Peat-based hairy root transformation using Rhizobium rhizogenes as a rapid and efficient tool for easily exploring...

Root-knot nematodes (RKNs) pose a worldwide threat to agriculture of many crops including cucumber. Genetic transformation (GT) has emerged as a powerful tool for exploration of plant-RKN interactions and genetic improvement of RKN resistance. However, it is usually difficult to achieve a highly efficient and stable GT protocol for most crops due to the complexity of this process.

Here we firstly applied the hairy root transformation system in exploring root-RKN interactions in cucumber plants and developed a rapid and efficient tool transformation using Rhizobium rhizogenes strain K599. A solid-medium-based hypocotyl-cutting infection (SHI) method, a rockwool-based hypocotyl-cutting infection (RHI) method, and a peat-based cotyledon-node injection (PCI) method was evaluated for their ability to induce transgenic roots in cucumber plants. The PCI method generally outperformed the SHI and RHI methods for stimulating more transgenic roots and evaluating the phenotype of roots during nematode parasitism. Using the PCI method, we generated the CRISPR/Cas9-mediated malate synthase (MS) gene (involved in biotic stress responses) knockout plant and the LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16, a potential host susceptibility gene for RKN) promoter-driven GUS expressing plant. Knockout of MS in hairy roots resulted in effective resistance against RKNs, while nematode infection induced a strong expression of LBD16-driven GUS in root galls. This is the first report of a direct link between these genes and RKN performance in cucumber.

Taken together, the present study demonstrates that the PCI method allows fast, easy and efficient in vivo studies of potential genes related to root-knot nematode parasitism and host response.