Background and Aims Plant acquisition of endogenous forms of soil phosphorus (P) could reduce external P requirements in agricultural systems. This study investigated the interaction of citrate and phytase exudation in controlling the accumulation of P and depletion of soil organic P by transgenic Nicotiana tabacum plants. Methods N. tabacum plant lines including wild-type, vector controls, transgenic plants with single-trait expression of a citrate transporter (A. thaliana frd3) or fungal phytases (phyA: A. niger, P. lycii) and crossed plant lines expressing both traits, were characterized for citrate efflux and phytase exudation. Monocultures and intercropped combinations of single-trait plants were grown in a low available P soil (12 weeks). Plant biomass, shoot P accumulation, rhizosphere soil pH and citrate-extractable-P fractions were determined. Land Equivalent Ratio and complementarity effect was determined in intercropped treatments and multiple-linear-regression was used to predict shoot P accumulation based on plant exudation and soil P depletion. Results Crossed plant lines with co-expression of citrate and phytase accumulated more shoot P than single-trait and intercropped plant treatments. Shoot P accumulation was predicted based on phytase-labile soil P, citrate efflux, and phytase activity (Rsq=0.58, P < .0001). Positive complementarity occurred between intercropped citrate- and phytase-exuding plants, with the greatest gains in shoot P occurring in plant treatments with A. niger phyA expression. Conclusions We show for the first time that trait synergism associated with the exudation of citrate and phytase by tobacco can be linked to the improved acquisition of P and the depletion of soil organic P.