ABSTRACTRNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next‐generation sequencing (NGS) approaches, such as rRNA‐depleted long non‐coding (lnc) RNA‐seq or targeted amplicon‐seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, I developed a rapid and cost‐effective long‐read sequencing approach, termed Target‐Indexed‐PCR (TIP) sequencing, for digital quantification of RNA editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high‐fidelity PCR amplification with Oxford Nanopore sequencing and custom in‐house Perl and Python scripts for streamlined data processing, including barcode‐based demultiplexing, strand reorientation, alignment to a pseudo‐genome, manual editing‐site inspection, and splicing variant identification. As a proof of concept, TIP sequencing was applied to ndhB and ndhD transcripts, two chloroplast NAD(P)H dehydrogenase genes with the highest number of known editing sites in Arabidopsis thaliana. These transcripts were analyzed both in an inducible CRISPR interference (iCRISPRi) system targeting MORF2, a key RNA‐editing factor, and in MORF2‐YFP transgenic lines with either overexpression or co‐suppression silencing. My findings revealed dose‐ and development‐dependent impacts of MORF2 on C‐to‐U editing efficiency. Moreover, I identified an accumulation of intron‐retaining ndhB transcripts, specifically in Dex‐treated iCRISPRi lines and in both MORF2‐YFP overexpression and silencing rosette leaves, indicating impaired chloroplast splicing functions when MORF2 expression is perturbed beyond an as‐yet‐undefined threshold. The platform achieves single‐molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA‐seq. Collectively, this study establishes TIP sequencing as a versatile, scalable, and affordable tool for targeted post‐transcriptional analysis in plant organelles and expands our understanding of MORF2's role in chloroplast RNA maturation. By overcoming key limitations of existing approaches, TIP sequencing enables routine, site‐specific quantification of post‐transcriptional regulation in organelles, including RNA editing and splicing, making it broadly accessible to researchers studying plastid biology, stress responses, and organelle–nucleus communication.

SUMMARYPlastoglobuli (PG) are plant lipoprotein compartments, present in plastid organelles. They are involved in the formation and/or storage of lipophilic metabolites. FIBRILLINs (FBNs) are one of the main PG‐associated proteins and are particularly abundant in carotenoid‐enriched chromoplasts found in ripe fruits and flowers. To address the contribution of different FBNs, independently and in combination, to isoprenoid formation and sequestration, a multiplex gene editing approach was undertaken in tomato. This approach generated a suite of single and high‐order fbn mutants that were shown to lack transcripts and respective protein products. The major PG‐related FBNs in tomato chosen for this study are SlFBN1, SlFBN2a, SlFBN4 and SlFBN7a. When knocked out independently, functional redundancy was revealed. However, paralog‐specific roles were detected regulating specific isoprenoids (e.g. plastochromanol 8) or plastidial esterification capability. In addition, high‐order fbn mutants displayed altered isoprenoid chromoplast sequestration patterns, notably with a significant reduction in carotenes (phytoene and phytofluene) in the PG fraction. Proteomic analysis confirmed the absence of PG‐core associated proteins, including NAD(P)H‐ubiquinone oxidoreductase C1, tocopherol cyclase (VTE1) and phytol esterase (PES1/PYP). Perturbations to the ultrastructure of the plastid were revealed, with aberrant PG formation and morphology predominating in high‐order mutants. Global lipidome profiles also highlighted broader changes directly affecting storage and plastid membrane lipids, for example, tri‐ and diacylglycerides and galactolipid species. Collectively, these results support both structural and metabolic roles of SlFBNs in PGs. The findings expose fundamental aspects of metabolic compartmentalisation in plant cells and the importance of lipoprotein particles for plastid integrity and functionality.

The apicoplast, a relic plastid organelle derived from secondary endosymbiosis, is crucial for many medically relevant Apicomplexa. While it no longer performs photosynthesis, the organelle retains several essential metabolic pathways. In this study, we examine the four primary metabolic pathways in the Toxoplasma gondii apicoplast, along with an accessory pathway, and identify conditions that can bypass these. Contrary to the prevailing view that the apicoplast is indispensable for T. gondii, we demonstrate that bypassing all pathways renders the apicoplast non-essential. We further show that T. gondii lacking an apicoplast (T. gondii−Apico) can be maintained indefinitely in culture, establishing a unique model to study the functions of this organelle. Through comprehensive metabolomic, transcriptomic, and proteomic analyses of T. gondii−Apico we uncover significant adaptation mechanisms following loss of the organelle and identify numerous putative apicoplast proteins revealed by their decreased abundance in T. gondii−Apico. Moreover, T. gondii−Apico parasites exhibit reduced sensitivity to apicoplast targeting compounds, providing a valuable tool for discovering new drugs acting on the organelle. The capability to culture T. gondii without its plastid offers new avenues for exploring apicoplast biology and developing novel therapeutic strategies against apicomplexan parasites.

