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

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.


INTRODUCTION
Soybean [Glycine max L. (Merr)] is an important and frequently consumed crop and serves as a major source of protein and oil worldwide. However, its yield is strongly affected by various pests and pathogens, including plant-parasitic nematodes (PPN). The soybean cyst nematode, Heterodera glycines Ichinohe, a sedentary and endoparasitic nematode, is one of the most damaging pests attacking soybean (Niblack et al., 2006). An estimated annual soybean yield loss by H. glycines has been reported to be around US$1 billion in the United States (Koenning and Wrather, 2010). This nematode is also a major constraint to soybean production in China (Ou et al., 2008;Zheng et al., 2009;Wang et al., 2015;Peng et al., 2016). The conventional management strategies rely heavily on the combination of non-host crop rotation (e.g., maize, Zea mays L.) and the use of resistant host cultivars in the field. However, the limited availability of genetic resources for resistance to H. glycines and the genetic variation in the field population of H. glycines restrict the application of this method (Chen et al., 2001;Koenning, 2004;Niblack et al., 2008). In addition, the use of highly effective nematocide is prohibited in some regions because of environmental health and safety concerns (Mereu and Chapman, 2010;UNEP, 2015). Recently, some biocontrol agents were reported to reduce H. glycines reproduction in field conditions (Jensen et al., 2018;Lund et al., 2018;Zhao et al., 2019Zhao et al., , 2020, but the efficacy of the biological agents may be easily affected by environmental conditions in the field. Thus, alternative new approaches are needed for controlling this widespread and destructive pest. The neuromuscular system is considered as the potential target of the effective chemotherapeutic treatments for animalparasitic nematode management because a series of normal parasite biological functions is governed through the nervemuscle coordinated function (Maule et al., 2002;McVeigh et al., 2012). The role of the neuromuscular system in PPN has also been demonstrated in various facets of parasitic activities, such as host location, penetration, and migration inside and outside of host tissues, and even slight head motion at the feeding site (Perry, 1996;Maule et al., 2002;Curtis, 2008;Reynolds et al., 2011;Han et al., 2018). There is an increasing interest in understanding the potential for exploiting key regulatory genes embedded within the neuromuscular system as a target resource to develop PPN control strategies in the agricultural system. Several neuron-related genes have been identified and cloned from PPN (Yan and Davis, 2002;Costa et al., 2009;Kang et al., 2011;Shivakumara et al., 2019). For example, acetylcholinesterases (ACEs) are important in the regulation of neuronal synaptic transmission, in vitro RNA interference, or plant-expressing dsRNAs to effect silencing of ace genes significantly inhibited the invasion, development, and parasitic abilities of PPN (Jae et al., 2011;Cui et al., 2017). Most importantly, transgenic expression of the synthetic peptides, which can disrupt nerve impulse transmission dependent on ACE, conferred plant resistance to Globodera pallida, Radopholus similis, and Meloidogyne incognita in potato, plantain, and eggplant, respectively (Liu et al., 2005;Lilley et al., 2011;Roderick et al., 2012;Papolu et al., 2020). In vitro silencing of two neuropeptide genes, via RNA interference (RNAi) also displayed a significant reduction in attraction and penetration of M. incognita in tomato root in the Pluronic gel system (Dash et al., 2017). Some neuropeptide-like proteins (NLP) identified from PPN were found to negatively regulate chemosensation, host invasion, and stylet thrusting of M. incognita and G. pallida, and exogenous application of the transgenic Bacillus subtilis and Chlamydomonas reinhardtii secreting these NLP can protect tomato from M. incognita and G. pallida invasion (Warnock et al., 2017). The recently reported silencing of chemosensory genes, , which are located in the amphidial neuron and phasmid of M. incognita, resulted in defects in the host recognition and invasion (Shivakumara et al., 2019). In this direction, the neuromuscular system may provide attractive and alternative targets for H. glycines control as part of an integrated management strategy.
To date, studies of FLP functions in PPN have primarily focused on root-knot nematodes, M. incognita and M. graminicola; only in vitro physiological experiments have tested the proteolysis of two synthetic peptides  and their effects on the behavior of H. glycines J2s (Masler, 2010;Masler et al., 2012). Chromatographic analysis of the FaRP immunoactivity from H. glycines further indicated that FLPs are present in all development stages and the highest FLP immunoactivity occurs in the J2 stage (Masler et al., 1999). Early studies also revealed the obvious immunoactivity to FMRFamide in the nervous system of H. glycines (Atkinson et al., 1988). These observations suggested that the flp family may have an important role in the behavior and parasitism of H. glycines. Previously, in order to understand the molecular basis of H. glycines recognizing host signals before J2s penetrate soybean roots and to identify new genes potentially involved in nematode chemoreception, we generated the transcriptome sequence data from H. glycines J2s attracted to host roots in the PF-127 gel system. Based on previous reports (McVeigh et al., 2006;Li and Kim, 2014) and our transcriptome annotation data of H. glycines J2s (unpublished), at least 20 flp genes are present in H. glycines. However, little is known about the functional characteristics of flp genes in H. glycines. In the present study, Hg-FLP-22 encoding gene was identified and cloned from H. glycines J2s, and its expression pattern was analyzed by quantitative real time (qRT)-PCR in different development stages. The functional characterization of Hg-flp-22 was studied in behavior, infection, and development of H. glycines via RNAi and exogenous application of synthetic peptides.

