FUNCTIONAL ANALYSIS OF THE ARABIDOPSIS DOWNY MILDEW RESISTANCE GENE RPP5 BY DOMAIN SWAPPING AND YEAST TWO-HYBRID ANALYSIS
E VAN DER BIEZEN, M COLEMAN, M BOTELLA, J PARKER and J JONES
Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK
Background and objectives
The Arabidopsis RPP5 gene confers resistance to the oomycete Peronospora parasitica (Noco2) and has sequence and structural homology with other plant resistance (R) genes, including the N gene for tobacco mosaic virus (TMV) resistance, and the Arabidopsis RPM1 gene for resistance to the bacterium Pseudomonas syringae avrB . These R gene products carry leucine-rich repeats (LRRs) and have nucleotide-binding (NB) domains. In contrast to RPM1 that has a leucine zipper (LZ) motif at the N-terminus, this region of RPP5 and N shares sequence homology with the cytoplasmic signalling domains of the Drosophila and human Toll receptors and the human Interleukin-1 receptor (TIR domain). The R gene products may function as controlling adaptors in a protein complex which becomes activated by pathogen-derived avirulence signals . The LRRs could play a role in ensuring that activation of the complex is recognition-dependent. The TIR and LZ domains may activate distinct downstream pathways following conformation-dependent ATP or GTP hydrolysis at the NB sites. Mutational analyses in Arabidopsis have indeed identified genes that define different pathways such as the EDS1 gene that is required for RPP5 but not for RPM1 function, and the NDR1 gene that is essential for RPM1 but not for RPP5 function. The hypothesis that (part of) the LRRs interact with the respective pathogen signals and that the TIR and LZ domains are involved in subsequent downstream signalling was tested by domain swap experiments. To further identify RPP5 domains that interact with Arabidopis or P. parasitica proteins the yeast two-hybrid sytem was employed.
Results and conclusions
Swapping domains between different R genes was predicted to change pathogen specificity and to converse activation of the EDS1 and NDR1 pathways. Recombinant R genes were made consisting of exchanged LRR, TIR and LZ domains of RPP5, N and RPM1, and were transferred to the Arabidopsis Col-0 line rps3-3 that is susceptible to P. parasitica (Noco2), TMV and P. syringae (avrB). Infections of the transgenic plants expressing the recombinant R genes with these pathogens showed that all chimaeric genes lost resistance function; several explanations are discussed.
Using different yeast two-hybrid bait constructs consisting of the TIR, NB and/or LRR domains, prey-cDNA libraries have been screened for interacting clones that activate reporter genes. The characterization of genes encoding proteins that specifically interact with RPP5 in yeast, and potentially involved in RPP5-mediated resistance in Arabidopsis, are presented.
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