Arbuscular mycorrhizal (AM) symbiosis between plants and AM fungi is conserved across the land plants and is the default nutrient uptake strategy for plants in nature. Plant recognition and accommodation of AM fungi requires signalling through two evolutionarily ancient pathways: the common symbiosis signalling pathway (CSSP) and the DWARF14-LIKE (D14L) pathway. D14L has diverse roles in plant development, including inhibition of mesocotyl elongation during seedling development and symbiotic perception of AM fungi in the soil. During D14L signalling in rice, the negative repressor SMAX1 is degraded and CSSP components are transcriptionally upregulated, indicating that the symbiotic function of D14L is to modulate the CSSP. Meanwhile, the transcription factor NSP2, which is implicated in AM symbiosis and is a target of the phosphate starvation response, has been found to operate upstream of D14L in Medicago. In this study, the functional conservation of D14L and its symbiotic role relative to the CSSP and NSP2 were defined in rice.

To investigate the evolutionary history of D14L, trans-species complementation of the rice d14l mutant by D14L homologues from a range of AM and nonmycorrhizal plants was performed. This analysis confirmed the highly conserved nature of the D14L protein and revealed that D14L does not specify the mycorrhizal status of angiosperms. To assess the role of NSP2 and closely related NSP1 in AM symbiosis in rice, CRISPR mutants were generated and investigated for defects in fungal colonisation and mesocotyl development. It was found that both NSP1 and NSP2 are essential for symbiosis and mesocotyl development. Furthermore, the mesocotyl phenotype of nsp mutants could be rescued by application of D14L signalling chemicals, providing evidence that both NSP1 and NSP2 are upstream of D14L in rice. To examine the downstream components of D14L signalling, complementation lines of rice d14l with the CSSP component CCaMK were generated. It was found that a gain-of-function CCaMK could complement the d14l mutant, revealing that D14L signalling regulates the CSSP for AM symbiosis. These results place the D14L signalling pathway at the centre of NSP and CSSP function. Transcriptome analysis of gain-of-function CCaMK lines demonstrated that CCaMK upregulates the expression of potential presymbiosis genes and genes relevant to conditioning a plant for symbiosis. Overall, a model is proposed in which D14L signalling functions as a master switch that can integrate the plant nutrient status into a decision to rapidly launch or block symbiosis.