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Effect of knockdown of genes involved in the RNAi pathway on Root-knot nematodes

Iqbal, Sadia (2015) Effect of knockdown of genes involved in the RNAi pathway on Root-knot nematodes. PhD thesis, Murdoch University.

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Plant parasitic nematodes are economically important crop pests: of these root-knot nematodes (Meloidogyne spp.) have the widest host range and are recognised as the most significant nematode pests of crop plants worldwide. Current control methods have serious limitations: natural resistance genes, chemicals and cultural practices are often not effective or are too expensive for large scale application. An alternative control strategy chosen here was to target and silence a conserved mechanism (RNA interference – RNAi) in the nematodes via transgenic plants. The RNAi pathway itself was studied for its potential to provide new gene targets for nematode control, with potentially wider applications to control of other plant nematodes.

In this study the overall aim was to undertake in silico identification of genes and ‘effectors’ of the RNAi pathway of root-knot nematodes, to use RNAi to silence some of these target genes, and to determine the effects on their parasitic success after both in vitro and in planta RNAi treatments. Twenty-seven genes were identified in the RNAi pathways of M. incognita, and selected for further study. In vitro RNAi experiments (‘soaking’ of J2 nematodes in dsRNA homologous to the target gene) to down-regulate expression of the 27 selected genes caused significant effects on the infectivity and development of the nematodes when they were used to infect susceptible tomato plants. Up to a 90% reduction in infection was observed for dcr-1 targeted nematodes. Down-regulation of effectors of the miRNA pathway (drsh-1, pash-1, alg-1, xpo-1, xpo-2) and dicer complex (drh-1, drh-3) had the greatest effect on nematode viability and/or development.

Seven of the cloned genes (dcr-1, drh-3, vig-1, mut-7, drsh-1, pash-1, rha-1) were chosen after in vitro screening for in planta analysis, and hairpin constructs for each were successfully transformed into A. thaliana plants. Challenge of the heterozygous T2 transgenic plants with M. incognita J2s exhibited significant reductions in infection parameters: 31 transgenic events of the 7 genes showed a reduction of infection of 50% or more when compared with controls, and the greatest reduction was 89%, for plants targeting drsh-1of M. incognita.

Another in vitro RNAi experiment targeting 7 different regions of the same gene, dcr-1, was conducted to evaluate gene silencing in relation to different regions of the same target. The results showed that there was variable target expression and RNAi effects depending on the target region used, with higher impact for sequences near the 5′ end of the targeted transcript. Targeting dcr-1 resulted in reduced nematode infection and reproduction: abnormal nematode development was also observed.

This project provides new information on genes involved in small RNA pathways of M. incognita, resulting in identification of novel targets for its control by gene silencing technology. It also provides additional data to improve design of more effective RNAi triggers related to the target region chosen. The in planta RNAi results generated provide further evidence of the potential of RNAi as a nematode control strategy based on results using the model plant A. thaliana: it is likely that these results are translatable to protect crop plants from nematode attack in the future.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Jones, Michael and Fosu-Nyarko, John
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