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The document discusses new tools for controlling gene expression in plants using artificial small RNAs, focusing on gene silencing processes essential for plant growth and disease resistance. It highlights the applications, advantages, and limitations of artificial small RNAs, including their potential for specific gene regulation and the complexities involved in their design and expression. The conclusion emphasizes the necessity of artificial small RNAs in advancing gene function studies and enhancing plant traits.

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Nuevas herramientas para el control de la expresión génica en plantas basadas en pequeños RNAs artificiales Alberto Carbonell www.slideshare.net/AlbertoCarbonell1 acarbonell@ibmcp.upv.es @A_Carbonell_
Gene Silencing  Eukaryotic evolutionarily conserved, sequence-specific, RNA-based gene-inactivation system that regulates key biological processes Development Stress response Chromatin structure Pathogen defense Gene Silencing
Gene Silencing is important for plant growth Normal RNA silencing Defective RNA silencing
Gene Silencing is necessary for proper leaf shape Defective RNA silencing Normal RNA silencing
Gene Silencing controls developmental timing Normal RNA silencing Defective RNA silencing
Gene Silencing defends plants against diseases Normal RNA silencing Defective RNA silencing Plant resistant to virus Plant susceptible to virus
Classes of Gene Silencing Transcriptional Gene Silencing (TGS) An * X Gene An mRNA Protein No Gene Silencing Post-Transcriptional Gene Silencing (PTGS) An* X
PTGS in Plants AGO dsRNA target RNA ssRNA Intramolecular (folding) RNA-dependent RNA polymerase DCL RDR .............. AGO .............. AGO .............. An sRNA An An Translational repression ............. RNA-dependent RNA polymerase target RNA RDR
Small RNA (sRNA) Silencing Pathways |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| HYL1 DCL1 An MIRNA primary transcript |||||| |||||| An miRNA targetAGO1 AGO1 SE miRNA foldback miRNA pathway HEN1miRNA An tasiRNA target |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An AGO1 DRB4 DCL4 DRB4 DCL4 SDE5 RDR6 SGS3 RDR6 TAS primary transcript AGO1 tasiRNA pathway HEN1 AGO dsRNA tasiRNA miRNA
Artificial sRNA Silencing Pathways hpRNA pathway hpRNA transgene An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! hpRNA primary transcript An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! HEN1# AGO# dsRNA! tasiRNA! ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||DCL4 DRB4 An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# ||||||||||||||| |||||||||||||||||||||| ||||||||||||||| |||||||||||||||||||||| HYL1% DCL1% An% ed'MIRNA'primary%transcript' |||||| |||||| An% amiRNA% target%RNA'AGO1% AGO1% SE% miRNA% oldback' HEN1% amiRNA' amiRNA*' miRNA% miRNA*% An An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| hpRNA siRNA |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| HYL1% DCL1% An% Modified'MIRNA'primary%transcript' |||||| |||||| An% amiRNA% target%RNA'AGO1% AGO1% SE% amiRNA% foldback' HEN1% amiRNA' amiRNA*' amiRNA% amiRNA*% s amiRNA pathway |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| HYL1% DCL1% An% Modified'MIRNA'primary%transcript' |||||| |||||| An% amiRNA% target%RNA'AGO1% AGO1% SE% amiRNA% foldback' HEN1% amiRNA' amiRNA*' amiRNA% amiRNA*% amiRNA transgene amiRNA amiRNA* An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! syn-tasiRNA pathway An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# syn-tasiRNA transgene syntasiR-1 syntasiR-2 An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! amiRNA pathway |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| HYL1% DCL1% An% Modified'MIRNA'primary%transcript' |||||| |||||| An% amiRNA% target%RNA'AGO1% AGO1% SE% amiRNA% foldback' HEN1% amiRNA' amiRNA*' amiRNA% amiRNA*% amiRNA transgene amiRNA amiRNA* An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA!
