Characterization of major resistance genes to Tomato Yellow Leaf Curl Virus (2013)

Tomato yellow leaf curl disease, a devastating disease of tomato, is caused by a complex of begomoviruses generally referred to as Tomato yellow leaf curl virus (TYLCV). Almost all breeding for TYLCV resistance has been based on the introgression of the Ty-1 and Ty-3 resistance loci derived from Solanum chilense LA1969 and LA1932/LA2779 respectively. The aim of this thesis was to fine map, clone and characterize these two TYLCV resistance genes. The Ty-1 gene has been used in tomato breeding already for almost 20 years. Its exact genetic location was however unknown which made precise marker assisted breeding difficult. Here we have analysed the recombination behavior of the chromosomal region where Ty-1 is introgressed by applying newly developed molecular markers in two F2 populations obtained from two commercial Ty-1 carrying hybrids. A big S. chilense introgression was detected in both populations that coveralmost the whole short arm and a part of the long arm of chromosome 6. In this introgression recombination suppression was detected and Fluorescence in situ Hybridization (FISH) analysis revealed two chromosomal rearrangements between S. lycopersicum and S. chilense LA1969. These rearrangements are most likely the cause of the observed recombination suppression. Using disease tests on progeny of informative recombinants Ty-1 was mapped to a region of approximately 600 kb which partly overlapped with the mapped region for Ty-3, which led to the indication that Ty-1 and Ty-3 could be allelic. Altogether these results nicely demonstrate the usefulness of FISH as a powerful tool to aid in the accurate mapping of genes that are introgressed from wild species into cultivated tomato (Chapter 2). To further fine map and ultimately clone Ty-1 and Ty-3 more plants were screened for recombination events and consequently recombinant inbred lines were generated. By developing new markers in combination with disease tests both genes were fine mapped to a very small, almost similar genomic region (approximately 70 kb). Using a Tobacco Rattle Virus-Virus Induced Gene Silencing approach, the resistance genes were finally identified. It was shown that Ty-1 and Ty-3 are allelic and that they code for a RNA-dependent RNA polymerase (RDR) belonging to the RDRɣ type which has an atypical DFDGD motif in the catalytic domain. In contrast to the RDRαtype, characterized by a catalytic DLDGD motif, no clear function has yet been described for the RDRɣ type. With the identification of Ty-1/Ty-3, a completely new class of resistance genes was unveiled (Chapter 3). The Ty-1/Ty-3 allele is characterized by a 4 amino acid insertion at the 5-prime part of the protein and by a catalytic DFDGD motif. The allelic variation of this gene was examined using cDNA from five S. chilense derived lines and using draft assemblies of whole genome sequences from more than 50 tomato cultivars, landraces and related wild species. Tobacco Rattle Virus induced gene silencing was used to silence Ty-1/Ty-3 and altogether showed that resistance was compromised in three out of five S. chilense derived lines tested. One line with resistance derived from S. chilense LA1971 remained resistance after silencing of Ty-1/Ty-3. For another line (8783, derived from LA1932) only 4 out of 13 plants showed symptoms after silencing,but silencing in this line was inefficient because only one out of three PDS controls showed photobleaching. Comparison of the two typical features of the Ty-1/Ty-3 gene showed no sequence variation amongst S. chilense derived lines. The catalytic domain was found to be conserved among all tomato lines and species analysed, while the characteristic 4 amino acid insertion was also observed in three species closely related to S. chilense, e.g.Solanum corneliomulleri, Solanum peruvianum and Solanum huaylasense. This indicated that most S. chilense accessions most likely carry a functional TYLCV resistance locus on chromosome 6, allelic to Ty-1/Ty-3, and Solanum species related to S. chilense could possibly be useful for future TYLCV resistance breeding (Chapter 4). The Ty-1 gene encoded an RDR and for this reason most likely conferred resistance involving amplification of the siRNA signal. In the last experimental chapter (Chapter 5) this hypothesis was tested. It was shown that upon TYLCV challenging of resistant Ty-1 and Ty-3 lines low virus titers were detected concomitant with the production of relatively high levels of siRNAs. In contrast to the situation in susceptible tomato Moneymaker where high virus titers were observed, but the amount of siRNAs produced lower compared to those in Ty-1 and Ty-3. Analysis of the spatial genomic siRNA distribution showed a consistent and subtle enrichment for siRNAs derived from the CP (V1) and C3 gene in Ty-1 and Ty-3 lines compared with Moneymaker. In tomato plants containing the Ty-2 resistance gene, included as a control and not an RDR, the virus was hardly detectable but the siRNA profile similar to the one observed in TYLCV-challenged susceptible tomato Moneymaker. Furthermore, genome methylation analysis revealed a relative hypermethylation of the TYLCV CP (V1) promoter region in genomic DNA collected from Ty-1 in comparison to susceptible tomato Moneymaker. Altogether this thesis describes the mapping, cloning and characterization of the TYLCV resistance genes Ty-1 and Ty-3. Future breeding efforts for TYLCV resistance can now exploit in-gene markers and the insights obtained can possibly direct future research and breeding efforts on plant virus resistance (Chapter 6).