Plants are continually exposed to a vast array of pathogens, and generally succeed in defending themselves. An important part of this process is the induction of defence gene products that hinder pathogen growth and delay or prevent disease development. The way in which plants do this is multi-factorial, and includes regulatory proteins known as transcription factors (TFs) which directly regulate the expression of defence-related genes. The expression and activity of these TFs can be differentially regulated by pathogen challenge, as well as following exposure to defence-related signalling molecules such as methyl jasmonate (MeJA). To identify TFs potentially involved in MeJA signalling and plant defence, the expression of all 1,534 putative Arabidopsis (Arabidopsis thaliana) TF genes identified in the genome (at the time) were screened by Real-Time Quantitative PCR (RT-Q-PCR) for altered transcript expression 6 h following either MeJA treatment or inoculation with the incompatible fungal pathogen Alternaria brassicicola. This primary screen identified 134 TF genes that showed a significant change in expression compared to mock-treated plants, and included many genes of previously unknown function belonging to APETALA2/Ethylene Response Factor (AP2/ERF), MYB, WRKY, and NAC TF gene families. Twenty-four of these TF genes were shown to be repressed or induced by MeJA, as well as repressed or induced by A. brassicicola (i.e. co-regulated). A high proportion of these co-regulated genes were members of either the AP2/ERF or WRKY TF gene families. Selected TF genes that showed significant differential regulation following MeJA exposure or A. brassicicola inoculation were profiled in a four-point timecourse over a 24 h period. It was found that the AP2/ERF TF genes identified in the primary screen were the most consistently and reproducibly regulated family of genes. The majority of these regulated TF genes belong to two distinct subclusters of AP2/ERFs in an amino acid based sequence similarity tree, implicating the B3 and B1a subclusters in MeJA signalling and A. brassicicola defence. One gene from each subcluster was chosen for further analysis, based on its consistent pattern of induction by MeJA and A. brassicicola as well as the compatible fungal pathogen Fusarium oxysporum. These two TFs were AtERF2 (B3 cluster) and AtERF4 (B1a cluster). Transgenic Arabidopsis plants were generated that stably overexpress the AtERF2 TF gene. Analysis of these plants revealed that this TF is a positive regulator of a subset of MeJA-responsive defence genes encoding the antimicrobial proteins PDF1.2 and CHIB. Additionally, AtERF2-overexpressing lines displayed increased resistance to the fungal pathogen F. oxysporum compared to wild-type plants, as well as enhanced sensitivity to the MeJA-inhibition of root elongation. In contrast to the positive regulation displayed by AtERF2, functional analysis of AtERF4 by both stable overexpression and insertional inactivation in Arabidopsis clearly demonstrated that AtERF4 acts as a negative regulator of the MeJAresponsive expression of PDF1.2. Furthermore, it was demonstrated that AtERF4 acts as a negative regulator of resistance to F. oxysporum and antagonizes the MeJAinhibition of root elongation. Overall, the work undertaken during this PhD has identified two conserved clusters of AP2/ERF TFs with members that display contrasting roles in the regulation of defence gene expression, along with resistance to F. oxysporum and root sensitivity to MeJA. These results suggest that plants co-ordinately express multiple repressor- and activator-type AP2/ERFs during pathogen challenge to modulate defence gene expression and hence the development of disease. By identifying and characterising selected members of the AP2/ERF TF family, this work has provided a greater understanding of the mechanisms of the plant defence response leading to disease resistance. In doing so, it has provided novel tools to assist in the development of pathogen-resistant plants for use by the agricultural industry.

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Plants are continually exposed to a vast array of pathogens, and generally succeed in defending themselves. An important part of this process is the induction of defence gene products that hinder pathogen growth and delay or prevent disease development. The way in which plants do this is multi-factorial, and includes regulatory proteins known as transcription factors (TFs) which directly regulate the expression of defence-related genes. The expression and activity of these TFs can be differentially regulated by pathogen challenge, as well as following exposure to defence-related signalling molecules such as methyl jasmonate (MeJA). To identify TFs potentially involved in MeJA signalling and plant defence, the expression of all 1,534 putative Arabidopsis (Arabidopsis thaliana) TF genes identified in the genome (at the time) were screened by Real-Time Quantitative PCR (RT-Q-PCR) for altered transcript expression 6 h following either MeJA treatment or inoculation with the incompatible fungal pathogen Alternaria brassicicola. This primary screen identified 134 TF genes that showed a significant change in expression compared to mock-treated plants, and included many genes of previously unknown function belonging to APETALA2/Ethylene Response Factor (AP2/ERF), MYB, WRKY, and NAC TF gene families. Twenty-four of these TF genes were shown to be repressed or induced by MeJA, as well as repressed or induced by A. brassicicola (i.e. co-regulated). A high proportion of these co-regulated genes were members of either the AP2/ERF or WRKY TF gene families. Selected TF genes that showed significant differential regulation following MeJA exposure or A. brassicicola inoculation were profiled in a four-point timecourse over a 24 h period. It was found that the AP2/ERF TF genes identified in the primary screen were the most consistently and reproducibly regulated family of genes. The majority of these regulated TF genes belong to two distinct subclusters of AP2/ERFs in an amino acid based sequence similarity tree, implicating the B3 and B1a subclusters in MeJA signalling and A. brassicicola defence. One gene from each subcluster was chosen for further analysis, based on its consistent pattern of induction by MeJA and A. brassicicola as well as the compatible fungal pathogen Fusarium oxysporum. These two TFs were AtERF2 (B3 cluster) and AtERF4 (B1a cluster). Transgenic Arabidopsis plants were generated that stably overexpress the AtERF2 TF gene. Analysis of these plants revealed that this TF is a positive regulator of a subset of MeJA-responsive defence genes encoding the antimicrobial proteins PDF1.2 and CHIB. Additionally, AtERF2-overexpressing lines displayed increased resistance to the fungal pathogen F. oxysporum compared to wild-type plants, as well as enhanced sensitivity to the MeJA-inhibition of root elongation. In contrast to the positive regulation displayed by AtERF2, functional analysis of AtERF4 by both stable overexpression and insertional inactivation in Arabidopsis clearly demonstrated that AtERF4 acts as a negative regulator of the MeJAresponsive expression of PDF1.2. Furthermore, it was demonstrated that AtERF4 acts as a negative regulator of resistance to F. oxysporum and antagonizes the MeJAinhibition of root elongation. Overall, the work undertaken during this PhD has identified two conserved clusters of AP2/ERF TFs with members that display contrasting roles in the regulation of defence gene expression, along with resistance to F. oxysporum and root sensitivity to MeJA. These results suggest that plants co-ordinately express multiple repressor- and activator-type AP2/ERFs during pathogen challenge to modulate defence gene expression and hence the development of disease. By identifying and characterising selected members of the AP2/ERF TF family, this work has provided a greater understanding of the mechanisms of the plant defence response leading to disease resistance. In doing so, it has provided novel tools to assist in the development of pathogen-resistant plants for use by the agricultural industry.