Rcr3 is a secreted protease of tomato that is targeted by fungal effector Avr2, a secreted protease inhibitor of the fungal pathogen Cladosporium fulvum. The Avr2-Rcr3 complex is recognized by receptor-like protein Cf-2, triggering hypersensitive cell death (HR) and disease resistance. Avr2 also targets Rcr3 paralog Pip1, which is not required for Avr2 recognition but contributes to basal resistance. Thus, Rcr3 acts as a guarded decoy in this interaction, trapping the fungus into a recognition event. Here we show that Rcr3 evolved > 50 million years ago (Mya), whereas Cf-2 evolved <6Mya by co-opting the pre-existing Rcr3 in the Solanum genus. Ancient Rcr3 homologs present in tomato, potato, eggplants, pepper, petunia and tobacco can be inhibited by Avr2 with the exception of tobacco Rcr3. Four variant residues in Rcr3 promote Avr2 inhibition, but the Rcr3 that co-evolved with Cf-2 lacks three of these residues, indicating that the Rcr3 co-receptor is suboptimal for Avr2 binding. Pepper Rcr3 triggers HR with Cf-2 and Avr2 when engineered for enhanced inhibition by Avr2. Nicotiana benthamiana (Nb) is a natural null mutant carrying Rcr3 and Pip1 alleles with deleterious frame-shift mutations. Resurrected NbRcr3 and NbPip1 alleles were active proteases and further NbRcr3 engineering facilitated Avr2 inhibition, uncoupled from HR signalling. The evolution of a receptor co-opting a conserved pathogen target contrasts with other indirect pathogen recognition mechanisms.

Plant immunity can be observed in a qualitative manner, in which plants are either completely resistant or susceptible, or in a quantitative way, in which certain plants of the same species are more resistant and others are less resistant. Such intraspecific quantitative variation in resistance was formally introduced as horizontal resistance in the field of phytopathology by Vanderplank in 1963, and he further extended his concept in 1968. By his definition, horizontal resistance is a product of multiple underlying genes, that all have minor effects on the amount of resistance observed. This may explain why quantitative resistance is observed to be pathogen genotype independent in many cases. This is in contrast to vertical resistance, also called qualitative resistance, that depends on a single major resistance (R) gene and is pathogen genotype dependent.Here, the product of specific R gene recognizes a specific pathogenic

9Natural plant populations encounter strong pathogen pressure and defense-associated genes are 2 0 known to be under different selection pressure dependent on the pressure by the pathogens. Here 2 1 we use wild tomato Solanum chilense populations to investigate natural resistance against 2 2 Cladosporium fulvum, a well-known pathogenic fungus of domesticated tomatoes. We show that 2 3 populations of S. chilense differ in resistance against the pathogen. Next, we explored the 2 4underlying molecular processes in a species wide-context. Then, focusing on recognition of the 2 5 two prominent avirulence factors secreted by C. fulvum (Avr4 and Avr9) in central and northern 2 6 populations of S. chilense we observed high complexity in the cognate homologues of 2 7Cladosporium resistance (Hcr9) locus underlying the recognition of these effectors. Presence of 2 8 canonical genomic regions coding for Cf-4 and Cf-9, two major dominant resistance genes in the 2 9Hcr9 locus recognizing Avr4 and Avr9, respectively, does not meet prediction from Avr 3 0 response phenotypes. We find both genes in varying fractions of the plant populations and we 3 1show possible co-existence of two functionally active resistance genes, previously thought to be 3 2 allelic. Additionally, we observed the complete local absence of recognition of additional Avr 3 3 proteins of C. fulvum. In the southern populations we attribute this to changes in the coregulatory 3 4 network. As a result of loss of pathogen pressure or adaptation to extreme climatic conditions. 5This may ultimately explain the observed pathogen susceptibility in the southern populations. 6This work puts major gene mediated disease resistance in an ecological context. 3 7 3 8 3 9

The wild tomato species Solanum chilense is divided in geographically and genetically distinct populations that show signs of defense gene selection and differential phenotypes when challenged with several phytopathogens, including the oomycete causal agent of late blight Phytophthora infestans. To better understand the phenotypic diversity of this disease resistance in S. chilense and to assess the effect of plant genotype vs. pathogen isolate, respectively, we evaluated infection frequency in a systematic approach and with large sample sizes. We studied 85 genetically distinct individuals representing nine geographically separated populations of S. chilense. This showed that differences in quantitative resistance properties can be observed between but also within populations at the level of individual plants. Data also did not reveal clear indications for complete immunity in any of the genotypes. We further evaluated the resistance of a subset of the plants against P. infestans isolates with diverse virulence properties. This confirmed that the relative differences in resistance phenotypes between individuals were mainly determined by the plant genotype under consideration with modest effects of pathogen isolate used in the study. Thus, our report suggest that quantitative resistance against P. infestans in natural populations of a wild tomato species S. chilense is likely not the result of specific adaptations of hosts to the pathogen but of basal defence responses that depend on the host genotype and are pathogen isolate-unspecific.

Natural plant populations encounter strong pathogen pressure and defence-associated genes are known to be under selection dependent on the pressure by the pathogens. Here, we use populations of the wild tomato Solanum chilense to investigate natural resistance against Cladosporium fulvum , a well-known ascomycete pathogen of domesticated tomatoes. Host populations used are from distinct geographical origins and share a defined evolutionary history. We show that distinct populations of S. chilense differ in resistance against the pathogen. Screening for major resistance gene-mediated pathogen recognition throughout the whole species showed clear geographical differences between populations and complete loss of pathogen recognition in the south of the species range. In addition, we observed high complexity in a homologues of Cladosporium resistance ( Hcr ) locus, underlying the recognition of C. fulvum, in central and northern populations. Our findings show that major gene-mediated recognition specificity is diverse in a natural plant-pathosystem. We place major gene resistance in a geographical context that also defined the evolutionary history of that species. Data suggest that the underlying loci are more complex than previously anticipated, with small-scale gene recombination being possibly responsible for maintaining balanced polymorphisms in the populations that experience pathogen pressure.