Frontiers Media SA Acta Medica Okayama 1664-462X 17 2026 Quantitative resistance to Rhizoctonia solani AG-4 develops independently in leaves and roots of Brachypodium distachyon 1875734 EN Rozi Fernanda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Niranjan Mahadevan Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Natsuka Kohno School of Agriculture, Okayama University Reiko Nagao School of Agriculture, Okayama University Megumi Watanabe Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Nanami Sakata Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hidenori Matsui Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yusuke Kouzai Crop Stress Management Group, Division of Plant Molecular Regulation Research, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO) Keiichi Mochida RIKEN Center for Sustainable Resource Science Yoshiteru Noutoshi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Rhizoctonia solani is a soil-borne phytopathogen with a broad host range, classified into multiple anastomosis groups (AGs). While the rice sheath blight pathogen AG-1 IA is well-studied, defense mechanisms against AG-4, which infects diverse monocots and dicots, remain unclear. Here, we screened 153 Brachypodium distachyon accessions for resistance to AG-4 HG-I+II using both leaf and soil inoculation assays. The accessions exhibited a quantitative resistance spectrum in both assays. Some accessions were resistant to both methods, while others showed resistance in only one, indicating that leaf and root defenses operate independently. Unlike the salicylic acid (SA)-dependent resistance observed in several accessions against AG-1 IA, AG-4 leaf resistance appears to be largely SA-independent. Among 22 resistant accessions, only eight induced the SA marker BdWRKY38, whereas twelve induced the jasmonic acid (JA) marker BdAOS. Additionally, resistance in Bd3–1 was unaffected by expression of the SA hydroxylase NahG. These results are consistent with previous findings that exogenous application of SA, JA, or ethylene fails to confer AG-4 resistance. Instead, most AG-4-resistant accessions showed common upregulation of cell wall biosynthesis-related genes following leaf infection, suggesting that cell wall fortification may contribute to defense against AG-4. These findings establish B. distachyon as a valuable model for dissecting resistance mechanisms against R. solani AG-4. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0032-0781 63 12 2022 Plant Hormonomics: A Key Tool for Deep Physiological Phenotyping to Improve Crop Productivity 1826 1839 EN Takashi Hirayama Institute of Plant Science and Resources, Okayama University Keiichi Mochida RIKEN Center for Sustainable Resource Science Agriculture is particularly vulnerable to climate change. To cope with the risks posed by climate-related stressors to agricultural production, global population growth, and changes in food preferences, it is imperative to develop new climate-smart crop varieties with increased yield and environmental resilience. Molecular genetics and genomic analyses have revealed that allelic variations in genes involved in phytohormone-mediated growth regulation have greatly improved productivity in major crops. Plant science has remarkably advanced our understanding of the molecular basis of various phytohormone-mediated events in plant life. These findings provide essential information for improving the productivity of crops growing in changing climates. In this review, we highlight the recent advances in plant hormonomics (multiple phytohormone profiling) and discuss its application to crop improvement. We present plant hormonomics as a key tool for deep physiological phenotyping, focusing on representative plant growth regulators associated with the improvement of crop productivity. Specifically, we review advanced methodologies in plant hormonomics, highlighting mass spectrometry- and nanosensor-based plant hormone profiling techniques. We also discuss the applications of plant hormonomics in crop improvement through breeding and agricultural management practices. No potential conflict of interest relevant to this article was reported.