Applications of Plant Artificial sRNAs  Study gene function CONCLUSION: Gene A is necessary for chlorophyll synthesis Normal function of plant gene A Artificial small RNAs shut down function of plant gene A
Applications of Plant artificial sRNAs  Induce antiviral resistance Niu et al. Nature Biotechnology (2006)
Advantages of Plant Artificial sRNAs  Spacio and temporal regulation of gene expression -Tissue specific expression of artificial sRNAs -Inducible expression of artificial sRNAs  Study of lethal genes  Simultaneous silencing of multiple: -Sequence related genes (e.g. gene family) -Sequence unrelated genes  Generation of allelic series with different silencing levels -Transformation process -Use of expression promoters of distinct strength -Fine tune regulation of the artificial sRNA efficacy by modifying base- pairing interactions between the artificial sRNA and target
Limitations of Artificial sRNA Systems 1. Design (WMD3): -Non-intuitive interface -Relatively slow -No syn-tasiRNA design tool http://wmd3.weigelworld.org/ 1st PCR amiRNA insert 2nd PCR BamHI cut EcoRI cut Gel purification Gel purification Entry vector Cut pBSK vector with EcoRI Alkaline Phosphatase treatment Gel purification BamHI cut Entry plasmid Mini-prep Pick up positive colony Confirm the sequencing Ligation Transform into E. coli Entry plasmid Binary vector Binary plasmid Cut with restriction enzyme(s) Gel purification Mini-prep Pick up positive colony Confirm the replacement of the fragment Transform into Agro Ligation Transform into E. coli Cut with restriction enzyme(s) Alkaline Phosphatase treatment Gel purification2. Cloning: -Long and slow (multi-step) -Non cost-effective -Non-high throughput capability -Lack of convenient syn-tasiRNA cloning systems Schwabb et al., Plant Cell (2006) 3. Expression: -Frequent miss-processing of amiRNAs (-> off target effects!) 1 2 3 4 5 6 7 8 9 amiRNAs -21 nt
This platform includes: a) Web-based tools for the design of artificial sRNAs b) A new generation of artificial sRNA vectors GOAL: To develop a new platform for the: 1. Design 2. Cloning (high-throughput) and 3. Expression of plant amiRNAs and syn-tasiRNAs in a simple, fast, cost- effective and effective manner for specific gene silencing in plants.
P-SAMS http://p-sams.carringtonlab.org Fahlgren et al. Bioinformatics (2016)
P-SAMS Computational Design of Artificial sRNAs  Step 1: Identification of all possible target sites in target transcript(s) by cataloguing all possible 21-nucleotide sequences  Step 2: Remove target sites that contain 15-nt sequence form positions 6-20 (core target pairing sequence) that perfectly match a non-target transcript  Step 3: Target sites are grouped by the core target pairing sequence, only target site groups that contain all input genes are considered further.  Step 4: Grouped sites are scored and ranked based on group-wise similarity and the identity of nucleotides at positions 1, 2, 3 and 21.  Step 5: For each group site, a guide RNA is designed to target all sites with the additional criteria that position 1 and 19 are a U and a C, and that position 21 is mismatched  Step 6: P-SAMS uses TargetFinder to predict target RNAs for each guide RNA. -Optimal Results: include guide RNAs predicted to target exclusively transcripts from input genes -Sub-Optimal Results: guide RNAs predicted to target transcripts from input genes AND from non-input genes
Precursor Selection For AmiRNA Vectors Ath-MIR390a For Eudicots A B Carbonell et al. Plant Physiology (2014) Osa-MIR390 For Monocots A B Carbonell et al. Plant Journal (2015)
