FMRFamide-Like Peptide 22 Influences the Head Movement, Host Finding, and Infection of Heterodera glycines

Abstract

The FMRFamide-like peptides (FLPs) represent the largest family of nematode neuropeptides and are involved in multiple parasitic activities. The immunoreactivity to FMRFamide within the nervous system of Heterodera glycines, the most economically damaging parasite of soybean [Glycine max L. (Merr)], has been reported in previous research. However, the family of genes encoding FLPs of H. glycines were not identified and functionally characterized. In this study, an FLP encoding gene Hg-flp-22 was cloned from H. glycines, and its functional characterization was uncovered by using in vitro RNA interference and application of synthetic peptides. Bioinformatics analysis showed that flp-22 is widely expressed in multiple nematode species, where they encode the highly conserved KWMRFamide motifs. Quantitative real-time (qRT)-PCR results revealed that Hg-flp-22 was highly expressed in the infective second-stage juveniles (J2s) and adult males. Silencing of Hg-flp-22 resulted in the reduced movement of J2s to the host root and reduced penetration ability, as well as a reduction in their subsequent number of females. Behavior and infection assays demonstrated that application of synthetic peptides Hg-FLP-22b (TPQGKWMRFa) and Hg-FLP-22c (KMAIEGGKWVRFa) significantly increased the head movement frequency and host invasion abilities in H. glycines but not in Meloidogyne incognita. In addition, the number of H. glycines females on the host roots was found to be significantly higher in Hg-FLP-22b treated nematodes than the ddH₂O-treated control J2s. These results presented in this study elucidated that Hg-flp-22 plays a role in regulating locomotion and infection of H. glycines. This suggests the potential of FLP signaling as putative control targets for H. glycines in soybean production.

Keywords: FMRFamide-like peptides; Heterodera glycines; neuropeptides; soybean; soybean cyst nematode.

Figure 1

(A) PCR amplification of flp-22 cDNA fragment of Heterodera glycines. M: 100 bp marker. (B) Multiple sequence alignment of FLP-22 peptide from H. glycines (GenBank Accession No. MW645239) with homologs from other nematode species using MULTALIN online tool (http://multalin.toulouse.inra.fr/multalin/). The predicted signal peptide of Hg-FLP-22 is marked by a green box. Residues identical to those of FLP-22 are indicated by the identical color of amino acids. Conserved Hg-FLP-22-like motifs are flanked by mono (R) or dibasic (KR) cleavage sites. (C) Phylogenetic tree of nematode flp-22 genes. Putative FLP-22 peptides from H. glycines and other 41 nematode species were obtained with BLAST using Hg-flp-22 sequence as a query. The evolutionary tree was generated by using the Neighbor-Joining method based on Poisson distance correction. The bootstrap analysis consisted of 1,000 replicates, and the cutoff value for a condensed tree was 38%.

Figure 2

The expression levels of Hg-flp-22 by quantitative real-time (qRT)-PCR analysis in eggs, pre-parasitic J2s (pre-J2s), parasitic J2s (par-J2s), J3s/J4s, and adult females and males. Gene expressions were calculated by using the 2^−ΔΔCT method and were presented relative to those of eggs (normalized with an endogenous reference gene Hg-GAPDH). Values are means ± SD from three biological replicates. Bars with the same letters indicate no significant difference at p > 0.05 (One-way AVOVA, Tukey’s HSD test).

Figure 3

(A) Fluorescence microscopy indicating uptake of fluorescein isothiocyanate (FITC) in dsRNA buffer by H. glycines J2s after 24 h incubation. (B) Pre-parasitic J2s were incubated in the socking buffer, Hg-flp-22 dsRNA, or gfp dsRNA for 24 h. Then, the Hg-flp-22 transcript levels in nematodes were analyzed by qRT-PCR. The results are the means ± SD of three biological replicates shown relative to expression in J2s maintained in the socking buffer. The same letters indicate no significant differences between treatments (p > 0.05, Tukey’s HSD test).

Figure 4

Effect of in vitro RNAi of Hg-flp-22 on H. glycines attraction and penetration in PF-127 medium. (A) The number of nematodes touching or within 5 mm of the root was counted at 4 h after starting the assay. Data are the means ± SD (n = 10) and are representative of three independent experiments. Statistical analysis was performed using Tukey’s HSD test. The same letters indicate no significant differences between treatments (p > 0.05). (B) The number of nematodes around the root within the terminal 5 mm was counted at 4 h after starting the assay. Data are the means ± SD (n = 10), and bars with the same letter indicate no significant difference at p > 0.05 (Tukey’s HSD test). J2s soaked with gfp dsRNA were used as the control. (C) Representative images showing the attraction of Hg-flp-22 dsRNA-treated J2s toward soybean root tips in PF-127 medium at 4 h after starting the assay. Scale bar = 500 μm. (D) The number of stained J2s inside the root was counted at 7 and 16 h after starting the assay. Values are the mean ± SD from one representative experiment (n = 10). The same letter indicates no significant differences at p > 0.05 (Tukey’s HSD test). J2s treated with gfp dsRNA were used as the control. (E) Stained J2s in soybean roots. Scale bar = 1 mm.

Figure 5

Effect of in vitro RNAi of Hg-flp-22 on H. glycines infection and development in soil. (A) The penetration ability of Hg-flp-22-silenced or gfp dsRNA-treated nematodes in soybean roots grown in pots containing sterilized sand and soil. Each plant was inoculated with 200 J2s. The number of nematodes in soybean roots was counted at 24 h after inoculation (hai) through acid fuchsin staining. Data are the means ± SD from eight plants and are representative of two independent experiments. The same letter denotes no significantly difference in the number of Hg-flp-22 dsRNA-treated J2s (p > 0.05, Tukey’s HSD test) compared with that of nematodes soaked with gfp dsRNA. (B) Females in soybean roots were observed 21 days after inoculation (dai) in two independent experiments. Values are the means ± SD from eight plants. Bars with the same letter indicate no significant difference at p > 0.05 (Tukey’s HSD test). J2s treated with gfp dsRNA were used as the control.

Figure 6

Effect of three mature Hg-FLP-22 peptides on the in vitro behavior of H. glycines J2s. (A) Effect of Hg-FLP-22 peptides on the frequency of J2 head movements per minute. Each value represented the mean head movement frequency ± SD from 60 individual nematodes. The same letters indicate no significant differences between the peptide treatments and ddH₂O control (p > 0.05, Dunnett’s test). (B) Effect of Hg-FLP-22 peptide treatments on chemotaxis indices of H. glycines J2s. Each bar represents the mean ± SD of the data from the two independent experiments. The same letters indicate no significant differences from ddH₂O control (p > 0.05, Dunnett’s test).

Figure 7

Effect of Hg-FLP-22 peptides on infection and development of H. glycines in soil. (A) Host invasion ability of Hg-FLP-22 peptides or ddH₂O-treated nematodes in soybean roots in pots containing sterilized sand and soil. Each plant was inoculated with 200 J2s. The number of nematodes in soybean roots was counted at 24 hai through acid fuchsin staining. Bars are the mean ± SD from one representative experiment (n = 8). These experiments were repeated three times with similar results. (B) Females in soybean roots were counted 21 dai. Values are the means ± SD from eight plants and are representative of three independent experiments. The same letters indicate no significant differences between the peptide treatments and ddH₂O control as determined by Dunnett’s test at p > 0.05.