In symbiotic plant-microbe interactions, the host invests considerable amounts of resources in the microbial partner. If the microbe does not reciprocate with a comparable symbiotic benefit it is regarded as a cheater. The host responds to cheaters with negative feedback mechanisms (sanctions) to prevent fitness deficits resulting from being exploited. Here, we studied sanctioning in the symbiosis between Medicago truncatula and its nitrogen-fixing partner Sinorhizobium meliloti by manipulating the exchange of resources between the partnersin three ways, by (i) using mutant rhizobia defective in nitrogenase, (ii) replacing nitrogen in the atmosphere with argon gas; and (iii) supplying rich nitrogen fertilizer to the host. We followed the consequences of simulated cheating by examining the metabolome and proteome of both partners. We find that cheating conditions result in sanctioning of the bacterial partner at different levels. In particular, we observe repression of essential symbiotic functions (e.g. leghemoglobins) and changes in central metabolism that are likely to be relevant for microbial fitness and that could therefore contribute to sanctioning. In addition, sanctioning triggers a broad panel of defense markers such as lignin biosynthesis, and immune receptors. Using deep genome sequencing of the microbial partner (1000x coverage), we observed reproducible high sequence diversity at specific loci related to the stringent response. Taken together, these results suggest that the host has ways to enforce mutualism, and to sanction selfish behaviour of the rhizobial partner with a multilayered antagonistic programme.
