Prime editing: Mechanism insight and recent applications in plants.

Prime editing (PE) technology utilizes an extended prime editing guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) to a specific location in the genome. This enables the installation of base changes at the targeted site using the extended portion of the pegRNA through RT activity. The resulting product of the RT reaction forms a 3' flap, which can be incorporated into the genomic site through a series of biochemical steps involving DNA repair and synthesis pathways. PE has demonstrated its effectiveness in achieving almost all forms of precise gene editing, such as base conversions (all types), DNA sequence insertions and deletions, chromosomal translocation and inversion and long DNA sequence insertion at safe harbour sites within the genome. In plant science, PE could serve as a groundbreaking tool for precise gene editing, allowing the creation of desired alleles to improve crop varieties. Nevertheless, its application has encountered limitations due to efficiency constraints, particularly in dicotyledonous plants. In this review, we discuss the step-by-step mechanism of PE, shedding light on the critical aspects of each step while suggesting possible solutions to enhance its efficiency. Additionally, we present an overview of recent advancements and future perspectives in PE research specifically focused on plants, examining the key technical considerations of its applications.

C-Type LECTIN receptor-like kinase 1 and ACTIN DEPOLYMERIZING FACTOR 3 are key components of plasmodesmata callose modulation.

Plasmodesmata (PDs) are intercellular organelles carrying multiple membranous nanochannels that allow the trafficking of cellular signalling molecules. The channel regulation of PDs occurs dynamically and is required in various developmental and physiological processes. It is well known that callose is a critical component in regulating PD permeability or symplasmic connectivity, but the understanding of the signalling pathways and mechanisms of its regulation is limited. Here, we used the reverse genetic approach to investigate the role of C-type lectin receptor-like kinase 1 (CLRLK1) in the aspect of PD callose-modulated symplasmic continuity. Here, we found that loss-of-function mutations in CLRLK1 resulted in excessive PD callose deposits and reduced symplasmic continuity, resulting in an accelerated gravitropic response. The protein interactome study also found that CLRLK1 interacted with actin depolymerizing factor 3 (ADF3) in vitro and in plants. Moreover, mutations in ADF3 result in elevated PD callose deposits and faster gravitropic response. Our results indicate that CLRLK1 and ADF3 negatively regulate PD callose accumulation, contributing to fine-tuning symplasmic opening apertures. Overall, our studies identified two key components involved in the deposits of PD callose and provided new insights into how symplasmic connectivity is maintained by the control of PD callose homoeostasis.

Generation of parthenocarpic tomato plants in multiple elite cultivars using the CRISPR/Cas9 system.

Tomato (Solanum lycopersicum L.) is one of the most important crops in the world for its fruit production. Advances in cutting-edge techniques have enabled the development of numerous critical traits related to the quality and quantity of tomatoes. Genetic engineering techniques, such as gene transformation and gene editing, have emerged as powerful tools for generating new plant varieties with superior traits. In this study, we induced parthenocarpic traits in a population of elite tomato (ET) lines. At first, the adaptability of ET lines to genetic transformation was evaluated to identify the best-performing lines by transforming the SlANT1 gene overexpression cassette and then later used to produce the SlIAA9 knockout lines using the CRISPR/Cas9 system. ET5 and ET8 emerged as excellent materials for these techniques and showed higher efficiency. Typical phenotypes of knockout sliaa9 were clearly visible in G0 and G1 plants, in which simple leaves and parthenocarpic fruits were observed. The high efficiency of the CRISPR/Cas9 system in developing new tomato varieties with desired traits in a short period was demonstrated by generating T-DNA-free homozygous sliaa9 knockout plants in the G1 generation. Additionally, a simple artificial fertilization method was successfully applied to recover seed production from parthenocarpic plants, securing the use of these varieties as breeding materials. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01452-1.

α1-COP modulates plasmodesmata function through sphingolipid enzyme regulation.

Callose, a ß-1,3-glucan plant cell wall polymer, regulates symplasmic channel size at plasmodesmata (PD) and plays a crucial role in a variety of plant processes. However, elucidating the molecular mechanism of PD callose homeostasis is limited. We screened and identified an Arabidopsis mutant plant with excessive callose deposition at PD and found that the mutated gene was α1-COP, a member of the coat protein I (COPI) coatomer complex. We report that loss of function of α1-COP elevates the callose accumulation at PD by affecting subcellular protein localization of callose degradation enzyme PdBG2. This process is linked to the functions of ERH1, an inositol phosphoryl ceramide synthase, and glucosylceramide synthase through physical interactions with the α1-COP protein. Additionally, the loss of function of α1-COP alters the subcellular localization of ERH1 and GCS proteins, resulting in a reduction of GlcCers and GlcHCers molecules, which are key sphingolipid (SL) species for lipid raft formation. Our findings suggest that α1-COP protein, together with SL modifiers controlling lipid raft compositions, regulates the subcellular localization of GPI-anchored PDBG2 proteins, and hence the callose turnover at PD and symplasmic movement of biomolecules. Our findings provide the first key clue to link the COPI-mediated intracellular trafficking pathway to the callose-mediated intercellular signaling pathway through PD.

