Exploitation of pathogen quantitative resistance diversity to improve disease tolerance in crops – RIPOSTE

Based on RKS1 that was recently identified as a quantitative resistance gene in Arabidopsis thaliana (i) conferring broadspectrum resistance to several races and pathovars of the bacterial vascular pathogen Xanthomonas campestris and (ii) under balancing selection with the maintenance of a long-lived polymorphism in natural populations, the main scientific aims and technologies of RIPOSTE project are:
(i). To integrate our recent knowledge of RKS1 dependent QDR effective against Xc with the existing defense pathways known in plants, and to characterize the regulatory pathway(s) controlled by RKS1. To test whether RKS1-dependent resistance pathway(s) operate(s) independently or through common regulatory nodes with other forms of resistance, this
part of the project will combine complementary methodologies: transcriptomic analysis, mapping-by-sequencing, deep candidate resequencing(dCARE), search for protein interactors by yeast two hybrid (Y2H) Arabidopsis cDNA library, FRET-FLIM technique.
(ii) To elucidate the forces promoting the maintenance of the RKS1 polymorphism by (1) estimating the cost/benefit tradeoffs along a range of infection intensities using an innovative approach of laboratory natural selection based on interconnected experimental populations, and (2) developing realistic models to understand and predict the adaptive
dynamics of quantitative resistance alleles in natural populations by analytical models and individual-based simulations. This part of the project will evaluate the suitability of the RKS1 gene (and other genes identified in (i)) for disease management in crops. (iii) To engineer three important agronomic crops (Brassica, tomato and pepper) for durable and broad-spectrum resistance to Xc, by using complementary methods to identify RKS1 homologs and homologs of genes involved in RKS1- dependent pathway: phylogeny and synteny, screening of tilled lines, phenotype-candidate gene association mapping.

Task1A: For molecular validation of the putative role of the regulatory regions/polymorphisms responsible for resistance/susceptibility, all the constructs by swapping promoters, ORFs and 5’ UTR regions from resistant and susceptible alleles of RKS1, have been generated, and are being tested by complementation of the rks1-1 KO mutant. Task1B: The phenotype associated with the EMS mutant pad-1 ¬results from a single nuclear gene, while the phenotype associated the EMS mutant eds-8 seems to result from two co-dominant genes. The phenotyping of 1200 F2 plants segregating for the EMS causal mutations is underway. Task 1C: The statistical analyses on the transcriptomic data previously obtained, has been achieved. A list of 28 candidate genes was first generated, 121 mutant lines ordered from which 35 have been (i) tested for their response to Xcc. At this point, 26 mutants (corresponding to 17 candidate genes) showed an altered phenotype in response to Xcc.Task 1D. For the identification of RKS1 protein interactors, a yeast 2 hybrid system screening has been recently performed using a cDNA library from A. thaliana infected with Xcc 147. From a first round, 72 clones have been found and sequenced. Five of them are currently under validation.
Task 2: Eight lines misregulated for RKS1 have been grown in absence and presence of the competitor grass Poa annua along a gradient of infection with the strain Xcc568. The phenotyping of more than 20 adaptive phenotypic traits is underway.
Task 3. The building of the model of coevolution between quantitative resistance and virulence in an isolated population has recently started.
Task 4. RKS1 candidates in cauliflower and tomato have been identified by sequence comparison (BLAST) and phylogenetic analyses. As planned in the proposal, tomato results will be used to identify candidate genes in pepper. A tomato candidate gene has been sent to partner 2 for tilling analyses (task4B).

Each of the tasks will be pursued as indicated in the project objectives.

1. Roux, F. and J. Bergelson. 2016. The genetics underlying natural variation in the biotic interactions of Arabidopsis thaliana: the challenges of linking evolutionary genetics and community ecology. Current Topics in Developmental Biology (in press).