Transient molecular chimerism for exploiting xenogeneic organelles

Domain: biology
Published: 2026-03-24
Editorial outcome: minor_revision

Abstract
The symbiogenetic origin of organelles, such as chloroplasts, is established, and organelle genomes provide evidence of prokaryotic ancestry. Nevertheless, most organelle proteins are nuclear-encoded and function in concert with those expressed from the organelle genomes, representing constitutive molecular chimerism. The evolutionary forces generating chimerism have been widely discussed, but without much evidence. Here we provide biochemical evidence of transient molecular chimerism in nature, along with a possible mechanistic explanation for chimerism. In the flagellate, Rapaza viridis, nuclear-encoded proteins support photosynthesis in transient, xenogeneic chloroplasts (kleptoplasts) acquired from the green alga Tetraselmis sp. We focused on two putative kleptoplast-targeted proteins: a RuBisCO small subunit-like protein (RvRbcS-like) and a RuBisCO activase homologue. Immunofluorescence microscopy confirmed the kleptoplast localization of the proteins, and the knockdown and knockout experiments demonstrated impaired photosynthesis, particularly for RvRbcS-like. The unique carboxyl-terminal extension of the RvRbcS-like protein suggests that it has an additional role in pyrenoid reorganization, a key step in kleptoplast remodelling. Protein translocation into kleptoplasts requires rapid, de novo assembly of transport systems after each acquisition, unlike the constitutive chimerism of established organelles. This previously unreported phenomenon in eukaryotes positions R. viridis as a unique, genetically tractable model for investigating the molecular and evolutionary origins of organelles.

Introduction
Eukaryotes are inherently chimeric. Although the core system originated in archaea, many eukaryote proteins were derived from different bacteria of another domain, and eukaryote organelles, such as mitochondria and chloroplasts, resulted from the cellular-level integration of other bacteria. Each integration arose in a specific physiological context, which was probably endosymbiotic, where selection operated at that moment, and the chimeric states found in modern eukaryotes are constitutive. Therefore, it is only natural that theories on organellogeneses hinge on two extremes: modern endosymbiotic examples with little molecular-level integration and genetically fully integrated organelles, although the proposed evolutionary processes are not well evidenced. Nevertheless, there are some exceptional and unique opportunities for evolutionary studies into the transient integrations of xenogeneic bodies in some eukaryotic cells, such as the phenomenon called kleptoplasty.

Kleptoplasty represents a distinct form of algal exploitation, in which the host cells temporarily acquire chloroplasts from other algal cells, presumably to exploit them for phototrophy, and the stolen plastids are called kleptoplasts. Kleptoplasty has been reported in a wide range of eukaryotes and, importantly, demonstrates a wide variety of symbiogenetic modes. Some well-known examples of multicellular host organisms include the sacoglossan sea slugs. However, unicellular hosts are more common and have been reported in taxa with diverse dinoflagellates, ciliates, and foraminiferans, a kathablepharid, a centrohelid, and euglenozoan. Unlike mutualistic algal endosymbiosis, the kleptoplasts that confer phototrophy to the host are extracted from the original algal cells and can be considered as symbiogenetic in nature, functioning as transient organelles. However, the molecular basis of kleptoplast maintenance differs among cases. For example, in the ciliate Mesodinium rubrum and the dinoflagellates Nusuttodinium aeruginosum and Durinskia capensis, the nuclei of the kleptoplast donor algae are retained in the host cytoplasm along with the kleptoplasts. These kleptoplasts seem to be temporarily maintained by the algal nuclei-encoded proteins, whose expression may or may not be modulated by the host.

In other intriguing cases, even without algal nuclei, partial genetic integrations have been proposed based on transcriptomic data to be involved in prolonged maintenance of active kleptoplasts by asserting the expression of host nuclear-encoded proteins in the xenogeneic kleptoplast interior. However, these hypotheses lacked biochemical confirmation. Approximately 60 candidate kleptoplast-targeted proteins were proposed in Dinophysis acuminate, which included several components of the thylakoid photosynthetic apparatus, sugar transporter, and some enzymes involved in pigment biosynthesis. In the Ross Sea dinoflagellate, some photosynthetic proteins encoded by the host nuclear genome may target and function in the kleptoplasts, especially the photosystem I subunits, allowing temporary establishment of cyclic electron transport.

Recent findings have revealed an outstanding case of kleptoplasty in the euglenozoan flagellate Rapaza viridis. This organism obtains its kleptoplasts from the green alga Tetraselmis sp. through phagocytosis. Although it retains the chloroplasts, the algal nucleus is expelled together with the cytoplasm shortly after its ingestion. The resulting kleptoplast is then split into smaller pieces and distributed to the daughter cells, without being enlarged, maintaining high photosynthetic performance for about 2 weeks before declining. Most importantly, the R. viridis transcriptome contained more chloroplast-related genes than ever reported, with 274 identified according to the most conservative estimate, each encoding distinctive amino-terminal (N-terminal) sequences similar to the bipartite targeting signals found in Euglenophyceae algae, the sister clade of R. viridis possessing secondary chloroplasts derived from Pyramimonas. Therefore, the phenomena observed in the kleptoplasty of R. viridis, such as pyrenoid reorganization and multiplication, persistence of photosynthetic activity, and accumulation of photosynthetic products in the host cytoplasm, were tentatively attributed to genes that are presumably acquired by horizontal gene transfer from various algae. However, in silico predictions alone are insufficient to prove protein import and elucidate its mechanistic underpinnings, highlighting the need for cellular-level biochemical investigations.

Here, we investigated kleptoplasty in R. viridis at the protein level, focusing on the intracellular localization and function of host nuclear-encoded genes. Using genetic engineering and biochemical approaches, we show that selected highly expressed proteins with predicted chloroplastic functions are translocated into kleptoplasts, where they are crucial for the effective operation of this xenogeneic organelle.

Results
Host chloroplastic gene expression
From the time-series transcriptome data taken from four distinct kleptoplastic stages, we identified 37 R. viridis transcripts encoding proteins with conserved domains indicative of chloroplastic functions. The expression levels of these transcripts were comparable to those of key mitochondrial metabolic genes. The presence of a distinctive euglenid 5′-spliced leader sequence in these transcripts and the mapping of their open reading frames (ORFs) to contigs in the draft genome, with or without introns conforming to the canonical GT(GC)-AG splice rule, verifies their genuine expression from the R. viridis nuclear genome. Phylogenetic analysis of the translated peptide sequences showed that about 40% clustered with Euglenophyceae proteins, consistent with previous reports, and about 20% with Tetraselmis spp. However, none of the 37 sequences were identical or highly homologous to the current kleptoplast donor strain Tetraselmis sp. NIES-4478. This suggests any horizontal transfers of these important genes occurred in the distant past, and the genes evolved as unique components of R. viridis.

These putative proteins include core components of the thylakoid membrane photosynthetic machinery, including light-harvesting proteins and subunits of photosystem II, cytochrome b6f, and ATP synthase. Transcripts were also identified for proteins involved in the Calvin–Benson cycle, including ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its related factors, thylakoid membrane proteases (FtsH homologs), a triose-phosphate/phosphate translocator (TPT), pentose-phosphate/phosphate translocators, and other enzymes. The peak transcript levels varied by gene. Although many transcripts, including those for the electron transport chain, peaked during the growth or stationary phases of the phototrophic stage, others peaked in the late kleptoplast transformation stage. From the late kleptoplast transformation stage, we selected two genes for further analysis: a homolog of the RuBisCO small subunit (RvRbcS-like) and of RuBisCO activase (RvRca-like). Quantitative polymerase chain reaction (qPCR) confirmed that RvRbcS-like expression peaked during the transformation stage, whereas RvRca-like expression peaked in the early to mid-growth phase. If transported into kleptoplasts and functionally active, these proteins are likely to be essential for carbon fixation in kleptoplastic photosynthesis.

Host protein kleptoplast localization
We confirmed the expression of RvRbcS-like and RvRca-like in R. viridis and their absence in their respective knockout strains by immunoblotting, with Tetraselmis sp. as a control. Custom anti-peptide antibodies were synthesized to recognize the corresponding amino acid sequences from R. viridis NIES-4477 specifically, and both proteins were detected in wild-type R. viridis but not in the knockout strains (ΔRvRbcS-like and ΔRvRca-like, respectively).

The observed peptide sizes were much smaller than predicted. RvRbcS-like encodes a 942-residue protein composed of four tandem RbcS domains flanked by a 177-residue N-terminal low-complexity domain (NtLCD) and a 228-residue C-terminal low-complexity domain (CtLCD), with an expected molecular weight of ~105 kDa. However, immunoblotting revealed a predominant band at ~90 kDa, consistent with the removal of the ~18 kDa NtLCD. Full-length peptides were rarely observed; only one instance of a ~105 kDa band was found in an early experiment. Similarly, RvRca-like encodes a 542-residue protein containing a conserved Rca domain (PLN00020) and a 171-residue NtLCD, with a predicted size of ~59 kDa. However, the detected band was ~42 kDa, consistent with cleavage of the ~17 kDa NtLCD.

These findings strongly suggest that both proteins undergo NtLCD cleavage during or after translocation into the kleptoplast. Because chloroplast-targeting sequences are typically removed during import, our data imply that these nuclear-encoded peptides are translocated into kleptoplasts via their NtLCDs and subsequently processed to their mature forms.

