Fig 1.
Reserpine inhibits G. pallida host plant invasion behaviour.
A. J2s were collected at 24 h post hatching and pre-incubated with 24 h in water without (control), or with the addition of reserpine (100 μM). J2s were applied to individual potato hairy root cultures at 3 infection points with 25 J2s per infection point. 13 days later roots were stained with acid fuchsin and parasitic nematodes visualised. Data are mean ± s.e.m. for 20 plants from two independent experiments; **** p<0.0001 unpaired Student's t-test. B. The effect of reserpine on J2 motility was tested in a dispersal assay in which their ability to move away from the central point of an agar arena was determined. For each assay about 50 to 100 J2s were pre-soaked in water (control), or water with 50 μM reserpine for 18 h. They were then pipetted onto the centre of an agar plate in a minimum (~ 5 μL) volume of liquid. The plate was demarcated with concentric circles with the centre of the plate labelled '0' and the outermost circle as '5'. The percentage of J2s in each position was determined after 2 h. Data are mean ± s.e.m. of 10 plates for each experimental group from 2 independent experiments. There is a significant increase in J2 distribution at positions '0' and '1' with reserpine treatment compared to control (F(5,54) = 97.62, P<0.0001; two way ANOVA).
Fig 2.
Reserpine inhibits stylet thrusting triggered by endogenous, but not exogenous, serotonin.
A. The cartoon provides a rationale for the differential effect of reserpine on the response to serotonin and fluoxetine. It depicts a serotonergic synapse, with the pre-synapse as the site of serotonin synthesis and release and the post-synapse which harbours serotonin receptors. In mammalian systems reserpine acts presynaptically to deplete the storage of the neurotransmitter serotonin (shown as open red circles) by preventing its uptake into vesicles (shown as solid red circles) by inhibition of the vesicular monoamine transporter, VMAT, present on the vesicle membrane [14]. This prevents storage of serotonin and results in a lack of serotonin presynaptically and an inability of the presynaptic terminal to release serotonin. Fluoxetine (Prozac) on the other hand blocks the plasma membrane serotonin transporter, SERT. This prevents the reuptake of serotonin into the presynaptic terminal following its release. Thus, fluoxetine increases synaptic levels of serotonin (shown by open red circles) and in this way fluoxetine can act indirectly to stimulate transmission at the serotonergic synapse. This effect of fluoxetine is susceptible to block by reserpine as it requires endogenous serotonin to elicit its effect. However, the response to exogenous serotonin circumvents reserpine inhibition as it acts directly on the postsynaptic receptors. B. The G. pallida stylet is a lance-like structure that can be thrust out of the mouth of the nematode (in this image it is shown in the retracted position) in a rhythmic manner to initiate hatching and root invasion. Inside the root it is used for migration and to support feeding and interaction with the host. The activity of the stylet can be visually scored by counting the number of thrusts made in 1 min. Scale bar ~ 20 μm. C. J2s were incubated in either serotonin or fluoxetine at the concentrations indicated for 1 h and then the number of stylet thrusts made in 1 min was counted. Data are mean ± s.e.m.; n = 8 to 17 J2s for each time point. D. Reserpine blocked the stylet response to fluoxetine but not serotonin. J2s were pre-soaked in reserpine at the concentration indicated for 24 h and subsequently immersed in either 10 mM serotonin or 2 mM fluoxetine for 30 min and stylet thrusting scored for 1 min. Data are mean ± s.e.m.; n = 10 J2s for each data point.
Fig 3.
Functional characterisation of a gene encoding G. pallida VMAT, gpa-cat-1.
A. Expression of G. pallida cat-1 (gpa-cat-1) rescues the pharyngeal phenotype of C. elegans cat-1(ok411). A comparison of the pumping rate on food for wild-type N2, cat-1(ok411) and transgenic lines of cat-1(ok411) expressing either wild type C. elegans cat-1, cat-1(+) or gpa-cat-1 behind a pan-neuronal promoter (Psnb-1). Four stable lines for each transgenic strain were tested and the data are pooled for presentation. Data are mean ± s.e.m; 'n' is shown in brackets where 'n' is the number of C. elegans. *** p<0.001; **** p<0.0001; One way ANOVA with Bonferroni's multiple comparisons. There was no significant difference between N2 and Psnb-1::cat-1a(+);cat-1(ok411), p = 0.7.84. There was a significant difference between N2 and Psnb-1::gpa-cat-1;cat-1(ok411) p = 0.002, indicating that the rescue for G. pallida cat-1 is partial. B. Expression of gpa-cat-1 in C. elegans cat-1(ok411) reinstated sensitivity to fluoxetine. One day old hermaphrodite C. elegans were placed on agar plates that either had no drug, 'control', or had been prepared with fluoxetine (5 to 500 μM). After 1 h the rate of pharyngeal pumping was scored in each nematode for 1 min. N2 wild type C. elegans pumped at a low rate which increased in a concentration-dependent manner in the presence of fluoxetine. Fluoxetine did not stimulate pumping in cat-1(ok411) but the response was restored in the transgenic C. elegans expressing either cat-1(+) or gpa-cat-1 behind a pan-neuronal promoter (psnb-1). Two stable lines for each transgenic strain were tested and the data are pooled. Data are the mean ± s.e.m.; n ≥ 17 where 'n' is the number of C. elegans; **p<0.01; *** p<0.001; Two way ANOVA with Bonferroni's multiple comparisons. For the sake of clarity only one comparison is shown for each strain between control and 5 μM fluoxetine.
