There is a popular demand for more natural means of pest management, including weed management, as well as a demand by farmers for herbicides with new chemistries and/or new modes of action to which current weed resistances do not apply. Natural compounds offer a source compounds that can either meet these needs in their natural state or as templates for herbicides with better physicochemical properties for field use. In some cases, simply identifying a good herbicide target site with [...] Abstract There is a popular demand for more natural means of pest management, including weed management, as well as a demand by farmers for herbicides with new chemistries and/or new modes of action to which current weed resistances do not apply. Natural compounds offer a source compounds that can either meet these needs in their natural state or as templates for herbicides with better physicochemical properties for field use. In some cases, simply identifying a good herbicide target site with

Summary: Sorgoleone, a major component of the hydrophobic root exudates of Sorghum spp., is probably responsible for many of the allelopathic properties attributed to members of this genus. Much of the biosynthetic pathway for this compound has been elucidated, with the exception of the enzyme responsible for the catalysis of the addition of two hydroxyl groups to the resorcinol ring. A library prepared from isolated Sorghum bicolor root hair cells was first mined for P450‐like sequences, which were then analyzed by quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) to identify those preferentially expressed in root hairs. Full‐length open reading frames for each candidate were generated, and then analyzed biochemically using both a yeast expression system and transient expression in Nicotiana benthamiana leaves. RNA interference (RNAi)‐mediated repression in transgenic S. bicolor was used to confirm the roles of these candidates in the biosynthesis of sorgoleone in planta. A P450 enzyme, designated CYP71AM1, was found to be capable of catalyzing the formation of dihydrosorgoleone using 5‐pentadecatrienyl resorcinol‐3‐methyl ether as substrate, as determined by gas chromatography‐mass spectroscopy (GC‐MS). RNAi‐mediated repression of CYP71AM1 in S. bicolor resulted in decreased sorgoleone contents in multiple independent transformant events. Our results strongly suggest that CYP71AM1 participates in the biosynthetic pathway of the allelochemical sorgoleone.

Knowledge of the mode of action of an allelochemical can be valuable for several reasons, such as proving and elucidating the role of the compound in nature and evaluating its potential utility as a pesticide. However, discovery of the molecular target site of a natural phytotoxin can be challenging. Because of this, we know little about the molecular targets of relatively few allelochemicals. It is much simpler to describe the secondary effects of these compounds, and, as a result, there is much information about these effects, which usually tell us little about the mode of action. This review describes the many approaches to molecular target site discovery, with an attempt to point out the pitfalls of each approach. Clues from molecular structure, phenotypic effects, physiological effects, omics studies, genetic approaches, and use of artificial intelligence are discussed. All these approaches can be confounded if the phytotoxin has more than one molecular target at similar concentrations or is a prophytotoxin, requiring structural alteration to create an active compound. Unequivocal determination of the molecular target site requires proof of activity on the function of the target protein and proof that a resistant form of the target protein confers resistance to the target organism.

Summary: Sorgoleone, a hydrophobic compound exuded from root hair cells of Sorghum spp., accounts for much of the allelopathic activity of the genus. The enzymes involved in the biosynthesis of this compound have been identified and functionally characterized. Here, we report the successful assembly of the biosynthetic pathway and the significant impact of in vivo synthesized sorgoleone on the heterologous host Nicotiana benthamiana. A multigene DNA construct was prepared for the expression of genes required for sorgoleone biosynthesis in planta and deployed in N. benthamiana leaf tissues via Agrobacterium‐mediated transient expression. RNA‐sequencing was conducted to investigate the effects of sorgoleone, via expression of its biosynthesis pathway, on host gene expression. The production of sorgoleone in agroinfiltrated leaves as detected by gas chromatography/mass spectrometry (GC/MS) resulted in the formation of necrotic lesions, indicating that the compound caused severe phytotoxicity to these tissues. RNA‐sequencing profiling revealed significant changes in gene expression in the leaf tissues expressing the pathway during the formation of sorgoleone‐induced necrotic lesions. Transcriptome analysis suggested that the compound produced in vivo impaired the photosynthetic system as a result of downregulated gene expression for the photosynthesis apparatus and elevated expression of proteasomal genes which may play a major role in the phytotoxicity of sorgoleone.