Nematode Culture
Heterodera glycines race 5 was cultured on a susceptible commercial soybean cultivar "DongSheng1" in a greenhouse at 22-28°C in the Northeast Institute of Geography and Agroecology, Harbin, China. Cysts were collected from the soil and soybean roots from the culture pots. Eggs were released by manually crushing cysts with a rubber stopper and then harvested by pouring the solution through a 25 μm pore sieve. Collected eggs were further isolated from debris through sucrose (100%, w/v) centrifugation and thoroughly washed in distilled water. Eggs were kept in 3 mM ZnSO 4 solution in an incubator at 28°C for hatching. Fresh J2s were collected at 3-4 days and then used immediately in each experiment. A pure culture of M. incognita was maintained on tomato cv. "Zhongshu-4" in the greenhouse under the same conditions as mentioned above. Nematode eggs and J2s were collected as previously described by Hu et al. (2017a).

Isolation of Hg-flp-22 Gene From H. glycines
A putative full-length cDNA encoding FLP-22, designated Hg-flp-22, was identified from this study of transcriptome sequence data of H. glycines J2s. Specific primers (Supplementary Table 1) were designated to amplify Hg-flp-22 cDNA containing the predicted full-length open reading frame (ORF) and partial 3' untranslated region (UTR) using Phanta Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). The cycling conditions consisted of an initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 30 s, followed by a final extension at 72°C for 5 min. The amplified PCR product was purified and cloned into pLB fast cloning vector (TIANGEN, Beijing, China), and then transformed into Escherichia coli DH5α chemically competent cells (Biomed, Beijing, China). Recombinant plasmids were isolated from at least four positive clones and sequenced by TSINGKE biological Technology (Harbin, China). Return sequences were analyzed by using BioEdit version 7.2.5.

Bioinformatic Analysis
To perform sequence homology comparisons, Basic Local Alignment Search Tool (BLAST) searches were conducted in the nematode non-redundant and expressed sequence tag (EST) databases at the National Centre for Biotechnology Information (NCBI) BLAST server by using BLASTX and BLASTN. The predicted protein sequences of Hg-flp-22 and its homologs across the nematode species were aligned using online multiple sequence alignment tools with default settings. 1 A phylogenetic tree was constructed using MEGA6 software with the neighborjoining algorithm based on the Poisson distance correction, and the bootstrap test was performed with 1,000 replicates.

