1.2.13
CHARACTERIZATION OF THE TOMATO ASC LOCUS, THAT DETERMINES RESISTANCE OR SUSCEPTIBILITY TO THE FUNGAL PATHOGEN ALTERNARIA ALTERNATA F. SP. LYCOPERSICI

BF BRANDWAGT, PL LAURENT, LA MESBAH, TJA KNEPPERS, HJJ NIJKAMP and J HILLE

Department of Genetics, Institute for Molecular Biological Sciences (IMBW), BioCentrum Amsterdam, Free University, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands

Background and objectives
Tomato plants homozygous for the Asc locus are resistant (Asc11/Asc11 and Asc12/Asc12) or sensitive (asc/asc) to the fungal pathogen Alternaria alternata f. sp. lycopersici and its host-specific AAL-toxins. Heterozygous plants are resistant to the fungus, but resistant (Asc12/asc) or sensitive (Asc11/asc) to high toxin concentrations [1]. In susceptible plant tissues, AAL toxins and the structurally related fumonisins of Fusarium moniliforme lead to the accumulation of free sphingoid bases by inhibition of ceramide synthase, a key enzyme in de novo sphingolipid biosynthesis and cause apoptosis-like symptoms. Recently, free sphingoid bases and their metabolites have been shown to function as second messengers that regulate cell death [2]. Molecular isolation and characterization of the locus will probably provide evidence on the mechanism(s) by which plants are sensitive or resistant to these toxins.

Results and conclusions
A 310-kb yeast artificial chromosome (Y442A) that encompasses the Asc locus (Asc11) has been isolated. 31 plants with a recombination between Asc and the closest RFLP markers (0.7 and 1.4 cM) were identified, supplying on average one recombinant per 15 kb in the Asc region. To identify transcribed genes on Y442A, it was hybridized to a tomato cDNA library. Three cDNA clones were identified, which are members of a small multigene family with no similarity to any known genes. Based on the position near the left arm of Y442A, it was concluded that these genes do not represent Asc. From two new markers flanking the Asc locus, Y442A has almost (a maximum of 5 kb has to be covered) been sub-cloned into a 85-kb contig of lambda clones that contains four recombination positions. The chromosomal region between the last recombination positions that should contain Asc is now used for complementation experiments. Transgenic tomato tissues were generated by Agrobacterium tumefaciens and A. rhizogenes T-DNA transformation and subsequently tested by AAL toxin assays. Since we do not know unequivocally which of the Asc alleles is active in planta, the asc allele will be isolated from a susceptible genomic library and also used for complementation assays. In addition, cDNA libraries of susceptible and resistant tomato lines are screened with chromosomal region to identify and sequence transcriptionally active genes at the Asc locus. These data will indicate which gene determines resistance or susceptibility to AAL-toxins and should give clues about the biochemical function of this gene.

References
1. Van der Biezen EA, Glagotskaya T, Overduin B, Nijkamp HJJ, Hille J, 1995. Molecular and General Genetics 247, 453-461.
2. Merrill AH Jr, Schmelz EM, Dillehay DL et al.., 1997. Toxicology and Applied Pharmacology 142, 208-225.