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A synthetic peptide shows retro- and anterograde neuronal transport before disrupting the chemosensation of plant-pathogenic nematodes.

Wang D, Jones LM, Urwin PE, Atkinson HJ - PLoS ONE (2011)

Bottom Line: The peptide takes 2.5 h to reach the neuronal cell bodies.The peptide probably undergoes transport along the dye-filling non-cholinergic chemoreceptive neurons to their synapses where it is taken up by the interneurons to which they connect.Coordinated responses to chemoreception are disrupted when the sub-set of cholinergic interneurons secrete the peptide at synapses that have post-synaptic nicotinic acetylcholine receptors.

View Article: PubMed Central - PubMed

Affiliation: Centre for Plant Science, University of Leeds, Leeds, United Kingdom.

ABSTRACT
Cyst nematodes are a group of plant pathogens each with a defined host range that cause major losses to crops including potato, soybean and sugar beet. The infective mobile stage hatches from dormant eggs and moves a short distance through the soil to plant roots, which it then invades. A novel strategy for control has recently been proposed in which the plant is able to secrete a peptide which disorientates the infective stage and prevents invasion of the pathogen. This study provides indirect evidence to support the mechanism by which one such peptide disrupts chemosensory function in nematodes. The peptide is a disulphide-constrained 7-mer with the amino acid sequence CTTMHPRLC that binds to nicotinic acetylcholine receptors. A fluorescently tagged version of this peptide with both epifluorescent and confocal microscopy was used to demonstrate that retrograde transport occurs from an aqueous environment along bare-ending primary cilia of chemoreceptive sensilla. The peptide is transported to the cell bodies of these neurons and on to a limited number of other neurons to which they connect. It appears to be localised in both neuronal processes and organelles adjacent to nuclei of some neurons suggesting it could be transported through the Golgi apparatus. The peptide takes 2.5 h to reach the neuronal cell bodies. Comparative studies established that similar but less abundant uptake occurs for Caenorhabditis elegans along its well studied dye-filling chemoreceptive neurons. Incubation in peptide solution or root-exudate from transgenic plants that secrete the peptide disrupted normal orientation of infective cyst nematodes to host root diffusate. The peptide probably undergoes transport along the dye-filling non-cholinergic chemoreceptive neurons to their synapses where it is taken up by the interneurons to which they connect. Coordinated responses to chemoreception are disrupted when the sub-set of cholinergic interneurons secrete the peptide at synapses that have post-synaptic nicotinic acetylcholine receptors.

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Neuronal uptake of Alexa Fluor 488-labelled nAChRbp by Heterodera schachtii.A) After 16 hours incubation in 183 µM peptide-dye conjugate the peptide was detected in J2 of H. schachtii by epifluorescence as in Figure 1. B) After a similar incubation in labelled peptide the region of neuronal cell bodies posterior to the nerve ring was observed using confocal microscopy. C) Epifluorescent detection of the labelled peptide 48 h after removal of the H. schachtii from the peptide solution. All images are lateral views of the nematodes. Key: c, cephalic framework; s, stomatostylet (both showing autofluorescence); a, amphidial pouch; d, tract of amphidial dendrites; n, nerve ring; g, region of the lateral and ventral ganglia. The scale bar is 10 µm and the dorsal side is that nearer to the scale bar.
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pone-0017475-g003: Neuronal uptake of Alexa Fluor 488-labelled nAChRbp by Heterodera schachtii.A) After 16 hours incubation in 183 µM peptide-dye conjugate the peptide was detected in J2 of H. schachtii by epifluorescence as in Figure 1. B) After a similar incubation in labelled peptide the region of neuronal cell bodies posterior to the nerve ring was observed using confocal microscopy. C) Epifluorescent detection of the labelled peptide 48 h after removal of the H. schachtii from the peptide solution. All images are lateral views of the nematodes. Key: c, cephalic framework; s, stomatostylet (both showing autofluorescence); a, amphidial pouch; d, tract of amphidial dendrites; n, nerve ring; g, region of the lateral and ventral ganglia. The scale bar is 10 µm and the dorsal side is that nearer to the scale bar.

