Mapping and functional characterization of the tomato I-7 gene for fusarium wilt resistance

Abstract

The I-7 gene, which confers resistance to Fol race 3, was introgressed from Solanum pennellii and recently recognised as a new resistance gene distinct from the I-3 gene. Since the chromosomal location of I-7 was unknown, no markers were available for the marker-assisted breeding of I-7 genotypes. Therefore, one aim of this project was to identify the chromosomal location of the I-7 gene and develop reliable PCR-based markers suitable for the marker-assisted breeding of I-7. Further aims were to identify and characterise the I-7 gene, and examine the resistance phenotype conferred by I-7. GFP-tagged derivatives of Fol race 3 were generated (Chapter 2) to observe the infection process and determine the timing and location of resistance conferred by I-7. Previously, using a gene replacement strategy, the promoter from the Fol Avr3 effector gene was used to drive GFP expression and show that the Avr3 promoter was only expressed in fungal hyphae growing inside the plant root (van der Does et al., 2008a). Unfortunately, Avr3 replacement reduces pathogenicity, so new transformants were generated with ectopic insertions carrying a Avr3 promoter:GFP reporter construct. Microscopic analysis of tomato roots challenged with these transformants showed that fluorescent hyphae could be imaged successfully, but unfortunately these transformants also showed reduced virulence. After an intensive but unproductive marker-based search for the S. pennellii introgression carrying I-7 (Chapter 3), an RNA-seq experiment was conducted to identify Single Nucleotide Polymorphisms (SNPs) in root transcripts derived from genes in the introgressed region (Chapter 4). Sequencing of root transcripts from the tomato cultivars Tristar (carrying I-7) and M82 (lacking I-7) enabled detection of a large number of SNPs. A plot of SNP frequency against gene position revealed a higher frequency of SNPs in 18 transcripts encoded by a cluster of genes on chromosome 8. CAPS markers based on these SNPs showed strong linkage with I-7. An orthologue of Solyc08g077740 was identified as a candidate for I-7. Transcriptome sequencing was also used to identify genes responsive to Fol race 3 infection in Tristar (Chapter 4). Analysis of the RNA-seq data revealed 38 genes that were significantly upregulated in Tristar but not the susceptible cultivar M82, including a number of genes reported to have a role in plant defence. Alleles of the I-7 candidate gene were cloned and sequenced from I-7 resistant (Tristar) and susceptible (M82) cultivars of tomato (Chapter 5). Transgenic plants expressing these alleles were generated from susceptible tomato lines and tested for resistance to Fol. The Tristar transgene but not the M82 transgene conferred resistance to Fol race 3, showing that the S. pennellii allele of Solyc08g077740 is I-7. I-7 was also found to confer resistance to Fol races 1 and 2. Based on predicted amino acid sequence, I-7 possesses a domain structure typical of extracellular leucine-rich repeat-receptor-like proteins. Further work to characterise I-7 (Chapter 6) found that I-7 resistance is dependent on the downstream signalling gene EDS1, and that I-7 confers resistance to Fol race 3 through recognition of an effector protein other than Avr3.