Characterisation of the SIX6 effector protein from Fusarium oxysporum f. sp. lycopersici

Abstract

The phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici (Fol) causes vascular wilt disease in tomato. During host colonisation, Fol secretes multiple effector proteins into the xylem sap, including fourteen SIX (Secreted In Xylem) proteins. Three of these SIX proteins function as the avirulence determinants Avr1 (SIX4), Avr2 (SIX3) and Avr3 (SIX1) recognised by tomato resistance proteins I, I-2 and I-3, respectively. Despite identifying the I-7 resistance gene, which encodes a leucine-rich repeat receptor protein, its corresponding avirulence gene remains undetermined. This work initially aimed to identify Avr7 by evaluating untested SIX genes and novel effector candidates. Effector candidates from transcriptomic analysis of infected tomato roots were assessed via Agrobacterium-mediated co-expression with I-7 in Nicotiana benthamiana. I-7-dependent leaf cell death would indicate recognition. None of the examined candidates exhibited I-7-dependent cell death, but SIX6 induced cell death independently of I-7 (Chapter 2). This observation prompted experiments to characterise SIX6 function in planta.

In collaboration with Daniel Yu from the Williams Laboratory at ANU, Fol SIX6 was expressed in E. coli and recombinant protein purified for functional analyses. Purified recombinant Fol SIX6 induced cell death in N. benthamiana and tomato (Chapter 3). Subsequently, SIX6 homologues from F. oxysporum ff. spp. cubense TR4, melonis and vasinfectum (Foc, Fom and Fov SIX6) were expressed and purified, and found to induce cell death responses in N. benthamiana and tomato (Chapter 3). Testing purified Fol, Foc and Fom SIX6 proteins in foliar tissues of representatives from multiple taxonomic families (Solanaceae, Cucurbitaceae, Brassicaceae and Leguminosae) revealed broad yet differential plant sensitivity (Chapter 3). Notably, Fol and Fov SIX6 induced stronger cell death in the corresponding tomato and cotton hosts than vice versa.

VIGS of known plant defence-signalling components in N. benthamiana was used to test whether Fol SIX6-induced cell death might constitute a plant immune response. VIGS targets included pattern recognition co-receptors BAK1 and SOBIR1, and mitogen-activated protein kinases WIPK and SIPK. VIGS-mediated depletion of these signalling components failed to attenuate Agrobacterium-mediated Fol SIX6-induced cell death (Chapter 4). These experiments were complemented by challenging mutant tomato lines compromised in defence signalling with Fol SIX6 protein. The ethylene-insensitive Never Ripe mutant and sun1-1 mutant deficient in EDS1-mediated resistance exhibited cell-death responses comparable to wild-type controls following SIX6 infiltration (Chapter 4). These findings suggested SIX6 induced cell death through a mechanism distinct from these plant defence pathways.

A microscopic investigation of cellular responses leading to SIX6-induced cell death revealed that Fol SIX6 protein causes stomatal opening in tomato and N. benthamiana before visible cell death (Chapter 5). Physiological measurements demonstrated that Fol SIX6 infiltration significantly increased stomatal conductance and transpiration rates in N. benthamiana leaves, regardless of light conditions (Chapter 5). Confocal fluorescence microscopy following Agrobacterium-mediated expression of a vacuole-targeted fluorescent protein revealed that SIX6 treatment caused vacuolar shrinkage and leakage in N. benthamiana leaf epidermal cells (Chapter 5).

An examination of the effect of Fol SIX6 on N. benthamiana and tomato roots showed that Fol SIX6 protein caused electrolyte leakage (Chapter 6). In vitro growth assays on medium supplemented with purified Fol SIX6 protein resulted in significant inhibition of primary root elongation in tomato seedlings (Chapter 6). These findings suggest SIX6 exerts physiological effects on root tissues that may contribute to Fol pathogenicity during infection. Show more