High-quality chromosome-level genome assembly of Nicotiana benthamiana.

Nicotiana benthamiana is a fundamental model organism in plant research. Recent advancements in genomic sequencing have revealed significant intraspecific genetic variations. This study addresses the pressing need for a precise genome sequence specific to its geographic origin by presenting a comprehensive genome assembly of the N. benthamiana LAB strain from the Republic of Korea (NbKLAB). We compare this assembly with the widely used NbLAB360 strain, shedding light on essential genomic differences between them. The outcome is a high-quality, chromosome-level genome assembly comprising 19 chromosomes, spanning 2,762 Mb, with an N50 of 142.6 Mb. Comparative analyses revealed notable variations, including 46,215 protein-coding genes, with an impressive 99.5% BUSCO completeness score. Furthermore, the NbKLAB assembly substantially improved the QV from 33% for NbLAB360 to 49%. This refined chromosomal genome assembly for N. benthamiana, in conjunction with comparative insights, provides a valuable resource for genomics research and molecular biology. This accomplishment forms a strong foundation for in-depth exploration into the intricacies of plant genetics and genomics, improved precision, and a comparative framework.

Enhanced binding and inhibition of SARS-CoV-2 by a plant-derived ACE2 protein containing a fused mu tailpiece.

Infectious diseases such as Coronavirus disease 2019 (COVID-19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The angiotensin-converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS-CoV-2 infection. Inhibition of the interaction between SARS-CoV-2 and ACE2 has been proposed as a target for the prevention and treatment of COVID-19. We produced four recombinant plant-derived ACE2 isoforms with or without the mu tailpiece (µ-tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant-derived ACE2 isoforms bound whole SARS-CoV-2 virus and the isolated receptor binding domains of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of µ-tp and KDEL to the ACE2 protein (ACE2 µK) had enhanced binding activity with SARS-CoV-2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant-derived ACE2 µK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. The efficient and rapid scalability of plant-derived ACE2 µK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.

Two long read-based genome assembly and annotation of polyploidy woody plants, Hibiscus syriacus L. using PacBio and Nanopore platforms.

Improvements in long read DNA sequencing and related techniques facilitated the generation of complex eukaryotic genomes. Despite these advances, the quality of constructed plant reference genomes remains relatively poor due to the large size of genomes, high content of repetitive sequences, and wide variety of ploidy. Here, we developed the de novo sequencing and assembly of high polyploid plant genome, Hibiscus syriacus, a flowering plant species of the Malvaceae family, using the Oxford Nanopore Technologies and Pacific Biosciences Sequel sequencing platforms. We investigated an efficient combination of high-quality and high-molecular-weight DNA isolation procedure and suitable assembler to achieve optimal results using long read sequencing data. We found that abundant ultra-long reads allow for large and complex polyploid plant genome assemblies with great recovery of repetitive sequences and error correction even at relatively low depth Nanopore sequencing data and polishing compared to previous studies. Collectively, our combination provides cost effective methods to improve genome continuity and quality compared to the previously reported reference genome by accessing highly repetitive regions. The application of this combination may enable genetic research and breeding of polyploid crops, thus leading to improvements in crop production.

Construction of deep learning-based disease detection model in plants.

Accurately detecting disease occurrences of crops in early stage is essential for quality and yield of crops through the decision of an appropriate treatments. However, detection of disease needs specialized knowledge and long-term experiences in plant pathology. Thus, an automated system for disease detecting in crops will play an important role in agriculture by constructing early detection system of disease. To develop this system, construction of a stepwise disease detection model using images of diseased-healthy plant pairs and a CNN algorithm consisting of five pre-trained models. The disease detection model consists of three step classification models, crop classification, disease detection, and disease classification. The 'unknown' is added into categories to generalize the model for wide application. In the validation test, the disease detection model classified crops and disease types with high accuracy (97.09%). The low accuracy of non-model crops was improved by adding these crops to the training dataset implicating expendability of the model. Our model has the potential to apply to smart farming of Solanaceae crops and will be widely used by adding more various crops as training dataset.

Genetic diversity assessment and genome-wide association study reveal candidate genes associated with component traits in sweet potato (Ipomoea batatas (L.) Lam).