Fig 4.
Functional characterisation of a gene encoding G. pallida tryptophan hydroxylase, gpa-tph-1.
A. Expression of gpa-tph-1 rescued the pharyngeal phenotype of C. elegans tph-1(mg280) similar to expression of a wild type copy of C. elegans tph-1, tph-1(+). Both genes were expressed from the pan-neuronal promoter Psnb-1. Pharyngeal pumping rate of one day old adult hermaphrodites on food was scored for 1 min for each nematode. Four stable lines for each transgenic strain were tested and the data are pooled. Data are mean ± s.e.m; 'n' is indicated on the graph where 'n' is the number of C. elegans; **** p<0.0001; one way ANOVA with Bonferroni's multiple comparisons. There was a significant difference between N2 and Psnb-1::C. elegans tph-1 (p = 0.008) and between Psnb-1::G. pallida tph-1 (p = 0.001) indicating the rescue is not complete. B. Expression of either C. elegans tph-1(+) or gpa-tph-1 in the C. elegans mutant tph-1(mg280) conferred sensitivity to the tryptophan hydroxylase inhibitor, CPA. One day old adult hermaphrodites were incubated for 2 h on agar plates with food (control), or with food and 10 mM CPA. Two stable lines were tested for each transgenic strain and the data are pooled. Data are the mean ± SEM of n ≥ 10 where 'n' is the number of C. elegans; ***p<0.001; one way ANOVA with Bonferroni's multiple comparisons.
Fig 5.
The role of TPH-1 in host plant invasion behaviour.
A. G. pallida J2s were soaked in either water (control) or water with the TPH inhibitor CPA (10 mM) for 24 h. This was followed by the addition of either serotonin (10 mM) or fluoxetine (2 mM) for 30 min and stylet thrusting was scored for 1 min. Data are mean ± s.e.m. n = 27 for serotonin and n = 40 for fluoxetine. *** p<0.001; Two way ANOVA with Bonferroni's multiple comparisons. B. CPA impaired the ability of J2s in root invasion. J2s were collected at 24 h post hatching and pre-incubated for 24 h in water without (control), or with the addition of CPA (100 μM). J2s were applied to individual potato hairy root cultures at 3 infection points with 25 J2s per infection point. 13 days later roots were stained with acid fuchsin and nematodes visualised. Data are mean ± s.e.m. for 19 plants; *** p<0.001 unpaired Student's t-test.
Table 1.
Identification of C. elegans serotonin receptor homologues in Globodera spp.
Globodera pallida and G. rostochiensis homologues of the five C. elegans genes encoding serotonin receptors were identified by reciprocal best BLAST hit analysis of the G. pallida [4] and G. rostochiensis [3] predicted gene complements.* All are G-protein coupled receptors with the exception of MOD-1 which is a serotonin-gated chloride channel. The likely gpa-ser-1 orthologue is incomplete in the genome assembly and encompassed by two adjacent gene models; this was confirmed by analysis of a transcriptome assembly produced using existing J2 RNAseq data [4]. # The complete sequence for gpa-ser-5 was not present in the genome assembly, but was identified from the transcriptome assembly.
Fig 6.
Characterisation of G. pallida SER-7 A.