Peptide Synthesis and Treatment
Peptides QPAGGVKWMRFa (Hg-FLP-22a), TPQGKWMRFa (Hg-FLP-22b), and KMAIEGGKWVRFa (Hg-FLP-22c) were synthesized by SangonBiotech (Shanghai, China) at 95% purity. Each synthesized peptide was dissolved to sterilized ddH 2 O to make a 10 mM stock solution and was then aliquoted and stored at −20°C. For nematode behavioral assay, J2s of H. glycines or M. incognita were incubated for 15 min in 10 μM, 100 μM, or 1 mM solution of peptides in a PCR tube. For infection assay, J2s were added to 400 μl of peptides solution and incubated for 16 h at room temperature in the darkness. All of the peptides stock stored at −20°C were used within 1 week.  Table 1). dsRNA of an unrelated gene gfp (720 bp) was synthesized from a plasmid containing gfp fragment and used as a non-native negative control. dsRNA soaking of J2s was performed as previously described by Urwin et al. (2002). Approximately 4,000 freshly hatched J2s were soaked in the soaking buffer containing 2 mg/ml dsRNA, 3 mM spermidine, and 50 mM octopamine at room temperature on a rotator in the dark. J2s incubated in dsRNA of gfp gene and soaking buffer (without dsRNA) were set as the control. After 24 h incubation, J2s were washed four times with sterile distilled water through centrifugation to remove the external dsRNA. Additionally, dsRNA uptake efficiency was assessed by using fluorescein isothiocyanate (FITC, 0.1 mg/ml, Sigma, St. Louis, MO, USA) as a tracer in dsRNA buffer, and then, nematodes were observed under a fluorescence microscope (LEICA DM2500, Wetzlar, Germany).

In vitro
Approximately 500 dsRNA-treated J2s were used for total RNA isolation and qRT-PCR analysis of the transcribed abundance of the target gene. The remaining J2s were used to assess behavior, infection, and development of nematode.

Analysis of H. glycines J2 Behavior
For head movement assay of nematode, approximately 100 J2s were incubated in peptides solution for 15 min. Then, J2s were transferred to a cell counting chamber (75 mm × 25 mm × 1.8 mm; chamber depth = 100 μm) with four rooms (10 μl loading volume), and head movements were observed for a randomly selected individual nematode under a stereomicroscope (OLYMPUS SZX-16, Tokyo, Japan). Head movement frequencies of J2s were determined following a definition previously described by Masler et al. (2012). Head movements of each J2 were counted for 1 min. At least 60 J2s from three independent experiments were counted for each peptide treatment.
In the present study, root extracts from 8-day-old soybean plants were used as a source of an attractant to test the chemotaxis effect. Briefly, 0.5 g fresh root tips (0.5 cm) were homogenized in 4 ml sterile distilled water, and homogenates were centrifuged at 12,000 g for 10 min at 4°C. The supernatant was then collected, filter sterilized (0.22 μm, Merck Millipore, Billerica, MA, USA), and used for chemotaxis assay (Hu et al., 2019). Fresh root extracts were collected from each independent experiment. Chemotaxis assays were performed in a 60 mm Petri dish according to the methods previously described (Hu et al., 2019). The dish was filled with 5 ml 1% agar (DAISHIN, Dublin, OH, USA) and allowed to solidify. Two points (A and B) were marked on the back of the dish 2 cm on either side of the dish center. Root extracts of 5 μl were applied at the A point and 5 μl of sterile distilled water (as a control) was added at the B point. After 10 min, approximately 100 J2s were added to the center of the plate, which is also the midpoint of the two marked points. The Petri dishes were placed at room temperature for 4 h in an incubator, and the number of J2s in the vicinity of the injection site of extracts or control was counted. Chemotaxis index was calculated as the number of J2s near the A point minus the number of J2s near the control B point divided by the total number of J2s present at A and B. The experiment was carried out two times with eight replicates each time.
Heterodera glycines attraction assays were conducted in six-well tissue culture plates containing Pluronic F-127 gel (NF Prill Poloxamer 407, BASF, Mount Olive, NJ, United States; Hu et al., 2017b). About 3 ml of 23% (w/v) Pluronic F-127 was added into each well at 4°C, and a 1 cm root piece (with an intact tip) of 4-day-old soybean seedling was placed in the gel. The culture plates were then transferred to room temperature conditions. After solidification, approximately 200 dsRNA-treated J2s were added into each well, and the injection site was about 1 cm from the root tip. The attractiveness of soybean roots to H. glycines was observed using a dissecting microscope, and the number of J2s touching the root surface or within 5 mm of the root tip was counted at 4 h. In addition, the root pieces were collected from gel at 7 and 16 h after starting the assay, and the number of J2s which had penetrated the root was determined by root staining with acid fuchsin (Byrd et al., 1983).