Mentions: Uptake of nAChRbp was visualized after conjugating it to Alexa Fluor 488. J2 of H. schachtii were placed in 183 µM of this fluorescent version of the peptide. The fluorescence detected was more extensive than for the fluorophore alone and at least as extensive as that for FITC. All nematodes examined revealed similar patterns of fluorescence. The region of the amphidial pouch was highly fluorescent for the nAChRbp/Alex Fluor 488 conjugate and it was present locally along the route of the dendrites of the amphidial neurons and also in the region just behind the weakly positive nerve ring to about 40 µm posterior to it (Fig. 3a). A key difference between uptake of labelled peptide and FITC dye staining was the aggregation of the peptide behind the nerve ring. Confocal microscopy confirmed the discrete nature of the fluorescence emissions (Fig. 3b). Fluorescence was lost from the amphidial pouches 48 h after removal of nematodes from the peptide solution but it remained extensive in the region of the amphidial cell bodies (Fig. 3c). The hermaphrodite of C. elegans also took up the peptide to the region of its lateral ganglia (Fig. 4) but to a lesser extent than occurred for H. schachtii. Uptake in the region of the neuronal cell bodies was studied further by placing J2 H. schachtii in a solution containing both 183 µM labelled peptide and 1 mM bisbenzimide. The region 5–30 µm posterior to the nerve ring showed two green fluorescent features. There was a reticulate pattern that is probably formed by neuronal processes passing among cells and intensively stained spheroids of 1.0–1.5 µm in diameter (Fig. 5a). Merging of this image with that for emission from bisbenzimide (Fig. 5c) established that the spheroids are closely associated with, but not integral to the nuclei (Fig. 5b).


A synthetic peptide shows retro- and anterograde neuronal transport before disrupting the chemosensation of plant-pathogenic nematodes.

Wang D, Jones LM, Urwin PE, Atkinson HJ - PLoS ONE (2011)

Neuronal uptake of Alexa Fluor 488-labelled nAChRbp by Heterodera schachtii.A) After 16 hours incubation in 183 µM peptide-dye conjugate the peptide was detected in J2 of H. schachtii by epifluorescence as in Figure 1. B) After a similar incubation in labelled peptide the region of neuronal cell bodies posterior to the nerve ring was observed using confocal microscopy. C) Epifluorescent detection of the labelled peptide 48 h after removal of the H. schachtii from the peptide solution. All images are lateral views of the nematodes. Key: c, cephalic framework; s, stomatostylet (both showing autofluorescence); a, amphidial pouch; d, tract of amphidial dendrites; n, nerve ring; g, region of the lateral and ventral ganglia. The scale bar is 10 µm and the dorsal side is that nearer to the scale bar.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3049761&req=5

