Divergent roles of <i>ent</i> ‐kaurene oxidase paralogs in rice momilactone biosynthesis

Rice (Oryza sativa) is a critically important food crop and a model cereal, with its momilactones among the first discovered phytoalexins. Notably, while momilactone biosynthesis is related to that of the gibberellin (GA) phytohormones, it was acquired via lateral gene transfer of an associated biosynthetic gene cluster (mBGC), but has been suggested to depend on paralog(s) of the ent-kaurene oxidase (KO) required for GA production. Rice contains a five-gene tandem array of the KO family, with three acting in GA biosynthesis (OsKO1-3), while the divergent KO-like OsKOL4 has been shown to catalyze reactions consistent with a role in the production of phytoalexins, such as the phytocassanes and momilactones. Here, building on biochemical characterization of distinct activity for OsKOL5, genetic evidence is provided indicating OsKOL4 is much more important for momilactone biosynthesis, although both play a role in the production of the phytocassanes. Indeed, knock-out lines (kol4, kol5, and kol4/5) are more susceptible, while overexpression increases resistance, to both the fungal blast pathogen Magnaporthe oryzae and bacterial blight pathogen Xanthomonas oryzae. Intriguingly, consistent with previously reported phytotoxicity of momilactone intermediates, a lesion mimic phenotype is observed with kol4 and kol4/5 but not kol5 lines. Concordantly, phylogenomic analysis suggests acquisition of mBGC was preceded by tandem duplication of KO to generate KOL4, with subsequent duplication generating KOL5. Thus, despite the deleterious effects of KOL4 loss, it can be speculated that linkage to the essential KOs may have alleviated such negative selection leading to mBGC recruitment, potentially relevant to plant BGC evolution more generally.