Abstract
<jats:p> 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 C₃ 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 <jats:italic>N. benthamiana</jats:italic> to make its chloroplast Rubisco holoenzyme compatible with <jats:italic>C. reinhardtii</jats:italic> pyrenoid proteins. Disruption of native <jats:italic>N. benthamiana</jats:italic> RBCS genes and complementation with the <jats:italic>C. reinhardtii</jats:italic> 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 <jats:italic>C. reinhardtii</jats:italic> pyrenoid regulation. Together, these results establish engineered <jats:italic>N. benthamiana</jats:italic> as a tractable platform for rapidly characterizing candidate pyrenoid proteins and provide a step toward reconstructing pyrenoid architecture and regulation in plants. </jats:p>