The Korean sweet potatoes were bred by various cultivars introduced from Japanese, American, Porto Rico, China, and Burundi. This issue enriched their genetic diversity but also resulted in a mixture of cultivars. For genotyping, we collected and sequenced 66 sweet potato germplasms from different localities around Korea, including 36 modern cultivars, 5 local cultivars, and 25 foreign cultivars. This identified 447.6 million trimmed reads and 324.8 million mapping reads and provided 39,424 single nucleotide polymorphisms (SNPs) markers. Phylogenetic clustering and population structure analysis distinctly classified these germplasms into 5 genetic groups, group 1, group 2, group 3, group 4, and group 5, containing 20, 15, 10, 7, and 14 accessions, respectively. Sixty-three significant SNPs were selected by genome-wide association for sugar composition-related traits (fructose, glucose, and total sugars), total starch, amylose content, and total carotenoid of the storage root. A total of 37 candidate genes encompassing these significant SNPs were identified, among which, 7 genes were annotated to involve in sugar and starch metabolism, including galactose metabolism (itf04g30630), starch and sucrose metabolism (itf03g13270, itf15g09320), carbohydrate metabolism (itf14g10250), carbohydrate and amino acid metabolism (itf12g19270), and amino sugar and nucleotide sugar metabolism (itf03g21950, itf15g04880). This results indicated that sugar and starch are important characteristics to determine the genetic diversity of sweet potatoes. These findings not only illustrate the importance of component traits to genotyping sweet potatoes but also explain an important reason resulting in genetic diversity of sweet potato.

High-quality genome assembly and genetic mapping reveal a gene regulating flesh color in watermelon (Citrullus lanatus).

The unique color and type characteristics of watermelon fruits are regulated by many molecular mechanisms. However, it still needs to be combined with more abundant genetic data to fine-tune the positioning. We assembled genomes of two Korean inbred watermelon lines (cv. 242-1 and 159-1) with unique color and fruit-type characteristics and identified 23,921 and 24,451 protein-coding genes in the two genomes, respectively. To obtain more precise results for further study, we resequenced one individual of each parental line and an F2 population composed of 87 individuals. This identified 1,539 single-nucleotide polymorphisms (SNPs) and 80 InDel markers that provided a high-density genetic linkage map with a total length of 3,036.9 cM. Quantitative trait locus mapping identified 15 QTLs for watermelon fruit quality-related traits, including ß-carotene and lycopene content in fruit flesh, fruit shape index, skin thickness, flesh color, and rind color. By investigating the mapping intervals, we identified 33 candidate genes containing variants in the coding sequence. Among them, Cla97C01G008760 was annotated as a phytoene synthase with a single-nucleotide variant (A → G) in the first exon at 9,539,129 bp of chromosome 1 that resulted in the conversion of a lysine to glutamic acid, indicating that this gene might regulate flesh color changes at the protein level. These findings not only prove the importance of a phytoene synthase gene in pigmentation but also explain an important reason for the color change of watermelon flesh.

Genome sequence and characterization of a novel fabavirus infecting Cirsium setidens (gondre) in South Korea.

The complete nucleotide sequence of a novel gondre (Cirsium setidens)-infecting virus, provisionally named "cirsium virus A" (CiVA), was determined by high-throughput and Sanger sequencing, revealing a genome organization typical of fabaviruses. RNA1 and RNA2 are 5,828 and 3,478 nucleotides long, excluding the 3'-terminal poly(A) tails, each containing a single open reading frame. The highest sequence identity values for the CiVA coat protein and proteinase-polymerase, compared with known fabavirus sequences, were 59.09% and 69.68%, respectively, falling below the current thresholds for Fabavirus species demarcation. Our findings support classifying CiVA as a novel putative member of the genus Fabavirus, subfamily Comovirinae, family Secoviridae.

Complete genome sequence and genome characterization of a novel potyvirus from Lamprocapnos spectabilis.

The genome of a new potyvirus from a Lamprocapnos spectabilis plant in South Korea was sequenced by high-throughput sequencing and confirmed by Sanger sequencing. The new potyvirus was tentatively named "lamprocapnos virus A" (LaVA); its complete genome contains 9,745 nucleotides, excluding the 3'-terminal poly(A) tail. The LaVA genome structure is similar to that of members of the genus Potyvirus and contains an open reading frame encoding a large putative polyprotein of 3,120 amino acids (aa) with conserved motifs. The complete genome shared 48%-56% nucleotide sequence identity and the polyprotein shared 41%-52% aa sequence identity with those of other potyviruses. These values are below the standard thresholds for potyvirus species demarcation. Phylogenetic analysis based on polyprotein sequences showed that LaVA belongs to the genus Potyvirus. To our knowledge, this is the first report of the complete genome sequence and genome characterization of a potyvirus infecting Lamprocapnos spectabilis.

