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
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.