Frontiers Media S.A. Acta Medica Okayama 1664-462X 13 2022 Time-series transcriptome of Brachypodium distachyon during bacterial flagellin-induced pattern-triggered immunity 1004184 EN Tsubasa Ogasahara Graduate School of Environmental and Life Science, Okayama University Yusuke Kouzai Graduate School of Environmental and Life Science, Okayama University Megumi Watanabe Graduate School of Environmental and Life Science, Okayama University Akihiro Takahashi Graduate School of Environmental and Life Science, Okayama University Kotaro Takahagi Kihara Institute for Biological Research, Yokohama City University June-Sik Kim Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University Keiichi Mochida Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0140-7791 45 11 2022 FE UPTAKE�]INDUCING PEPTIDE1 maintains Fe translocation by controlling Fe deficiency response genes in the vascular tissue of Arabidopsis 3322 3337 EN Satoshi Okada Group of Environmental Stress Response Systems, Institute of Plant Science and Resources, Okayama University Gui J. Lei Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Naoki Yamaji Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Sheng Huang Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Jian F. Ma Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Keiichi Mochida Crop Design Research Team, Institute of Plant Science and Resources, Okayama University Takashi Hirayama Group of Environmental Stress Response Systems, Institute of Plant Science and Resources, Okayama University FE UPTAKE-INDUCING PEPTIDE1 (FEP1), also named IRON MAN3 (IMA3) is a short peptide involved in the iron deficiency response in Arabidopsis thaliana. Recent studies uncovered its molecular function, but its physiological function in the systemic Fe response is not fully understood. To explore the physiological function of FEP1 in iron homoeostasis, we performed a transcriptome analysis using the FEP1 loss-of-function mutant fep1-1 and a transgenic line with oestrogen-inducible expression of FEP1. We determined that FEP1 specifically regulates several iron deficiency-responsive genes, indicating that FEP1 participates in iron translocation rather than iron uptake in roots. The iron concentration in xylem sap under iron-deficient conditions was lower in the fep1-1 mutant and higher in FEP1-induced transgenic plants compared with the wild type (WT). Perls staining revealed a greater accumulation of iron in the cortex of fep1-1 roots than in the WT root cortex, although total iron levels in roots were comparable in the two genotypes. Moreover, the fep1-1 mutation partially suppressed the iron overaccumulation phenotype in the leaves of the oligopeptide transporter3-2 (opt3-2) mutant. These data suggest that FEP1 plays a pivotal role in iron movement and in maintaining the iron quota in vascular tissues in Arabidopsis. No potential conflict of interest relevant to this article was reported.

BMC Acta Medica Okayama 1474-760X 22 1 2021 Cellular and transcriptomic analyses reveal two-staged chloroplast biogenesis underpinning photosynthesis build-up in the wheat leaf EN Naresh Loudya Department of Biological Sciences, Royal Holloway University of London Priyanka Mishra Department of Biological Sciences, Royal Holloway University of London Kotaro Takahagi RIKEN Center for Sustainable Resource Science Yukiko Uehara-Yamaguchi RIKEN Center for Sustainable Resource Science Komaki Inoue RIKEN Center for Sustainable Resource Science Laszlo Bogre Department of Biological Sciences, Royal Holloway University of London Keiichi Mochida Institute of Plant Science and Resources, Okayama University Enrique Lopez-Juez Department of Biological Sciences, Royal Holloway University of London Background The developmental gradient in monocot leaves has been exploited to uncover leaf developmental gene expression programs and chloroplast biogenesis processes. However, the relationship between the two is barely understood, which limits the value of transcriptome data to understand the process of chloroplast development. Results Taking advantage of the developmental gradient in the bread wheat leaf, we provide a simultaneous quantitative analysis for the development of mesophyll cells and of chloroplasts as a cellular compartment. This allows us to generate the first biologically-informed gene expression map of this leaf, with the entire developmental gradient from meristematic to fully differentiated cells captured. We show that the first phase of plastid development begins with organelle proliferation, which extends well beyond cell proliferation, and continues with the establishment and then the build-up of the plastid genetic machinery. The second phase is marked by the development of photosynthetic chloroplasts which occupy the available cellular space. Using a network reconstruction algorithm, we predict that known chloroplast gene expression regulators are differentially involved across those developmental stages. Conclusions Our analysis generates both the first wheat leaf transcriptional map and one of the most comprehensive descriptions to date of the developmental history of chloroplasts in higher plants. It reveals functionally distinct plastid and chloroplast development stages, identifies processes occurring in each of them, and highlights our very limited knowledge of the earliest drivers of plastid biogenesis, while providing a basis for their future identification. No potential conflict of interest relevant to this article was reported.

