Main conclusion <p>Our work highlights that glutamine plays a central role in contributing to the outcome of disease in the <i>Medicago truncatula-Aphanomyces euteiches</i> interaction when modulating plant N supply.</p> Abstract <p>Nitrogen (N) is essential for the growth of plants and microorganisms. The quantity and quality of N supply can impact plant development but also its interaction with pathogens. Our previous work showed that N modulated <i>Medicago truncatula</i> (Mt) susceptibility to the oomycete pathogen <i>Aphanomyces euteiches</i> (Ae) when plants were grown in vitro and glutamine (Gln) was proposed to mediate this effect of N on plant disease. Using more than 30 lines representative of Mt diversity, we show here that pathogen-induced root Gln concentrations are correlated with higher susceptibility to Ae. N modulation of the response to Ae of the partially resistant Mt A17 genotype was associated with changes in the expression of <i>MtGS1</i> genes encoding cytosolic glutamine synthetases (GSs). This raises the question of the importance of Gln during Mt/Ae interaction and a possible role of cytosolic GS in mediating Mt susceptibility to Ae. Interestingly, exogenous Gln induced a higher susceptibility of the A17 line to Ae and induced a metabolic profile of inoculated A17 roots similar to that of a susceptible genotype. RNAseq experiments highlighted a higher expression of numerous plant defense genes in non-inoculated roots on Gln. On the pathogen side, a higher expression of genes encoding proteases and a lower expression of genes encoding elicitins as well as a better growth of Ae on Gln could explain the higher susceptibility of Mt on Gln. Altogether our results highlight the delicate balance between plant immunity, pathogen growth and virulence in contributing to the outcome of disease when modulating N supply and that Gln plays a central role in this process.</p>