Next, we demonstrated the localization of these peptides within the kleptoplasts using immunofluorescence microscopy. We generated a genome-edited strain in which an epitope tag was fused to RvRbcS-like because the custom anti-peptide antibody targeting RvRbcS-like used above is unlikely to recognize the protein in its natural conformation, forming the RuBisCO complex. A 3×HA tag was inserted immediately downstream of the fourth putative RbcS domain, ensuring that the tag was exposed outside the putative complex (strain RvRbcS-uCt-insHA3). The resulting peptide, ~90 kDa after NtLCD removal, was detected by immunoblotting of the total cellular proteins using an anti-HA antibody, confirming successful tag insertion. After staining with the anti-HA antibody, immunofluorescence microscopy of the HA-tagged RvRbcS strain revealed fluorescent signals localized just inside the kleptoplasts, demonstrating RvRbcS-like translocation into the kleptoplast. Multiplex observations using a generic anti-RuBisCO large subunit (RbcL) antibody showed significant overlap between the HA-tagged protein and RbcL signals. This observation strongly suggests that RvRbcS-like may function together with RuBisCO in the kleptoplast pyrenoid. No HA signal was detected in wild-type cells, validating the specificity of the reporter immunofluorescence.

Similarly, immunofluorescence microscopy using the anti-RvRca-like antibody revealed signals localized in the kleptoplasts in wild-type cells, but not in ΔRvRca-like cells, confirming that the detected signal represented naturally folded RvRca-like. These findings demonstrate that RvRca-like is also translocated into the kleptoplasts.

Function of host-derived proteins
Next, we examined the phenotypic consequences of knocking out RvRbcS-like and RvRca-like by genome editing to assess their functional importance. In batch cultures of the ΔRvRbcS-like strain with precisely equal initial cell concentrations at the ingestion event, ΔRvRbcS-like exhibited markedly reduced growth, reaching less than half of the wild-type level. Most ΔRvRbcS-like cells died by day 22, much earlier than the wild-type survival of approximately 5 weeks. Additionally, the accumulation of cytoplasmic polysaccharide grains was markedly diminished in ΔRvRbcS-like compared with that in wild-type cells. Photosynthetic activity per cell in ΔRvRbcS-like was suppressed to about half of that of the wild-type activity. Dark oxygen consumption, a measure of respiratory activity, was also significantly reduced, suggesting suppressed cellular activity due to the reduced supply of photosynthetic products. Supporting these findings, RNA interference targeting RvRbcS-like also impaired photosynthesis. Although gene silencing was incomplete, the light–response curves showed that the net oxygen evolution per cell in the RNA interference-treated culture was significantly reduced to about half of the control level.

These results highlight the critical role of RvRbcS-like in maintaining photosynthetic efficiency in kleptoplasts. Because orthodox RbcS is an essential subunit of RuBisCO in algal and plant chloroplasts, RvRbcS-like is likely to be involved in carboxylation activity in kleptoplasts. Although RbcL is encoded by the chloroplast genome and may continue to be expressed in kleptoplasts, the original Tetraselmis sp. nuclear-encoded RbcS protein (TsRbcS) is unlikely to be produced after the kleptoplast transformation stage, during which the algal nucleus is eliminated from the R. viridis cells.

A time-series immunoblot analysis of total cellular proteins using a general plant RbcL antibody showed no significant changes in the RbcL levels in the first week after the ingestion event in wild-type cells, indicating maintenance of intact RuBisCO. Because RbcL and RbcS are balanced in a stoichiometric ratio to form the RbcL8RbcS8 heterohexadecameric RuBisCO complex, the RbcL levels are expected to be regulated by the availability of RbcS. Thus, the maintenance of RbcL levels after the ingestion event is expected to depend on RbcS or its functional equivalent. Indeed, in wild-type cells, TsRbcS levels gradually declined, whereas RvRbcS-like levels increased over 7 days, as detected using anti-peptide antibodies specific to each protein. This interpretation was further supported when the immunoblot signals were evaluated in terms of protein abundance per culture volume (i.e., the total amount of each protein within a unit volume of culture), a measure independent of cell proliferation and associated variation in total protein content. By contrast, in the ΔRvRbcS-like cells, which lacks the supply of RvRbcS-like, RbcL levels also decreased, strongly indicating the effective loss of RuBisCO.

The phenotype of another knockout strain, ΔRvRca-like, was considerably milder, despite the well-recognized essential role of RuBisCO activase in RuBisCO function. The growth rate of ΔRvRca-like cells during the first week after the ingestion event was nearly comparable to that of wild-type cells, with only a slightly lower final cell concentration. However, phenotypic differences became apparent during the stationary phase, because the ΔRvRca-like cells did not show the accelerated accumulation of cytoplasmic polysaccharide grains that was observed in wild-type cells. Notably, the photosynthetic rate of ΔRvRca-like cells on day 8 was significantly lower than that of wild-type cells. These observations suggest that the ΔRvRca-like phenotype manifests more gradually than the ΔRvRbcS-like phenotype, reflecting a delayed but substantial reduction in RuBisCO efficiency.

NtLCDs in kleptoplast-targeted proteins
We demonstrated that the NtLCD of RvRbcS-like alone is sufficient to mediate the translocation of a downstream peptide into the kleptoplast. Specifically, based on the results indicating that the NtLCDs of RvRbcS-like function as translocation signals and are cleaved during maturation, we tested this hypothesis using a NanoLuc luciferase reporter system. We generated a transformant line expressing luciferase without an NtLCD and another line expressing luciferase fused to the NtLCD of RvRbcS-like at the N-terminus. In the absence of the NtLCD, luciferase bioluminescence was detected throughout the cytosol but not in kleptoplasts. In contrast, when fused to the NtLCD, luciferase was mainly localized in kleptoplasts. These results provide direct evidence that the NtLCD is sufficient for transporting nuclear-encoded proteins into kleptoplasts.

Importantly, NtLCDs ranging from 134 to 320 amino acids (mean 194 ± 37) were identified in 35 of the 37 putative kleptoplast-targeted proteins listed above. These NtLCDs structurally resemble chloroplast-targeting presequences in Euglena gracilis, which typically include one or two transmembrane helices (TMHs). Based on this similarity, the R. viridis NtLCDs could be classified into four types: the most common RvClass IA, which features two TMHs flanking a 90–100 residue hydrophilic segment enriched in hydroxylated residues at both ends, basic residues in the center, and acidic residues toward the C-terminus; RvClass IB, which includes an additional TMH in the C-terminal extension; RvClass IC, which has a shorter spacer (40–80 residues) lacking the acidic C-terminal region; and RvClass II, which lacks a second TMH and has a simplified hydrophilic structure similar to RvClass IC. The NtLCD of RvRbcS-like falls into RvClass IA.

These findings suggest that at least 20 candidate proteins with RvClass IA NtLCDs, including RvRbcS-like and RvRca-like, and possibly three with RvClass IB NtLCDs, are likely transported into kleptoplasts via an evolutionarily conserved but unidentified translocation mechanism. All NtLCD classes identified in R. viridis are structurally comparable to the N-terminal targeting signals of E. gracilis chloroplast proteins. Based on this similarity, the remaining 12 proteins with RvClass IC or II NtLCDs are also likely to be transported into kleptoplasts, because Class II signals similar to RvClass II were sufficient for chloroplast import in E. gracilis. By contrast, the two proteins lacking extended NtLCDs showed constitutive expression patterns across all kleptoplastic stages. RvTPT1 encodes a predicted TPT, which may function on the outermost kleptoplast membrane of phagosomal origin, rather than within the kleptoplast itself. Among four additional TPT genes with lower expression, three (RvTPT2–4) carry RvClass IA NtLCDs, suggesting that they are directed to inner membranes derived from algal chloroplasts.

Methods
Cell culture
R. viridis (NIES-4477) and Tetraselmis sp. (NIES-4478) were cultured under axenic conditions in Daigo IMK medium (Nihon Pharmaceutical, Tokyo, Japan; maintenance medium) at 20°C under illumination of 100–150 μmol photons m−2 s−1 with a 14-h light/10-h dark cycle (14L:10D). R. viridis cultures were generally established by mixing Tetraselmis sp. cells into fresh medium at a ratio of 1:1–1:3 (depending on the experiment) as a source of new kleptoplasts. After inoculation, the Tetraselmis sp. cells were typically completely consumed by R. viridis within 0.5–3 h, depending on the initial ratio, resulting in monospecific cultures containing only R. viridis. The maintenance culture conditions were described in previous reports. Experimental R. viridis cultures used for the time-series transcriptome and qPCR analysis were initiated by mixing Tetraselmis sp. cells at a 1:1 ratio, resulting in rapid consumption of the kleptoplast donor within 0.5 h. The remaining experiments were initiated by mixing Tetraselmis sp. cells at a 1:3 ratio. The cell numbers were counted using a particle counter/analyzer (CDA-1000, Sysmex, Kobe, Japan).

Microscopy
Differential interference contrast images were obtained using an inverted microscope (IX71, Olympus, Tokyo, Japan) equipped with a color CCD camera (FX630, Olympus) and a Flovel Image Filing System (Flovel, Chofu, Japan). Live cells were placed between two glass coverslips (0.13–0.17 mm) and observed.

Time-series transcriptomic analysis
To obtain the transcriptomes of R. viridis NIES-4477, we also analyzed the transcriptome of the kleptoplast donor alga Tetraselmis sp. NIES-4478 from the unialgal culture. This was done to subtract any potential contaminating sequences, although all Tetraselmis cells are typically consumed shortly after inoculation, and the cytoplasm and nucleus are eliminated early during kleptoplast formation. R. viridis cells were sampled from quintuplicate batch cultures at 8, 18, 120, and 360 h after initial inoculation, and Tetraselmis was sampled from quintuplicate unialgal cultures. The collected cells were pelleted by centrifugation and immediately frozen in liquid nitrogen. Total RNA was extracted from all samples using the NucleoSpin RNA XS kit (TaKaRa, Kusatsu, Japan), and the three replicates with the highest RNA yields were selected for downstream experiments. Then, 2.0‒22.4 μg of the total RNA samples were sent to Macrogen Co. (Tokyo, Japan) for library construction and sequencing using a sequencing system (HiSeq X, Illumina, San Diego, CA, USA).

