SOBIR1-containing immune complexes at the plant cell surface: partners and signalling

We can learn from nature, by studying the mechanisms by which plants defend themselves against pathogens. This knowledge can be applied to improve crops in a sustainable fashion. Plants are sessile organisms with multiple layers of defence against pathogens. A prominent first layer of defence, which is similar to the innate immune system of mammalian cells, is provided by transmembrane (TM)-receptors, which are present at the plant cell surface. Upon pathogen invasion of the extracellular (apoplastic) space, TM-receptors mediate the recognition of pathogen- and/or host derived invasion patterns (IPs) in the extracellular space using their extracellular domain (ECD), which often consists of leucine-rich repeats (LRRs). Next to the ECD, TM-receptors have a single pass TM domain, and an intracellular domain. TM-receptors can be receptor-like kinases (LRR-RLKs), which contain an intracellular kinase domain to enable cytoplasmic signalling, or receptor-like proteins (LRR-RLPs), which contain only a small cytoplasmic tail and no signalling domain. LRR-RLKs and LRR-RLPs are here further referred to as RLKs and RLPs. The regulatory RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR, further referred to as SOBIR1) was recently found to constitutively interact with RLPs, and thereby support RLP accumulation as a kind of scaffold protein. Additionally, SOBIR1 was proposed to provide a signalling domain to RLPs, to result in bimolecular RLKs. RLKs, as well as bimolecular RLP/SOBIR1 complexes, both recruit the co-receptor Brassinosteroid-Insensitive 1 - Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3, further referred to as BAK1) upon ligand recognition by the primary ligand receptor. This BAK1 recruitment is thought to activate the TM-receptor complex for downstream signalling. Cultivated tomato (Solanum lycopersicum, Sl) possesses cell surface receptors, introgressed from wild tomato varieties, that provide resistance to the biotrophic leaf pathogen Cladosporium fulvum. These so-called Cf proteins mediate recognition of secreted effectors (also known as avirulence factors (Avrs)) of the pathogen in the apoplast. These Cf proteins are RLPs, and have recently been shown to constitutively interact with the RLK SOBIR1. The work in this thesis was initiated to elucidate the nature of the signalling steps that take place downstream of RLP/SOBIR1 bimolecular RLKs. To this aim, the signalling events that take place upon activation of the Cf-4/SOBIR1 complex by the matching C. fulvum effector Avr4 were studied. In Chapter 1, the plant innate immune system is introduced, and an overview of the current knowledge is given. The main focus lays on cell surface receptor complexes with an ECD consisting of LRRs, on their regulation, and on the immune responses that they trigger. Moreover, the tomato – C. fulvum pathosystem is introduced, with emphasis on the Cf-4/Avr4 gene-for-gene pair. SOBIR1 is required for RLP-mediated resistance to a wide range of pathogens, and it is hypothesized that SOBIR1 is targeted by effector proteins of pathogens to suppress host defence responses. In Chapter 2 it is shown that AvrPto, an effector of the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, interacts with SOBIR1 from Arabidopsis thaliana (At, further referred to as Arabidopsis) and with various Solanaceous SOBIR1 orthologues. This interaction is independent of SOBIR1 kinase activity. Interestingly, AvrPto suppresses AtSOBIR1-induced constitutive immunity, which is observed as cell death (the hypersensitive response (HR)) upon overexpression of AtSOBIR1 in N. benthamiana and tobacco. Additionally, AvrPto compromises the Avr4-triggered HR in Cf-4-transgenic N. benthamiana, without affecting Cf-4/SOBIR1/BAK1 complex formation. These results demonstrate that the bacterial effector AvrPto targets the regulatory RLK SOBIR1, and thereby compromises SOBIR1-mediated defence responses. In Chapter 3 it is shown that kinase activity of SOBIR1 is not essential for its scaffold function, as the kinase-dead mutant SlSOBIR1D473N also stabilizes Cf-4, similar to wild-type SOBIR1. However, kinase activity of SOBIR1 is crucial for downstream immune signalling, and therefore it is hypothesised that SOBIR1 transphosphorylates downstream signalling partners to initiate the activation of defence responses. Phosphorylation of signalling partners is an important molecular switch in various cellular processes, including plant defence. It was observed that AtSOBIR1, which constitutively activates immune responses upon its overexpression in N. benthamiana and tobacco, is highly phosphorylated in planta. Moreover, next to the required kinase activity of SOBIR1, kinase-active BAK1 is also essential for AtSOBIR1-induced constitutive immunity and for the phosphorylation of AtSOBIR1. Furthermore, the activation of a defence response upon perception of Avr4 by Cf-4 depends on signalling-competent BAK1. These results, in addition to observations described in literature about other RLK signalling partners, suggest that SOBIR1 likely first transphosphorylates BAK1 upon its recruitment to the ligand-activated RLP/SOBIR1 complex, after which activated BAK1 transphosphorylates SOBIR1 to subsequently together initiate downstream signalling for immunity. Phosphorylation of SOBIR1 appears to be important for its role in signalling for defence and Chapter 4 elaborates on the findings described in Chapter 3. In Chapter 4, amino acids of the kinase domain of SOBIR1 that could potentially be phosphorylated upon pathogen recognition are identified, and the role of these potential phosphorylation sites in signalling for defence is analysed. Mutational analyses and three-dimensional modelling showed that the threonine (Thr, T) residues T519, T523, and