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Rapid reconstitution and kinase-controlled regulation of algal pyrenoid condensates in engineered Nicotiana benthamiana

Yana KazachkovaiD, Jason PunskovskyiD, Ashwani K. Rai, Eric FrankliniD, Martin C. JonikasiD

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Abstract

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.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (21 Jul 2026) · CC BY 4.0

Knocking out Nicotiana benthamiana RBCS genes and complementing with algal RBCS2 enables rapid EPYC1 condensate formation, and the kinase KEY1 consolidates condensates to one per chloroplast.

Testing candidate pyrenoid components in plants has been slow: Arabidopsis stable transformation limits iteration to 4-6 months, and native N. benthamiana RBCS lacks the algal-specific residues needed to bind pyrenoid proteins. This work builds a Rubisco-recombinant N. benthamiana background that supports transient-expression screening, with cloning to results within 14 days.

Key results

  1. ShownMobile-sgRNA CRISPR-Cas9 editing of the eight photosynthetic NbRBCS loci produced white tissue. A single chimeric regenerant had white leaves with no detectable NbRBCS or NbRbcL protein, and its progeny did not grow beyond the cotyledon stage.

    Four sgRNAs, each locus covered by at least two guides; immunoblots show wild type has robust NbRBCS and NbRbcL while Nbrbcs white leaves show none. Stable Cas9/sgRNA transformation gave chlorotic T0 plants that did not survive tissue culture. · CRISPR/Cas9-mediated genome editing disrupts…; Discussion

  2. ShownExpressing C. reinhardtii RBCS2 in the Nbrbcs background restored green, autotrophic, seed-producing plants in three independent lines. Photosynthetic capacity stayed below wild type, with the weakest growth in Line 1.

    Apparent Vcmax was 22%, 33% and 29% of wild type in Lines 1, 2 and 3. Lines 2 and 3 had fresh weight comparable to wild type but reduced height. Stomatal conductance was comparable across genotypes; gas exchange used 5-7 replicates per genotype. · Chlamydomonas reinhardtii RBCS complements…; Photosynthetic measurements; Tables

  3. ShownTransient EPYC1-GFP expression in Nbrbcs;CrRBCS2 chloroplasts produced multiple discrete condensates. Wild-type plants showed only diffuse stromal signal. CrRBCS2-mCherry co-localized with the condensates, indicating Rubisco enrichment.

    Condensates appeared in all three lines and as electron-dense structures by transmission electron microscopy. Two independent stable EPYC1-GFP lines (Nbrbcs;CrRBCS2-2 background) showed the same multi-condensate phenotype. No chlorosis or necrosis was seen. · Transient expression of EPYC1 reconstitutes…

  4. ShownChloroplast-targeted KEY1-mScarlet co-expressed with EPYC1-GFP shifted the multi-condensate state to predominantly one condensate per chloroplast.

    Median condensates per chloroplast was 4 for EPYC1-GFP alone (n = 130) vs 1 with KEY1-mScarlet (n = 153); Mann-Whitney U test, p < 0.0001; consistent across three independent experiments. · Pyrenoid kinase KEY1 promotes single condensate…; Figure legends

  5. SuggestedKEY1 co-expression slowed EPYC1 migration on Phos-tag gels, consistent with KEY1-dependent EPYC1 phosphorylation in planta. The authors infer this phosphorylation drives condensate consolidation.

    Anti-EPYC1 immunoblots after Phos-tag SDS-PAGE showed decreased mobility with KEY1 relative to EPYC1 alone. No quantification is given, and mobility shift is indirect evidence of phosphorylation. · Pyrenoid kinase KEY1 promotes single condensate…; Discussion; Immunoblot analysis

Methods, data and reproducibility

Endogenous NbRBCS loci were disrupted in Cas9-expressing N. benthamiana by virus-delivered mobile sgRNAs, followed by stable CrRBCS2 complementation. Pyrenoid proteins were then expressed transiently and stably and assessed by confocal and electron microscopy, Phos-tag immunoblotting and leaf gas exchange.

  • Cas9-expressing N. benthamiana edited with four NbRBCS sgRNAs; one chimeric white/green regenerant (Nbrbcs) used as source tissue · Nicotiana benthamiana · n = 1 chimeric regenerant
  • Nbrbcs;CrRBCS2 complemented lines (Lines 1-3) vs Cas9 wild type: growth and leaf gas exchange (A/Ci curves, Vcmax, compensation point) · Nicotiana benthamiana · n = 3 independent lines; 5-6 replicates (growth), 5-7 replicates (gas exchange) · Cre02.g120150 (CrRBCS2)
  • Transient and stable EPYC1-GFP expression, with CrRBCS2-mCherry co-expression, imaged by confocal microscopy and TEM · Nicotiana benthamiana (C. reinhardtii proteins) · n = 2 independent stable EPYC1-GFP lines
  • Condensate counts per chloroplast, EPYC1-GFP alone vs EPYC1-GFP + KEY1-mScarlet in Nbrbcs;CrRBCS2-2 · Nicotiana benthamiana (C. reinhardtii proteins) · n = n = 130 and n = 153 chloroplasts, 3 independent experiments
  • N. benthamiana genome assembly queried with A. thaliana RBCS sequences to identify photosynthetic RBCS loci · Nicotiana benthamiana · n = 8 photosynthetic RBCS loci
Methods & tools
CRISPR-Cas9 with mobile sgRNAs (pEE83 vector), Phytozome N. benthamiana genome, GoldenBraid cloning, Agrobacterium-mediated transient and stable transformation, VT-iSIM confocal microscopy, Fiji with Microvolution deconvolution, Transmission electron microscopy, Phos-tag SDS-PAGE and immunoblotting, LI-6800 gas exchange, plantecophys v1.4 (FvCB model), Mann-Whitney U test, Dunnett's T3 post-hoc test
Reagents & environment
R package plantecophys v1.4 for FvCB fitting; Rubisco kinetic constants from Bernacchi et al. (2001); Agrobacterium tumefaciens GV3101.
Reproducibility gaps
No code or data availability statement; raw images, gas-exchange data and the engineered plant lines have no stated deposition. Source constructs came from Addgene and the McCormick laboratory.
Limitations (5)
  • The reduced Vcmax and elevated CO2 compensation point in complemented lines could reflect lower Rubisco abundance, altered hybrid enzyme catalysis, or both; the authors state this is undetermined. · stated by the authors
  • Vcmax was computed with tobacco Rubisco kinetic parameters, so values are comparative estimates of leaf-level assimilation rather than hybrid-enzyme kinetics. · stated by the authors
  • Chlorophyll bleedthrough prevented distinguishing cells with fully dissolved condensates from cells not expressing KEY1, so full dissolution could not be assessed. · stated by the authors
  • The Nbrbcs background derives from a single serendipitous chimeric regenerant, so the knockout was not independently replicated and the exact edits at each locus are not reported. · noted in this summary
  • Phosphorylation is inferred only from an unquantified Phos-tag mobility shift, without a phosphatase control, a kinase-dead KEY1 or mass spectrometry; the link to condensate consolidation is correlative. · noted in this summary

Read this if you engineer algal pyrenoid components into plants and want a fast N. benthamiana screening platform with Rubisco-EPYC1 condensates and KEY1-regulated condensate number.

Citations
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