Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A) Workflow for MutChromSeq. (B) Infection responses, disease scorings, and quantification of fungal biomass in Bowman and six barley EMS M1 lines after inoculation with Bipolaris sorokiniana ND90Pr. Photos were taken at seven days after inoculation. The 1-9 rating scale of Fetch and Steffenson was used to rate the spot blotch disease. Fungal biomass was quantified for Bowman and three EMS M1 lines using quantitative PCR. (C) Gene structure and EMS mutations in Scs6 , a gene encoding a canonical coiled-coiled-type NLR (CNL).

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Infection, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A) The coding sequence of Scs6 was expressed under the maize Ubi promoter and NOS terminator (NOSt), and cloned into the backbone of binary vector pANIC12A . (B) The coding sequence of Scs6 was expressed under the promoter+5’UTR (pMla6+5’ UTR) and 3’UTR+terminator (3’ UTR+tMla6) of Mla6 , which was assembled into binary vector pBract202 . Both constructs use hptII driven by the 35S Cauliflower Mosaic Virus (35S) promoter for plant selection during transformation (shown in purple). Left and right T-DNA borders (LB and RB) are shown with filled grey and black blocks, respectively.

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Construct, Virus, Selection, Transformation Assay

Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A-B) Representative images of infection responses of Golden Promise (GP), SxGP DH47 (DH47) and derived transgenic Scs6 T1 plants to B. sorokiniana ND90Pr, seven days after inoculation.

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Infection, Derivative Assay, Transgenic Assay

Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A) PCR amplification of Scs6 with genomic DNA and cDNA from Bowman (Bow_DNA and Bow_cDNA), Golden Promise (GP_DNA and GP_cDNA) and three T0 transgenic barley plants (GP_Scs6-1, GP_Scs6-2, and GP_Scs6-3) using primers Scs6-F4+Scs6-R1 . (B) PCR amplification of Scs6 with cDNA derived from Bowman (Bow_cDNA), Golden Promise (GP_cDNA), and five T1 plants derived from each of the two T0 transgenic barley plants (GP_Scs6-4 and GP_Scs6-5) using primers Scs6-F4+Scs6-R1 . * (+) indicated that PCR was positive and (-) indicated that PCR was negative.

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Amplification, Transgenic Assay, Derivative Assay

Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A) Barley genotypes that express Scs6 (Bowman) or negative control (ND B112) were infiltrated with intercellular washing fluid (IWF) that was isolated from Bowman leaves infected either with wild type B. sorokiniana ND90Pr (IWF ND90Pr ) or mutant B. sorokiniana Δ nps1 (IWF Δ nps1 ), as indicated. (B-C) N. benthamiana plants were transformed transiently, as indicated. Genes were fused in between the 35S promotor sequence and 4xmyc (receptors) or mYFP (AVR A6 without signal peptide) epitope sequences. Twenty-four hours after Agrobacterium -mediated gene delivery, IWF ND90Pr , IWF Δ nps1 or water was infiltrated, as indicated. Cell death phenotypes were assessed and documented at two or four days after agroinfiltration for IWF-triggered cell death or effector-triggered cell death, respectively. Representative pictures of at least six biological replicates (indicated in brackets) are shown and combinations that resulted in cell death are highlighted with a red box. (D) For determination of protein levels of receptor-4xMyc (approx. 114 kDa and AVR A6 -mYFP (39 kDA) in N. benthamiana , leaf tissue was harvested two days post Agrobacterium infiltration.

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Negative Control, Isolation, Infection, Mutagenesis, Transformation Assay, Sequencing

Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) mScarlet expression in the 6 indicated KOs, rescued with their respective V5-tagged protein-coding sequence (CDS), or with an empty vector. Each data point is an individual clone (n=3 clones per KO). (B) Hygromycin resistance after rescue. One representative clone is shown for each KO. (C) Dazl expression after rescue (n=3 rescued clones per KO). (D) Western blot analysis of DAZL and the indicated V5-tagged CDS. (E) RT-qPCR assays showing the expression changes of Dazl , differentiation ( Fgf5 ) and pluripotency ( Prdm14, Pou5f1 ) markers upon spontaneous differentiation after withdrawal of LIF. (F) MeDIP assay showing the relative levels of 5mC at the Dazl promoter for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO). (G) LUMA assay showing the changes in the global level of DNA methylation for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO). (H and I) LC-MS/MS assay showing the changes in the global level of 5mdC/C (H) and 5hmdC/C (I) for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO).