The RNA-seq raw reads were cleaned using cutadapt ver. 2.8 by trimming low-quality-value ends (<QV20) and adapter sequences and discarding any reads shorter than 50 bp. The trimmed reads from the R. viridis samples were assembled de novo using Trinity ver. 2.11.0 in the paired-end mode with the option “--min_contig_length 300”. When splicing variants of a gene were found, the longest transcript was selected as the representative mRNA sequence. The ORFs were predicted using TransDecoder ver. 5.5.0 (http://transdecoder.github.io). The de novo transcriptome sequences were then filtered against the genome data from Tetraselmis sp. (methods described below; Supplementary data deposited in Figshare), although reads attributable to the Tetraselmis sp. genome were, in fact, miscellaneous in the R. viridis RNA-seq reads. Therefore, the contigs with 97% identity to Tetraselmis nucleotide sequences evaluated by BLASTN search were omitted, and the remaining contigs were used as a reference for the R. viridis transcripts. We confirmed that none of the omitted sequences contained the euglenid 5′-spliced leader sequence. To estimate the function of the predicted orf, BLASTP searches were conducted using the Uniprot/Swiss-Prot database (Uniprot release 2020_6, downloaded from https://www.uniprot.org/uniprotkb). To obtain gene expression scores, one side of the trimmed reads was mapped to the reference using Bowtie2 ver. 2.4.2. SAMtools ver. 1.11, BEDtools ver. 2.92.2, and R ver. 4.0.3 were used to calculate the number of reads mapped to contigs (raw count) and the reads per kilobase of transcript per million mapped reads. To analyze the time-series transcriptomic changes of R. viridis, genes with low mapped read counts were omitted using the filterLowCountGenes command (low.count=10) in the TCC package ver. 1.30.0 in R. The data were then normalized using the calcNormFactors command, and differentially expressed genes (DEGs) were identified using the estimateDE command of TCC in R. R. viridis contigs were defined as DEGs if the false discovery rate was <0.05 (Supplementary data deposited in Figshare). The normalized count was obtained using the getNormalizedData command of TCC.

For genome assembly of Tetraselmis sp. NIES-4478 in association with the above R. viridis RNA-seq analysis, genomic DNA was extracted from a stationary-phase monoxenic culture using the DNeasy Plant Mini Kit (QIAGEN, Venlo, Netherlands). The purified DNA was sent to Macrogen Co. (Tokyo, Japan) for library preparation and sequencing on an Illumina HiSeq X system. Raw reads were processed with cutadapt ver. 2.1, trimming adapter sequences and low-quality bases (<QV20), and discarding reads shorter than 50 bp. A total of 19 million paired-end reads were assembled using SPAdes version 3.13.0, and the resulting scaffold FASTA file was used to eliminate Tetraselmis contigs from the R. viridis transcriptome reference, as described above.

Nuclear genomic DNA sequencing and assembling
Genomic DNA was purified from a stationary phase monoxenic culture of R. viridis containing Tetraselmis sp. using the standard phenol/chloroform/isoamyl alcohol method. The purified DNA sample was sent to Macrogen Co. for library construction and sequencing using a sequencing system (NovaSeq 6000, Illumina). After trimming and quality control procedures using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/), 152 million paired-end reads were retained. Genome assembling was carried out using MaSuRCA version 4.0.7 as follows. To determine the reliable insert size and standard deviation (SD) of the insert size for paired-end reads, a preliminary scaffold was constructed using 10% of paired-end reads with SPAdes version 3.15.4 and default setting. Mapping analysis against the preliminary scaffold was performed using Bowtie2, and the insert size and SD were estimated to be 120 bp and 110 bp, respectively. The MaSuRCA assembly was subsequently performed using all 152 million quality-controlled paired-end reads with the CABOG assembly option. Finally, a total primary scaffold length of 508 Mbp with an average coverage depth of 91× was obtained.

Amplification and sequencing of specific cDNA and gDNA loci
The full-length cDNA and genomic DNA (gDNA) sequences of RvRbcS-like and RvRca-like, including the flanking regions, were amplified and sequenced (DDBJ accession numbers: LC877979‒LC877982). Total RNA (>200 bases) was extracted from R. viridis using ISOGEN II (Nippon Gene, Toyama, Japan) and reverse-transcribed with SuperScript IV (Thermo Fisher Scientific, Waltham, MA, USA) and the GeneRacer oligo-dT24 primer (Thermo Fisher Scientific). Rapid amplification of cDNA ends (RACE) was performed to determine the 5′ and 3′ termini using primers for the spliced leader sequence and the GeneRacer adapter, as described by Nakazawa et al. The target gDNA regions were identified by BLASTN searches against the DNA-seq contigs, and amplicons spanning from the first to the last exon were cloned and sequenced by next-generation sequencing (Plasmid-EZ, Azenta, South Plainfield, NJ, USA), which also identified missing gaps in the DNA-seq data, typically enriched short repeats. The RvPsbR gene was also partially re-sequenced due to discrepancies between its cDNA and contig sequences. All primers used in this study are listed in Supplementary Table 6. All resulting sequenced data have been deposited in Figshare.

Phylogenetic analysis
BLASTP searches were conducted against the NCBI nr database using the translated peptide sequences of each highly expressed gene listed in Supplementary Table 1 as queries. Homologs from the chloroplast donor Tetraselmis sp. and the Euglenophyceae algae Eutreptiella spp. and E. gracilis were retrieved from in-house RNA-seq data and manually incorporated into the dataset. Sequence alignment was performed using the MAFFT algorithm with default parameters (MAFFT package v7.520), followed by trimming with trimAl 1.2rev57. Short and ambiguously aligned positions were manually removed. Maximum likelihood trees were inferred using IQ-TREE software v1.6.12, and the number of nonparametric bootstrap replicates and substitution models are specified in each figure. The resulting trees were manually inspected to assess the relationships between R. viridis sequences and those of other organisms in the database.

qPCR analysis
Wild-type R. viridis cells were sampled from a batch cultures at 1, 6, 12, 18, 24, 48, 72, 96, 144, 192, 240, 288, and 336 h after initial inoculation. The cells were pelleted by centrifugation and immediately frozen in liquid nitrogen. Total RNA was extracted using ISOGEN II (Nippon Gene), and cDNA was synthesized by reverse transcription using the PrimeScript RT Reagent Kit (TaKaRa). The RvRbcS-like and RvRca-like transcript levels were quantified using a real-time PCR system (LightCycler 96 system, NIPPON Genetics, Tokyo, Japan) with KAPA SYBR Fast Universal (NIPPON Genetics) with the primers listed in Supplementary Table 6.

RNAi
Silencing experiments targeting RvRbcS-like were performed following the method described by Maruyama et al. Double-stranded DNA templates were amplified from R. viridis cDNA using specific primers, each containing a 5′ extension with a T7 promoter sequence (Supplementary Table 6). Double-stranded RNA (dsRNA) corresponding to a 434 bp fragment of RvRbcS-like was synthesized and purified using the MEGAscript RNAi Kit (Thermo Fisher Scientific). The dsRNA was introduced into R. viridis cells through two consecutive electroporation treatments. For the first electroporation, R. viridis cells 5 days after inoculation with the prey Tetraselmis sp. were used. A total of 4 × 106 cells were resuspended in 200 μL of the RNAi electroporation buffer (1/20-strength artificial seawater (ASW; 5% (v/v)) with 500 mM trehalose) containing 15 μg of dsRNA and electroporated using a GENE PULSER II system (Bio-Rad, Hercules, CA, USA) at 0.45 kV and 50 μF in a 0.2 cm gap cuvette. Immediately afterward, the cells were transferred to fresh IMK medium and incubated for 2 days (half a day in the dark followed by a light–dark cycle at 20°C). Negative control experiments were conducted in parallel without dsRNA. The electroporation was repeated once, 2 days later, using the same procedure. The treated cells were again kept in the dark for half a day and then fed with Tetraselmis sp. to acquire new kleptoplasts, thereby establishing RNAi-knockdown batch cultures.

CRISPR/Cas9 genome editing
CRISPR/Cas9 genome editing experiments were performed following the general procedure described by Maruyama et al., with the following design-specific modifications. Target CRISPR RNAs (crRNAs) were designed using the CHOPCHOP (version 3) webtool and are listed in Supplementary Table 7. Custom crRNAs were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Guide RNAs were prepared by hybridizing each crRNA with tracrRNA (Alt-R CRISPR-Cas9 tracrRNA, Integrated DNA Technologies) and then were incubated with Alt-R S.p. HiFi Cas9 Nuclease V3 (Integrated DNA Technologies) to assemble the ribonucleoprotein (RNP) complexes. The expected cleavage sites in the RvRbcS-like and RvRca-like coding regions of the gDNA are shown in Supplementary Fig. 5.

To generate knockout strains (ΔRvRbcS-like and ΔRvRca-like), pairs of RNPs targeting two distinct loci in the first exon of each gene (Supplementary Fig. 5), together with a single-stranded oligonucleotide (ssODN) donor (Supplementary Table 7), were introduced into R. viridis cells through an electroporation treatment described in the next section. This procedure enabled precise deletions of 200 and 120 bp at the target sites in RvRbcS-like and RvRca-like, respectively, while simultaneously introducing stop codons at the resulting junctions (Supplementary Fig. 5). To generate the HA-tagged RvRbcS-like strain, an RNP targeting a single site on the 3′ side of the sequence encoding the fourth RbcS domain in the second exon was used (Supplementary Table 7). In this case, four partially overlapping ssODN fragments were introduced together with the RNPs, allowing for seamless in-frame integration of the HA tag sequence at the cleavage site through complementary base pairing (Supplementary Fig. 5).