Nematode Infection in Soil
Soybean seeds were surface-sterilized in 1.5% sodium hypochlorite for 15 min, rinsed thoroughly with tap water, and germinated for 4 days in darkness at 26°C, after which they were transferred to pots containing sterilized sand and soil (2:1) and grown in a growth chamber with a photoperiod of 16 h light (26°C) and 8 h dark (20°C) cycle. After 8 days, soybean seedlings were inoculated with 200 H. glycines J2s per plant, the J2s have been pretreated with peptides or dsRNA. All plants were placed in a completely randomized design in a growth chamber and eight biological replicates of each treatment per time-point were used. At 1 day after the nematode inoculation (dai), root systems were harvested and the penetration ability of the nematodes was determined under a stereoscope through acid fuchsin staining. The number of females on the roots was counted under a stereomicroscope using a counter at 21 dai.
For M. incognita inoculation, a similar procedure was used but with a tomato host plant. About 200 J2s were applied on each 2-week-old tomato seedling (cv. Zhongshu-4) which was grown and maintained in a sterilized soil mixture (1:1 mixture of sand and soil) in a 10 cm × 9.5 cm tray, and then, the number of nematodes that had penetrated the host roots was counted at 1 dai. During the course of the experiment, plants were watered four times a week, with one of the watering consisting of Hoagland's nutrient solution (Hoagland and Arnon, 1950). The experiment was conducted three times with at least eight replicates for each time.

RNA Extraction, cDNA Synthesis and qRT-PCR Analysis
For development expression level assay, individuals (1,000-2,000) in different life stages of H. glycines (including eggs, pre-parasitic J2s, parasitic J2s, mixed J3s/J4s, adult females, and males) were used for RNA extraction. The infected roots were harvested at 3 and 12 dai, and the parasitic J2s and mixed J3s/J4s were extracted according to the method as described by Wang et al. (2014). Briefly, the roots were homogenized for 20 s in a juice extractor, and the homogenate was washed through successive 850, 90, and 25 μm pore sieves. Then, the filtrate containing nematodes was purified by a sucrose (50%, w/v) gradient. Males and adult females were obtained from soybean roots at 16 and 21 dai, respectively, in the pouch system (You et al., 2018). Total RNA of nematode samples was purified using an RNAPrep Pure Micro Kit (TianGen Biotech, Beijing, China) following the instructions of the manufacturer including a step DNase treatment. First-strand cDNA was synthesized from 0.5 μg of total RNA using FastKing gDNA Dispelling RT SuperMix FastKing Kit (TianGen Biotech, Beijing, China).

qRT-PCR analysis was carried out in the LightCycler ® 480
System with AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) according to the procedure described by the manufacturer. Cycle conditions were 95°C for 5 min and next 40 cycles of 10 s at 95°C and 30 s at 60°C. The 2 −∆∆Ct method was applied to calculate the related expression of Hg-flp-22 in different life stages and dsRNA treatments (Hu et al., 2017b). Hg-GAPDH was used as a reference control to normalize all qRT-PCR results (Wang et al., 2014). All experiments were performed with three independent biological replicates, each with three technical replicates. The primers used for qRT-PCR were listed in Supplementary Table 1.

Data Analysis
Data were subjected to one-way ANOVA using SPSS version 17.0 software (Chicago, IL, United States). Tukey's HSD test (p < 0.05) was used for overall pairwise comparisons. The error bars in the figures represent the SD of means, and the significance level was set at p < 0.05.

Identification and Sequence Analysis of Hg-flp-22 From H. glycines
A putative flp-22 gene of H. glycines was identified based on our transcriptome sequence data of H. glycines J2s. The cDNA fragment of Hg-flp-22 (479 bp) containing the predicted fulllength ORF and partial 3' UTR was amplified using specific primers ( Figure 1A; Supplementary Table 1). The ORF (GenBank Accession MW645239) encodes a protein precursor of 129 amino acids, including a secretion signal peptide of 38 amino acids at its N-terminal ( Figure 1B). BLAST analysis was conducted in the available nematode EST, genomic, and transcriptomic databases using Hg-flp-22 as a search query, and Hg-flp-22 complements were identified in 42 nematode species, including free-living and animal-and plant-parasitic (Supplementary Table 2). Multiple sequence alignment results showed a considerable sequence diversity of FLP-22 among these species (Figure 1B). In free-living nematodes, Caenorhabditis spp., except C. bovis, flp-22 gene encodes a single peptide SPSAKWMRF-NH 2 with three copies, while flp-22 genes from other nematodes are translated to three distinct active peptides with a common C-terminal KWMRF-NH 2 and a poorly conserved N-terminal. It is worth noting that Hg-FLP-22 was more similar to a homolog from Globodera rostochiensis (Gr-FLP-22, BM343164.1), sharing 72% sequence identity; indeed, the identity of two peptides, QPAGGVKWMRFa and TPQGKWMRFa, in H. glycines and G. rostochiensis were 100% ( Figure 1B). Phylogenetic analysis showed that flp-22 of H. glycines was clustered with the flp-22 complements of PPN and animal-parasitic nematodes, Strongyloides stercoralis, Strongyloides ratti, Parastrongyloides trichosuri, and Steinernema carpocapsae ( Figure 1C).

