Arabidopsis thaliana Xylem Cysteine Protease 1 Gene Regulates Xylem Bridge Reconnection and Delayed Incompatibility in Arabidopsis/Nicotiana Interfamilial Grafts

XYLEM CYSTEINE PROTEASE 1 (XCP1) is a cysteine protease that plays a critical role in xylem differentiation and tracheary element (TE) formation. Our previous study demonstrated that TE remodeling occurs at the graft union in the Arabidopsis thaliana (At)/Nicotiana benthamiana (Nb) interfamilial graft. Here, we identify that the AtXCP1 transcript is specifically localized in TEs at the graft interface of the incompatible At/Nb interfamilial grafts, and its expression is reduced in these incompatible grafts compared to the compatible grafts. Analysis of AtXCP1pro::GFP reporter lines revealed that AtXCP1 expression is rapidly induced by wounding at the graft interface in At/Nb interfamilial grafts during the early grafting stage. Notably, AtXCP1 expression was significantly stronger in At/Nb heterografts than in At/At homografts, and GFP fluorescence was observed in the stock xylem at 7 days after grafting (DAG) in heterografts, a dynamic process absent in At/At homografts. We found that the Atxcp1 mutant promoted the survival of At/Nb interfamilial grafts during the early grafting stage but decreased the survival after several months, indicating delayed incompatibility. Anatomical examination revealed that large cellular deposits accumulated at the graft interface in Atxcp1/Nb interfamilial grafts and exhibited abnormal TE morphology at later stages. Our findings identify AtXCP1 as a key regulator of xylem reconnection and delayed incompatibility in At/Nb interfamilial grafts.