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

Sugano, Shigeo S.; Nishihama, Ryuichi; Shirakawa, Makoto; Takagi, Junpei; Matsuda, Yasutaka; Ishida, Susumu; Shimada, Tomoo; Hara‐Nishimura, Ikuko; Osakabe, Keishi; Kohchi, Takayuki

doi:10.1371/journal.pone.0205117

Cited by 185 publications

(135 citation statements)

References 52 publications

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“…Marchantia is an excellent research model: it is small; has a rapid growth rate; can asexually reproduce in large numbers through single-cell-derived clonal gemmae; and, has a small genome (approximately 220 Mb) which, although larger than that of Arabidopsis (at 135 Mb), contains significantly fewer genes (around 19,000 gene models compared with around 28,000 protein-coding genes in Arabidopsis) (Ishizaki et al, 2015;Bowman et al, 2016;Shimamura, 2016;Bowman et al, 2017). It also offers efficient CRISPR/Cas9 mutagenesis in a dominant haploid gametophytic generation (Sugano et al, 2018).…”

Section: Flavonoids and Tolerance To Ultraviolet B Lightmentioning

confidence: 99%

The Evolution of Flavonoid Biosynthesis: A Bryophyte Perspective

et al. 2020

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The flavonoid pathway is one of the best characterized specialized metabolite pathways of plants. In angiosperms, the flavonoids have varied roles in assisting with tolerance to abiotic stress and are also key for signaling to pollinators and seed dispersal agents. The pathway is thought to be specific to land plants and to have arisen during the period of land colonization around 550-470 million years ago. In this review we consider current knowledge of the flavonoid pathway in the bryophytes, consisting of the liverworts, hornworts, and mosses. The pathway is less characterized for bryophytes than angiosperms, and the first genetic and molecular studies on bryophytes are finding both commonalities and significant differences in flavonoid biosynthesis and pathway regulation between angiosperms and bryophytes. This includes biosynthetic pathway branches specific to each plant group and the apparent complete absence of flavonoids from the hornworts.

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Section: Flavonoids and Tolerance To Ultraviolet B Lightmentioning

confidence: 99%

The Evolution of Flavonoid Biosynthesis: A Bryophyte Perspective

et al. 2020

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“…The sequence information of the M. polymorpha genome portal site MarpolBase (http://marchantia.info; JGI 3.1) was used for plasmid construction. Constructs for the CRISPR/Cas9-target mutagenesis of MpRRT1 were generated by annealing oligonucleotide pairs with MpRRT1_CR1_F and MpRRT1_CR1_R ( Supplementary Table 1) and the ligation of these annealed products into the BsaI site of a pMpGE_En03 vector (Addgene) (Sugano et al, 2018) to produce plasmids En03_MpRRT1_CR1 including gRNA expression cassettes. The sequence between attL1 and attL2 within En03_MpRRT1_CR1 was inserted into the binary vector pMpGE011 (Addgene) (Sugano et al, 2018) via an LR reaction using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific).…”

Section: Functional Analyses Of Mprrt1 In Liverwortmentioning

confidence: 99%

“…Constructs for the CRISPR/Cas9-target mutagenesis of MpRRT1 were generated by annealing oligonucleotide pairs with MpRRT1_CR1_F and MpRRT1_CR1_R ( Supplementary Table 1) and the ligation of these annealed products into the BsaI site of a pMpGE_En03 vector (Addgene) (Sugano et al, 2018) to produce plasmids En03_MpRRT1_CR1 including gRNA expression cassettes. The sequence between attL1 and attL2 within En03_MpRRT1_CR1 was inserted into the binary vector pMpGE011 (Addgene) (Sugano et al, 2018) via an LR reaction using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific). The resulting construct, GE011_MpRRT1_CR1, was introduced into Tak-1 using A. tumefaciens strain GV2260 as described previously (Kubota et al, 2013).…”