Expression of Hg-flp-22 in Different Development Stages of H. glycines
The transcript levels of Hg-flp-22 were analyzed at six different parasitic-stages (eggs, pre-parasitic J2s, parasitic J2s, J3s/J4s, and adult females and males) using the expression levels in eggs as a reference by qRT-PCR. Hg-flp-22 was expressed at the highest levels in pre-parasitic J2s and adult males compared with other life stages. Although its transcript levels declined during the parasitic J2 and J3/J4 stages, it also exhibited, respectively, approximately 2.7-and 2.6-fold higher transcripts than those of the eggs. The lowest expression levels of Hg-flp-22 were found in adult females in contrast to its upregulation in early parasitic-stages of H. glycines (Figure 2). We tried to use in situ hybridization assay to detect the tissue localization of Hg-flp-22 in the neuron system of pre-parasitic J2s, but DIG-labeled probes specific to Hg-flp-22 did not exhibit specific neural or non-neural tissue expression pattern even though a detectable signal was found in the anterior and tail regions of J2s (Supplementary Figure 1).

In vitro RNAi of Hg-flp-22 Affects Infection and Development of H. glycines
To study the role of Hg-flp-22 in H. glycines parasitism, the universal RNAi technology in vitro was used to investigate whether silencing of Hg-flp-22 expression can affect mobility, host recognition, penetration, and development of H. glycines. Fluorescence microscopy indicated that dsRNA was efficiently absorbed into J2 bodies ( Figure 3A). qRT-PCR analysis of efficiency for gene-silencing showed that the transcript levels of Hg-flp-22 were significantly decreased by 54% in H. glycines J2s after soaking in target dsRNA (2 mg/ml) when compared with those in the soaking buffer without dsRNA. Non-native control dsRNA-gfp treatment did not result in a statistically significant reduction in the target transcript of Hg-flp-22 ( Figure 3B). The host-searching behavior of J2s toward soybean root tips, which requires a combination of abilities of nematode locomotion and chemosensation, was assessed in Pluronic F-127 (PF-127) gel system (Hu et al., 2017b). Normally, infective J2s of H. glycines can recognize the presence of signals from the host root exudates and are preferentially attracted to soybean root tips, especially the elongation zone (Figure 4). Although, no significant difference was observed in the number of nematodes touching the root between the target gene dsRNAtreated group and the gfp dsRNA-treated control 4 h postexposure ( Figure 4A). Silencing of Hg-flp-22 resulted in lower J2 attraction to the vicinity of the soybean root tip (Figures 4B,C). The average number of J2s treated with Hg-flp-22 dsRNA surrounding the root tip was 43.4 ± 8.8, whereas it was 27.4 ± 4.7 for gfp dsRNA-treated nematodes (Figures 4B,C). Once they reach the host root tips, J2s of H. glycines use their stylet to invade the host root within 2 h after the assay started in PF-127 medium (Hu et al., 2017b). Therefore, the effect of in vitro RNAi of Hg-flp-22 on the penetration ability of J2s was further evaluated at 7 and 16 h post-exposure. Compared to the gfp dsRNA-socking nematodes, a significantly lower number of target dsRNA-treated J2s were observed to penetrate the host root at 7 and 16 h (Figures 4D,E).
Because H. glycines are soil-borne in nature, we further compared the infection and development of the host root by the Hg-flp-22 dsRNA-treated J2s to the gfp dsRNA-treated nematodes in soil assay. Results showed that silencing of Hg-flp-22 decreased the number of the stained J2s inside roots by 49.9% at 24 h post inoculation (hpi; Figure 5A). A significant reduction in the number of adult females on the root system at 21 days after the nematode inoculation (dpi) of target dsRNA-treated J2s was found in one independent biological experiment assay compared with that of the gfp dsRNA-treated J2s ( Figure 5B).

