1 Supplementary Figure Legends
2
3 Figure S1.
Alignment of the predicted ubiquitin carboxyl extension protein of Globodera
4 rostochiensis (GrUBCEP12) with homologues from Heterodera nematode species,
5 Saccharomyces cerevisiae , Caenorhabditis elegans , and Arabidopsis thaliana .
6 Unlike the canonical ubiquitin carboxyl extension proteins that contain a relatively long carboxyl
7 extension protein (CEP) of either 52-amino acid (aa) (CEP52) or 76- to 80-aa (CEP76-80),
8 UBCEPs from cyst nematodes form a unique class that contain an N-terminal signal peptide for
9 secretion, a monoubiquitin domain, and a short CEP domain showing no similarity to CEP52 and
10 CEP76-80. Included ubiquitin carboxyl extension proteins are CeUBIB (L31492) from C.
11 elegans , ScUBI1 (P0CH09) and ScUBI3 (P05759) from S. cerevisiae , AtUBQ1 (AAA32904)
12 and AtUBQ5 (AAA32906) from A. thaliana , HgUBI1 (AAN32889) from H. glycines , and
13 HsUBI1 (AAP30081) from H. schachtii . Each domain in these proteins is indicated and identical
14 and similar aa residues are shaded in black and gray, respectively.
15
16 Figure S2. In situ mRNA hybridization on nematode specimen.
17 The digoxigenin-labeled sense GrUBCEP12 cDNA probe did not give any hybridization signal
18 in both preparasitic (a) and parasitic (b) life stages of Globodera rostochiensis . Scale bars, 10 µm;
19 DG, dorsal gland; M, metacorpus; S, stylet.
20
21 Figure S3. Immunofluorescence labeling on nematode specimen.
22 Immunofluorescence labeling using an antiubiquitin antibody was observed in the tail region of a
23 preparasitic second-stage juvenile of Globodera rostochiensis . Scale bars, 50 µm.
1

24
25 Figure S4. Subcellular localization of a control GFP:GUS fusion protein in the plant cell.
26 The empty vector control (GFP:GUS alone) was transformed into onion epidermal cells by
27 biolistic bombardment. The GFP signal was only observed in the cell cytoplasm.
28
29 Figure S5.
Schematic view of the RNAi construct and the GrUBCEP12 gene.
30 (a) Schematic structure of the hairpin double-stranded RNA (dsRNA) construct targeting
31
32
GrUBCEP12 . Both the sense and antisense sequences corresponding to the CEP domain and the
3’untranslated region of
GrUBCEP12 were cloned into the pSUPERMD-RNAi vector for
33 producing the hairpin dsRNA in transgenic potato plants.
34 (b) Schematic structure of GrUBCEP12 . Open boxed areas represent GrUBCEP12 encoded
35 protein and the thick line indicates the 3’ untranslated region (UTR) of GrUBCEP12 . The
36 specific region targeted for RNAi is indicated as shown. SP, signal peptide; UB, ubiquitin
37 domain; CEP, carboxyl extension protein domain.
38
39 Figure S6. Expression of non-RNAi target genes in feeding nematodes.
40 qPCR was used to examine the expression of GrCM1 (Lu et al ., 2008) (a) and GrUBCEP52 (b),
41 a canonical UBCEP gene, in nematodes recovered from RNAi lines in comparison with those
42 recovered from the control lines. Expression levels of GrCM1 and GrUBCEP52 in nematodes
43 recovered from the independent RNAi lines were not significantly different from those in
44 nematodes recovered from the control lines. Values are means ± SDs of two independent
45 experiments.
2