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Plants are constantly exposed to microbial populations in roots and leaf surfaces, some of which can cause disease and result in infection, which causes crop losses. The study of the natural co-evolution of plants with pathogens provides new leads for pest regulation and ideally, anticipate evolving pathogen strategies that avoid recognition. Studies of many host-pathogen associations suggest that a pathogen strain becomes adapted to a particular plant host by evolving virulence factors, called effectors. Plant nucleotide binding-leucine rich repeat (NB-LRR) receptors recognize such effectors and act as molecular switches that trigger a strong resistance response. Remarkably, studies demonstrate that moderate changes in temperature influence some NB-LRR mediated defense responses. Studies of the variability of NB-LRR genes between and within populations can be used to understand the effect of local environments on NB-LRR diversification and/or selection. Important new information linking plant immunity with environmental cues is given by the analysis of naturally occurring temperature-dependent genetic interactions that activate defense in the absence of pathogen challenge. Such cases, referred to as ‘incompatible hybrids’ often involve NB-LRR genes and are suitable models to decipher the evolution of NB-LRR genes in nature and intersections between NB-LRR signaling and temperature.

This project studies a genetic interaction involving NB-LRR genes of the RPP1-like cluster in the Arabidopsis thaliana accession Landsberg (Ler), which triggers a temperature-conditioned activation of defense in combination with Kas-2 and Kond central Asian alleles of the receptor-like kinase SRF3 in Ler/Kas-2 and Ler/Kond Arabidopsis hybrids (Alcázar et al., Proc Natl Acad Sci U S A. 2009 106:334-339 and Alcázar et al. Nat. Genet. 2010 42:1135-1139). The incompatibility is extended to population scales, and therefore it has a true impact on evolutionary scales.
Using this case as model, the project follows three main objectives: (1) Characterization of RPP1-like gene divergence at global and local scales. We aim at establishing which is the natural distribution of RPP1-like alleles which are causal for incompatibility with SRF3, and perform population genetics analyses in local populations. (2) Identify RPP1-like genes and polymorphisms driving incompatibility with SRF3 and (3) study the temperature-dependency of the RPP1-SRF3 interaction at molecular level.

Regarding to the task 1 of the project, we previously collected and genetically characterized an Arabidopsis local population of Landsberg relatives or descents (called Gorzów population, Gw) collected in the area where Landsberg was originally found in 1939. Using genome-wide SNP genotyping, structure and PCA analyses, we identified that Gw individuals share between 58 – 72 % SNP with Ler. Sequencing of the locus in Gw individuals revealed that that 30 % of the population carries a conserved RPP1-like Ler haplotype. We demonstrate by crossing that Gw individuals carrying RPP1-like Ler haplotype are incompatible with Kas-2 and Kond. Therefore, we extend the Ler/Kas-2 and Ler/Kond incompatibilities to population scales which allow the determination of evolutionary forces shaping the composition of NB-LRR genes in nature. Genome sequencing of Gorzów individuals containing a conserved RPP1-like Ler haplotype revealed the absence of polymorphism flanking the RPP1-like locus, which is compatible with signatures of selection and/or reduced recombination. In addition, sequencing data revealed the origins of RPP1-like Ler locus genes by complex rearrangements, and the different evolutionary origins associated with the capacity or not of NB-LRR proteins to trigger cell death. Through complementation and mutagenesis approaches we have identified that co-action of certain NB-LRR genes is required for incompatibility with Kas-2, thus suggesting the need of NB-LRR protein receptor associations.

Regarding to the task 2 of the project, we previously generated transgenic lines in neutral backgrounds which carry each of the eight RPP1-like Ler genes (R1 – R8) and observed that RPP1-like Ler R3 gene overexpression is sufficient to condition the balance between growth and defense, and that high R3 expression correlated with incompatibility in Ler/Kas-2 recombinant inbred lines. This correlation was also obtained by artificial microRNA silencing lines that suppress incompatibility. However, our data indicated that R3 requires the co-action of another member within the cluster for incompatibility. In this period, we have generated double RPP1-like transgenics in all combinations, which have been crossed to Kas-2 to identify the minimal combination of RPP1-like Ler genes triggering incompatibility. This approach has been complemented by the identification of intragenic suppressors of Ler/Kas-2 incompatibility by EMS-mutagenesis and the generation of CRISPR/Cas9 induced mutations of RPP1-like Ler genes.

Regarding to task 3, we previously tested interactions between TIR and LRR domains of RPP1-like R1-R8 genes in yeast and performed yeast two-hybrid screens with TIR and full-length proteins R3 and R4 using cDNA libraries from the reference accession Col-0. Results indicated absence of direct interaction between RPP1-like members, but it remained to be studied whether such were indirect. The generation of new libraries using incompatible hybrid cDNAs and the use of co-IP approaches coupled to GC/MS has enabled the identification of proteins interacting with RPP1-like Ler receptors and with SRF3, thus giving clue to the signaling components underlying the Ler/Kas-2 incompatibility.

Finally, the use of local pathogens indicates that the presence of incompatible alleles might confer a selective advantage in terms of disease resistance, thus providing a biological meaning for their occurrence in nature.

From a socio-economic impact, we have analyzed the metabolic costs from immune activation aiming at the amelioration of the growth defects derived from immune activation. We expect that these investigations will contribute to the development of crops with broad spectrum of disease resistance and no deleterious effects on yield.