A multiple shoot induction system for peptide-mediated gene delivery into plastids in Arabidopsis thaliana, Nicotiana benthamiana, and Fragaria×ananassa.

The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.

Functional peptide-mediated plastid transformation in tobacco, rice, and kenaf.

In plant engineering, plastid transformation is more advantageous than nuclear transformation because it results in high levels of protein expression from multiple genome copies per cell and is unaffected by gene silencing. The common plastid transformation methods are biolistic bombardment that requires special instruments and PEG-mediated transformation that is only applicable to protoplast cells. Here, we aimed to establish a new plastid transformation method in tobacco, rice, and kenaf using a biocompatible fusion peptide as a carrier to deliver DNA into plastids. We used a fusion peptide, KH-AtOEP34, comprising a polycationic DNA-binding peptide (KH) and a plastid-targeting peptide (AtOEP34) to successfully deliver and integrate construct DNA into plastid DNA (ptDNA) via homologous recombination. We obtained transformants in each species using selection with spectinomycin/streptomycin and the corresponding resistance gene aadA. The constructs remained in ptDNA for several months after introduction even under non-selective condition. The transformants normally flowered and are fertile in most cases. The offspring of the transformants (the T1 generation) retained the integrated construct DNA in their ptDNA, as indicated by PCR and DNA blotting, and expressed GFP in plastids from the integrated construct DNA. In summary, we successfully used the fusion peptide method for integration of foreign DNA in tobacco, rice, and kenaf ptDNA, and the integrated DNA was transmitted to the next generations. Whereas optimization is necessary to obtain homoplasmic plastid transformants that enable stable heterologous expression of genes, the plastid transformation method shown here is a novel nanomaterial-based approach distinct from the conventional methods, and we propose that this easy method could be used to target a wide variety of plants.

A collection of inducible transcription factor-glucocorticoid receptor fusion lines for functional analyses in Arabidopsis thaliana.

Arabidopsis possesses approximately 2000 transcription factors (TFs) in its genome. They play pivotal roles in various biological processes but analysis of their function has been hampered by the overlapping nature of their activities. To uncover clues to their function, we generated inducible TF lines using glucocorticoid receptor (GR) fusion techniques in Arabidopsis. These TF-GR lines each express one of 1255 TFs as a fusion with the GR gene. An average 14 lines of T2 transgenic TF-GR lines were generated for each TF to monitor their function. To evaluate these transcription lines, we induced the TF-GR lines of phytochrome-interacting factor 4, which controls photomorphogenesis, with synthetic glucocorticoid dexamethasone. These phytochrome-interacting factor 4-GR lines showed the phenotype described in a previous report. We performed screening of the other TF-GR lines for TFs involved in light signaling under blue and far-red light conditions and identified 13 novel TF candidates. Among these, we found two lines showing higher anthocyanin accumulation under light conditions and we examined the regulating genes. These results indicate that the TF-GR lines can be used to dissect functionally redundant genes in plants and demonstrate that the TF-GR line collection can be used as an effective tool for functional analysis of TFs.

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts.

Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.

Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation.

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

Efficient callus induction and a temperature condition for flowering and seed setting in kenaf Hibiscus cannabinus.

Kenaf, Hibiscus cannabinus, is a fiber-enriched plant belonging to Malvaceae and is an important fiber crop. The features of kenaf of being fast-growing and fiber-enriched suggest the potential for the use of kenaf in biomass and materials. Here, we modified procedures for regeneration from kenaf explants in order to establish efficient genetic modification for improving the property of kenaf as a material. We tested several tissues of kenaf seedlings for callus induction and subsequent shoot regeneration by supplying several combinations of plant growth factors. We show the cotyledons to be the best tissue for efficient callus induction, and the rate for callus induction reached ∼98% on an improved callus inducing medium. We also show the efficient regeneration of kenaf from cotyledon-derived calli achieved by shoot induction on a shoot-inducing medium. We also achieved seed setting of the regenerated kanaf plants under a regulated growth condition in a chamber. Our efficient regeneration method and seed setting condition will enable the production of stably transformed kenaf that can improve the properties of kenaf as a material.

Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus.

Cell-penetrating peptides (CPPs) are used for various applications, especially in the biomedical field. Recently, CPPs have been used as a part of carrier to deliver proteins and/or genes into plant cells and tissues; hence, these peptides are attractive tools for plant biotechnological and agricultural applications, but require more efficient delivery rates and optimization by species before wide-scale use can be achieved. Here, we developed a library containing 55 CPPs to determine the optimal CPP characteristics for penetration of BY-2 cells and leaves of Nicotiana benthamiana, Arabidopsis thaliana, tomato (Solanum lycopersicum), poplar (hybrid aspen Populus tremula × tremuloides line T89), and rice (Oryza sativa). By investigating the cell penetration efficiency of CPPs in the library, we identified several efficient CPPs for all the plants studied except rice leaf. In the case of rice, several CPPs showed efficient penetration into rice callus. Furthermore, we examined the relationship between cell penetration efficiency and CPP secondary structural characteristics. The cell penetration efficiency of Lys-containing CPPs was relatively greater in plant than in animal cells, which could be due to differences in lipid composition and surface charge of the cell membranes. The variation in optimal CPPs across the plants studied here suggests that CPPs must be optimized for each plant species and target tissues of interest.