RING-Type E3 Ubiquitin Ligases AtRDUF1 and AtRDUF2 Positively Regulate the Expression of PR1 Gene and Pattern-Triggered Immunity.

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae.

Complete nucleotide sequence of hemisteptia virus A, a polero-like virus.

The complete genomic nucleotide sequence of hemisteptia virus A (HemVA) from a Hemisteptia lyrata Bunge plant in South Korea was identified by high-throughput sequencing. The HemVA genome consists of 6,122 nucleotides and contains seven putative open reading frames, ORF0-5 and ORF3a, encoding the putative proteins P0-P5 and P3a, respectively. Pairwise amino acid sequence analysis shows that the HemVA P1-P5 proteins have the highest sequence identity (23.68%-54.15%) to the corresponding proteins of members of the families Solemoviridae and Tombusviridae. Phylogenetic analysis of the P1-P2 and P3 amino acid sequences indicated that HemVA should be classified as a member of a distinct species in the genus Polerovirus.

Identification and molecular characterization of a novel kudzu-infecting virus of the family Betaflexiviridae.

A new virus, tentatively named "kudzu virus D" (KuVD) was discovered in kudzu (Pueraria montana var. lobata) in South Korea. Its complete genome comprises 7,922 nucleotides, excluding the poly(A) tail, and contains five open reading frames (ORFs) encoding, from 5' to 3', a replicase (ORF1), three triple gene block proteins TGB1-3 (ORF2-ORF4), and a coat protein (ORF5). This genome organization is typical of members of the subfamily Quinvirinae of the family Betaflexiviridae. Pairwise alignment analysis revealed that the nucleotide sequences of the replicase and coat protein of KuVD were 12.13-54.46% and 24.03-50.67% identical, respectively, to those of other members of the family Betaflexiviridae. These values are far below the current species ICTV demarcation threshold. Consequently, KuVD should be considered a member of a new species in the subfamily Quinvirinae.

Identification of accession-specific variants and development of KASP markers for assessing the genetic makeup of Brassica rapa seeds.

BACKGROUND: Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed purity has been assessed with a grow-out test (GOT) in the field, a time consuming and costly venture. Early in the last decade, seed testing with molecular markers was introduced as a replacement for GOT, and Kompetitive allele specific PCR (KASP) markers were recognized as promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and applicable to only a small number of accessions or varieties due to the limited genetic information and reference genomes available. RESULTS: We identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. From these, we identified 2,925 SNPs as accession-specific, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by the Next Generation Sequencing (NGS) analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguised individuals from the mixed population including 50 target accessions from B. rapa core collection and the outgroup. Additionally, the marker set we developed here discriminated F1 hybrids and their parental lines with distinct clusters. CONCLUSIONS: This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).

Temporally distinct regulatory pathways coordinate thermo-responsive storage organ formation in potato.

Tuberization is an important developmental process in potatoes, but it is highly affected by environmental conditions. Temperature is a major environmental factor affecting tuberization, with high temperatures suppressing tuber development. However, the temporal aspects of thermo-responsive tuberization remain elusive. In this study, we show that FT homolog StSP6A is suppressed by temporally distinct regulatory pathways. Experiments using StSP6A-overexpressing plants show that post-transcriptional regulation plays a major role at the early stage, while transcriptional regulation is an important late-stage factor, suppressing StSP6A at high temperatures in leaves. Overexpression of StSP6A in leaves restores tuber formation but does not recover tuber yield at the late stage, possibly because of suppressed sugar transport at high temperatures. Transcriptome analyses lead to the identification of potential regulators that may be involved in thermo-responsive tuberization at different stages. Our work shows that potato has temporally distinct molecular mechanisms that finely control tuber development at high temperatures.

Complete genome sequence of a putative novel alphaendornavirus isolated from Fagopyrum esculentum in South Korea.

The complete genome sequence of a putative new virus isolate, provisionally named "Fagopyrum esculentum endornavirus 2" (FeEV2), is 15,706 nucleotides long with a single, large open reading frame and a typical endornavirus genome organization. FeEV2 shares 19.4%-22.1% nucleotide sequence identity with other known endornavirus genome sequences. The putative polyprotein, RNA-dependent RNA polymerase (RdRp), helicase, and glycosyltransferase (GT) share 10.6%-24.3%, 30.4%-66.1%, 16.3%-45.7%, and 10.1%-21.6% amino acid sequence identity, respectively, with the homologous sequenced proteins from known endornaviruses. This suggests that it is a member of a new, distinct species. Phylogenetic analysis of RdRp sequences places FeEV2 with other Alphaendornavirus genus members (family Endornaviridae). This is the first report of the complete genome sequence of FeEV2, which was isolated from Fagopyrum esculentum in South Korea.

