Following pathogen challenge, plants can develop a disease resistant state in distant, non-inoculated (systemic) tissues following an initial pathogen challenge. This increased resistance to subsequent pathogen challenge is associated with the up-regulation of defence marker genes such as the PDF 1.2, Thi2.1, PR1 and BGL2 genes in Arabidopsis each of which encodes various types of antimicrobial proteins. Prior to the up-regulation of these defence marker genes, a complex signalling network appears to transduce the perceived signal in locally infected and systemic, non-infected tissues. It may be possible to increase disease resistance by manipulation of this defence signalling network, to produce a broad-spectrum defence response. It was hypothesised that genes that are activated early during the response may encode key components that initiate or regulate the defence response. The main aim of this thesis was to identify novel elements involved in the regulation of the systemic defence response and characterise the function of the encoded proteins.
It was first useful to define the time frame for the initiation of the systemic defence response. For this purpose the expression profile of the gene PDF1.2, encoding an antimicrobial plant defensin, was analysed following inoculation of Arabidopsis with the incompatible fungal pathogen, Altenaria brassicicola. The up-regulation of a directly antimicrobial component of the defence response was used as a temporal marker to define the beginning of the "maintenance phase" of the defence response. Genes activated prior to the up-regulation of PDF1.2 were thought to be more likely to be involved in the "initiation phase" of the defence response and therefore possibly regulate the expression of PDF 1.2 and other defence marker genes encoding antimicrobial components. This work suggested that some induction of PDF 1.2 occurred as early as 5 hours post inoculation (PI) in local tissue and 10 hours in systemic tissue but statistically significant induction in four replicate experiments was only observed at 10 hours PI in local tissue and 15 hours PI in systemic tissue.
A multilayered screening process involving microarray, macroarray and quantitative realtime PCR gene expression analyses, and seven independent replicates of a time course inoculation experiment, was employed to analyse gene expression within the 15 hour PI time frame defined as the "initiation" phase. The application of multiple screening layers identified a small number of genes having a robust association with the initiation of the systemic defence response. Three genes, Rap-1 encoding a bHLH transcription factor, TT4 encoding chalcone synthase and At2g05940 (called Pipk1) encoding a protein kinase, were selected for further functional analysis based on significant induction at 5 hours PI. An additional gene, Zat10, was selected for further analysis based on significant induction prior to 21/2 hours PI in local tissue and 10 hours PI in systemic tissue, and the reported induction of gene expression by various other stress responses in other published microarray studies. Analysis of the pathogen-responsive induction of these four genes in selected defence signalling mutants provided information on their regulation and placed them in the context of current knowledge of the defence signalling network.
Disruption of the genes by T-DNA insertion led to the constitutive up-regulation of the PDF 1.2 and Thi2.1 defence marker genes in the untreated mutant plants and revealed that both Rap-1 and Zat10 negatively regulate the expression of the jasmonic acid-dependent defence pathway. Transfection of protoplasts with constructs for over-expression of Rap- 1 and a transcriptional activator of PDF 1.2, revealed that Rap-1 mediated negative regulation of PDF1.2 occurred through transcriptional suppression of activating transcription factors including ERFl. Stable transformation of Arabidopsis with the Zat10 gene led to suppression of PDF1.2 induction following treatment of plants with hormone inducers or inoculation with A. brassicicola, a result complementary to the TDNA insertion line and confirming the negative regulatory function of the Zat10 protein. The protein kinase encoded by At2g05940 (Pipk1) was found to affect the expression of the defence marker genes PDF 1.2, Thi2.1, ChiB and BGL2 in transgenic over-expressing Arabidopsis lines suggesting the involvement of the kinase in the defence signalling network.
The apparent regulation of defence marker genes by the identified regulatory proteins, and the presence of putative orthologues in crop species, suggests these genes may be useful for engineering increased broad-spectrum disease resistance in crop species.