Sorghum is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8',l l ', 14'- pentadecatrieneJ-^-benzoquinone) which likely accounts for much of its allelopathic properties. Prior investigations into the biosynthesis of sorgoleone have suggested the participation of one or more alkylresorcinol synthases (ARS), which are type III polyketide synthases (PKS) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells, involving the synthesis of a 5- pentadecatrienyl resorcinol intermediate derived from an unusual 16:3 fatty acyl-CoA starter unit. To characterize the enzymes responsible for the biosynthesis of this alkylresorcinol intermediate, a previously-described expressed sequence tag (EST) database prepared from isolated root hairs was first mined for all PKS-I ike sequences. Quantitative real-time RT- PCR analyses revealed that two of these sequences were preferentially expressed in root hairs, and recombinant enzyme studies demonstrated that both sequences (designated ARSl and ARS2) encode ARS enzymes capable of accepting a variety of fatty acyi-CoA starter units. Furthermore, RNA interference (RNAi) experiments directed against ARSl and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARSl and ARS2 are likely to participate in the biosynthesis of sorgoleone inpJαntα. The sequences oϊARSl and ARS2 were also used to identify several rice genes encoding ARSs, which are likely involved in the production of defense-related alkyiresorcinols.