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Soil-borne cyst nematodes are obligatory sedentary parasites that cause severe losses to cultivation of major crops such as potato and soybean. Cyst nematodes establish specialised permanent feeding sites within the roots of their host by manipulating plant morphology and physiology through secreted effectors. Here we identified host targets of effector GpRbp-1 and studied their roles in plant-nematode interactions. GpRbp-1 was found to interact in yeast and in planta with the potato and Arabidopsis homologues of Siz/PIAS-type E3 SUMO ligase SIZ1. Our results show that a pathogen effector targets the master regulator SIZ1 in plant cells, which has not been demonstrated earlier to our knowledge. The interaction of GpRbp-1 and SIZ1 localizes to the plant nucleus, suggesting that the nuclear functions of SIZ1 as regulator of plant immunity and physiology may be modulated by GpRbp-1. Furthermore, nematode infection assays and transcriptomic profiling indicate that SIZ1 is required for susceptibility to cyst nematodes. So, these data indicate that E3 SUMO ligases may play an important role in plant-nematode interactions. Based on the prediction of SUMO acceptor and interaction sites in GpRbp-1, a model is proposed in which the effector may recruit SIZ1 to be SUMOylated for full functionality in host cells.

The DOG box is not a quantitative predictor of temporal expression. A. In G. pallida, genes with four or more motifs and a signal peptide are primarily expressed during the infective stage. Each line represents the expression pattern of a different gene. B. The number of motifs does not correlate with gene expression at J2 (black, all numbers of motifs; red, four or more motifs). (PDF 1707 kb)

Phylogenetic analysis of G. rostochiensis pathotypes. A. A maximum likelihood phylogeny based on 730,705 genome wide SNPs. Two distinct groups of Ro1 are together separated from Ro2, Ro3, Ro4 and Ro5. Node labels indicate bootstrap support values for 100 iterations. B. Principle component analysis (PCA) based on the same dataset suggest that for pathotype Ro1, intra-pathotype variation is similar to inter-pathotype variation. REF Ro1 = Reference strain Ro1 assembly, MAR149 Ro1 = British Columbia, QC Ro1 = Quebec, Ro19 Ro1 = European, NFLD Ro1 = Newfoundland, SCRI_Ro2, SCRI_Ro3, SCRI_Ro4, SCRI_Ro5 are pathotype populations from the James Hutton Institute collection. (PDF 1636 kb)

Comparison of gene expression between parasitic G. pallida life stages. The key transitions to be captured in terms of gene expression of all genes, and in particular for effectors, is from outside the plant (J2) compared to inside the plant (sedentary female). There is almost no difference in global gene expression between the early sedentary time points [22]. A. 84 % of the variation in expression at 7 days post infection (dpi) is explained by variation in expression at 14 dpi. B. This correlation is even more profound if the analysis is restricted to only the effectors (89 %). Similar correlations are possible between 14 and 21 dpi, albeit of lesser magnitude but an identical trend (60 % correlation for all genes, and 64 % correlations for specifically effectors). This is not the case, however, when comparing J2 and 7 dpi (44 % for all genes, and zero correlation for all effectors). Fourteen dpi provides an ideal intermediate for the sedentary stages. (PDF 4379 kb)

Supplementary methods and results. (DOCX 28 kb)

Differential expression super-clusters. Ninety-four percent of all differentially expressed genes are manually grouped into 25 biologically relevant expression super-clusters. For each super-cluster, individual cluster graphs are shown where for all expression displayed as centred log fold-change is in the order, Cyst, Cyst, Egg, Egg, J2, J2, 14 dpi, 14 dpi. (PDF 1669 kb)

Phylum wide analysis of GC/AG splice sites in nematodes. The percentage of GC/AG splices sites with associated consensus sequences are shown for 17 species against a schematic phylogeny of the phylum Nematoda (adapted from [89]). Red numbers indicate those which likely represent under reporting due to over-strict parameter settings during gene prediction. (PDF 1371 kb)

Background: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes.Results: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative ‘effector islands’ in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking.Conclusions: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.

Phylogenetic analysis of Worm-specific Argonauts (WAGOs). A. A total of 604 nematode and non-nematode argonaute proteins drawn as an unrooted phylogram. It is assumed that each subtree (Bâ F) is effectively rooted by the other subtrees; however, the extreme divergence between these proteins yields low support for some subtrees. Bâ F Subtrees containing G. rostochiensis WAGOs (GrWAGOs). Branch widths of subtrees are drawn proportional to branch support. Coloured boxes indicate membership of taxa to phylogenetic groups (nematode Clade I, Clade III, Clade IV and Clade V, non-nematode taxa) and coloured stars indicate clades composed entirely of Heteroderidae-WAGOs (Globodera spp., Meloidogyne spp., Heterodera glycines). Orange dots indicate GrWAGOs in differential expression super-clusters 19, 20 or 21. GrWAGOs are placed within Nematode WAGO-subclades ALG1/ALG2 (1), RDE1/ERGO1/PRG1/2 (2), WAGO1/2/4/5 (7), CSR/WAGO-III/WAGO-IV (8) and NRDE/WAGO-10/11 (5), sensu Buck and Blaxter, 2013 [30]. As expected, no GrWAGOs were observed in WAGO-subclades SAGO2/PPW, WAGO-III/-V and ALG3/ALG4. Subclades NRDE/WAGO-10/11, WAGO1/2/4/5 and CSR/WAGO-III/WAGO-IV show increased numbers of paralogous expansion of these gene families within Clade IV in general and Heteroderidae in particular. B All Globodera spp. and Meloidogyne spp. exhibit one ALG1/ALG2 orthologue each, which form a clade. C The RDE1/ERGO1/PRG1/2 subtree contains another Heteroderidae-specific clade; however, one M. incognita sequence is sister to a subclade of Clade III taxa. This is surprising since the Clade III parasites A. suum and B. pahangi are thought to have lost the piRNA pathway [30] and may be an artefact. D The NRDE/WAGO-10/WAGO-11 subtree shows an expansion of paralogous Heteroderidae-WAGOs, with three GrWAGOs expressed at 14 dpi. E The WAGO1/2/4/5 subtree depicts two Heterodera-specific expansions, of which the larger subclade contains four GrWAGOs expressed at 14 dpi. F The CSR/WAGO-III/WAGO-IV subtree contains another two Heteroderidae-specific expansion, of which one also includes a sequence from the Clade V nematode Dictyocaulus viviparus (ochre star). (PDF 64 kb)

BACKGROUND: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. RESULTS: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative ‘effector islands’ in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. CONCLUSIONS: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.