Oxford Univ Press Acta Medica Okayama 2631-9268 2 3 2020 Parental legacy and regulatory novelty in Brachypodium diurnal transcriptomes accompanying their polyploidy lqaa067 EN Komaki Inoue RIKEN Center for Sustainable Resource Science Kotaro Takahagi RIKEN Center for Sustainable Resource Science Yusuke Kouzai RIKEN Center for Sustainable Resource Science Satoru Koda Graduate School of Mathematics, Kyushu University Minami Shimizu RIKEN Center for Sustainable Resource Science Yukiko Uehara-Yamaguchi RIKEN Center for Sustainable Resource Science Risa Nakayama RIKEN Center for Sustainable Resource Science Toshie Kita RIKEN Center for Sustainable Resource Science Yoshihiko Onda RIKEN Center for Sustainable Resource Science Toshihisa Nomura RIKEN Center for Sustainable Resource Science Hidetoshi Matsui Faculty of Data Science, Shiga University Kiyotaka Nagaki Institute of Plant Science and Resources, Okayama University Ryuei Nishii School of Information and Data Science, Nagasaki University Keiichi Mochida Institute of Plant Science and Resources, Okayama University Polyploidy is a widespread phenomenon in eukaryotes that can lead to phenotypic novelty and has important implications for evolution and diversification. The modification of phenotypes in polyploids relative to their diploid progenitors may be associated with altered gene expression. However, it is largely unknown how interactions between duplicated genes affect their diurnal expression in allopolyploid species. In this study, we explored parental legacy and hybrid novelty in the transcriptomes of an allopolyploid species and its diploid progenitors. We compared the diurnal transcriptomes of representative Brachypodium cytotypes, including the allotetraploid Brachypodium hybridum and its diploid progenitors Brachypodium distachyon and Brachypodium stacei. We also artificially induced an autotetraploid B. distachyon. We identified patterns of homoeolog expression bias (HEB) across Brachypodium cytotypes and time-dependent gain and loss of HEB in B. hybridum. Furthermore, we established that many genes with diurnal expression experienced HEB, while their expression patterns and peak times were correlated between homoeologs in B. hybridum relative to B. distachyon and B. stacei, suggesting diurnal synchronization of homoeolog expression in B. hybridum. Our findings provide insight into the parental legacy and hybrid novelty associated with polyploidy in Brachypodium, and highlight the evolutionary consequences of diurnal transcriptional regulation that accompanied allopolyploidy. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 22 3 2021 Genetic Elucidation for Response of Flowering Time to Ambient Temperatures in Asian Rice Cultivars 1024 EN Kiyosumi Hori National Agriculture and Food Research Organization, Institute of Crop Science Daisuke Saisho Institute of Plant Science and Resources, Okayama University Kazufumi Nagata National Agriculture and Food Research Organization, Institute of Crop Science Yasunori Nonoue National Agriculture and Food Research Organization, Institute of Crop Science Yukiko Uehara-Yamaguchi RIKEN Center for Sustainable Resource Science Asaka Kanatani RIKEN Center for Sustainable Resource Science Koka Shu National Agriculture and Food Research Organization, Institute of Crop Science Takashi Hirayama Institute of Plant Science and Resources, Okayama University Jun-Ichi Yonemaru National Agriculture and Food Research Organization, Institute of Crop Science Shuichi Fukuoka National Agriculture and Food Research Organization, Institute of Crop Science Keiichi Mochida Institute of Plant Science and Resources, Okayama University Climate resilience of crops is critical for global food security. Understanding the genetic basis of plant responses to ambient environmental changes is key to developing resilient crops. To detect genetic factors that set flowering time according to seasonal temperature conditions, we evaluated differences of flowering time over years by using chromosome segment substitution lines (CSSLs) derived from japonica rice cultivars "Koshihikari" x "Khao Nam Jen", each with different robustness of flowering time to environmental fluctuations. The difference of flowering times in 9 years' field tests was large in "Khao Nam Jen" (36.7 days) but small in "Koshihikari" (9.9 days). Part of this difference was explained by two QTLs. A CSSL with a "Khao Nam Jen" segment on chromosome 11 showed 28.0 days' difference; this QTL would encode a novel flowering-time gene. Another CSSL with a segment from "Khao Nam Jen" in the region around Hd16 on chromosome 3 showed 23.4 days" difference. A near-isogenic line (NIL) for Hd16 showed 21.6 days' difference, suggesting Hd16 as a candidate for this QTL. RNA-seq analysis showed differential expression of several flowering-time genes between early and late flowering seasons. Low-temperature treatment at panicle initiation stage significantly delayed flowering in the CSSL and NIL compared with "Koshihikari". Our results unravel the molecular control of flowering time under ambient temperature fluctuations. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 0032-0781 61 8 2020 Decoding Plant–Environment Interactions That Influence Crop Agronomic Traits 1408 1418 EN Keiichi Mochida Institute of Plant Science and Resources, Okayama University Ryuei Nishii School of Information and Data Sciences, Nagasaki University Takashi Hirayama Institute of Plant Science and Resources, Okayama University To ensure food security in the face of increasing global demand due to population growth and progressive urbanization, it will be crucial to integrate emerging technologies in multiple disciplines to accelerate overall throughput of gene discovery and crop breeding. Plant agronomic traits often appear during the plants�f later growth stages due to the cumulative effects of their lifetime interactions with the environment. Therefore, decoding plant–environment interactions by elucidating plants�f temporal physiological responses to environmental changes throughout their lifespans will facilitate the identification of genetic and environmental factors, timing and pathways that influence complex end-point agronomic traits, such as yield. Here, we discuss the expected role of the life-course approach to monitoring plant and crop health status in improving crop productivity by enhancing the understanding of plant–environment interactions. We review recent advances in analytical technologies for monitoring health status in plants based on multi-omics analyses and strategies for integrating heterogeneous datasets from multiple omics areas to identify informative factors associated with traits of interest. In addition, we showcase emerging phenomics techniques that enable the noninvasive and continuous monitoring of plant growth by various means, including three-dimensional phenotyping, plant root phenotyping, implantable/injectable sensors and affordable phenotyping devices. Finally, we present an integrated review of analytical technologies and applications for monitoring plant growth, developed across disciplines, such as plant science, data science and sensors and Internet-of-things technologies, to improve plant productivity. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0960-7412 104 4 2020 BdWRKY38 is required for the incompatible interaction of Brachypodium distachyon with the necrotrophic fungus Rhizoctonia solani 995 1008 EN Yusuke Kouzai Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Minami Shimizu Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Komaki Inoue Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Yukiko Uehara�]Yamaguchi Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Kotaro Takahagi Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Risa Nakayama Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Takakazu Matsuura Institute of Plant Science and Resources (IPSR), Okayama University Izumi C. Mori Institute of Plant Science and Resources (IPSR), Okayama University Takashi Hirayama Institute of Plant Science and Resources (IPSR), Okayama University Sobhy S. H. Abdelsalam Graduate School of Environmental and Life Science, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Keiichi Mochida Institute of Plant Science and Resources (IPSR), Okayama University Rhizoctonia solani is a soil�]borne necrotrophic fungus that causes sheath blight in grasses. The basal resistance of compatible interactions between R. solani and rice is known to be modulated by some WRKY transcription factors (TFs). However, genes and defense responses involved in incompatible interaction with R. solani remain unexplored, because no such interactions are known in any host plants. Recently, we demonstrated that Bd3�]1, an accession of the model grass Brachypodium distachyon, is resistant to R. solani and, upon inoculation with the fungus, undergoes rapid induction of genes responsive to the phytohormone salicylic acid (SA) that encode the WRKY TFs BdWRKY38 and BdWRKY44. Here, we show that endogenous SA and these WRKY TFs positively regulate this accession�]specific R. solani resistance. In contrast to a susceptible accession (Bd21), the infection process in the resistant accessions Bd3�]1 and Tek�]3 was suppressed at early stages before the development of fungal biomass and infection machinery. A comparative transcriptome analysis during pathogen infection revealed that putative WRKY�]dependent defense genes were induced faster in the resistant accessions than in Bd21. A gene regulatory network (GRN) analysis based on the transcriptome dataset demonstrated that BdWRKY38 was a GRN hub connected to many target genes specifically in resistant accessions, whereas BdWRKY44 was shared in the GRNs of all three accessions. Moreover, overexpression of BdWRKY38 increased R. solani resistance in Bd21. Our findings demonstrate that these resistant accessions can activate an incompatible host response to R. solani, and BdWRKY38 regulates this response by mediating SA signaling. