Targeted modification of CmACO1 by CRISPR/Cas9 extends the shelf-life of Cucumis melo var. reticulatus melon.

The gaseous plant hormone ethylene is a regulator of fruit shelf-life, one of the essential traits in fruits. Extending fruit shelf-life reduces food loss, thereby expected to contribute to food security. The enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) is the final step of the ethylene production pathway. Its suppression via antisense technology has been demonstrated to extend the shelf-life of melon, apple, and papaya. Genome editing technology is an innovative technique for plant breeding. Because the genome editing technology would not leave the exogenous genes in the final crop products, the crops via genome editing can be considered non-genetically modified yields; compared to conventional breeding, such as mutation breeding, the breeding term would be expected to be relatively short. These points include the advantage of this technique in utilization for commercial applications. We attempted to extend the shelf-life of the Japanese luxury melon (Cucumis melo var. reticulatus, 'Harukei-3') via modification of the ethylene synthesis pathway with the genome editing technology, CRISPR/Cas9 system. The Melonet-DB (https://melonet-db.dna.affrc.go.jp/ap/top) showed that the melon genome had the five CmACOs and the gene CmACO1 predominantly expressed in harvested fruits. From this information, CmACO1 was expected to be a key gene for shelf-life in melons. Based on this information, the CmACO1 was selected as the target of the CRISPR/Cas9 system and introduced the mutation. The final product of this melon did not have any exogenous genes. The mutation was inherited for at least two generations. In the T2 generation, the fruit phenotypes 14 days after harvest were as follows: ethylene production was reduced to one-tenth that of the wild type, pericarp colour remained green, and higher fruit firmness. Early fermentation of the fresh fruit was observed in the wild-type fruit but not in the mutant. These results show that CmACO1 knockout via CRISPR/Cas9 extended the melon's shelf-life. Moreover, our results suggest that genome editing technology would reduce food loss and contribute to food security.

Modification of tomato breeding traits and plant hormone signaling by target-AID, the genome-editing system inducing efficient nucleotide substitution.

Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar "Micro-Tom" and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both "Micro-Tom" and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.

The accumulation of recombinant miraculin is independent of fruit size in tomato.

The taste-modifying protein miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety 'Micro-Tom,' it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). 'Micro-Tom' is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background 'Micro-Tom'). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B ('Moneymaker' background, which exhibits the highest miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.

Genome editing in PDS genes of tomatoes by non-selection method and of Nicotiana benthamiana by one single guide RNA to edit two orthologs.

The CRISPR/Cas9 system is widely used for targeted mutagenesis in many organisms including plants. For application of this system, tissue culture methods need to be established. In this study, detailed methods for introduction of mutations in tomato and Nicotiana benthamiana plants using the CRISPR/Cas9 system are described. The methods include tissue culture protocols for tomato and N. benthamiana. We also demonstrate the methodology to generate Cas9-free genome edited tomato plants and use of one single guide RNA (sgRNA) to edit two orthologs in N. benthamiana. The examples of editing the PHYTOENE DESATURASE (PDS) genes in these plants are also provided. The Cas9-free tomato line was obtained when tomato plants were cultured on a non-selective medium after transformation with the CRISPR/Cas9 system. Two orthologs of PDS in N. benthamiana were mutated using a sgRNA, because these orthologs contain the same nucleotide sequences with PAM motif. These mutations were inherited to the next generation. The mutations in the PDS genes resulted in an albino phenotype in tomato and N. benthamiana plants. These results demonstrate that the non-selective method is one of the ways to obtain Cas9-free genome editing in tomato plants and that the two orthologs can be edited by one sgRNA in N. benthamiana.

Comparative genomics of muskmelon reveals a potential role for retrotransposons in the modification of gene expression.

