Co-Silencing of the Voltage-Gated Calcium Channel β Subunit and High-Voltage Activated α1 Subunit by dsRNA Soaking Resulted in Enhanced Defects in Locomotion, Stylet Thrusting, Chemotaxis, Protein Secretion, and Reproduction in Ditylenchus destructor

Abstract

The voltage-gated calcium channel (VGCC) β subunit (Ca_vβ) protein is a kind of cytosolic auxiliary subunit that plays an important role in regulating the surface expression and gating characteristics of high-voltage-activated (HVA) calcium channels. Ditylenchus destructor is an important plant-parasitic nematode. In the present study, the putative Ca_vβ subunit gene of D. destructor, namely, DdCa_vβ, was subjected to molecular characterization. In situ hybridization assays showed that DdCa_vβ was expressed in all nematode tissues. Transcriptional analyses showed that DdCa_vβ was expressed during each developmental stage of D. destructor, and the highest expression level was recorded in the third-stage juveniles. The crucial role of DdCa_vβ was verified by dsRNA soaking-mediated RNA interference (RNAi). Silencing of DdCa_vβ or HVA Ca_vα₁ alone and co-silencing of the DdCa_vβ and HVA Ca_vα₁ genes resulted in defective locomotion, stylet thrusting, chemotaxis, protein secretion and reproduction in D. destructor. Co-silencing of the HVA Ca_vα₁ and Ca_vβ subunits showed stronger interference effects than single-gene silencing. This study provides insights for further study of VGCCs in plant-parasitic nematodes.

Keywords: Cavβ; Ditylenchus destructor; RNAi; plant-parasitic nematode; voltage-gated calcium channels.

Figure 1

DdCa_vβ sequence analysis. (A), The DdCa_vβ cDNA sequence and deduced amino acid sequence. The start codon (ATG) and the stop codon (TAA) are indicated in boxes. The fragment that is not translated before the start codon is the 3’ UTR, and the fragment that is not translated after the termination codon is the 5’ UTR. The underlined area represents SL1 at the front end of the 5’ UTR and the poly-A tail at the end of the 3’ UTR. The asterisk (*) indicates the stop codon (TAA). The light blue and brown colors indicate the NH₂ terminus and the COOH terminus, respectively; yellow, the SH3 domain; purple, the HOOK region; and green, the GK domain. The residues that are involved in interactions with the AID are marked with the “•” symbol. (B), The predictive structure of DdCa_vβ.

Figure 2

Molecular phylogenetic analysis of DdCa_vβ via the neighbor-joining method. The species that were used in phylogenetic tree construction are summarized in Table 1. The phylogram was constructed based on the amino acid sequences of 16 Ca_vβ proteins using MEGA 7.0. The numbers below the branches indicate the bootstrap values.

Figure 3

Localization of the expressed DdCa_vβ gene in D. destructor via in situ hybridization. (A–D) In situ hybridization of sense probes for DdCa_vβ in D. destructor. (E–H) In situ hybridization of antisense probes for DdCa_vβ in D. destructor. st: stylet; mb: median bulb; e.g., esophageal gland; an: anus; vd: vas deferens; sp: spicules. Scale bar = 20 μm.

Figure 4

The relative expression levels of DdCa_vβ at different developmental stages of D. destructor. The expression level in J2s was used as the standard value. The data are presented as the mean ± s.d of three biological replicates and three technical replicates (n = 9). Asterisks indicate a significant difference at the level of p < 0.01, as tested by Duncan’s new complex difference method.

Figure 5

The relative expression levels of DdCa_vβ, DdCα1D, and DdCα1A in D. destructor after soaking with specific dsRNA. (A) The relative expression levels of the DdCa_vβ, DdCα1D, and DdCα1A genes after dsCa_vβ treatment. (B) The relative expression levels of the DdCa_vβ, DdCα1D, and DdCα1A genes after dsCα1D treatment; (C) The relative expression levels of the DdCa_vβ, DdCα1D, and DdCα1A genes after dsCα1A treatment; (D) The relative expression levels of the DdCa_vβ, DdCα1D, and DdCα1A genes after dsCα1D + dsCa_vβ treatment; (E) The relative expression levels of the DdCa_vβ, DdCα1D, and DdCα1A genes after dsCα1A + dsCa_vβ treatment. Significant differences between the treatment and control are indicated with a line with asterisks (** p < 0.01; Student’s t test). “NS” indicates that there was no significant difference between the samples.

Figure 6

Silencing of Ca_vβ or HVA Ca_vα₁ and co-silencing of Ca_vβ and HVA Ca_vα₁ affected the locomotory activity of D. destructor. Nematodes that were treated with gfp dsRNA were used as controls. Different lowercase letters indicate a significant difference at the level of p < 0.05, as tested by Duncan’s new complex difference method.

Figure 7

Silencing of Ca_vβ or HVA Ca_vα₁ and co-silencing of Ca_vβ and HVA Ca_vα₁ caused defects in the chemotaxis of D. destructor toward sweet potato slices. Nematodes that were treated with gfp dsRNA were used as controls. Different lowercase letters indicate a significant difference at the level of p < 0.05, as tested by Duncan’s new complex difference method.

Figure 8

Silencing of Ca_vβ or HVA Ca_vα₁ and co-silencing of Ca_vβ and HVA Ca_vα₁ affected the stylet thrusting of D. destructor. Nematodes that were treated with gfp dsRNA were used as controls. Different lowercase letters indicate a significant difference at the level of p < 0.05, as tested by Duncan’s new complex difference method.

Figure 9

Silencing of Ca_vβ or HVA Ca_vα₁ and co-silencing of Ca_vβ and HVA Ca_vα₁ affected the protein secretion by D. destructor. Nematodes that were treated with gfp dsRNA were used as controls. Different lowercase letters indicate a significant difference at the level of p < 0.05, as tested by Duncan’s new complex difference method.

Figure 10

Silencing of Ca_vβ or HVA Ca_vα₁ and co-silencing of Ca_vβ and HVA Ca_vα₁ affected the reproduction rate of D. destructor. Nematodes that were treated with gfp dsRNA were used as controls. Different lowercase letters indicate a significant difference at the level of p < 0.05, as tested by Duncan’s new complex difference method.