An efficient root transformation system for CRISPR/Cas9-based analyses of shoot-root communication in cucurbit crops

Cucurbit crops are suitable models for studying long-distance signaling in horticultural plants. Although thousands of substances are graft transmissible in cucurbits, functional studies have been hampered by the lack of efficient genetic transformation systems. Here, we report a convenient and efficient root transformation method for several cucurbit crops that will facilitate studies of functional genes and shoot-root crosstalk. We obtained healthy plants with completely transformed roots and non-transgenic shoots within 6 weeks. Furthermore, we combined this root transformation method with grafting, which allowed for gene manipulation in the rootstock. We validated our system by exploring salt tolerance mechanisms using a cucumber (Cucumis sativus)/pumpkin (Cucurbita moschata Duch.) (scion/rootstock) graft in which the sodium transporter gene High-affinity K⁺ transporter1 (CmoHKT1;1) was edited in the pumpkin rootstock and by overexpressing the pumpkin tonoplast Na⁺/H⁺ antiporter gene Sodium hydrogen exchanger4 (CmoNHX4) in cucumber roots.