Wild-type allele of TaHRC suppresses calcium-mediated plant immune response by hijacking TaCAXIP4 to trigger FHB susceptibility in wheat

Fusarium head blight (FHB) is one of the most destructive diseases of wheat worldwide. Epidemics of FHB cause a serious reduction in grain yield and quality of wheat and result in significant economic losses to wheat producers. Recently, we have cloned a histidine-rich calcium-binding protein gene (TaHRC) as the causal gene for Fhb1 and demonstrated that the wild-type allele of TaHRC conditions FHB susceptibility and a large deletion including the start codon resulted in FHB resistance. However, the molecular mechanisms on how TaHRC regulating FHB susceptibility remains unknown. In this study, we conducted yeast two-hybrid screening (Y2H) against the wheat cDNA expression libraries using TaHRC as bait and identified a cation exchanger (CAX)-interacting protein 4 (TaCAXIP4) as the candidate protein that interacts with TaHRC to affect calcium transport activity. The strong interaction was further confirmed by Bimolecular fluorescence complementation (BiFC) assays. Using gene editing, we edited three different sites (one before and one within and one after the NLS domain) of TaHRC in a susceptible wheat cultivar Bobwhite using the CRISPR/Cas9 gene editing technology and demonstrated the N-terminus carrying NLS domain of TaHRC plays a critical role for the interaction and conditions TaHRC function on FHB susceptibility. We determined that the interaction between TaCAXIP4 and TaHRC occurs in the nuclei of cells by subcellular colocalization assay. Intriguingly, we found TaHRC can sequester TaCAXIP4 to suppress the Ca2+ transporting activity of TaCAX1 (a H+/Ca2+ antiporter) through yeast calcium suppression assay and suggested wild-type TaHRC may hijack TaCAXIP4 to suppresses calcium-mediated plant immune response during Fusarium infection in wheat. Furthermore, we performed the reactive oxygen species (ROS) assays and further showed that TaHRC might suppress the chitin-triggered plant immune responses during Fusarium infection by sequestering TaCAXIP4 to trigger FHB susceptibility, which facilitates the pathogen spread within a wheat spike. This work provides first line of evidence to support wild type Fhb1 is a susceptible gene and how Fhb1 wild type allele regulate FHB susceptibility.