Specific missense alleles of the arabidopsis jasmonic acid co-receptor COI1 regulate innate immune receptor accumulation and function.

Plants utilize proteins containing nucleotide binding site (NB) and leucine-rich repeat (LRR) domains as intracellular innate immune receptors to recognize pathogens and initiate defense responses. Since mis-activation of defense responses can lead to tissue damage and even developmental arrest, proper regulation of NB-LRR protein signaling is critical. RAR1, SGT1, and HSP90 act as regulatory chaperones of pre-activation NB-LRR steady-state proteins. We extended our analysis of mutants derived from a rar1 suppressor screen and present two allelic rar1 suppressor (rsp) mutations of Arabidopsis COI1. Like all other coi1 mutations, coi1(rsp) missense mutations impair Jasmonic Acid (JA) signaling resulting in JA-insensitivity. However, unlike previously identified coi1 alleles, both coi1(rsp) alleles lack a male sterile phenotype. The coi1(rsp) mutants express two sets of disease resistance phenotypes. The first, also observed in coi1-1 null allele, includes enhanced basal defense against the virulent bacterial pathogen Pto DC3000 and enhanced effector-triggered immunity (ETI) mediated by the NB-LRR RPM1 protein in both rar1 and wild-type backgrounds. These enhanced disease resistance phenotypes depend on the JA signaling function of COI1. Additionally, the coi1(rsp) mutants showed a unique inability to properly regulate RPM1 accumulation and HR, exhibited increased RPM1 levels in rar1, and weakened RPM1-mediated HR in RAR1. Importantly, there was no change in the steady-state levels or HR function of RPM1 in coi1-1. These results suggest that the coi1(rsp) proteins regulate NB-LRR protein accumulation independent of JA signaling. Based on the phenotypic similarities and genetic interactions among coi1(rsp), sgt1b, and hsp90.2(rsp) mutants, our data suggest that COI1 affects NB-LRR accumulation via two NB-LRR co-chaperones, SGT1b and HSP90. Together, our data demonstrate a role for COI1 in disease resistance independent of JA signaling and provide a molecular link between the JA and NB-LRR signaling pathways.

A chemical screen for suppressors of the avrRpm1-RPM1-dependent hypersensitive cell death response in Arabidopsis thaliana.

Arabidopsis thaliana RPM1 encodes an intracellular immune sensor that conditions disease resistance to Pseudomonas syringae expressing the type III effector protein AvrRpm1. Conditional expression of this type III effector in a transgenic line carrying avrRpm1 under the control of a steroid-inducible promoter results in RPM1-dependent cell death that resembles the cell death response of the incompatible RPM1-avrRpm1 plant-bacterium interaction. This line was previously used in a genetic screen, which revealed two genes that likely function in the folding of pre-activation RPM1. We established a chemical screen for small molecules that suppress steroid-inducible and RPM1-avrRpm1-dependent cell death in Arabidopsis seedlings. Screening of a library comprising 6,800 compounds of natural origin identified two trichothecene-type mycotoxins, 4,15-diacetoxyscirpenol (DAS) and neosolaniol (NEO), which are synthesized by Fusarium and other fungal species. However, protein blot analysis revealed that DAS and NEO inhibit AvrRpm1 synthesis rather than suppress RPM1-mediated responses. This inhibition of translational activity likely explains the survival of the seedlings under screening conditions. Likewise, flg22-induced defense responses are also impaired at the translational, but not the transcriptional, level by DAS or NEO. Unexpectedly, both compounds not only prevented AvrRpm1 synthesis, but rather caused an apparent hyper-accumulation of RPM1 and HSP70. The hyper-accumulation phenotype is likely unrelated to the ribotoxic function of DAS and NEO and could be due to an inhibitory activity on the proteolytic machinery of Arabidopsis or elicitor-like activities of type A trichothecenes.

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

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.

A duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and RPS2-mediated hypersensitive response.

