Strategies to Circumvent Herbicide Resistance Through the Inhibition of Lysine Biosynthesis

Weeds imperil the productivity and profitability of agricultural industries. Thus, effective management strategies are critical to mitigate crop yield and quality losses due to weeds. The most effective and economical weed control method is through the use of herbicides. However, the rapid emergence and spread of weeds that are resistant to our current herbicides is threatening their utility. New modes of action to which there are no existing resistance mechanisms are therefore urgently needed. Several of our most widely used herbicides interfere with the endogenous synthesis of amino acids in plants. However, there are no commercial herbicides that inhibit plant enzymes within lysine biosynthesis. This thesis has therefore focussed on exploring the potential of these enzymes as herbicide targets, and innovative strategies to develop herbicidal inhibitors against them. The essentiality of the genes encoding the lysine biosynthesis enzymes dihydrodipicolinate reductase (DHDPR)1 and DHDPR2 was examined to determine their suitability as herbicide targets. To do so, a hybrid CRISPR-Cas9 methodology was employed to generate mutations in these genes. Analyses of the inheritance of these mutations indicated that the lack of a functional DHDPR-encoding allele is lethal at either the gametophytic or embryonic stage. This finding was supported by the observation of structural abnormalities in both the pollen and fruit of mutant plants. These results strongly suggested that a functional DHDPR enzyme is essential in plants, and thus, that it may represent a promising herbicide target. Recently, the first herbicidal inhibitors of lysine biosynthesis were developed by our research group against the enzyme dihydrodipicolinate synthase. The present study found that these inhibitors possess an additional site of action through their inhibition of DHDPR. These inhibitors therefore represent the first synthetic multi-target herbicidal compounds, which may have a reduced propensity to generate resistance in weeds. Having validated that inhibitors of DHDPR can have herbicidal properties, other inhibitors of this enzyme were developed. To do so, an inhibitor of the bacterial DHDPR orthologue was repurposed as a plant-specific scaffold for the generation of analogues with improved herbicidal efficacy. These inhibitors were found to have activity against both monocotyledonous and dicotyledonous weed species of economic significance. These findings provide proof-of-concept for a novel repurposing strategy that could fast-track the development of plant-specific herbicide leads. The potential of another plant lysine biosynthesis enzyme, diaminopimelate aminotransferase (DAPAT), was investigated by assessing the suitability of in vitro DAPAT inhibitors as scaffolds for the development of herbicide leads. These compounds were found to be the most potent herbicidal inhibitors of lysine biosynthesis reported to date. Furthermore, this study demonstrated that lysine biosynthesis inhibitors could be utilised as both pre- and post-emergence treatments. The compounds targeting DAPAT were used to probe the systemic effects of lysine biosynthesis inhibitors that result in phytotoxicity. Plants treated with DAPAT inhibitors exhibited dysregulated amino acid biosynthesis pathway networks leading to broad impairments in respiration, photosynthesis and metabolism. Ultimately, this work has contributed significant insights into new targets, strategies and lead inhibitors that could aid in the development of herbicides with new modes of action.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 202