Final Activity Report Summary - RAR1/SGT1 (Dynamics of RAR1-SGT1 complexes in disease resistance)
SGT1 and RAR1 are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90. In plants, SGT1, RAR1 and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. The overall aim of this fellowship was to assess the role of RAR1-SGT1 complexes in disease resistance.
Although RAR1, SGT1 and HSP90 interact with each other, it remains to be shown whether RAR1 and/or SGT1 associate together with HSP90 in a single complex. Using in vitro pull-down I have been able to demonstrate that RAR1 CHORDII domain and HSP90 can interact simultaneously with SGT1 through the CS domain forming a ternary complex. Furthermore, NMR studies using SGT1 CS domain have been performed in collaboration with Raphael Guerois (SBFM-DBJC, CEA Saclay, France) allowing to model the CS domain structure in solution and revealed that CHORDII domain of RAR1 and the N-terminal domain of HSP90 interact with opposite surfaces of the CS domain further supporting the existence of a ternary complex containing the three proteins. Using recombinant SGT1, RAR1 and HSP90 I also demonstrated that RAR1 is able to enhance SGT1-HSP90 interaction. SGT1 mutants impaired either in RAR1 binding (G190D) or HSP90 binding (K229E) revealed that RAR1 effect on SGT1-HSP90 binding depends on SGT1-RAR1 interaction and also on SGT1-HSP90 interaction.
In order to study interactions between R proteins and SGT1-RAR1-HSP90 complexes I successfully expressed several R proteins including Rx, Mla1 and Mla6 using a wheat germ based TNT. In vitro pull-down experiments showed that both SGT1 and RAR1 are able to interact with Rx, Mla1 and Mla6 through the CS and CHORDI domains respectively. Furthermore, as in the case of SGT1-HSP90 interaction, RAR1 was also able to enhance SGT1-Rx interaction. At the same time, in vitro studies have been combined with functional analysis of SGT1 CS mutations in vivo using the previously established Rx-PVX system in Nicotiana benthamiana (Peart et al., 2002). Functional analysis of CS mutations impairing either RAR1 or HSP90 interaction indicated that the interaction between SGT1 and HSP90, but not SGT1 and RAR1, is required for SGT1 function in Rx-mediated resistance against PVX.
Although RAR1, SGT1 and HSP90 interact with each other, it remains to be shown whether RAR1 and/or SGT1 associate together with HSP90 in a single complex. Using in vitro pull-down I have been able to demonstrate that RAR1 CHORDII domain and HSP90 can interact simultaneously with SGT1 through the CS domain forming a ternary complex. Furthermore, NMR studies using SGT1 CS domain have been performed in collaboration with Raphael Guerois (SBFM-DBJC, CEA Saclay, France) allowing to model the CS domain structure in solution and revealed that CHORDII domain of RAR1 and the N-terminal domain of HSP90 interact with opposite surfaces of the CS domain further supporting the existence of a ternary complex containing the three proteins. Using recombinant SGT1, RAR1 and HSP90 I also demonstrated that RAR1 is able to enhance SGT1-HSP90 interaction. SGT1 mutants impaired either in RAR1 binding (G190D) or HSP90 binding (K229E) revealed that RAR1 effect on SGT1-HSP90 binding depends on SGT1-RAR1 interaction and also on SGT1-HSP90 interaction.
In order to study interactions between R proteins and SGT1-RAR1-HSP90 complexes I successfully expressed several R proteins including Rx, Mla1 and Mla6 using a wheat germ based TNT. In vitro pull-down experiments showed that both SGT1 and RAR1 are able to interact with Rx, Mla1 and Mla6 through the CS and CHORDI domains respectively. Furthermore, as in the case of SGT1-HSP90 interaction, RAR1 was also able to enhance SGT1-Rx interaction. At the same time, in vitro studies have been combined with functional analysis of SGT1 CS mutations in vivo using the previously established Rx-PVX system in Nicotiana benthamiana (Peart et al., 2002). Functional analysis of CS mutations impairing either RAR1 or HSP90 interaction indicated that the interaction between SGT1 and HSP90, but not SGT1 and RAR1, is required for SGT1 function in Rx-mediated resistance against PVX.