Back to the roots: how an underutilised biotechnological tool can support research to improve grapevine resilience against biotic stressors in an unpredictable future

Abstract

Hairy roots (HRs) are a symptom of a natural genetic modification by the soil-borne phytopathogen Rhizobium rhizogenes. Transfer DNA is stably integrated into the hosts genome and subsequently expressed by the plant, leading to the characteristic prolific non-geotropic root growth. This infection strategy can be hijacked to introduce DNA of interest and establish HR cultures (HRCs) which can be used for functional characterisation of genes, the bioproduction of metabolites or recombinant proteins, root-pathogen studies and for biotechnological improvement. To date, HRCs have been generated from numerous plant species, including over 50 Vitis species, rootstock hybrids or V. vinifera scion cultivars, highlighting the scientific interest and feasibility of this biotechnological tool. Our work on grapevine HRCs started with an attempt to establish a standardised Vitis HRC induction, maintenance, and validation protocol for the reliable, reproducible, and routine cross-cultivar use of the HR system in grapevine. The optimisations included the analysis of two infection protocols for HR induction, three R. rhizogenes strains, seven Vitis cultivars (three V. vinifera cultivars and four Vitis rootstock hybrids), a range of explant tissue types, different co-cultivation times, growth media with various anti-browning agents, and the design of universal multiplex primer pairs for HR confirmation. Three visual reporter genes (DsRed, eyGFPuv and VvMYBA1) and two antibiotic resistance marker genes (nptII and hptII) were evaluated for selection of HRs. The next phase of the research employs a bioprospecting approach to identify and test potentially useful grapevine antimicrobial peptides (AMPs). Computational analyses were used to identify AMPs by utilising core amino acid motifs, length of peptides and predicted tertiary structures in combination with available grapevine genomic resources. HRCs overexpressing candidate AMPs from different grapevine genotypes will be used to assess the bioproduction capacity and evaluate whether the system can produce functional antimicrobial compounds. Furthermore, the AMP overexpressing HRCs are useful to analyse the impact newly identified AMPs have on grapevine pathogens in bioassays. The presented research aims to connect science with industry by showcasing how biotechnological tools can be applied to generate sustainable and eco-friendly biopesticides, to combat emerging pests and pathogens, by harnessing what nature has provided.