α-Tomatine, synthesized by Lycopersicon species, is toxic to a broad range of fungi and has long been considered a potential barrier to microbial infection. I hypothesize that a successful tomato pathogen must overcome this toxic compound in order to parasitize the plant. In this study, I have examined a number of saprophytes, non-pathogens of tomato, and tomato pathogens for their tolerance to α-tomatine and their ability to enzymatically degrade this compound. Although, overall, fungal saprophytes and non-pathogens of tomato were sensitive to α-tomatine while tomato pathogens were very tolerant, several tomato pathogens were quite sensitive. All tomato pathogens, except the Pythiaceous fungi, were able to degrade α-tomatine to the less toxic compounds β₂-tomatine or tomatidine. Many saprophytes and non-pathogens were sensitive to these degradation products while tomato pathogens were tolerant suggesting that tomato pathogens may have also acquired a non-degradative means to tolerate α-tomatine and its derivatives. As an initial step to evaluate the importance of detoxification of α-tomatine, I have purified to homogeneity a β-1,2-D glucosidase from the tomato leaf pathogen Septoria lycopersici that hydrolyzes the β-1,2-D glucosyl bond on the tetrasaccharide moiety of α-tomatine to produce β₂-tomatine and glucose. Little to no enzyme activity was detected when other β-1,2-D glycosides were used as substrates suggesting that this organism has evolved a special means to overcome α-tomatine. The gene encoding this protein, called β₂-tomatinase, was isolated from S. lycopersici and a β₂-tomatinase homologue from the green fruit pathogen Colletotrichum coccodes. The β₂-tomatinase genes in C. coccodes and S. lycopersici were mutated via transformation-mediated gene disruption and the β₂-tomatinase mutants were analyzed for their tolerance to α-tomatine and their ability to parasitize tomato. The S. lycopersici β₂-tomatinase mutant became 12 times more sensitive to α-tomatine. The C. coccodes β₂-tomatinase mutant still retained its tolerance to α-tomatine, its ability to degrade α-tomatine to the aglycone tomatidine, and its ability to parasitize the tomato fruit but was deficient in β₂-tomatinase activity. Expression of the S. lycopersici β₂-tomatinase gene in N. haematocacca, a pea pathogen, increased its tolerance to α-tomatine and these transformants acquired the ability to form lesions on green tomato fruit, an organ containing α-tomatine.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/282156 |
| Date | January 1996 |
| Creators | Sandrock, Robert Wayne, 1966- |
| Contributors | Etten, Hans Van |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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