Profiling of conserved orthologs and miRNAs for understanding their role in salt tolerance mechanism of Sesuvium portulacastrum L.

BACKGROUND: Sesuvium portulacastrum is a facultative halophyte capable of thriving in a saline environment. Despite molecular studies conducted to unravel its salt adaptation mechanism, there is a paucity of information on the role of salt-responsive orthologs and microRNAs (miRNAs) in this halophyte. Here, we searched the orthology to identify salt-responsive orthologs and miRNA targets of Sesuvium using the Arabidopsis genome. METHODS: The relative fold change of orthologs, conserved miRNAs, and miRNA targets of Sesuvium was analyzed under 100 mM (LS) and 250 mM NaCl (HS) treatment at 24 h using qRT-PCR. The comparison between the expression of Sesuvium orthologs and Arabidopsis orthologs (Arabidopsis eFP browser database) was used to identify differentially expressed genes. RESULTS: Upon salt treatment, we found that SpCIPK3 (1.95-fold in LS and 2.90-fold in HS) in Sesuvium roots, and SpNHX7 (1.61-fold in LS and 6.39-fold in HS) and, SpSTPK2 (2.54-fold in LS and 7.65-fold in HS) in Sesuvium leaves were upregulated in a salt concentration-specific manner. In Arabidopsis, these genes were either downregulated or did not show significant variation, implicating its significance in the halophytic nature of Sesuvium. Furthermore, miRNAs like miR394a, miR396a, and miR397a exhibited a negative correlation with their targets-Frigida interacting protein 1, Cysteine proteinases superfamily protein, and Putative laccase, respectively under different salt treatments. CONCLUSION: The study revealed that the high salt tolerance in Sesuvium is associated with distinct transcriptional reprogramming, hence, to gain holistic mechanistic insights, global-scale profiling is required.

Genome editing and beyond: what does it mean for the future of plant breeding?

MAIN CONCLUSION: Genome editing offers revolutionized solutions for plant breeding to sustain food production to feed the world by 2050. Therefore, genome-edited products are increasingly recognized via more relaxed legislation and community adoption. The world population and food production are disproportionally growing in a manner that would have never matched each other under the current agricultural practices. The emerging crisis is more evident with the subtle changes in climate and the running-off of natural genetic resources that could be easily used in breeding in conventional ways. Under these circumstances, affordable CRISPR-Cas-based gene-editing technologies have brought hope and charged the old plant breeding machine with the most energetic and powerful fuel to address the challenges involved in feeding the world. What makes CRISPR-Cas the most powerful gene-editing technology? What are the differences between it and the other genetic engineering/breeding techniques? Would its products be labeled as "conventional" or "GMO"? There are so many questions to be answered, or that cannot be answered within the limitations of our current understanding. Therefore, we would like to discuss and answer some of the mentioned questions regarding recent progress in technology development. We hope this review will offer another view on the role of CRISPR-Cas technology in future of plant breeding for food production and beyond.

In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components.

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.

CRISPR/Cas-based precision genome editing via microhomology-mediated end joining.

Gene editing and/or allele introgression with absolute precision and control appear to be the ultimate goals of genetic engineering. Precision genome editing in plants has been developed through various approaches, including oligonucleotide-directed mutagenesis (ODM), base editing, prime editing and especially homologous recombination (HR)-based gene targeting. With the advent of CRISPR/Cas for the targeted generation of DNA breaks (single-stranded breaks (SSBs) or double-stranded breaks (DSBs)), a substantial advancement in HR-mediated precise editing frequencies has been achieved. Nonetheless, further research needs to be performed for commercially viable applications of precise genome editing; hence, an alternative innovative method for genome editing may be required. Within this scope, we summarize recent progress regarding precision genome editing mediated by microhomology-mediated end joining (MMEJ) and discuss their potential applications in crop improvement.

Sphingolipids Modulate Secretion of Glycosylphosphatidylinositol-Anchored Plasmodesmata Proteins and Callose Deposition.

