Heat shock protein 90 and its co-chaperone protein phosphatase 5 interact with distinct regions of the tomato I-2 disease resistance protein

Recent data suggest that plant disease resistance (R) proteins are present in multi-protein complexes. Tomato R protein 1-2 confers resistance against the fungal pathogen Fusarium oxysporum. To identify components of the 1-2 complex, we performed yeast two-hybrid screens using the 1-2 leucine-rich repeat (LRR) domain as bait, and identified protein phosphatase 5 (PP5) as an 1-2 interactor. Subsequent screens revealed two members of the cytosolic heat shock protein 90 (HSP90) family as interactors of PP5. By performing in vitro protein-protein interaction analysis using recombinant proteins, we were able to show a direct interaction between 1-2 and PP5, and between 1-2 and HSP90. The N-terminal part of the LRR domain was found to interact with HSP90, whereas the C-terminal part bound to PP5. The specific binding of HSP90 to the N-terminal region of the 1-2 LRR domain was confirmed by co-purifying HSP90 from tomato lysate using recombinant proteins. Similarly, the interaction between PP5 and HSP90 was established. To investigate the role of PP5 and HSP90 for 1-2 function, virus-induced gene silencing was performed in Nicotiana benthamiana. Silencing of HSP90 but not of PP5 completely blocked cell death triggered by 1-2, showing that HSP90 is required for 1-2 function. Together these data suggest that R proteins require, like steroid hormone receptors in animal systems, an HSP90/PP5 complex for their folding and functioning.