Knockdown of Lotus japonicus ROP3 alters the root symbiotic phenotype and alters the expression of genes involved in nutrient acquisition during both rhizobial and mycorrhizal symbioses

Abstract

The rhizobial and the arbuscular mycorrhizal symbioses are present on legume roots and lead to local and systemic transcriptional changes of common and specific plant genes. Among them, some small GTPase proteins called ROPs (Rho of plants) have been shown to be involved in the establishment of the legume-rhizobia interaction. In this study, we aimed to characterise the effects of LjROP3 knockdown in Lotus japonicus on plant physiology and expression of symbiosis-related genes after single and dual inoculation with rhizobia and arbuscular mycorrhizal fungus. In wild-type (Gifu) plants, the dual inoculation increased the shoot and root dry weight, nitrogen (derived from symbiosis) and phosphate content, and the number of arbuscules or nodules compared with single inoculation treatments. In addition, we observed a decrease in the expression of genes encoding the mycorrhizal transcription factors LjRAM1 and LjRAM2, and the downstream genes involved in ammonium (LjAMT2.2) and phosphate (LjPT4 and LjPT8) uptake by the plant at the arbuscule level when the dual inoculation was compared with fungal inoculation. An alteration in the expression of the Nod factor receptor LjNFR1, but not of LjNFR5, was measured in wild-type (Gifu) L. japonicus plants compared to rop3 plants under dual inoculation. We have also measured a reduction in the expression of genes encoding rhizobial and mycorrhizal transcription factors (LjNIN and LjRAM1), and of the downstream mycorrhizal genes involved in ammonium (LjAMT2.2) and phosphate (LjPT4 and LjPT8) uptake by the plant at the arbuscule level. In addition, the expression of AM fungal genes encoding nutrient transporters (known to be expressed at the arbuscule level) was also altered. In conclusion, despite altered expression of plant genes involved in the functioning of the symbioses, and associated with a reduction in the number of nodules and arbuscules, knockdown of LjROP3 did not alter plant growth and nutrition under dual inoculation, suggesting that the beneficial effects of the dual symbiosis were maintained.