Plasma membranes encapsulated in the symplasmic nanochannels of plasmodesmata (PD) contain abundant lipid rafts, which are enriched with sphingolipids (SLs) and sterols. Reduction of sterols has highlighted the role played by lipid raft integrity in the intercellular trafficking of glycosylphosphatidylinositol (GPI)-anchored PD proteins, particularly in affecting callose enhancement. The presence of callose at PD is strongly attributed to the regulation of callose accumulation and callose degradation by callose synthases and ß-1,3-glucanases (BGs), respectively. SLs are implicated in signaling and membrane protein trafficking; however, the underlying processes linking SL composition to the control of symplasmic apertures remain unknown. The wide variety of SLs in plants prompted us to investigate which SL molecules are important for regulating symplasmic apertures in Arabidopsis (Arabidopsis thaliana). We introduced several potential SL pathway inhibitors and genetically modified SL contents using two independent SL pathway mutants. We were able to modulate callose deposition to control symplasmic connectivity through perturbations of SL metabolism. Alteration in glucosylhydroxyceramides or related SL composition particularly disturbed the secretory machinery for the GPI-anchored PdBG2 protein, resulting in an overaccumulation of callose. Moreover, our results revealed that SL-enriched lipid rafts link symplasmic channeling to PD callose homeostasis by controlling the targeting of GPI-anchored PdBG2. This study elevates our understanding of the molecular linkage underlying intracellular trafficking and precise targeting of GPI-anchored PD proteins incorporating glucosyl SLs.

CRISPR/Cas9-based precise excision of SlHyPRP1 domain(s) to obtain salt stress-tolerant tomato.

KEY MESSAGE: CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.

Conserved Opposite Functions in Plant Resistance to Biotrophic and Necrotrophic Pathogens of the Immune Regulator SRFR1.

Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.

CRISPR/Cas9-Mediated Generation of Pathogen-Resistant Tomato against Tomato Yellow Leaf Curl Virus and Powdery Mildew.

Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.

Highly efficient homology-directed repair using CRISPR/Cpf1-geminiviral replicon in tomato.

Genome editing via the homology-directed repair (HDR) pathway in somatic plant cells is very inefficient compared with error-prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR-based genome editing efficiency approximately threefold compared with a Cas9-based single-replicon system via the use of de novo multi-replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon-free but stable HDR alleles. The efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31 °C under a light/dark cycle after Agrobacterium-mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single-replicon system into a multi-replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR-based genome editing of a salt-tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self-pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mm NaCl. Our work may pave the way for transgene-free editing of alleles of interest in asexually and sexually reproducing plants.

Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in Arabidopsis.

The expression of CBF (C-repeat-binding factor) genes is required for freezing tolerance in Arabidopsis thaliana. CBFs are positively regulated by INDUCER OF CBF EXPRESSION1 (ICE1) and negatively regulated by MYB15. These transcription factors directly interact with specific elements in the CBF promoters. Mitogen-activated protein kinase (MAPK/MPK) cascades function upstream to regulate CBFs. However, the mechanism by which MPKs control CBF expression during cold stress signaling remains unknown. This study showed that the activity of MYB15, a transcriptional repressor of cold signaling, is regulated by MPK6-mediated phosphorylation. MYB15 specifically interacts with MPK6, and MPK6 phosphorylates MYB15 on Ser168. MPK6-induced phosphorylation reduced the affinity of MYB15 binding to the CBF3 promoter and mutation of its phosphorylation site (MYB15S168A) enhanced the transcriptional repression of CBF3 by MYB15. Furthermore, transgenic plants overexpressing MYB15S168A showed significantly reduced CBF transcript levels in response to cold stress, compared with plants overexpressing MYB15. The MYB15S168A-overexpressing plants were also more sensitive to freezing than MYB15-overexpressing plants. These results suggest that MPK6-mediated regulation of MYB15 plays an important role in cold stress signaling in Arabidopsis.

Callose balancing at plasmodesmata.

In plants, communication and molecular exchanges between different cells and tissues are dependent on the apoplastic and symplastic pathways. Symplastic molecular exchanges take place through the plasmodesmata, which connect the cytoplasm of neighboring cells in a highly controlled manner. Callose, a ß-1,3-glucan polysaccharide, is a plasmodesmal marker molecule that is deposited in cell walls near the neck zone of plasmodesmata and controls their permeability. During cell differentiation and plant development, and in response to diverse stresses, the level of callose in plasmodesmata is highly regulated by two antagonistic enzymes, callose synthase or glucan synthase-like and ß-1,3-glucanase. The diverse modes of regulation by callose synthase and ß-1,3-glucanase have been uncovered in the past decades through biochemical, molecular, genetic, and omics methods. This review highlights recent findings regarding the function of plasmodesmal callose and the molecular players involved in callose metabolism, and provides new insight into the mechanisms maintaining plasmodesmal callose homeostasis.

