Phenotypic basis of balanced polymorphism is not due to interaction with Avr

Based on the presence of positive Tajima’s D values concentrated in the intron of the Rcr3

gene, three potential targets of selection can be envisioned: 1) selection on different regulatory motifs in the intron, 2) selection for different splicing variants or 3) selection on one or more amino acid polymorphism(s) in linkage with the intron. Since the first two alternatives could experimentally be excluded (see Chapter 3.2), it is most likely that balancing selection is acting on amino acid polymorphism(s) linked to the intron.

To evaluate functional differences between sequence types, I took a four-pronged approach. Using an over-expression vector in planta, I first evaluated whether protein

accumulated equally from all sequence types. Of the 54 different alleles tested, protein was not detected for seven variants by Western Blots with Rcr3-specific antibodies. In all of these cases in which no protein accumulated, the candidate (putatively deleterious) substitutions could be identified. Since these seven alleles appear to be pseudogenes, they were excluded from population genetic analyses.

The second assay was a protease enzymatic assay. I found that all 47 alleles with detectable protein levels encoded functional proteases. The third and fourth functional assays were to detect differences among alleles in their sensitivity to Avr2 and in their ability to elicit HR upon co-infiltration with Avr2 into Rcr3-mutant tomato plants. Most of the tested alleles

were inhibited by Avr2, resulting in the activation of the defence response in planta. The

amino acid polymorphism (N194D) is significantly associated with variation in inhibition by Avr2. Alleles, which carry the N194D mutation, fail to activate the defence response in planta, in the presence of Avr2. This supports previous results using site directed mutagenesis by Shabab et al. (2008).

Due to the large sample size used in this study (54 alleles), I had sufficient power to detect epistatic interactions between amino acid variants. One such case is the presence of a second substitution (R151Q) in an allele carrying the N194D mutation. All other alleles with the D variant at site 194 failed to be inhibited by Avr2. However, the single allele with the Q variant at site 151 and D variant at site 194 was inhibited by Avr2, implicating potential epistatic interactions between these two polymorphisms.

Two additional polymorphisms were associated with variation in sensitivity to Avr2: a synonymous polymorphism at bp 717 and a nonsynonymous polymorphism at bp 750. However, since alleles, which have the polymorphism at bp 750 (R213S), but not N194D can

be inhibited by Avr2 and elicit HR in planta, it is likely that the association between

phenotype and sequence variation for this polymorphism is due to linkage disequilibrium. In this data set encompassing nine Solanum species, the amino acid substitution

N194D was found exclusively in individuals of S. peruvianum and S. chilense. Since the

substitution was not detected in any other closely related species or the outgroup

S. lycopersicoides, this suggests that this mutation is derived. However, as only few

individuals carry this mutation, it is unlikely that the maintenance of these Rcr3 variants

accounts for the evidence of a balanced polymorphism at the Rcr3 locus.

4.2.2 Interspecific functional diversification at the Rcr3 locus is likely, but the balanced

polymorphism is not based on recognition of differential pathogen effectors

Since Rcr3 is known to be targeted by several effectors secreted by different pathogens, one possibility could be that the balanced polymorphism at the Rcr3 locus underlies differential

recognition of different effectors. To test this hypothesis, I investigated the effect of three additional effectors on Rcr3 protease function and performance in planta: Epic1, Epic2B and

Rip1. These additional effectors secreted by different pathogen species differed in their interaction with different Rcr3 variants.

Nearly all except nine Rcr3 variants were not inhibited by Epic1, while the opposite was true for Epic2B, i.e. most but four variants were inhibited. In these cases the potentially causative mutations could be pinpointed. There seems to be an overlap between the mutations causing sensitivity to inhibition by Epic1 and insensitivity to inhibition by Epic2B. In both cases, the substitutions H148N and D283N were found to be significantly associated with the alternate phenotype. Both effectors are secreted by the same pathogen, P. infestans. Since

these effectors belong to the same effector gene family and are believed to originate from gene duplication, it is possible that they contribute to different host specificities (Tian et al.

2007). For example, it is possible that the two effectors employ complementary functions and are selectively maintained in the pathogen population. Perhaps, natural selection on the effector target in the host led to changes at the Epic interacting site, which in turn led to compensatory changes in the effector. Duplication of the effector gene in the pathogen and subsequent functional diversification in both interacting effector types would therefore be advantageous for the pathogen (Michelmore & Meyers 1998).

