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.