Molecular handshakes: going deep into the identification of proteins interacting with the tomato NB-LRR NRC1

PO-46 Molecular handshakes: going deep into the identification of proteins interacting with the tomato NB-LRR NRC1 Daniela SUELDO1, Marcel PRINS2, Matthieu H. A. J. JOOSTEN1 and Wladimir I.L. TAMELING1 1Wageningen UR, the Netherlands ; 2 Keygene, the Netherlands Plant disease resistance (R) proteins confer immunity to pathogens possessing the corresponding avirulence (Avr) proteins. Most R proteins localize intracellularly and only few (e.g. Cfs) are extracellular transmembrane proteins. Most intracellular R proteins belong to the nucleotide-binding and leucine-rich repeat (NB-LRR) class. Activation of NB-LRRs is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. NRC1 (NB-LRR required for HR-associated cell death-1) is an NB-LRR from tomato, which participates in the signaling cascade required for Cf-mediated resistance to Cladosporium fulvum. Silencing of NRC1 in Nicotiana benthamiana results in an impaired HR induced by extracellular and intracellular R proteins. To understand how NRC1 regulates the defense response induced by multiple R proteins, we focus on the identification of interacting proteins. To this end, a yeast two-hybrid screening is currently in progress. A tomato cDNA library was constructed, derived from tomato ‘dying seedlings’ that undergo a synchronized HR; as they produce both Cf-4 as well as its cognate effector Avr4. Different domains of NRC1 are used as baits (NRC1-CC, NRC1-NB and NRC1-NB-ARC-LRR). In parallel, various tagged versions of NRC1 will be expressed in N. benthamiana for subsequent affinity-purification and identification of associated proteins by mass spectrometry. In most cases only a slight inhibition of NRC1 activity was observed. With this research we want to elucidate whether NRC1 is a true signaling hub for multiple R proteins, required to induce defense responses