For electroporation, R. viridis cells 1 week after inoculation with the prey Tetraselmis sp. were used. A total of 2 × 105 cells were resuspended in 50 μL of the CRISPR/Cas9 electroporation buffer (1/10-strength ASW (10% (v/v)) supplemented with 450 mM trehalose) containing 100 μM of each RNP and 200 μM of each ssODN, and electroporated using a GENE PULSER II system at 0.45 kV and 50 μF in a 0.2 cm gap cuvette. Immediately afterward, the cells were transferred to fresh IMK medium and incubated at 26°C in the dark for 24 h. The RNP-transfected R. viridis cells were then fed with an equal number of Tetraselmis sp. cells and further cultured for 72 h in a 14L:10D cycle at 26°C before being transferred to the standard culture condition at 20°C (primary culture). Clonal strains were generated from the primary culture by aseptically separating individual R. viridis cells into culture plates using a microcapillary method using glass tubes. The targeted genomic region of each clone was PCR-amplified and sequenced to identify successfully edited clones.

Immunoblotting
R. viridis cultures were harvested by centrifugation at designated time points following the kleptoplast acquisition and unialgal Tetraselmis sp. cultures, and the cell pellets were immediately snap frozen in liquid nitrogen. For protein extraction, the pellets were resuspended in lysis buffer containing 1 mM phenylmethanesulfonyl fluoride and 5 mM 6-aminocaproic acid, followed by sonication. The lysates were centrifuged at 20,000 × g for 1 min, and the resulting supernatants were mixed with 2× sodium dodecyl sulfate (SDS) sample buffer (500 mM Tris-HCl, pH 6.8, 10% [w/v] SDS, 0.01% [w/v] bromophenol blue, 10% [v/v] β-mercaptoethanol, 20% [v/v] glycerol). The samples were heat-denatured for 10 min at 90°C or 3 min at 100°C. Protein concentrations were determined by the Bradford assay (XL-Bradford KY-1040; Apro Science Inc., Tokushima, Japan). Equal amounts (3 μg) of total protein were loaded onto SDS-polyacrylamide gels (12.5%) and electrophoresed. The proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Merck Millipore, Burlington, MA, USA) by electroblotting. The membranes were probed with the following antibodies: rabbit polyclonal anti-RvRbcS-like (epitope: HSLAKAHRREGGET, amino acids [aa] 491–505; Eurofins Genomics KK, Tokyo, Japan), rabbit polyclonal anti-RvRca-like (epitope: AIASDDNVERFGKK, aa 181–189; Eurofins Genomics KK), rabbit polyclonal anti-TsRbcS (epitope: RISTALPIEKRSVA, aa 131–144; Eurofins Genomics KK), rabbit polyclonal anti-RbcL (#AS03-037; Agrisera, Vännäs, Sweden), and mouse monoclonal anti-HA (clone 16B12, #901513; BioLegend, San Diego, CA, USA). For reprobing, antibodies were stripped from the membranes using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific), followed by incubation with monoclonal anti-α-tubulin antibody produced in mouse (clone B-5-1-2, #T5168; Sigma-Aldrich). Immunoreactive bands were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse immunoglobulin [Ig]G[H+L] or anti-rabbit IgG[H+L]; Cell Signaling Technology, Danvers, MA, USA) and Immobilon western chemiluminescent HRP substrate (Merck KGaA, Darmstadt, Germany). Signals were visualized with an image analyzer (LAS-4000, GE Healthcare, Chicago, IL, USA) and a multi imager (MultiImager II ChemiBOX, BioTools, Gunma, Japan). The signal intensities were quantified using ImageJ software.

Immunofluorescence microscopy
Immunostaining of fixed cells was performed using mouse monoclonal anti-HA (clone 16B12, #901513; BioLegend) and rabbit polyclonal anti-RbcL (#AS03-037; Agrisera) antibodies to examine the colocalization of HA-tagged RvRbcS-like and RbcL, and with rabbit polyclonal anti-RvRca-like (epitope: AIASDDNVERFGKK, Eurofins Genomics KK) antibody to examine the localization of RvRca-like. Secondary antibodies included Alexa Fluor 555-conjugated anti-mouse IgG and Alexa Fluor 488-conjugated anti-rabbit IgG (Thermo Fisher Scientific). Images for colocalization analysis of HA-tagged RvRbcS-like and RbcL, as well as for RvRca-like localization, were acquired using a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany) equipped with a 100× oil-immersion objective lens (EC Plan-Neofluar 100×/1.3 Oil Ph3 M27, Zeiss). Alexa Fluor 488-labeled RbcL or RvRca-like was excited with a 488 nm laser, and fluorescence was recorded between 505 and 550 nm. Alexa Fluor 555-labeled HA-tagged RvRbcS-like was excited at 555 nm, and fluorescence was recorded between 560 and 615 nm. Chlorophyll autofluorescence was excited at 488 nm, and fluorescence above 650 nm was recorded. Z-stack images were captured at 0.4-μm intervals and processed by maximum intensity projection using ImageJ software.

Photosynthetic activity measurement
Wild-type and knockout strains of R. viridis (2 × 106 cells), as well as RNAi-knockdown cells, were harvested by gentle centrifugation from batch cultures at 8 days after initial inoculation and immediately resuspended in fresh IMK medium (2 mL). To avoid carbon limitation during measurement, 1 M NaHCO3 (10 μL) was added to the suspension. Oxygen evolution was then measured using a temperature-controlled liquid-phase oxygen electrode system (OXYT-1, Hansatech Instruments, Kings Lynn, UK).

Quantification of polysaccharide grains
Polysaccharide grains were extracted from R. viridis cells and purified using a previously described method. The resulting insoluble polysaccharide precipitate was dissolved in 1 M NaOH, and its concentration was determined using the phenol–sulfuric acid assay, with glucose solution as the standard reference.

NanoLuc reporter assay
Transformants expressing NanoLuc luciferase were generated using the procedure described by Nakazawa et al. For the construct lacking an NtLCD sequence, a DNA cassette was assembled consisting of the E. gracilis PNO promoter region (−118 to −1), the NanoLuc gene codon-optimized for E. gracilis (optNluc), and a G418 resistance gene (neor) fused downstream via a linker encoding the peptide GSSGAIA, followed by the Arabidopsis thaliana heat shock protein (AtHSP) terminator. For the construct containing the NtLCD of RvRbcS-like, the same expression cassette was used, with the NtLCD sequence inserted upstream of the optNluc gene. Both constructs were introduced into R. viridis cells using the electroporation protocol described by Nakazawa et al. Following electroporation, the cells were inoculated into Daigo IMK medium (15 mL), cultured for 10 days, and then transferred twice at weekly intervals. After complete consumption of Tetraselmis cells in the third culture, G418 was added at a final concentration of 10 μg/mL. Eight days later, a fourth transfer was performed, and the G418 concentration was increased to 20 μg/mL. Weekly transfers continued for 1 month, after which the clonal strains were isolated using glass microcapillaries and maintained in G418-free medium. To screen for successful transformants, 25 μL of the supernatant from the R. viridis lysates were assessed using a luciferase assay system (Nano-Glo, Promega, Madison, WI, USA) with a 20/20n Luminometer (Promega). Positive clones were further validated by cDNA PCR amplification of the inserted gene constructs. For luminescence imaging, the cells were briefly suspended in 100% methanol to immobilize them and increase substrate permeability, and then they were resuspended in IMK medium before imaging with a luminescence imaging system (LV200, Olympus).