Oligonucleotide Design for Direct AmiRNA Cloning in MIR390-Based Vectors Carbonell et al. Plant Physiology (2014)
AmiRNA Cloning in B/c Vectors Carbonell et al. Plant Physiology (2014)
Plant expression vectors AmiRNA B/c Vectors Carbonell et al. Plant Physiology (2014) GATEWAY-compatible entry vectors Carbonell et al. Plant Journal (2015)
Functionality Of AmiRNA Vectors For Eudicots Carbonell et al. Plant Physiology (2014) G - U6 - amiRNA amiR- Trich - + amiR- Lfy - + amiR- Ch42 - + amiRNA accumulation in Arabidopsis transgenic lines amiR- Ft - + 21- 24- - amiRNA amiR-CH42 - + - U6 amiR- CH42 Severe Inter- mediate Weak No phenotype C Weak 35S:GUS Intermediate Severe No phenotype Silencing of CHLORINA 42 (CH42) LFY - + amiR-Lfy TargetmRNA relativeexpression 0 0.2 0.4 0.6 0.8 1 1.2 CH42 - + amiR-Ch42 RNAaccumulation amiR-CH42 - + 62/101 25/101 10/101 vector 48/48 35S:GUS 35S:AtMIR390a-Trich G - U6 - amiRNA amiR- Trich - + amiR- Lfy - + amiR- Ch42 - + amiRNA accumulation in Arabidopsis transgenic lines amiR- Ft - + 21- 24- - amiRNA - + - U6 amiR-Trich amiR-Trichvector TRY CPC ETC2 - + - + - + amiR-Trich RNAaccumulation LFY - + amiR-Lfy TargetmRNA relativeexpression 0 0.2 0.4 0.6 0.8 1 1.2 amiR-Trich - + - + - + 52/5333/33 Silencing of TRICH (TRY, CPC, ETC2)
UC U AG C U A GACAGGCGUAAGAUUGCG CGCAAUCUU C GCC UGCUC A U G C 35S:OsMIR390-AtL- Bri1 16/20 AC U UG C U A GACAGGCGUAAGAUUGCG CGCAAUCUU C GCC UGCUC A U G C 35S:OsMIR390- Bri1vector 0/7 7/11 Silencing of BRASSINOSTEROID- INSENSITIVE 1 (BRI1) Carbonell et al. Plant Journal (2015) Functionality Of AmiRNA Vectors For Monocots 0 0.2 0.4 0.6 0.8 1 1.2 1.4 amiR-Bri1 - + BRI1 RNA - 35S:OsMIR390 35S:OsMIR390- AtL + vector 35S:OsMIR390- Bri1 AtL-Bri1 21 - 24 - - amiRNA - U6 amiR-Spl11 35S: OsMIR390- Spl11 35S: OsMIR390-AtL- Spl11vector 0/33 8/8 23/23 AC U UG C U A UGGCUACUGCUCAGAUCG CGAUCUGAG A GUA GCCCA U A G A C UG C U A UGGCUACUGCUCAGAUCG CGAUCUGAG A GUA GCCCA U A G A U A vector 35S:OsMIR390- Spl11 AtL-Spl11 Silencing of SPOTTED LEAF 11 (SPL11) - + + SPL11 RNA 21 - 24 - - amiRNA - U6 LFY - + amiR-Lfy TargetmRNA relativeexpression 0 0.2 0.4 0.6 0.8 1 1.2 vector 35S:OsMIR390 35S:OsMIR390- AtL
Carbonell et al. Plant Physiology (2014) Precursor Selection For Syn-tasiRNA Vectors Oligonucleotide Design for Cloning in AtTAS1c- based Vectors
Syn-tasiRNA Cloning in B/c Vectors Carbonell et al. Plant Physiology (2014)
pMDC123SB-AtTAS1c-B/c Gateway-compatible entry clonePlant expression vectors pMDC32B-AtTAS1c-B/c pENTR-TAS1c-B/c ccdB BsaI BsaIAtTAS1c 5’ 3’ KanRattL2attL 1 ccdB BsaI BsaIAtTAS1c 5’ 3’ HygRNosR B L B 2x35S BsaI KanR ccdB BsaI BsaIAtTAS1c 5’ 3’ BastaRNosR B L B 2x35S BsaI KanR Syn-tasiRNA B/c Vectors Carbonell et al. Plant Physiology (2014)
AtTAS1c-D3&D4-Trich target mRNA 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||||||:| 3’ AGGGTAAGCTATGACGAGTGA TRY TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||||||:| AGGGTAAGCTATGACGAGTGA 5’ AtTAS1c-D3&D4-Ft target mRNA 5’ TTGGTTATAAAGGAAGAGGCC |||||||||||||||||:||| 3’ AACCAATATTTCCTTCTTCGG TTGGTTATAAAGGAAGAGGCC 3’ |||||||||||||||||:||| AACCAATATTTCCTTCTTCGG 5’ AtTAS1c-D3Trich-D4Ft target mRNA 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||||||:| 3’ AGGGTAAGCTATGACGAGTGA TTGGTTATAAAGGAAGAGGCC 3’ |||||||||||||||||:||| AACCAATATTTCCTTCTTCGG 5’ FT TRY TRY FT FT CPC 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG ETC2 5’ TCCCATTCGATACTGCTCGCC ||||||||||| ||:|||:|| 3’ AGGGTAAGCTACGATGAGTGA CPC TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| AGGGTAAGCTATGATGAGTGG 5’ ETC2 