So far it has only been demonstrated that Rcr3 plays a role in tomato resistance to

study, I show that inhibition of Rcr3 by Epic1 or Epic2B does not cause HR in tomato plants expressing Cf-2. The reason for this result may be that inhibition of Rcr3 by Epic1/2B does not activate the defence response. As known from experiments using the protease inhibitor E- 64, inhibition of Rcr3 alone is not sufficient to elicit the Cf-2 dependent defence response (Rooney et al. 2005). Putative conformational changes in Rcr3, which may be caused by

inhibition through Avr2, elicit the Cf-2 dependent defence response (Krüger et al. 2002). It is

possible that inhibition by Epic1/2B does not cause the same conformational changes and another mechanism between Rcr3 and Epic1/2B is responsible for the increased resistance of Rcr3-plants to P. infestans. Alternatively, the defence response activated by Epic1/2B may

not be Cf-2 dependent, but could rely on other molecules, which were not present in the tested plant genotype.

Although most tested Rcr3 variants did not interact with Rip1, eleven Rcr3 variants were inhibited by this effector. However, a role of Rcr3 in resistance of tomatoes to P. syringae has not been demonstrated so far. Rip1 is a protease inhibitor and has been detected

through interaction with the Rcr3-like protease C14, its host target in potato (M. Ilyas personal communication). In tomato C14 is one of the papain-like cysteine proteases residing in the tomato apoplast (like Rcr3) and has significant sequence homologies to Rcr3 (Shabab et al. 2008). Therefore, it is likely that the Rip1 effect on Rcr3 is merely a side effect and happens because Rcr3 and C14 are related to one another. When Rcr3 alleles were coinfiltrated together with Rip1 in tomato plants expressing Cf-2, no HR phenotype could be observed. The reason for this could be that inhibition of Rcr3 by Rip1 does not activate the defence response because it does not cause the same conformational changes as Avr2 (see paragraph above). Alternatively, the defence response activated by Rip1 is not Cf-2 dependent, but relies on other molecules, which were not present in the tested plant genotype. No nucleotide positions within the Rcr3 gene could be significantly associated with

inhibition by Rip1. The reason therefore may be that Rip1 functions differently from other protease inhibitors such as Avr2 or Epic1/2B. These three inhibitors seem to target very specific sites within the Rcr3 protease domain. Alteration of these sites will immediately prevent inhibition of protease activity by these effectors. Rip1 binding might be less specific (perhaps because Rcr3 is not its original target) and depend more on the entire structure of the protease. Many amino acid residues in the Rcr3 protease domain may interact epistatically and in different combinations and create a protein structure resembling the C14 structure, which by chance can be targeted by Rip1.

Mutations that are associated with differential behaviour of Rcr3 alleles regarding interaction with the different effectors were found among the different tomato species, but also within S. peruvianum. It is of course possible that this functional divergence between

species is due to diversifying selection at the Rcr3 locus as recently suggested (Shabab et al.

2008). Rcr3 would then adopt different functions in different lineages depending on the corresponding environmental conditions, i.e. the corresponding pathogen pressure and

effector repertoire. This hypothesis may apply in this study to the tested aspects of Rcr3 function. For instance, differential sensitivity for inhibition by Avr2 was only found in individuals from the sister species S. peruvianum and S. chilense, but not in individuals from

other tomato species or the outgroup S. lycopersicoides (Figure 4.2). The mutation was caused

by the same nonsynonymous substitution and may likely have arisen in the common ancestor of the two species. Alternatively, the presence in both lineages could also be explained by convergent evolution. It is possible that this mutation confers selective advantage in the environmental conditions these two species encounter or has been introgressed by gene flow, but does not have the same effect in other species. Also, the fact that the mutation was only found in S. peruvianum and S. chilense could be due to the sampling effect, because more

individuals from S. peruvianum and S. chilense were sampled than individuals from the other species. Thus it is not certain whether this mutation occurs in other species as well.

Concerning inhibition by Epic1, the alleles, which are inhibited, originate mostly from the species S. habrochaites, S. pennellii, S. chmielewskii, S. chilense and the outgroup

S. lycopersicoides, but in general most Rcr3 alleles are not inhibited. Among the large dataset from S. peruvianum, only few alleles were inhibited by Epic1. Since this phenotypic

characteristic is found in several species (from which a small sample size was recovered) and in the outgroup, but not abundant in the recently diverged tomato species, it is likely that the state of being inhibited is thus ancestral at Rcr3 alleles. Insensitivity to Epic1 inhibition would

then be the derived state. This may have arisen because it gives a potential selective advantage, perhaps in pathogen resistance. In summary, the functional tests with different Rcr3 alleles uncovered inter-species phenotypic variation regarding interaction with four different effectors, but this variation is unlikely the cause for the signature of balancing selection observed in S. peruvianum.

Figure 4.2: Phylogeny of the tomato clade showing differential behaviour of Rcr3

variants. The tree was adopted from Peralta et al. (2008). Alleles were tested from species

indicated in bold. The coloured dots indicate differential phenotypic behaviour regarding the different tested effectors. Differential behaviour is defined as behaviour being different from the phenotype as known from the cultivated tomato S. lycopersicum.

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