Tomatinase from Fusarium oxysporum f. sp. lycopersici defines a new class of saponinases.
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Plants produce a variety of secondary metabolites, many of which have antifungal activity. Saponins are plant glycosides that may provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici and other tomato pathogens produce extracellular enzymes known as tomatinases, which deglycosylate alpha-tomatine to yield less toxic derivatives. We have cloned and characterized the cDNA and genomic DNA encoding tomatinase from the vascular pathogen of tomato F. oxysporum f. sp. lycopersici. This gene encodes a protein (FoTom1) with no amino acid sequence homology to any previously described saponinase, including tomatinase from Septoria lycopersici. Although FoTom1 is related to family 10 glycosyl hydrolases, which include mainly xylanases, it has no detectable xylanase activity. We have overexpressed and purified the protein with a bacterial heterologous system. The purified enzyme is active and cleaves alpha-tomatine into the less toxic compounds tomatidine and lycotetraose. Tomatinase from F. oxysporum f. sp. lycopersici is encoded by a single gene whose expression is induced by alpha-tomatine. This expression is fully repressed in the presence of glucose, which is consistent with the presence of two putative CREA binding sites in the promoter region of the tomatinase gene. The tomatinase gene is expressed in planta in both roots and stems throughout the entire disease cycle of F. oxysporum f. sp. lycopersici. (+info)
A point mutation in the ethylene-inducing xylanase elicitor inhibits the beta-1-4-endoxylanase activity but not the elicitation activity.
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Ethylene-inducing xylanase (EIX) elicits plant defense responses in certain tobacco (Nicotiana tabacum) and tomato cultivars in addition to its xylan degradation activity. It is not clear, however, whether elicitation occurs by cell wall fragments released by the enzymatic activity or by the xylanase protein interacting directly with the plant cells. We cloned the gene encoding EIX protein and overexpressed it in insect cells. To determine the relationship between the two activities, substitution of amino acids in the xylanase active site was performed. Substitution at glutamic acid-86 or -177 with glutamine (Gln), aspartic acid (Asp), or glycine (Gly) inhibited the beta-1-4-endoxylanase activity. Mutants having Asp-86 or Gln-177 also lost the ability to induce the hypersensitive response and ethylene biosynthesis. However, mutants having Gln-86, Gly-86, Asp-177, or Gly-177 retained ability to induce ethylene biosynthesis and the hypersensitive response. Our data show that the xylanase activity of EIX elicitor can be separated from the elicitation process, as some of the mutants lack the former but retain the latter. (+info)
The enzymatic activity of fungal xylanase is not necessary for its elicitor activity.
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Fungal xylanases from Trichoderma spp. are potent elicitors of defense responses in various plants. To determine whether enzymatic activity is necessary for elicitor activity, we used site-directed mutagenesis to reduce the catalytic activity of xylanase II from Trichoderma reesei. For this, the glutamic acid residue at position 210, which is part of the active center in this family of enzymes, was changed to either aspartic acid (E210D) or serine (E210S). Wild-type and mutated forms of xylanase II were expressed in yeast cells and purified to homogeneity. Compared with the wild-type form of xylanase II, E210D had >100-fold and E210S 1,000-fold lower enzymatic activity. In contrast, these mutated forms showed no comparable drop in elicitor activity. They fully stimulated medium alkalinization and ethylene biosynthesis in suspension-cultured tomato (Lycopersicon esculentum) cells, as well as hypersensitive necrosis in leaves of tomato and tobacco (Nicotiana tabacum) plants. These results provide direct evidence that enzyme activity is not necessary for elicitor activity of fungal xylanase. (+info)
Expression of a polygalacturonase associated with tomato seed germination.
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Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth. (+info)
Glucose polyester biosynthesis. Purification and characterization of a glucose acyltransferase.
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Glandular trichomes of the wild tomato species Lycopersicon pennellii secrete 2,3,4-O-tri-acyl-glucose (-Glc), which contributes to insect resistance. A Glc acyltransferase catalyzes the formation of diacyl-Glc by disproportionating two equivalents of 1-O-acyl-beta-Glc, a high-energy molecule formed by a UDP-Glc dependent reaction. The acyltransferase was purified 4,900-fold from L. pennellii leaves by polyethylene glycol fractionation, diethylaminoethyl chromatography, concanavalin A affinity chromatography, and chromatofocusing. The acyltransferase possesses an isoelectric point of 4.8, a relative molecular mass around 110 kD, and is composed of 34- and 24-kD polypeptides as a heterotetramer. The 34- and 24-kD proteins were partially sequenced. The purified enzyme catalyzes both the disproportionation of 1-O-acyl-beta-Glcs to generate 1,2-di-O-acyl-beta-Glc and anomeric acyl exchange between 1-O-acyl-beta-Glc and Glc. (+info)
Cloning, expression, and molecular characterization of a small pea gene family regulated by low levels of ultraviolet B radiation and other stresses.
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A pea (Pisum sativum) DNA fragment (termed MB3) was isolated by differential display of cDNAs obtained from total leaf RNA of ultraviolet B (UV-B) radiation-treated plants. Longer cDNAs were cloned by rapid amplification of cDNA ends in the 3' to 5' direction. Three different, but very similar, cDNAs were cloned, sadA, sadB, and sadC, the major difference between them being a 36-bp deletion in the coding region of sadB. Southern blotting confirmed the occurrence of at least three genes in the pea genome. Database comparisons of the SAD protein sequences revealed high identity (46%) and similarity (77%) with a putative tomato (Lycopersicon esculentum) short-chain alcohol dehydrogenase. Very low levels of UV-B radiation (the biologically effective radiation normalized to 300 nm = 0.08 W m(-2)) was shown to up-regulate expression, a dose considerably lower than that needed to induce expression of the well-known UV-B defensive chalcone synthase and phenylalanine ammonia lyase genes. RNase protection assay revealed that primarily sadA and sadC mRNA accumulation was enhanced by UV-B. In addition to UV-B irradiation, ozone fumigation, wounding, aluminum stress, and salt stress induced increased transcript levels of the sad genes in pea. (+info)
Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato.
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It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding. (+info)
A proline-, threonine-, and glycine-rich protein down-regulated by drought is localized in the cell wall of xylem elements.
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A cDNA clone encoding a proline-, threonine-, and glycine-rich protein (PTGRP) was isolated from a wild tomato species (Lycopersicon chilense) (L.X. Yu, H. Chamberland, J.G. Lafontain, Z. Tabaeizadeh [1996] Genome 39: 1185-1193). Northern-blot analysis and in situ hybridization studies revealed that PTGRP is down-regulated by drought stress. The level of the mRNA in leaves and stems of 8-d drought-stressed plants decreased 5- to 10-fold compared with that in regularly watered plants. The mRNA re-accumulated when drought-stressed plants were rewatered. Antibodies raised against a glutathione S-transferase/PTGRP fusion protein were used to elucidate the subcellular localization of the protein by immunogold labeling. In regularly watered L. chilense plants, PTGRP protein was found to be localized in xylem pit membranes and disintegrated primary walls. Examination of sections from drought-stressed plants revealed a significant decrease in the levels of labeling. In these samples, only a few scattered gold particles were detected in the same areas. In the leaf tissues of plants that had been rewatered for 3 d following an 8-d drought stress, the labeling pattern was similar to that of the regularly watered plants. To our knowledge, PTGRP is the first drought-regulated protein that has been precisely localized in the cell wall. (+info)