Abstract
Gene tagging utilizing the heterologous integration of plasmids is now widely used to clone genes where little biochemical information is available. The development of the so-called REMI (restriction enzyme mediated integration) method has substantially improved this technique and some genes important for fungal pathogenicity have already been tagged and cloned. We demonstrate here the isolation of host-specific toxin deficient mutants from Alternaria alternata by the REMI method. The apple and tomato pathotypes of A. alternata that are producers of AM- and AAL-toxin, respectively, were first examined for the effect of addition of restriction enzyme to the transformation. A significant increase in transformation frequency by addition of enzyme was observed in these pathotypes, and a simple integration profile was confirmed in the tomato pathotypes. AAL-toxin deficient mutants were isolated from a library of transformants obtained with the addition of enzymes. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity. Detailed molecular analysis of these mutants, rescue of flanking genomic sequence, and gene disruption experiments are now in progress.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Akamatsu, H., Itoh, Y., Kodama, M., Otani, H. and Kohmoto, K. (1997). AAL-toxin deficient mutants of Alternaria alternata tomato pathotype by restriction enzyme-mediated integration. Phytopathology (in press).
Azcona-Olivera, J.I., Abouzied, M.M., Plattner, R.D., Norred, W. P., Pestka, J.J. (1992) Generation of antibodies reactive with fumonisins B1, B2 and B3 by using cholera toxin as carrier-adjuvant. Appl. Environ. Microbiol. 58:169–173.
Bölker, M., Böhnert, H.U., Braun, K.H., Görl, J. and Kahmann, R. (1995). Tagging pathogenicity genes in Ustilago maydis by restriction enzyme-mediated integration (REMI). Mol. Gen. Genet. 248:547–552.
Ge, Y., Rose, M.S., Turgeon, B.G., Yoder, O.C. (1996). A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin. Plant Cell 8:2139–2150.
Hayashi, N., Tanabe, K., Tsuge, T., Nishimura, S., Kohmoto, K. and Otani, H. (1990). Determination of host-selective toxin production during spore germination of Alternaria alternata by high-performance liquid chromatography. Phytopathology 80:1088–1091.
Jones, B.N. and Gilligan, J.P. (1983). o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J. Chromatogr. 266:471–482.
Kistler, H.C. and Miao, V.P.W. (1992). New modes of genetic change in filamentous fungi. Annu. Rev. Phytopathol. 30:131–152.
Kodama M., Otani H., Kohmoto K. (1995). Rapid and sensitive procedure for the quantitative detection of AL-toxin by fluorescence derivatization and separation by high performance liquid chromatography. Ann. Phytopath. Soc. Jpn. 61:477–480.
Kohmoto, K. (1992). Determination of Host-Selective Toxins. In: Modern Methods of Plant Analysis. Volume 13, Plant Toxin Analysis,. (H. F. Linskens and J. F. Jackson, eds.). Springer-Verlag, Berlin Heidelberg New York, pp.51–73.
Kohmoto, K. and Otani, H. (1991). Host recognition by toxigenic plant pathogen. Experientia 47:755–764.
Kuspa, A. and Loomis, W.F. (1992). Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc. Natl. Acad. Sci. USA 89:8803–8807.
Lu, S., Lyngholm, L., Yang, G., Bronson, C.R. and Yoder, O.C. (1994). Tagged mutations at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration. Proc. Natl. Acad. Sci. USA 91:12649–12653.
Mills, D. and McCluskey, K. (1990). Electrophoretic karyotypes of fungi: The new cytology. Mol. Plant-Microbe Interact. 6:351–357.
Otani, H., Kohmoto, K. and Kodama, M. (1995). Alternaria toxins and their effects on host plants. Can. J. Bot. 73:S453–S458.
Otani, H., Kohmoto, K., Kodama, M. and Nishimura, S. (1991). Role of host-specific toxins in the pathogenesis of Alternaria alternata. Pages 139-149 in: Molecular Strategies of Pathogens and Host Plants. Patil, S.S., Ouchi, S., Mills, D. and Vance, C., eds. Springer-Verlag, New York.
Panaccione, D.G., Scott-Craig, J.S., Pocard, J.A. and Walton, J.D. (1992). A cyclic peptide synthetase gene required for pathogenicity of the fungus Cochliobolus carbonum on maize. Proc. Natl. Acad. Sci. USA 89:6590–6594.
Pringle, R.B. and Scheffer, R.P. (1964). Host-specific plant toxins. Annu. Rev. Phytopath. 2:133–156.
Punt, P.J., Oliver, R.P., Dingemanse, M.A., Pouwels, P.H. and Van Den Hondel, C.A.M.J.J. (1987). Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117–124.
Schiestl, R.H. and Petes, T.D. (1991). Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 88:7585–7589.
Shi, Z., Christian, D. and Leung, H. (1995). Enhanced transformation in Magnaporthe grisea by restriction enzyme mediated integration of plasmid DNA. Phytopathology 85:329–333.
Siler, D.J. and Gilchrist, D.G. (1982). Determination of host-selective phytotoxin from Alternaria alternata f. sp. lycopersici as their maleyl derivatives by high-performance liquid chromatography. J. Chromatogr. 238:167–173.
Taga, M. and Murata, M. (1994). Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization. Chromosoma 103:408–413.
Tsuge, T., Kobayashi, H. and Nishimura, S. (1989). Organization of ribosomal RNA genes in Alternaria alternata Japanese pear pathotype, a host-selective AK-toxin-producing fungus. Curr. Genet. 16:267–272.
Tsuge, T., Nishimura, S. and Kobayashi, H. (1990). Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences. Gene 90:207–214.
Turgeon, B.G., Kodama, M., Yang, G., Rose, M.S., Lu, S.W. and Yoder, O.C. (1995). Function and chromosomal location of the Cochliobolus heterostrophus TOX1 locus. Can. J. Bot. 73:S1071–S1076.
Walton, J.D. (1987). Two enzymes involved in biosynthesis of the host-selective phytotoxin HC-toxin. Proc. Natl. Acad. Sci. USA 84:8444–8447.
Watanabe, Y. and Imai, K. (1984). Sensitive detection of amino acids in human serum and dried blood disc of 3mm diameter for diagnosis of inborn errors of metabolism. J. Chromatogr. 309:279–286.
Yang, G., Turgeon, B.G. and Yoder, O.C. (1994). Toxin-deficient mutants from a toxin-sensitive transformant of Cochliobolus heterostrophus. Genetics 137:751–757.
Yoder, O.C., Yang, G., Rose, M.S., Lu, S.W. and Turgeon, B.G. (1994). Complex genetic control of polyketide toxin production by Cochliobolus heterostrophus. In Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. 3. M.J. Daniels et al. (eds.), Kluwer Academic Publishers, Dordrecht, pp. 223–230.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Kodama, M. et al. (1998). Host-Specific Toxin Deficient Mutants of the Tomato Pathotype of Alternaria Alternata Obtained by Restriction Enzyme-Mediated Integration. In: Kohmoto, K., Yoder, O.C. (eds) Molecular Genetics of Host-Specific Toxins in Plant Disease. Developments in Plant Pathology, vol 13. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-5218-1_4
Download citation
DOI: https://doi.org/10.1007/978-94-011-5218-1_4
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-010-6197-1
Online ISBN: 978-94-011-5218-1
eBook Packages: Springer Book Archive