Rhizophagus irregularis regulates RiCPSI and RiCARI expression to influence plant drought tolerance.

Arbuscular mycorrhizal fungi (AMF) can transfer inorganic nitrogen (N) from the soil to host plants to cope with drought stress, with arginine synthesis and NH4+ transport being pivotal processes. However, the regulatory mechanism underlying these processes remains unclear. Here, we found that drought stress upregulated expression of genes involved in the N transfer pathway and putrescine and glutathione synthesis in the mycorrhizal structures of Rhizophagus irregularis within alfalfa (Medicago sativa) roots, i.e. carbamoyl phosphate synthase (RiCPSI), arginase (RiCARI), urease (RiURE), ornithine decarboxylase (RiODC), and glutamate-cysteine ligase (RiGCL). Furthermore, we confirmed that RiCPSI is a carbamoyl phosphate synthase. Silencing RiCARI via host-induced gene silencing inhibited arbuscule formation, suppressed putrescine and glutathione synthesis, and altered arginine metabolism within R. irregularis-plant symbiosis, leading to a substantial reduction in the drought tolerance of M. sativa. Conversely, silencing RiCPSI decreased arginine, putrescine, and glutathione synthesis in R. irregularis but did not adversely affect NH4+ transfer from fungi to the host plant and drought tolerance of M. sativa. Interestingly, overexpressing RiCPSI via our host-induced gene overexpressing system enhanced arginine, putrescine, and glutathione synthesis in R. irregularis, reduced arbuscule abundance, and improved drought tolerance of M. sativa. Our findings demonstrate that under drought stress, the nitrogen transfer from AMF to the host plant was improved. This is accompanied by increased arginine, putrescine, and glutathione synthesis within R. irregularis, driven by the upregulation of RiCPSI and RiCARI expression in mycorrhizal structures within the roots. These molecular adjustments collectively contribute to enhanced drought tolerance in R. irregularis-plant symbiosis.