Final Activity Report Summary - RLP-FUNCTION (Structure / Function analysis of RLPs) When I first start in 2004, some work done in the Jones lab on Arabidopsis homolog of Cf-9 revealed conserved amino acids in various domains of Cf-9. Our hypothesis was that this conservation might play a role in Cf-9 function.The first part of my project was to investigate the functional significance of amino acids conserved between Cf-9 and Arabidopsis RLPs. I focused my research on two main areas: C2, TM and juxta-TM regions. I used transient assays of mutated Cf-9 alleles in Nicotiana benthamiana (Nb) lines that express Avr9. My results clearly showed that the expression of most of the mutants did not show any HR after transient expression in plants carrying Avr9 gene. This demonstrated that those conserved amino acids are crucial for Cf-9 function. In order to follow the accumulation of the protein, and its membrane association, I designed a new antibody raised against C2 domain of Cf-9, which is as well able to detect Cf-4 (protein very close to Cf-9). As bioinformatics study in Jones lab of Cf-9 TM region revealed 2 GxxxG motifs, involved in protein homo- or hetero-dimerisation, it is likely that Cf-9 is involved in a protein complex to be functional. Moreover, I showed by site-directed mutagenesis that some amino acids in the C2 domain must have an interaction with other partner to be able to induce HR. So we decided to try to identify partner proteins by virus-induced gene silencing (Vigs) approach on 11000 cDNA clones from an Nb library. The silencing of 4 genes out of the 11 000 gave us not HR upon co-infiltration of Cf4 and Avr4, which indicate they are involved in the HR pathway. The silencing of the tomato homolog of those genes in Cf-4 expressing tomato plants showed for one of them and in a less extend for the 3 others a development of C.fulvum carrying Avr4. This, indicate that the disruption of these genes made Avr4 not able to be recognised by Cf-4. The same has been shown for Cf-9.The second part of my project was a new and risky approach to the study of the function of RLPs. The goal was to establish a Cf-/Avr response in Arabidopsis that can be used for genetic studies on Cf- signal transduction. I made chimeras between Cf- proteins and different Arabidopsis RLPs, and tested them by Agrobacterium transient assays in leaves of turnip (Brassica rapa), a system routinely used in the Jones lab to analyse RPS4 and AvrRPS4 function and Arabidopsis. The lab previously established a functional chimera between Cf-9 and Cf-2 (1). In this study, domain C1 and C2 of Cf-2 was fused to domain C3 and the rest of Cf-9. The chimera carried the Cf-2 recognition specificity (and requirement for Rcr3), but signaled using the Cf-9 C-terminal domain. This strategy was also used by He and co-worker (2) in the study of brassinosteroid perception by extra cellular domain of the receptor kinase Bri1. This was a risky approach and unfortunately we have no clear indication of success yet.