Multiple plastid losses within photosynthetic stramenopiles revealed by comprehensive phylogenomics.

Complete plastid loss seems to be very rare among secondarily non-photosynthetic eukaryotes. Leukarachnion sp. PRA-24, an amoeboid colourless protist related to the photosynthetic algal class Synchromophyceae (Ochrophyta), is a candidate for such a case based on a previous investigation by transmission electron microscopy. Here, we characterize this organism in further detail and describe it as Leucomyxa plasmidifera gen. et sp. nov., additionally demonstrating it is the first known representative of a broader clade of non-photosynthetic ochrophytes. We recovered its complete plastid genome, exhibiting a reduced gene set similar to plastomes of other non-photosynthetic ochrophytes, yet being even more extreme in sequence divergence. Identification of components of the plastid protein import machinery in the L. plasmidifera transcriptome assembly corroborated that the organism possesses a cryptic plastid organelle. According to our bioinformatic reconstruction, the plastid contains a unique combination of biosynthetic pathways producing haem, a folate precursor and tocotrienols. As another twist to its organellar biology, L. plasmidifera turned out to contain an unusual long insertion in its mitogenome related to a newly discovered mitochondrial plasmid exhibiting unprecedented features in terms of its size and coding capacity. Combined, our work uncovered further striking outcomes of the evolutionary course of semiautonomous organelles in protists.

The plastid organelle is vital for photosynthesis and energy production. Advances in sequencing technology have enabled the exploration of plastomic resources, offering insights into plant evolution, diversity, and conservation. As an important group of horticultural ornamentals in the Crassulaceae family, Sempervivum plants are known for their unique rosette-like structures and reproduction through offsets. Despite their popularity, the classification status of Sempervivum remains uncertain, with only a single plastome sequence currently available. Furthermore, codon usage bias (CUB) is a widespread phenomenon of the unbalanced usage of synonymous codons in the coding sequence (CDS). However, due to the limited available plastid data, there has been no research that focused on the CUB analysis among Sempervivum until now. To address these gaps, we sequenced and released the plastomes of seven species and one subspecies from Sempervivum, revealing several consistent patterns. These included a shared 110 bp extension of the rps19 gene, 14 hypervariable regions (HVRs) with distinct nucleotide diversity (π: 0.01173 to 0.02702), and evidence of selective pressures shaping codon usage. Notably, phylogenetic analysis robustly divided the monophyletic clade into two sections: Jovibarba and Sempervivum. In conclusion, this comprehensive plastomic resource provides valuable insights into Sempervivum evolution and offers potential molecular markers for DNA barcoding.

Intracellular cargo transport is a ubiquitous cellular process in all eukaryotes. In many cell types, membrane bound cargo is associated with molecular motors which transport cargo along microtubule and actin tracks. In Toxoplasma gondii (T. gondii), an obligate intracellular parasite in the phylum Apicomplexa, organization of the endomembrane pathway depends on actin and an unconventional myosin motor, myosin F (MyoF). Loss of MyoF and actin disrupts vesicle transport, organelle positioning, and division of the apicoplast, a nonphotosynthetic plastid organelle. How this actomyosin system contributes to these cellular functions is still unclear. Using live-cell imaging, we observed that MyoF-EmeraldFP (MyoF-EmFP) displayed a dynamic and filamentous-like organization in the parasite cytosol, reminiscent of cytosolic actin filament dynamics. MyoF was not associated with the Golgi, apicoplast or dense granule surfaces, suggesting that it does not function using the canonical cargo transport mechanism. Instead, we found that loss of MyoF resulted in a dramatic rearrangement of the actin cytoskeleton in interphase parasites accompanied by significantly reduced actin dynamics. However, actin organization during parasite replication and motility was unaffected by the loss of MyoF. These findings revealed that MyoF is an actin organizing protein in Toxoplasma and facilitates cargo movement using an unconventional transport mechanism.