The Arabidopsis cyclophilin CYP18-1 facilitates PRP18 dephosphorylation and the splicing of introns retained under heat stress.

In plants, heat stress induces changes in alternative splicing, including intron retention; these events can rapidly alter proteins or downregulate protein activity, producing nonfunctional isoforms or inducing nonsense-mediated decay of messenger RNA (mRNA). Nuclear cyclophilins (CYPs) are accessory proteins in the spliceosome complexes of multicellular eukaryotes. However, whether plant CYPs are involved in pre-mRNA splicing remain unknown. Here, we found that Arabidopsis thaliana CYP18-1 is necessary for the efficient removal of introns that are retained in response to heat stress during germination. CYP18-1 interacts with Step II splicing factors (PRP18a, PRP22, and SWELLMAP1) and associates with the U2 and U5 small nuclear RNAs in response to heat stress. CYP18-1 binds to phospho-PRP18a, and increasing concentrations of CYP18-1 are associated with increasing dephosphorylation of PRP18a. Furthermore, interaction and protoplast transfection assays revealed that CYP18-1 and the PP2A-type phosphatase PP2A B'η co-regulate PRP18a dephosphorylation. RNA-seq and RT-qPCR analysis confirmed that CYP18-1 is essential for splicing introns that are retained under heat stress. Overall, we reveal the mechanism of action by which CYP18-1 activates the dephosphorylation of PRP18 and show that CYP18-1 is crucial for the efficient splicing of retained introns and rapid responses to heat stress in plants.

First Report of Cotton Leafroll Dwarf Virus Infecting Hibiscus syriacus in South Korea.

Three cotton leafroll dwarf virus (CLRDV; genus Polerovirus, family Solemoviridae) genotypes have recently been identified (Tabassum et al., 2021; Ramos-Sobrinho et al., 2021). This virus is widespread in the United States (Thiessen et al., 2020; Aboughanem-Sabanadzovic et al., 2019; Tabassum et al., 2020) and has also been reported to infect chickpea (Cicer arietinum) in Uzbekistan (Kumari et al., 2020). As well, CLRDV was detected from 23 weed species (16 families), including Hibiscus sabdariffa (Sedhain et al., 2021, Hagan et al., 2019). From June to September 2019, virus-like symptoms, including mild leaf stunting, crinkling, and deformation, were observed in multiple plants (n=14) in several provinces of South Korea (e-Xtra Table. 1). To characterize the associated viruses, pooled leaf tissues from all 14 samples were used for total RNA isolation, followed by paired-end high-throughput sequencing (HTS) on the Illumina NovaSeq 6000 platform (Macrogen, South Korea). A total of 614,424,952 trimmed and high-quality reads were assembled into 506,024 contigs using Trinity de novo transcriptome assembly. The resulting contigs were compared with viral sequences in the GenBank database using BLASTx analysis. Several viral contigs were identified, including cucumber mosaic virus, apple stem pitting virus, apple stem grooving virus, cherry virus A, and CLRDV. The CLRDV contig of 5,800 nucleotides (nt) with an average coverage of 307x shared 92.1% identity (query coverage: 99%) with the CLRDV isolate CN-S5 (KX588248). To confirm CLRDV infection and to obtain its complete genome sequence, total RNA was extracted from each of the 14 samples and used for reverse transcription (RT)-PCR with six overlapping sets of primers designed from the HTS contig (e-Xtra Table. 2). The expected product sizes were obtained only for the Hibiscus syriacus L. (family: Malvaceae) sample showing foliar mild vein clearing symptoms on the leaves (e-Xtra Fig.1). All RT-PCR products were cloned using the RBC TA Cloning vector (Taipei, Taiwan) and at least five positive clones per cloned DNA fragment were sequenced. The 5 and 3 termini sequences were determined as described previously (Zhao et al. 2016). The complete genome of CLRDV isolate SK (OK073299) was determined to be 5,862 nt and it shared 89-91% complete genome identity with 12 other CLRDV isolates based on pairwise comparisons (e-Xtra Table. 3). Maximum likelihood phylogenetic analysis based on the complete genome and P3-CP aa sequences showed that CLRDV-SK is more closely related CN-S5 (e-Xtra Fig. 2). In the fall of 2021, additional H. syriacus samples (n=18) with mild chlorosis, blistering and crinkling symptoms were collected from 2 provinces of South Korea and tested by RT-PCR using the primers: CLRDV-SK-101-120 For & CLRDV-SK-1021-1040 Rev targeting a region of the ORF0. Two of 18 samples (11.1%) tested positive for CLRDV. The 16 negative samples only showed symptoms of mild yellowing. The RT-PCR products were cloned and sequenced. In pairwise comparisons, the obtained sequences (OM339522-23) were 95.85% and 96.06% identical to the corresponding sequences of CLRDV isolate SK. This is the first report of CLRDV occurrence in H. syriacus in South Korea to the best of our knowledge. Our findings will assist further studies on the epidemiology and sustainable management of diseases caused by CLRDV. Acknowledgments This work was supported by IPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries; Project No. AGC1762111), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. References Tabassum, A., et al., 2021. PloS One. 16: e0252523 Ramos-Sobrinho, R., et al., 2021. Viruses. 13:2230 Thiessen, L.D., et al. 2020. Plant Dis. 104:3275 Aboughanem-Sabanadzovic, N., et al. 2019. Plant Dis. 103:1798 Tabassum, A., et al. 2020. Microbiol. Res. Announce. 9:e00812-20 Kumari, S.G., et al. 2021. Plant Dis. 104:2532 Sedhain, N.P., et al. 2021. Crop protection 144:105604 Hagan, A., er al. 2019. Alabama Cooperative Extension System. ANR:2539 Zhao, F., et al. 2016. Arch. Virol. 161:2047 Conflict of interest The authors declare that they have no conflict of interest.