Melon exhibits substantial natural variation especially in fruit ripening physiology, including both climacteric (ethylene-producing) and non-climacteric types. However, genomic mechanisms underlying such variation are not yet fully understood. Here, we report an Oxford Nanopore-based high-grade genome reference in the semi-climacteric cultivar Harukei-3 (378 Mb + 33,829 protein-coding genes), with an update of tissue-wide RNA-seq atlas in the Melonet-DB database. Comparison between Harukei-3 and DHL92, the first published melon genome, enabled identification of 24,758 one-to-one orthologue gene pairs, whereas others were candidates of copy number variation or presence/absence polymorphisms (PAPs). Further comparison based on 10 melon genome assemblies identified genome-wide PAPs of 415 retrotransposon Gag-like sequences. Of these, 160 showed fruit ripening-inducible expression, with 59.4% of the neighboring genes showing similar expression patterns (r > 0.8). Our results suggest that retrotransposons contributed to the modification of gene expression during diversification of melon genomes, and may affect fruit ripening-inducible gene expression.

Author Correction: Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis.

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

Super-Agrobacterium ver. 4: Improving the Transformation Frequencies and Genetic Engineering Possibilities for Crop Plants.

Agrobacterium tumefaciens has been utilized for both transient and stable transformations of plants. These transformation methods have been used in fields such as breeding GM crops, protein production in plant cells, and the functional analysis of genes. However, some plants have significantly lower transient gene transfer and stable transformation rates, creating a technical barrier that needs to be resolved. In this study, Super-Agrobacterium was updated to ver. 4 by introducing both the ACC deaminase (acdS) and GABA transaminase (gabT) genes, whose resultant enzymes degrade ACC, the ethylene precursor, and GABA, respectively. A. tumefaciens strain GV2260, which is similar to other major strains (EHA105, GV3101, LBA4404, and MP90), was used in this study. The abilities of the Super-Agrobacterium ver. 4 were evaluated in Erianthus ravennae, Solanum lycopersicum "Micro-Tom," Nicotiana benthamiana, and S. torvum. Super-Agrobacterium ver. 4 showed the highest T-DNA transfer (transient transformation) frequencies in E. ravennae and S. lycopersicum, but not in N. benthamiana and S. torvum. In tomato, Super-Agrobacterium ver. 4 increased the stable transformation rate by 3.6-fold compared to the original GV2260 strain. Super-Agrobacterium ver. 4 enables reduction of the amount of time and labor required for transformations by approximately 72%, and is therefore a more effective and powerful tool for plant genetic engineering and functional analysis, than the previously developed strains. As our system has a plasmid containing the acdS and gabT genes, it could be used in combination with other major strains such as EHA105, EHA101, LBA4404, MP90, and AGL1. Super-Agrobacterium ver. 4, could thus possibly be a breakthrough application for improving basic plant science research methods.

Efficient transient protein expression in tomato cultivars and wild species using agroinfiltration-mediated high expression system.

KEY MESSAGE: The new transient protein expression system using the pBYR2HS vector is applicable to several tomato cultivars and wild species with high level of protein expression. Innovation and improvement of effective tools for transient protein expression in plant cells is critical for the development of plant biotechnology. We have created the new transient protein expression system using the pBYR2HS vector that led to about 4 mg/g fresh weight of protein expression in Nicotiana benthamiana. In this study, we validated the adaptability of this transient protein expression system by agroinfiltration to leaves and fruits of several tomato cultivars and wild species. Although the GFP protein was transiently expressed in the leaves and fruits of all tomato cultivars and wild species, we observed species-specific differences in protein expression. In particular, GFP protein expression was higher in the leaves and fruits of Micro-Tom, Solanum pimpinellifolium (0043) and S. pimpinellifolium (0049-w1) than in those of cultivars and wild species. Furthermore, Agrobacterium with GABA transaminase enhanced transient expression in tomato fruits of Micro-Tom. Taken together with these results, our system is applicable to several tomato cultivars and species as well as a model tomato, even though characteristics are often different among tomato cultivars or species. Thus, the system is an effective, simple, and valuable tool to achieve rapid transgene expression to examine gene function in tomato plant cells.

Ethylene biosynthesis controlled by NON-RIPENING: A regulatory conflict between wounding and ripening.

