INAUGURAL ARTICLE by a Recently Elected Academy Member:Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation

Plants have evolved a highly complex immune system centered on pathogen recognition via the evolutionarily-conserved NB-LRR proteins. Pathogen-triggered activation of NB-LRR proteins leads to several responses, including cell wall strengthening, transcriptional reprogramming, and a form of programmed cell death termed the hypersensitive response (HR). Because their function often results in cell death, proper maintenance of NB-LRR protein levels and activation state are vital to the health of the plant (1). NB-LRR proteins can be divided into 2 structural subgroups based on the presence of either a likely coiled-coil (CC) or Toll interleukin-1 receptor (TIR) domain at their N termini. Either of these N-terminal domains is followed in both subgroups by a middle nucleotide binding (NB) site and a C-terminal leucine-rich repeat (LRR). This general structure is not only conserved across all plants but extends to NOD/Caterpiller/NLR proteins that mediate various processes in mammalian innate immunity (2). Just as the domain composition of these intracellular receptors is conserved from plants to animals, so is the regulation of their steady-state accumulation. Cytosolic HSP90 and the cochaperone SGT1 have been previously demonstrated to not only be important for regulation of NB-LRR proteins in plants, but also in regulation of NLR function in animals (3). A third protein called RAR1 appears to play a role in innate immunity specifically in plants (4). All 3 of these proteins can independently interact with one another; the CS domain of SGT1b, or the CHORDI domain of RAR1, can interact with the N-terminal ATPase domain of HSP90; the CHORDII domain of RAR1 also interacts with the CS domain of SGT1 (5). The interaction of SGT1 with HSP90 has been shown to be required for SGT1 function (6). Mutation of SGT1 can suppress rar1 for some NB-LRR functions, but not all (7). However, the relationship between RAR1 and HSP90 is less understood. We present and characterize specific missense alleles of HSP90.2 in the reference plant, Arabidopsis, that suppress rar1. These hsp90.2 alleles are uniquely interesting in that they can bypass the requirement for a cochaperone and result in recovery of client protein accumulation and function. We used genetic and biochemical analyses to demonstrate that these hsp90.2 mutant proteins act on NB-LRR proteins affected by rar1, suppressing all identified rar1 phenotypes. We further show that these mutations are functionally distinct from previously-identified hsp90.2 mutations (8), including a null allele. These specific missense changes in hsp90.2 enable a separation of HSP90 ATP hydrolysis activity and HSP90 function in client protein accumulation. By recapitulating the activity of RAR1 in its absence, the phenotypes of these hsp90.2 mutants strongly suggest that RAR1 physically enhances the transition state of HSP90 as it moves from a “lid open” ADP-bound conformation to a “lid closed” ATP-bound conformation.