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)
Effects of targeted replacement of the tomatinase gene on the interaction of Septoria lycopersici with tomato plants.
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Many plants produce constitutive antifungal molecules belonging to the saponin family of secondary metabolites, which have been implicated in plant defense. Successful pathogens of these plants must presumably have some means of combating the chemical defenses of their hosts. In the oat root pathogen Gaeumannomyces graminis, the saponin-detoxifying enzyme avenacinase has been shown to be essential for pathogenicity. A number of other phytopathogenic fungi also produce saponin-degrading enzymes, although the significance of these for saponin resistance and pathogenicity has not yet been established. The tomato leaf spot pathogen Septoria lycopersici secretes the enzyme tomatinase, which degrades the tomato steroidal glycoalkaloid alpha-tomatine. Here we report the isolation and characterization of tomatinase-deficient mutants of S. lycopersici following targeted gene disruption. Tomatinase-minus mutants were more sensitive to alpha-tomatine than the wild-type strain. They could, however, still grow in the presence of 1 mM alpha-tomatine, suggesting that nondegradative mechanisms of tolerance are also important. There were no obvious effects of loss of tomatinase on macroscopic lesion formation on tomato leaves, but trypan blue staining of infected tissue during the early stages of infection revealed more dying mesophyll cells in leaves that had been inoculated with tomatinase-minus mutants. Expression of a defense-related basic beta-1,3 glucanase gene was also enhanced in these leaves. These differences in plant response may be associated with subtle differences in the growth of the wild-type and mutant strains during infection. Alternatively, tomatinase may be involved in suppression of plant defense mechanisms. (+info)
HISTAMINE PROTECTION PRODUCED BY PLANT TUMOUR EXTRACTS. THE ACTIVE PRINCIPLE OF TOMATO PLANTS INFECTED WITH GROWN-GALL.
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Guinea-pigs were protected against the lethal effects of a histamine aerosol by intraperitoneal injection of stable extracts of normal tomato plants and of tomato plants infected with crown-gall tumours. The protection was short-lasting. No difference was observed between the activities of extracts of normal and of infected plants. The active principle of the extracts was isolated, and identified as the steroid alkaloid glycoside, tomatine. (+info)
Chemosensory tuning to a host recognition cue in the facultative specialist larvae of the moth Manduca sexta.
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Larvae of Manduca sexta are facultative specialists on plants in the family Solanaceae. Larvae reared on solanaceous foliage develop a strong preference for their host; otherwise, they remain polyphagous. The host-specific recognition cue in potato foliage for Manduca larvae is the steroidal glycoside, indioside D. Two pairs of galeal taste sensilla, the lateral and medial sensilla styloconica, are both necessary and sufficient for the feeding preferences of host-restricted larvae. We conducted electrophysiological tip recordings from sensilla of solanaceous or wheat germ diet-reared larvae. For each animal, recordings of the responses to indioside D, glucose, tomatine and KCl were compared. All responses included both phasic and tonic portions. The sensilla styloconica of solanaceous-reared larvae were tuned to indioside D, defined as maintaining a high sensitivity to indioside D, while showing lower sensitivity to other plant compounds. Half of the sensillar neurons of solanaceous-reared larvae were 'tuned' to indioside D, whereas those of wheat germ diet-reared larvae were not. The different responses between the two types of animals were a result of changes of individual receptor cells' responses in the sensilla. Feeding on solanaceous foliage therefore appears to result in a modification of the physiological responses of individual taste receptor cells that causes them to be tuned to the host-recognition cue indioside D. We propose that this tuning is the basis for the host-restricted larvae's strong behavioral preferences for solanaceous foliage. (+info)
Tomatidine and lycotetraose, hydrolysis products of alpha-tomatine by Fusarium oxysporum tomatinase, suppress induced defense responses in tomato cells.
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Many fungal pathogens of tomato produce extracellular enzymes, collectively known as tomatinases, that detoxify the preformed antifungal steroidal glycoalkaloid alpha-tomatine. Tomatinase from the vascular wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici cleaves alpha-tomatine into the aglycon tomatidine (Td) and the tetrasaccharide lycotetraose (Lt). Although modes of action of alpha-tomatine have been extensively studied, those of Td and Lt are poorly understood. Here, we show that both Td and Lt inhibit the oxidative burst and hypersensitive cell death in suspension-cultured tomato cells. A tomatinase-negative F. oxysporum strain inherently non-pathogenic on tomato was able to infect tomato cuttings when either Td or Lt was present. These results suggest that tomatinase from F. oxysporum is required not only for detoxification of alpha-tomatine but also for suppression of induced defense responses of host. (+info)
Identification of a tomatinase in the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382.
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The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin alpha-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by alpha-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of alpha-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker. (+info)
Dual effects of plant steroidal alkaloids on Saccharomyces cerevisiae.
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Many plant species accumulate sterols and triterpenes as antimicrobial glycosides. These secondary metabolites (saponins) provide built-in chemical protection against pest and pathogen attack and can also influence induced defense responses. In addition, they have a variety of important pharmacological properties, including anticancer activity. The biological mechanisms underpinning the varied and diverse effects of saponins on microbes, plants, and animals are only poorly understood despite the ecological and pharmaceutical importance of this major class of plant secondary metabolites. Here we have exploited budding yeast (Saccharomyces cerevisiae) to investigate the effects of saponins on eukaryotic cells. The tomato steroidal glycoalkaloid alpha-tomatine has antifungal activity towards yeast, and this activity is associated with membrane permeabilization. Removal of a single sugar from the tetrasaccharide chain of alpha-tomatine results in a substantial reduction in antimicrobial activity. Surprisingly, the complete loss of sugars leads to enhanced antifungal activity. Experiments with alpha-tomatine and its aglycone tomatidine indicate that the mode of action of tomatidine towards yeast is distinct from that of alpha-tomatine and does not involve membrane permeabilization. Investigation of the effects of tomatidine on yeast by gene expression and sterol analysis indicate that tomatidine inhibits ergosterol biosynthesis. Tomatidine-treated cells accumulate zymosterol rather than ergosterol, which is consistent with inhibition of the sterol C(24) methyltransferase Erg6p. However, erg6 and erg3 mutants (but not erg2 mutants) have enhanced resistance to tomatidine, suggesting a complex interaction of erg mutations, sterol content, and tomatidine resistance. (+info)
alpha-Tomatine, the major saponin in tomato, induces programmed cell death mediated by reactive oxygen species in the fungal pathogen Fusarium oxysporum.
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The tomato saponin alpha-tomatine has been proposed to kill sensitive cells by binding to cell membranes followed by leakage of cell components. However, details of the modes of action of the compound on fungal cells are poorly understood. In the present study, mechanisms involved in alpha-tomatine-induced cell death of fungi were examined using a filamentous pathogenic fungus Fusarium oxysporum. alpha-Tomatine-induced cell death of F. oxysporum (TICDF) occurred only under aerobic conditions and was blocked by the mitochondrial F(0)F(1)-ATPase inhibitor oligomycin, the caspase inhibitor D-VAD-fmk, and protein synthesis inhibitor cycloheximide. Fungal cells exposed to alpha-tomatine showed TUNEL-positive nuclei, depolarization of transmembrane potential of mitochondria, and reactive oxygen species (ROS) accumulation. These results suggest that TICDF occurs through a programmed cell death process in which mitochondria play a pivotal role. Pharmacological studies using inhibitors suggest that alpha-tomatine activates phosphotyrosine kinase and monomeric G-protein signaling pathways leading to Ca(2+) elevation and ROS burst in F. oxysporum cells. (+info)