The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice.

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYß genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYß mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.

LysM-mediated signaling in Marchantia polymorpha highlights the conservation of pattern-triggered immunity in land plants.

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in angiosperms. The model liverwort Marchantia polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M. polymorpha and the significance of PTI in bryophytes have not yet been described. We here show that M. polymorpha has four lysin motif (LysM)-domain-containing receptor homologs, two of which, LysM-receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR, are responsible for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M. polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M. polymorpha and highlights conserved elements and new aspects of pattern-triggered immunity in land plants.

Zinc deficiency-induced defensin-like proteins are involved in the inhibition of root growth in Arabidopsis.

The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn-deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin-like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn-depleted conditions. DEFLs are small cysteine-rich peptides involved in defense responses, development and excess metal stress in plants. However, the functions of DEFLs in the Zn-deficiency response are largely unknown. Here, phylogenetic tree analysis revealed that seven DEFLs (DEFL202-DEFL208) were categorized into one subgroup. Among the seven DEFLs, the transcripts of five (not DEFL204 and DEFL205) were upregulated by Zn deficiency, consistent with the presence of cis-elements for basic-region leucine-zipper 19 (bZIP19) or bZIP23 in their promoter regions. Microscopic observation of GFP-tagged DEFL203 showed that DEFL203-sGFP was localized to the apoplast and plasma membrane. Whereas a single mutation of the DEFL202 or DEFL203 genes only slightly affected root growth, defl202 defl203 double mutants showed enhanced root growth under all growth conditions. We also showed that the size of the root meristem was increased in the double mutants compared with the wild type. Our results suggest that DEFL202 and DEFL203 are redundantly involved in the inhibition of root growth under Zn-deficient conditions through a reduction in root meristem length and cell number.

Single-Cell RNA Sequencing of Arabidopsis Leaf Tissues Identifies Multiple Specialized Cell Types: Idioblast Myrosin Cells and Potential Glucosinolate-Producing Cells.

The glucosinolate-myrosinase defense system (GMDS), characteristic of Brassicales, is involved in plant defense. Previous single-cell transcriptomic analyses have reported the expression profiles of multiple GMDS-related cell types (i.e. myrosinase-rich myrosin idioblasts and multiple types of potential glucosinolate synthetic cells as well as a candidate S-cell for glucosinolate accumulation). However, differences in plant stages and cell-type annotation methods have hindered comparisons among studies. Here, we used the single-cell transcriptome profiles of extended Arabidopsis leaves and verified the distribution of previously used markers to refine the expression profiles of GMDS-associated cell types. Moreover, we performed beta-glucuronidase promoter assays to confirm the histological expression patterns of newly obtained markers for GMDS-associated candidates. As a result, we found a set of new specific reporters for myrosin cells and potential glucosinolate-producing cells.

Viscosity-aided electromechanical poration of cells for transfecting molecules.

Cell poration technologies offer opportunities not only to understand the activities of biological molecules but also to investigate genetic manipulation possibilities. Unfortunately, transferring large molecules that can carry huge genomic information is challenging. Here, we demonstrate electromechanical poration using a core-shell-structured microbubble generator, consisting of a fine microelectrode covered with a dielectric material. By introducing a microcavity at its tip, we could concentrate the electrical field with the application of electric pulses and generate microbubbles for electromechanical stimulation of cells. Specifically, the technology enables transfection with molecules that are thousands of kDa even into osteoblasts and Chlamydomonas, which are generally considered to be difficult to inject. Notably, we found that the transfection efficiency can be enhanced by adjusting the viscosity of the cell suspension, which was presumably achieved by remodeling of the membrane cytoskeleton. The applicability of the approach to a variety of cell types opens up numerous emerging gene engineering applications.

DNA-functionalized colloidal crystals for macromolecular encapsulation.