TCCCATTCGATACTGCTCGCC 3’ ||||||||||| ||:|||:|| AGGGTAAGCTACGATGAGTGA 5’ CPC 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG 5’ 2x35S:AtTAS1c miR173 target site miR173 5’ GUGAUUUUUCUCUACAAGCGAA 3’ |||||||:||||| |||||||| 3’ CACUAAAGAGAGACGUUCGCUU 5’ syn-tasiRNAs syn-tasiRNA-1 (3’D3[+]) syn-tasiRNA-2 (3’D4[+]) AtTAS1c-D3&D4-Trich target mRNA 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||||||:| 3’ AGGGTAAGCTATGACGAGTGA TRY TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||||||:| AGGGTAAGCTATGACGAGTGA 5’ AtTAS1c-D3&D4-Ft target mRNA 5’ TTGGTTATAAAGGAAGAGGCC |||||||||||||||||:||| 3’ AACCAATATTTCCTTCTTCGG TTGGTTATAAAGGAAGAGGCC 3’ |||||||||||||||||:||| AACCAATATTTCCTTCTTCGG 5’ AtTAS1c-D3Trich-D4Ft target mRNA 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||||||:| 3’ AGGGTAAGCTATGACGAGTGA TTGGTTATAAAGGAAGAGGCC 3’ |||||||||||||||||:||| AACCAATATTTCCTTCTTCGG 5’ FT AtTAS1c-D3Ft-D4Trich target mRNA 5’ TTGGTTATAAAGGAAGAGGCC |||||||||||||||||:||| 3’ AACCAATATTTCCTTCTTCGG FT TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||||||:| AGGGTAAGCTATGACGAGTGA 5’ TRY TRY TRY FT FT CPC 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG ETC2 5’ TCCCATTCGATACTGCTCGCC ||||||||||| ||:|||:|| 3’ AGGGTAAGCTACGATGAGTGA CPC TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| AGGGTAAGCTATGATGAGTGG 5’ ETC2 TCCCATTCGATACTGCTCGCC 3’ ||||||||||| ||:|||:|| AGGGTAAGCTACGATGAGTGA 5’ CPC 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG 5’ ETC2 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||| ||:|||:|| 3’ AGGGTAAGCTACGATGAGTGA 5’ CPC 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG 5’ ETC2 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||| ||:|||:|| 3’ AGGGTAAGCTACGATGAGTGA 5’ miR173 5’ GUGAUUUUUCUCUACAAGCGAA 3’ |||||||:||||| |||||||| 3’ CACUAAAGAGAGACGUUCGCUU 5’ syn-tasiRNA-1 (3’D3[+]) syn-tasiRNA-2 (3’D4[+]) Functionality of Syn-tasiRNA Vectors 35S:GUS Trich Ft 35S:AtMIR390- 35S:GUS Trich Ft D3&D4-Trich D3&D4-Ft D3Trich-D4Ft D3Ft-D 35S:AtMIR390- 35S:AtTAS1c- vector syntasiRNA-Trich syntasiRNA-Ft 35S:GUS Trich Ft D3&D4-Trich D3&D4-Ft D3Trich-D4Ft D3Ft-D4Trich 35S:AtMIR390- 35S:AtTAS1c- Carbonell et al. Plant Physiology (2014)
 syn-tasiRNA vectors for targeting single or multiple (sequence unrelated) genes -Arabidopsis (and close species) vectors: AtTAS1c-based -In other species if MIR173 is co-expressed  amiRNA vectors for targeting single or multiple (sequence related) genes: -Eudicot vectors: AtMIR390a-based -Monocot vectors: OsMIR390-AtL-based Development of a new platform to design, clone and express plant artificial small RNAs in a simple, fast, cost-effective and effective manner to silence single or multiple genes in plants Summary  P-SAMS webtool with two apps (P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer) for the automated design of amiRNAs and syn-tasiRNAs, respectively AtMIR390a-B/c vector amiRNA insert Oligo annealing BsaI digestion + ligation E. coli transformation Plasmid purification Sequencing amiRNA construct 1 h 5 min Plant transformation Day 6 Agrobacterium liquid culture Day 7 Day 2 amiRNA oligos Agrobacterium transformation Day 4 E. coli liquid culture Day 1 P-SAMS amiRNA Design Oligo ordering Day 3 1-10 min amiRNA sequence
World-Wide Usage Of P-SAMS Number of Sessions
B/c Vectors are Available @ www.addgene.org
Some Institutions Having Requested B/c Vectors
