Fig 1.
Identification of selected candidate effector gene families of Radopholus similis.
(A) Maximum-likelihood phylogenetic tree of glutathione synthetase (GS) proteins of R. similis with GS sequences of other plant-parasitic nematodes (adapted from [41]). Clade 1 (green) contains the “progenitor” GS sequence of each nematode species included in the tree. Clade 2 (blue) represents the first expansion of GS genes in plant-parasitic nematodes and contains 9 GS coding genes from R. similis. Arrows indicate putative secreted GS-like effectors of R. similis. Clade 3 (red) represents the second GS genes expansion, and contains all known GS-like effectors of cyst and reniform nematodes. (B) Number of genes encoding putative cell wall-degrading proteins in R. similis. GH: glycosyl hydrolyse; PL: pectate lyase; EXPB: expansin-like.
Fig 2.
Identification of known candidate effector genes identified in the esophageal gland cell library of Radopholus similis by in silico analyses.
(A) Top most abundant PFAM protein domains represented within the list of 542 transcripts coding putative secreted proteins. (B) List of selected known candidate effector genes present in the esophageal glands library of R. similis. (C-J) Detection of gene transcripts encoding putative secreted proteins in the esophageal glands of R. similis by in situ hybridization. (C) ß-1,4-endoglucanase (positive control, g11661.t1), (D) ß-1,4- endoglucanase (g4814.t1), (E) pectate lyase (g460.t1), (F) expansin-like (g15237.t1), (G) venom allergen-like (g14608.t1), (H) ShK-Like domain protein (g11934.t1), (I) glutathione synthetase (g13505.t1), (J) Extracellular solute-binding protein (g10597.t1). (K) Example of a control image obtained using the sense probe (e.g. g460.t1). Bars = 20 μm.
Fig 3.
Detection of Radopholus similis gene transcripts encoding pioneer secreted proteins by in situ hybridization.
(A-S) Transcripts encoding 19 different genes were localized in the nematode esophageal glands of Radopholus similis using the corresponding anti-sense DIG-labeled cDNA probes. (A) g9016.t1, (B) g5306.t1, (C) g14603.t1, (D) g10323.t1, (E) g2910.t1, (F) g10498.t1, (G) g4420.t1, (H) g1733.t1, (I) g8454.t1, (J) g3018.t1, (K) g14271.t1, (L) g13627.t1, (M) g2281.t1, (N) g11602.t1, (O) g7057.t1, (P) g4680.t1, (Q) g11095.t1, (R) g8376.t1, (S) g3015.t1. Due to the high variability of the esophageal gland size among different specimens and nematode stages, both dorsal and subventral glands were labeled as esophageal glands. Details regarding each gene abundance within the glands library is presented in S2 Table. (T) Example of a control image obtained using the sense probe (e.g. g14603.t1). g: esophageal glands; m: median bulb; s: stylet. Bars = 20 μm.
Fig 4.
Detection of gene transcripts encoding putative secreted proteins and localized in different Radopholus similis tissues by in situ hybridization.
(A) g9088.t1, (B) g1157.t1, (C) g12860.t1, (D) g12895.t1, (E) g5235.t1, (F) g11460.t1 and (G) g13141.t1. White arrows in images C-G indicate “cell-like bodies” along the anterior region of the nematode. (H) Example of a control image obtained using the sense probe (e.g. g9088.t1). (ed): excretory duct; m: median bulb; s: stylet. Bars = 20 μm.
Fig 5.
Identification of a non-coding motif associated with subventral gland cell expression in Radopholus similis: the Rs-SUG box.
(A) Graphic representation of the strategy applied to identify a highly enriched consensus motif of 10 nucleotides (CTG[T|A|C][T|G|A]CAAAC) in the promoter region of gland effector genes. (B) Number of genes coding predicted secreted proteins containing at least one motif in the promoter region (n = 149). (C) The more motifs present in the promoter region, the more likely is that the corresponding gene will contain a signal peptide for secretion. (D) The frequency of the motif peaks mainly in the 100–300 bp upstream the start codon and is non-strand specific. (E) The distribution of the motif within the 1,500 bp upstream the start codon correlates with genes coding putative secreted proteins. (F) Graphic representation of the motif variant combinations. Seven (black bars) out of the nine variants showed enrichment for putatively secreted proteins.
Fig 6.
Identification of candidate effector genes through promoter motif searches in the genome of Radopholus similis.