Measurements of pharyngeal pumping in the intact nematode: Expression of gpa-ser-7 rescued the pharyngeal phenotype of C. elegans ser-7(tm1325) similar to expression of a wild type copy of C. elegans ser-7a, ser-7a(+). Both genes were expressed from the pan-neuronal promoter Psnb-1. Pharyngeal pumping rate of one day old adult hermaphrodites after 20 min off food was scored for 1 min for each nematode either in the absence (control) or presence (SER) of 10 mM serotonin. Three stable lines for each transgenic strain were tested and the data are pooled. Data are mean ± s.e.m; n ≥ 20; **** p<0.0001; one way ANOVA with Bonferroni's multiple comparisons. Note that whilst the pumping rate of ser-7(tm1325) was increased by 10 mM serotonin this response was significantly lower than that for wild type and both transgenic strains.The stimulation of pumping rate by serotonin in the rescue lines Psnb-1::C. elegans ser-7 and Psnb-1::G. pallida ser-7 was significantly different from both N2 (p = 0.0001) and from ser-7 (p = 0.001). B. An image of the cut head preparation that was used for the experiments shown in C and D. Cutting the head exposes the pharynx to the external solution allowing ready access of applied drugs to the receptors regulating the pharyngeal network. Pharyngeal pumps were recorded visually by counting the contraction-relaxation cycles of the terminal bulb. Scale bar ~25 μm. C. Concentration-response of the pumping rate of the cut head pharyngeal preparations to serotonin for wild type (N2), ser-7(tm1325) and transgenic lines of ser-7(tm1325) expressing either C. elegans or G. pallida ser-7 behind a pan-neuronal promoter (psnb-1). Note that in the dissected pharyngeal preparation ser-7(tm1325) mutants do not respond to even 100 μM serotonin. Data are the mean ± s.e.m.; n ≥ 20. D. Methiothepin (MET) blocked the response to serotonin in both transgenic strains. Cut heads were pre-incubated with methiothepin at the concentrations indicated for 5 min after which 100 μM serotonin was added and after a further 10 min the pumping rate was scored for 1 min. Two stable lines for each transgenic strain were tested and the data are pooled. Data are the mean ± s.e.m; n ≥ 5.
Fig 7.
Characterisation of G. pallida MOD-1.
A. Expression of gpa-mod-1 rescued the motility phenotype of C. elegans mod-1(ok103) similar to expression of a wild type copy of C. elegans mod-1, mod-1(+). The control expressed the transformation marker only. Both genes were expressed from the putative native mod-1 promoter, Pmod-1. Approximately 10 one day old adult hermaphrodite C. elegans were dispensed into each well of a 96-well microtitre plate. At time 0, 33 mM serotonin dissolved in M9 buffer was added to each well and the percentage of immobilised nematodes in each well was scored every min for up to 20 min. Three stable lines for each transgenic strain were tested and the data are pooled. Data are mean ± s.e.m; n = 3 independent experiments; **** p<0.01; two way ANOVA with Bonferroni's multiple comparisons.There was no significant difference between N2 and either Pmod-1::C. elegans mod-1 or Pmod-1::G. pallida mod-1. B. The serotonin-induced paralysis was blocked by methiothepin (MET) in both wild type (N2) and in the transgenic strain expressing gpa-mod-1 in mod-1(ok103). The experiment was performed as in A with the addition of a preincubation in 10 μM MET on an agar plate seeded with OP50 for 2 h and the inclusion of 10 μM MET with serotonin in the microtitre plate wells. Data are mean ± s.e.m; n = 3 independent experiments; ** p<0.01; two way ANOVA with Bonferroni's multiple comparisons.
Fig 8.
Methiothepin is a potent inhibitor of G. pallida behaviours, hatching and root invasion, that are dependent on serotonin regulated stylet thrusting.
A. G. pallida J2s were soaked in either a control solution of 0.5% ethanol or methiothepin at different concentrations for 24 h. They were transferred to 2 mM fluoxetine and after 30 min had elapsed the rate of stylet thrusting was counted for 1 min. Data are mean ± s.e.m.; n = 12; p<0.0001; one-way ANOVA with Bonferroni's multiple comparisons. B. Methiothepin (MET) impaired the ability of J2s to invade the host root. J2s were collected at 24 h post hatch and pre-incubated for 24 h in water without (control), or with the addition of MET (100 μM). J2s were applied to individual potato hairy root cultures at 3 infection points with 25 J2s per infection point. 13 days later roots were fuschin stained and visualised for J2. Data are mean ± s.e.m. for 20 plants for control and 21 plants for MET; **** p<0.001 unpaired Student's t-test. C. MET blocked hatching of J2s from cysts. G. pallida cysts were soaked in a 24-well plate with 1 cyst per well in the presence of potato root diffusate modified with either vehicle (PRD), 10 μM methiothepin (M) or 100 μM methiothepin and hatching of J2 juveniles was scored. At 24 days, the cysts were placed into PRD alone to assess recovery of hatching. Methiothepin inhibited hatching and hatching did not recover on removal from drug (P<0.0001). D. Representative images of unhatched eggs from cysts treated with a control solution and 100 μM methiothepin. Note that methiothepin treatment does not appear to have negatively impacted the integrity of the unhatched J2 (egg dimensions 120 μm by 60 μm).
Table 2.
Primers used for amplification of cDNA from C. elegans and G. pallida.
These primers were used to amplify cDNA for genes of interest as indicated in the left-hand column of the table.