Viroids  Single-stranded circular RNA (246-401 nt)  High secondary structure content  Do not code for proteins  Need host factors for replication G CUGA 120 C U U C A G G U C U CG A C U G . . . . . . . . . . . . G A 160 180 200 240 380 GG C ACCUGA U G C C U A G C G U C C A A C C C C G G G A G G AAA GGGGUUGGG A C G G G C C A G U C C C A G U C G G U U C G C U C UCG U A G U C A C A G C C A C U G G G GA A CC U A G G C AG A U GGCUGGACG G A U G U C U A G U C C CC G A GC .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 U C C U U U G G A G UA C U C C A G A G G A . . . . . . . . . . . CCGGAA C G UU C CG . . . . . .. U U G G G U U UGA A A C C C C C A A A G G U A A A UA C C U C . . . . . C U C U A A G G G A G . . . . A GG U C G U A A A A C U U C C. . . . . 140 60 100 U C C A U U U C U C A U CA G G A A A C C C A . . . . . .. . . . . . . . .. . . . U G G C U G U G C G . 20 U C GGU G UCUGA A GAAAC CUC U 40 G A U C C A U GA C A G A U C . . . . . 80 U A A A G G A G G C U U U C C C C C U C C A A G G C U U C . . . . . . . . . . . . . . 320 340 360 280 C C A U U U C GAGAUGG .. . . . . . . 300 U C U GA A G G . . U A G CA A . C G U U G G A A U U C U C C UCGG 260 . C U A 220 A U G G G CUGA 120 C U U C A G G U C U CG A C U G . . . . . .. . . . . . . . . . . .. . . . . . G A 160 180 200 240 380 GG C ACCUGA U G C C U A G C G U C C A A C C C C G G G A G G AAA GGGGUUGGG A C G G G C C A G G G C C A G U C C C A G U U C C C A G U C G G U U G G U U C G C U G C U C UCG U A G U C A C A G C C A C U G G G GA A CC U A G G C AG A U GGCUGGAC GGCUGGACG G A U G U C U A G U CG U C U A G U C C CC G A GC .. . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . .. . . . . 1 U C C U U U G G A G U U C C U U U G G A G UA C U C C A G A G G A . . . . . . . . . . . CCGGAA C G UU C CG . . . . . .. U U G G G U U UGA A A C C C C C A A A G G U A A A UA C C U C . . . . . C U C U C U C U A A G G G A G G G A G . . . . . . . . A GG U C G U A A A A C U U C C. . . . . 140 60 100 U C C A U U U C U C A U CA G G A A A C C C A . . . . . .. . . . . ... . . . . .. . . . . . . .. . . .. . .. . . . U G G C U G G G C U G U G C G . 20 U C GGU G UCUGA A GAAAC CUC U 40 G A U C C A U GA C A G A U C . . . . . 80 U A A A G G A G G C G G A G G G G A G G C U U U C C C C C U C C A A G G C U U C C C C U C C A A G G C U U C C C U C C A A G G C U U C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320320 340340 360360 280 C C A U U U C GAGAUGG .. . . . . . ... . . . . . . 300300 U C U GA A G G . . U A G CA A . C G U U G G A A U U C U C C UCGG 260 . C U A 220 A U G G GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CGG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGUUUCG U CCUUUCGG A CUCAUCAGGG AAA GUACACA GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CGG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGUUUCG U CCUUUCGG A CUCAUCAGGG AAA GUACACACU U UCC GAC GG U GGG U UCGUC G A C A C C UC U C C C C C U C C C A G G U A C U A U C C C C U U U C A A G G A U G U G U U C C C U A G G A G G G U G G G U G U A C CUCUUUUGGAU UGC U C C GGCCUUCCAGGAGAG A U AGA G GACGACCUC U CC C C A1 204060 80 100 120 140 160 180 200 220 240 260 280 300320 CU U UCC GAC GG U GGG U UCGUC G A C A C C UC U C C C C C U C C C A G G U A C U A U C C C C U U U C A A G G A U G U G U U C C C U A G G A G G G U G G G U G U A C CUCUUUUGGAU UGC U C C GGCCUUCCAGGAGAG A U AGA G GACGACCUC U CC C CAUAU A1 4060 80 100 120 140 160 180 200 220 240 260 280 300320 GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CGG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGU GGG U GGUGUGUGC CAC CCCGAUG A CCGAAAGG U CAAAU GAAAU GG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGU GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGUUUCG U CCUUUCG A CUCAUCAGGG AAA GUACACA GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGU G U CCUUU A CUCAUCAGGG AAA GUACAC CU U UCC GAC GG U GGG U UCGUC G A C A C C UC U C C C C C U C C C A G G U A C U A U C C C C U U U C A A G G A U G U G U U C C C U A G G A G G G U G G G U G U A C CUCUUUUGGAU UGC U C C GGCCUUCCAGGAGAG A U AGA G GACGACCUC U CC C C A1 4060 80 100 120 140 160 180 200 220 240 260 280 300320 U U UCC GAC GG U GGG U UCGUC G C A C C UC U C C C C C U C C C A G G U A C U A U C C C C U U U C A A G G A U G U G U U C C C U A G G A G G G U G G G U G U A C CUC UUUGGAU UGC U C C GGCCUUCCAGGAGAG A U AGA G GACGACCUC U CC C CAUAU A1 4060 80 100 120 140 160 180 200 220 240 260 280 300320 GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAG U CG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGU GGG U GGUGUGUGC CAC CCCGAUG A CCGAAAG U CAAA GAGG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CC  Plant pathogens Trifoliate orange Semancik and Weathers Virology (1972) Gross et al. Europ. J. Biochem. (1982) Tomato Chrysanthemum
Potato Tuber Spindle Viroid (PSTVd) Potato Tomato 1 C G GA CUAA A UU C ACACCU GA CCUC CU GAGCAG AA AAGA A AA A AGAAGGCGG CUCGG A GG A G C UCCCGAG AA CCGCUUUUU C U C U A UCUU AC UGCUU C GGGG C G A GGGUGU UU AGCC C U U GGAACCGCAGUUGGUUC C U GCUUCAG G G A UCC C G U GGA A A C A A CUGAAGC CGGG G A A A C CUGGAGCGA A C U GGC A A A GC GCUGUCGCUUCGG C U AC U ACCCG AAAGG AC C CCUUU GGUGGGGAGUG CACCCCUCGCC C AC CCAGCGGCCG CGCCCGCAGG AC CG AGGAG UUCCU UA CC AUUCCCG CGGGUGU CC UU GAAA C AGGGU U U U C ACCCU U C C UUUC 20 40 60 80 100 120 140 160 180200220240 260 280300320340 U A C C GUGGUUCC U G U GGU 1 C G GA CUAA A UU C ACACCU GA CCUC CU GAGCAG AA AAGA A AA A AGAAGGCGG CUCGG A GG A G C UCCCGAG AA CCGCUUUUU C U C U A UCUU AC UGCUU C GGGG C G A GGGUGU UU AGCC C U U GGAACCGCAGUUGGUUC C U GCUUCAG G G A UCC C G U GGA A A C A A CUGAAGC CGGG G A A A C CUGGAGCGA A C U GGC A A A GC GCUGUCGCUUCGG C U AC U ACCCG AAAGG AC C CCUUU GGUGGGGAGUG CACCCCUCGCC C AC CCAGCGGCCG CGCCCGCAGG AC CG AGGAG UUCCU UA CC AUUCCCG CGGGUGU CC UU GAAA C AGGGU U U U C ACCCU U C C UUUC 20 40 60 80 100 120 140 160 180200220240 260 280300320340 U A C C GUGGUUCC U G U GGU N. benthamiana Gross et al. Nature (1978) Diener Virology (1971)
Goal: To induce PSTVd resistance in tomato plants using efficient plant artificial sRNAs: -amiRNAs -syn-tasiRNAs
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| A Modified TA primary tran syn-tasiRNAs TAS1c- tasiRNAs (…) miR173 target site miR173 dsR DCL4 Virus A An RDR6 AGO1 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||RDR6 ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| DCL4 AGO1 AGO1 AGO1 AGO1 AGO1 TAS1c-bas syn-tasiRN B. B. P-SAMS design of multiple amiRNAs against a given plant virus (repeat this process for each target virus) amiRNA constructs A. syn-tasiRNA construct Cloning in B/c amiRNA vector Plant transformation Rapid amiRNA screening (e.g. agroinfiltration in N. benthamiana) Selected sRNAs Cloning in B/c syn-tasiRNA vector Transgenic plant expressing antiviral syn-tasiRNAs Analysis of the plant multiviral resistance Transgenic plant long lasting resistant to multiple viruses sRNA sequences Carbonell et al. RNA&Disease (2016) Methodology