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

Techniques: Expressing, Sequencing, Plasmid Preparation, Clone Assay, Western Blot, Quantitative RT-PCR, Methylated DNA Immunoprecipitation, DNA Methylation Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) RT-qPCR analysis of the expression of 2C-specific genes in Spop KO, Mcm3ap KO, Kdm5c KO, and Zbtb14 KO, compared to the parental DASH mES cells (n=3 clones per KO). (B) Western blot analysis of ZSCAN4 and MERVL-Gag for Spop KO, Mcm3ap KO, Kdm5c KO, Zbtb14 KO, and parental DASH mES cells cultured in Serum or 2i. (C) RT-qPCR analysis of the expression of 2C-specific genes for Spop KO, Mcm3ap KO, Kdm5c KO, and Zbtb14 KO, transfected with either an empty plasmid or their respective CDS, compared to the parental DASH mES cells (n=3, clones per KO). (D) Western blot analysis of V5, ZSCAN4, and MERVL-Gag in 4 candidates KO, transfected with an empty plasmid or their respective V5-tagged CDS. (E) Model for role of novel epigenetic factors ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D in the regulation of germline genes, and 2 cell-like cell state.

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

Techniques: Quantitative RT-PCR, Expressing, Clone Assay, Western Blot, Cell Culture, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) RT-qPCR analysis of the expression of 2C-specific genes in DASH mES cells cultured in 2i, Bend3 KO, and Kmt2d KO. (B) Western blot analysis of ZSCAN4 and MERVL-Gag for Bend3 KO, Kmt2d KO, and parental DASH mES cells cultured in Serum or 2i. (C) RT-qPCR analysis of the expression of 2C-specific genes for Bend3 KO (n=3, clones), transfected with an empty plasmid or their respective CDS, compared to the parental DASH mES cells. (D) Western blot analysis of ZSCAN4 and MERVL-Gag for Kdm5c KO clones, transfected with an empty plasmid, or a wild-type Kdm5c CDS, or a catalytically dead Kdm5c CDS (H514A). (E) RT-qPCR analysis of the expression of 2C-specific genes for Kdm5c KO clones, transfected with an empty plasmid, or a wild-type Kdm5c CDS, or a catalytically dead Kdm5c CDS (H514A).

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Clone Assay, Transfection, Plasmid Preparation

Journal: Horticulture Research

Article Title: Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

doi: 10.1093/hr/uhab045

Figure Lengend Snippet: Sequence analysis of oft3. a The genomic DNA structure of SlIDI1 and the deleted fragment in oft3. Black boxes and gray lines represent the exons and introns of SlIDI1, respectively. The deleted fragment encompassed 55 bp in exon 5 (underlined) and 61 bp in intron 5. b Genotyping of tomato varieties using the markers developed based on the SlIDI1 deletion mutation of oft3. One 520 bp band was amplified by PCR in five orange-fruited inbred lines (Lanes 1–5), and their corresponding F1 hybrids (Lanes 11–14) were obtained by crossing with oft3, whereas a 636 bp band was amplified in five wild-type red-fruited inbred lines (Lanes 6–10). The two bands were produced by PCR in the three F1 hybrids between oft3 and the r mutant TB0040 (Lane 15), t mutant TB0017 (Lane 16) and wild-type AC (Lane 17). c Deduced protein sequence of SlIDI1 in oft3. SlIDI1 in oft3 was deduced to produce a truncated protein (157 aa) terminated by the premature translational stop signal resulting from the deletion mutation.

Article Snippet: The full-length CDS and the upstream 1500 bp genomic sequence of SlIDI1 from AC (WT) were artificially synthesized by GenScript Biotechnology Co., Ltd. (Nanjing, China).

Techniques: Sequencing, Mutagenesis, Amplification, Produced

Journal: Horticulture Research

Article Title: Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

doi: 10.1093/hr/uhab045

Figure Lengend Snippet: Functional complementation and knockout analysis of oft3. Functional complementation analysis was conducted in oft3 by transforming oft3 plants with SlIDI1 driven by its native promoter, and all the transformants (pIDI:IDI1 #1 ~ 12) were restored to the red-fruited phenotype, as observed in wild-type AC (left). CR-idi1 #1 was generated by knocking out SlIDI1 in wild-type AC using the CRISPR–Cas9 system. CR-idi1 #1 plants from the T1 generation that were shown to be homozygous for mutated SlIDI1 by genotyping showed an orange-fruited phenotype, similar to that of oft3 (right). Fruit color and longitudinal sections from four different ripening stages are shown. DPA: days post-anthesis.

