Structure-function analysis of an autoactive derivative of the tomato Cf-9 disease resistance protein

Abstract

An autoactive chimera of the tomato extracellular leucine-rich repeat receptor-like protein Cf-9, designated Hcr9-M205 has been characterized previously as exhibiting characteristics of constitutive defence activation (Barker et al., 2006b). The initial work of this thesis (Chapter 3) involved generation and assessment of transgenic tobacco containing an E22 (PR-5) promoter: gusA reporter construct as a quantitative reporter for Hcr9-M205 autoactivity in Agrobacterium-mediated transient expression (agroinfiltration) assays. Time course analysis showed that the induction of E22 promoter preceded the necrotic response induced by Hcr9-M205, providing an early indication of defence activation. Further characterization of the E22 promoter (Chapter 4) by incubating the E22: gusA tobacco leaf disks in different defence-inducing compounds using a multi-well plate set-up indicated the defence-inducible nature of E22 promoter including antagonistic regulation by salicylic acid and jasmonic acid, activation by ethylene and synergistic activation by salicylic acid and cytokinin; demonstrating the applicability of the leaf disks assays in screening potential plant defence activators.

Chapter 5 presents the structure-function analysis of the Hcr9-M205 protein. Previously, domain swapping analysis identified key regions involved in the control of Hcr9-M205 autoactivity namely a mismatch between LRRs 10-17 of Hcr9-9A (an upstream Cf-9 paralogue) and Cf-9 LRR 18 required for basal level of autoactivity and an additional Cf-9 C-terminal region comprising the loop-out domain and LRRs 24-26 for complete level of autoactivity (Anderson et al. in preparation). This thesis focuses on the proposed signalling repression domain in LRRs 10-17. Domain swapping analysis showed that an Hcr9-9A substitution in Cf-9 LRRs 15-17 was sufficient to cause autoactivity, suggesting that LRRs 15-17 and LRR 18 normally interacts for Cf-9 autoinhibition. The specificity-determining residues located at the solvent-exposed positions in the concave β-sheet surface of Cf-9 LRRs 13-16 required for Avr9 recognition (Wulff et al., 2009b) lie in the signalling repression domain and overlap the polymorphic positions involved in autoactivity, providing a basis for site-directed mutagenesis analysis. Introduction of these residues into the corresponding positions in Hcr9-M205 via site-directed mutagenesis revealed that those located the closest to LRR 18 had the greatest effects in signalling repression: Y389 of LRR 13 and E411 of LRR 14 did not significantly affect autoactivity, A433 of LRR 15 marginally repressed autoactivity whereas L457 of LRR 16 completely abolished autoactivity, similar to L481 of LRR 17 shown by Anderson et al. (in preparation). These findings were consistent with the notion that Cf-9 is autoinhibited by interactions between LRRs 15-17 and LRR 18. Unexpectedly, introduction of C387 of LRR 13 into Hcr9-M205 enhanced autoactivity. Sequence analysis comparing the Hcr9-M205(L389C) mutant containing C387 in Hcr9-M205, the CLB103V(14) domain swap that exhibited enhanced autoactivity and domain swaps that did not indicated that this phenomenon only occurred with an additional Hcr9-9A substitution spanning LRRs 14-17, suggesting that C387 may enhance signal activation upon Avr9-induced derepression and a possible role of E411 of LRR 14 in signalling repression. The data revealing some of the specificity-determining residues in signalling repression suggest that Avr9 recognition may directly compete with the autoinhibitory interactions mediated by these residues for Cf-9 activation.