The wheat dioxygenase BX6 is involved in the formation of benzoxazinoids in planta and contributes to plant defense against insect herbivores

Benzoxazinoids are plant specialized metabolites with defense properties, highly abundant in wheat (Triticum), one of the world's most important crops. The goal of our study was to characterize dioxygenase BX6 genes in tetraploid and hexaploid wheat genotypes and to elucidate their effects on defense against herbivores. Phylogenetic analysis revealed four BX6 genes in the hexaploid wheat T. aestivum, but only one ortholog was found in the tetraploid (T. turgidum) wild emmer wheat and the cultivated durum wheat. Transcriptome sequencing of durum wheat plants, damaged by either aphids or caterpillars, revealed that several BX genes, including TtBX6, were upregulated upon caterpillar feeding, relative to the undamaged control plants. A virus-induced gene silencing approach was used to reduce the expression of BX6 in T. aestivum plants, which exhibited both reduced transcript levels and reduced accumulation of different benzoxazinoids. To elucidate the effect of BX6 on plant defense, bioassays with different herbivores feeding on BX6-silenced leaves were conducted. The results showed that plants with silenced BX6 were more susceptible to aphids and the two-spotted spider mite than the control. Overall, our study indicates that wheat BX6 is involved in benzoxazinoid formation in planta and contributes to plant resistance against insect herbivores.