Microbial eukaryotes are important components of marine ecosystems, and the Marine Alveolates (MALVs) are consistently both abundant and diverse in global environmental sequencing surveys. MALVs are dinoflagellates that are thought to be parasites of other protists and animals, but the lack of data beyond ribosomal RNA gene sequences from all but a few described species means much of their biology and evolution remain unknown. Using single-cell transcriptomes from several MALVs and their free-living relatives, we show that MALVs evolved independently from two distinct, free-living ancestors and that their parasitism evolved in parallel. Phylogenomics shows one subgroup (MALV-II and -IV, or Syndiniales) is related to a novel lineage of free-living, eukaryovorous predators, the eleftherids, while the other (MALV-I, or Ichthyodinida) is related to the free-living predator Oxyrrhis and retains proteins targeted to a non-photosynthetic plastid. Reconstructing the evolution of photosynthesis, plastids, and parasitism in early-diverging dinoflagellates shows a number of parallels with the evolution of their apicomplexan sisters. In both groups, similar forms of parasitism evolved multiple times and photosynthesis was lost many times. By contrast, complete loss of the plastid organelle is infrequent and, when this does happen, leaves no residual genes.

Kekuatan tanah dasar berkaitan dengan daya dukung tanah, bergantung pada kandungan air yang ada dalam tanah, jenis tanah, dan keadaan asal dari tanah. Pemadatan adalah salah satu faktor paling pending dalam menambah kekuatan tanah. Karakteristik pemadatan tanah diperoleh dari pengujian di laboratorium untuk mendapatkan nilai kadar air optimum (OMC) dan berat kering maksimum (ɤdmaks). Meskipun penetuan parameter pengujian pemadatan laboratorium sederhana, pada saat di lapangan pengujian dalam jumlah besar untuk tanah timbunan sulit untuk mendapatkan jenis tanah yang dibutuhkan dalam satu area. Sehingga perlu dicari korelasi untuk memahami karakteristik tersebut. Data – data yang digunakan dari hasil pengujian di laboratorium menggunakan sampel tanah Kampar, Duri, dan Kulim. Tujuan penelitian adalah untuk melihat korelasi anatar optimum moisture content(OMC) dengan batas plastis pada proses pemadatan tanah timbunan. Metode yang digunakan adalah pengujian di laboratorium yaitu pengujian pemadatan laboraorium, analisa butiran, dan atterberg. Hasil yang diperoleh korelasi OMC dan nilai batas plastis (PL) korelasi yang diperoleh adalah sangat kuat 0.80 – 1.00, karena nilai R = 0.946. Kesimpulannya korelasi antara optimum moisture content dengan batas plastis (PL) pada tanah berbutir halus tanah timbunan pada Quarry Kampar, Kulim, dan Duri adalah sangat kuat. Saran perlu penelitian lebih lanjut dengan jumlah sampel yang lebih banyak lagi.

Isoprenoid precursor synthesis is an ancient and fundamental function of plastid organelles and a critical metabolic activity of the apicoplast in Plasmodium malaria parasites [1-3]. Over the past decade, our understanding of apicoplast properties and functions has increased enormously [4], due in large part to our ability to rescue blood-stage parasites from apicoplast-specific dysfunctions by supplementing cultures with isopentenyl pyrophosphate (IPP), a key output of this organelle [5,6]. In this Pearl, we explore the interdependence between isoprenoid metabolism and apicoplast biogenesis in P. falciparum and highlight critical future questions to answer.

Toxoplasma gondii and most other parasites in the phylum Apicomplexa contain an apicoplast, a non-photosynthetic plastid organelle required for fatty acid, isoprenoid, iron-sulfur cluster, and heme synthesis. Perturbation of apicoplast function results in parasite death. Thus, parasite survival critically depends on two cellular processes: apicoplast division to ensure every daughter parasite inherits a single apicoplast, and trafficking of nuclear encoded proteins to the apicoplast. Despite the importance of these processes, there are significant knowledge gaps in regards to the molecular mechanisms which control these processes; this is particularly true for trafficking of nuclear-encoded apicoplast proteins. This study provides crucial new insight into the timing of apicoplast protein synthesis and trafficking to the apicoplast. In addition, this study demonstrates how apicoplast-centrosome association, a key step in the apicoplast division cycle, is controlled by the actomyosin cytoskeleton.

Iron-sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite Plasmodium falciparum. Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.