Statistics and reproducibility
Statistics and reproducibility. No statistical method was used to predetermine sample size. No outliers were excluded from the analyses. For the time-series RNA-seq experiment, three biological replicates with the highest RNA yields were selected from five independent cultures for library preparation and sequencing. The experiments were not randomized. Investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present exiting work on Rapaza viridis, a unique marine euglenid that retains chloroplasts only by constant\nreplenishment of their organellar complement through kleptoplasty. The biology of this system is in istelf thrilling.\nCommendably, Kashiyama and colleagues have taken the investigation into this system to the next level by presenting\ngenetic studies on the joint action of the genetic compartmetns -- host nucleus and kleptoplast -- to maintain kleptoplast\nfunction in an essential process.\nThis is outstanding and the work is of extremely high quality. I just have a few remarks.\nThe interaction between plastid and nucleus is often considered as one of the otustanding features of streptophytes,\nespecially land plants; see e.g.:\nGoldbecker, E. S. & de Vries, J. Systems Biology of Streptophyte Cell Evolution. Annual Review of Plant Biology 76, 493-\n522 (2025). https://doi.org:10.1146/annurev-arplant-083123-060254\nThere are also some avenues in chloropytes:\nDuanmu, D. et al. Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival. Proc Natl Acad\nSci U S A 110, 3621-3626 (2013). https://doi.org:10.1073/pnas.1222375110\nI think unpacking this, summarizing what we know in streptophyte and chlorophytes in light of their evolution, and how the\nneeds of the kleptoplasts might be communicated to the host in this unique setting would make a very interesting additioanl\ndiscussion point.\nAdding a bit of a summary of which key genes are ecnoded in which compartment (e.g. to Figure 1) would help a lot making\nthis work more easily accessible.", "reviewer_2": "First, I would like to congratulate the authors on this very comprehensive and elegant piece of work that provides important\ninsights into the biology of kleptoplasts, providing clear evidence for the import of host-encoded proteins. The work\nbeautifully demonstrates that integration of kleptoplasts can evolve beyond the transient survival of the “stolen plastids”\nsupported solely by the proteins they bring along and highlights kleptoplasts as a possible intermediate on the route to\nacquisition of a novel complex plastid that is permanently retained in the “host” species.\nDespite my overall enthusiasm for the work, I have a number of issues (mostly minor) that need to be addressed before I can\nrecommend accepting the manuscript for publication.\nMajor issues\n• Fig. 3 c-e and Fig. 4e: Coomassie staining is generally considered incompatible with downstream Western blotting as it\nfixes the proteins in the gel. Nevertheless, the authors chose Coomassie staining of the gel as a loading control for Western\nblotting (Extended Data Fig. 1). The Methods section on Immunoblotting does not mention a Coomassie staining step. Thus,\nit is unclear what is shown in Extended Data Fig. 1. Are these the gels before transfer using a Western blot-compatible\nCoomassie staining protocol? If so, details have to be provided in the methods. Or are these parallelly loaded SDS-PAGE\ngels with the same samples. If so, this is not a suitable control for equal loading. Either a staining compatible with\ndownstream Western blot (such as TCE staining) or immune detection of a house-keeping protein on the same membrane is\nrequired. Since protein amounts are critical for the interpretation of the experiments (especially in Fig. 4e), proper loading\ncontrols are essential here.\n• Lines 112-117: “Phylogenetic analysis of the translated peptide sequences showed that […] about 20% [clustered] with\nTetraselmis spp.. However, none were identical or highly homologous to the current kleptoplast donor strain Tetraselmis sp.\nNIES-4478. This suggests any horizontal transfers of these important genes occurred in the distant past and the genes\nevolved as unique components of R. viridis.” This statement seems to be in contradiction with the information that de novo\ntranscriptome sequences were filtered against the genome data from Tetraselmis sp. and contigs with 97% identity were\nomitted” (lines 579-584). -> Since contigs identical or very similar to Tetraselmis were actively removed following\ntranscriptome assembly, it is not surprising that such transcripts are missing in the final dataset. An important question that\nshould be addressed to answer the question if recent gene transfers occurred would be if any of the transcripts identical or\nvery similar to Tetraselmis contain the host-specific SL at their 5’end.\n• An interesting question that is not addressed by the study concerns the extent of protein import into the kleptoplasts.\nCan the identified features in the different classes of targeting signals be used to bioinformatically predict further kleptoplast-\ntargeted proteins? Please explain why not or include this analysis in the revised version of the manuscript.\nMinor issues\n• Title and throughout the text: The expression “organelloid” is uncommon and appears inapt to describe a kleptoplast.\nThe term “organoid” (meaning organ-like) is commonly used for synthetic miniature models of organs that replace the actual\norgans in research. A kleptoplast, however, would rather resemble a transplanted organ, not a synthetic miniature replica.\nTherefore, I would recommend that the authors stay with the established term kleptoplast and specify that this kleptoplast is\ntransiently operated with the help of nucleus-encoded kleptoplast-targeted proteins.\n• Line 95: “insufficient to resolve the intricacies of these unique systems” -> This is a rather vague statement. Please explain\nprecisely for what the predictions are insufficient for? E.g., insufficient to prove protein import and unravel its mechanistic\nunderpinnings.\n• Line 105-108: These transcripts were homologous to those found in the chloroplast-targeted proteins of photosynthetic\neukaryotes based on the time-series transcriptome data (Supplementary Data) from four distinct kleptoplastic stages (Fig. 1),\nwhose expression levels were comparable to key mitochondrial metabolic genes (Extended Data Table 1, Fig. 2a). ->\nAwkward, long sentence. I can only guess its meaning. Please rephrase.\n• Line 167: Replace “Fig. 3h” and “Fig. 3i” by “Fig. 3g”\n• Lines 217-219: “Based on the results suggesting that the NtLCDs of RvRbcS-like and RvRca-like act as translocation\nsignals and are cleaved upon maturation (Fig. 3a, Extended Data Table 2)” -> Incomplete sentence and unclear how\nExtended Data Table 2 supports this statement. Correct citation?\n• Line 226: Change reference to Extended Data Table 2 to Extended Data Table 3.\n• Line 251: Term “structural recruitment” is not clear.\n• Line 307: Expression “permanent transient kleptoplasts” is contradictory in itself. Please rephrase.\n• Lines 341-344: “R. viridis remains highly photosynthetic for approximately 2 weeks, remaining virtually autotrophic through\na 1-week growth phase and a 1-week stationary phase. This is supported by sustained growth without external inorganic\nnitrogen9, culture decline in the absence of light8, and cytosolic polysaccharide accumulation in stationary phase cells8,9.” -\n> It is unclear why independence of inorganic nitrogen supports high photosynthetic activity or autotrophy. Please rephrase\nor explain.\n• Line 621: Please provide database name along with accession numbers.\n• Lines 660-668: The description of the RNAi procedure is not very clear. Please rephrase the whole section. Are 15 µg\ndsRNA introduced in total or 15 µg per electroporation? When was the electroporation procedure repeated? 2 d after the first\nround? When was the experiment evaluated? 3 d after the second electroporation round? I have to guess these parameters.\nThey are not very clear from the text. What is the source of the seawater? Does 5% seawater mean 5% (v/v) seawater in\ndH2O or does 5% indicate the salinity of the seawater?\n• Line 672: Maruyama et al is not reference 8. Please correct.\n• Line 681: …using previously described electroporation conditions8. Please check if 8 is the correct reference or if it should\nbe 9.\n• Lines 670-688: For the CRISPR/Cas9 genome editing section, information on how cultures were handled following\nelectroporation is missing. Please include a detailed description of procedures and timing between electroporation and\nanalysis of the generated cells.\n• Fig. 1: What are the large light grey structures in cells in the declining state?\n• Fig. 3 g: I assume, the labelling of  RvRca and Wt are inverted.\n• Fig. 4d: Scale bars are required for the microscopic images shown.\n• Fig. 6: Cys is listed as polar as well as non-polar amino acid. The numbers in the figure legend don’t fit to the main text and\nthe proteins shown in the figure. Legend: “Of 29 sequences examined, 27 exhibited extended NtLCDs”. Figure and main\ntext: 37 proteins analyzed in total, 35 with classifiable NtLCDs. Furthermore, the actual alignment underlying the NtLCD\nanalysis shown in Fig. 6 should be provided in the supplemental material.\n• Extended Data Table 3: Define “unread gap”.\n• Extended Data Table 4: Definition required for the numbers provided in brackets behind the “Yes” and “Possible” entries\nunder “Phylogenetic analysis” (bootstrap support, I assume(?)).\n• Extended Data Fig. 4: Legend doesn’t fit to figure. The described panel a is missing. What are orange and blue in panel b?", "reviewer_3": "This study by Kashiyama et al. is logical and technically sound. Controls are properly employed, and the results support the\nmain experimental claims. The main contribution of this study is the experimental confirmation of the targeting and\ntranslocation of two functionally important kleptoplast-localized nucleus-encoded proteins in Rapaza viridis. This is an\nimportant technical advance and is much welcomed.\nFrom a biological and physiological perspective, neither the questions nor the results are novel or unexpected. The proteins\nchosen are expected to be targeted to the kleptoplasts given an abundance of circumstantial evidence provided in previous\nstudies. The study is technically impressive, but the technical advancements (i.e., knock-down, CRISPR-Cas9) had\npreviously been developed and reported by Maruyama et al.\nIn my opinion, the framing of the manuscript is misleading. The title is rather cryptic and overhyped. These concerns are\nmostly restricted to the title and the introduction. Unnecessary neologism such as “xenogeneic organelloids” are introduced\nto presumably exaggerate the importance of this contribution.\nThe statement in L319-323 is somewhat confusing and not well-informed based on evolutionary theory. Extant living species\ncannot be considered evolutionary intermediates, but there are characters that can be considered as such. Species express\ncombination of characters that can be considered ancestral or derived. In this way, R. viridis is informative as it retains\nsystems that may have been ancestral to photosynthetic euglenids and may therefore inform the evolutionary origin of\nphototrophy in this group."