The protein kinase TOUSLED facilitates RNAi in Arabidopsis.

RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA. These data suggest a possible role for TSL phosphorylation in the specific regulation of exogenous and antiviral RNA silencing in Arabidopsis and identify TSL as an intrinsic regulator of RNA interference.

Cell-to-cell movement of viruses via plasmodesmata.

Plant viruses utilize plasmodesmata (PD), unique membrane-lined cytoplasmic nanobridges in plants, to spread infection cell-to-cell and long-distance. Such invasion involves a range of regulatory mechanisms to target and modify PD. Exciting discoveries in this field suggest that these mechanisms are executed by the interaction between plant cellular components and viral movement proteins (MPs) or other virus-encoded factors. Striking working analogies exist among endogenous non-cell-autonomous proteins and viral MPs, in which not only do they all use PD to traffic, but also they exploit same regulatory components to exert their functions. Thus, this review discusses on the viral strategies to move via PD and the PD-regulatory mechanisms involved in viral pathogenesis.

N-glycan maturation is crucial for cytokinin-mediated development and cellulose synthesis in Oryza sativa.

To explore the physiological significance of N-glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N-acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N-glycan maturation and accumulated high-mannose N-glycans. Phenotypic analyses revealed that gnt1 shows defective post-seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark-induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A-type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N-glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.

Transcription factor-mediated cell-to-cell signalling in plants.

Plant cells utilize mobile transcription factors to transmit intercellular signals when they perceive environmental stimuli or initiate developmental programmes. Studies on these novel cell-to-cell signals have accumulated multiple pieces of evidence showing that non-cell-autonomous transcription factors play pivotal roles in most processes related to the formation and development of plant organs. Recent studies have explored the evolution of mobile transcription factors and proposed mechanisms for their trafficking through plasmodesmata, where a selective system exists to facilitate this process. Mobile transcription factors contribute to the diversity of the intercellular signalling network, which is also established by peptides, hormones, and RNAs. Crosstalk between mobile transcription factors and other intercellular molecules leads to the development of complex biological signalling networks in plants. The regulation of plasmodesmata appears to have been another major step in controlling the intercellular trafficking of transcription factors based on studies of many plasmodesmal components. Furthermore, diverse omics approaches are being successfully applied to explore a large number of candidate transcription factors as mobile signals in plants. Here, we review these fascinating discoveries to integrate current knowledge of non-cell-autonomous transcription factors.

RNA-seq analysis of Rubus idaeus cv. Nova: transcriptome sequencing and de novo assembly for subsequent functional genomics approaches.

KEY MESSAGE: Using Illumina sequencing technology, we have generated the large-scale transcriptome sequencing data containing abundant information on genes involved in the metabolic pathways in R. idaeus cv. Nova fruits. Rubus idaeus (Red raspberry) is one of the important economical crops that possess numerous nutrients, micronutrients and phytochemicals with essential health benefits to human. The molecular mechanism underlying the ripening process and phytochemical biosynthesis in red raspberry is attributed to the changes in gene expression, but very limited transcriptomic and genomic information in public databases is available. To address this issue, we generated more than 51 million sequencing reads from R. idaeus cv. Nova fruit using Illumina RNA-Seq technology. After de novo assembly, we obtained 42,604 unigenes with an average length of 812 bp. At the protein level, Nova fruit transcriptome showed 77 and 68 % sequence similarities with Rubus coreanus and Fragaria versa, respectively, indicating the evolutionary relationship between them. In addition, 69 % of assembled unigenes were annotated using public databases including NCBI non-redundant, Cluster of Orthologous Groups and Gene ontology database, suggesting that our transcriptome dataset provides a valuable resource for investigating metabolic processes in red raspberry. To analyze the relationship between several novel transcripts and the amounts of metabolites such as γ-aminobutyric acid and anthocyanins, real-time PCR and target metabolite analysis were performed on two different ripening stages of Nova. This is the first attempt using Illumina sequencing platform for RNA sequencing and de novo assembly of Nova fruit without reference genome. Our data provide the most comprehensive transcriptome resource available for Rubus fruits, and will be useful for understanding the ripening process and for breeding R. idaeus cultivars with improved fruit quality.