CmCNIH1 improves salt tolerance by influencing the trafficking of CmHKT1 ;1 in pumpkin

Pumpkin is often used as a rootstock for other Cucurbitaceae crops due to its resistance to soil-borne diseases and abiotic stress. Pumpkin rootstocks use a sodium transporter (CmHKT1;1) to promote the transport of Na+ from the shoot to the root effectively and improve the salt tolerance of the scion. However, the molecular regulatory mechanisms that influence the activity of CmHKT1;1 during salt stress response remain unknown. In this study, CmCNIH1, a cornichon homolog, was identified as a potential cargo receptor for CmHKT1;1. Yeast two-hybrid, biomolecular fluorescence complementation and luciferase complementary assays demonstrated that CmCNIH1 and CmHKT1;1 could interact. CmCNIH1 was a key component of the cellular vesicle transport machinery located in the endoplasmic reticulum (ER), ER export site and Golgi apparatus. A CmCNIH1 knockout mutant was more sensitive to salt stress than the wild-type (WT). In addition, ion homeostasis was disrupted in cmcnih1 mutants, which had higher Na+ and lower K+ content in shoots and roots than the WT. Two-electrode voltage-clamp experiment displayed that CmCNIH1 could not influence the Na+ current that passed through the plasma membrane (PM) in CmHKT1;1-expressing Xenopus laevis oocytes. Data from co-localization assays indicated that intact CmCNIH1 protein could alter the subcellular localization of CmHKT1;1 in tobacco leaf, pumpkin root and yeast. In summary, CmCNIH1 may function as a cargo receptor that regulates the localization of CmHKT1;1 to the PM to improve salt tolerance in pumpkin.