著者

Shuhei Yasumoto Satoru Sawai Hyoung Jae Lee Masaharu Mizutani Kazuki Saito Naoyuki Umemoto Toshiya Muranaka

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.37, no.2, pp.205-211, 2020-06-25 (Released:2020-06-25)

参考文献数

24

被引用文献数

22

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Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat–CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.

著者

Naoyuki Umemoto Shuhei Yasumoto Muneo Yamazaki Kenji Asano Kotaro Akai Hyoung Jae Lee Ryota Akiyama Masaharu Mizutani Yozo Nagira Kazuki Saito Toshiya Muranaka

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.211-218, 2023-09-25 (Released:2023-09-25)

参考文献数

19

被引用文献数

1

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Genome editing is highly useful for crop improvement. The method of expressing genome-editing enzymes using a transient expression system in Agrobacterium, called agrobacterial mutagenesis, is a shortcut used in genome-editing technology to improve elite varieties of vegetatively propagated crops, including potato. However, with this method, edited individuals cannot be selected. The transient expression of regeneration-promoting genes can result in shoot regeneration from plantlets, while the constitutive expression of most regeneration-promoting genes does not result in normally regenerated shoots. Here, we report that we could obtain genome-edited potatoes by positive selection. These regenerated shoots were obtained via a method that combined a regeneration-promoting gene with the transient expression of a genome-editing enzyme gene. Moreover, we confirmed that the genome-edited potatoes obtained using this method did not contain the sequence of the binary vector used in Agrobacterium. Our data have been submitted to the Japanese regulatory authority, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and we are in the process of conducting field tests for further research on these potatoes. Our work presents a powerful method for regarding regeneration and acquisition of genome-edited crops through transient expression of regeneration-promoting gene.

著者

Masahiro Nishihara Toshiya Muranaka

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.181-184, 2023-09-25 (Released:2023-09-25)

参考文献数

30

著者

Ayumi Kawasaki Ayaka Chikugo Keita Tamura Hikaru Seki Toshiya Muranaka

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.2, pp.205-218, 2021-06-25 (Released:2021-06-25)

参考文献数

72

被引用文献数

2

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Uridine 5′-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1–4) showed UGD activity in vitro. GuUGD1–4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1–4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.

著者

Atsushi Okazawa Takatoshi Wakabayashi Toshiya Muranaka Yukihiro Sugimoto Daisaku Ohta

出版者

Pesticide Science Society of Japan

雑誌

Journal of Pesticide Science (ISSN:1348589X)

巻号頁・発行日

vol.45, no.4, pp.230-237, 2020-11-20 (Released:2020-11-20)

参考文献数

48

被引用文献数

5

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Orobanchaceae root parasitic weeds cause serious agricultural damage worldwide. Although numerous studies have been conducted to establish an effective control strategy for the growth and spread of root parasitic weeds, no practical method has been developed so far. Previously, metabolomic analyses were conducted on germinating seeds of a broomrape, Orobanche minor, to find novel targets for its selective control. Interestingly, planteose metabolism was identified as a possible target, and nojirimycin (NJ) selectively inhibited the germination of O. minor by intercepting planteose metabolism, although its precise mode of action was unclear. Here, transcriptome analysis by RNA-Seq was conducted to obtain molecular insight into the effects of NJ on germinating O. minor seeds. Differential gene expression analysis results suggest that NJ alters sugar metabolism and/or signaling, which is required to promote seed germination. This finding will contribute to understanding the effect of NJ and establishing a novel strategy for parasitic weed control.