Construction of SARS-CoV-2 virus-like particles in plant.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

Complete genome sequence of Plantago asiatica virus A, a novel putative member of the genus Polerovirus.

Here, we report the complete genome sequence of a novel polerovirus, "Plantago asiatica virus A" (PlaVA), detected in Plantago asiatica using high-throughput RNA sequencing and validated by Sanger sequencing. The complete PlaVA genome contains 5,881 nucleotides and has seven open reading frames (ORF0-5 and ORF3a) encoding putative proteins (P0-5 and P3a, respectively) in an arrangement that is similar to that of typical Polerovirus members. Pairwise sequence comparisons revealed that P0 to P5 encoded by PlaVA had the highest sequence identity (25.48%-79.21%) to the corresponding proteins of previously reported poleroviruses. A phylogenetic analysis using the PlaVA P1-2 and P3 amino acid sequences and those of members of the family Solemoviridae (formerly Luteoviridae) indicated that although PlaVA belongs to the genus Polerovirus, it does not represent a known species. Consequently, PlaVA should be considered a member of a new species within the genus Polerovirus.

Suppression of Phytochrome-Interacting Factors Enhances Photoresponses of Seedlings and Delays Flowering With Increased Plant Height in Brachypodium distachyon.

Phytochromes are red and far-red photoreceptors that regulate plant growth and development under ambient light conditions. During phytochrome-mediated photomorphogenesis, phytochrome-interacting factors (PIFs) are the most important signaling partners that regulate the expression of light-responsive genes. However, the function of PIFs in monocots has not been studied well. In this study, using RNA interference (RNAi), we investigated the functions of BdPIL1 and BdPIL3, two PIF-like genes identified in Brachypodium distachyon, which are closely related to Arabidopsis PIF1 and PIF3. The expression of their genes is light-inducible, and both BdPIL1 and BdPIL3 proteins interact with phytochromes in an active form-specific manner. Transgenic Brachypodium seedlings with the RNAi constructs of BdPIL1 and BdPIL3 showed decreased coleoptile lengths and increased leaf growth when exposed to both red and far-red light. In addition, the transgenic plants were taller with elongated internodes than wild-type Bd21-3 plant, exhibiting late flowering. Moreover, RNA-seq analysis revealed downregulation of many genes in the transgenic plants, especially those related to the regulation of cell number, floral induction, and chlorophyll biosynthesis, which were consistent with the phenotypes of increased plant height, delayed flowering, and pale green leaves. Furthermore, we demonstrated the DNA-binding ability of BdPIL1 and BdPIL3 to the putative target promoters and that the DNA-binding was inhibited in the presence of phytochromes. Therefore, this study determines a molecular mechanism underlying phytochrome-mediated PIF regulation in Brachypodium, i.e., sequestration, and also elucidates the functions of BdPIL1 and BdPIL3 in the growth and development of the monocot plant.