Exogenous Application of Synthetic Peptides Affects the Behavior of H. glycines
We, next, determined whether synthetic Hg-FLP-22 peptide treatments could produce the opposite phenotype of (1 mM) led to approximately 27.3% increase in the mean head movement frequency compared with the ddH 2 O-treated nematodes. Significant stimulatory effects of FLP-22c on the head movement of H. glycines were found at both 10 μM and 1 mM peptide solutions. In contrast, FLP-22a had no significant effect on head movement frequency ( Figure 6A). Chemotaxis assay suggested that none of the synthetic Hg-FLP-22 peptides treatments can affect the normal chemotaxis toward soybean root exudate (Figure 6B). Infection assay indicated that many more FLP-22b-(82 ± 12, p < 0.05) and FLP-22c-treated (86 ± 12, p < 0.05) J2s penetrated into soybean roots compared with the ddH 2 O-treated control nematodes (51 ± 12, p < 0.05; Figure 7A). FLP-22b treatment also caused a significant increase in the number of females on the host roots relative to the ddH 2 O-treated control at 21 dpi ( Figure 7B).
In PPN, flp-22 gene encodes three mature peptides with the most highly conserved C-terminal motif (-GVKWMRFG, -QG/SKWMRFG, and -GKWM/VRFG; Supplementary Figure 2A). We, therefore, investigated whether the other PPN have similar behavioral responses to the synthetic Hg-FLP-22 peptides. The same head movement and infection assays were performed in M. incognita J2s exposure to 1 mM FLP-22a, FLP-22b, or FLP-22c. When compared to H. glycines J2s, three synthetic Hg-FLP-22 peptides failed to significantly stimulate the head movement of M. incognita J2s, and only FLP-22b displayed a slight stimulatory effect on the frequency of head movement (Supplementary Figure 2B). Additionally, the number of synthetic peptides-treated and ddH 2 O-treated J2s of M. incognita in tomato roots was not significantly different (Supplementary Figure 2C).