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance.

The recent developments of transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) have expanded plant breeding technology. One technical issue related to the current genome editing process is residual transgenes for TALEN and CRISPR/Cas9 left in plant genomes after the editing process. Here, we aim to add transient kanamycin resistance into apple leaf cells by introducing neomycin phosphotransferase II (NPTII) into apple leaf cells using the fusion peptide system. At 75 mg/L of kanamycin for 2 days, apple JM1 leaf cells infiltrated with NPTII could be selected. Thus, we successfully demonstrated the first transient selection system of plant cells using a fusion peptide-mediated protein delivery system.

Small open reading frames associated with morphogenesis are hidden in plant genomes.

It is likely that many small ORFs (sORFs; 30-100 amino acids) are missed when genomes are annotated. To overcome this limitation, we identified ∼8,000 sORFs with high coding potential in intergenic regions of the Arabidopsis thaliana genome. However, the question remains as to whether these coding sORFs play functional roles. Using a designed array, we generated an expression atlas for 16 organs and 17 environmental conditions among 7,901 identified coding sORFs. A total of 2,099 coding sORFs were highly expressed under at least one experimental condition, and 571 were significantly conserved in other land plants. A total of 473 coding sORFs were overexpressed; ∼10% (49/473) induced visible phenotypic effects, a proportion that is approximately seven times higher than that of randomly chosen known genes. These results indicate that many coding sORFs hidden in plant genomes are associated with morphogenesis. We believe that the expression atlas will contribute to further study of the roles of sORFs in plants.

A comprehensive analysis of interaction and localization of Arabidopsis SKP1-like (ASK) and F-box (FBX) proteins.

F-Box (FBX) proteins are encoded by a multigene family present in major lineages of eukaryotes. A number of FBX proteins are shown to be subunits of SCF complex, a type of E3 ligases composed of SKP1, CULLIN, FBX and RBX1 proteins. The Arabidopsis SKP-LIKE (ASK) proteins are also members of a family and some of them interact with FBX proteins directly. To clarify how FBX and ASK proteins combine, we carried out a large-scale interaction analysis between FBX and ASK proteins using yeast two-hybrid assay (Y2H) in Arabidopsis thaliana. FBX proteins randomly chosen from those proteins that interacted with more than one ASK protein were further analyzed for their subcellular localization and in vivo interaction with ASK proteins. Furthermore, the expression profiles of FBX and ASK genes were compared. This work reveals that FBX proteins had a preference for interacting with ASK proteins depending on the domains they contain such as the FBX-associated (FBA) domain, the Kelch domain and leucine rich repeat (LRR). In addition, it was found that a single FBX protein could form multiple SCF complexes by interacting with several ASK proteins in many cases. Furthermore, it was suggested that the variation of SCF complexes were especially abundant in tissues related to male gametophyte and seed development. More than half of the FBX proteins studied did not interact with any of the ASK proteins, implying the necessity for certain regulations for their interaction in vivo and/or distinct roles from subunits of the SCF complex.

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice.

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.

Metabolomic screening applied to rice FOX Arabidopsis lines leads to the identification of a gene-changing nitrogen metabolism.

Plant metabolomics developed as a powerful tool to examine gene functions and to gain deeper insight into the physiology of the plant cell. In this study, we screened Arabidopsis lines overexpressing rice full-length (FL) cDNAs (rice FOX Arabidopsis lines) using a gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS)-based technique to identify rice genes that caused metabolic changes. This screening system allows fast and reliable identification of candidate lines showing altered metabolite profiles. We performed metabolomic and transcriptomic analysis of a rice FOX Arabidopsis line that harbored the FL cDNA of the rice ortholog of the Lateral Organ Boundaries (LOB) Domain (LBD)/Asymmetric Leaves2-like (ASL) gene of Arabidopsis, At-LBD37/ASL39. The investigated rice FOX Arabidopsis line showed prominent changes in the levels of metabolites related to nitrogen metabolism. The transcriptomic data as well as the results from the metabolite analysis of the Arabidopsis At-LBD37/ASL39-overexpressor plants were consistent with these findings. Furthermore, the metabolomic and transcriptomic analysis of the Os-LBD37/ASL39-overexpressing rice plants indicated that Os-LBD37/ASL39 is associated with processes related to nitrogen metabolism in rice. Thus, the combination of a metabolomics-based screening method and a gain-of-function approach is useful for rapid characterization of novel genes in both Arabidopsis and rice.

Systematic approaches to using the FOX hunting system to identify useful rice genes.

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23,000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis. This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.