Novel DNA-based structures with the ability to encapsulate nanoscale molecules, such as proteins, can be applied to a wide range of areas, including reaction fields and micro/nano drug carriers. DNA-functionalized nanoparticle (DNA-NP) colloidal crystals have emerged as a new class of programmable DNA-based structures harboring metal nanoparticles with improved mechanical properties. The encapsulation of guest molecules into empty spaces in lattice structures is theoretically possible. However, due to the lack of a strategy for versatile encapsulation of guest molecules, the feasibility of nanoscale encapsulation by DNA-NP crystals is unclear. In this study, we developed DNA-functionalized gold nanoparticle (DNA-AuNP) crystals with tunable interparticle spacing for molecular encapsulation. We demonstrated that the modification of DNA-AuNP crystals with functional moieties, that is, biotin molecules, was effective in retaining molecules in the crystals. The crystallinities before and after encapsulation of the molecules were confirmed using small-angle X-ray scattering. We also succeeded in encapsulating CRISPR/Cas9 ribonucleoproteins into DNA-AuNP crystals by harnessing their affinity for target molecules. These findings demonstrated the potential use of metal-DNA hybrid crystals as carriers for direct protein delivery via biolistic bombardment. Thus, this study provides an attractive strategy for creating a new class of DNA-based structures for macromolecular encapsulation, and an alternative research direction toward colloidal crystal engineering using DNA.

Comprehensive analysis of peptide-coding genes and initial characterization of an LRR-only microprotein in Marchantia polymorpha.

In the past two decades, many plant peptides have been found to play crucial roles in various biological events by mediating cell-to-cell communications. However, a large number of small open reading frames (sORFs) or short genes capable of encoding peptides remain uncharacterized. In this study, we examined several candidate genes for peptides conserved between two model plants: Arabidopsis thaliana and Marchantia polymorpha. We examined their expression pattern in M. polymorpha and subcellular localization using a transient assay with Nicotiana benthamiana. We found that one candidate, MpSGF10B, was expressed in meristems, gemma cups, and male reproductive organs called antheridiophores. MpSGF10B has an N-terminal signal peptide followed by two leucine-rich repeat (LRR) domains and was secreted to the extracellular region in N. benthamiana and M. polymorpha. Compared with the wild type, two independent Mpsgf10b mutants had a slightly increased number of antheridiophores. It was revealed in gene ontology enrichment analysis that MpSGF10B was significantly co-expressed with genes related to cell cycle and development. These results suggest that MpSGF10B may be involved in the reproductive development of M. polymorpha. Our research should shed light on the unknown role of LRR-only proteins in land plants.

Higher Stomatal Density Improves Photosynthetic Induction and Biomass Production in Arabidopsis Under Fluctuating Light.

Stomatal density (SD) is closely associated with photosynthetic and growth characteristics in plants. In the field, light intensity can fluctuate drastically within a day. The objective of the present study is to examine how higher SD affects stomatal conductance (g s ) and CO2 assimilation rate (A) dynamics, biomass production and water use under fluctuating light. Here, we compared the photosynthetic and growth characteristics under constant and fluctuating light among three lines of Arabidopsis thaliana (L.): the wild type (WT), STOMAGEN/EPFL9-overexpressing line (ST-OX), and EPIDERMAL PATTERNING FACTOR 1 knockout line (epf1). ST-OX and epf1 showed 268.1 and 46.5% higher SD than WT (p < 0.05). Guard cell length of ST-OX was 10.0% lower than that of WT (p < 0.01). There were no significant variations in gas exchange parameters at steady state between WT and ST-OX or epf1, although these parameters tended to be higher in ST-OX and epf1 than WT. On the other hand, ST-OX and epf1 showed faster A induction than WT after step increase in light owing to the higher g s under initial dark condition. In addition, ST-OX and epf1 showed initially faster g s induction and, at the later phase, slower g s induction. Cumulative CO2 assimilation in ST-OX and epf1 was 57.6 and 78.8% higher than WT attributable to faster A induction with reduction of water use efficiency (WUE). epf1 yielded 25.6% higher biomass than WT under fluctuating light (p < 0.01). In the present study, higher SD resulted in faster photosynthetic induction owing to the higher initial g s . epf1, with a moderate increase in SD, achieved greater biomass production than WT under fluctuating light. These results suggest that higher SD can be beneficial to improve biomass production in plants under fluctuating light conditions.

Author Correction: Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Manganese Treatment Alleviates Zinc Deficiency Symptoms in Arabidopsis Seedlings.

