Functional analysis of tomato immune receptor Ve1 and recognition of Verticillium effector Ave1

Similar to the animal innate immune system, plants employ extracellular leucine rich repeat (eLRR)-containing cell surface receptors to recognize conserved molecular structures that are derived from microbial pathogens. A number of these immune receptors, as well as the corresponding pathogen ligands, have been characterized. The interaction between the tomato Ve1 immune receptor and the Ave1 effector from the pathogenic fungus Verticillium serves as a model system for the study of plant innate immunity. The research described in this thesis was aimed at a further understanding of how the eLRR-containing cell surface receptor Ve1 confers recognition of the Ave1 ligand and how it activates downstream immune signaling. It has been shown that eLRR-containing cell surface receptors play important roles in development and innate immunity in various plant species.Chapter 1 gives an overview on the current status of research on eLRR-containing cell surface receptors, their co-receptors and corresponding ligands, with emphasis on structural aspects. The functions of distinct eLRR receptor domains, their role in structural conformation, ligand perception, signal transduction and receptor complex formation are extensively discussed. To facilitate studies on the Ve1-Ave1 model system, we describe the establishment of protocols to investigate Ve1-mediated recognition of Ave1 and immune signaling in tobacco in Chapter 2. We optimized an Agrobacterium tumefaciens transient expression assay (agroinfiltration) by testing various over-expression vectors, and found that co-expression of Ve1 and Ave1 leads to hypersensitive response (HR) only in particular tobacco species. We further report on virus-induced gene silencing (VIGS) in Nicotiana tabacum cv. Samsun that allows investigating signaling components involved in Ve1-mediated resistance. Collectively, we established N. tabacum as a model plant to study Ve1-mediated immunity. In Chapter 3, we further investigated whether co-expression of Ve1 and Ave1 in Arabidopsis leads to an HR, which may potentially be used as a straightforward screening method upon a random mutagenesis. However, although Ave1 is able to trigger an HR in resistant tomato and tobacco plants, co-expression of Ve1 and Ave1 did not activate an HR in Arabidopsis. These results suggest that the HR occurs as a consequence of Ve1-mediated resistance signaling, and it is not absolutely required for Verticillium resistance. In Chapter 4 we investigated the contribution of particular regions of Ve1 to the activation of immune signaling through domain swaps between Ve1 with its non-functional homolog Ve2. Agroinfiltration, as well as stable Arabidopsis transformation, revealed that chimeras in which the first thirty eLRRs of Ve1 were replaced with those of Ve2 remain able to induce HR and activate Verticillium resistance. However, a truncated Ve1 protein that lacks the first 30 eLRRs is no longer functional. We speculate that the non-functional Ve2 receptor may still interact with the Ave1 effector in the eLRR domain, but fails to activate immune signaling due to a non-functional C-terminus. In Chapter 5, site-directed mutagenesis was employed to further investigate the eLRR domain of Ve1. We designed alanine scanning mutants in the solvent-exposed residues across the convex surface of the eLRR domain. In each mutant, two of the five solvent-exposed residues in β-sheet of a single eLRR were substituted into alanines. Functionality of the mutants through agroinfiltration and stable transformation of Arabidopsis revealed three eLRR regions that are potentially required for ligand specificity and for co-receptor interaction. In addition, alanine substitution was employed to evaluate role of putative protein-protein interaction and endocytosis motifs in the transmembrane domain and the cytoplasmic tail of the Ve1 protein. However, no requirement of these domains for Ve1 functionality could be demonstrated. It has been demonstrated that eLRR-containing cell-surface immune receptors often recognize short peptide sequence stretches as epitopes of their ligands. In Chapter 6, we aimed to identify the surface epitope of the Verticilliumeffector Ave1 that is recognized by Ve1. Firstly, we assessed whether various Ave1 homologs are recognized by Ve1. Since we found that C-terminal fusion of a GFP tag to Ave1 compromised its recognition, we hypothesized that accessibility of the Ave1 C-terminus is essential for Ve1-mediated recognition. Ave1 truncations and domain swaps with Ave1 homologs that are not recognized by Ve1 showed that a nine amino acid sequence derived from the C-terminus of Ave1 is essential for recognition by Ve1. This nine amino acid epitope is sufficient to activate Ve1-mediated immunity. In Chapter 7 the highlights of the thesis are discussed and placed in a broader perspective. The current understanding of eLRR-containing cell surface receptors is discussed, taking the findings of this thesis into account, with specific emphasis on ligand perception and receptor complex formation. In addition, future perspectives on the future are sketched, and novel research questions are posed aimed to obtain further insights into how Ve1 may form complexes with various co-receptors and how Ave1 contributes to Verticillium pathogenicity.