The levamisole sensitive nicotinic acetylcholine receptor of the potato cyst nematode Globodera pallida

The potato cyst nematode Globodera pallida costs the UK potato industry over £50 million per annum. In order to invade a host plant, the infective J2 stage must hatch from eggs within the soil and migrate towards the root system. Orthologues of Caenorhabditis elegans genes involved in neurotransmission were identified in the G. pallida and G. rostochiensis genome assemblies. The complement of cys loop ligand gated ion channel genes was distinct compared to C. elegans and other parasitic nematodes. Orthologues of genes encoding subunits which comprise the C. elegans levamisole sensitive nicotinic acetylcholine receptor (cel-lev 1, cel-lev 8, cel-unc 29, cel-unc 63 and cel-unc 38) were searched for, and cel-lev 1 and cel-lev 8 orthologues were absent in both Globodera spp. Two orthologues were identified for cel-unc 29 and cel-unc 38. This suggested that the composition of the G. pallida L nAChR may differ. The use of C. elegans as a heterologous system to study the expression pattern of G. pallida nAChR genes was explored. GFP expressing lines were created using promoter regions of gpa acr 2 and gpa unc 63. Expression was observed in the ventral nerve cord and nerve ring for pgpa-acr 2. Expression of pgpa-unc 63 was variable, but was found in the head and tail region and along the ventral side of the body. The impact of this distinct complement of cys loop subunits on anthelmintic sensitivity was demonstrated by the increased resistance of both G. pallida and G. rostochiensis J2s to levamisole. The EC50 of G. pallida and G. rostochiensis was 19.7 mM and 5.6 mM respectively, compared to the EC50 of 9 µM for C. elegans, representing a 500 – 2000 fold increase in levamisole resistance. This increased resistance to levamisole was associated with an orthologue of cel-unc 38 identified in G. pallida, gpa unc 38.1. Rescue of C. elegans unc 38(x20) mutants with gpa unc 38.1 restores normal movement suggesting a functional reconstitution of the L nAChR, but full sensitivity to levamisole is not restored. Gpa unc 38.1 was expressed with the remaining four subunits from C. elegans in Xenopus oocytes to produce a chimeric receptor. The EC50 of the response to acetylcholine and levamisole of the chimeric receptor and the native receptor was comparable and had similar opening responses to different agonists. Chimeric genes were created to analyse key motifs in gpa unc 38.1 that may affect receptor function and levamisole sensitivity. Gpa unc 38.1 was necessary for structural reformation of the receptor, but not acetylcholine binding. Removal or addition of a loop B glutamate residue, previously associated with levamisole sensitivity of Cel UNC 38, did not affect levamisole sensitivity of Cel-UNC 38 or Gpa UNC 38.1. An amino acid change (I>M) in TM2 of Cel-UNC 38 increased levamisole sensitivity and basal thrashing rate. The reciprocal change (M>I) in Gpa UNC 38.1 comprised basal thrashing rescue. The basis of increased levamisole resistance of gpa unc 38.1 was not identified, as all gpa unc 38.1 chimeric genes retained a higher resistance to levamisole than cel-unc 38. This works reveals that the nAChRs of plant parasitic nematodes have distinct pharmacological characteristics.