LRRs accumulated changes due to positive selection, as by averaging over all domains we loose power to detect positive selection affecting only certain GALA proteins. To test this we subdivided the aligned LRR sequences, so that only sequences from the orthologous GALAs were analyzed together. Parameter estimates from these alignments showed that for all GALA proteins 50–70% of the LRR positions are rather conserved while substitutions at remaining sites generally have neutral effect on the fitness of the protein. However for GALA2 both LRTs for positive selection were highly significant (with P-values ,0.01). Estimates suggested that 8% of sites evolved under positive selection. For GALA2 LRRs, the Bayesian approach detected positions 8 and 15 (numbering as in Figures 1 and 2) with high probability (e.g., using model M8, the corresponding probabilities were 0.97 and 0.99, see Methods and Table S1 of Supporting Information for details). In accordance with the modeled GALA-LRR structure these residue positions are located in the a-helical region and exposed to the solution (Figure 2A). In the LRR domains these positions are located on the convex surface of the horseshoe shaped structure (Figure 2B). For GALA7 LRRs, only the LRT comparing M7 vs. M8 supported positive selection (P-value ,0.05), but the estimate of the v ratio (describing selective pressure) was only slightly higher than 1 (v<1.15), indicating the lack of clear support for positive selection signal. Model M1a that does not allow positive selection described data equally as well as model M2a that allows positive selection. The Bayesian inference suggested that positions 4, 8, 11, 15 and 17 had a slightly elevated ratio of nonsynonymous to synonymous changes. Changes at these sites at the very least should be neutral to the fitness of the protein but may have a mild advantageous effect, possibly indicating a recent increase of adaptive pressure. The same can be concluded about position 4 in GALA1 and GALA3 LRRs and position 11 in GALA5 LRRs. If mapped on the structural model of the GALA-LRR, most of them (positions 8, 11, 15 and 17) are located on the external side of the a-helix and on the convex surface of the LRR solenoid. The side-chain in position 4 belongs to the loop connecting b- strand with the a-helix and also is exposed to the solvent (Figure 2A). To see if signature of positive selection on GALA 2 and 7 is detectable on the level of the entire LRR domain of these proteins, we analyzed separately the groups of four GALA2 and GALA7 orthologous sequences from the different strains of R. solanacearum. Analysis of both GALA2 and GALA7 LRRs returned highly significant results for both tests (with P-values from 0.0014 to 0.025), providing the evidence of positive selection on both genes. The lack of the strong evidence for positive selection in GALA7 LRRs in the previous analysis suggests that positive selection may affect only certain (orthologous) repeats of GALA7 while the homologous sites in other repeats of this protein evolve neutrally. In this last analysis the number of sequences is too low for the Bayesian prediction to be accurate, and so the results of such inference are used only in an explorative manner, to see if the predicted positive selection sites correspond to any particular repeats and where such sites could be located. Mapping of the predicted sites of GALA2 onto the repeats of the LRR domain (with probability .0.85 from BEB) shows that they are located in position 15 in four LRRs and in position 21 of one LRR and dispersed over the LRR domain mostly on the convex surface (see Figures S2 and S3 of Supporting Information). Interestingly, the analysis of individual LRRs of GALA2 also pointed at position 15, that is the most represented in the analysis of the entire LRR domain. In the GALA7 LRR domain, these sites are also dispersed over the convex surface of the protein and are found in exactly the Evolution of GALA Proteins same positions as predicted in individual LRRs of GALA7 (positions 4, 11, 15 and 17). Thus, it is encouraging to observe that most inferred positions throughout the LRR domain of orthologous GALAs coincide with those inferred in individual LRR repeats analysis on groups of GALA orthologues. It is important to mention that all positions, that are inferred to be under positive selection, are found on the surface of the structural model of GALA LRR domain. This grants additional support for the GALA-LRR structure prediction described above. Furthermore, our analysis suggests that the convex surface of the horse-shoe shaped GALA-LRR domain is more prone to positive selection than its concave one. It is tempting to propose that the selective pressure leading to an increase of variability on such residues could be the site of protein binding, relevant to the adaptor function of the F-box GALA proteins in the SCF-type E3 ubiquitin ligase. This study provides a strong background for further functional studies. Conclusions The GALA-LRR example shows that the differences in LRR consensus motifs can be not only mutually exclusive in terms of inter-LRR packing as it was observed in LRRs from different subfamilies [3], but also permissive as we found in the case of GALA-LRRs and CC-LRRs. The permissive packing may serve as a criterion for the affiliation of LRRs having different consensus sequences with the same LRR subfamily. Therefore, one may expect to find a subfamily of evolutionary related proteins that share similar functions, cellular location, globular domains and, at the same time, having quite different repetitive consensus patterns. The relationships between GALA-, GL- and the other CC-LRRs suggest that structural constraints, namely, permissive packing of repeats may control the limits of the LRR variability within a subfamily. This result provides new insight into the fascinating interplay between the structural constraints and unusual evolu- tionary dynamics of LRRs and can be used to classify other newly identified LRR domains. The R. solanacearum GALA proteins are bacterial F-box proteins containing a new kind of LRR, which can be found in other bacteria, plants and unicellular eukaryotes. These GALA-LRRs and related GL-LRRs are part of the CC-LRR subfamily, which is generally associated with an F-box domain. The presence of this F- box domain in GALA proteins is indicative of the probable ancient lateral transfer from eukaryotic (possibly plant) genes into a R. solanacearum ancestral recipient strain. We further looked into the GALA-LRR in all the GALA sequences available and found that for some GALA proteins there is a strong signature of positively selected residues and only on the convex side of the GALA-LRR structure. This suggests that the GALA proteins, probable E3- ubiquitin ligase adaptors, necessary for the virulence of R. solanacearum on its host plants, could bind their potential protein ligand on the convex side of the LRR domains, similarly to the A. thaliana TIR1 F-box type E3 ubiquitin ligase [33]. As we have an experimental system that enables us to test for the functionality of GALA proteins in virulence [4], this current study provides a strong theoretical background for testing the relevance of specific GALA-LRR residues to pathogenesis. Methods Sequence profile search The generalized sequence profile method and the pftools package [7] were used. Since a single LRR would be unlikely to form a stable structure on its own, we limited the search to proteins containing at least three tandem repeats, thus increasing the selectivity of the search. Several profiles corresponding to different PLoS ONE | www.plosone.org 6 February 2008 | Volume 3 | Issue 2 | e1694