Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation

Abstract

Both plants and animals require the activity of proteins containing nucleotide binding (NB) domain and leucine-rich repeat (LRR) domains for proper immune system function. NB-LRR proteins in plants (NLR proteins in animals) also require conserved regulation via the proteins SGT1 and cytosolic HSP90. RAR1, a protein specifically required for plant innate immunity, interacts with SGT1 and HSP90 to maintain proper NB-LRR protein steady-state levels. Here, we present the identification and characterization of specific mutations in Arabidopsis HSP90.2 that suppress all known phenotypes of rar1. These mutations are unique with respect to the many mutant alleles of HSP90 identified in all systems in that they can bypass the requirement for a cochaperone and result in the recovery of client protein accumulation and function. Additionally, these mutations separate HSP90 ATP hydrolysis from HSP90 function in client protein folding and/or accumulation. By recapitulating the activity of RAR1, these novel hsp90 alleles allow us to propose that RAR1 regulates the physical open-close cycling of a known "lid structure" that is used as a dynamic regulatory HSP90 mechanism. Thus, in rar1, lid cycling is locked into a conformation favoring NB-LRR client degradation, likely via SGT1 and the proteasome.

Fig. 1.

rsp alleles suppress all known rar1 phenotypes. (A–D) Bacterial growth assays comparing rar1 mutants to hsp90.2^rsp rar1 double mutants. Note the logarithmic scale. hsp90.2^rsp mutants suppress rar1 phenotypes for disease resistance mediated by RPS5 (A), RPM1 (B), and RPS2 (C). (D) hsp90.2^rsp mutants suppress the rar1 phenotype of decreased basal resistance to Pto DC3000 (7). (E) hsp90.2^rsp alleles suppress the rar1 phenotype of loss of RPM1-mediated HR. An increase in conductivity is indicative of the release of ions from cells undergoing HR. (F) hsp90.2^rsp alleles suppress the rar1 phenotype of lowered steady-state accumulation of RPM1-myc protein.

Fig. 2.

hsp90.2^rsp mutants are phenotypically distinct from an hsp90.2^lra single mutant and an hsp90.2 T-DNA insertion null mutant. hsp90.2^rsp, hsp90.2^lra, and hsp90.2^KO single mutant plants are compared with each other and Col-0 and rpm1 plants. (A) Bacterial growth assay for recognition of Pto DC3000(avrRpm1) by RPM1. (B) Conductivity assay measuring the HR triggered by RPM1 activation after recognition of AvrRpm1.

Fig. 3.

Neither an hsp90.2^lra allele nor the hsp90.2^KO null allele suppress rar1. (A) Bacterial growth assay measuring disease resistance to Pto DC3000 (avrRpm1) mediated by RPM1. Wild-type Col-0 and rar1 mutant plants are compared with hsp90.2–3^lra rar1 and hsp90.2^KO rar1 double mutants. (B) Conductivity assay measuring the HR to Pto DC3000 (avrRpm1).

Fig. 4.

Interactions between hsp90.2 mutant proteins and RAR1 or SGT1b does not correlate with phenotype. (A–C) β-Galactosidase assay quantification of the results of yeast 2-hybrid interaction measurements between HSP90.2 and mutant variants with RAR1 (A), SGT1a (B), or SGT1b (C). (D) HSP90.2 and mutant variants accumulate to equivalent levels in yeast as measured by Western blot. RAR1 interacts normally with SGT1a in this assay.

Fig. 5.

HSP90 ATPase activity does not predict hsp90.2 mutant phenotype. In vitro ATPase activity of HSP90.2 and mutant variants with a range of ATP concentrations was used to determine the K_cat. HSP90 concentration was 5 μM, and ATP concentrations ranged between 0 and 1.2 mM (see Materials and Methods).

Fig. 6.

HSP90.2^rsp mutant proteins retain dimerization capability. Chemical cross-linking of wild-type and mutant forms of HSP90.2 in the presence of ADP or the nonhydrolysable ATP analog AMP-PNP. All variants of HSP90.2 are unable to dimerize in the presence of ADP. However, although no lra mutant variants (A) are able to dimerize even in the presence of AMP-PNP, both rsp mutant variants (B) can dimerize in the presence of AMP-PNP. The experiment was performed with an HSP90 concentration of 0.25 mg/mL, 15 molar equivalents of DMS, and 10 mM nucleotide.

Fig. 7.

rsp mutations affect residues in the lid region of HSP90 in the closed conformation. Ribbon structures of yeast HSP90 (Protein Data Bank ID code 2CG9) bound to ATP (light gray). This lid (red) is hinged at G95 and G122 and swings 180° to fold over the nucleotide-binding pocket (yeast G94 and G121). (A) R337 (yellow) coordinates interaction of the central client-binding domain (purple, left) with the flexible lid (red) by interacting with V115 and S116 in the lid region and E363 within the middle domain of HSP90 (yeast R346, V114, S115, and E372). (B) A11 (yeast A10) from 1 monomer (green) interacts directly with T96 (yeast T95; red side chain) within the hinge of the other monomer. Black arrows indicate the locations of the hinges of the lid.