DISCUSSION
Increasing evidence for FLP-mediated biological functions in host finding, invasion, and reproduction of PPN support the potential valuable repository of FLP genes as possible targets for the management of plant pathogenic nematodes (Kimber et al., 2007;Dalzell et al., 2010;Papolu et al., 2013;Dong et al., 2014;Kumari et al., 2017;Banakar et al., 2020) Quantitative real time-PCR analysis demonstrated that Hg-flp-22 is predominantly expressed in pre-parasitic J2s and adult males but that it showed a significantly reduced expression in adult females compared with other stages. This expression profile may be associated with the remodeling of the neuromuscular structure during the mobile stage of H. glycines transition to immobility and the resumption of mobility in males (Han et al., 2018). Similar expression profiles in mobile J2s and adult males were also reported in other flp genes in M. graminicola (flp-1, flp-3, flp-6, flp-7, flp-11, flp-12, flp-14, flp-16, and flp-18;Kumari et al., 2017) and M. incognita (flp-14 and flp-18; Papolu et al., 2013). The degeneration of specific neuromuscular cells may lead to the lower transcript levels of Frontiers in Plant Science | www.frontiersin.org these flp genes in the adult female stage of PPN (Bird, 1967;Han et al., 2018). These observations suggest the important role of the flp family in the early parasitic process and the male mating behavior of PPN. Before the onset of the sedentary stage, mobile parasitic-J2s travel toward the vascular cylinder of host roots and initiate the formation of a specialized feeding site, from which nematode withdraws nutrients throughout its life (Davis et al., 2004). All of these continuous actions, such as stylet movement, effector secretion from an esophageal gland, and swallowing nutrients, are dependent on the function of motor neurons. Despite the emergence of cell-specific body muscle atrophy in H. glycines during the sedentary stages, the atrophy in head and esophageal muscles is not observed in the immobile nematodes, suggesting that the persistent head movement and stylet thrusting are also essential for H. glycines feeding behavior from syncytium (Han et al., 2018). The data showed that Hg-flp-22 displayed relatively high transcripts in parasitic-J2 and J3/J4 stages, together with the expression pattern of flp-22 in many cell-types, such as muscle cells, as well as the physiological phenotypes of FLP-22 peptide-treated nematodes (Moffett et al., 2003;Kim and Li, 2004;Papaioannou et al., 2005), and it is possible that Hg-flp-22 may also exert its functions on the development of the sedentary H. glycines. A remarkable abundance of flp genes is expressed in the nervous system of all nematodes, including motor neurons, sensory neurons, and interneurons (Li and Kim, 2014;Peymen et al., 2014). The wide distribution pattern of the flp genes family endows them the functional flexibility in multiple behaviors, as well as supporting muscle and somatic cells. However, the localization of Hg-flp-22 did not display the specific expression sites of the neurons in the anterior and tail regions of H. glycines J2s except for a diffuse ISH staining pattern. The expression levels of Hg-flp-22 are below the detectable threshold using the DIG-labeled probe in the present study. However, a previous study reported by Kim and Li (2004) found that the expression pattern of flp-22 in C. elegans using the GFP reporter construct is widely distributed in the whole body. Most importantly, the tissue-specific location of flp-22 transcripts of C. elegans was observed in multiple neuronal cell types (AIM, ASG, AVA, AVG, AVL, CEP, PVD, PVW, RIC, AIZ, RIV, and SMD) and non-neuronal cells (URA and uv1) and was mainly focused on the anterior region. Notably, published data indicated that exogenous FLP-22 peptide of C. elegans has markedly excitatory effect on the pharyngeal action (Papaioannou et al., 2005); other evidence suggested that FLP-22 can cause muscle contraction in the ovijector of Ascaris suum (Moffett et al., 2003). In C. elegans, body movement is dependent on muscle control, which is innervated by a series of motor neurons in the ventral nerve cord (VNC), CNR, and other nerve structures (White et al., 1986;Francis and Waterston, 1991). Considering that the neural connectivity in H. glycines J2s is similar to C. elegans (Gendrel et al., 2016;Han et al., 2018) and that Hg-FLP-22 peptides displayed the obvious stimulatory effects on the anterior movement of H. glycines, the distribution of Hg-flp-22 transcript in J2 body may be involved in regulating muscle contraction and head movement of H. glycines.
Applications of synthetic Hg-FLP-22 peptides (FLP-22b and FLP-22c) significantly increased the head movement frequency of H. glycines. This is similar to the observation which showed that FLP-6 and FLP-14, both highly conserved single-copy peptides in nematode species (Peymen et al., 2014), also have significant stimulatory effects on the anterior movement frequency of H. glycines and M. incognita (Masler et al., 2012). Once hatched from eggs, the infective J2s of PPN need to quickly move toward host roots and penetrate the cortex layer cells via thrusting their stylet before their energy reserves are depleted (Perry, 1996). Therefore, we summarize that the high head movement frequency of pre-parasitic J2s in the soil is in favor of body motility and root penetration. Nematode infection assay indicated that H. glycines J2s treated with FLP-22b or FLP-22c are more quickly attracted to soybean roots and exhibited higher infection rates compared to the control treatments; this information supports the above hypothesis. Conversely, our results confirmed that loss of Hg-flp-22 by in vitro RNAi decreased the J2s movement to root tips and the penetration activity in PF-127 medium and soil assay. Similar inhibitory effects on the