Activity of nitric oxide is dependent on, but is partially required for function of, salicylic acid in the signaling pathway in tobacco systemic acquired resistance.

When tobacco plants were treated by injection with nitric oxide (NO)-releasing compounds, the sizes of lesions caused by Tobacco mosaic virus (TMV) on the treated leaves and on upper nontreated leaves were significantly reduced. The reduction in TMV lesion size was caused by NO released from the NO-releasing compounds; the byproduct formed after release of NO from the NO-releasing compound NOC-18, diethylenetriamine, did not itself alter lesion size. Treatment of tobacco plants with inhibitors of nitric oxide synthase or an NO scavenger attenuated but did not abolish the systemic acquired resistance (SAR) induced by salicylic acid (SA). In NahG transgenic tobacco plants, NO had no effect on lesion size following TMV infection. These results are consistent with the hypothesis that NO plays an important role in SAR induction in tobacco and that NO is required for the full function of SA as an SAR inducer. The activity of NO is fully dependent on the function of SA in the SAR signaling pathway in tobacco.     

Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.     

cDNA cloning of six mRNAs induced by TMV infection of tobacco and a characterization of their translation products

A cDNA library was constructed to 10-15 S poly(A) RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco. By differential colony hybridization of 1400 transformants, 32 clones were obtained corresponding to TMV-inducible tobacco mRNAs. These clones were subdivided into six clusters on the basis of cross-hybridization of the inserts. By Northern blot hybridization it was shown that three of the corresponding mRNAs were strongly induced by spraying tobacco plants with salicylic acid, whereas one mRNA was weakly induced by this treatment. All mRNAs were systemically induced in plants in which only the lower leaves were locally infected by TMV. Hybrid-selected translation was performed, using six clones representing one cluster each, followed by immunoprecipitation using an antiserum to purified pathogenesis-related (PR) proteins. Four clones yielded precipitable translation products. One of these clones represented a cluster of PR-1 clones, another clone encoded the thaumatin-like (TL) protein of tobacco which may correspond to PR-P or −Q.     

Molecular characterization of messenger RNAs for 'pathogenesis related' proteins la, lb and lc, induced by TMV infection of tobacco

A cDNA library was made to poly(A)-containing RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco plants and clones corresponding to mRNAs for the `pathogenesis-related' (PR) proteins 1a, 1b and 1c were identified. One clone was found to contain a complete copy of PR-1b mRNA. The structural organization of this RNA is: a leader sequence of 29 nucleotides, an open reading frame of 504 nucleotides encoding a 30 amino acid long signal peptide and a 138 amino acid long mature protein, and a 3'-non-coding region of 235 nucleotides. Two other clones were found to contain partial copies of PR-1a and PR-1c mRNAs. The data indicate an ~90% homology between the amino acid sequences of PR-1a, -1b and -1c. Using one of the clones as probe it was shown that in the TMV-inoculated lower leaves and the non-inoculated upper leaves of a tobacco plant, the PR-1 mRNAs become detectable from 2 and 8 days after inoculation, respectively.     

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria ( P.  syringae ) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.     

Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

Replication in the phloem is not necessary for efficient vascular transport of tobacco mosaic tobamovirus

Plant viruses move systemically from one leaf to another via phloem. However, the viral functions needed for systemic movement are not fully elucidated. An experimental system was designed to study the effects of low temperature on the vascular transport of the tobacco mosaic tobamovirus (TMV). Vascular transport of TMV from lower inoculated leaves to upper non-inoculated leaves via a stem segment kept at low temperature (4 degrees C) was not affected. On the other hand, several experiments were performed on tobacco leaves to demonstrate that virus replication did not occur at the same temperature. The data suggest that replication of TMV in the phloem of wild-type tobacco plants is not necessary for the vascular transport of TMV, and that the virus moves with photoassimilates as suggested previously.     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.     

Tobacco proteinase inhibitor I genes are locally,but not systemically induced by stress

A cDNA library of tobacco mosaic virus (TMV)-infected tobacco was screened with polymerase chain reaction products obtained using a degenerate primer corresponding to proteinase inhibitor I (PI-I) of tomato and potato. The resulting clones encoded two highly similar, putative tobacco PI-I proteins, indicating that both genes identified in tobacco are probably expressed. The tobacco PI-I's were approximately 50% identical to wound-inducible potato and tomato PI-I and 80% identical to an ethylene-regulated tomato PI-I. Northern blot analyses indicated that healthy tobacco leaf contains only minor amounts of PI-I mRNA, and that the inhibitor genes are induced by TMV infection, salicylate treatment, ethephon spraying, UV light irradiation and wounding. The results indicate that the tobacco PI-I genes are coordinately expressed with the genes for the basic pathogenesis-related proteins. Contrary to PI-I genes of tomato and potato, wound induction of the tobacco genes occurs only locally; the upper, unwounded leaves do not show any wound-induced PI-I gene expression.     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene

Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.     

Expression of tobacco mosaic virus RNA in transgenic plants

Summary Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in planta.     

Synthesis of the tobacco mosaic virus in blocked spreading of infection

The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.     

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.     

Molecular cloning and characterization of a cDNA for a novel ethylene receptor, NT-ERS1, of tobacco (Nicotiana tabacum L.)

The cDNA encoding a novel member (NT-ERS1) of ethylene receptor family of tobacco (Nicotiana tabacum L.) was obtained by a combination of RT-PCR and 5'-/3'-RACE cloning. The cDNA was 2,092 nucleotides long and had an open reading frame of 1,911 bp encoding 637 amino acids. The deduced polypeptide lacked a response regulator domain, indicating that the ethylene receptor belongs to an ERS-group. The amino acid sequence was similar to respective members of the tobacco ethylene receptor family: 67.8% to NT-ETR1, 39.1% to NTHK1 and 31.1% to NTHK2. Comparison of amino acid sequence suggested that NT-ERS1 is the counterpart of Nr in the ethylene receptor family of tomato, which belongs to Solanaceae as does tobacco. Northern blot analysis showed that mRNA of NT-ERS1 was present in leaf, shoot and root tissues, and accumulated in leaves treated with exogenous ethylene. A mutated NT-ERS1 cDNA transgene, obtained by introducing one nucleotide substitution into NT-ETR1 cDNA, conferred ethylene insensitivity in tobacco plants, indicating that the translation product of the cDNA actually functioned in the plants.