ORIGINAL ARTICLE Nutrient demand and fungal access to resources control the carbon allocation to the symbiotic partners in tripartite interactions of Medicago truncatula Arjun Kafle 1 | Kevin Garcia 1 | Xiurong Wang 1,2 | Philip E. Pfeffer 3 | Gary D. Strahan 3 | Heike Bücking 1 1 South Dakota State University, Biology and Microbiology Department, Brookings, South Dakota 2 South China Agricultural University, State Key Laboratory for Conservation and Utilization of Subtropical Agro‐Bioresources, Root Biology Center, Guangzhou, China 3 Eastern Regional Research Center, USDA, Agricultural Research Service, Wyndmoor, Pennslyvania Correspondence H. Bücking, Biology and Microbiology Department, South Dakota State University, Brookings, SD 57007. Email: heike.bucking@sdstate.edu Funding information USDA, Grant/Award Number: 2017‐67014‐ 26530; SD Soybean Research and Promotion Council; Agricultural Experiment Station at SDSU Abstract Legumes form tripartite interactions with arbuscular mycorrhizal fungi and rhizobia, and both root symbionts exchange nutrients against carbon from their host. The carbon costs of these interactions are substantial, but our current understanding of how the host controls its carbon allocation to individual root symbionts is limited. We examined nutrient uptake and carbon allocation in tripartite interactions of Medicago truncatula under different nutrient supply conditions, and when the fungal partner had access to nitrogen, and followed the gene expression of several plant transporters of the Sucrose Uptake Transporter (SUT) and Sugars Will Eventually be Exported Transporter (SWEET) family. Tripartite interactions led to synergistic growth responses and stimulated the phosphate and nitrogen uptake of the plant. Plant nutrient demand but also fungal access to nutrients played an important role for the carbon transport to different root symbionts, and the plant allocated more carbon to rhizobia under nitrogen demand, but more carbon to the fungal partner when nitrogen was available. These changes in carbon allocation were consistent with changes in the SUT and SWEET expression. Our study provides important insights into how the host plant controls its carbon allocation under different nutrient supply conditions and changes its carbon allocation to different root symbionts to maximize its symbiotic benefits. KEYWORDS arbuscular mycorrhizal symbiosis, carbon transport, Ensifer meliloti, legumes, nitrogen uptake, rhizobia, Rhizophagus irregularis, sucrose transport, sucrose uptake transporter (SUT), sugars will eventually be exported transporter (SWEET) 1 | INTRODUCTION Legumes, such as soybean, cowpea, and Medicago, are among the most important crop species worldwide. They account for 27% of the world's primary crop production, for 33% of the dietary nitrogen (N) needs of humans (Vance, 2001), and play a significant role in crop rotations and in the soil N cycle. The majority of legumes form tripar- tite interactions and are simultaneously colonized with N‐fixing bacte- ria and arbuscular mycorrhizal (AM) fungi. It is well known that these interactions can substantially contribute to the nutrient acquisition of legumes and increase the fitness of both the host and the different root symbionts (Afkhami & Stinchcombe, 2016; Mortimer, Pérez‐ Fernández, & Valentine, 2009; Ossler, Zielinski, & Heath, 2015). N‐fixing rhizobia bacteria reside within specialized root nodules that provide them with an oxygen‐reduced environment for biological N 2 ‐fixation (BNF). Within nodules, rhizobia differentiate into bacte- roids that are able to convert atmospheric N 2 to NH 3 through their nitrogenase complex. NH 3 is exported together with amino acids Received: 27 April 2018 Revised: 27 May 2018 Accepted: 28 May 2018 DOI: 10.1111/pce.13359 270 © 2018 John Wiley & Sons Ltd Plant Cell Environ. 2019;42:270–284. wileyonlinelibrary.com/journal/pce