P-SAMS-Based amiRNA Design 164 optimal results PSTVd(+)-amiRNAs 6 amiRNAs selected* PSTVd(-)-amiRNAs GUS-amiRNAs 2 amiRNAs selected 148 optimal results 3 optimal results 6 amiRNAs selected* *Criteria: -Low score -Target different genomic locations -With non-overlapping target sites 1 C G G A A C U A AAC U C G U G G U U C C U G U G G U U C A C A C C U G A C C U C C U G A C A A G A A A A G A A A A A A G A A G G C G G C U C G G A G G A G C UCCCGAG AA CCGCUUUUU C U C U A UCUU A CUG C UC C GGGG C G A GGGUGU UU AGCC C U U GGAACCGCAGUUGGUUC C U G C U U C A G G G A U C C C G U GGA A A C A A CUGAAGC C G G G G A A A C C U G G A G C G A A C U G G C A A A GC GCUGUCGCUUCGG C U AC U A CCCG A A A G G A C C CCUUU G G U G G G G A G U G CACCCCUCGCC C AC C C A G C G G C C G CGCCCGCAGG A C CG A G G A G UUCCU U A CC A U U C C C G CGGGUGU C C UU G A A A C A G G G U U U U C ACCCU U C C UUUC 20 40 60 80 100 120 140 160 180200220 260 280300320 340 U G U U C U C A A AA AU U A U U TCR (11-26) CCR (81-108;258-283) (+)-4 (+)-1 (+)-2(+)-3(-)-1 (-)-3 (-)-6 (-)-4 (-)-5 (+)-5 (+)-6 (-)-2 Location of PSTVd-amiRNA Target Sites
0 0.5 1 1.5 2 1 2 3 4 5 6 7 8 9 10 amiR-PSTVd(+) 1 2 3 4 5 6- 1 GUS 2 amiR-GUS PSTVd * * * - PSTVd 0 0.5 1 1.5 1 2 3 4 5 6 7 8 9 10 amiR-PSTVd(-) 1 2 3 4 5 6- 1 GUS 2 amiR-GUS PSTVd * * * - PSTVd Screening of AmiRNA Anti-PSTVd Activity Nicotiana benthamiana transient assays amiRNA 2x35S T PSTVd 2x35S T -Collect leaves 2 dpi -RNA extraction -Northern-blot analysis From Wikipedia.org amiRNAs against PSTVd(-)amiRNAs against PSTVd(+)
Syn-tasiRNAs Against PSTVd |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| An PSTVd syn-tasiRNAs miR173 target site miR173 DCL4 RDR6 AGO1 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||RDR6 ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| DCL4 AGO1 AGO1 AGO1 AGO1 AGO1 syn-tasiRNA construct PSTVd
Acknowledgements Mockler lab Todd Mockler Skyler Mitchell Kevin Cox Kevin Reilly DDPSC Bioinformatics Noah Fahlgren Steven Hill Carrington lab Jim Carrington Atsushi Takeda Josh T. Cuperus Daròs lab José Antonio Daròs Teresa Cordero Verónica Aragonés

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060516_UMH_AC

  • 1.
    Nuevas herramientas parael control de la expresión génica en plantas basadas en pequeños RNAs artificiales Alberto Carbonell www.slideshare.net/AlbertoCarbonell1 acarbonell@ibmcp.upv.es @A_Carbonell_
  • 2.
    Gene Silencing  Eukaryoticevolutionarily conserved, sequence-specific, RNA-based gene-inactivation system that regulates key biological processes Development Stress response Chromatin structure Pathogen defense Gene Silencing
  • 3.
    Gene Silencing is importantfor plant growth Normal RNA silencing Defective RNA silencing
  • 4.
    Gene Silencing is necessaryfor proper leaf shape Defective RNA silencing Normal RNA silencing
  • 5.
    Gene Silencing controls developmentaltiming Normal RNA silencing Defective RNA silencing
  • 6.
    Gene Silencing defends plantsagainst diseases Normal RNA silencing Defective RNA silencing Plant resistant to virus Plant susceptible to virus
  • 7.
    Classes of GeneSilencing Transcriptional Gene Silencing (TGS) An * X Gene An mRNA Protein No Gene Silencing Post-Transcriptional Gene Silencing (PTGS) An* X
  • 8.
    PTGS in Plants AGO dsRNA targetRNA ssRNA Intramolecular (folding) RNA-dependent RNA polymerase DCL RDR .............. AGO .............. AGO .............. An sRNA An An Translational repression ............. RNA-dependent RNA polymerase target RNA RDR
  • 9.
    Small RNA (sRNA)Silencing Pathways |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| HYL1 DCL1 An MIRNA primary transcript |||||| |||||| An miRNA targetAGO1 AGO1 SE miRNA foldback miRNA pathway HEN1miRNA An tasiRNA target |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An AGO1 DRB4 DCL4 DRB4 DCL4 SDE5 RDR6 SGS3 RDR6 TAS primary transcript AGO1 tasiRNA pathway HEN1 AGO dsRNA tasiRNA miRNA
  • 10.
