A simple test for the cleavage activity of customized endonucleases in plants

Budhagatapalli, Nagaveni; Schedel, Sindy; Gurushidze, Maia; Pencs, Stefanie; Hiekel, Stefan; Rutten, Twan; Kusch, Stefan; Morbitzer, Robert; Lahaye, Thomas; Panstruga, Ralph; Kumlehn, Jochen; Hensel, Goetz

doi:10.1186/s13007-016-0118-6

Cited by 34 publications

(46 citation statements)

References 38 publications

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“…The generation of the HvCKX1 -specific Cas9 construct was essentially done as described previously ( Budhagatapalli et al, 2016 ). The target sequence for the guide RNA has been selected within the first exon of HvCKX1 gene using DESKGEN KNOCKIN tool 2 .…”

Section: Methodsmentioning

confidence: 99%

Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches

et al. 2018

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Barley is one of the most important cereals, which is used for breweries, animal and human feeds. Genetic manipulation of plant hormone cytokinins may influence several physiological processes, besides others stress tolerance, root formation and crop yield. In planta, endogenous cytokinin status is finely regulated by the enzyme cytokinin dehydrogenase (EC 1.5.99.12; CKX), that irreversible degrades the side chain of adenine-derived isoprenoid cytokinins. Increasing grain yield by mean of manipulation of endogenous cytokinin content was assayed by the silencing of the HvCKX1 gene. Moreover, to elucidate the putative role of HvCKX1 gene on grain production, knocked-out Hvckx1 mutant plants were generated using the RNA-guided Cas9 system. Homozygote transgenic plants with silenced HvCKX1 gene and azygous knock-out Hvckx1 mutants have been selected and analyzed. Both reduced expression of HvCKX1 gene and CKX activity were measured in different stages of barley grain development. Phenotyping of the transgenic lines revealed reduced root growth, however, plants produced more tillers and grains than azygous wild-type controls and the total yield was increased up to 15 per cent. Although plant productivity was increased, total grain biomass was decreased to 80% of WT grains. RNA-seq analysis of knock-down transgenic lines revealed that several important macronutrient transporters were downregulated in the stage of massive starch accumulation. It suggests that local accumulation of cytokinins negatively affected nutrients flow resulting in reduced grain biomass. Obtained results confirmed the key role of HvCKX1 for regulation of cytokinin content in barley.

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Section: Methodsmentioning

confidence: 99%

Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches

et al. 2018

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“…Next, the HvCMF3 -specific protospacer sequences were synthesized by introducing proper overhangs to facilitate downstream cloning steps (gRNA1 forward: 5’- GGCG TCGTCGTCCCGCCGCTTGA-3’ and reverse: 5’- AAAC TCAAGCGGCGGGACGACGAC-3’; gRNA2 forward: 5’- GGCG TGACCGTGATGTCCTTCAC-3’ and reverse: 5’- AAAC GTGAAGGACATCACGGTCAC-3’). The protospacer sequence (i.e., annealed oligonucleotides) was then cloned into vector pSH91 (Budhagatapalli et al, 2016). The derived vector was designated as pGH379-7 for gRNA1 and pGH380-12 for gRNA2.…”

Section: Methodsmentioning

confidence: 99%

Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley due to Reduced Plastid Translation

Hensel²,

Melzer³

et al. 2019

Preprint

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Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained only if at least one gene of the pair undergoes neo- or subfunctionalization. Sequence-based phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed that they belong to a newly identified subfamily of genes encoding CCT domain proteins with putative N-terminal chloroplast transit peptides. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a xantha phenotype and successively develop pale green leaves. Compared to the wild type, plastids of the mutant seedlings show decreased PSII efficiency and lower amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional region, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs localized to plastids. Hvcmf3Hvcmf7 double mutants indicated epistatic activity of HvCMF7 over HvCMF3. The chloroplast ribosome deficiency is discussed as the primary defect of the Hvcmf3 mutants. Our data suggests that HvCMF3 and HvCMF7 have similar but not identical functions.One-sentence summaryPhylogenetic and mutant analyses of the barley protein HvCMF3 (ALBOSTRIANS-LIKE) identified, in higher plants, a subfamily of CCT domain proteins with essential function in chloroplast development.

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“…The search for genomic target motifs was focused to the region surrounding the 4 bp deletion carried by the original albostrians mutant and one proper target on each side of the deletion was selected through in silico analysis using the online prediction tool (https://www.deskgen.com/guidebook/; the ‘KNOCKIN’ panel was chosen for gRNA design) (Doench et al, 2014). A synthetic double-stranded oligonucleotide carrying the target-specific part of the gRNA was inserted between the Os U3 (RNA polymerase III) promoter and the downstream gRNA scaffold present in the monocot-compatible intermediate vector pSH91 (Budhagatapalli et al, 2016). Next, the fragment containing the expression cassettes of gRNA and Cas9 was introduced into the binary vector p6i-d35S-TE9 (DNA-Cloning-Service, Hamburg, Germany) using the Sfi I restriction sites.…”

Section: Methodsmentioning

confidence: 99%

“…The physical parameters, 1100 psi helium pressure and 27 inch Hg vacuum had been set to reach efficient bombardment conditions for barley epidermal cells. Preparation and delivery of DNA-coated gold particles was performed according to (Budhagatapalli et al, 2016). The sample was then scanned for the presence of fluorescence signals by help of a Confocal Laser Scanning Microscopy LSM 780 (Carl Zeiss MicroImaging GmbH, Jena, Germany).…”

Section: Methodsmentioning

confidence: 99%

Leaf Variegation and Impaired Chloroplast Development Caused by a Truncated CCT Domain gene in albostrians Barley

Hensel

Mascher

et al. 2019

Preprint

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Chloroplasts fuel plant development and growth by converting solar into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated albostrians mutant of barley has greatly broadened the field of chloroplast biology including the discovery of retrograde signaling. Here, we report the identification of the ALBOSTRIANS gene HvAST by positional cloning as well as its functional validation based on independently induced mutants by TILLING and RNA-guided Cas9 endonuclease mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvAST in the original albostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. HvAST is a homolog of the Arabidopsis thaliana CCT Motif transcription factor gene AtCIA2, which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Interestingly, in barley we localized HvAST to the chloroplast indicating novel without any clear evidence of nuclear localization.One-sentence summaryLeaf variegation in the barley mutant albostrians is caused by mutation of a single CCT-domain containing gene with residual activity, which is directed to the chloroplast.

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A simple test for the cleavage activity of customized endonucleases in plants

Cited by 34 publications

References 38 publications

Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches

Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches

Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley due to Reduced Plastid Translation

Leaf Variegation and Impaired Chloroplast Development Caused by a Truncated CCT Domain gene in albostrians Barley

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