The balanced polymorphism is based on differences in activation of the defence response

According to the Guard-Hypothesis the defence response relies upon two different events: modification of the guardee by the effector and activation of the defence signalling through the guard molecule (Dangl & Jones 2001; Jones & Dangl 2006; van der Biezen & Jones 1998). The in vitro results did not reveal substantial differences in the interaction between

different effectors and guardee. However, the in planta assays enabled me to also investigate

the second step of defence response activation. Indeed, Rcr3 alleles differ substantially in the

strength of the defence response they elicit in planta. Variation in this trait is significantly

associated with three amino acid polymorphisms at nucleotide positions 728, 775 and 1099 encoding the amino acid changes I 206K, Q222E and S330A and two synonymous changes at positions 102 and 144 (Figure 3.14). In a structural model of the Rcr3 protease domain, these three substitutions are located in close proximity to positions with importance in protease

function or compatibility between Rcr3 and Cf-2 (Figure 3.15, Krüger et al. 2002; Shabab et al. 2008). All five mutations correlated with the weak HR phenotype are in linkage with one

another and with the intron, despite frequent gene conversion at the locus and are responsible for the peculiar sequence type structure (Figure 3.14, B7). These are the positions showing positive Tajima’s D values in the sliding window analysis and may thus contribute to the

signature of balancing selection at the Rcr3 locus. It is possible that one or all of the amino

acid changes is involved in the variation in the defence response. The other two polymorphisms (both at synonymous sites) are not likely the causative mutations and rather in linkage disequilibrium with the amino acid polymorphisms.

Additionally, to complete the link between genotype to phenotype I infiltrated the effector Avr2 into wild tomato plants grown from seeds, which were collected at the same sites as the plants I used for the population genetic and functional assays. In doing so, I tried to recover similar Rcr3 genotypes to those that had previously been tested in the functional

assays previously. In these assays, no HR phenotype was observed even at 10 days after inoculation with the effector. Several reasons might explain this observation. First, it is possible that Rcr3 was not expressed in these plants and therefore no Avr2-Rcr3 interaction could take place to elicit the defence response. Second, these plants may not carry or express functional Cf-2 molecules, which would prevent activation of the defence signalling. Third, other molecules in the pathway leading to HR are absent. Fourth, other stimuli such as PAMP-triggering are needed to activate the full defence machinery including ETI. Fifth, Rcr3 expressed in these plants is insensitive to the Avr2 used in this study and therefore no defence response is activated. It remains to be demonstrated which of these five explanations is the likely cause of these results.

Previous studies on R gene evolution demonstrated the maintenance of variation for

pathogen recognition (for example in the case of Pto) or presence/absence polymorphism (for

example in the case of Rpm1) (Bakker et al. 2006b; Bergelson et al. 2001b; Rose et al. 2004;

Stahl et al. 1999). In contrast, the balanced polymorphism in the case of the Rcr3 locus, – a

guardee, not an R gene – seems to have an effect on the activation of the defence response. A

possible explanation of this effect might involve a resistant/susceptible polymorphism at the

Rcr3 locus (Tellier & Brown 2007b), associated with a potential cost of resistance (Stahl et al.

1999; Tian et al. 2003). Alternatively, the diversity measured at the Rcr3 locus might also be

due to coevolution of Rcr3 with allelic types of Avr2 or of other pathogen effectors, which

were not tested in this study. Based on the results revealing differences in the outcome of the defence response – the HR – the attenuated defence response may be a by-product of the

interaction between Rcr3 and Cf-2. This interaction transmits the signal upon pathogen recognition and initiates the defence response. Members of the Rcr3 and Cf-2 gene families

seem to be tightly coevolving. This interaction requires a precise matching between allelic variants. A mismatch, such as when a variant of Rcr3 from one Solanum species is paired

with a variant of Cf-2 from another but nevertheless closely related Solanum species, results

in an autonecrotic response (Krüger et al. 2002) and could be an example of Dobzhansky-

Muller incompatibility between tomato species (Bomblies et al. 2007). An attenuated

response due to incompatibility between guard and guardee may decrease the risk of auto immune responses and can therefore be advantageous when the corresponding pathogen is absent (Ispolatov & Doebeli 2009). Since both genes evolve in gene families, it may be advantageous for a plant to have different alleles of both genes, which would form matching partners. One of the amino acid changes (Q222E) associated with the attenuated HR phenotype differs between Rcr3 from S. lycopersicum and S. pimpinellifolium and could

potentially contribute to the reported incompatibility between Rcr3 and Cf-2 likely causing attenuated HR in incompatible genetic backgrounds (Krüger et al. 2002). In this study, most

of the individuals carry both Rcr3 phenotypic types. Since I tested all Rcr3 alleles in identical genetic backgrounds, some may not be matched with their optimal Cf-2 partner, explaining attenuated response for some pairings of Rcr3 with Cf-2.

This combination of population genetic, computational and statistical methods and functional assays on the molecular level provided a more complete understanding of the role of the guardee in plant immune system evolution. I show that natural selection appears to maintain diversity at the effector target Rcr3 through balancing selection and gene

duplication. These are mechanisms, which have been shown to play a key role in R gene

evolution (Michelmore & Meyers 1998; Stahl et al. 1999). This study reveals that in contrast

to R gene evolution, evolutionary forces shaping the guardee rather act on the guard-guardee

than on the guardee-effector interface and propose that diversity at genes involved in immunity is not only created by natural selection for pathogen recognition, but also for improved transduction of the defence signal or avoidance of autoimmune response.