b c vectors pentr slmir482bts b c  (Addgene inc)

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: B/c -based vectors for direct cloning of syn-tasiRNAs downstream SlmiR482b target site (TS). ( A ) TS is in orange, the spacer sequence derived from AtTAS1c is in blue. Top, diagram of the Gateway-compatible entry vectors. Bottom, diagram of binary vector for in plant expression of syn-tasiRNAs. RB: right border; 35S: Cauliflower mosaic virus promoter; Bsa I: Bsa I recognition site, ccd B: gene encoding the gyrase toxin, in pink; LB: left border; attL1 and attL2: GATEWAY recombination sites. Kan R : kanamycin resistance gene; Hyg R : hygromycin resistance gene. ( B ) Organization of SlmiR482b-based syn-tasiRNA constructs. Base pairing between SlmiR482b (dark orange) and its target site (light orange) nucleotides is shown. Curved black arrows indicate DCL4 processing sites. Black linear arrows indicate sRNA-guided cleavage sites. TS refers to target site. In the example diagram, one single 21-nt guide syn-tasiRNA sequence was introduced at the 3’D2[+] position in SlmiR482bTS -based precursors.

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Cloning, Sequencing, Derivative Assay, Plasmid Preparation, Expressing, Virus, Construct

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: Functional analysis of Solanum lycopersicum T1 transgenic lines expressing syn-tasiR-SlSFT, a syn-tasiRNA targeting SINGLE FLOWER TRUSS ( SlSFT ). ( A ) Schematic representation of the anti- SlSFT syn-tasiRNA construct, 35S:SlmiR482bTS-SlSFT , engineered to express syn-tasiR-SlSFT (light blue) from a minimal precursor containing the SlmiR482b target site (TS) (orange) and a 11-nt spacer derived from AtTAS1c (dark blue). Other details are as described in . ( B ) Photographs taken at 5 weeks post-transplanting (wpt) of representative transgenic tomato lines expressing anti- SlSFT syn-tasiRNA compared to a non-transgenic control (NTC) plant. Top panel: whole plants. Bottom panel: detail of the apical region, with arrows marking the first emerging inflorescence (I1) and the last leaf (L), numbered, before it. ( C ) Phenotypic analysis of flowering time in NTC and syn-tasiRNA transgenic lines, showing the number of leaves present at the time of the first emerging inflorescence in each plant. ( D ) Accumulation of SlSFT mRNA in tomato plants. Data are presented as the mean +SE relative expression levels of SlSFT mRNA at 12 wpt after normalization to ACTIN (SlACT), as determined by RT–qPCR (NTC=1 in all comparisons). The asterisk indicates a significant difference from the NTC samples (P<0.05; pairwise Student’s t -test comparison). The NTC sample corresponds to a pooled sample from five NTCs. ( E ) 5′-RLM-RACE analysis of syn-tasiR-SlSFT-guided cleavage of SlSFT . Upper panel: predicted base pairing between syn-tasiR-SlSFT and SlSFT mRNA, with the expected cleavage site indicated by an arrow. The proportion of cloned 5′-RLM-RACE products at the expected cleavage site is shown for syn-tasiR-SlSFT-expressing lines. Lower panel: ethidium bromide-stained gel showing 5′-RLM-RACE products corresponding to the 3′ cleavage product from syn-tasiR-SlSFT-guided cleavage (top), along with RT– PCR products for the target SlSFT (middle) and the control SlACT genes (bottom). The position and expected sizes of syn-tasiRNA-based 5′-RLM-RACE products and control RT-PCR products are indicated.

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Functional Assay, Transgenic Assay, Expressing, Construct, Derivative Assay, Control, Quantitative RT-PCR, Comparison, Clone Assay, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: Accumulation and processing of syn-tasiR-SlSFT expressed from SlmiR482bTS -based precursors in Solanum lycopersicum T1 transgenic lines. ( A ) Northern blot detection of syn-tasiR-SlSFT in RNA preparations from apical leaves collected 12 weeks post-transplanting from six independent transgenic lines and one non-transgenic control (NTC). Each sample represents a pool of two apical leaves. Other details are as described in . ( B ) sRNA profile of 19-24 nt [+] reads mapping to each of the 54 nucleotide positions within the SlmiR482bTS-SlSFT precursor from plants expressing 35S:SlmiR482bTS-SlSFT . Orange, dark blue and light blue boxes represent nucleotides corresponding to NbmiR482aTS , the AtTAS1c -derived spacer and syn-tasiR-SlSFT, respectively. ( C ) Pie chart showing the percentage of reads corresponding to accurately processed 21-nt authentic syn-tasiR-SlSFT (blue) versus other 19-24-nt sRNAs (gray). ( D ) Radar plot displaying the distribution of 21-nt reads across the 21 registers of the precursor transcripts, with position 1 designated immediately after the SlmiR482b-guided cleavage site.

