Comparative and functional analysis of the spliceosome units of the cyst and root lesion nematodes

The root lesion nematode Pratylenchus thornei and the cyst nematode Heterodera schachtii belong to the top three plant parasitic nematode groups with regards to scientific and economic importance. RNA interference (RNAi) technology is a potential strategy for delivering transgenic resistance for nematode management in agriculture. The natural cellular mechanism of gene silencing, RNAi, is a sequence specific mRNA degradation process induced by the presence of double stranded RNA (dsRNA). The aim of this research was to investigate whether genes involved in cis and trans-splicing of mRNA in plant parasitic nematodes present suitable targets for nematode control using RNAi. In plant parasitic nematodes, RNAi was induced either through ingestion of dsRNA of genes involved in splicing with the help of a neurostimulant or via in planta delivery. Comparative analysis with Caenorhabditis elegans identified 55 spliceosome genes in the root lesion nematodes P. thornei, P. zeae and P. coffeae and 44 genes in the cyst nematodes H. glycines and H. schachtii. Identification of spliced leader (SL) RNA specific sut-1, sut-2, sna-1 and sna-2 genes suggests the occurrence of SL trans-splicing of mRNA in these nematodes. In vitro RNAi induced by soaking mixed stages of P. thornei and second stage juveniles of H. schachtii in 1 mg/mL dsRNA for selected spliceosome genes for 16 hours in some cases affected nematode movement causing paralysis, ‘knots’ and loss of direction, compared to those fed with dsRNA of green fluorescent protein (gfp) or controls not exposed to dsRNA. When dsRNA treated nematodes were transferred to carrot mini discs or Arabidopsis thaliana plants, after 5 weeks compared to control treatments there was a 55-99% reduction in the number of RNAi-treated P. thornei cultured on the carrot mini discs, and a 9-81% reduction in the number of H. schachtii cysts which developed on wild type A. thaliana, demonstrating that silencing of specific target genes reduced nematode survival and reproduction for both species. Reduction in transcript levels of some genes was also evident when analysed by semi-quantitative PCR. When carrot hairy roots were generated which expressed sequences of dsRNA for SL specific genes of P. thornei, there was a wide variation in the number of nematodes extracted per gram root, between different transgenic events. H. schachtii cyst development over 5 weeks was reduced by 3-100% on transgenic A. thaliana plants transformed to express dsRNA of target 8 genes. The results suggest that some of the target genes studied are potential candidates for further development of nematode resistance in crop plants, and could contribute to the on-going search for delivering sustainable control of these and related nematodes using RNAi technology.