potential LGT. Apart from the F-box domain, the F-box- containing proteins are constituted of repeat domains (mostly LRRs). Thus phylogenetic inference on the whole protein for large numbers of divergent taxa appears highly problematic. Instead we focused the analysis on the F-box domain, using 50 aa on their own, as well as together with 150 aa from the downsteam F-box- adjacent region or a shorter region containing 2 or 3 LRRs. Fifteen large datasets were assembled to include homologues from the broadest possible species range. The datasets varied by the similarity thresholds used in the profile searches [7], criteria used to align the sequences (profile, penalties), amount of gaps, and included from 217 to 2853 taxa (and from 64 to 217 aa in the alignment). Trees were inferred with neighbour-joining and maximum likelihood methods (see methods section). The resulted (approx.) 30 inferred phylogenies had low average branch supports, which demonstrated that phylogenetic inference for our data (with many short sequences of deep divergences and large percentage of gaps) is indeed problematic, even when considering the conserved F-box domain. The choice of the analysis model did not influence the inference much but considerably more variation in branching order was observed when analyzing different sets of sequences and alignments. In particular, varying the amount of gaps in the alignment had an impact. However, most phylogenies favoured the scenario where all R. solanacearum GALA genes clustered together and with Arabidopsis thaliana or with Oryza sativa as their closest basal lineages (for a representative example of an inferred Maximum Likelihood (ML) tree see Figure S1 that is a supplemental file phymlGALA.tre, which can be viewed with ATV-forester [22] from www.phylosoft.org/atv/). Only in some rare cases we also observed that one or two GALA genes were grouped with a non-plant lineage (but with low clade supports). Overall, the underlying phylogenetic signal appears to be in favour of the postulated LGT from a plant lineage. At the same time, the limited accuracy of inference does not enable us to confidently suggest a putative donor. Our conclusion that the GALA-LRRs belong to the CC-LRR subfamily is consistent with the hypothesis of the lateral transfer. The updated CC-LRR subfamily has proteins from a very heterogeneous group of organisms including animals, plants, fungi, protista, and bacteria. By its wide taxonomic distribution it resembles the TpLRR subfamily [1] that includes proteins found in all three domains of life [23]. The broad distribution of TpLRR proteins also has been explained by LGT. Furthermore, the analysis of TpLRRs suggested that genes linked to pathogenicity can be shared between parasitic bacteria and parasitic eukaryotes [23]. The results of our present analysis of CC-LRRs agree with this hypothesis. The updated CC-LRR subfamily includes many proteins from bacteria (Gemmata sp., Parachlamydia sp., Legionella pneumophila, Rhodopirellula baltica, Ralstonia solanacearum), protista (Entamoeba histolytica, Leishmania, Trypanosoma, Dictyostelium) and fungi (Cryptococcus, Candida albicans, Candida glabrata, Neurospora), among which many are parasitic organisms colonizing plants and animals. Despite the similarity of GALA-LRRs and CC-LRRs, they have some systematic differences (Figure 1B) and it needs to be explained. Usually, only about half of residue positions of LRRs remain conserved over a long evolutionary period [3]. The conservation usually reflects the importance of these residues for the preservation of the structure. However, GALA-LRRs are nearly perfectly repeated and this suggests that they emerged relatively recently. On the other hand, our molecular modeling indicates that GALA- and CC-LRRs fold in very similar structures that can be compatible and well-packed together, if a CC-LRR is inserted between GALA-LRR or visa versa (Figure 3). This Evolution of GALA Proteins conclusion is supported by the fact that two plant F-box-LRR proteins have a couple of GALA-LRRs inserted in GL-LRR tandem arrays (Figure 1A). Considering that CC-LRRs are much more abundant in plants than GALA-LRRs, and based on the above-mentioned facts, we propose the following sequence of evolutionary events that could ‘‘transform’’ the CC-LRR into GALA-LRR tandem arrays. First, the accumulation of point mutations may lead to the spontaneous occurrence of the first GALA-LRR and due to the structural complementarities between this new LRR and the CC-LRRs (see previous section) the occurrence of GALA-LRR does not significantly affect the overall structure and stability of the CC-LRR domain. Second, it is known that repetitive sequences can evolve more rapidly than non-repetitive ones [24,25]. This applies both to the repeat multiplication and to the repeat deletion. Therefore, once appeared, GALA-LRRs can multiply and CC-LRRs disappear. As a result the plant CC-LRR genes, being acquired by a bacterium, may shed their CC-LRRs replacing them by GALA- LRRs seeded in their original sequences. Currently it is not clear why R. solanacearum may prefer to generate arrays of GALA-LRRs instead of CC-LRRs. It has been shown that GALA are type III effectors required for virulence of R. solanacearum on three different plants, namely Arabidopsis, Tomato and Medicago truncatula. [13]. Furthermore it is very likely that the GALA type III effectors participate in virulence through their action in plant cells as the adaptors in SCF-type E3- ubiquitine ligases [4,26]. In the SCF-type E3 ubiquitin ligase complex F-box containing proteins interact through their LRR (or other protein-protein interaction domains) with the protein targets to be ubiquitinated. We propose that a possible conversion from an original F-box and CC-LRR protein to an F-box and GALA- LRR protein was dictated by functional constrains. It is possible that the new GALA-LRRs have better plant-protein target recognition and are more versatile adaptors suitable to detect protein targets from diverse host plants. Testing GALA-LRRs for positive selection in an attempt to establish their functional binding sites To gain insight into the function of the GALA proteins we examined whether adaptation could have acted on a proportion of protein residues during the evolution of GALA LRRs and identified positions of such sites, using likelihood ratio tests (LRTs) and the Bayesian prediction [27,28]. In the agreement with the evolutionary scenario suggested by us for GALA-LRRs, data sets containing aligned LRRs were used to analyze the strength of selective pressure across its residues since their common ancestor sequence was acquired by the bacterium. To some extent, the evolution of LRRs in one particular GALA protein may be likened to the evolution of the gene family members after a duplication event whereby paralogous genes originate from the common single ancestral sequence [29]. Using this analogy we studied the process of the accumulation of substitutions at each site in a single LRR by comparing the codon substitutions at the homologous sites in other repeat sequences from the group of orthologous GALA proteins in four different R. solanacearum strains: GMI1000 [4,20], RS1000 [30], UW551 [31] and MolK2 (C. Boucher and S. Genin, personal communication). The first two strains belong to the phylotype I and the others to the phylotype II, among the four phylotypes defined previously for this species complex [32]. The full coding DNA alignment of all LRRs from all the available GALA proteins (.400 sequences) was analyzed and none of the tests gave a significant evidence for positive selection, although parameter estimates hinted that this possibility could not be ruled out. This could mean that only for some GALAs the PLoS ONE | www.plosone.org 5 February 2008 | Volume 3 | Issue 2 | e1694