Aims to:

• Identify, through the use of functional genomics technologies, novel genes involved in defence signalling that may be used to enhance resistance to economically important pathogens
• Test the principle that these genes will confer resistance in transgenic plants using Arabidopsis as a model system and apply this knowledge to crop plants
• Further evaluate selected plant defence gene promoters patented by the CRC for Tropical Plant Protection as specific disease resistance switches to drive expression of defence transgenes in crop plants while minimising impact on crop yield
• Genetically map and characterise jasmonate-signalling mutants (under- or over-expressing PDF1.2 marker gene after methyl jasmonate treatment) identified earlier in this sub-project

 Where we're at:

During 2001-02, sixty (60) candidate genes, selected from a detailed time course of the defence response on 2500 Arabidopsis genes, were analysed in greater detail using the reverse northern technique. Several genes were subsequently selected for functional analysis in transgenic plants. The expression profiles of 44 genes identified by microarray analysis were confirmed during 2002-03 using independent biological replicates for real-time quantitative RT-PCR. Detailed time-course studies at time points between 1 and 72 hours after pathogen inoculation or treatment with different signalling compounds have been completed on both wild-type plants and known defence-signalling mutants.

• Over-expressing transgenic Arabidopsis plants for a number of putative key regulatory genes (e.g. genes encoding different transcription factors, including WRKY; AP2 domain containing transcriptions factors) were produced during 2002-03. These over-expressing lines, and T-DNA insertional mutants (knock-out mutants) for these genes, have been analysed for changes in the plant defence response. Significant changes in expression of plant defence genes such as PDF1.2 and PR4 were identified for lines containing these putative regulatory genes, providing insight into the possible regulatory functions of these genes. Strategies to increase disease resistance by manipulating the expression of these regulatory genes are in development.
• Functional analysis, using different experimental approaches, of the putative key regulatory genes encoding two transcription factors was undertaken during 2002-03. Detailed expression profiles were obtained for both genes using time-course studies of plants that were either inoculated with Alternaria brassicola, or treated with defence signalling compounds. The results showed an up-regulation as early as one hour after treatment, suggesting early involvement in the response. Similar studies were also carried out on known defence mutants to identify which pathways are affected. A transient protoplast assay, together with over-expressing transgenic Arabidopsis plants, allowed the comparison of expression profiles of defence marker genes (e.g. PDF1.2) with those in wild-type plants and T-DNA insertional mutants. Results showed that, when over-expressed under certain conditions, both regulatory genes led to the down-regulation of jasmonate-dependent defence genes. In contrast, in insertional (knock-out) mutants, up-regulation was measured, suggesting the role of these genes as negative regulators of plant defence. Results on pathogen challenged plants suggest increased resistance in plants harbouring a dysfunctional transcription factor. Currently, this strategy is being tested using orthologous genes from crop species, including wheat, maize, rice, barley and cotton.
• During 2002-03, detailed expression profiles using time-course studies were obtained for non-regulating genes, e.g. those encoding a lectin-like protein, a senescence-associated protein, and an ABC transporter. Purification and assessment of antimicrobial activity of some of these proteins is currently being tested in combination with pathogen challenge experiments.
• A variety of plants containing hormone response mutations were tested for an altered pathogen response profile using real-time PCR of selected signal response pathway genes in Arabidopsis, to identify cross-communication between hormonal and developmental signalling pathways.

A transgenic ‘indicator plant’ line expressing the plant defensin gene PDF1.2 promoter linked to a GUS reporter gene was used to rapidly identify plants with disrupted ability to defend themselves against pathogens so that the corresponding defence signalling genes could be identified and studied. One of the putative defence signalling mutants showed levels of PDF1.2 transcript significantly lower than in wild-type plants. In addition, the inducibility of PDF1.2 by methyl jasmonate in this mutant was significantly lower at 24 hours after treatment compared with wild-type plants. The location of the mutation is currently being mapped. Pathogen inoculation experiments are currently being optimised to further characterise the mutant.

Scientific Highlights:

• A quick transient assay has been developed to functionally characterise putative signalling genes identified by microanalysis
• Functional confirmation of putative defence genes by over-expression in transgenic Arabidopsis plants, and comparison to T-DNA insertional (knock-out) mutants
• Development of a new strategy for disease resistance engineering using a novel defence gene identified in this sub-project
• Characterisation of inducible promoters from defence-associated genes, including the PDF1.2 gene of Arabidopsis
• Establishment of a large cDNA sample collection as a valuable resource for rapid expression profiling of any Arabidopsis gene under investigation

Potential Industry Outcomes:

• Defence signalling genes will provide novel, generic tools for engineering a wide range of crops for resistance against a wide range of pathogens
• Precise engineering of crops with optimised defence response
• Detailed knowledge of cross-communication between signalling pathways will assist in the production of plants with robust disease resistance that are not compromised by other unexpected outcomes, e.g. triggering of stress response

For more information contact:

Dr Paul Ebert
The University of Queensland
Phone: +61 (0)7 3365 2973
Email: p.ebert@mailbox.uq.edu.au