pone-0017475-g003: Neuronal uptake of Alexa Fluor 488-labelled nAChRbp by Heterodera schachtii.A) After 16 hours incubation in 183 µM peptide-dye conjugate the peptide was detected in J2 of H. schachtii by epifluorescence as in Figure 1. B) After a similar incubation in labelled peptide the region of neuronal cell bodies posterior to the nerve ring was observed using confocal microscopy. C) Epifluorescent detection of the labelled peptide 48 h after removal of the H. schachtii from the peptide solution. All images are lateral views of the nematodes. Key: c, cephalic framework; s, stomatostylet (both showing autofluorescence); a, amphidial pouch; d, tract of amphidial dendrites; n, nerve ring; g, region of the lateral and ventral ganglia. The scale bar is 10 µm and the dorsal side is that nearer to the scale bar.
Mentions: Uptake of nAChRbp was visualized after conjugating it to Alexa Fluor 488. J2 of H. schachtii were placed in 183 µM of this fluorescent version of the peptide. The fluorescence detected was more extensive than for the fluorophore alone and at least as extensive as that for FITC. All nematodes examined revealed similar patterns of fluorescence. The region of the amphidial pouch was highly fluorescent for the nAChRbp/Alex Fluor 488 conjugate and it was present locally along the route of the dendrites of the amphidial neurons and also in the region just behind the weakly positive nerve ring to about 40 µm posterior to it (Fig. 3a). A key difference between uptake of labelled peptide and FITC dye staining was the aggregation of the peptide behind the nerve ring. Confocal microscopy confirmed the discrete nature of the fluorescence emissions (Fig. 3b). Fluorescence was lost from the amphidial pouches 48 h after removal of nematodes from the peptide solution but it remained extensive in the region of the amphidial cell bodies (Fig. 3c). The hermaphrodite of C. elegans also took up the peptide to the region of its lateral ganglia (Fig. 4) but to a lesser extent than occurred for H. schachtii. Uptake in the region of the neuronal cell bodies was studied further by placing J2 H. schachtii in a solution containing both 183 µM labelled peptide and 1 mM bisbenzimide. The region 5–30 µm posterior to the nerve ring showed two green fluorescent features. There was a reticulate pattern that is probably formed by neuronal processes passing among cells and intensively stained spheroids of 1.0–1.5 µm in diameter (Fig. 5a). Merging of this image with that for emission from bisbenzimide (Fig. 5c) established that the spheroids are closely associated with, but not integral to the nuclei (Fig. 5b).

Bottom Line: The peptide takes 2.5 h to reach the neuronal cell bodies.The peptide probably undergoes transport along the dye-filling non-cholinergic chemoreceptive neurons to their synapses where it is taken up by the interneurons to which they connect.Coordinated responses to chemoreception are disrupted when the sub-set of cholinergic interneurons secrete the peptide at synapses that have post-synaptic nicotinic acetylcholine receptors.

View Article: PubMed Central - PubMed

Affiliation: Centre for Plant Science, University of Leeds, Leeds, United Kingdom.

ABSTRACT
Cyst nematodes are a group of plant pathogens each with a defined host range that cause major losses to crops including potato, soybean and sugar beet. The infective mobile stage hatches from dormant eggs and moves a short distance through the soil to plant roots, which it then invades. A novel strategy for control has recently been proposed in which the plant is able to secrete a peptide which disorientates the infective stage and prevents invasion of the pathogen. This study provides indirect evidence to support the mechanism by which one such peptide disrupts chemosensory function in nematodes. The peptide is a disulphide-constrained 7-mer with the amino acid sequence CTTMHPRLC that binds to nicotinic acetylcholine receptors. A fluorescently tagged version of this peptide with both epifluorescent and confocal microscopy was used to demonstrate that retrograde transport occurs from an aqueous environment along bare-ending primary cilia of chemoreceptive sensilla. The peptide is transported to the cell bodies of these neurons and on to a limited number of other neurons to which they connect. It appears to be localised in both neuronal processes and organelles adjacent to nuclei of some neurons suggesting it could be transported through the Golgi apparatus. The peptide takes 2.5 h to reach the neuronal cell bodies. Comparative studies established that similar but less abundant uptake occurs for Caenorhabditis elegans along its well studied dye-filling chemoreceptive neurons. Incubation in peptide solution or root-exudate from transgenic plants that secrete the peptide disrupted normal orientation of infective cyst nematodes to host root diffusate. The peptide probably undergoes transport along the dye-filling non-cholinergic chemoreceptive neurons to their synapses where it is taken up by the interneurons to which they connect. Coordinated responses to chemoreception are disrupted when the sub-set of cholinergic interneurons secrete the peptide at synapses that have post-synaptic nicotinic acetylcholine receptors.

Show MeSH
Related in: MedlinePlus