Assoc Prof Kathleen Soole

Contact Details

Key Responsibilities

• Deputy Head of School

Teaching

• Biochemistry

Research

The following projects will be available in my laboratory. My projects range from ones involving plant, marine invertebrates and mammalian cell culture as I am interested in stress response in a wide range of organisms.

A. Respiratory Responses to Stress – Biotic and Abiotic.

It is now clear that mitochondria, the powerhouse of the cell, play key roles in oxidative stress and programmed cell death. Plant mitochondria have a number of unique enzymes and pathways that are not present in mammalian cells, which are not involved in ATP production. Evidence now suggests that these unique enzymes respond or change expression during stresses such as cold temperatures, pathogen attack, and nutrient limitation. Therefore, identification and characterisation of these enzymes such as Alternative Oxidase and the ND2 family of NADH dehydrogenases, may allow manipulation to improve post-harvest physiology or aid in understanding the cellular responses to pathogen attack or the processes in the initiation of cell death. These projects are funded by the Australian Research Council.

1. Role of oxidative metabolism in plant cell death/pathogen attack.

• Alternative Oxidase expression in tobacco challenged by Phytophthora spp. (Collaborator, Dr. Amanda Able, Plant Sciences Waite Institute, Univ. of Adelaide)

Resistant tobacco (Nicotiana tabacum) suspension cells challenged with zoospores from the Oomycete pathogen, Phytophthora nicotianae undergo a programmed cell death event called the hypersensitive response (HR) and produce the reactive oxygen species (ROS), superoxide and hydrogen peroxide. These ROS are produced in two bursts specific to resistance. The first burst between 0 and 2 hours after challenge appears to be involved in signalling while the much larger second burst between 8 and 12 hours after challenge is responsible for induction of the HR (Able et al., 1998). ROS production and the HR are not observed in susceptible plant cells challenged with Phytophthora or in controls.

Alternative Oxidase (AOX) responds to H2O2 levels in plant cells and is hypothesized to play a role in limiting the production of ROS. Interestingly and perhaps contrary to what is expected, potential AOX inhibitors prevent the HR and ROS production during the resistant response in the tobacco-Phytophthora interaction (Able et al., 2000).

While the use of inhibitors may be informative, actual detection of gene induction or protein levels is preferable. In this project, the expression and activity of AOX will be assessed during the resistant and susceptible responses of tobacco suspension cells (Zhang et al, 2001a&b) and plants to Phytophthora nicotianae.

In addition to this approach, we are interested in generating AOX knock-out mutants of the resistant and susceptible tobacco lines to further elucidate AOX’s potential role in the response of the plant to invasion. This will be done using post-transcriptional gene silencing or RNAi technology as developed by CSIRO (Waterhouse, P. et al, 2001).

• Does expression of Alternative Oxidase alter during Barley Scald infection? Collaborator – Dr. Peter Anderson

We also wish to determine whether AOX activity and expression alters in other plant-pathogen interactions. One interaction that is being studied at Flinders and the Waite Institute is the Barley scald disease. This is caused by an infection with the fungus, Rhynchosporium. To look at AOX expression in the Barley scald infection we wish to link the barley AOX promoter to a reporter gene. We already have some sequence information for a Barley AOX gene and this can be used to screen a barley genomic library which is available. This promoter would then be linked to an easily visualized reporter protein, such as the green fluorescent protein (GFP) or GUS and barley transformed with this construct. The transformed barley would be exposed to the fungus that causes Barley Scald (Rhynchosporium) and changes in GFP or GUS expression observed. Any changes would be indicative of changes in AOX expression.

2. Molecular Identification of the ND2 family of NADH dehydrogenases in plant mitochondria and elucidation of their physiological role.

Currently, a lot of information exists about AOX and its identity and role in stress response, relatively little is known about the other non-energy producing pathways in plant mitochondria, i.e. the NADH dehydrogenases. These enzymes do respond to stresses as does AOX, however, very little is known about their molecular composition. From enzyme activity analyses, we know that there are potentially four of these enzymes linked to the inner membrane electron transport chain. Recently, some sequence information has become available about these dehydrogenases with the sequencing of the Arabidopsis genome. From this database, five putative NADH dehydrogenases have been identified but it is not clear whether they are the mitochondrial proteins. It is the aim of this project to obtain the full-length sequence of the Arabidopsis cDNA of these dehydrogenases, and attempt to identify it using

1. yeast complementation using mutant yeast lines where a mitochondrial NADH dehydrogenase has been disrupted. the response of AOX

2. Investigating whether the cDNA encodes for a protein that can be imported into mitochondria.

3. Obtain the tobacco homologue using either RT-PCR or screening a library for future generation of a tobacco transformant as a null mutant using the RNAi technology mentioned above.  Currently, we have one tobacco homologue and are using gene silencing to knock out this gene. Analysis of the mitochondria will reveal which NADH DH this gene encodes.

B. Projects within the CRC for Tissue Growth and Repair.

1. Identification of Insulin-like peptides (ILP) from Invertebrates and investigation of their use as indicator of stress and/or growth rate (in collaboration with Dr. John Carragher and Ass. Prof. Jon Havenhand ).

Recently, we have cloned an ILP from abalone, which may be related to changes in growth rate or health status of the organisms. We are now producing this protein recombinantly to assess its impact on cell growth and glucose metabolism on cells in culture and for the development of a biological assay to determine the amount of this protein. Currently , there is a lot on Industry interest in this project as it may prove useful as stress indicator in the Abalone industry. Further, we have identified that this protein may also be present in a number of other invertebrates, which are also important in the aquaculture industry. Thus, we may be able to develop a ‘generic’ assay for this protein if there is a high homology in sequence among the invertebrate organisms.

Project: Identification of an ILP in Oyster.

This project is using the use preliminary sequence information gained for this protein in Abalone and Oyster to obtain the full length Oyster cDNA sequence and then examine the expression of this protein between different tissues and under different growth conditions.

2. Identification and characterization of TIMP-2 interacting proteins. (in collaboration with Prof. Wheldrake, Dr Dyer – FUSA and Dr. C. Goddard and Dr. A. Dunbar, GroPeP ).

The tissue inhibitor of metalloproteinase-2 (TIMP-2) protein regulates extracellular matrix stability by inhibiting the proteolytic activity of metalloproteinases. TIMP-2 also has a role in promoting the growth of certain cell types. The metalloproteinase inhibitory activity and the growth-promoting activity of TIMP-2 are thought to act via independent mechanisms. It is believed that the metalloproteinase inhibitory activity of TIMP-2 is confined to the N-terminal domain of the protein, however the growth-promoting activity of TIMP-2 is poorly understood.

It is the aim of this project to elucidate the mechanism of the growth-promoting action of TIMP-2. Specifically, to identify and characterise proteins that have been shown to interact with TIMP-2. Some candidates have been identified using the yeast 2-hybrid system and their interaction is being confirmed in mammalian cell systems. A more detailed study of the interactions, each unknown has with TIMP-2 is being investigated by producing mutant cDNA constructs and expressing the mutant proteins in the 2-hybrid system.

See Publication List

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