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Transgenic Assay, Northern Blot, Control, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: Functional analysis of potato virus X (PVX) constructs expressing syn-tasiRNAs against tomato spotted wilt virus (TSWV) in S. lycopersicum . ( A ) Schematic representation of PVX-based constructs. Nucleotides of anti-TSWV art-sRNA sequences syn-tasiR-TSWV-1, syn-tasiR-TSWV-2, syn-tasiR-TSWV-3 and syn-tasiR-TSWV-4 are in red, dark brown, light brown and yellow, respectively. Nucleotides of control anti-GUS art-sRNA sequences syn-tasiR-GUS Sl -1 and syn-tasiR-GUS Sl -2 are in dark and light grey, respectively. Nucleotides of AtmiR173a target site (TS) are in red and brown, respectively. Other details are as in . ( B ) Diagram of PVX-based constructs expressing anti-TSWV or anti- GUS syn-tasiRNAs. Color coding for the syn-tasiRNA sequences is consistent with panel (A). Other details are as in . ( C ) Two-dimensional line graph showing, for each of the six-plant sets listed, the percentage of symptomatic plants per day during 28 days. ( D ) Photographs taken at 14 days post-inoculation (dpi) of plants agroinoculated with the different constructs and inoculated (+TSWV) or not (mock) with TSWV. ( E ) Western blot detection of TSWV in protein extracts from apical leaves collected at 14 dpi. A Ponceau-stained membrane is shown as a loading control, highlighting the large subunit of Rubisco (ribulose1,5-biphosphate carboxylase/oxygenase). ( F ) RT-PCR detection at 14 dpi of SlmiR482bTS -based precursors and PVX coat protein fragment (PVX-CP) in apical leaves agroinoculated plants. RT-PCR amplification of SlTYP41 is included as a control. ( G ) Northern blot detection of anti-TSWV art-sRNAs in RNA preparations from apical leaves collected at 14 dpi. A cocktail of probes to simultaneously detect syn-tasiR-TSWV-1, syn-tasiR-TSWV-2, syn-tasiR-TSWV-3 and syn-tasiR-TSWV-4 was used. Other details are as in .

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Functional Assay, Virus, Construct, Expressing, Control, Western Blot, Staining, Membrane, Reverse Transcription Polymerase Chain Reaction, Amplification, Northern Blot

Journal: bioRxiv

Article Title: Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

doi: 10.1101/2025.04.10.648111

Figure Lengend Snippet: Transgene-free gene silencing through PVX-based syn-tasiR-VIGS in Solanum lycopersicum . ( A ) Schematic representation of PVX-based constructs. Nucleotides of anti- SlSu syn-tasiRNAs (syn-tasiR-SlSu-1 and syn-tasiR-SlSu-2) are shown in dark and light blue, respectively. Nucleotides of anti- SlDXS syn-tasiRNAs (syn-tasiR-SlDXS-1 and syn-tasiR-SlDXS-2) are shown in dark and light green, respectively. Other details are as in and . ( B ) Diagram of PVX-based constructs expressing anti- SlSu , anti- SlDXS or anti- GUS syn-tasiRNAs. Color coding for the syn-tasiRNA sequences is consistent with panel (A). Other details are as in and . ( C ) Experimental procedure for transgene-free syn-tasiR-VIGS in S. lycopersicum . Left: crude extracts are prepared from Nicotiana benthamiana plants previously agroinfiltrated with the corresponding syn-tasiR-VIGS construct. Right: young tomato plants are spray-inoculated with syn-tasiR-VIGS crude extracts to induce bleaching associated with SlSu or SlDXS silencing. ( D ) Representative photographs of tomato leaves at 14 days post-spray (dps), from plants sprayed with different crude extracts obtained from agroinoculated N. benthamiana plants. ( E ) Accumulation of SlSu and SlDXS mRNA in tomato plants treated with syn-tasiR-VIGS crude extracts. Data are presented as the mean +SE relative expression levels of SlSu or SlDXS mRNA at 14 dps after normalization to ACTIN ( SlACT ), as determined by RT–qPCR (Mock=1 in all comparisons). The asterisk indicates a significant difference from the mock samples (P<0.05; pairwise Student’s t -test comparison). ( F ) RT-PCR detection at 14 dps of SlmiR482bTS -based precursors and PVX coat protein fragment (PVX-CP) in apical leaves of sprayed plants. RT-PCR amplification of SlACT is included as a control. ( G ) Northern blot detection of anti- SlSu and anti- SlDXS syn-tasiRNAs in RNA preparations from apical leaves collected at 14 dps. A cocktail of probes to simultaneously detect syn-tasiR-SlSu-1, syn-tasiR-SlSu-2, syn-tasiR-SlDXS-1 and syn-tasiR-SlDXS-2 was used. Other details are as described in .

Article Snippet: These new B/c vectors pENTR-SlmiR482bTS-B/c (Addgene plasmid #234368) and pMDC32B-SlmiR482bTS-B/c (Addgene plasmid #234369) were deposited in Addgene.

Techniques: Construct, Expressing, Quantitative RT-PCR, Comparison, Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Northern Blot