locomotion and invasion behaviors were found in flp-6 and flp-14 dsRNA-treated J2s of the other PPN (Kimber et al., 2007;Papolu et al., 2013;Kumari et al., 2017), suggesting that Hg-flp-22 may possess similar actions on neuromuscular control of H. glycines motor functions associated with movement and host invasion. Lower infection ability of the RNAi-treated J2s may lead to a lower proportion of nematodes at the mature female stage. We found that silencing of Hg-flp-22 reduced the number of females in only one independent assay, indicating that the efficacy of the RNAi experiment may be transient. Although Hg-FLP-22b treatment increased the number of females, it is difficult to interpret this information as indicative of Hg-flp-22 function in parasitic stages because of the transient efficacy and short durability of RNAi and synthetic peptides, which have been reported in several studies (Kimber et al., 2007;Roderick et al., 2012;Kumari et al., 2017). Unfortunately, hostinduced RNAi of Hg-flp-22 was not effective in expressing Hg-flp-22-RNAi constructs of soybean hairy roots (data not shown), and further functional studies of Hg-flp-22 during H. glycines parasitism are needed.
FMRFamide-like peptides are thought to exert most of their basic functions through the activation of cognate-receptors, and several FLP receptors have been identified in C. elegan by using heterologous cellular systems and genetic experiments (Li and Kim, 2014;McCoy et al., 2014;Peymen et al., 2014). In this study, the corresponding phenotypes of H. glycines J2s exposed to Hg-FLP-22 peptide imply that exogenous FLP peptide can be transported to neural or non-neural cells of nematode, where they can activate their cognate receptors and ultimately form the neuronal circuitry. However, the behavior potency of H. glycines responses to three peptides encoded on the same gene Hg-flp-22 shows subtle differences. Hg-FLP-22b and Hg-FLP-22c displayed significant stimulatory effects on head movement and stylet penetration ability compared with Hg-FLP-22a, suggesting the two peptides may be significantly more active isoforms of Hg-FLP-22 for activation of putative cognate receptors. Similar observations were also reported by previous studies indicating obvious differences in physiological function and potency between FLP peptides encoded on the same gene in H. glycines, Panagrellus redivivus, or C. elegans (Masler et al., 2012;Atkinson et al., 2016). It has been documented that a single FLP receptor can be activated by multiple FLPs, whereas the potency of receptor activation by structurally similar FLPs is different (Rogers et al., 2003;Mertens et al., 2005;Cohen et al., 2009). Thus, the sequence variation of Hg-FLP-22 peptides at the N-terminal may affect ligands binding with receptor and potency, eventually resulting in the differential regulation of nematode behavior. Notably, Hg-FLP-22b and Hg-FLP-22c did not produce a significant impact on head movement frequency and infection behaviors of M. incognita. These results indicated that the sensitivity of nematodes to exogenous FLP-22 peptides treatment appears to be speciesspecific. Indeed, sequence dissimilarity of N-terminal residues is present between three FLP-22 peptides from H. glycines and M. incognita. This further supports our speculation that the conservation of the N-terminal amino acid of FLP-22 seems to be most essential for receptor activation. Another possible explanation is the difference in the metabolic levels of three peptides in vivo. Masler et al. (2012) reported that the in vitro digestion rates of FLP-14a and FLP-14b encoded by flp-14 are different for the nematode proteolytic extraction from the same species. In fact, the posttranslational amidation of the FLP at the C-terminal is required for preventing their immediate degradation and biological function (Bowman et al., 1996). Hg-FLP-22 peptides used in the present study are in their unamidated forms and need further amidation in neuron cells following their uptake. Therefore, the lower Hg-FLP-22a amidation could result in a shorter half-life in vivo, which in turn leads to the low potential for regulating physiological function.
Identification of FLP-receptor couples is very important for functional studies on FLPs in nematodes. Until now, FLPs that functionally activate cognate receptors have not been reported in PPN. However, Atkinson et al. (2016) presumed that Gp-flp-32R is the cognate receptor of Gp-flp-32 based on their matching RNAi phenotypes and expression pattern, which may not fully reflect the characterization of the FLP-GPCR relationship; more detailed receptor pharmacology experiments are necessary to further verify the observations. The Y59H11AL.1 receptor can be activated by several C. elegans FLP peptides including FLP-22; however, among those peptides, FLP-7 is the most active peptide (Mertens et al., 2005). In addition, knockdown of the Y59H11AL.1 receptor did not result in any defect phenotypes, such as locomotion (Keating et al., 2003). These studies suggested that Y59H11AL.1 may not be a putative receptor of FLP-22. Therefore, identification of Hg-FLP-22 receptors by using bioinformatics and receptor pharmacology experiments is required to exploit its functional characterization, as this would boost the utility of FLP-22-mediated signaling as a target for H. glycines control.
In conclusion, this is the first functional characterization of FLP encoding genes in H. glycines (via RNAi). These data indicate a positive regulation of bioactive FLP-22 peptides in H. glycines behaviors in terms of increased body movement, penetration, and development. This study also deepens the basic understanding of FLP signaling in the early parasitism of H. glycines, which is the basis for the exploitation of the FLP system as putative control targets.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.