Article Snippet: The full-length CDS and the upstream 1500 bp genomic sequence of SlIDI1 from AC (WT) were artificially synthesized by GenScript Biotechnology Co., Ltd. (Nanjing, China).

Techniques: Functional Assay, Knock-Out, Generated, CRISPR

Journal: Horticulture Research

Article Title: Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

doi: 10.1093/hr/uhab045

Figure Lengend Snippet: Carotenoid contents of the flowers of AC (WT) and the CRISPR–Cas9-generated SlIDI1 mutant

Article Snippet: The full-length CDS and the upstream 1500 bp genomic sequence of SlIDI1 from AC (WT) were artificially synthesized by GenScript Biotechnology Co., Ltd. (Nanjing, China).

Techniques: CRISPR

Journal: Horticulture Research

Article Title: Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

doi: 10.1093/hr/uhab045

Figure Lengend Snippet: Spatiotemporal specific expression analysis of SlIDI1 in wild-type AC by qRT–PCR. a Tissue-specific expression analysis of SlIDI1 in different tissues of wild-type AC. b Stage-specific expression analysis of SlIDI1 in the fruit, petals and stamens of wild-type AC at different ripening or maturation stages. Values are the means of four biological replicates ± SD. For stage-specific expression analysis, values were compared among different stages of each tissue, and asterisks denote significance by Student’s t-test (*P < 0.05, **P < 0.01). DPA: days post-anthesis, DBA: days before anthesis.

Article Snippet: The full-length CDS and the upstream 1500 bp genomic sequence of SlIDI1 from AC (WT) were artificially synthesized by GenScript Biotechnology Co., Ltd. (Nanjing, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Horticulture Research

Article Title: Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

doi: 10.1093/hr/uhab045

Figure Lengend Snippet: Alternative transcripts of SlIDI1 identified by RACE-PCR. a RACE-PCR products amplified from the leaves, fruit, petals and anthers of wild-type AC. For 5’ RACE-PCR (left), two identical short products were amplified from leaves and fruit. For 3’ RACE-PCR (right), one short product was amplified from fruit. b Schematic representation of the structures of DNA, the full-length cDNA of SlIDI1 (SlIDI1-L) and two short RACE-PCR products (SlIDI1–5’S and SlIDI1–3’S) identified by 5’ RACE-PCR and 3’ RACE-PCR. SlIDI1-L: long transcripts with the longest 5’-UTR (44 bp) and 3’-UTR (338 bp). Exons and introns are represented by boxes and lines. The 5′ and 3’ UTR are represented by gray boxes. The two specific primers used for 5’-RACE and 3’-RACE (5’RACE-GSP and 3’RACE-GSP) are represented by red boxes. In alternative transcription-generated SlIDI1–5’S, a 274 bp deletion occurred in the CDS region, including exon 1 and part of exon 2. In alternative-splicing-generated SlIDI1–3’S, 254 bp of exon 6 was replaced by 165 bp of intron 5 (white box). c Aligned sequences of the full-length cDNA of SlIDI1 (SlIDI1-L) and two short RACE-PCR products (SlIDI1–5’S and SlIDI1–3’S). The putative initiation codons and stop codons are indicated in red. The retained 165 bp of intron 5 in SlIDI1–3’S is boxed. d Aligned protein sequences deduced from SlIDI1-L, SlIDI1–5’S and SlIDI1–3’S. SlIDI1 is 294 amino acids in length. The putative CTP and type 1 peroxisome targeting sequence (PTS1) are underlined.

Article Snippet: The full-length CDS and the upstream 1500 bp genomic sequence of SlIDI1 from AC (WT) were artificially synthesized by GenScript Biotechnology Co., Ltd. (Nanjing, China).

