Reverse‐genetic approach to verify physiological roles of rice phytoalexins: characterization of a knockdown mutant of OsCPS4 phytoalexin biosynthetic gene in rice

A variety of labdane‐related diterpenoids, including phytocassanes, oryzalexins and momilactones, were identified as phytoalexins in rice ( Oryza sativa L.). Momilactone B was also isolated as an allelochemical exuded from rice roots. The biosynthetic genes of these phytoalexins have been identified, including six labdane‐related diterpene cyclase genes such as OsCPS2 , OsCPS4 , OsKSL4 , OsKSL7 , OsKSL8 and OsKSL10 . Here we identified an OsCPS4 knockdown mutant, cps4‐tos , by screening Tos17 mutant lines using polymerase chain reaction. OsCPS4 encodes a syn ‐copalyl diphosphate synthase responsible for momilactones and oryzalexin S biosynthesis. Because Tos17 was inserted into the third intron of OsCPS4 , the mature OsCPS4 mRNA was detected in the cps4‐tos mutant as well as the wild type. Nevertheless, mature OsCPS4 transcript levels in the cps4‐tos mutant were about one sixth those in the wild type. The cps4‐tos mutant was more susceptible to rice blast fungus than the wild type, possibly due to lower levels of momilactones and oryzalexin S in the mutant. Moreover, co‐cultivation experiments suggested that the allelopathic effect of cps4‐tos against some kinds of lowland weeds was significantly lower than that of the wild type, probably because of lower momilactone content exuded from cps4‐tos roots. A reverse‐genetic strategy using the cps4‐tos mutant showed the possible roles of momilactones not only as phytoalexins but also as allelopathic substances.