Section: Functional Analyses Of Mprrt1 In Liverwortmentioning

confidence: 99%

Diversity of Pectin Rhamnogalacturonan I Rhamnosyltransferases in Glycosyltransferase Family 106

et al. 2020

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Rhamnogalacturonan I (RG-I) comprises approximately one quarter of the pectin molecules in land plants, and the backbone of RG-I consists of a repeating sequence of [2)-a-L-Rha(1-4)-aD -GalUA(1-] disaccharide. Four Arabidopsis thaliana genes encoding RG-I rhamnosyltransferases (AtRRT1 to AtRRT4), which synthesize the disaccharide repeats, have been identified in the glycosyltransferase family (GT106). However, the functional role of RG-I in plant cell walls and the evolutional history of RRTs remains to be clarified. Here, we characterized the sole ortholog of AtRRT1-AtRRT4 in liverwort, Marchantia polymorpha, namely, MpRRT1. MpRRT1 had RRT activity and genetically complemented the AtRRT1-deficient mutant phenotype in A. thaliana. However, the MpRRT1-deficient M. polymorpha mutants showed no prominent morphological changes and only an approximate 20% reduction in rhamnose content in the cell wall fraction compared to that in wild-type plants, suggesting the existence of other RRT gene(s) in the M. polymorpha genome. As expected, we detected RRT activities in other GT106 family proteins such as those encoded by MpRRT3 in M. polymorpha and FRB1/AtRRT8 in A. thaliana, the deficient mutant of which affects cell adhesion. Our results show that RRT genes are more redundant and diverse in GT106 than previously thought.

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“…S2a) using the Gateway LR Clonase™ II Enzyme Mix. For construction of the genome-editing vectors, the target sequences were selected using CRISPR direct (https://crispr.dbcls.jp/) 36 , and double-stranded oligonucleotides of the target sequences were inserted into the pMpGE_En03 vector 37 . The resultant gRNA cassettes were introduced into the pMpGE010 or pMpGE011 vectors 37 using the Gateway LR Clonase II Enzyme Mix.…”

Section: Methodsmentioning

confidence: 99%

“…For construction of the genome-editing vectors, the target sequences were selected using CRISPR direct (https://crispr.dbcls.jp/) 36 , and double-stranded oligonucleotides of the target sequences were inserted into the pMpGE_En03 vector 37 . The resultant gRNA cassettes were introduced into the pMpGE010 or pMpGE011 vectors 37 using the Gateway LR Clonase II Enzyme Mix. For construction of the homologous recombination-mediated gene targeting vector, the 3.5-kb homologous genomic sequences were amplified from the Tak-1 genome and inserted at Pac I and Asc I sites of the pJHY-TMp1 vector 38 using the In-Fusion HD Cloning System.…”

Section: Methodsmentioning

confidence: 99%

Switching secretory pathway direction for organelle acquisition in plants

Kanazawa

Morinaka

Ebine

et al. 2020

Preprint

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26Eukaryotic cells acquired novel organelles during evolution through mechanisms that 27 remain largely obscure. The existence of the unique oil body compartment is a 28 synapomorphy of liverworts that represents lineage-specific acquisition of this organelle 29 during evolution, although its origin, biogenesis, and physiological function are yet 30 unknown. We found that two Syntaxin 1 paralogs in the liverwort, Marchantia 31 polymorpha, are distinctly targeted to forming cell plates and the oil body, suggesting 32 these structures share some developmental similarity. Oil body formation is under the 33 regulation of an ERF/AP2-type transcription factor and loss of the oil body increased M. 34 polymorpha herbivory. These findings highlight a common strategy for the acquisition 35 of organelles with distinct functions in plants, via periodical switching in secretion 36 direction depending on cellular phase transition. 37 38 Main text: 39 Eukaryotic cells originated from prokaryotes by expanding their 40 endomembrane network during evolution, with the last eukaryotic common 41 ancestor (LECA) likely possessing a complex set of organelles 1 . New membrane 42 trafficking pathways were added to the LECA endomembrane network, some of 43 which were secondarily lost in a lineage-specific manner, resulting in divergent 44 and organism-specific membrane trafficking systems and organelle compositions 45 of extant eukaryotes 2-4 . It remains mostly unknown how organelles were acquired 46during evolution. We report that the liverwort-specific oil body was acquired by 47

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

Cited by 185 publications

References 52 publications

The Evolution of Flavonoid Biosynthesis: A Bryophyte Perspective

The Evolution of Flavonoid Biosynthesis: A Bryophyte Perspective

Diversity of Pectin Rhamnogalacturonan I Rhamnosyltransferases in Glycosyltransferase Family 106

Switching secretory pathway direction for organelle acquisition in plants

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