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

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene.

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to "GS-like effectors". Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function

Data from: Effector gene birth in plant parasitic nematodes: neofunctionalization of a housekeeping glutathione synthetase gene

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function

All GS-like nucleotide sequences from 10 nematode species

176 GS-like nucleotide sequences from 10 nematode species across the phylum. Sequences are not available for M. incognita. sequences encode the entire transcript, and in some cases 5' and/or 3' UTR

All GS-like protein sequences from 11 nematode species.

180 GS-like protein sequences from 11 nematode species across the phylum. Deduced amino acid sequences from genome and/or transcriptome assemblies

Data from: Effector gene birth in plant parasitic nematodes: neofunctionalization of a housekeeping glutathione synthetase gene

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function

The detection of novel thiols in syncytial feeding sites induced by cyst nematodes.

<p><b>A</b>) Free thiols, stained with ThiolTracker Violet, accumulate in the cytoplasm of syncytia induced by the cyst nematode <i>Heterodera schachtii</i> in <i>Arabidopsis</i> roots throughout the infection process (7, 14 and 21 days post infection). N indicates nematode. Scale bars = 100 μm. <b>B</b>) Optical cross section through the feeding site using the same stain. Arrows indicate partially dissolved cell wall. <b>C</b>) Analysis of low molecular weight thiols extracted from syncytia formed in potato by <i>Globodera pallida</i> (blue), control uninfected potato root (red), female nematodes (orange), and glutathione standard (black), by hydrophilic interaction liquid chromatography. Example plant-specific peaks are indicated with red arrows, syncytia-specific peaks with blue arrows, and a plant-specific peak increased in abundance in syncytia with a black arrow. <b>D</b>) The same sample used in panel C was separated at higher resolution using a shallower elution gradient. The peak at approximately 1.8 minutes in panel C (asterisk) corresponds to approximately 4 minutes in panel D (asterisk). This peak is absent in control roots, absent in nematode tissue, and highly abundant in syncytial feeding site material. No corresponding peak in the mass spectrum trace was identified (bottom).</p

The crystal structure of <i>Globodera pallida</i> GS-like effector Gpa-GSS22.

<p><b>A)</b> The crystal structure of Gpa-GSS22 is composed of a homodimeric molecule in both its open (apo (red)) and ADP-bound closed (gold) conformations. <b>B)</b> The two helix bundle that constitutes the ATP grasp fold undergoes a 7.8 Å conformational change on binding ADP to close over the active site. <b>C)</b> The presence of electron density (blue mesh) in the active site is consistent with a single ADP molecule and two magnesium ions per subunit.</p

Cyst nematode GS-like genes are re-purposed to carry out a novel function.

<p><b>A)</b> Purified protein for St-GSS1, Gpa-GSS1, 5, 12, 17, 22, 24 and 30 were tested for glutathione synthetase activity by measuring phosphate release from ATP in the presence of canonical substrates (γ-EC, glycine and ATP). To determine specific activity, rates are presented with subtraction of buffer controls in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007310#pgen.1007310.s009" target="_blank">S2 Table</a>. <b>B)</b> and <b>C)</b> Comparison of residues in the di-peptide binding pocket of Gpa-GSS22-closed and St-GSS-closed, with inset the variation at these positions in all other <i>G</i>. <i>pallida</i> Clade 3 GS-like effectors. <b>B)</b> In St-GSS1, the cysteine of the di-peptide substrate (γ-EC) is coordinated by the side chain of an arginine (top left), and the backbone of two serines (bottom left and bottom right). The arginine is conserved in Gpa-GSS22 and all GS-like effectors (inset). The two serines are not conserved in sequence in either Gpa-GSS22 or the remainder of Clade 3 (inset), but the equivalent residues are preferentially small and uncharged amino acids that do not vary greatly in the remainder of Clade 3 (inset). <b>C)</b> In St-GSS1, the glutamic acid of γ-EC is coordinated exclusively by side chain interactions with a number of charged residues. All of these residues are different in Gpa-GSS22, and these positions are highly variable across Clade 3 (inset).</p

Comparison of residues in the glutamate binding pocket of canonical GS with the same positions in nematode GS-like effectors.

<p>The canonical arrangement in St-GSS1 surrounding the glutamic acid of γ-EC has been conserved for ~1 billion years of evolution in three kingdoms: Plantae (<i>Solanum tuberosum</i> St-GSS1-closed, PDB 5OES), Fungi (<i>Saccharomyces cerevisiae</i>, PDB 1M0T), and Animalia (<i>Homo sapiens</i> GSS1, PDB 2HGS). Both solved structures of GS-like effectors (Gpa-GSS22 and Gpa-GSS30) show a non-canonical arrangement that is highly unusual among Eukaryotes. Together with the conservation in the remainder of the di-peptide substrate binding pocket, and the conservation in the entire ATP binding pocket, this diversification likely indicates novel substrate usage.</p