SUMMARY Mitogen-activated protein kinases (MAPKs) play pivotal roles in the signal transduction pathway of plant defence responses against pathogens. A search for MAPK-interacting proteins revealed an interaction between a Nicotiana benthamiana MAPK, SIPK (NbSIPK) and cytosolic Hsp90 (NbHsp90c-1) in yeast two-hybrid assay. To study the function of Hsp90 in disease resistance, we silenced NbHsp90c-1 in N. benthamiana by virus-induced gene silencing (VIGS) with Potato virus X (PVX). NbHsp90c-1 silenced plants exhibited: (1) a stunted phenotype, (2) no hypersensitive response (HR) development after infiltration with the Phytophthora infestans protein INF1 and a non-host pathogen Pseudomonas cichorii that normally triggers HR in N. benthamiana, (3) compromised non-host resistance to P. cichorii, and (4) consistently reduced transcription levels of PR (pathogenesis related) protein genes. Similar phenotypes were observed also for plants in which a cytosolic Hsp70 (NbHsp70c-1), a gene for another class of molecular chaperon, was silenced. Hsp90 was isolated as a MAPK-interacting protein in yeast two-hybrid assay, therefore we tested the effect of NbHsp90c-1 silencing as well as NbHsp70c-1 silencing on the HR development caused by infiltration of a hyperactive potato MAPKK (StMEK1(DD)). No difference in the timing or extent of HR was found among NbHsp90c-1 silenced, NbHsp70c-1 silenced and control plants. This result indicates that observed impairment of INF1- and P. cichorii-mediated HR development in NbHsp90c-1 silenced and NbHsp70c-1 silenced plants was not caused by the abrogation in MAPK function downstream of active MAPKK that leads to HR. These findings suggest essential roles of Hsp90 and Hsp70 in plant defence signal transduction pathway upstream or independent of the MAPK cascade.