}, "author_response": "RESPONSES TO REVIEWER COMMENTS\nReviewer #1:\nThe authors present exiting work on Rapaza viridis, a unique marine euglenid that retains chloroplasts only by\nconstant replenishment of their organellar complement through kleptoplasty. The biology of this system is in itself\nthrilling. Commendably, Kashiyama and colleagues have taken the investigation into this system to the next level\nby presenting genetic studies on the joint action of the genetic compartments -- host nucleus and kleptoplast -- to\nmaintain kleptoplast function in an essential process.\nWe are honored by your kind words. We are pleased that you have taken an interest in this study.\nThis is outstanding and the work is of extremely high quality. I just have a few remarks.\n The interaction between plastid and nucleus is often considered as one of the outstanding features of\nstreptophytes, especially land plants; see e.g.:\nGoldbecker, E. S. & de Vries, J. Systems Biology of Streptophyte Cell Evolution. Annual Review of Plant Biology 76,\n493-522 (2025). https://doi.org:10.1146/annurev-arplant-083123-060254\nThere are also some avenues in chlorophytes:\nDuanmu, D. et al. Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival. Proc Natl\nAcad Sci U S A 110, 3621-3626 (2013). https://doi.org:10.1073/pnas.1222375110\nI think unpacking this, summarizing what we know in streptophyte and chlorophytes in light of their evolution,\nand how the needs of the kleptoplasts might be communicated to the host in this unique setting would make\na very interesting additional discussion point.\nYour comment was refreshing and drew attention to aspects we had overlooked. The papers you recommended,\nespecially the first one, offer stimulating reviews with important implications for evolutionary thinking.\nIn fact, most kleptoplasty studies, including ours, have mainly focused on direct structural control by\nnucleus-encoded proteins acting on kleptoplasts but not much about communications. Regarding Rapaza\nviridis, two issues would be central: (1) anterograde control by the nucleus over transcription and translation\nof the kleptoplast genome and (2) whether retrograde signals from the chloroplast are actively received and\nutilized by the nucleus, potentially through factors in the R. viridis cytosol or the outermost envelope (i.e., the\nphagosomal membrane). Such control is crucial for permanent integration, particularly in the presence of\nreactive oxygen species.\nHowever, we consider it premature to address these topics here. First, our comparative transcriptomics\nwere not designed around light–dark cycles that critically affect photosynthetic physiology. All samples were\ncollected during the light phase in order to exclude diurnal effects but to isolate kleptoplast maturation. Thus,\nour data are not suited to detect diurnally varying nucleus-chloroplast communications. Second, Tetraselmis\n(Chlorodendrophyceae) is phylogenetically distant from the commonly used chlorophyte model,\nChlamydomonas (Chlorophyceae). Simple extrapolation of retrograde signaling of the chlorophyte would not\nbe very appropriate. For example, Tetraselmis possesses phytochromes, analogous to those found in\nstreptophytes, and therefore likely employs signaling pathways distinct from those of chlorophytes.\nnot detect indicative factors of such a communication among the highly expressed genes we analyzed. At\nmoderate expression levels, however, we find suggestive candidates, such as a putative kleptoplast-targeted\nheme oxygenase and sequences compatible with chloroplast σ factors. Selectively including these candidates\nwould, however, blur the focus of the current manuscript.\nFor these reasons, we have decided to only briefly touch on bidirectional communication between the\nkleptoplast and the R. viridis nucleus as a topic for future research. Accordingly, we have inserted the following\nsentence into the latter half of the final paragraph (P14-15; L324-327) as a proposal for future research.\nBeyond the direct structural manipulation by host-supplied proteins, the interaction\nmay involve precise anterograde and retrograde signalling between the host nucleus and\nthe organelle51-53, which would be essential for regulating photosynthesis to prevent the\nxenogeneic object from merely becoming a source of reactive oxygen species.\n Adding a bit of a summary of which key genes are encoded in which compartment (e.g. to Figure 1) would\nhelp a lot making this work more easily accessible.\nWe found this to be a very good suggestion. We have now added a Venn diagram showing the genes encoding\nsubunits of key photosynthetic complexes in Fig. 1e (P16).\nReviewer #2 (Remarks to the Author):\nFirst, I would like to congratulate the authors on this very comprehensive and elegant piece of work that provides\nimportant insights into the biology of kleptoplasts, providing clear evidence for the import of host-encoded proteins.\nThe work beautifully demonstrates that integration of kleptoplasts can evolve beyond the transient survival of the\n“stolen plastids” supported solely by the proteins they bring along and highlights kleptoplasts as a possible\nintermediate on the route to acquisition of a novel complex plastid that is permanently retained in the “host” species.\nDespite my overall enthusiasm for the work, I have a number of issues (mostly minor) that need to be\naddressed before I can recommend accepting the manuscript for publication.\nMajor issues\n Fig. 3 c-e and Fig. 4e: Coomassie staining is generally considered incompatible with downstream Western\nblotting as it fixes the proteins in the gel. Nevertheless, the authors chose Coomassie staining of the gel as a\nloading control for Western blotting (Extended Data Fig. 1). The Methods section on Immunoblotting does not\nmention a Coomassie staining step. Thus, it is unclear what is shown in Extended Data Fig. 1. Are these the\ngels before transfer using a Western blot-compatible Coomassie staining protocol? If so, details have to be\nprovided in the methods. Or are these parallelly loaded SDS-PAGE gels with the same samples. If so, this is\nnot a suitable control for equal loading. Either a staining compatible with downstream Western blot (such as\nTCE staining) or immune detection of a house-keeping protein on the same membrane is required. Since\nprotein amounts are critical for the interpretation of the experiments (especially in Fig. 4e), proper loading\ncontrols are essential here.\nsame volumes of identical cell lysates for each sample and electrophoresed simultaneously in the same chamber.\nWe understand that equal loading should be particularly critical for the Western blot analyses shown in Fig. 4e\nand thus decided to take your comment seriously there. Following your suggestion, we prepared another set of\nparallel time-course samples and repeated the entire experiment. This included additional immunoblotting\nusing an anti-α-tubulin antibody to verify protein loading in each lane.\nBecause our samples contain two distinct organisms, the alga Tetraselmis and the flagellate R. viridis,\nas well as representing different cellular stages of the latter, we think the minor variation observed among the\ntubulin signal observed across the series was an understandable consequence. Indeed, this occurred even\nthough we estimated equal protein masses from each sample tube. Importantly, the new results with the α-\ntubulin signals reinforce our previous interpretation. In particular, in the ΔRvRbcS-like time series samples,\nthe\nTsRbcS and RbcL signals decreased on days 4 and 7, while the α-tubulin signal increased slightly.\nAccordingly, we replaced Fig. 4e with the new data (P19). In addition, we conducted two more\nimmunoblotting experiments as independent triplicates, starting from separate culture batches, and obtained\ndensitometry data (n = 3), including the blot shown in Fig. 4e. Using these data, we updated Fig. 4f. (P19).\nDuring this process, we discovered an error in the previous Excel-based calculations, which had inadvertently\nexaggerated the apparent decrease in TsRbcS levels. The newly generated plots, prepared with greater care,\nsupport what we consider to be the appropriate trend. In the new Fig. 4f, we also present the time-series changes\nin RbcL. Corresponding revisions have been made to the main text (P9; L195–202), as well as to the figure\ncaption and Methods section.\n Lines 112-117: “Phylogenetic analysis of the translated peptide sequences showed that […] about 20%\n[clustered] with Tetraselmis spp.. However, none were identical or highly homologous to the current\nkleptoplast donor strain Tetraselmis sp. NIES-4478. This suggests any horizontal transfers of these important\ngenes occurred in the distant past and the genes evolved as unique components of R. viridis.” This statement\nseems to be in contradiction with the information that de novo transcriptome sequences were filtered against\nthe genome data from Tetraselmis sp. and contigs with 97% identity were omitted” (lines 579-584). -> Since\ncontigs identical or very similar to Tetraselmis were actively removed following transcriptome assembly, it is\nnot surprising that such transcripts are missing in the final dataset. An important question that should be\naddressed to answer the question if recent gene transfers occurred would be if any of the transcripts identical\nor very similar to Tetraselmis contain the host-specific SL at their 5’end.\nFirst, we discussed in the paragraph you mentioned regarding exclusively on the 37 highly expressed genes.\nSo, what we meant was that none of these apparently important genes are of recent horizontal gene transfer\nfrom the donor Tetraselmis. Thus, we slightly modified the phrase of the main text to clarify our intent (P5;\nL113).\nMoreover, following your comment, we actually examined whether any sequences matching Tetraselmis\nat over 97% similarity (i.e., those excluded from the reference dataset) contained Euglenid-type spliced leader\nsequences at their ends. We found none. This information has now been added to the text in Methods section.\n An interesting question that is not addressed by the study concerns the extent of protein import into the\nkleptoplasts. Can the identified features in the different classes of targeting signals be used to bioinformatically\n3\npredict further kleptoplast-targeted proteins? Please explain why not or include this analysis in the revised\nversion of the manuscript.\nIn our earlier paper (Karnkowska et al., 2023, PNAS), we extrapolated the features of the Euglena gracilis\nchloroplast-targeting signal to search for sequences in Rapaza viridis that might be directed to kleptoplasts and\ndiscussed these candidates. At that stage, however, kleptoplast targeting remained purely hypothetical: there\nwas no experimental evidence and no quantitative transcript data. In the present study, therefore, we conducted\nbiochemical experiments supported by genetic manipulations to demonstrate that at least two of these\ncandidates are indeed transported to kleptoplasts. Importantly, the candidates in this study were not selected\nsimply by checking for “signal-like” motifs. Instead, we first identified chloroplast-related genes that are highly\nexpressed in R. viridis despite its lack of a bona fide chloroplast (the kleptoplasty itself having been confirmed\nin the above paper), and then biochemically verified that their products are actually transported. We\nsubsequently analyzed in detail the N-termini of other highly expressed chloroplast-related genes (a point not\nexamined in depth in our previous study) and found that, in the majority of cases, these sequences share features\nwith, although are not identical to, the E. gracilis chloroplast-targeting signal. Thus, this study represents the\nfirst deductive approach to identify and evaluate kleptoplast-targeting signals.