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Improving the effectiveness and delivery of gene silencing triggers to control plant parasitic nematodes

Naz, Fareeha (2017) Improving the effectiveness and delivery of gene silencing triggers to control plant parasitic nematodes. PhD thesis, Murdoch University.

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Abstract

Plant parasitic nematodes (PPNs) are a major group of crop pests that decrease yields by 7-15%, and locally up to 50% or more. The most economically important groups are the sedentary endoparasites which invade roots and form distinct feeding sites, and the migratory endoparasites which feed from individual cells. Gene silencing through RNA interference (RNAi) could be applied to control a range of plant pests. The overall aim of this study was to try to enhance the effectiveness of RNAi to control PPNs. The research included: targeting multiple genes simultaneously in the sedentary endoparasitic cyst-nematode H. schachtii, investigating the role of splice-leaders as RNAi targets for PPNs, and seeking alternative modes of delivery of double-stranded RNA (dsRNA) (e.g. by spraying) to bypass the need to generate transgenic plants. This could be applied when transgenic technology is not acceptable or financially viable.

After H. schachtii had been soaked in dsRNA of different combinations of two different genes (vha-3-vha-8, vha-3-pat-10, and vha3-prp-21), there was around 9-31% greater reduction in their growth and development compared to soaking in mixtures of single genes (vha-3 & vha- 8; vha-3 & pat-10; vha-3 & prp-21). Similarly, in ‘Host-Induced Gene Silencing ‘(HIGS), transgenic A. thaliana plants expressing dsRNA of H. schachtii combinations of vha-3-vha-8, exhibited up to >80% reduction in the development of nematodes compared to transgenic plants expressing genes singly (vha-3: up to ~70% and vha-8: up to ~50%).

Splice-leader (SL) sequences were identified for the PPNs P. thornei and H. schachtii. Soaking of mixed stages of P. thornei in SL siRNA significantly reduced penetration of nematodes into wheat roots compared to untreated nematodes. In contrast, J2s of H. schachtii soaked in SL siRNA did not significantly affect their development on A. thaliana roots.

Ectopic delivery of fluorescent oligonucleotides (with a phosphorothioate morpholino oligomer (PMO) backbone) to A. thaliana demonstrated systemic movement, but delivery of naked dsRNA did not appear to initiate endogenous gene silencing. The topically applied nematode dsRNA (dsvha-3) on A. thaliana leaves did not reduce the nematode development on the roots as compared to the controls (dsgfp and no dsRNA).

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Western Australian State Agricultural Biotechnology Centre
UNSD Goals: Goal 15: Conserve Life on Land
Supervisor: Jones, Michael
URI: http://researchrepository.murdoch.edu.au/id/eprint/40748
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