Further analysis showed that the interaction between CmNAC100L1 and CmCalS1 in the IG plants enhanced, but decreased the activity of CmCalS1 in the CG plants. Interestingly, CmNAC100L1 interacted with CmCalS1 to regulate its activity. Furthermore, IAA negatively regulated the expression of Cm-miR164a, which directly targeted cleavage of CmNAC100L1. Treatment with IAA polar transport inhibitor in the root of the IG plants decreased CmCalS activity and callose content. The incompatible combination (IG) grafting interface accumulated more callose, and the activity of callose synthase (CmCalS1) and IAA content were significantly higher than those in compatible combination (CG). In this study, we found that IAA-miR164a-NAC100L1 module induced callose deposition to mediate the symbiotic incompatibility of cucumber/pumpkin grafted seedlings. The specific molecular regulation mechanism of graft incompatibility is still largely unclear. However, the symbiotic incompatibility between rootstock and scion affects the normal growth and development of grafted seedlings after survival. Grafting is one of the key technologies to overcome the obstacles of continuous cropping, and improve crop yield and quality.
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