Mapping, isolation and characterization of genes responsible for late blight resistance in potato.

Late blight (LB), caused by the oomycete Phytophthora infestans, is one of the most devastating diseases on potato. Resistance (R) genes from the wild species Solanum demissum have been used by breeders to generate late blight resistant cultivars, but resistance was soon overcome by the pathogen. A more recent screening of a large number of wild species has led to the identification of novel sources of resistance, many of which are currently being characterized. R-gene based resistance to any plant pathogen has been conceptualized according to a model known as gene-for-gene interaction. When matching avirulence (Avr) and resistance (R) proteins are produced by the pathogen and the plant respectively, a resistance response is triggered resulting in a hyper-sensitive response (HR) causing necrosis and cell death at the infection site. If one of these components is missing the plant-pathogen interaction is compatible and will result in completion of the life cycle of the pathogen. This thesis describes the cloning and the characterization of the resistant alleles Rpi-vnt1.1, Rpivnt1.2 and Rpi-vnt1.3 from Solanum venturii and their counterpart Avr-vnt1 from Phytophthora infestans. The Rpi-/Avr- genes pair Rpi-vnt1/Avr-vnt1 along with R3a/Avr3a and Rpi-blb3/Avr2 have been used to study the genetic and molecular mechanisms behind tuber blight resistance. The cloning of Rpi-vnt1 alleles (Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3) was achieved by the combination of long range PCR (Chapter 2) and a classical map based cloning strategy (Chapter 3). The long range PCR made use of Tm2 homologous PCR primers, upon identification of Tm2 sequence homology in associated markers generated with an NBStargeted fingerprinting technique. Rpi-vnt1 alleles belong to the CC-NBS-LRR class of plant R genes and encode predicted peptides of 891 and 905 amino acids, respectively, which share 75% amino acid (a.a.) identity with the ToMV resistance protein Tm-22 from tomato. Compared to Rpi-vnt1.1, the allele Rpi-vnt1.3 harbors a 14 amino acid insertion in the Nterminal region of the protein and two different amino acids in the LRR domain. Despite these differences, Rpi-vnt1.1 and Rpi-vnt1.3 genes have the same resistance spectrum. An allele mining study of Rpi-vnt1 alleles across Solanum section Petota showed that the three functional alleles were confined within S. venturii as only two accessions from the closely related species S. weberbaueri and S. mochiquense carried Rpi-vnt1.1 (Chapter 4). Subsequent alignment of Rpi-vnt1-like homologs with Rpi-vnt1 alleles revealed the presence of illegitimate recombination (IR) signatures suggesting that two successive deletion events might have occurred in the CC domain. Meanwhile, the construction of a Neighbor Joining tree, based on AFLP data from all the accessions carrying Rpi-vnt1 alleles or Rpi-vnt1-like homologs showed that Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 alleles belong to a monophyletic clade. Signatures of illegitimate recombination and the monophyletic grouping of Rpi-vnt1 alleles suggested how Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 could have evolved. Extensive phenotyping with various Phytophthora isolates identified another Rpi gene in S. venturii named Rpi-vnt2, complementing the Rpi-vnt1 allelic resistance spectrum. The genetic position of this second independent locus is not yet identified. The identification of the matching avirulence factor from the pathogen, Avr-vnt1, was achieved by using an efficient and high throughput effector screen of resistant wild potato species (Chapter 5). Avr-vnt1 encodes a typical RXLR-EER effector which expression is induced 2 days post inoculation. Avr-vnt1 is located on a single locus in the reference strain T30-4. Among nine isolates, four alleles were identified. The virulent strain EC1 carries a functional coding sequence of Avr-vnt1 but fails to express the gene. In Chapter 6, the genetic and molecular mechanisms of tuber late blight have been investigated. Using transgenic cv. Desiree plants transformed with Rpi-vnt1.1, R3a or Rpiblb3 tuber blight resistance could be studied in an identical genetic background. First, we demonstrated that transient co-expression of the matching Avr- genes in these transgenic tuber slices trigger a hypersensitive responses (HR), showing that the presence and interaction of both proteins is sufficient to establish tuber blight resistance. Second, phenotypic and molecular analysis of a panel of transformants for Rpi-vnt1.1, R3a and Rpi-blb3, and transcriptional analysis of the corresponding effectors (Avr-vnt1, Avr3a and Avr2 respectively) during leaf and tuber infection showed that the expression level of a given Rgene should equal or exceed the expression level of the matching effector in order to trigger an efficient resistance response in the tuber. Therefore, foliar and tuber late blight resistance are controlled by similar genetic mechanisms. The perceived lack of phenotypic correlation between foliage and tuber blight resistance is thus solely due to the tissue specific expression level of the Rpi-gene. In the general discussion (Chapter 7), results from the experimental chapters are discussed in a broader perspective.