The phytohormone ethylene is involved in multiple aspects of morphological and physiological processes in plants. Tomato rapidly and transiently increases ethylene production during fruit ripening and in plant defense responses. The transcription factor non-ripening (NOR) has significant effects on fruit ripening via regulation of ethylene biosynthesis-related genes. The nor loss-of-function allele produces a basal level of ethylene during ripening, in contrast to the induced ethylene evolution observed upon Agrobacterium tumefaciens infection. The use of ACC deaminase represses ethylene production and significantly improves the efficiency of Agrobacterium-mediated T-DNA transfer in nor plants. Analyses of the transcription levels of the ethylene biosynthesis genes ACC synthase (ACS) and ACC oxidase (ACO) in nor plants revealed that the induced ethylene production was largely due to transcriptional accumulation of ACS2 and ACO1. Accumulation of ACS2 and ACO1 mRNA opposes NOR-mediated regulation in tomato fruit during ripening, and the feedback regulation of NOR is rendered ineffective by defense responses, thereby precluding the control of its own expression. The ethylene synthesis mechanisms respond properly to NOR-mediated transcriptional regulation that is differed through the wound-induced and ripening-induced signaling pathway.

Improved G-AgarTrap: A highly efficient transformation method for intact gemmalings of the liverwort Marchantia polymorpha.

Liverworts are key species for studies of plant evolution, occupying a basal position among the land plants. Marchantia polymorpha has emerged as a highly studied model liverwort, and many relevant techniques, including genetic transformation, have been established for this species. Agrobacterium-mediated transformation is widely used in many plant species because of its low cost. Recently, we developed a simplified Agrobacterium-mediated method for transforming M. polymorpha, known as AgarTrap (agar-utilized transformation with pouring solutions). The AgarTrap procedure, which involves culturing the liverwort tissue in various solutions on a single solid medium, yields up to a hundred independent transformants. AgarTrap is a simple procedure, requiring minimal expertise, cost, and time. Here, we investigated four factors that influence AgarTrap transformation efficiency: (1) humidity, (2) surfactant in the transformation buffer, (3) Agrobacterium strain, and (4) light/dark condition. We adapted the AgarTrap protocol for transforming intact gemmalings, achieving an exceptionally high transformation efficiency of 97%. The improved AgarTrap method will enhance the molecular biological study of M. polymorpha. Furthermore, this method provides new possibilities for improving transformation techniques for a variety of plant species.

An improved assembly and annotation of the melon (Cucumis melo L.) reference genome.

We report an improved assembly (v3.6.1) of the melon (Cucumis melo L.) genome and a new genome annotation (v4.0). The optical mapping approach allowed correcting the order and the orientation of 21 previous scaffolds and permitted to correctly define the gap-size extension along the 12 pseudomolecules. A new comprehensive annotation was also built in order to update the previous annotation v3.5.1, released more than six years ago. Using an integrative annotation pipeline, based on exhaustive RNA-Seq collections and ad-hoc transposable element annotation, we identified 29,980 protein-coding loci. Compared to the previous version, the v4.0 annotation improved gene models in terms of completeness of gene structure, UTR regions definition, intron-exon junctions and reduction of fragmented genes. More than 8,000 new genes were identified, one third of them being well supported by RNA-Seq data. To make all the new resources easily exploitable and completely available for the scientific community, a redesigned Melonomics genomic platform was released at http://melonomics.net . The resources produced in this work considerably increase the reliability of the melon genome assembly and resolution of the gene models paving the way for further studies in melon and related species.

Comparative effects of ethylene inhibitors on Agrobacterium-mediated transformation of drought-tolerant wild watermelon.

Ethylene (C2H4), a phytohormone that is produced in response to both abiotic and biotic stresses, is an important factor influencing the efficiency of Agrobacterium-mediated transformation. In this study, effects of various ethylene inhibitors on the efficiency of Agrobacterium-mediated genetic transformation in drought-tolerant wild watermelon was comparatively examined. Consequently, in comparison to the application of chemical inhibitors such as AgNO3 and aminoethoxyvinylglycine (AVG), lower ethylene level was observed when the infecting Agrobacterium contained a gene for 1-aminocyclopropane-carboxylic acid (ACC) deaminase (acdS), which cleaves ethylene precursor ACC into α-ketobutyrate and ammonia. GUS histochemical and spectrophotometric enzyme assays showed that acdS was more effective in enhancing gene transfer than the chemical ethylene inhibitors. Efficiency of transgenic shoots formation was higher in acdS- and AVG-treated explants. These observations demonstrated that controlling the ethylene level during co-cultivation and shoot formation, particularly using the acdS-harboring Agrobacterium, is advantageous for enhancing the transformation efficiency in this plant.