The Arabidopsis RPM1 protein confers resistance to disease caused by Pseudomonas syringae strains delivering either the AvrRpm1 or AvrB type III effector proteins into host cells. We characterized two closely related RPM1-interacting proteins, RIN2 and RIN3. RIN2 and RIN3 encode RING-finger type ubiquitin ligases with six apparent transmembrane domains and an ubiquitin-binding CUE domain. RIN2 and RIN3 are orthologs of the mammalian autocrine motility factor receptor, a cytokine receptor localized in both plasma membrane caveolae and the endoplasmic reticulum. RIN2 is predominantly localized to the plasma membrane, as are RPM1 and RPS2. The C-terminal regions of RIN2 and RIN3, including the CUE domain, interact strongly with an RPM1 N-terminal fragment and weakly with a similar domain from the Arabidopsis RPS2 protein. RIN2 and RIN3 can dimerize through their C-terminal regions. The RING-finger domains of RIN2 and RIN3 encode ubiquitin ligases. Inoculation with P. syringae DC3000(avrRpm1) or P. syringae DC3000(avrRpt2) induces differential decreases of RIN2 mobility in SDS-PAGE and disappearance of the majority of RIN2. A rin2 rin3 double mutant expresses diminished RPM1- and RPS2-dependent hypersensitive response (HR), but no alteration of pathogen growth. Thus, the RIN2/RIN3 RING E3 ligases apparently act on a substrate that regulates RPM1- and RPS2-dependent HR.

Arabidopsis RIN4 negatively regulates disease resistance mediated by RPS2 and RPM1 downstream or independent of the NDR1 signal modulator and is not required for the virulence functions of bacterial type III effectors AvrRpt2 or AvrRpm1.

Bacterial pathogens deliver type III effector proteins into the plant cell during infection. On susceptible (r) hosts, type III effectors can contribute to virulence. Some trigger the action of specific disease resistance (R) gene products. The activation of R proteins can occur indirectly via modification of a host target. Thus, at least some type III effectors are recognized at site(s) where they may act as virulence factors. These data indicate that a type III effector's host target might be required for both initiation of R function in resistant plants and pathogen virulence in susceptible plants. In Arabidopsis thaliana, RPM1-interacting protein 4 (RIN4) associates with both the Resistance to Pseudomonas syringae pv maculicola 1 (RPM1) and Resistance to P. syringae 2 (RPS2) disease resistance proteins. RIN4 is posttranslationally modified after delivery of the P. syringae type III effectors AvrRpm1, AvrB, or AvrRpt2 to plant cells. Thus, RIN4 may be a target for virulence functions of these type III effectors. We demonstrate that RIN4 is not the only host target for AvrRpm1 and AvrRpt2 in susceptible plants because its elimination does not diminish their virulence functions. In fact, RIN4 negatively regulates AvrRpt2 virulence function. RIN4 also negatively regulates inappropriate activation of both RPM1 and RPS2. Inappropriate activation of RPS2 is nonspecific disease resistance 1 (NDR1) independent, in contrast with the established requirement for NDR1 during AvrRpt2-dependent RPS2 activation. Thus, RIN4 acts either cooperatively, downstream, or independently of NDR1 to negatively regulate RPS2 in the absence of pathogen. We propose that many P. syringae type III effectors have more than one target in the host cell. We suggest that a limited set of these targets, perhaps only one, are associated with R proteins. Thus, whereas any pathogen virulence factor may have multiple targets, the perturbation of only one is necessary and sufficient for R activation.

Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein.

The Arabidopsis protein RPM1 activates disease resistance in response to Pseudomonas syringae proteins targeted to the inside of the host cell via the bacterial type III delivery system. We demonstrate that specific mutations in the ATP-binding domain of a single Arabidopsis cytosolic HSP90 isoform compromise RPM1 function. These mutations do not affect the function of related disease resistance proteins. RPM1 associates with HSP90 in plant cells. The Arabidopsis proteins RAR1 and SGT1 are required for the action of many R proteins, and display some structural similarity to HSP90 co-chaperones. Each associates with HSP90 in plant cells. Our data suggest that (i) RPM1 is an HSP90 client protein; and (ii) RAR1 and SGT1 may function independently as HSP90 cofactors. Dynamic interactions among these proteins can regulate RPM1 stability and function, perhaps similarly to the formation and regulation of animal steroid receptor complexes.