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Towards engineering host resistance to root-knot nematodes (Meloidogyne spp)

Washer, Stewart James (1996) Towards engineering host resistance to root-knot nematodes (Meloidogyne spp). PhD thesis, Murdoch University.

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Abstract

Root-knot nematodes (Meloidogyne spp.) are serious root pathogens that affect commercial crops world-wide. The overall aim of this study was to develop techniques to identify plant genes expressed in specialised cells induced to feed these nematodes in order to develop a novel approach to host resistance. Little data exists on which root-knot nematode species are present in Western Australia. An initial study was carried out to identify species present in the major horticultural areas of WA. M. incognita, M. hapla, M. arenaria and M. javanica were isolated from a variety of crops with M. javanica found to be the most prevalent and widespread species. As a result, M. javanica was used as the nematode to develop an infection system for the molecular study of the host/parasite interaction with tomato as the host plant.

Several molecular approaches for developing host resistance to root knot nematodes have been suggested and are discussed. Root-knot nematodes parasitise the host plant root by modifying host cells from which the nematode feeds. These feeding structures are large, multinucleate cells termed "giant cells". Knowledge of giant cell-specific gene expression in the host plant is needed to develop synthetic resistance genes and to aid understanding of the host/nematode interaction. In an attempt to clone giant cell specific transcripts, a published PCR-based method for creating cDNA libraries from limited amounts of plant tissue was followed without success. As a result, considerable effort was expended to generate a new method for creating subtracted cDNA libraries. This new method involved the use of paramagnetic oligo-dT Dynabeads for the mRNA isolation directly from microgram quantities of crude giant cell enriched tissue homogenate, first strand synthesis on the beads using reverse transcriptase then tailing of the cDNA strand with dGTP so that a pool of second strand cDNA could be generated with a novel primer. This pool of second strand cDNA was then subtracted with a healthy root cDNA driver bound to Dynabeads for easy removal after each of three rounds of subtraction. The remaining giant cell enriched cDNA transcripts were then PCR amplified by use of primers with engineered restriction digest sites that allow direct cloning to create the cDNA library. Development of this original protocol and its benefits over existing techniques are discussed.

Thirteen clones from a giant cell enriched cDNA library were analysed by sequencing and hybridisation to DNA and RNA from plants and nematodes. Several clones were identified which have homology to known sequences.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: repository@murdoch.edu.au. Thank you.
Supervisor(s): Jones, Michael
URI: http://researchrepository.murdoch.edu.au/id/eprint/51678
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