Peroxisomal 4-coumaroyl-CoA ligases participate in shikonin production in Lithospermum erythrorhizon.

4-Coumaroyl-CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway, which is involved in the biosynthesis of various specialized metabolites such as flavonoids, coumarins, lignans, and lignin. Plants have several 4CLs showing divergence in sequence: class I 4CLs involved in lignin metabolism, class II 4CLs associated with flavonoid metabolism, and atypical 4CLs and 4CL-like proteins of unknown function. Shikonin, a Boraginaceae-specific specialized metabolite in red gromwell (Lithospermum erythrorhizon), is biosynthesized from p-hydroxybenzoic acid, and the involvement of 4CL in its biosynthesis has long been debated. In this study, we demonstrated the requirement of 4CL for shikonin biosynthesis using a 4CL-specific inhibitor. In silico analysis of the L. erythrorhizon genome revealed the presence of at least eight 4CL genes, among which the expression of three (Le4CL3, Le4CL4, and Le4CL5) showed a positive association with shikonin production. Phylogenetic analysis indicated that Le4CL5 belongs to class I 4CLs, while Le4CL3 and Le4CL4 belong to clades that are distant from class I and class II. Interestingly, both Le4CL3 and Le4CL4 have peroxisome targeting signal 1 in their C-terminal region, and subcellular localization analysis revealed that both localize to the peroxisome. We targeted each of the three Le4CL genes by CRISPR/Cas9-mediated mutagenesis and observed remarkably lower shikonin production in Le4CL3-ge and Le4CL4-ge genome-edited lines compared with the vector control. We therefore conclude that peroxisomal Le4CL3 and Le4CL4 are responsible for shikonin production and propose a model for metabolite-specific 4CL distribution in L. erythrorhizon.

Disruption of p-coumaroyl-CoA:monolignol transferases in rice drastically alters lignin composition.

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants' cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries.

SULTR2;1 Adjusts the Bolting Timing by Transporting Sulfate from Rosette Leaves to the Primary Stem.

Sulfur (S) is an essential macronutrient for plant growth and metabolism. SULTR2;1 is a low-affinity sulfate transporter facilitating the long-distance transport of sulfate in Arabidopsis. The physiological function of SULTR2;1 in the plant life cycle still needs to be determined. Therefore, we analyzed the sulfate transport, S-containing metabolite accumulation and plant growth using Arabidopsis SULTR2;1 disruption lines, sultr2;1-1 and sultr2;1-2, from seedling to mature growth stages to clarify the metabolic and physiological roles of SULTR2;1. We observed that sulfate distribution to the stems was affected in sultr2;1 mutants, resulting in decreased levels of sulfate, cysteine, glutathione (GSH) and total S in the stems, flowers and siliques; however, the GSH levels increased in the rosette leaves. This suggested the essential role of SULTR2;1 in sulfate transport from rosette leaves to the primary stem. In addition, sultr2;1 mutants unexpectedly bolted earlier than the wild-type without affecting the plant biomass. Correlation between GSH levels in rosette leaves and the bolting timing suggested that the rosette leaf GSH levels or limited sulfate transport to the early stem can trigger bolting. Overall, this study demonstrated the critical roles of SULTR2;1 in maintaining the S metabolite levels in the aerial part and transitioning from the vegetative to the reproductive growth phase.

The trans-zeatin-type side-chain modification of cytokinins controls rice growth.

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.

Lignocellulose molecular assembly and deconstruction properties of lignin-altered rice mutants.

Bioengineering approaches to modify lignin content and structure in plant cell walls have shown promise for facilitating biochemical conversions of lignocellulosic biomass into valuable chemicals. Despite numerous research efforts, however, the effect of altered lignin chemistry on the supramolecular assembly of lignocellulose and consequently its deconstruction in lignin-modified transgenic and mutant plants is not fully understood. In this study, we aimed to close this gap by analyzing lignin-modified rice (Oryza sativa L.) mutants deficient in 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (CAldOMT) and CINNAMYL ALCOHOL DEHYDROGENASE (CAD). A set of rice mutants harboring knockout mutations in either or both OsCAldOMT1 and OsCAD2 was generated in part by genome editing and subjected to comparative cell wall chemical and supramolecular structure analyses. In line with the proposed functions of CAldOMT and CAD in grass lignin biosynthesis, OsCAldOMT1-deficient mutant lines produced altered lignins depleted of syringyl and tricin units and incorporating noncanonical 5-hydroxyguaiacyl units, whereas OsCAD2-deficient mutant lines produced lignins incorporating noncanonical hydroxycinnamaldehyde-derived units. All tested OsCAldOMT1- and OsCAD2-deficient mutants, especially OsCAldOMT1-deficient lines, displayed enhanced cell wall saccharification efficiency. Solid-state nuclear magnetic resonance (NMR) and X-ray diffraction analyses of rice cell walls revealed that both OsCAldOMT1- and OsCAD2 deficiencies contributed to the disruptions of the cellulose crystalline network. Further, OsCAldOMT1 deficiency contributed to the increase of the cellulose molecular mobility more prominently than OsCAD2 deficiency, resulting in apparently more loosened lignocellulose molecular assembly. Such alterations in cell wall chemical and supramolecular structures may in part account for the variations of saccharification performance of the OsCAldOMT1- and OsCAD2-deficient rice mutants.

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems.

Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.

Expanding the plant genome editing toolbox with recently developed CRISPR-Cas systems.