Plastids, similar to mitochondria, are organelles of endosymbiotic origin, which retained their vestigial genomes (ptDNA). Their unique architecture, commonly referred to as the quadripartite (four-part) structure, is considered to be strictly conserved; however, the bulk of our knowledge on their variability and evolutionary transformations comes from studies of the primary plastids of green algae and land plants. To broaden our perspective, we obtained seven new ptDNA sequences from freshwater species of photosynthetic euglenids-a group that obtained secondary plastids, known to have dynamically evolving genome structure, via endosymbiosis with a green alga. Our analyses have demonstrated that the evolutionary history of euglenid plastid genome structure is exceptionally convoluted, with a patchy distribution of inverted ribosomal operon (rDNA) repeats, as well as several independent acquisitions of tandemly repeated rDNA copies. Moreover, we have shown that inverted repeats in euglenid ptDNA do not share their genome-stabilizing property documented in chlorophytes. We hypothesize that the degeneration of the quadripartite structure of euglenid plastid genomes is connected to the group II intron expansion. These findings challenge the current global paradigms of plastid genome architecture evolution and underscore the often-underestimated divergence between the functionality of shared traits in primary and complex plastid organelles.

Penelitian ini dilakukan dengan menggunakan metode eksperimental yaitu percobaan di laboratorium. Benda uji tanah asli dan tanah dengan variasi garam dapur rata 5% dengan variasi serbuk bata merah 3%, 6%, 9%, 12% dari berat tanah dibuat sebanyak 15 benda uji. Pengujian CBR laboratorium tanpa perendaman (unsoaked) dengan pemeraman selama 1 hari. Hasil penelitian didapatkan kadar air pada sampel tanah asli 50,42%, berat jenis 2,68, batas cair (LL) 52,94%, batas plastis (PL) 37,89%, dan indeks plastisitas (IP) 15,05%. Pada pengujian pemadatan dengan modified proctor, didapat nilai kepadatan kering maksimum tanah asli sebesar 1,369 gr/cm3 dengan kadar air optimum 32,80%, nilai kepadatan kering maksimum terbesar terjadi pada penambahan 9% serbuk bata merah dengan berat isi kering 1,47 gr/cm3 dengan kadar air optimum 28,70%. Hasil pengujian CBR tanpa perendaman pada sampel tanah asli sebesar 17,72%. Pada variasi campuran 3%, 6%, 9%, 12% didapatkan nilai CBR sebesar 26,90%, 30,75%, 33,95%, 27,60%.Nilai optimum CBR terjadi pada penambahan 9% serbuk bata merah yaitu sebesar 33,95% meningkat 91,59% dari nilai CBR tanah asli sedangkan untuk nilai rata-rata selisih kenaikan nilai CBR dari tanah asli yaitu sebesar 68,17%. Hasil pengujian menunjukkan bahwa penambahan bahan campuran serbuk bata merah dan garam dapur dapat meningkatkan nilai CBR tanah.

The plastid organelle is essential for many vital cellular processes and the growth and development of plants. The availability of a large number of complete plastid genomes could be effectively utilized to understand the evolution of the plastid genomes and phylogenetic relationships among plants. We comprehensively analyzed the plastid genomes of Viridiplantae comprising 3,654 taxa from 298 families and 111 orders and compared the genomic organizations in their plastid genomic DNA among major clades, which include gene gain/loss, gene copy number, GC content, and gene blocks. We discovered that some important genes that exhibit similar functions likely formed gene blocks, such as the psb family presumably showing co-occurrence and forming gene blocks in Viridiplantae. The inverted repeats (IRs) in plastid genomes have doubled in size across land plants, and their GC content is substantially higher than non-IR genes. By employing three different data sets [all nucleotide positions (nt123), only the first and second codon positions (nt12), and amino acids (AA)], our phylogenomic analyses revealed Chlorokybales + Mesostigmatales as the earliest-branching lineage of streptophytes. Hornworts, mosses, and liverworts forming a monophylum were identified as the sister lineage of tracheophytes. Based on nt12 and AA data sets, monocots, Chloranthales and magnoliids are successive sister lineages to the eudicots + Ceratophyllales clade. The comprehensive taxon sampling and analysis of different data sets from plastid genomes recovered well-supported relationships of green plants, thereby contributing to resolving some long-standing uncertainties in the plant phylogeny.

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host–plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.

