Abstract
Activating chloroplast immunity to enhance host resistance offers a novel and sustainable approach for the effective control of kiwifruit bacterial canker. Chloroplasts serve as a central hub for ROS, SA, and Ca(2+) signaling. As a chloroplast-localized protein, CaS participates in Ca(2+)-signaling pathways. However, the mechanisms underlying CaS-mediated immune regulation and whether to be attacked by pathogens remain unclear. Here, we created AcCaS-overexpressing transgenic plants, then we found that AcCaS activates chloroplast reactive oxygen species (ROS) bursts and enhances resistance against Pseudomonas syringae pv. actinidiae (Psa). Mutational analysis revealed that the chloroplast transit peptide (cTP) of AcCaS is essential for its immune function, and deletion of cTP abolished ROS production and disease resistance. Yeast two-hybrid reveals that Psa employs the effector HopAU1 targets AcCaS in kiwifruit. Luciferase complementation imaging, and microscale thermophoresis assays identified Asn-121 of AcCaS as the critical residue mediating both HopAU1 binding and Ca(2+) sensing. Strikingly, molecular modeling and competitive binding experiments showed that HopAU1 directly occupies the Ca(2+)-binding site at Asn-121, thereby blocking calcium signaling and suppressing chloroplast immunity. In summary, this study uncovers that AcCaS enhances resistance against Psa by activating chloroplast ROS and binding with Ca(2+). The Asn-121 residue plays a pivotal role in Ca(2+)-binding and HopAU1-mediated immune suppression, as mutations at this site abolish both activities. These findings revealed the battle of chloroplast Ca(2) signaling in plant-pathogen conflicts and provide a mechanistic basis for engineering AcCaS-centered resistance in kiwifruit.