Colwyn M. Thomas

Novel Disease Resistance Specificities Result from Sequence Exchange between Tandemly Repeated Genes at the Cf-4/9 Locus of Tomato

Tomato Cf genes confer resistance to C. fulvum, reside in complex loci carrying multiple genes, and encode predicted membrane-bound proteins with extracytoplasmic leucine-rich repeats. At least two Cf-9 homologs confer novel C. fulvum resistance specificities. Comparison of 11 genes revealed 7 hypervariable amino acid positions in a motif of the leucine-rich repeats predicted to form a beta-strand/beta-turn in which the hypervariable residues are solvent exposed and potentially contribute to recognition specificity. Higher nonsynonymous than synonymous substitution rates in this region imply selection for sequence diversification. We propose that the level of polymorphism between intergenic regions determines the frequency of sequence exchange between the tandemly repeated genes. This permits sufficient exchange to generate sequence diversity but prevents sequence homogenization.

The Tomato Cf-2 Disease Resistance Locus Comprises Two Functional Genes Encoding Leucine-Rich Repeat Proteins

In plants, resistance to pathogens is frequently determined by dominant resistance genes, whose products are proposed to recognize pathogen-encoded avirulence gene (Avr) products. The tomato resistance locus Cf-2 was isolated by positional cloning and found to contain two almost identical genes, each conferring resistance to isolates of tomato leaf mould (C. fulvum) expressing the corresponding Avr2 gene. The two Cf-2 genes encode protein products that differ from each other by only three amino acids and contain 38 leucine-rich repeat (LRR) motifs. Of the LRRs, 20 show extremely conserved alternating repeats. The C-terminus of Cf-2 carries regions of pronounced homology to the protein encoded by the unlinked Cf-9 gene. We suggest that this conserved region interacts with other proteins involved in activating plant defense mechanisms.

Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction

We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis.

Dispersion of the Cf-4 disease resistance gene in Lycopersicon germplasm

In the past, numerous Cf genes have been reported in tomato (Lycopersicon esculentum Mill.) that confer resistance against leaf mould (Cladosporium fulvum Cke.). We are interested in genetic variation at Cf loci. Therefore, previously uncharacterized Cf genes were further analysed. Recognition of the AVR4 elicitor, DNA gel blot analysis, PCR analysis and sequencing of part of the Cf-4 locus showed that a large proportion of the accessions tested harboured the Cf-4 resistance gene. We concluded that despite differences in nomenclature, all these accessions harbour the same Cf-4 locus, probably introgressed from the same donor. The origin of the Cf-4 locus and the reasons for discrepancies with earlier reports are discussed.

Sugarbeet minicircular mitochondrial DNAs: high-resolution transcript mapping, transcript abundance and copy number determination

SummaryThree minicircular mitochondrial DNAs have been studied to address several aspects of transcription in sugarbeet mitochondria. High-resolution transcript mapping experiments have shown that sequences at the 5′ termini of minicircle transcripts are highly homologous and resemble sequences at the 5′ termini of sugarbeet mainband mitochondrial genes (atpA, atp6). In addition, they show homology to transcript termini of mitochondrial genes from other dicotyledonous plants, suggesting they may function as promoter sequences. Conserved sequences, which most probably act as RNA processing signals, were also identified at the 3′ termini of minicircle transcripts. An oligonucleotide probe to a 14 base conserved sequence was used to determine the relative copy numbers of the three minicircle components in male-fertile mitochondria. Copy numbers were roughly equivalent, suggesting minicircles are replicated and/or transmitted with nearly equal efficiency, at least in sugarbeet taproots. Mc.a and Mc.c transcript levels are equivalent, consistent with their template copy number, however; Mc.d transcript levels were significantly lower than expected, implicating additional factors such as promoter strength and/or transcript stability in determining transcript levels in sugarbeet mitochondria, as recently demonstrated in maize.

Strategies for the Cloning of Genes in Tomato for Resistance to Fulvia Fulva

We are developing two alternative strategies to clone one or more of the Cf2, Cf4, Cf5, Cf9 or Cf11 genes of tomato (Lycopersicon esculentum) that confer resistance to Fulvia fulva (syn. Cladosporium fulvum). The first approach is tagging with the maize transposon Ac. Since Ac preferentially transposes to closely linked sites in both maize and tobacco, close linkage should improve tagging efficiency. We are therefore RFLP mapping T-DNAs carrying Ac which have been transformed into tomato using as probes inverse polymerase chain reaction (IPCR) amplified tomato sequences adjacent to the T-DNA ends. Similarly, we are mapping the Cf genes on the RFLP map, so that we can identify for further analysis those transformants with T-DNAs closely linked to Cf genes. The second strategy involves the development of a technique of subtractive cDNA cloning, in which cDNA from a Cm plant (plant with no detectable resistance genes) is used to subtract cDNA which is common to a near-isogenic Cf2 plant, leaving Cf2 specific cDNA which can be cloned, and used as a probe for differential screening.