Molecular analysis of plant innate immunity triggered by secreted effectors from bacterial and fungal pathogens of apple : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, Institute of Agriculture and Environment, Massey University, New Zealand

Abstract

In comparison to animals, plants do not have a dedicated immune system with mobile immune cells to protect themselves. Instead they rely on the innate immunity of each cell. Plant immunity branches into two classical layers: PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). PTI detects the conserved molecular patterns (PAMPs) associated with pathogens and often can be overcome by pathogens translocating effector molecules into plant cells to inhibit the PTI. ETI, in turn, relies on intracellular receptors that can specifically recognize effectors or their activity and activate a rapid and robust response. The research presented in this thesis is focused on two pathogens of apple plants: the bacterial pathogen Erwinia amylovora (the causal agent of fire blight) and fungal pathogen Venturia inaequalis (the causal agent of apple scab disease). As both bacterial and fungal pathogens deliver effector molecules in order to promote their virulence, ETI engineering is a promising universal strategy to control these pathogens. In Chapter 3, the main aim was to elucidate the requirements and precise mechanism of how an important effector of E. amylovora, AvrRpt2, is recognized by the MR5 disease resistance (R) protein, derived from a hybrid apple Malus x robusta 5. I identified that a fragment of the guardee apple protein RIN4 was required and sufficient and required for MR5 activation. I further identified crucial amino acid residues responsible for this activation. Interestingly, cognate residues in RIN4 guardee homolog from Arabidopsis thaliana are responsible for suppression of the autoactivity of R protein RPS2. These findings led to the proposal of a novel hypothesis for evolutionary guardee adaption to the pool of R proteins present in plants. In Chapter 4, the main focus was to apply newly acquired whole-genome sequencing data of V. inaequalis for identifying the previously mapped AvrRvi8 effector, as well as several novel effectors predicted in silico. The sequences of these effectors were validated by amplification and resequencing of candidate genes from V. inaequalis cDNA. Further functional analysis of the selected gene candidates was performed. In addition, a library of constructs for generating V. inaequalis knock-out strains was prepared for future work. The findings from this thesis expected to be useful for breeders of apple to battle two economically important pathogens devastating the industry. Deployment of the MR5 system in apples should facilitate fire blight resistance in pipfruit and offers the opportunity for further engineering of MR5 to detect other pathogens. Furthermore, the effector library developed for V. inaequalis offers a novel tool for studying both virulence and avirulence mechanisms present in the applescab pathosystem. It is envisaged that further effector research will elucidate authentic targets critical for resistance development in apple.