The Arabidopsis TIR-NBS-LRR protein CSA1 guards BAK1-BIR3 homeostasis and mediates convergence of pattern- and effector-induced immune responses.

Arabidopsis BAK1/SERK3, a co-receptor of leucine-rich repeat pattern recognition receptors (PRRs), mediates pattern-triggered immunity (PTI). Genetic inactivation of BAK1 or BAK1-interacting receptor-like kinases (BIRs) causes cell death, but the direct mechanisms leading to such deregulation remains unclear. Here, we found that the TIR-NBS-LRR protein CONSTITUTIVE SHADE AVOIDANCE 1 (CSA1) physically interacts with BIR3, but not with BAK1. CSA1 mediates cell death in bak1-4 and bak1-4 bir3-2 mutants via components of effector-triggered immunity-(ETI) pathways. Effector HopB1-mediated perturbation of BAK1 also results in CSA1-dependent cell death. Likewise, microbial pattern pg23-induced cell death, but not PTI responses, requires CSA1. Thus, we show that CSA1 guards BIR3 BAK1 homeostasis and integrates pattern- and effector-mediated cell death pathways downstream of BAK1. De-repression of CSA1 in the absence of intact BAK1 and BIR3 triggers ETI cell death. This suggests that PTI and ETI pathways are activated downstream of BAK1 for efficient plant immunity.

Specifying the role of BAK1-interacting receptor-like kinase 3 in brassinosteroid signaling.

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1-associated kinase 1 (BAK1)-interacting receptor-like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.

The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1.

BAK1 is a coreceptor and positive regulator of multiple ligand binding leucine-rich repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity, and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here, we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand binding receptors to prevent BAK1 receptor complex formation.

Identification of trichlormethiazide as a Mdr1a/b gene expression enhancer via a dual secretion-based promoter assay.

Transporters of the ATP-binding cassette (ABC) family such as MDR1 play a pivotal role in persistence of brain homeostasis by contributing to the strict permeability properties of the blood-brain barrier. This barrier on one hand compromises treatment of central nervous system diseases by restricting access of drugs; on the other hand, an impaired or altered function of barrier building cells has been described in neurological disorders. The latter might contribute to increased vulnerability of the brain under pathological conditions or even enforce pathogenesis. Here, we present a novel approach for a systematic examination of drug impact on Mdr1 gene expression by establishing a dual reporter gene assay for the murine upstream core promoters of Mdr1a and b. We validated the time-resolved assay in comparison with single reporter gene constructs and applied it to analyze effects of a Food and Drug Administration (FDA)-approved drug library consisting of 627 substances. The chemo-preventive synthetic dithiolethione oltipraz was reidentified with our assay as an already known inducer of Mdr1 gene expression. Together with two newly characterized modifiers - gemcitabine and trichlormethiazide - we prove our findings in a blood-brain barrier culture model as well as in wild-type and Mdr1 knockout mice. In sum, we could demonstrate that our dual reporter gene assay delivers results, which also persist in the living animal and consequently is applicable for further analysis and prediction of Mdr1 regulation in vivo.