tobacco with any of the other tobamoviruses. The induction of X-bodies appears to be characteristic of some tobamovirus only and, at best, can only be associated with, rather than causative of, the severity of symptoms induced by those viruses.

Amino Acid Sequence↗
PubMed1989

Spurious cross-reactions between plant viruses and monoclonal antibodies can be overcome by saturating ELISA plates with milk proteins.

It has been claimed recently [Dietzgen (1986) Arch Virol 91: 163-173] that a series of monoclonal antibodies (Mabs) produced against the nepovirus, arabis mosaic virus (ArMV) cross-reacted with the tobamovirus, tobacco mosaic virus (TMV). In the present report, this alleged cross-reactivity was re-examined by two ELISA procedures using Mabs produced against each of the two viruses. It was found that when highly concentrated preparations of Mabs were used, all antibodies reacted in a nonspecific manner with several plant viruses. However, when defatted milk instead of bovine serum albumin was used both as blocking agent and as diluent for the Mabs, the spurious cross-reactions between unrelated viruses were abolished. The use of milk as blocking agent did not prevent the detection of genuine cross-reactions between related nepoviruses.

Antibodies, Monoclonal↗
PubMed1989

Limitations of different ELISA procedures for localizing epitopes in viral coat protein subunits.

The reactivity of monoclonal antibodies (McAbs) to the coat protein of tobacco mosaic virus (TMVP) with the isolated coat protein, disks, virions and a number of antigenic variants of TMV was tested in eight different ELISA procedures. Although certain McAbs, when used as detecting antibody in the liquid phase, did not react with some of these antigens, they were able to bind to them when used as the capturing antibody on the solid phase. This finding was attributed to the ability of the trapping McAb to induce a complementary conformation in the antigen presented in the liquid phase. In many cases, the reactivity of the McAbs was found to depend on the format of the ELISA. This finding together with the presence of oligomers in viral coat protein preparations made it impossible to map TMVP epitopes on the surface of the viral subunit by means of competitive ELISA.

Antibodies, Monoclonal↗
PubMed1995

The complete nucleotide sequence and genome organization of odontoglossum ringspot tobamovirus RNA.

The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot tobamovirus (ORSV) was determined. The RNA genome of ORSV is 6618 nucleotides long and contains five open reading frames (ORFs 1 to 5) coding for proteins of M(r) 126 K, 181 K, 34 K, 18 K and 52 K, respectively. This is the longest RNA of the known viruses of the Tobamovirus genus. The sequences of the ORSV RNA encoded proteins exhibit high homology to the proteins of the members of the Tobamovirus genus. The genomic organization and sequence analysis showed that ORSV is more closely related to tobacco mild green mosaic virus (TMGMV), pepper mild mottle virus (PMMV), tomato mosaic virus (ToMV) and TMV than to cucumber green mottle mosaic virus (CGMMV) and sunn-hemp mosaic virus (SHMV).

Amino Acid Sequence↗
PubMed1992

Biophysical and biochemical properties of baculovirus-expressed CaMV P1 protein.

Cauliflower mosaic virus (CaMV) gene I encodes a protein (P1) that has been implicated in the control of virus movement in infected plants. To assist in the characterization of the mechanism of action of P1, gene I has been expressed efficiently in Spodoptera frugiperda (Sf) cells using recombinant baculovirus. Control of the expression of CaMV gene I by the polyhedrin late promoter in the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) resulted in very high levels of P1 accumulation late in the infection cycle. This was predominantly as insoluble inclusion bodies within the cytoplasm of infected Sf cells, and not extracellularly. Evidence from anomalous gel migration and sequence homology with an analogous viral protein (tobacco mosaic virus 30K) indicated that P1 may be post-translationally processed. However, neither phosphorylation nor glycosylation of P1 occurred in this system, suggesting a functional distinction between P1 and TMV 30K. P1 from insect cells and native P1 from infected plants were immunologically related, allowing the expressed product to be used in the preparation of anti-P1 serum for detecting P1 in plant extracts. The full-size (46 kD) P1 product from insect cells, from plants, and from in vitro translations of in vitro gene I transcripts all showed similar behavior on two-dimensional protein gels, with a major pI of 7.0. Using a combination of 4 M urea, 1 M NaCl, and high temperature, P1 was solubilized. Approximately 5% of the starting material remained in solution after dialysis and remained stable to freeze/thawing. This preparation should enable us to identify the biochemical function of P1 and to resolve its role in controlling virus spread.

Animals↗
PubMed1976

Changes in soluble proteins in callus cells of hypersensitive tobacco inoculated with tobacco mosaic virus.

Protein changes occurred in callus cells of hypersensitive tobacco (Nicotiana tabacum var. Xanthi-nc) 72 hr after inoculation with tobacco mosaic virus and incubation on a minimal growth medium. Two protein bands, serologically related to viral coat protein, were obtained from extracts of infected cells following electrophoresis on 7% and 10% polyacrylamide gels. An additional, slower migrating protein, perhaps due to virus-induced stimulation of a host protein, also was detected. Although local lesions appeared on callus after 40 hr of incubation, four proteins previously reported in lesion-bearing hypersensitive tobacco leaves were not found. The possible significance of this and the usefulness of a callus-TMV system as a tool to study virus-induced protein changes are discussed.

Cells, Cultured↗
PubMed2003

Construction of a deep coverage BAC library from Capsicum annuum, 'CM334'.