T529, which are all highly conserved in the activation segment of the kinase domain of SOBIR1, are important residues for the role of SOBIR1 in immune signalling. Phosphorylation of these sites likely locks SOBIR1 in an active conformation by controlling the conformation of the activation loop. Phosphorylation of these amino acids likely stimulates the interaction of T523 and T529 with the arginine (Arg, R) residue and the catalytic aspartic acid (Asp, D) residue of the ‘RD’ motif, respectively. Moreover, phosphorylation on T522, and the tyrosine (Tyr, Y) residues 532 and 538, which are also highly conserved, is likely generating substrate specificity and differential affinity for interacting partners. Co-immunoprecipitation of Cf-4-associated SOBIR1, through a pull-down of Cf-4 in the resting state and in the Avr4-activated state from N. benthamiana:Cf-4-eGFP plants, and subsequent analysis via mass spectrometry (MS), did not identify differential phosphorylation of the SOBIR1 kinase domain. However, in planta overexpression of AtSOBIR1-eGFP, followed by its immunoprecipitation and analysis via MS, revealed that AtSOBIR1 is phosphorylated on several serine (Ser, S) and Thr residues of its kinase domain, including T519. It is concluded that specific phosphorylation of the kinase domain of SOBIR1 likely enables this regulatory RLK to specifically switch on immune signalling downstream of RLPs. Directly downstream of RLKs, plants employ receptor-like cytoplasmic kinases (RLCKs) to signal for defence. Botrytis-Induced Kinase 1 (BIK1) is a central RLCK that signals downstream of several RLKs, including Flagellin-Sensing 2 (FLS2) in Arabidopsis. In Chapter 5, BIK1 is shown to be also important for defence signalling downstream of RLP/SOBIR1-containing complexes, as AtBIK1 was found to interact with AtSOBIR1, as well as with SlSOBIR1. Moreover, overexpression of the closest Solanaceous BIK1 orthologues enhanced the HR triggered by Cf-4 upon recognition of Avr4. On the contrary, overexpression of AtBIK1 appeared to suppress the Cf-4/Avr4-triggered HR. Although a silencing screen of a broad set of N. benthamiana BIK1-homologues did not point to a clear Solanaceous RLCK involved in the Cf-4/SOBIR1-mediated HR, a split-luciferase assay showed the interaction between several tomato BIK1-homologues and SlSOBIR1 and AtFLS2. Furthermore, the tomato RLCK Tomato Protein Kinase 1b (SlTPK1b) was shown to specifically interact with SlSOBIR1. Together, these data suggest that RLCKs play a role in signalling downstream of RLP/SOBIR1 complexes. TM-receptors need to be tightly controlled and regulated to ensure the triggering of a robust defence signal, and at the same time prevent false activation of defence responses. Physical separation of receptors and their co-receptors on the PM by negative regulators helps to keep signalling for defence in check, and thereby retain plant homeostasis concerning growth and development on the one hand and immunity on the other hand. The BAK1-Interacting RLK (BIR) protein family of Arabidopsis is a group of RLKs that negatively regulate immunity by interfering with the hetero-dimerization of the co-receptor BAK1 with ligand-binding immune-receptors. In Chapter 6, it is shown that the BIR protein family is conserved in Solanaceous plants. Gene silencing, overexpression, and protein-protein interaction studies show that the Solanaceous BIR1 orthologues are, similar to AtBIR1, negative regulators of cell death, and that BIR1 might suppress the Avr4-triggered HR in tomato containing Cf-4. Additionally, BIR2 orthologues of tomato and N. benthamiana seem not to be involved in modulating the Cf-4/Avr4-triggered HR. From these results it is concluded that SlBIR1 might be a negative regulator of SOBIR1-mediated defence, possibly through its interaction with SlBAK1, whereas SlBIR2 appears not to be involved in the regulation of Cf-4/SOBIR1-mediated defence. This leads to the hypothesis that BIR1 and BIR2 each likely interact with a different pool of BAK1 present at the plasma membrane, and that each pool is probably involved in a different defence pathway. Chapter 7 summarizes and discusses the major findings described in this thesis. The results of this thesis, together with other recent fundamental discoveries describing plant-microbe interactions on a molecular level, support a refinement of the invasion model that was developed to describe plant-microbe interactions. All TM-receptors with an LRR ECD recruit BAK1 upon their activation by the matching ligand, leading to transphosphorylation events and the initiation of downstream defence responses. These defence responses can be either mild or strong. Strikingly, there are RLP/SOBIR1 complexes that signal for a strong defence response including an HR, and also RLP/SOBIR1 complexes that signal for basal defence responses. All RLP/SOBIR1 complexes tested to far require BAK1 recruitment, and probably transphosphorylation of the kinase domains of SOBIR1 and BAK1 to initiate defence signalling. Therefore, as all signalling initiated by TM-receptors seems to be similar on the molecular level, this supports a spatial division rather than a division based on the intensity of the generated defence responses, which was up till now mostly used. In the ‘spatial invasion model’, IPs are therefore proposed to be classified as either extracellular IPs (ExIPs) or intracellular IPs (InIPs). As a consequence, recognition of ExIPs by TM-receptors leads to extracellularly-triggered immunity (ExTI), and recognition of InIPs by cytoplasmic receptors (which are mostly nucleotide-binding leucine-rich repeat receptors, NB-LRRs) leads to intracellularly-triggered immunity (InTI). Using this spatial dichotomy, the spatial invasion model facilitates a broadly including, but clearly distinguishing nomenclature to describe plant-microbe interactions