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.innate immunity ͉ Pseudomonas syringae ͉ SGT1 ͉ STAND ATPase protein P lants 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 wi...

R proteins as fundamentals of plant innate immunity

“…Both interacting proteins might be involved in building a recognition complex that consists of R and RAR1 proteins and recognized effectors [95,96]. Interestingly, it was recently discovered that in Arabidopsis, specific mutations in HSP90, referred as hsp90.2, suppress the rar1 mutation and restore the accumulation and function of R proteins [97]. It was observed that SGT1 interacts with another co-chaperone, HSC70, in both the nucleus and cytosol.…”

HSP90 functions in the circadian clock through stabilization of the client F-box protein ZEITLUPE

“…HSP90 acts as a dimer and in an ATPase-dependent cycle alternately complexes with and separates from additional factors and cochaperones to effect a kinetically dynamic process of client protein maturation. In plants, HSP90 is best characterized as associating with the cochaperone SGT1 to stabilize NLR proteins, which mediate plant defense mechanisms (28)(29)(30). Additionally, HSP90 is important in phenotypic plasticity, developmental stability, and buffering of genetic variation (31)(32)(33).…”

Effect of extreme temperatures on powdery mildew development and Hsp70 induction in tomato and wild Solanum spp.

“…ATP-dependent Hsp70 chaperones, originally discovered in Drosophila as "puffing pattern" (Ritossa 1962(Ritossa , 1996, are known to be associated with numerous cellular roles like protein translation and translocation, protein folding or chaperoning, suppression of aggregation and re-activation of denatured proteins (Zhang et al 2005;Hubert et al 2009). Plant Hsp70s, together with their cochaperones DnaJ/Hsp40 and GrpE, are localised in the cytosol, endoplasmic reticulum, mitochondria, chloroplasts, and peroxisomes (Vierling 1991;Boston et al 1996;Bukau & Horwich 1998).…”