Utilization of a Genome-Edited Tomato (Solanum lycopersicum) with High Gamma Aminobutyric Acid Content in Hybrid Breeding.

γ-Aminobutyric acid (GABA) is a nonproteogenic amino acid with health-promoting functions. Although tomato fruits have a relatively high GABA content compared with other crops, levels must be further increased to effectively confer the health-promoting functions. In this study, we evaluated the potential of the genome-edited tomato as a breeding material for producing high-GABA hybrid tomatoes. Hybrid lines were produced by crossing the genome-edited tomato with a pure line tomato cultivar, "Aichi First", and were evaluated for GABA accumulation and other fruit traits. The hybrid lines showed high GABA accumulation in the fruits, which was sufficiently high for expecting health-promoting functions and had minimal effects on other fruit traits, suggesting that the high GABA is a dominant trait and that the genome-edited tomato would be useful as a parental line of hybrid cultivars. These results also indicate that genome editing technology is useful for the rapid breeding of high-GABA hybrid tomato cultivars.

Melonet-DB, a Grand RNA-Seq Gene Expression Atlas in Melon (Cucumis melo L.).

Melon (Cucumis melo L.) is an important Cucurbitaceae crop produced worldwide, exhibiting wide genetic variations and comprising both climacteric and non-climacteric fruit types. The muskmelon cultivar "'Earl's favorite Harukei-3 (Harukei-3)"' known for its sweetness and rich aroma is used for breeding of high-grade muskmelon in Japan. We conducted RNA sequencing (RNA-seq) transcriptome studies in 30 different tissues of the 'Harukei-3' melon. These included root, stems, leaves, flowers, regenerating callus and ovaries, in addition to the flesh and peel sampled at seven stages of fruit development. The expression patterns of 20,752 genes were determined with fragments per kilobase of transcript per million fragments sequenced (FPKM) >1 in at least one tissue. Principal component analysis distinguished 30 melon tissues based on the global gene expression profile and, further, the weighted gene correlation network analysis classified melon genes into 45 distinct coexpression groups. Some coexpression groups exhibited tissue-specific gene expression. Furthermore, we developed and published web application tools designated "'Gene expression map viewer"' and "'Coexpression viewer"' on our website Melonet-DB (http://melonet-db.agbi.tsukuba.ac.jp/) to promote functional genomics research in melon. By using both tools, we analyzed melon homologs of tomato fruit ripening regulators such as E8, RIPENING-INHIBITOR (RIN) and NON-RIPENING (NOR). The "'Coexpression viewer"' clearly distinguished fruit ripening-associated melon RIN/NOR/CNR homologs from those expressed in other tissues. In addition, several other MADS-box, NAM/ATAF/CUC (NAC) and homeobox transcription factor genes were identified as fruit ripening-associated genes. Our tools provide useful information for research not only on melon but also on other fleshy fruit plants.

Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis.

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects. Tomato (Solanum lycopersicum L.) is among the most widely cultivated and consumed vegetables in the world and contains higher levels of GABA than other major crops. Increasing these levels can further enhance the blood pressure-lowering function of tomato fruit. Glutamate decarboxylase (GAD) is a key enzyme in GABA biosynthesis; it has a C-terminal autoinhibitory domain that regulates enzymatic function, and deleting this domain increases GAD activity. The tomato genome has five GAD genes (SlGAD1-5), of which two (SlGAD2 and SlGAD3) are expressed during tomato fruit development. To increase GABA content in tomato, we deleted the autoinhibitory domain of SlGAD2 and SlGAD3 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 technology. Introducing a stop codon immediately before the autoinhibitory domain increased GABA accumulation by 7 to 15 fold while having variable effects on plant and fruit size and yield. This is the first study describing the application of the CRISPR/Cas9 system to increase GABA content in tomato fruits. Our findings provide a basis for the improvement of other types of crop by CRISPR/Cas9-based genetic modification.

