Uncrossing the ‘X’: Characterization of alternative alleles for KSLX in Oryza

The widely cultivated Asian rice (Oryza sativa) produces a variety of physiologically relevant diterpenoid products, which range in effect from the phytohormone gibberellin, derived from ent-kaurene, to phytoalexins such as the momilactones, derived from syn-pimara-7,15-diene. Previous reports have shown functional variation in the kaurene synthase-like (KSL) genes responsible for synthesizing diterpene precursors to more specialized metabolites, leading to the creation of distinct diterpenoids from allelomorphic genes. Here is reported the product of two previously discovered but uncharacterized alleles of the unusual KSLX, representing a cross between (fusion of) the tandem pair KSL8-KSL9p found in most cultivars. The previously characterized allele (KSLXo) was reported to act on syn-copalyl pyrophosphate (syn-CPP) to produce syn-abieta-7,12-diene, precursor to the phytoalexin oryzalactone. However, at least one other functionally distinct allele was reported from the O. sativa pan-genome (KSLXn), along with another phylogenetically distinct allele found in Oryza barthii (KSLXb), but these were not further characterized. Here both KSLXn and KSLXb were found to selectively react with syn-CPP and produce syn-pimara-9(11),15-diene, a novel diterpene in rice. Additionally, evolution of this locus was investigated, with KSLXb hypothesized to be a functional KSL9. The striking complexity of this locus, which includes distinct composition (KSL8-KSL9(p) or KSLX) as well as allelomorphism of both KSL8 and KSLX, suggests it is subject to balancing selection, consistent with the competing pressures exerted on phytoalexin biosynthesis. Regardless, the studies reported here clarify this additional example of allelomorphic variation in the rice KSL family, providing insight into the rice pan-genomic diterpenoid arsenal.