Abstract:
Perception of bacterial flagellin by the receptor kinase AtFLS2 is a classic model for studying the molecular functioning of plant immunoreceptors. Although FLS2 receptors with a conserved ectodomain consisting of 28 leucine-rich repeats (LRRs) occur in a broad range of plant species, these FLS2 homologs display species-specific characteristics with respect to the affinity and specificity for the peptide ligand flg22 and derivatives thereof. SlFLS2 from tomato, for example, has higher affinity for flg22 than AtFLS2 and is able to recognize the shortened ligand peptide flg15. In previous work, chimeric receptors with LRR subdomain swaps between AtFLS2 and SlFLS2 were generated to investigate the LRRs responsible for the species-specific features (Mueller et al., 2012). Most of these chimeric receptors indeed proved functional and exhibited characteristics for either SlFLS2 or AtFLS2.
However, Sl15-24, one of the hybrids with the LRRs 15-24 from SlFLS2 replacing the corresponding LRRs in AtFLS2, showed constitutive activation of defence responses even in the absence of ligand. Interestingly, this autoactivation depended strictly on the co-receptor AtBAK1/AtSERK3. None of the other SERKs from A. thaliana, even when overexpressed, did cause autoactivation. Intriguingly also, in mutants lacking AtBAK1 the Sl15-24 hybrid behaved as a functional flg22-receptor much like AtFLS2, inducing a full set of responses when treated with its ligand flg22. Additionally, Sl15-24-induced autoactivation could be supressed by the negative regulators BIR2, BIR3 and BIR4 which are known to negatively regulate AtBAK1 and prevent flg22-dependent activation of AtFLS2. These results strongly suggest that the Sl15-24 hybrid interacts with AtBAK1 to trigger cellular responses in the absence of ligand. However, Sl15-24 and AtBAK1 do not form a stable complex in a manner comparable to the ligand (flg22)-induced complex between AtFLS2 and AtBAK1. Since the autoactivation process depends on functional kinase domains on Sl15-24 and AtBAK1, this suggests transient interaction between these partners to be sufficient for the induction process.
With further swapping constructs we narrowed the LRRs required from SlFLS2 for causing autoactivation to the LRRs 18-24. However, importantly, extending the swap to the LRRs 11-24 abolished the autoactivation effect and resulted in fully functional, ligand-dependent FLS2 receptor chimeras. Thus, autoactivation occurs only in chimeras that have 18-24 from SlFLS2 in combination with LRRs 7-14 from AtFLS2.
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