Techniques: Amplification, Generated, Alternative Splicing, Sequencing

Journal: Horticulture Research

Article Title: Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

doi: 10.1093/hr/uhab045

Figure Lengend Snippet: Subcellular location of SlIDI1. 35S:SlIDI1-EGFP, with the full-length SlIDI1 CDS, and 35S:SlIDI1t-EGFP, with a truncated SlIDI1 CDS lacking the 59-amino acid extension sequence at the N-terminus, were agroinfiltrated into tobacco leaves, and the agroinfiltrated leaf epidermal cells were examined under a confocal microscope. 35S:EGFP served as the vector control

Article Snippet: The full-length CDS and the upstream 1500 bp genomic sequence of SlIDI1 from AC (WT) were artificially synthesized by GenScript Biotechnology Co., Ltd. (Nanjing, China).

Techniques: Sequencing, Microscopy, Plasmid Preparation, Control

Journal: Horticulture Research

Article Title: Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way.

doi: 10.1093/hr/uhab045

Figure Lengend Snippet: Expression analysis of SlBCH1 and SlBCH2 by qRT–PCR in the fruit of the SlIDI1 mutant at four ripening stages. a and b Expression analysis of SlBCH1 and SlBCH2 in two wild-type (WT-1 and WT-2) and two oft3 genotyped individuals (oft3–1 and oft3–2) from the segregating BC1F2 population. c and d Expression analysis of SlBCH1 and SlBCH2 in the CRISPR–Cas9-generated SlIDI1 mutant CR-idi1 #1 compared with its parental line AC. Values are the means of four biological replicates ± SD and were compared between genotypes at the same ripening stage. Asterisks denote significance by Student’s t-test (*P < 0.05, **P < 0.01). DPA: days post-anthesis.

Article Snippet: The full-length CDS and the upstream 1500 bp genomic sequence of SlIDI1 from AC (WT) were artificially synthesized by GenScript Biotechnology Co., Ltd. (Nanjing, China).

Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, CRISPR, Generated

Journal: Data in Brief

Article Title: Dataset from de novo transcriptome assembly of Myristica fatua leaves using MinION nanopore sequencer

doi: 10.1016/j.dib.2022.108838

Figure Lengend Snippet: Read and assembly statistics of Myristica fatua leaves.

Article Snippet: • This data provides Myristica fatua coding sequence (CDS) as the first transcriptome reference using Oxford Nanopore Technologies of long-read sequencing • This data could benefit studies to identify full-length transcripts related to flavonoid biosynthesis for molecular biologists that are used for downstream analysis in Myristica fatua and related genera.

Techniques:

Journal: Data in Brief

Article Title: Dataset from de novo transcriptome assembly of Myristica fatua leaves using MinION nanopore sequencer

doi: 10.1016/j.dib.2022.108838

Figure Lengend Snippet: Functional annotation summary of Myristica fatua transcripts.

Article Snippet: • This data provides Myristica fatua coding sequence (CDS) as the first transcriptome reference using Oxford Nanopore Technologies of long-read sequencing • This data could benefit studies to identify full-length transcripts related to flavonoid biosynthesis for molecular biologists that are used for downstream analysis in Myristica fatua and related genera.

Techniques: Functional Assay

Journal: Data in Brief

Article Title: Dataset from de novo transcriptome assembly of Myristica fatua leaves using MinION nanopore sequencer

doi: 10.1016/j.dib.2022.108838

Figure Lengend Snippet: Gene Ontology (GO) classification of Myristica fatua for (a) Biological Process, (b) Molecular Function, (c) Cellular Component.

Article Snippet: • This data provides Myristica fatua coding sequence (CDS) as the first transcriptome reference using Oxford Nanopore Technologies of long-read sequencing • This data could benefit studies to identify full-length transcripts related to flavonoid biosynthesis for molecular biologists that are used for downstream analysis in Myristica fatua and related genera.

Techniques:

Journal: Data in Brief

Article Title: Dataset from de novo transcriptome assembly of Myristica fatua leaves using MinION nanopore sequencer

doi: 10.1016/j.dib.2022.108838

Figure Lengend Snippet: Number of microsatellite regions observed in Myristica fatua transcripts.

Article Snippet: • This data provides Myristica fatua coding sequence (CDS) as the first transcriptome reference using Oxford Nanopore Technologies of long-read sequencing • This data could benefit studies to identify full-length transcripts related to flavonoid biosynthesis for molecular biologists that are used for downstream analysis in Myristica fatua and related genera.

Techniques: