Identification and regulation of receptors involved in plant immunity

Abstract

Plants sense invading microbes using surface-localised transmembrane receptors (pattern recognition receptors, PRRs) and intracellular nucleotide-binding - leucine-rich repeats proteins (NB-LRR). PRRs detect elicitors conserved amongst whole classes of microbes (pathogen-associated molecular patterns, PAMPs), such as bacterial flagellin. PAMP perception initiates cellular reactions collectively called PAMP-triggered immunity (PTI). Considering the number of transmembrane receptors present in plant genomes, only few PRRs have been identified to date. Identification of novel PRRs is important from a basic knowledge standpoint and because interfamily transfer of PRRs can confer broad-spectrum disease resistance. I used the common PRR co-factor BAK1 as bait to purify new PRRs after PAMP treatment. The success of this novel strategy was confirmed by the identification of the receptor for the bacterial PAMP cold shock protein (CSP) from Nicotiana benthamiana (Cold Shock Protein Receptor; NbCSPR). Perception of CSP is potentiated by earlier flagellin recognition, confers resistance to bacteria in an age-dependent manner, and limits Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transfer of NbCSPR to Arabidopsis thaliana confers CSP recognition, suggesting that the gene can be transferred into other plant species to confer anti-bacterial resistance. I further studied recognition of the fungal pathogen Puccinia striiformis f.sp. tritici in non-host species. Treatment of N. benthamiana and A. thaliana with stripe rust PAMP-preparations suggested the existence of PRRs that recognise this wheat pathogen. Using the above BAK1 strategy, I identified candidates for such PRRs and their potential roles in restricting rust diseases in non-host plants through PTI may now be determined. PTI can be supressed by pathogenic microbes through the secretion of virulence effector proteins, which target defence components and lead to disease development in susceptible plants. The recognition of effectors by intracellular NB-LRR Resistance (R) protein can however initiate effector-triggered immunity (ETI), usually associated with a localised cell death that can prevent pathogen spread. How effector recognition translates to cell death is not well understood. Because of the dramatic outcome of ETI, R proteins have to be tightly regulated, and understanding these mechanisms is crucial for engineering durable resistance to crop pathogens. The tomato R protein complex composed of the Pto kinase and the NB-LRR protein Prf confers resistance to the bacterium Pseudomonas syringae pathovar tomato (Psto), the causal agent of bacterial speck disease. The Pto/Prf complex is tightly regulated: In the absence of the Psto effectors AvrPto and AvrPtoB, Pto negatively regulates Prf and vice versa. Effector binding to Pto triggers a number of conformational changes within the complex for which the structural basis is largely undetermined. The unique N-terminal domain of Prf (N) is the Pto binding site, and must play an important role in regulation of the complex. I analysed the interactions between Pto and N using a co-immunoprecipitation strategy. I developed a schematic model of the complex, which includes a novel interaction between the N and LRR domains. Finally, using the Pto homolog Fen, I develop a model suggesting that Fen (and by analogy Pto) together with N, encodes the module for downstream signalling, leading to cell death and resistance.