Kazachkova, Y.; Punskovsky, J.; Rai, A. K.; Franklin, E.; Jonikas, M. C.

The pyrenoid is an organelle found in nearly all algae that concentrates CO2 around Rubisco to enhance photosynthetic carbon fixation. Engineering a functional pyrenoid into C plants is a promising route to improve plant photosynthetic performance and yields. However, progress has been limited by the lack of a plant platform for rapidly testing candidate components. Here, we engineered N. benthamiana to make its chloroplast Rubisco holoenzyme compatible with C. reinhardtii pyrenoid proteins. Disruption of native N. benthamiana RBCS genes and complementation with the C. reinhardtii RBCS2 generated a Rubisco-recombinant background where transient expression of the Rubisco linker protein EPYC1 was sufficient to drive the formation of Rubisco-EPYC1 condensates. Co-expression with the algal pyrenoid kinase KEY1 shifted the multi-condensate state toward a single condensate per chloroplast by altering EPYC1 phosphorylation in planta, recapitulating C. reinhardtii pyrenoid regulation. Together, these results establish engineered N. benthamiana as a tractable platform for rapidly characterizing candidate pyrenoid proteins and provide a step toward reconstructing pyrenoid architecture and regulation in plants.