Differential regulation in tobacco cell suspensions of genes involved in plant-bacteria interactions by pathogen-related signals

Six cDNA clones whose corresponding mRNAs accumulate early during the hypersensitive reaction in tobacco leaves have been classified into 2 groups according to their maximum levels of accumulation in an incompatible versus a compatible interaction withPseudomonas solanacearum. We present evidence that, at least in the first stages of the interaction, tobacco cell suspensions retain the ability to respond differentially to compatible and incompatible isolates ofP. solanacearum.In addition, studies on the effect of a fungal elicitor on the accumulation of the mRNAs corresponding to the cDNA clones in cell suspensions indicate that only one group of genes responds to this treatment.     

Characterization of hsr201 and hsr515, two tobacco genes preferentially expressed during the hypersensitive reaction provoked by phytopathogenic bacteria

During an incompatible interaction between tobacco and the bacterial phytopathogen Pseudomonas solanacearum, 2 classes of genes, the so-called hsr (hypersensitivity-related) genes, activated preferentially during the hypersensitive reaction, and the str (sensitivity-related) genes, expressed strongly during compatible and incompatible interactions, have been identified. In this report, two hsr cDNA clones, hsr515 and hsr201, as well as their expression patterns are presented. Hsr515 was found to encode a P450 monooxygenase and is most similar to the ripening-related avocado gene CYP71A1 (40.6% amino acid identity). Hsr201 presents 58.6% amino acid identity with pTom36, a tomato gene expressed during fruit maturation. The putative functions of the hsr gene products appear to be quite diverse and their characteristics of activation were found to be very conserved: accumulation of the corresponding mRNAs primarily in leaf areas in contact with the avirulent P. solanacearum strain or with a Pseudomonas fluorescens strain containing the hrpZ gene encoding a necrotizing polypeptide, harpin and absence of expression during normal plant development. Our results also suggest that, in a tobacco line expressing NahG, a lower level of salicylic acid, a compound associated with systemic acquired resistance, and also possibly involved in the development of necrotic lesions characteristic of the HR, does not affect the hsr gene expression.     

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.     

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.     

Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.     

Cloning of genes by mRNA differential display induced during the hypersensitive reaction of soybean after inoculation with Pseudomonas syringae pv. glycinea

Soybean (Glycine max[L.] Merr.) cell suspension cultures (cv. Williams 82) inoculated with the pathogenic bacteria Pseudomonas syringae pv. glycinea respond with a hypersensitive reaction (HR) when the bacteria express the avirulence gene avrA. A mRNA differential display was established for this system to allow the identification of genes induced during the HR. Six PCR-fragments (DD1–DD6) from the differential display analysis were identified, which are induced during the HR. Database searches revealed that the fragment DD1 encodes chalcone isomerase and DD2 was identified as ubiquitin. The fragment DD3 shares significant homology to the signalling molecule 14-3-3. The partial DD4 product is homologous to the enhancer of rudimentary from Drosophila and an uncharacterized homologue of it from Arabidopsis. The fragment DD5 is similar to glucose-6-phosphate dehydrogenase which provides NADPH to the cell. The PCR-product DD6 seems to be a new leucine-rich-repeat disease resistance gene from soybean, which is significantly induced during the HR. All of the identified genes are clearly induced during a HR in infected plants of the same cultivar, indicating that results from the cell culture model system can be transferred to intact plants. These studies show that complex mRNA differential display is a powerful tool to identify new induced gene in plant-pathogen interactions.     

Role of Ca2+ in the metabolism of phenolic compounds in tobacco leaves (Nicotiana tabacum L.)

Given the essential role played by phenol metabolism in many resistance responses to different types of stress, the aim of the present work was to determine how different application rates of calcium may influence this metabolic process. Increased calcium in the nutrient solution in which tobacco plants were grown considerably reduced the foliar concentration of phenolic compounds. Calcium clearly exerted a positive influence on the activities of enzymes (phenylalanine ammonia-lyase, polyphenol oxidase and peroxidase) involved in the metabolism of the phenolics. High dosages of calcium (5 mM) promoted more oxidation than synthesis of these compounds, thus explaining the lower concentration of the phenolics.     

Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. phaseolicola

Changes in the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) which regulate the persistence of active oxygen species (AOS) were examined in leaves of bean (Phaseolus vulgaris L. cv. Tendergreen) undergoing compatible and incompatible interactions to race 6 and race 3 strains, respectively, of the halo-blight bacterium Pseudomonas syringae pv. phaseolicola. Resistance of cv. Tendergreen to race 3 is determined by the R3 gene and was expressed by a hypersensitive reaction (HR) which was associated with a rapid increase in lipid peroxidation between 8 and 12 h after inoculation. Five main isoforms of SOD were resolved by native polyacrylamidegel electrophoresis (PAGE). Major changes were found in the activities of the cytosolic Cu, Zn-SOD3 and Cu, ZnSOD5 isoforms, which increased by 6 h after inoculation with race 3, and the possibly peroxisomal MnSOD2 isoform, which decreased rapidly in tissue undergoing the HR. Three further minor isoforms of SOD showed a strong increase in activity during the HR. A low level of extracellular SOD activity was also resolved; two isoforms, one of which increased dramatically in activity during the HR, were detected within intercellular fluids recovered from inoculation sites. Fewer changes in SOD activities were found during the compatible interaction to race 6, and they did not occur until 16 h after inoculation. In tissue around infiltration sites, no decrease in the activity of Mn-SOD2 was observed but slight increases in some other isoforms were found. Four groups of POD isoforms were detected in both 3,3-diaminobenzidine/H₂O₂-and o-dianisidine/H₂O₂-stained PAGE gels. Significant changes in activity were again associated with development of the HR. In particular, by 2 h after inoculation, increases in POD3a, b and c isoforms were detected within total soluble extracts and also in POD3c within intercellular fluids (no other isoform was found in the apoplasm). By contrast, POD1 and POD2 activities generally declined following inoculation. The principal change in activity in tissues surrounding infiltration sites was an increase in POD3 isoforms following inoculation with race 3. Measurements of total activity showed a decrease in CAT activity as early as 2 h after inoculation, followed by a recovery after 8 h and a further decrease as infiltrated tissue collapsed during the HR. A more-gradual decline in CAT activity was observed at sites undergoing the compatible interaction and also in tissue surrounding inoculation sites. The spatial and temporal changes detected in activities of CAT and isoforms of SOD and POD clearly demonstrate the complexity and potential subtlety of control of the production and persistence of AOS in bean following microbial challenge. The generation of AOS through HR-specific, early increases in extra-cellular POD and SOD isoforms is discussed.This work was supported in part by the scientific Research Foundaation (OTKA F 5082), the foundation for Hungarian science, a british council scolership to A.L.A and the U.K. Agricultural and food Reaserch council.     

Netuptake of sulphate and its transport to the shoot in tobacco plants fumigated with H2S or SO2

In tobacco plants the net uptake of sulphate and its transport to the shoot were determined after cultivation with low, normal, and high sulphate supply. The relative amount of the sulphate taken up that was transported to the shoot was used as a measure of xylem loading. Net uptake of sulphate and its transport to the shoot were low in tobacco plants grown with low sulphate, and high in plants cultivated with high sulphate. Xylem loading, however, was relatively low in tobacco plants grown with high sulphate and relatively high in tobacco plants grown with low sulphate supply. Pre-culture in low sulphate containing nutrient solution also resulted in a high proportion of the absorbed sulphate being transported into the xylem if normal sulphateconcentration was supplied afterwards. Fumigation with H₂S or SO₂ reduced net uptake of sulphate in tobacco plants grown with normal, but not with high sulphate supply. Sulphate transport to the shoots was diminished by H₂S or SO₂ fumigation in tobacco plants grown with normal and high sulphate supply. Also the relative amount of the sulphate taken up that was transported to the shoot was lowered by fumigation with H₂S or SO₂ in tobacco plants grown with normal sulphate supply. Apparently, the diminished sulphate transport to the shoot upon H₂S or SO₂ fumigation can only partially be explained by a smaller sulphate uptake. Sulphur nutrition of tobacco plants also seems to be controlled by xylem loading of sulphate. The possible role of glutathione as a signal regulating sulphur nutrition of tobacco plants upon fumigation with H₂S and SO₂ is discussed.     

Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination,callus and shoot formation

A novel acetyltransferase (Mpr1) found in Saccharomyces cerevisiae (strain 1278b) has been shown to specifically detoxify a proline analog, l-azetidine-2-carboxylic acid (A2C) in yeast cells [M. Shichiri et al. (2001) J Biol Chem 276: 41998–42002]. We investigated whether the yeast MPR1 gene would function similarly in a plant system and if its expression could confer resistance to proline analogs. The MPR1 gene coding sequence driven by two different constitutive promoters, with or without the 5- and 3-noncoding sequence from the MPR1 gene adjacent to the conventional NOS terminator, was transformed into tobacco (Nicotiana tabacum L. cv. Xanthi) plants via Agrobacterium tumefaciens infection. The presence of the yeast 5- and 3-noncoding sequences appeared to increase the likelihood of MPR1 gene expression in the transgenic plants. The kanamycin-selected transgenic plants with a high level of Mpr1 activity grew normally, and their progeny expressed acetyltransferase activity that could utilize A2C, azetidine-3-carboxylic acid and 4-hydroxy-l-proline as substrates. Resistance to A2C, but not to the other two analogs, was exhibited during leaf tissue culture and seed germination. The A2C toxicity to the wild-type plants was reversed by the addition of proline, suggesting that A2C acts as a proline analog. Our studies confirm that MPR1 can function in a similar fashion in tobacco as in yeast to detoxify the toxic proline analog A2C, so it could potentially be used as a new selectable marker for plant transformation. However, our attempts to utilize MPR1 as an efficient selectable marker gene for the A. tumefaciens-mediated transformation of tobacco were unsuccessful.Abbreviations A2C: l-Azetidine-2-carboxylic acid - A3C: Azetidine-3-carboxylic acid - Hyp: 4-Hydroxy-l-proline - hpt: Hygromycin phosphotransferase II - NPTII: Neomycin phosphotransferase II Communicated by H. Wang