The BIR3 interactome revealed the NLR CSA1 as a component necessary for BIR- and BAK1-mediated cell death

Abstract

BIR3 prevents BAK1 complex formation with ligand-binding receptors in the absence of a ligand. The presence and balanced levels of BIR3 and BAK1 are necessary to prevent cell death. To study how BIR3 adds to the cell death control and how it exerts its receptor regulatory function, we used ESI-LC-MS/MS in order to identify further components of the interactome of BIR3. We focused on candidates which might be involved in cell death. They were of strong interest as they could help elucidating the molecular mechanism underlying the cell death containment mediated by BAK1 or the BIR-family. The conducted MS-analysis revealed the NLR CONSTITUTIVE SHADE AVOIDANCE (CSA1), which we subsequently characterized for its involvement in BAK1- and BIR-mediated cell death. We pointed out that the knock-out of CSA1 in crossings with bir2-1 and bak1-4 did not interfere with flg22-mediated immune responses. Moreover, we observed a significant reduction of SA-levels connected with a partial reduction of local cell death symptoms in all mutants tested. We concluded (i) that CSA1 is a positive regulator of cell death, activated by the absence of BAK1 and BIR proteins, (ii) that the cell death induced by CSA1 is independent of PTI-triggered ROS-signaling and (iii) that the cell death conferred by CSA1 is associated with elevated SA levels. Since the BAK1- and BIR-cell-death responses are only partially rescued by CSA1 mutation, we concluded that other components might be involved. The second BIR3-interacting NLR candidate from our BIR3 interactome analysis was a CC-type NLR protein, which appears to be a promising candidate for further investigations. With this work we defined the BIR3 interactome which allowed us to reveal the NLR CSA1 as a component necessary for BIR- and BAK1-mediated cell death.