Editing out insecticide use: Use of gene editing Eruca sativa for more effective biofumigation

Biofumigation is sometimes used as an alternative to chemical insecticides, but efficacy is variable. Biofumigation relies on growing and incorporating insecticidal plants into the soil. This project uses gene editing techniques to improve existing biofumigant crops, altering their chemical composition, and developing our knowledge of glucosinolate metabolism and underlying genetics.

Use of biofumigants is increasing as it reduces the reliance on insecticide application to reduce nematode load as well as fungi, oomycetes, bacteria and dormant insects; limiting root damage, leaf damage and microbial/virus transfer. Its efficacy can be inconsistent owing to variable levels of bioactive metabolites. Glucosinolates are a stable precursor to a range of bioactive thiols, particularly isothiocyanates (ITCs).

When the plant cell is damaged by chewing insects or microbial breakdown, endogenous myrosinases, as well as those produced by some soil bacteria catalyse the production of bioactive ITCs.

Traditional soil treatments have considerable environmental impacts (e.g. methyl bromide, Dazomet, and fluorpyram). Fluorpyram is highly persistent (DT50 = 118 days) easily leached into waterways, and causes harm to earthworms, and mammals, bees, and significant harm to birds. By contrast natural isothiocyanates have a much shorter halflife in soil (DT50 = 3 days) but stored as the more stable glucosinolate precursor, released only via cell damage, leading to a longer lasting effect, and very high target specificity.

To improve the viability of biofumigants we will investigate glucosinolate composition of Eruca sativa and Diplotaxis tenuifolia, used both as a salad crop and as a commercial biofumigant. Using existing knowledge of the Eruca/Diplotaxis genomes and transcriptomes we will use gene editing to create knockouts, demonstrating genetic regulation, how chemical composition can be altered to fit the needs of the grower and end consumer, and elucidate any non-target effects. This has wider reaching implications in plant defence, flavour and nutrition.

Gene editing can result in a 'Precision Bred Organism'. Precision breeding was removed from the definition of Genetic Modification in legislation passed into UK law on 23rd of March this year.

By utilising knockouts we can alter the composition of glucosinolates produced by the plant. As there is a genetically controlled divergence of the indolic GSL pathways and the aliphatic GSL pathway, we hypothesise that we can reduce the total indolic content and shift plant resources into the aliphatic pathway, and more focussed alterations, beneficial to the role of biofumigation.

We hypothesise that changes in chemical composition will produce both a stable increase in the potency of the varieties and a wider range of effective compounds, increasing reliability.

We hypothesise that we can develop a suitable transformation protocol for our Eruca and Diplotaxis varieties based on the paper by Slater, Keller and Scoles,. This will enable the generation of stable mutations using VIGS (virus induced gene silencing) and CRISPR gene editing techniques. Transformation is highly cultivar and species dependent, for this reason we have access to a novel Agrobacterium strain expressing a type III secretion system to enhance transformation and range of cultivars and alternative brassica species from Elsoms seeds if a backup is required.

This will ensure the outcomes of the project remain relevant if preferred varieties cannot be transformed. Objectives

1.Develop a reliable transformation protocol for Eruca and Diplotaxis cultivars. Other Brassica crops (such as mustard and radish) will also be investigated if this fails. 2.Demonstrate function of genes in glucosinolate synthesis pathway through gene editing based gene knockouts 3.Analyse glucosinolate and isothiocyanate content in wild type and mutant crop

Assess biofumigant activity in gene knockouts.