(A) List of candidate effector genes encoding secreted proteins without transmembrane domains identified by promoter motif CTG[T|A|C][T|G|A]CAAAC searches. (B) Maximum-likelihood phylogenetic tree of R. similis GH62 protein sequence (in red) with representative sequences retrieved from bacteria at NCBI (100 top hits). Bootstrap values are presented in the corresponding branches. (C) Graphic representation of R. similis GH62 gene model and corresponding RNAseq coverage. Exons are represented by red boxes, and signal peptide by a black arrow. (D-G) Genes associated with the Rs-SUB box coding predictive secreted proteins were localized in the nematode esophageal glands by in situ hybridization: (D) α-L-arabinofuranosidase GH62 (g448.t1), (E) endo-1,4-ß-xylanase GH30 (g15208.t1), (F) ShK-domain protein (g6688.t1), (G) pioneer gene (g4323.t1). *Validated in the esophageal glands in Fig 2. **Validated in the esophageal glands after the Rs-SUB box identification. g: esophageal glands; m: median bulb; s: stylet. Bars = 20 μm.
Fig 7.
Gene expression analyses of candidate effector genes of Radopholus similis across different nematode developmental stages.
(A) Genes validated in the esophageal glands of the nematode as shown in Fig 2. (B) Genes validated in different nematode tissues as shown in Fig 3. Heat maps represent the expression levels calculated using the fragments per kilo base of transcript per million mapped reads (FPKM) of specific nematode developmental stages RNA-seq libraries (Bioproject PRJNA427497).
Fig 8.
Expression profile of Radopholus similis candidate effector genes in planta.
(A-D) Acid fuschin staining of nematodes (red color) in carrot hairy roots at 1 (A), 3 (B), 7 (C) and 30 (D) days after inoculation. (E) Semi-quantitative RT-PCR validating the expression of 20 candidate effectors in carrot hairy roots at 1, 3 and 7 DAI. As a positive control, all cDNA libraries were amplified with primers derived from the α-tubulin gene of R. similis or the translation elongation factor EF-1 alpha gene (EF-1α) of carrot. C corresponds to non-infected carrot hairy roots. (F) Heat map representing the expression profile of 30 candidate effectors validated herein in the esophageal glands of R. similis. The expression levels were calculated using the fragment per kilobase of transcript per million mapped reads (FPKM) values of two independent RNA-seq libraries generated from mixed nematode stages collected from carrot hairy roots at 30DAI.
Fig 9.
Phenotypic changes in Nicotiana benthamiana plants infected with recombinant potato virus X carrying cell wall-modifying genes of Radopholus similis.
(A-C) Expression of Rs-PEL gene induced large necrotic lesion and overall chlorosis in leaves (A-B) and stems (C) of the infected plants. (D-F) Expression of Rs-GH16 gene induced chlorotic and uneven appearance of the leaf tissue, as well as necrotic lesion in the leaves (D-E), and pitting or grooving-like phenotype in the stems ((indicated by arrows in F). (G-I) Plants expressing PVX/Pp-EXPB displayed necrotic spots in the leaves (G-H), and lesion-like spots in the stems (indicated by arrows in I). (J-L) Typical mosaic-like symptoms induced by PVX/RsGH62 and (M-N) PVX wild-type (PVX/WT) on leaves of N. benthamiana plants. All photos were taken 14 days after inoculation.
Fig 10.
Radopholus similis holds a diverse and emergent repertoire of effectors shaped by diverse evolutionary events.
Top panel corresponds to a schematic phylogeny of the phylum Nematoda based on 86 highly conserved genes among plant-parasitic nematodes with distinct lifestyles, and the free-living nematode Caenorhabditis elegans (adapted from [38]). The lower panel indicates the presence/absence of putative homologues through BLAST analyses (e-value < 10−5 and bit score > 50) using as query the full set of effectors identified so far for R. similis. The new candidate effectors identified in this study are represented in bold (n = 30). Numbers within the cells of R. similis correspond to the number of genes coding putative secreted proteins in relation to the total number of related genes found in the Rv genome. *Both GH30 and GH5 are represented by the corresponding genes (g15208.t1 and g4814.t1) validated in this study, while cathepsin proteinases are represented by g8452.t1. Legends: Pratylenchus penetrans; Pc: Pratylenchus coffeae; Pn: Pratylenchus neglectus; Pt: Pratylenchus thornei; Mi: Meloidogyne incognita; Mh: Meloidogyne hapla; Na: Nacobbus aberrans; Rs: Radopholus similis; Rr: Rotylenchulus reniformis; Hg: Heterodera glycines; Gr: Globodera rostochiensis; Gp: Globodera pallida; Dd: Ditylenchus destructor; Bx: Bursaphelenchus xylophilus; Ce: Caenorhabditis elegans. RKN: root-knot nematodes; CN: cyst nematodes.