A variant of the venom allergen-like protein, Dd VAP2, is required for the migratory endoparasitic plant nematode Ditylenchus destructor parasitism of plants

Abstract

The potato rot nematode, Ditylenchus destructor, poses a serious threat to numerous root and tuber crops, yet the functional characterization of effectors from this migratory endoparasitic plant nematode remains limited. Despite inhabiting distinct habitats, sedentary and migratory plant parasitic nematodes share the structurally conserved effectors, such as venom allergen-like proteins (VAPs). In this study, a variant of DdVAP2 was cloned from D. destructor. The transcription profile analysis revealed that DdVAP2 was higher expressed in D. destructor feeding on either potato or sweet potato compared to on fungus via qRT-PCR. And DdVAP2 was highly expressed at all life stages feeding on sweet potato, except for eggs. DdVAP2 was confirmed to be specifically expressed in the subventral esophageal glands of D. destructor through in situ hybridization assays. Combined with functional validation of the signal peptide of DdVAP2, it suggested that DdVAP2 could be secreted from nematode into host. Heterologous expression of DdVAP2 in Nicotiana benthamiana revealed that the protein localized in both cytosol and nuclei of plant cells. Knocking down DdVAP2 by RNAi in D. destructor resulted in infection and reproduction defects on plants. All the results suggest that DdVAP2 plays a crucial role in the interaction between D. destructor and plants by facilitating the nematode infection.

Keywords: Ditylenchus destructor; RNAi; effector; migratory endoparasitic plant nematode; venom allergen-like protein.

Figure 1

Sequencing and sorting intolerant from tolerant (SIFT) analysis of the sequenced DdVAP2. (A) ORF alignment of the sequence obtained and sequenced in this study with the published DdVAP2 (GU370352). GU370352: the sequence published on NCBI by a former study; sequenced: the sequence obtained and sequenced in this study. (B) Alignment of the amino acid sequences encoded by the DdVAP2 sequenced in this study and the published DdVAP2 (GU370352). GU370352: the sequence published on NCBI by a former study; sequenced: the sequence obtained and sequenced in this study. (C) The effects of the two amino acid substitutions between the two amino acid sequences of DdVAP2 were analyzed using SFIT4G (https://sift.bii.a-star.edu.sg/sift4g/AnnotateVariants.html).

Figure 2

Phylogenetic analysis of VAPs from Ditylenchus destructor and other plant parasitic nematodes. All the shaded sequences are from Ditylenchus destructor. Black circles indicate two VAPs cloned from Ditylenchus destructor before, and the red star indicate the DdVAP2 cloned and sequenced in this study.

Figure 3

Functional validation of the signal peptide of DdVAP2. YTK12 was untransformed Saccharomyces cerevisiae strain, and the YTK12 strain carrying the original pSUC2 vector were defined as pSUC2. The first 25 amino acids of Mg87 (no signal peptide function) from Magnaporthe oryzae was transformed into YTK12 strain with the pSUC2 vector to serve as a negative control. The signal peptides of Avr1b from Phytophthora sojae and DdVAP2 were cloned into pSUC2 for expression in YTK12 strains. CMD-W is the medium with 0.67% yeast nitrogen base without amino acids, 0.075% tryptophan dropout supplement, 2% sucrose, 0.1% glucose and 2% agar. And YPRAA is the medium with 1% yeast extract, 2% peptone, 2% raffinose, 2mg/ml antimycin A and 2% agar.

Figure 4

Transcription pattern of DdVAP2 in Ditylencus destructor. (A) Relative expression levels of DdVAP2 in (D) destructor populations cultured on either F. semitectum, potato or sweet potato. (B) Relative expression levels of DdVAP2 in different life stages of Ditylencus destructor. Vertical lines indicate standard errors of the mean from two independent biological replicates. Different letters indicate significant differences (P < 0.05).

Figure 5

Tissue localization of DdVAP2 in Ditylenchus destructor. (A, B) No hybridization signals were detected in negative controls with the digoxigenin-labeled sense DdVAP2 RNA probe. (C, D) Specific hybridization signals were detected in the esophageal glands of D. destructor with the digoxigenin-labeled antisense DdVAP2 RNA probe. S, stylet; M, median bulb; SVG, subventral oesophageal glands. Bars indicate 50 μm.

Figure 6

Subcellular localization of DdVAP2 in Nicotiana benthamiana. The original vector expressing only green fluorescent protein (GFP) was control. The transient expression of a DdVAP2ΔSP-GFP fusion protein was named as DdVAP2ΔSP. DIC is light field, and Merged is the combination of both fluorescence and light field. Bars indicate 20 μm.

Figure 7

Functional analysis of DdVAP2 during Ditylenchus destructor interaction with plants using RNAi. (A) Knock-down efficiencies of DdVAP2 after nematodes soaked in soaking buffer containing dsRNA of either GFP, RNAi-1 or RNAi-2 for 24, 48 and 72 h. Values are expressed relative to the reference gene actin (GenBank accession no. GQ327968), with the dsRNA of GFP treatment set to 1. Values represent mean ± SE of three independent replicates. Differences were assessed using ANOVA. Different letters indicate P < 0.05. (B, C) Number of nematodes and reproduction rate in different treatments. All the statistical results were obtained from five independent replicates and values represent mean ± SE of three independent biological replicates. Differences were assessed using ANOVA. Different letters indicate P < 0.05.