An Agrobacterium tumefaciens Strain with Gamma-Aminobutyric Acid Transaminase Activity Shows an Enhanced Genetic Transformation Ability in Plants.

Agrobacterium tumefaciens has the unique ability to mediate inter-kingdom DNA transfer, and for this reason, it has been utilized for plant genetic engineering. To increase the transformation frequency in plant genetic engineering, we focused on gamma-aminobutyric acid (GABA), which is a negative factor in the Agrobacterium-plant interaction. Recent studies have shown contradictory results regarding the effects of GABA on vir gene expression, leading to the speculation that GABA inhibits T-DNA transfer. In this study, we examined the effect of GABA on T-DNA transfer using a tomato line with a low GABA content. Compared with the control, the T-DNA transfer frequency was increased in the low-GABA tomato line, indicating that GABA inhibits T-DNA transfer. Therefore, we bred a new A. tumefaciens strain with GABA transaminase activity and the ability to degrade GABA. The A. tumefaciens strain exhibited increased T-DNA transfer in two tomato cultivars and Erianthus arundinacues and an increased frequency of stable transformation in tomato.

Jasmonate-Responsive ERF Transcription Factors Regulate Steroidal Glycoalkaloid Biosynthesis in Tomato.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced in species of the Solanaceae. Here, we report that a group of jasmonate-responsive transcription factors of the ETHYLENE RESPONSE FACTOR (ERF) family (JREs) are close homologs of alkaloid regulators in Cathranthus roseus and tobacco, and regulate production of SGAs in tomato. In transgenic tomato, overexpression and dominant suppression of JRE genes caused drastic changes in SGA accumulation and in the expression of genes for metabolic enzymes involved in the multistep pathway leading to SGA biosynthesis, including the upstream mevalonate pathway. Transactivation and DNA-protein binding assays demonstrate that JRE4 activates the transcription of SGA biosynthetic genes by binding to GCC box-like elements in their promoters. These JRE-binding elements occur at significantly higher frequencies in proximal promoter regions of the genes regulated by JRE genes, supporting the conclusion that JREs mediate transcriptional co-ordination of a series of metabolic genes involved in SGA biosynthesis.

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.

WIND1-based acquisition of regeneration competency in Arabidopsis and rapeseed.

Callus formation and de novo organogenesis often occur in the wounded tissues of plants. Although this regenerative capacity of plant cells has been utilized for many years, molecular basis for the wound-induced acquisition of regeneration competency is yet to be elucidated. Here we find that wounding treatment is essential for shoot regeneration from roots in the conventional tissue culture of Arabidopsis thaliana. Furthermore, we show that an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) plays a pivotal role for the acquisition of regeneration competency in the culture system. Ectopic expression of WIND1 can bypass both wounding and auxin pre-treatment and increase de novo shoot regeneration from root explants cultured on shoot-regeneration promoting media. In Brassica napus, activation of Arabidopsis WIND1 also greatly enhances de novo shoot regeneration, further corroborating the role of WIND1 in conferring cellular regenerative capacity. Our data also show that sequential activation of WIND1 and an embryonic regulator LEAFY COTYLEDON2 enhances generation of embryonic callus, suggesting that combining WIND1 with other transcription factors promote efficient and organ-specific regeneration. Our findings in the model plant and crop plant point to a possible way to efficiently induce callus formation and regeneration by utilizing transcription factors as a molecular switch.

Melon (Cucumis melo).

Genetic transformation is an important technique used in plant breeding and to functionally characterize genes of interest. The earliest reports of Agrobacterium-mediated transformation in the melon (Cucumis melo) were from the early 1990s (Fang and Grumet, Plant Cell Rep, 9: 160-164, 1990; Dong et al., Nat Biotechnol 9: 858-863, 1991; Valles and Lasa, Plant Cell Rep 13: 145-148, 1994). These early studies described three problems that decreased the efficiency of transformation: tetraploidy, chimeras, and escape. Using a liquid culture system for somatic embryogenesis, Akasaka-Kenedy et al. (Plant Sci 166: 763-769, 2004) overcame these problems and established an efficient transformation system; the protocol introduced in this chapter is based on this method.