\nWhile we agree that a more comprehensive and higher-precision bioinformatic search for kleptoplast-\ntargeting signals beyond our 2023 study may become feasible in the future, we consider it premature to make\nstrong claims at this stage. One reason is that no concrete information is yet available regarding the\ntranslocation machinery. Speculation based solely on sequence similarity is undesirable in the present situation,\nand we should bear in mind that the mechanism remains unresolved even in E. gracilis. Moreover, in 2023 we\nnecessarily adopted an empirical strategy for identifying E. gracilis chloroplast-targeted proteins; that is, by\ncombining an existing signal-peptide prediction model (notably, only the older version worked adequately,\nwhile newer versions did not) with an existing transit-peptide prediction model, both of which were exclusively\ndesigned for green plants. In contrast, the present study enables us to demonstrate kleptoplast-targeting\nsequence function deductively. We therefore consider the next logical step to be a comprehensive analysis of\nthe proteins that are actually transported and the characterization of their N-terminal architectures, which may\nultimately provide a more reliable, empirically grounded prediction model, even without a full understanding\nof the underlying machinery.\nGene-by-gene validation using genetic manipulations, as performed here, would be quite challenging,\nespecially for lower-expression candidates. We are therefore currently isolating and purifying mature\nkleptoplasts for proteomic analyses to conclusively distinguish the R. viridis-derived proteins localized inside\nthe kleptoplast from those that are not transported into it. We believe these analyses will provide much more\nreliable bioinformatic predictions. However, these ongoing results are beyond the scope of the present\nmanuscript, and that is precisely why we wish to refrain from discussing bioinformatic predictions at this time.\nMinor issues\n Title and throughout the text: The expression “organelloid” is uncommon and appears inapt to describe a\nkleptoplast. The term “organoid” (meaning organ-like) is commonly used for synthetic miniature models of\norgans that replace the actual organs in research. A kleptoplast, however, would rather resemble a transplanted\norgan, not a synthetic miniature replica. Therefore, I would recommend that the authors stay with the\nestablished term kleptoplast and specify that this kleptoplast is transiently operated with the help of nucleus-\nencoded kleptoplast-targeted proteins.\nanticipate that the category of biological phenomena termed “kleptoplasty” will need to be reevaluated in the\nnear future. Phenomena traditionally referred to as kleptoplasty are rather heterogeneous, ranging from systems\nthat transiently maintain only chloroplasts with an uncertain dependence on photosynthesis to systems that\nretain the entire algal cytoplasm. Adding R. viridis to this category increases its cell biological diversity even\nfurther. Most importantly, it is highly questionable whether these phenomena are evolutionarily homologous\nor even lie on the same evolutionary spectrum.\nSecond, we believe that the importance of R. viridis as a new experimental model lies in elucidating the\nunderlying (and likely universal) cellular mechanisms that enabled this phenomenon to evolve. For this reason,\nwe were keen to avoid using the term “kleptoplast” in the title, as it might suggest to readers that it is a special\nor idiosyncratic phenomenon.\nHowever, we understand your point: the term \"organelloid\" may also evoke something artificial.\nFurthermore, our frequent use of “kleptoplast(y)” in the main text makes our terminology self-contradictory.\nWe propose replacing “xenogeneic organelloid” with “xenogeneic organelle” wherever it appears (including\nin the title). We believe this term is not problematic and remains consistent with our intention to frame the\nphenomenon within the broader context of phagocytic interactions, as suggested in the Discussion.\n Line 95: “insufficient to resolve the intricacies of these unique systems” -> This is a rather vague statement.\nPlease explain precisely for what the predictions are insufficient for? E.g., insufficient to prove protein\nimport and unravel its mechanistic underpinnings.\nFollowing your suggestion, we have revised the wording to be more specific as shown below (P4-5: L94-96).\nHowever, in silico predictions alone are insufficient to prove protein import and elucidate\nits mechanistic underpinnings, highlighting the need for cellular-level biochemical\ninvestigations9.\n Line 105-108: These transcripts were homologous to those found in the chloroplast-targeted proteins of\nphotosynthetic eukaryotes based on the time-series transcriptome data (Supplementary Data) from four distinct\nkleptoplastic stages (Fig. 1), whose expression levels were comparable to key mitochondrial metabolic genes\n(Extended Data Table 1, Fig. 2a). -> Awkward, long sentence. I can only guess its meaning. Please rephrase.\nTogether with the previous sentence, we rephrased as follows (P5: L104-107).\nFrom the time-series transcriptome data taken from four distinct kleptoplastic stages (Fig.\n1; Supplementary Data), we identified 37 R. viridis transcripts encoding proteins with\nconserved domains indicative of chloroplastic functions37. The expression levels of these\ntranscripts were comparable to those of key mitochondrial metabolic genes (Extended\nData Table 1; Fig. 2a).\n Line 167: Replace “Fig. 3h” and “Fig. 3i” by “Fig. 3g”\n Lines 217-219: “Based on the results suggesting that the NtLCDs of RvRbcS-like and RvRca-like act as\ntranslocation signals and are cleaved upon maturation (Fig. 3a, Extended Data Table 2)” -> Incomplete\nsentence and unclear how Extended Data Table 2 supports this statement. Correct citation?\nThat was indeed an incorrect citation; it should be “(Fig. 3c, Extended Data Fig. 2)” instead. In addition, we\nhave made minor revisions to the wording to clarify the intent of the sentence (P10: L215-217).\n Line 226: Change reference to Extended Data Table 2 to Extended Data Table 3.\nCorrected (P10: L224).\n Line 251: Term “structural recruitment” is not clear.\nRephased as “structural incorporation” (P11: L249).\n Line 307: Expression “permanent transient kleptoplasts” is contradictory in itself. Please rephrase.\nRemoved “permanent” (P14: L305). It was an error.\n Lines 341-344: “R. viridis remains highly photosynthetic for approximately 2 weeks, remaining virtually\nautotrophic through a 1-week growth phase and a 1-week stationary phase. This is supported by sustained\ngrowth without external inorganic nitrogen9, culture decline in the absence of light8, and cytosolic\npolysaccharide accumulation in stationary phase cells8,9.” -> It is unclear why independence of inorganic\nnitrogen supports high photosynthetic activity or autotrophy. Please rephrase or explain.\nIt was another error; “sustained” should have been “suspended”. To make this clearer, we have rephrased the\nsentence as follows (P16: L343-345):\nThis autotrophy is supported by suspended growth without external inorganic nitrogen9,\nculture decline in the absence of light8, and cytosolic polysaccharide accumulation in\nstationary phase cells8,9.\n Line 621: Please provide database name along with accession numbers.\nThese sequences are from the DDBJ database, and we have added this information to the manuscript (P31:\nL630).\n6\n Lines 660-668: The description of the RNAi procedure is not very clear. Please rephrase the whole section.\nAre 15 µg dsRNA introduced in total or 15 µg per electroporation? When was the electroporation procedure\nrepeated? 2 d after the first round? When was the experiment evaluated? 3 d after the second electroporation\nround? I have to guess these parameters. They are not very clear from the text. What is the source of the\nseawater? Does 5% seawater mean 5% (v/v) seawater in dH2O or does 5% indicate the salinity of the seawater?\nWe have rewritten the entire “RNAi” section. Please refer to the revised manuscript (P32-33: L664-679). They\nmust be clear now. Also, we clarified the timing of the observations after these electroporation experiments in\nthe caption of Extended Data Fig. 4.\n Line 672: Maruyama et al is not reference 8. Please correct.\nCorrected to 9 (P33; L666).\n Line 681: …using previously described electroporation conditions8. Please check if 8 is the correct reference\nor if it should be 9.\nCorrected to 9 (P33; L682).\n Lines 670-688: For the CRISPR/Cas9 genome editing section, information on how cultures were handled\nfollowing electroporation is missing. Please include a detailed description of procedures and timing between\nelectroporation and analysis of the generated cells.\nWe have rewritten the entire “CRISPR/Cas9 genome editing” section (P33-34; L680-708). Please refer to the\nrevised manuscript. In this case, however, the “timing between electroporation” is not relevant, unlike in the\nRNAi experiments, because the genotypes of the edited clones are permanently modified.\n Fig. 1: What are the large light grey structures in cells in the declining state?\nThey are vacuoles. To make this clearer, we have modified the caption (P16; L345-347) as follows:\nd, In the absence of new kleptoplast acquisition, vacuoles begin to form after 3 weeks and\ngradually expand to occupy most of the cell. The cells ultimately die after 4‒5 weeks\n(declining stage).\n Fig. 3 g: I assume, the labelling of ΔRvRca and Wt are inverted.\nIt was indeed an error (inverted). We corrected in the revised figure (P18).\n7\n Fig. 4d: Scale bars are required for the microscopic images shown.\nWe added a scale bar to the lower right panel and included the scale information in the figure caption (P19).\n Fig. 6: Cys is listed as polar as well as non-polar amino acid. The numbers in the figure legend don’t fit to the\nmain text and the proteins shown in the figure. Legend: “Of 29 sequences examined, 27 exhibited extended\nNtLCDs”. Figure and main text: 37 proteins analyzed in total, 35 with classifiable NtLCDs. Furthermore, the\nactual alignment underlying the NtLCD analysis shown in Fig. 6 should be provided in the supplemental\nmaterial.\nFirst, Cys was treated as a polar amino acid in the calculation, and the caption has been corrected accordingly\n(P21; L426). The numerical values in the figure legend were erroneous and have now been revised (P21; L422-\n423). The actual alignments underlying the NtLCD analyses in Fig. 6b and 6c are now provided in the\nSupplementary Data65. The availability of the raw data has also been noted in the figure caption (P21; L431-\n432).\n Extended Data Table 3: Define “unread gap”.\nWe have added text explaining the definition of “unread gap” in the caption of Extended Data Table 3 as\nfollows.\n“Unread gap” indicates the number of regions within each RNA-seq contig for which no\ncorresponding sequence was found in the DNA-seq data and whose presence has not been\nexperimentally verified by Sanger sequencing.\n Extended Data Table 4: Definition required for the numbers provided in brackets behind the “Yes” and\n“Possible” entries under “Phylogenetic analysis” (bootstrap support, I assume(?)).\nThe caption of Extended Data Table 4 has been revised as follows to include definitions of the terms used.