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Stability and heritability of RNAi in generations of transgenic plants and nematodes

Akther, Jebin (2019) Stability and heritability of RNAi in generations of transgenic plants and nematodes. PhD thesis, Murdoch University.

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Plant parasitic nematodes (PPNs) are a major group of pests causing irreversible damage to crops worldwide. Damage caused by PPNs in Australia has been estimated at AU$ 300 million per annum. Two genera of sedentary endo-parasitic nematodes, with relatively wide host ranges, are major temperate/tropical crop pests in Australia. These are root-knot (Meloidogyne spp) and cyst nematodes (Heterodera, Globodera spp). They are difficult to control using crop rotation, chemical nematicides or biological control, and many nematicides have now been banned. RNA interference (gene silencing) technology is a potential alternative genetic approach for nematode control. A challenge of an RNAi-based transgene approach (‘Host-Induced Gene Silencing’) is to generate plants that express nematode control transgenes stably over many generations. Various factors can influence transgene stability (e.g., the target selected, transgene copy number, promoter methylation, sequence similarity). Therefore, in this project, the inheritance and effectiveness of RNAi in plants conferring resistance to PPNs and effects on PPNs themselves were studied to underpin knowledge of the application of RNAi as a strategy to confer nematode resistance in crop plants.

T2 seeds of different transgenic events in Arabidopsis thaliana plants expressing RNAi constructs for seven target genes of the root-knot nematode (Meloidogyne incognita)(dcr-1, drh-3, vig-1, mut-7, drsh-1, pash-1, rha-1) and two target genes of the beet cyst nematode (Heterodera schachtii) (sna-1, prp-4) were studied to determine the segregation patterns in subsequent generations and the stability of nematode resistance. Among 63 different transgenic events tested, 22 transgenic events segregated stably with simple Mendelian segregation patterns of 3:1 in three generations, whereas ten transgenic events consistently segregated for more than two T-DNA inserts. In the T2 generation of Mi-hp constructs, the pool of 52 individual transgenic events was positive for the presence of the selectable marker gene nptII that gave the expected PCR amplicons of 364 bp in transformed plants, whereas no amplification was detected from untransformed control plants. Similarly, in T2 and T3 generations, the stable integration of the nptII gene was confirmed by PCR, indicating that the transgenes were successfully transmitted through three generations. In the case of Hs-hp constructs, from 15 transgenic events, the pool of individual DNA samples was positive for the presence of nptII in T2 generation and T3 generations. q-PCR and ddPCR assays were performed to select single copy, and multiple copies of integrated RNAi hairpin constructs in transgenic plants, since copy number can affect the stability of transgenes. Selected transgenic plants of seven different genes were challenged with nematodes to determine the reduction of infection in two generations. Segregation patterns and copy number analysis showed different transgenic events containing single or multiple insertion patterns and copies respectively in the advanced generations. Copy number estimation by ddPCR and qPCR gave the same result. Three transgenic events (hp-gfp-7, Mi-hp-dcr-1.15, Mi-hp-rha-1.2) were confirmed as single copy over three generations both by ddPCR and qPCR. Infection assays of M. incognita showed significantly fewer (80%) galls and egg masses on the roots of infected transgenic plants than on wild type plants. Infection with H. schachtii resulted in significantly fewer (93 %) cysts compared with controls. Interestingly, nematodes that survived after feeding on transgenic plants had reduced egg hatching compared to nematodes fed on control plants. An additional infection assay was conducted to evaluate progeny nematodes for the maintenance of reproductive suppression. The results indicated that progeny were also impaired in their ability to reproduce successfully, as demonstrated by a 50% reduction in the cyst (H. schachtii) and 60% reduction in galls (M. incognita) when inoculated to wild type plants.

The outcomes of this study will help optimise and support an RNAi-based nematode control strategy for field application since stability in RNAi-based traits is vital for this strategy to be used to reduce the economic losses caused by PPNs.

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