Identification and functional analysis of Pseudomonas syringae pv. actinidiae effector-triggered immunity in Nicotiana spp. and Arabidopsis thaliana : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand

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Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker in commercially important cultivars of kiwifruit (Actinidia delicosa and A. chinensis) worldwide, including New Zealand. Like many gram-negative pathogens, Psa is expected to utilise type III effectors to promote virulence in host plants. In order to better understand Psa effector-triggered immunity and susceptibility, we aimed to investigate multiple molecular characteristics of Psa type III effectors and their recognition mechanisms in model plants, Nicotiana spp. and Arabidopsis thaliana. Nicotiana tabacum and N. benthamiana are widely-used model plants for Agrobacterium-mediated transient expression (agroinfiltration) of effectors for functional characterization. Firstly, we screened multiple characteristics of effectors from two Psa strains, Psa NZ V13 and Psa NZ LV5. The former is a strongly virulent and the latter is a weakly virulent strain in kiwifruit. By using agroinfiltration in Nicotiana spp. to express individual effector proteins, we observed diverse subcellular localisation for Psa effectors. Additionally, we identified multiple Psa effectors that can trigger HR-like cell death (HCD) in both N. tabacum and N. benthamiana. Using virus-induced gene silencing (VIGS), we identified that some Psa effector-triggered HCD requires the immunity regulator SGT1, suggesting that the Psa effector-triggered HCD could be a result of immunity activation. We focused on one Psa NZ V13 effector, HopZ5, which belongs to the YopJ-like acetyltransferase family. HopZ5 triggers hypersensitive response (HR) in Arabidopsis accession, Ct-1. Another Arabidopsis accession, Col-0, does not develop an HR but shows immunity in response to HopZ5. The gene that confers HopZ5-triggered HR in Ct-1 was identified as SOBER1 (SUPPRESSOR OF AVRBST-ELICITED RESISTANCE 1) by using recombinant inbred lines derived from two parental accessions, Ct-1 and Col-0. SOBER1 is a known suppressor of Xanthomonas effector AvrBsT-triggered immunity. Interestingly, AvrBsT also belongs to YopJ family. Uniquely, SOBER1 specifically suppressed HCD triggered by several YopJ-like acetyltransferase effectors in N. benthamiana, including HopZ5 and HopZ3 from Psa. This suggests a common mechanism shared between a subset of YopJ-like acetyltransferase effectors is suppressed by SOBER1. Finally, we identified one Arabidopsis accession, Ga-0, which carries a truncated SOBER1 variant but does not develop an HR upon HopZ5 delivery. Using bulked- segregant analysis of an F2 population derived from a cross between Ct-1 and Ga-0, we mapped the locus conferring HopZ5-recognition in Ct-1 to the upper arm of Chromosome 3.
Pseudomonas syringae, Phytopathogenic bacteria, Arabidopsis thaliana, Nicotiana benthamiana, Disease and pest resistance, Kiwifruit, Diseases and pests, Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::Immunology