all_cleaned_unique_cep_genomic_clone_sequence_new_names

genomic nucleotide sequence for all unique CEP genomic clones from R. reniformis

All_transcripts_from_the_same_comp_as_those_containing_cep-like_sequences_in_transcriptome

All transcripts from the same comp as those containing cep-like sequences in transcriptome

Unique_genomic_CEPs_cleaned_basian_new_names

Bayesian phylogenetic tree of cloned genomic CEP sequences

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

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

New GS-like paralogues are redeployed during parasitism as effectors.

<p><b>A</b>) Examples of spatial expression patterns (<i>in situ</i> hybridisation) of GS-like gene members of Clades 1, 2, and 3 (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007310#pgen.1007310.s003" target="_blank">S3 Fig</a> for additional examples and negative controls). <i>Globodera pallida</i> Clade 1 and 2 GS genes are expressed in the intestine (white arrowheads). Clade 3 GS-like genes are specifically expressed in the dorsal gland cell (Dg) of <i>G</i>. <i>pallida</i>, <i>G</i>. <i>rostochiensis</i> and <i>R</i>. <i>reniformis</i> (left to right). Scale bars = 100 μm. <b>B</b>) An affinity-purified antibody raised against <i>G</i>. <i>pallida</i> Clade 3 GS-like effector Gpa-GSS17 recombinant protein was used for immunolocalisation (green fluorescence). Gpa-GSS17 is localised in the dorsal gland cell (Dg) of parasitic nematodes, the cytoplasmic gland extension and the ampulla at the base of the stylet where secretions accumulate prior to their release (top) Scale bars = 50 μm. The same native protein was localised within the syncytial feeding site (asterisks) induced by the nematode in a potato root (green fluorescence, bottom left). No such localisation was seen with the 2^o antibody control (bottom right). Cell walls are stained blue. <b>C</b>) Western blot to determine antibody specificity. The anti-Gpa-GSS17 antibody specifically recognises Gpa-GSS17, and does not recognise other Clade 3 GS-like proteins tested, other plant-parasitic nematode GS proteins tested (Clades 1 and 2), nor the endogenous potato GS (St-GSS1).</p

Impairing plant glutathione synthetic capacity is independently detrimental to syncytium and cyst nematode development.

<p>Both syncytia <b>(A)</b> and nematodes (<b>B)</b> are significantly smaller on <i>pad2-1</i> mutant <i>Arabidopsis</i> roots than on wild-type (Student’s T-test p ≤ 0.001, n = 82 and 147 for syncytia and nematodes respectively). Error bars indicate standard error of the mean. <b>(C)</b> In both <i>pad2-1</i> and wild-type roots, syncytium and nematode size significantly co-vary (Pearson’s correlation, p ≤ 0.05 and 0.001, n = 49 and 66 respectively), however the correlation is weak and most of the variation in nematode size (83–89%) is not explained by syncytium size. <b>(D)</b> Example images of unsuccessful nematode infection on <i>pad2-1</i> roots (middle) and successful infection on both <i>pad2-1</i> (bottom) and wild-type roots (top). Black and white arrows indicate syncytial and nematode boundaries respectively, blue arrows indicate an aborted syncytium and surrounding areas, scale bars indicate 100 μm. <b>E)</b> and <b>F)</b> In the presence of 1 mM BSO, syncytia are initiated but not properly maintained. Apoptosis, indicated by blue arrows and evidenced by propidium iodide staining (red), occurs in the local area of the syncytium (blue arrows) and spreads often non-distal to the site of infection; a similar, but more severe and frequent phenotype than observed in <i>pad2-1</i> roots. Scale bars indicate 100 μm. <b>(G)</b> and <b>(H)</b> J2 stage nematodes incubated for the normal root invasion period (48 hours) on water agar plates containing 1 mM BSO are unaffected in <b>(G)</b> mortality (n = 68) or <b>(H)</b> motility (Mann-Whitney U Test, p = 0.408 n = 20).</p