Genetic evidence for natural product-mediated plant-plant allelopathy in rice (Oryza sativa)

Abstract

• There is controversy as to whether specific natural products play a role in directly mediating antagonistic plant-plant interactions - that is, allelopathy. If proved to exist, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa). • However, while substantiated by the presence of phytotoxic compounds at potentially relevant concentrations, demonstrating a direct role for specific natural products in allelopathy has been difficult because of the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied. • The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4 (OsKSL4)), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli). • Thus, our results not only provide novel genetic evidence for natural product-mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant.

Fig. 1

Rice momilactones. (a) Biosynthetic gene cluster; (b) biosynthetic pathway.

Fig. 2

Effect of OsCPS4 knock-out (cps4) relative to parental wild-type (WT). (a) Loss of momilactone A in root exudates. LC-MS selected ion (m/z = 315 → 271) chromatogram of root exudate extracts of eight 2-wk-old Oryza sativa seedlings for each genotype (as indicated). Note that these are shown on the same scale (see also Supporting Information Fig. S3). (b) Lack of effect on growth and development. Depicted are 4-wk-old seedlings: WT, green labels; cps4, white labels.

Fig. 3

Effect of OsCPS4 knock-out mutant (cps4) on blast disease resistance and allelopathy. (a) Parental wild-type (WT) and cps4 mutant (as indicated) rice (Oryza sativa) plant leaves and roots infected with Magnaporthe oryzae. (b) Lettuce (Lactuca sativa) seedlings germinated in the presence of WT or cps4 rice seedlings (as indicated). (c) Comparison of allelopathic effect of WT (open bars) versus cps4 (closed bars) rice seedlings on growth of lettuce, both roots and hypocotyls (measured with 10 plates, totaling 60 lettuce seedlings, for each genotype). (d) Comparison of allelopathic effect of WT versus cps4 rice seedlings on root length of germinated barnyard grass (Echinochloa crus-galli) seedlings (measured with 10 plates, totaling 90 barnyard grass seedlings, for each genotype). For both (c and d) the histograms depict mean length (± SE). P values: *, < 0.05; **, < 0.005.

Fig. 4

Effect of OsKSL4 knock-out mutant (ksl4) on allelopathy (histograms depict mean length, ± SE). All comparison between parental wild-type (WT) and ksl4 exhibit P-values < 0.001 (indicated by ***). (a) Comparison of allelopathic effect of WT (open bars) versus ksl4 (closed bars) rice (Oryza sativa) seedlings on growth of lettuce (Lactuca sativa), both roots and hypocotyls (measured with 10 plates, totaling 90 lettuce seedlings, for each genotype). (b) Comparison of allelopathic effect of WT versus ksl4 rice seedlings on root length of germinated barnyard grass (Echinochloa crus-galli) seedlings (measured with eight plates, totaling 80 barnyard grass seedlings, for each genotype).

Fig. 5

Effect of OsKSL4 knock-out mutant (ksl4) on germination and subsequent growth and development of rice (Oryza sativa). (a) Histogram showing reduced germination rate. This was measured in two separate trials, with 80 or 60 seeds, respectively, for each genotype (± SE). The associated P-value is < 0.001 (indicated by ***). (b) Lack of effect on subsequent growth and development. Depicted are mature 5-month-old plants: WT, left; ksl4, right (white labels).