A bacterial artificial chromosome (BAC) library consisting of 235,000 clones with an average insert size of 130 kb was constructed from Capsicum annuum, 'CM334'. Based on a pepper haploid genome size of 2,702 Mbp/C, the BAC library is estimated to contain approximately 12 genome equivalents and represents at least 99% of the pepper genome. Screening of the library with mitochondrial DNA probes (coxII, coxIII, atp6 and atp9) and chloroplast DNA probes (atpB, rbcL) indicated that contamination with cytoplasmic DNA was less than 0.5%. To estimate the possibility of isolating a specific clone, the library was screened with single or low-copy gene-specific probes and RFLP probes. Screening of high density BAC filters with RFLP markers linked to L (TMV resistance), y (fruit color), C2 (fruit color) and C (pungency) loci under high stringency conditions revealed that at least three positive BAC clones were found per each probe. This fact indicates that the library is highly reliable and represents a resource for map-based cloning, physical mapping, and characterization of upstream and downstream regulations of the chili pepper genes.

Capsicum↗
PubMed2004

A new selection method for pepper transformation: callus-mediated shoot formation.

We used two genes, TMV-CP and PPI1 (pepper-PMMV interaction 1 transcription factor), to transform commercially important chili pepper (Capsicum annuum) inbred lines (P915, P409) by means of Agrobacterium co-culture. Eighteen independently transformed T0 plants were obtained. The most critical point in the pepper transformation protocol was the selection of shoots growing on calli--referred to as callus-mediated shoot formation (indirect shooting)--because shoots not grown from the callus (direct shooting from the wounded surface) developed into non-transformants. Selection of the correct right callus type also proved to be an important requirement for obtaining transformed peppers. Six different types of callus developed during the selection process. Shoots regenerated from two of these types, while one type regenerated significantly more shoots than the other types, suggesting that the capacity for shoot formation is callus type-specific. Although the transformation rate was low, transformation via callus-mediated shoot formation proved to be reproducible and was confirmed by Southern and Northern blot analyses. Based on the experimental data, we have succeeded in developing a new protocol for the selection and transformation of pepper and expect that it will be used in the future for pepper transformation.

Capsicum↗
PubMed2006

Expression of a beta-glucosidase gene results in increased accumulation of salicylic acid in transgenic Nicotiana tabacum cv. Xanthi-nc NN genotype.

A beta-glucosidase gene (bglA) from Butyrivibrio fibrisolvens H17c was cloned into the binary vector pGA482 under the control of the 35S Cauliflower Mosaic Virus (CaMV) promoter. A second construct was generated for accumulation of the bglA gene product in the vacuole of transformed tobacco plants. Reverse transcription-polymerase chain reaction analysis demonstrated that the bglA gene was expressed in 71% of cytosol-targeted and 67% of vacuole-targeted transgenic tobacco T(1) plants. T(1) transgenic plants (pGLU100 and pGLU200) exhibited elevated levels of free salicylic acid (SA) with a concomitant significant decrease in the level of glucosylsalicylic acid (GSA) compared to the untransformed tobacco plants and tobacco plants transformed with the empty vector (pGA482). Following inoculation with Tobacco Mosaic Virus (TMV), lesion area was 51% smaller in pGLU100 plants and 60% smaller in pGLU200 plants compared to inoculated untransformed and negative control plants.

Gene Expression↗
PubMed2005

A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to Tobacco mosaic virus and Xanthomonas campestris.

Plant WRKY transcription factors were previously implicated in the alteration of gene expression in response to various pathogens. The WRKY proteins constitute a large family of plant transcription factors, whose precise functions have yet to be elucidated. Using a domain-specific differential display procedure, we isolated a WRKY gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) or Xanthomonas campestris pv . vesicatoria (Xcv). The full-length cDNA of CaWRKY-a (Capsicum annuum WRKY-a) encodes a putative polypeptide of 546 amino acids, containing two WRKY domains with a zinc finger motif. The expression of CaWRKY-a could be rapidly induced by not only chemical elicitor such as salicylic acid (SA) or ethephon but also wounding treatments. The nuclear localization of CaWRKY-a was determined in transient expression system using tobacco BY-2 cells by polyethylene glycol (PEG)-mediated transformation experiment. With oligonucleotide molecules containing the putative W-box sequences as a probe, we confirmed that CaWRKY-a protein had W-box-binding activity. These results suggest that CaWRKY-a might be involved as a transcription factor in plant defense-related signal transduction pathways.

Amino Acid Sequence↗
PubMed2006

Insight into Types I and II nonhost resistance using expression patterns of defense-related genes in tobacco.

Plants protect themselves against pathogens using a range of response mechanisms. There are two categories of nonhost resistance: Type I, which does not result in visible cell death; and Type II, which entails localized programmed cell death (or hypersensitive response) in response to nonhost pathogens. The genes responsible for these two systems have not yet been intensively investigated at the molecular level. Using tobacco plants (Nicotiana tabacum), we compared expression of 12 defense-related genes between a Type I (Xanthomonas axonopodis pv. glycines 8ra) nonhost interaction, and two Type II (Pseudomonas syringae pv. syringae 61 and P. syringae pv. phaseolicola NPS3121) nonhost interactions, as well as those expressed during R gene-mediated resistance to Tobacco mosaic virus. In general, expression of most defense-related genes during R gene-mediated resistance was activated 48 h after challenge by TMV; the same genes were upregulated as early as 9 h after infiltration by nonhost pathogens. Surprisingly, X. axonopodis pv. glycines (Type I) elicited the same set of defense-related genes as did two pathovars of P. syringae, despite the absence of visible cell death. In two examples of Type II nonhost interactions, P. syringae pv. phaseolicola NPS3121 produced an expression profile more closely resembling that of X. axonopodis pv. glycines 8ra, than that of P. syringae pv. syringae 61. These results suggest that Type I nonhost resistance may act as a mechanism providing a more specific and active defense response against a broad range of potential pathogens.

Cell Death↗