ABSTRACTThe apicoplast, which harbors key pathways involved in biosynthesis of vital metabolites, is a unique and essential nonphotosynthetic plastid organelle in apicomplexan parasites. Intriguingly, autophagy-related protein 8 (Atg8), a highly conserved eukaryotic protein, can localize to the outermost membrane of the apicoplast and modulate its inheritance in both Toxoplasma and Plasmodium parasites. The Atg8-Atg3 interaction plays a key role in Atg8 lipidation and localization, and our previously work in Toxoplasma has suggested that the core Atg8-family interacting motif (AIM) in TgAtg3, 239FADI242, and the R27 residue of TgAtg8 contribute to TgAtg8-TgAtg3 interaction in vitro. However, little is known about the function of this interaction or its importance in tachyzoite growth in Toxoplasma gondii. Here, we generated two complemented cell lines, TgAtg3F239A/I242A and TgAtg8R27E, based on the TgAtg3 and TgAtg8 conditional knockdown cell lines, respectively. We found that both mutant complemented cell lines were severely affected in terms of tachyzoite growth and displayed delayed death upon conditional knockdown of endogenous TgAtg3 or TgAtg8. Intriguingly, both complemented lines appeared to be defective in TgAtg8 lipidation and apicoplast inheritance. Moreover, we showed that the interaction of TgAtg8 and TgAtg3 is critical for TgAtg8 apicoplast localization. In addition, we found that the TgAtg3F239A/I242A complemented line exhibits an integral mitochondrial network upon ablation of endogenous TgAtg3, which is distinct from TgAtg3-depleted parasites with a fragmented mitochondrial network. Taken together, this work solidifies the contribution of the TgAtg8-TgAtg3 interaction to apicoplast inheritance and the growth of T. gondii tachyzoites.IMPORTANCEToxoplasma gondiiis a widespread intracellular parasite infecting a variety of warm-blooded animals, including humans. Current frontline treatment of toxoplasmosis suffers many drawbacks, including toxicity, drug resistance, and failure to eradicate tissue cysts, underscoring the need to identify novel drug targets for suppression or treatment of toxoplasmosis. TgAtg8 is thought to serve multiple functions in lipidation and is considered essential to the growth and development of both tachyzoites and bradyzoites. Here, we show that Toxoplasma gondii has adapted a conserved Atg8-Atg3 interaction, required for canonical autophagy in other eukaryotes, to function specifically in apicoplast inheritance. Our finding not only highlights the importance of TgAtg8-TgAtg3 interaction in tachyzoite growth but also suggests that this interaction is a promising drug target for the therapy of toxoplasmosis.

Isolation and characterization of sub-plastidial fractions from carotenoid rich fruits.

Eukaryote-eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms that act to stabilize emergent endosymbioses-between two fundamentally selfish biological organisms-are unclear. Theory suggests that enforcement mechanisms, which punish misbehavior, may act to stabilize such interactions by resolving conflict. However, how such mechanisms can emerge in a facultative endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model facultative endosymbiosis between Paramecium bursaria and Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived messenger RNA (mRNA) released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term "RNAi collisions," represents a mechanism that can promote stability in a facultative eukaryote-eukaryote endosymbiosis. Specifically, by imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions.

ABSTRACTMalaria parasites have three genomes: a nuclear genome, a mitochondrial genome, and an apicoplast genome. Since the apicoplast is a plastid organelle of prokaryotic origin and has no counterpart in the human host, it can be a source of novel targets for antimalarials. Plasmodium falciparum DNA gyrase (PfGyr) A and B subunits both have apicoplast-targeting signals. First, to test the predicted localization of this enzyme in the apicoplast and the breadth of its function at the subcellular level, nuclear-encoded PfGyrA was disrupted using CRISPR/Cas9 gene editing. Isopentenyl pyrophosphate (IPP) is known to rescue parasites from apicoplast inhibitors. Indeed, successful growth and characterization of PfΔGyrA was possible in the presence of IPP. PfGyrA disruption was accompanied by loss of plastid acyl-carrier protein (ACP) immunofluorescence and the plastid genome. Second, ciprofloxacin, an antibacterial gyrase inhibitor, has been used for malaria prophylaxis, but there is a need for a more detailed description of the mode of action of ciprofloxacin in malaria parasites. As predicted, PfΔGyrA clone supplemented with IPP was less sensitive to ciprofloxacin but not to the nuclear topoisomerase inhibitor etoposide. At high concentrations, however, ciprofloxacin continued to inhibit IPP-rescued PfΔGyrA, possibly suggesting that ciprofloxacin may have an additional nonapicoplast target in P. falciparum. Overall, we confirm that PfGyrA is an apicoplast enzyme in the malaria parasite, essential for blood-stage parasites, and a possible target of ciprofloxacin but perhaps not the only target.