The requirement for the molecular chaperone Hsp90 in N-mediated resistance to TMV

Tessa M. Burch-Smith, Yule Liu, Michael Schiff, Suhua Feng and S.P. Dinesh-Kumar

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven CT

The TIR-NBS-LRR R gene, N, confers resistance to the tobacco mosaic virus (TMV) in tobacco and in transgenic Nicotiana benthamiana. We performed a yeast three-hybrid screen to identify proteins that interact with the LRR domain of N. This screen of a tomato cDNA library identified LeHSP90 as interacting partner of the LRR of N. Concurrently, we identified LeHSP90 as an interacting partner of NbRar1 in a yeast two-hybrid screen. Rar1 is a conserved signaling component of several R gene pathways and we have previously shown that NbRar1 is required for N-mediated resistance to TMV. On the basis of these two interactions, we checked the interaction of LeHSP90 with NbSGT1, a protein partner of both N and NbRar1. LeHSP90 also interacts with NbSGT1. These yeast interactions were confirmed by in vitro pull down and biological significance was demonstrated by coimmunoprecipitating LeHSP90 with N, NbRar1 and NbSGT1 in turn. Further, we show by VIGS of LeHSP90 that it is required for N-mediated resistance. On the basis of our results and considering recently published results on other R genes, we conclude that the highly conserved chaperone HSP90 has an important in R gene signaling. In other efforts to understand the N-TMV interaction, we are also conducting studies to determine the cellular location of the interaction. Preliminary biochemical data indicates that N may be localized to the cytoplasm of uninfected cells.