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

Abstract

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 𝘗𝘴𝘢effector-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.