Plant phenotypes caused by mineral deficiencies differ depending on growth conditions. We recently reported that the growth of Arabidopsis thaliana was severely inhibited on MGRL-based zinc (Zn)-deficient medium but not on Murashige-Skoog-based Zn-deficient medium. Here, we explored the underlying reason for the phenotypic differences in Arabidopsis grown on the different media. The root growth and chlorophyll contents reduced by Zn deficiency were rescued by the addition of extra manganese (Mn) during short-term growth (10 or 14 d). However, this treatment did not affect the growth recovery after long-term growth (38 d). To investigate the reason for plant recovery from Zn deficiency, we performed the RNA-seq analysis of the roots grown on the Zn-basal medium and the Zn-depleted medium with/without additional Mn. Principal component analysis of the RNA-seq data showed that the gene expression patterns of plants on the Zn-basal medium were similar to those on the Zn-depleted medium with Mn, whereas those on the Zn-depleted medium without Mn were different from the others. The expression of several transcription factors and reactive oxygen species (ROS)-related genes was upregulated in only plants on the Zn-depleted medium without Mn. Consistent with the gene expression data, ROS accumulation in the roots grown on this medium was higher than those grown in other conditions. These results suggest that plants accumulate ROS and reduce their biomass under undesirable growth conditions, such as Zn depletion. Taken together, this study shows that the addition of extra Mn to the Zn-depleted medium induces transcriptional changes in ROS-related genes, thereby alleviating short-term growth inhibition due to Zn deficiency.

Two Y-chromosome-encoded genes determine sex in kiwifruit.

Dioecy, the presence of male and female individuals, has evolved independently in multiple flowering plant lineages1-3. Although theoretical models for the evolution of dioecy, such as the 'two-mutations' model, are well established4,5, little is known about the specific genes determining sex and their evolutionary history3. Kiwifruit, a major tree crop consumed worldwide, is a dioecious species. In kiwifruit we previously identified a Y-encoded sex-determinant candidate gene acting as the suppressor of feminization (SuF), named Shy Girl (SyGI)6. Here, we identify a second Y-encoded sex-determinant that we named Friendly Boy (FrBy), which exhibits strong expression in tapetal cells. Gene-editing and complementation analyses in Arabidopsis thaliana and Nicotiana tabacum indicated that FrBy acts for the maintenance of male (M) functions, independently of SyGI, and that these functions are conserved across angiosperm species. We further characterized the genomic architecture of the small (<1 megabase pairs (Mb)) male-specific region of the Y chromosome (MSY), which harbours only two genes expressed extensively in developing gynoecia and androecia, respectively: SyGI and FrBy. Re-sequencing of the genome of a natural hermaphrodite kiwifruit revealed that this individual is genetically male but carries deletion(s) of parts of the Y chromosome, including SyGI. Additionally, expression of FrBy in female kiwifruit resulted in hermaphrodite plants. These results clearly indicate that Y-encoded SyGI and FrBy act independently as the SuF and M factors in kiwifruit, respectively, and provide insight into not only the evolutionary path leading to a two-factor sex-determination system, but also a new breeding approach for dioecious species.

Chromatin-mediated feed-forward auxin biosynthesis in floral meristem determinacy.

In flowering plants, the switch from floral stem cell maintenance to gynoecium (female structure) formation is a critical developmental transition for reproductive success. In Arabidopsis thaliana, AGAMOUS (AG) terminates floral stem cell activities to trigger this transition. Although CRABS CLAW (CRC) is a direct target of AG, previous research has not identified any common targets. Here, we identify an auxin synthesis gene, YUCCA4 (YUC4) as a common direct target. Ectopic YUC4 expression partially rescues the indeterminate phenotype and cell wall defects that are caused by the crc mutation. The feed-forward YUC4 activation by AG and CRC directs a precise change in chromatin state for the shift from floral stem cell maintenance to gynoecium formation. We also showed that two auxin-related direct CRC targets, YUC4 and TORNADO2, cooperatively contribute to the termination of floral stem cell maintenance. This finding provides new insight into the CRC-mediated auxin homeostasis regulation for proper gynoecium formation.

Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha.

Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.

CRISPR/Cas9-Based Genome Editing of Transcription Factor Genes in Marchantia polymorpha.

