Peat-based hairy root transformation using Rhizobium rhizogenes as a rapid and efficient tool for easily exploring potential genes related to root-knot nematode parasitism and host response

Abstract

Background 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. Results 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. Conclusion 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.

Essential Oil Volatiles as Sustainable Antagonists for the Root-Knot Nematode Meloidogyne ethiopica

“…The use of in vitro roots transformed with R. rhizogenes is not new and was previously studied in combination with RKNs to uncover important mechanisms of plant-nematode interactions, e.g., in analyzing the biochemical mechanisms of RKN parasitism [38], for characterizing and/or validating candidate genes for host resistance to RKNs [39][40][41][42], in propagating RKN resistant plant genotypes [43], in understanding the processes of RKN infection after knocking out specific stress-related or putative susceptibility genes [44], or for the general maintenance of RKN collections [45][46][47]. However, resorting to plantnematode co-cultures as a tool to uncover bioactive compounds or mixtures for the design of safer biopesticides is less reported in the literature.…”

Highly efficient Agrobacterium rhizogenes-mediated transformation for functional analysis in woodland strawberry

“…Previous studies have reported that cotyledon as explants produce more hairy roots than hypocotyl segments in cotton and other plants [ 37 ]. For example, cotyledon node injection allows faster and more efficient in vivo studies of root development in cucumber than hypocotyl cutting injection [ 38 ]. However, in some cases, hypocotyls can yield a higher transformation efficiency that depends on plant species.…”

Metabolic variations in root tissues and rhizosphere soils of weak host plants potently lead to distinct host status and chemotaxis regulation of Meloidogyne incognita in intercropping

“…Cucumber hairy root transformation was performed using R. rhizogenes K599 as detailed in Zhang et al ( 2023 ). A promoter region of about 2000 bp of CsPTS (CsaV3_3G028840) and CsCHS (CsaV3_3G027830) was amplified and cloned into the pCAMBIA 1391‐GUS vector.…”