Since its first appearance, CRISPR-Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR-Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR-Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR-Cas systems have been reported. Among them, several CRISPR-Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR-Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR-Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR-Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR-Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR-Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR-Cas tools are helping expand our plant genome engineering toolbox.

Genome-edited rice deficient in two 4-COUMARATE:COENZYME A LIGASE genes displays diverse lignin alterations.

The 4-coumarate:coenzyme A ligase (4CL) is a key enzyme that contributes to channeling metabolic flux in the cinnamate/monolignol pathway, leading to the production of monolignols, p-hydroxycinnamates, and a flavonoid tricin, the major building blocks of lignin polymer in grass cell walls. Vascular plants often contain multiple 4CL genes; however, the contribution of each 4CL isoform to lignin biosynthesis remains unclear, especially in grasses. In this study, we characterized the functions of two rice (Oryza sativa L.) 4CL isoforms (Os4CL3 and Os4CL4) primarily by analyzing the cell wall chemical structures of rice mutants generated by CRISPR/Cas9-mediated targeted mutagenesis. A series of chemical and nuclear magnetic resonance analyses revealed that loss-of-function of Os4CL3 and Os4CL4 differently altered the composition of lignin polymer units. Loss of function of Os4CL3 induced marked reductions in the major guaiacyl and syringyl lignin units derived from both the conserved non-γ-p-coumaroylated and the grass-specific γ-p-coumaroylated monolignols, with more prominent reductions in guaiacyl units than in syringyl units. In contrast, the loss-of-function mutation to Os4CL4 primarily decreased the abundance of the non-γ-p-coumaroylated guaiacyl units. Loss-of-function of Os4CL4, but not of Os4CL3, reduced the grass-specific lignin-bound tricin units, indicating that Os4CL4 plays a key role not only in monolignol biosynthesis but also in the biosynthesis of tricin used for lignification. Further, the loss-of-function of Os4CL3 and Os4CL4 notably reduced cell-wall-bound ferulates, indicating their roles in cell wall feruloylation. Overall, this study demonstrates the overlapping but divergent roles of 4CL isoforms during the coordinated production of various lignin monomers.

Genome editing in mammalian cells using the CRISPR type I-D nuclease.

Adoption of CRISPR-Cas systems, such as CRISPR-Cas9 and CRISPR-Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I-the most abundant CRISPR system in bacteria-remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense ('Cascade': Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR-Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR-Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR-Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

OsMYB108 loss-of-function enriches p-coumaroylated and tricin lignin units in rice cell walls.

Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.

Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/Cas9 system.

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.

Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering.

Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants ("null segregants") in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research.

Downregulation of p-COUMAROYL ESTER 3-HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification.

p-Coumaroyl ester 3-hydroxylase (C3'H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3'H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3'H deficiency on the structure and properties of grass cell walls. C3'H-knockdown lines generated via RNA interference (RNAi)-mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3'H-knockout rice mutants generated via CRISPR/Cas9-mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3'H-knockdown RNAi lines revealed that their lignins were largely enriched in p-hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non-acylated lignin units, with grass-specific γ-p-coumaroylated lignin units remaining apparently unchanged. Suppression of C3'H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross-linking ferulates. Collectively, our data demonstrate that C3'H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross-linking. We also demonstrated that C3'H-suppressed rice displays enhanced biomass saccharification.

Lotus japonicus Triterpenoid Profile and Characterization of the CYP716A51 and LjCYP93E1 Genes Involved in Their Biosynthesis In Planta.

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted ß-amyrin into 24-hydroxy-ß-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.

MYB transcription factor gene involved in sex determination in Asparagus officinalis.

Dioecy is a plant mating system in which individuals of a species are either male or female. Although many flowering plants evolved independently from hermaphroditism to dioecy, the molecular mechanism underlying this transition remains largely unknown. Sex determination in the dioecious plant Asparagus officinalis is controlled by X and Y chromosomes; the male and female karyotypes are XY and XX, respectively. Transcriptome analysis of A. officinalis buds showed that a MYB-like gene, Male Specific Expression 1 (MSE1), is specifically expressed in males. MSE1 exhibits tight linkage with the Y chromosome, specific expression in early anther development and loss of function on the X chromosome. Knockout of the MSE1 orthologue in Arabidopsis induces male sterility. Thus, MSE1 acts in sex determination in A. officinalis.

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.

Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System.

Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution.

A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA.

Gene targeting (GT) is a useful technology for accurate genome engineering in plants. A reproducible approach based on a positive-negative selection system using hygromycin resistance and the diphtheria toxin A subunit gene as positive and negative selection markers, respectively, is now available. However, to date, this selection system has been applied exclusively in rice (Oryza sativa). To establish a universally applicable positive-negative GT system in plants, we designed a selection system using a combination of neomycin phosphotransferaseII (nptII) and an antisense nptII construct. The concomitant transcription of both sense and antisense nptII suppresses significantly the level of expression of the sense nptII gene, and transgenic calli and plants become sensitive to the antibiotic geneticin. In addition, we were able to utilize the sense nptII gene as a positive selection marker and the antisense nptII construct as a negative selection marker for knockout of the endogenous rice genes Waxy and 33-kD globulin through GT, although negative selection with this system is relatively less efficient compared with diphtheria toxin A subunit. The approach developed here, with some additional improvements, could be applied as a universal selection system for the enrichment of GT cells in several plant species.

Visualization of specific repetitive genomic sequences with fluorescent TALEs in Arabidopsis thaliana.

Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in sub-nuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms.

Precise marker excision system using an animal-derived piggyBac transposon in plants.

Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth-derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase-expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T0 plants without concomitant re-integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.