Improved resistance to root pests: final report to Grape and Wine Research & Development Corporation

There were two major aims of this project, which was jointly funded by GWRDC and CRCV2. The first aim was to identify and characterise grapevine genes involved in the plantâ s interaction with phylloxera and/or root-knot nematode. This information could be used to design strategies for engineering novel resistance to these pests in grapevine. The second aim of the project was to develop and use systems to rapidly assess candidate genes for preventing infestation of vine roots by phylloxera and/or root-knot nematode. Progress towards both goals was achieved despite premature termination of the project after 4, instead of 7 years.Project Leader: Dr. Robyn van Heeswijck until her retirement in July, 2002, then Prof. Steve Tyerman (in an administrative capacity); Author Details: Dr Tricia Franks (using text from the original application by Dr. Robyn van Heeswijck

Interaction between Vitis vinifera and grape phylloxera: Changes in root tissue during nodosity formation.

• Background and Aims The interaction between the gall‐forming grapevine parasite, phylloxera, and the susceptible grapevine species Vitis vinifera was investigated. • Methods Phylloxera and grapevines were cocultivated using both potted and micropropagated grapevines. Development of nodosities on primary roots was studied by microscopy and histochemistry, and nodosities were analysed for biochemical changes and changes in gene expression. • Key Results Within a nodosity, phylloxera fed at a site in the root cortex. Nodosity development was characterized by swelling of the root tissue distal to the feeding site, lack of development of a suberized endodermis, and starch and amino acid accumulation, and was eventually followed by root necrosis. No evidence of a defence response was observed in pre‐necrotic nodosities, but defence‐type responses were observed in tissue adjacent to necrotic regions. Changes in gene expression were not detected by northern hybridization using DNA probes encoding a range of V. vinifera transcripts. • Conclusions Nodosities on V. vinifera potentially function as nutrient reservoirs, and defence responses to phylloxera attack were not detected

Purification and characterization of ornithine acetyltransferase from Saccharomyces cerevisiae

Ornithine acetyltransferase has been purified 4000-fold to homogeneity from Saccharomyces cerevisiae. The enzyme catalyses the freely reversible interchange of an acetyl group between N-acetylornithine and glutamate and has a specific activity of 22 μmol · min−1· mg−1 at 37°C and pH 7.5. The Km values were determined for the substrates of the forward and reverse directions to be 1.0 mM for N-acetylornithine, 7.2 mM for glutamate, 1.5 mM for ornithine and 17.1 mM for N-acetylglutamate. The enzyme was localised to the mitochondrial matrix and was found to be a 57-kDa heterodimer consisting of subunits of 31 kDa and 26 kDa. Antibodies raised against the small subunit immunoprecipitated a single in vitro translation product of approximately 57 kDa suggesting that the subunits are processed from a single precursor protein. This is supported by N-terminal sequence analysis which shows that the 26-kDa subunit exhibits 40% sequence identity (8 out of 20) with the N-terminus of ornithine acetyltransferase from Neisseria gonorrhoeae whereas the N-terminus of the 31-kDa subunit exhibits 45% identity (9 out of 20) with a sequence located in the middle of the 60-kDa N. gonorrhoeae enzyme. The N-terminal sequence of the small subunit has the potential to form an amphiphilic helix, further suggesting that the precursor protein with the small subunit at its N-terminus could be targeted to mitochondria and processed into two subunits