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 0032-0781 61 8 2020 Life-Course Monitoring of Endogenous Phytohormone Levels under Field Conditions Reveals Diversity of Physiological States among Barley Accessions 1438 1448 EN Takashi Hirayama Institute of Plant Science and Resources, Okayama University Daisuke Saisho Institute of Plant Science and Resources, Okayama University Takakazu Matsuura Institute of Plant Science and Resources, Okayama University Satoshi Okada Institute of Plant Science and Resources, Okayama University Kotaro Takahagi RIKEN Center for Sustainable Resource Science Asaka Kanatani RIKEN Center for Sustainable Resource Science Jun Ito Kihara Institute for Biological Research, Yokohama City University Hiroyuki Tsuji Kihara Institute for Biological Research, Yokohama City University Yoko Ikeda Institute of Plant Science and Resources, Okayama University Keiichi Mochida Institute of Plant Science and Resources, Okayama University Agronomically important traits often develop during the later stages of crop growth as consequences of various plant–environment interactions. Therefore, the temporal physiological states that change and accumulate during the crop�fs life course can significantly affect the eventual phenotypic differences in agronomic traits among crop varieties. Thus, to improve productivity, it is important to elucidate the associations between temporal physiological responses during the growth of different crop varieties and their agronomic traits. However, data representing the dynamics and diversity of physiological states in plants grown under field conditions are sparse. In this study, we quantified the endogenous levels of five phytohormones �\ auxin, cytokinins (CKs), ABA, jasmonate and salicylic acid �\ in the leaves of eight diverse barley (Hordeum vulgare) accessions grown under field conditions sampled weekly over their life course to assess the ongoing fluctuations in hormone levels in the different accessions under field growth conditions. Notably, we observed enormous changes over time in the development-related plant hormones, such as auxin and CKs. Using 3�Œ RNA-seq-based transcriptome data from the same samples, we investigated the expression of barley genes orthologous to known hormone-related genes of Arabidopsis throughout the life course. These data illustrated the dynamics and diversity of the physiological states of these field-grown barley accessions. Together, our findings provide new insights into plant–environment interactions, highlighting that there is cultivar diversity in physiological responses during growth under field conditions. No potential conflict of interest relevant to this article was reported.

Nature Research Acta Medica Okayama 2045-2322 10 1 2020 Identification of effector candidate genes of Rhizoctonia solani AG-1 IA expressed during infection in Brachypodium distachyon 14889 EN Sobhy S. H. Abdelsalam Graduate School of Environmental and Life Science, Okayama University Yusuke Kouzai Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Megumi Watanabe Graduate School of Environmental and Life Science, Okayama University Komaki Inoue Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Seiji Tsuge Graduate School of Agriculture, Kyoto Prefectural University Keiichi Mochida Institute for Plant Science and Resources (IPSR), Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Rhizoctonia solani is a necrotrophic phytopathogen belonging to basidiomycetes. It causes rice sheath blight which inflicts serious damage in rice production. The infection strategy of this pathogen remains unclear. We previously demonstrated that salicylic acid-induced immunity could block R. solani AG-1 IA infection in both rice and Brachypodium distachyon. R. solani may undergo biotrophic process using effector proteins to suppress host immunity before necrotrophic stage. To identify pathogen genes expressed at the early infection process, here we developed an inoculation method using B. distachyon which enables to sample an increased amount of semi-synchronous infection hyphae. Sixty-one R. solani secretory effector-like protein genes (RsSEPGs) were identified using in silico approach with the publicly available gene annotation of R. solani AG-1 IA genome and our RNA-sequencing results obtained from hyphae grown on agar medium. Expression of RsSEPGs was analyzed at 6, 10, 16, 24, and 32 h after inoculation by a quantitative reverse transcription-polymerase chain reaction and 52 genes could be detected at least on a single time point tested. Their expressions showed phase-specific patterns which were classified into 6 clusters. The 23 RsSEPGs in the cluster 1-3 and 29 RsSEPGs in the cluster 4-6 are expected to be involved in biotrophic and necrotrophic interactions, respectively. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2666-1667 1 1 2020 Highly Efficient CRISPR-Associated Protein 9 Ribonucleoprotein-Based Genome Editing in Euglena gracilis 100023 EN Toshihisa Nomura RIKEN Center for Sustainable Resource Science Mizuki Yoshikawa RIKEN Baton Zone Program Kengo Suzuki RIKEN Baton Zone Program Keiichi Mochida Institute of Plant Science and Resources, Okayama University Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, and biofuels. However, targeted mutagenesis in this species has been a long-standing challenge. We recently developed a transgene-free, highly efficient, genome editing method for E. gracilis using CRISPR/Cas9 ribonucleoproteins (RNPs). Our method achieved mutagenesis rates of approximately 80% or more through an electroporation-based direct delivery of Cas9 RNPs. Therefore, this method is suitable for basic research and industrial applications, such as the breeding of Euglena. For complete details on the use and execution of this protocol, please refer to Nomura et al. (2019). No potential conflict of interest relevant to this article was reported.