Structural investigation of the interaction between SIX effectors and resistance proteins

Abstract

Fungal pathogens are the main causative agents of disease in plants. Fusarium oxysporum is a diverse fungal pathogen able to infect a wide range of hosts. It causes the disease known as Fusarium wilt that results in large economic losses in many important crop industries worldwide. During colonisation of the xylem vessels, F. oxysporum secretes a suite of virulence proteins known as effectors to promote pathogen virulence. However, effectors in general lack amino acid sequence similarity to proteins of known function hindering accurate structural and functional predictions based on amino acid sequence alone. Some SIX effectors can be recognised by tomato resistance proteins, which results in the initiation of a defence response and disease resistance. The molecular basis of this recognition is also poorly understood. In this project I applied a cross-disciplinary approach involving synthetic biology, structural biology (experimental and computational), protein biochemistry and plant biology to characterise effectors from Fusarium oxysporum f. sp. lycopersici (Fol) and recognition by immune receptors. Many fungal effectors have a high cysteine content, with these cysteines often forming disulfide bonds. My research approach requires access to high quantities (mg) of pure recombinant effector proteins, however, the production of disulfide-rich proteins in Escherichia coli is difficult due to the unfavourable redox potential when produced in the cytoplasm. In Chapter 3, I adopted and optimised a co-expression system to produce disulfide-rich fungal effectors in E. coli. I also naturalised the system specifically for SIX effectors from F. oxysporum. This optimised workflow has facilitated the production of effectors from F. oxysporum, Parastagonospora nodorum and Rhynchosporium commune, with yield increases ranging from 2 to 29-fold compared to previously used production systems. The yield increases also helped improve protein purity and homogeneity. Collectively, this system facilitated the structural and functional investigation of numerous effectors. Chapter 4 focuses on the structural characterisation of effectors from F. oxysporum f. sp. lycopersici using experimental and computational approaches, and the utilisation of these structures to understand effector-effector and effector-receptor interactions. I solved the crystal structures of Avr1 (SIX4), Avr3 (SIX1) and SIX6, which revealed that they adopt a similar structural fold despite sharing less than 20% amino acid similarity. They represent the founding members of the F. oxysporum f. sp. lycopersici dual-domain (FOLD) structural family. I utilised computational structural prediction to demonstrate that Fol secretes effectors that adopt a limited number of structural folds during infection of tomato. This analysis also revealed a structural relationship between transcriptionally co-regulated effector pairs. Finally, I make use of the Avr1 structure to understand its recognition by the I receptor. Mutagenesis studies reveal that Avr1 is recognised by IM82 at the C-domain, and surface exposed residues in the C-domain allow Avr1 to escape recognition by iMoneymaker. In Chapter 5, I sought to understand how mutagenesis of IM82 and iMoneymaker can modulate the recognition of Avr1. It demonstrated that polymorphic residues, between IM82 and iMoneymaker, within the island domain control Avr1 recognition specificity. A single amino acid mutation in iMoneymaker was sufficient to rescue the recognition of Avr1 to levels similar to IM82, when assessed using a transient expression system. Collectively, the work presented in this thesis lays the foundation for the engineering of novel immune receptors in tomato and aid future efforts to protect plants against Fusarium wilt disease. Show more