Engineering DMNT emission in cotton enhances direct and indirect defense against mirid bugs.

Introduction: As an important herbivore-induced plant volatile, (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) is known for its defensive role against multiple insect pests, including attracting natural enemies. A terpene synthase (GhTPS14) and two cytochrome P450 (GhCYP82L1, GhCYP82L2) enzymes are involved in the de novo synthesis of DMNT in cotton. We conducted a study to test the potential of manipulating DMNT-synthesizing enzymes to enhance plant resistance to insects.

Objectives: To manipulate DMNT emissions in cotton and generate cotton lines with increased resistance to mirid bug Apolygus lucorum.

Methods: Biosynthesis and emission of DMNT by cotton plants were altered using CRISPR/Cas9 and overexpression approaches. Dynamic headspace sampling and GC-MS analysis were used to collect, identify and quantify volatiles. The attractiveness and suitability of cotton lines against mirid bug and its parasitoid Peristenus spretus were evaluated through various assays.

Results: No DMNT emission was detected in knockout CAS-L1L2 line, where both GhCYP82L1 and GhCYP82L2 were knocked out. In contrast, gene-overexpressed lines released higher amounts of DMNT when infested by A. lucorum. At the flowering stage, L114 (co-overexpressing GhCYP82L1 and GhTPS14) emitted 10-15-fold higher amounts than controls. DMNT emission in overexpressed transgenic lines could be triggered by methyl jasmonate (MeJA) treatment. Apolygus lucorum and its parasitoid were far less attracted to the double edited CAS-L1L2 plants, however, co-overexpressed line L114 significantly attracted bugs and female wasps. A high dose of DMNT, comparable to the emission of L114, significantly inhibited the growth of A. lucorum, and further resulted in higher mortalities.

Conclusion: Turning down DMNT emission attenuated the behavioral preferences of A. lucorum to cotton. Genetically modified cotton plants with elevated DMNT emission not only recruited parasitoids to enhance indirect defense, but also formed an ecological trap to kill the bugs. Therefore, manipulation of DMNT biosynthesis and emission in plants presents a promising strategy for controlling mirid bugs.