Structural basis of disease resistance in flax against flax rust

Plant diseases are a major issue for economically important crops. Plant immunity is usually triggered by the recognition of a pathogen effector protein by a plant resistance (R) protein, leading to the activation of plant defenses and a localized cell death response. The effectors are structurally diverse and they have been shown to be involved in both virulence and suppression of basal immune responses. R proteins are structurally conserved and the majority of them have a central nucleotide binding (NB) domain, a C-terminal leucine rich repeat (LRR) domain and either a coiled coil (CC), or Toll/Interleukin-receptor like (TIR) domain at the N-terminus. The LRR domain appears to be involved in both recognition of the effector proteins and negative regulation, while the NB and TIR domains are required for R protein activation and initiation of defence signalling, respectively. The molecular and structural basis of R protein effector recognition, activation and signalling is poorly understood. In this study I have used the interaction between flax and the fungal pathogen flax-rust as a model system to characterize this process. This is a power full model system for studying the structural basis of plant disease resistance. Several flax R-genes and corresponding flax rust effector-genes have been identified, and the effector proteins AvrL567 and AvrM have been shown to interact directly with the R proteins L6 and M, respectively. The crystal structure of AvrL567 has also been solved. In the first part of this study I report on structural characterization of flax R proteins. The 2nd chapter of the thesis describes a high-throughput strategy that was employed to screen for constructs that can produce soluble R proteins and fragments in E. coli. This resulted in identification of several TIR domain constructs that yielded soluble protein in both sufficient quality and quantity required for structural studies. The 3rd and the 4th chapter describe crystallization, structure determination, and structural analysis of the L6 TIR domain. The structure reveals important differences from the structures of mammalian TIR domains, and highlights three separate functionally important protein surfaces, involved in dimerisation, interaction with a downstream signalling partner, and regulatory intramolecular interactions, respectively. In the second part of the thesis I report on the structural characterization of two different variants of the secreted flax-rust effector AvrM. One of these variants AvrM-A, is recognized by the M resistance protein in flax, which results in activation of a necrotic immune response. The second variant avrM, is not detected by M and promotes disease. The 5th chapter reveals that AvrM has a !-helical fold, exists as a dimer in solution and has a C-terminal structured domain. This chapter also describes crystallisation and structure determination of the C-terminal domain of both AvrM variants. In the 6th chapter refined structures of the two AvrM variants are reported. Both structures have a novel L-shaped helical fold, with two chains forming an anti-parallel dimer with an unusual non-globular shape. Analysis of the two structures provides insight into the molecular basis of cell entry, and the direct interaction with flax R proteins. In summary, this thesis has made a significant contribution towards our understanding of effector recognition, and the structural mechanisms behind initiation of R protein defence signalling. Our results bring us a step closer to understanding the molecular basis for the disease resistance process in plants, which is a prerequisite for the future engineering of novel resistance specificities into commercially important crops.