Our understanding of the mechanisms involved in Nod-factor perception was further refined through domain swap experiments using receptors from L. japonicus and L. filicaulis. Initially it was observed that L. filicaulis, in contrast to L. japonicus, did not develop root nodules after inoculation with the R. leguminosarum DZL strain (Pacios-Bras 2003). Analysis of transgenic plants subsequently traced this inability back to variations in the amino acid composition of the NFR1 or NFR5 receptors between L. japonicus and L. filicaulis (Radutoiu et al. 2007). Domain swaps combined with substitutions of single amino acids were then used to show that specific recognition of the DZL Nod-factor relied on the LysM containing domains of NFR1 and NFR5 and that the LysM2 domain of NFR5 played a major role in discriminating M. loti and R. leguminosarum DZL Nod-factors in L. filicaulis. A leucine adjacent to a putative Nod-factor binding groove located between the first p-strand and first helix of the L. japonicus NFR5 LysM2 domain, a position filled by a lysine in L. filicaulis, was found to be largely responsible for the recognition of the Nod-factor synthesised by the DZL strain (Radutoiu et al. 2007). However, presence of three LysM domains in the NFR5 and NFR1 receptors (Madsen et al. 2003; Radutoiu et al. 2003; Arrighi et al. 2006), suggests the involvement of more than one LysM domain in Nod-factor perception. Two lines of evidence supports this notion: (1) the non-nodulation phenotype caused by an amino acid substitution in the LysM1 domain of the M. trunctula homolog of NFR5 called NFP (Arrighi et al. 2006) and (2) the involvement of LysM1 of the pea SYM37 NFR1-like receptor in distinguishing "European" and "Middle East" R. leguminosarum bv. viciae strains (Zhukov et al. 2008).
Further insight into the functional role of individual amino acids in Nod-factor perception was obtained in a domain swap study using the extracellular domains of NFR1 and NFR5 from a more distantly related species, Lotus pedunculatus. This Lotus species is normally nodulated by a Bradyrhizobium spp. strain producing a Nod-factor with an additional carbamoyl group at the non-reducing moiety (Bek et al. 2010). It was found that the combined amino acid differences of the NFR1 and NFR5
extracellular domains of L. japonicus and L. pedunculatus were not influencing the recognition of the Nod-factor substituted with one or two carbamoyls at the non-reducing end. Considering that a high number of amino acid variations is found between L. japonicus and L. pedunculatus NFR proteins, this suggests the involvement of only a small fraction of amino acids in deciphering Nod-factor structure.
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