Identification and Characterisation of a Novel Glutathione Synthetase Gene Family in the Plant Parasitic Nematode Rotylenchulus reniformis

The reniform nematode, Rotylenchulus reniformis Linford & Oliveira, is a sedentary species of plant parasitic nematode that is widely distributed in tropical and subtropical regions and causes significant economic loss. There has been little molecular characterisation of R. reniformis, particularly in relation to the function of its effectors. Recent genomic and transcriptomic resources have become available that provide evidence of the complex suite of effector genes in R. reniformis. Expanded families of putative effector genes have been described for other plant parasitic nematodes. In particular it was noted that the Globodera pallida genome encoded a large number (30) of complete glutathione synthetase-like genes in comparison to the free-living nematode C. elegans which has a solitary glutathione synthetase (gs) gene. In this study, we have identified a profusion of 73 complete glutathione synthetase-like genes from the R. reniformis genome and transcriptomes. The phylogeny of R. reniformis GS-like genes divides this family into three major clades: Clade 1 contains only one sequence that is the likely ancestor of the R. reniformis GS gene family; Clades 2 and 3 represent two independent expansions that acquire their unique functions during evolution. In addition, most Clade 3 GS do carry a signal peptide for secretion while Clade 1 & 2 GS do not. Furthermore, most Clade 3 gs are most highly expressed in the parasitic female stage whereas Clade 1 & 2 gs are up-regulated in the non-parasitic stages. In situ analysis showed Clade 3 gs are expressed in the gland cell of R. reniformis which is a common site of nematode effector synthesis. In contrast, Clade 1 & 2 gs are expressed in the intestine tissues. Glutathione synthetase is a key enzyme in the second step of glutathione biosynthesis. Biochemical analysis of GS from R. reniformis confirmed the functional diversity between each clade. Clade 1 GS exhibited the canonical GS enzyme activity which was all-but lacking in Clade 2 & 3 GSs. Crystallography was then exploited to investigate the structural differences between canonical and non-canonical GSs, indicating that an alternative substrate may be accepted by non-canonical GS. This project also set out to investigate the functions of R. reniformis GS. None of the R. reniformis GS, including canonical GS could complement the Arabidopsis GS mutant gsh2. In addition, Arabidopsis overexpressing Clade 3 GS showed enhanced susceptibility to the cyst nematode Heterodera schachtii. In conclusion, this study revealed evolved functional diversity of this expanded large GS family by phylogenetic, biochemical, structural and functional evidence.