\nExtended Data Table 4 | Putative kleptoplast genes compared with the previous\ntranscriptome analysis by Karnkowska et al.8 and a brief summary of the molecular\nphylogenetic analysis for each translated peptide. For each R. viridis sequence,\nwhether it forms a monophyletic group with Euglenophyceae or Tetraselmis spp. is\nindicated; bootstrap values for such nodes are shown in parentheses (Yes: >90.0; Possible:\n>75.0). Grouping with other lineages is labeled “No,” and cases lacking sufficient\nbootstrap support are labeled “Poorly resolved.” Full phylogenetic trees for each gene\nproduct are available at Figshare65.\n8\n Extended Data Fig. 4: Legend doesn’t fit to figure. The described panel a is missing. What are orange and blue\nin panel b?\nThe caption of Extended Data Fig. 4 has been revised as follows. Please also note that we re-examined the\noriginal primary data obtained by the student co-author, and the plot has been updated accordingly.\nExtended Data Fig. 4 | RNA interference (RNAi) experiments targeting RvRbcS-like.\na, qPCR measurements showing time-series changes in RvRbcS-like expression levels,\nnormalized to those of the α-tubulin gene, of three independent experiments. b, Net\noxygen evolution rates in wild-type R. viridis cells (blue) and RNAi-treated cells (orange)\nmeasured on days 8, 12, and 16 after feeding on Tetraselmis sp., corresponding to 9, 13,\nand 17 days after the second electroporation introducing dsRNA into the cells.\nThis study by Kashiyama et al. is logical and technically sound. Controls are properly employed, and the results\nsupport the main experimental claims. The main contribution of this study is the experimental confirmation of the\ntargeting and translocation of two functionally important kleptoplast-localized nucleus-encoded proteins in Rapaza\nviridis. This is an important technical advance and is much welcomed.\nFrom a biological and physiological perspective, neither the questions nor the results are novel or unexpected.\nThe proteins chosen are expected to be targeted to the kleptoplasts given an abundance of circumstantial evidence\nprovided in previous studies.\nFor us, the primary purpose of this paper is to introduce Rapaza viridis as an experimental organism that anyone\ncan study, one that does not rely on “circumstantial evidence” with respect to the early evolution of organelles.\nOur previous report (Karnkowska et al., 2023, PNAS) was, we believe, novel and important for biological\nscience, but it had major drawbacks that could not be addressed at that time. For example, it did not allow\nquantitative assessment of gene expression levels from the RNA-seq data.\nBy generating new quantitative datasets that are independent of the previous report, we now know which\ngenes are truly significant and which are not. In fact, many of the genes turned out to be expressed only at low\nlevels, whereas others are expressed at very high levels. One important, if somewhat cryptic, message of this\npaper is that in silico approaches to poorly understood biological phenomena, when based on limited or\nsuperficial data (although often the only option for organisms that are difficult to culture), require very careful\ninterpretation. This was precisely why, in our previous paper, we deliberately restricted our discussion of the\ncDNA data to horizontal gene transfer and carefully avoided speculating about their actual functions.\nAt least in our view, there is no “circumstantial evidence provided in previous studies” that could\nconvincingly support the functional significance of such genes, because none of the kleptoplastic organisms or\nphotoendosymbiotic systems studied before R. viridis had provided biochemical, and therefore conclusive,\nevidence that endosymbionts or kleptoplasts are molecularly modified by host genes. Although we\nacknowledge that we ourselves previously provided substantial but still circumstantial evidence for targeting,\nthe present paper offers the first direct evidence of actual targeting.\nImportantly, the significance of this study goes beyond demonstrating that nucleus-encoded proteins are\ntransported to kleptoplasts and function there, as had been anticipated. Our data further show that the\nkleptoplast interior is actively remodeled by the host through the formation of a protein complex that differs\nfrom its original state, exemplified by the reconstituted RuBisCO complex. This indicates that the kleptoplast\nis not merely maintained but is molecularly reconfigured by host-derived components.\nWe are confident that this study will serve as a landmark, moving research beyond the predominantly in\nsilico-based cell–cell interaction studies that have accumulated so far and advancing it to biochemical-level\nanalyses that cannot simply be extrapolated from preexisting, minimalistic model organisms.\nThe study is technically impressive, but the technical advancements (i.e., knock-down, CRISPR-Cas9) had\npreviously been developed and reported by Maruyama et al.\nWe acknowledge that the technical advancement in genetic manipulation compared with our previous paper\n(Maruyama et al., 2023, PCP) may be minor, but this should not diminish the value of the present manuscript.\nWe believe that this study more effectively highlights the strong appeal of R. viridis as an organism that\n10\nresearchers will be motivated to study through biochemical and molecular-physiological investigations\nsupported by genetic manipulation.\nIn my opinion, the framing of the manuscript is misleading. The title is rather cryptic and overhyped. These\nconcerns are mostly restricted to the title and the introduction. Unnecessary neologism such as “xenogeneic\norganelloids” are introduced to presumably exaggerate the importance of this contribution.\nRegarding the title, your point may well be justified. In particular, on reflection we felt that the term\n“organelloid” could be misleading, and we have therefore reverted to “organelle”. What we wished to convey\nwith the original title was that R. viridis, which has finally become available as an experimental organism,\nshould not be confined to the narrow context of interest in kleptoplasty alone. As we also state in the Discussion,\nthe importance of being able to study such an experimental organism lies in the possibility of addressing more\ngeneral scientific questions, such as which combinations of molecular mechanisms present in ancestral\norganisms underlie innovations like kleptoplasty.\nTherefore, we intentionally avoided strongly foregrounding the term and concept of “kleptoplasty” as a\nwell-known phenomenon in the first half of the paper, so that even readers who are unfamiliar with, or not\nparticularly interested in, kleptoplasty would be encouraged to read the manuscript in depth.\nThe statement in L319-323 is somewhat confusing and not well-informed based on evolutionary theory.\nExtant living species cannot be considered evolutionary intermediates, but there are characters that can be\nconsidered as such. Species express combination of characters that can be considered ancestral or derived. In this\nway, R. viridis is informative as it retains systems that may have been ancestral to photosynthetic euglenids and\nmay therefore inform the evolutionary origin of phototrophy in this group.\nFirst, we apologize for having used wording that could lead to misunderstandings for readers. The intention\nbehind this phrasing was essentially that we did not want R. viridis to become widely framed in the public\nsphere as an organism “on its way to acquiring chloroplasts,” which would, in a sense, put it into a quasi-\npseudoscientific context. This concern was partially because this is a broad-audience journal that will also be\nread by people who are not very familiar with evolution.\nWe believe we understand your point.\nOne important issue, in my view, is that we cannot rule out the possibility that many of the elements\ninvolved in the kleptoplastic phenomenon of R. viridis are in fact “derived” characters. While we do think\nphagocytosis itself is an ancestral character (rather than something that was re-acquired), we cannot completely\nexclude the possibility that a cell that once possessed a permanent chloroplast through some other process later\nlost it and transitioned to a kleptoplastic state. Personally, I consider this less likely, but there are so many\nchloroplast-related genes, including many that are barely expressed, that one is tempted to suspect such a\nscenario, and it is also puzzling that the Tetraselmis nucleus is excluded from the outset. Also, taking this into\naccount, we think we should keep in mind the possibility that, in the future, “kleptoplasty” will no longer be\ntreated as a single homologous phenomenon grouped under one umbrella.\nIn any case, with the data currently available to us, it is not possible to discuss the common ancestor at a\nfiner resolution, as we also noted in the earlier part of the relevant paragraph. Essentially, we need to wait for\nthe further exploration and discovery of lineages that are sister to Rapaza and Euglenophyceae.\nportion you indicated and revised the text (P14; L317-324) as follows.\nAll organisms exist in evolutionarily transient states, shaped by continuous\nevolutionary refinements as they adapt to ever-changing conditions to ensure their\nsurvival through successive generations, and R. viridis is no exception to this. This species\ncannot be definitively placed on a trajectory toward acquiring chloroplasts. Nevertheless,\nR. viridis represents a new example of the unexplored ecophysiological potential of\neukaryotic cells. Thus, the introduction of R. viridis as an experimental model phototroph\nthat may be amenable to genetic engineering, alongside other kleptoplastic phototrophs\nthat have been and will be studied, will expand our understanding of the molecular\nmechanisms by which eukaryotes interact with the bacterium-derived photosynthetic\nmachinery.\n12"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my remarks to my fullest satisfaction. I do not have any further comments and congratulate\nthem on a landmark paper.", "reviewer_2": "The revised version of the manuscript has improved substantially, and the authors are to be congratulated on the new\nversion. Almost all of this reviewer’s previous criticisms have been addressed sufficiently. However, the terms “5% seawater”\nand “10% seawater” remain unclear and should be specified more precisely as requested previously.", "reviewer_3": "I stand by my previous comments, and I am not persuaded by the author's responses. I leave it to the editor to make a\ndecision based on the significance and novelty of the paper for the journal."}, "author_response": "RESPONSES TO REVIEWER COMMENTS\nReviewer #2:\nThe revised version of the manuscript has improved substantially, and the authors are to be congratulated on the\nnew version. Almost all of this reviewer’s previous criticisms have been addressed sufficiently. However, the terms\n“5% seawater” and “10% seawater” remain unclear and should be specified more precisely as requested previously.\nWe apologize for the lack of clarity regarding the “5%/10% seawater” conditions. We have now explicitly\ndefined these electroporation conditions in the Methods as 1/20-strength ASW (5% (v/v)) or 1/10-strength\nASW (10% (v/v)) supplemented with trehalose at the indicated final concentrations. Importantly, the RNAi\nelectroporation buffer is unchanged from Mariyama et al. (2023) (1/20-strength ASW with 500 mM trehalose).\nIn contrast, the CRISPR/Cas9 genome-editing workflow has since been optimized and currently uses a\ndifferent electroporation buffer (1/10-strength ASW with 450 mM trehalose). We have revised the manuscript\nto make this method-specific difference explicit and to avoid any impression of a typographical inconsistency.\n1"}]

Decision letter