Plant transcription factors (TFs) belong to a wide variety of gene families. The systematic and rapid establishment of knockout lines of TF genes is critical for functional genetics. Genome engineering techniques for dissecting out the molecular function of TFs have been dramatically improved by CRISPR/Cas9-based genome editing technology. In the CRISPR/Cas9 system, Cas9 functions as a Cas9-gRNA ribonucleoprotein complex and uses its DNA endonuclease activity to induce the cleavage of the genome, which is targeted by gRNA. Double-strand breaks sometimes induce insertions and deletions at the target site, leading to frameshift mutations of TF genes. In this chapter, we describe a detailed protocol for the targeted mutagenesis of TFs using the CRISPR/Cas9 system, specifically in the case of Marchantia polymorpha, an emerging model plant for functional genomics. The CRISPR/Cas9 system is highly versatile for targeting genomic sequences because only an alteration of the gRNA sequence is needed to change target sequences. The labor and cost required to establish genome-edited lines are low enough that multiple mutants of TF genes can be generated in one laboratory. The CRISPR/Cas9-based genome editing technique consists of four steps: (1) gRNA design; (2) vector construction; (3) transformation; and (4) isolation of genome-edited lines. This manuscript focuses mainly on the strategy of gRNA design, the workflow for off-target searches, and the selection and identification of genome-edited lines by genotyping. Although we describe a protocol for M. polymorpha, the basic strategy of generating genome-edited lines of TF genes should be applicable widely to other plants.

Development of versatile non-homologous end joining-based knock-in module for genome editing.

CRISPR/Cas9-based genome editing has dramatically accelerated genome engineering. An important aspect of genome engineering is efficient knock-in technology. For improved knock-in efficiency, the non-homologous end joining (NHEJ) repair pathway has been used over the homology-dependent repair pathway, but there remains a need to reduce the complexity of the preparation of donor vectors. We developed the versatile NHEJ-based knock-in module for genome editing (VIKING). Using the consensus sequence of the time-honored pUC vector to cut donor vectors, any vector with a pUC backbone could be used as the donor vector without customization. Conditions required to minimize random integration rates of the donor vector were also investigated. We attempted to isolate null lines of the VDR gene in human HaCaT keratinocytes using knock-in/knock-out with a selection marker cassette, and found 75% of clones isolated were successfully knocked-in. Although HaCaT cells have hypotetraploid genome composition, the results suggest multiple clones have VDR null phenotypes. VIKING modules enabled highly efficient knock-in of any vectors harboring pUC vectors. Users now can insert various existing vectors into an arbitrary locus in the genome. VIKING will contribute to low-cost genome engineering.

Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system.

Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.

Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9.

Parthenocarpy in horticultural crop plants is an important trait with agricultural value for various industrial purposes as well as direct eating quality. Here, we demonstrate a breeding strategy to generate parthenocarpic tomato plants using the CRISPR/Cas9 system. We optimized the CRISPR/Cas9 system to introduce somatic mutations effectively into SlIAA9-a key gene controlling parthenocarpy-with mutation rates of up to 100% in the T0 generation. Furthermore, analysis of off-target mutations using deep sequencing indicated that our customized gRNAs induced no additional mutations in the host genome. Regenerated mutants exhibited morphological changes in leaf shape and seedless fruit-a characteristic of parthenocarpic tomato. And the segregated next generation (T1) also showed a severe phenotype associated with the homozygous mutated genome. The system developed here could be applied to produce parthenocarpic tomato in a wide variety of cultivars, as well as other major horticultural crops, using this precise and rapid breeding technique.

The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.

Stem cell polarization is a crucial step in asymmetric cell division, which is a universal system for generating cellular diversity in multicellular organisms. Several conventional genetics studies have attempted to elucidate the mechanisms underlying cell polarization in plants, but it remains largely unknown. In plants, stomata, which are valves for gas exchange, are generated through several rounds of asymmetric divisions. In this study, we identified and characterized a chemical compound that affects stomatal stem cell polarity. High-throughput screening for bioactive molecules identified a pyridine-thiazole derivative, named bubblin, which induced stomatal clustering in Arabidopsis epidermis. Bubblin perturbed stomatal asymmetric division, resulting in the generation of two identical daughter cells. Both cells continued to express the stomatal fate determinant SPEECHLESS, and then differentiated into mispatterned stomata. Bubblin-treated cells had a defect in the polarized localization of BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), which is required for asymmetric cell fate determination. Our results suggest that bubblin induces stomatal lineage cells to divide without BASL-dependent pre-mitotic establishment of polarity. Bubblin is a potentially valuable tool for investigating cell polarity establishment in stomatal asymmetric division.

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.