    Artificial sRNA SilencingPathways hpRNA pathway hpRNA transgene An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! hpRNA primary transcript An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! HEN1# AGO# dsRNA! tasiRNA! ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||DCL4 DRB4 An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# ||||||||||||||| |||||||||||||||||||||| ||||||||||||||| |||||||||||||||||||||| HYL1% DCL1% An% ed'MIRNA'primary%transcript' |||||| |||||| An% amiRNA% target%RNA'AGO1% AGO1% SE% miRNA% oldback' HEN1% amiRNA' amiRNA*' miRNA% miRNA*% An An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| hpRNA siRNA |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| HYL1% DCL1% An% Modified'MIRNA'primary%transcript' |||||| |||||| An% amiRNA% target%RNA'AGO1% AGO1% SE% amiRNA% foldback' HEN1% amiRNA' amiRNA*' amiRNA% amiRNA*% s amiRNA pathway |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| HYL1% DCL1% An% Modified'MIRNA'primary%transcript' |||||| |||||| An% amiRNA% target%RNA'AGO1% AGO1% SE% amiRNA% foldback' HEN1% amiRNA' amiRNA*' amiRNA% amiRNA*% amiRNA transgene amiRNA amiRNA* An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! syn-tasiRNA pathway An# syntasiRNA+1# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# Modified#TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! syntasiRNA+1! syntasiRNA+2! syntasiR-1 syntasiR-2 syntasiR-1 syntasiR-2 An# syntasiRNA+2# target#RNA! AGO1# syn-tasiRNA transgene syntasiR-1 syntasiR-2 An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA! amiRNA pathway |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| |||||||||||||||||| |||||||||||||||||| |||||||||||||||||||||| HYL1% DCL1% An% Modified'MIRNA'primary%transcript' |||||| |||||| An% amiRNA% target%RNA'AGO1% AGO1% SE% amiRNA% foldback' HEN1% amiRNA' amiRNA*' amiRNA% amiRNA*% amiRNA transgene amiRNA amiRNA* An# tasiRNA# target#RNA! |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| An# AGO1# DRB4# DCL4# DRB4# DCL4# SDE5# RDR6# SGS3# RDR6# TAS!primary#transcript! AGO1# HEN1# AGO# dsRNA! tasiRNA!
  • 11.
    Applications of PlantArtificial sRNAs  Study gene function CONCLUSION: Gene A is necessary for chlorophyll synthesis Normal function of plant gene A Artificial small RNAs shut down function of plant gene A
  • 12.
    Applications of Plantartificial sRNAs  Induce antiviral resistance Niu et al. Nature Biotechnology (2006)
  • 13.
    Advantages of PlantArtificial sRNAs  Spacio and temporal regulation of gene expression -Tissue specific expression of artificial sRNAs -Inducible expression of artificial sRNAs  Study of lethal genes  Simultaneous silencing of multiple: -Sequence related genes (e.g. gene family) -Sequence unrelated genes  Generation of allelic series with different silencing levels -Transformation process -Use of expression promoters of distinct strength -Fine tune regulation of the artificial sRNA efficacy by modifying base- pairing interactions between the artificial sRNA and target
  • 14.
    Limitations of ArtificialsRNA Systems 1. Design (WMD3): -Non-intuitive interface -Relatively slow -No syn-tasiRNA design tool http://wmd3.weigelworld.org/ 1st PCR amiRNA insert 2nd PCR BamHI cut EcoRI cut Gel purification Gel purification Entry vector Cut pBSK vector with EcoRI Alkaline Phosphatase treatment Gel purification BamHI cut Entry plasmid Mini-prep Pick up positive colony Confirm the sequencing Ligation Transform into E. coli Entry plasmid Binary vector Binary plasmid Cut with restriction enzyme(s) Gel purification Mini-prep Pick up positive colony Confirm the replacement of the fragment Transform into Agro Ligation Transform into E. coli Cut with restriction enzyme(s) Alkaline Phosphatase treatment Gel purification2. Cloning: -Long and slow (multi-step) -Non cost-effective -Non-high throughput capability -Lack of convenient syn-tasiRNA cloning systems Schwabb et al., Plant Cell (2006) 3. Expression: -Frequent miss-processing of amiRNAs (-> off target effects!) 1 2 3 4 5 6 7 8 9 amiRNAs -21 nt
  • 15.
    This platform includes: a)Web-based tools for the design of artificial sRNAs b) A new generation of artificial sRNA vectors GOAL: To develop a new platform for the: 1. Design 2. Cloning (high-throughput) and 3. Expression of plant amiRNAs and syn-tasiRNAs in a simple, fast, cost- effective and effective manner for specific gene silencing in plants.
  • 16.
  • 17.
    P-SAMS Computational Design ofArtificial sRNAs  Step 1: Identification of all possible target sites in target transcript(s) by cataloguing all possible 21-nucleotide sequences  Step 2: Remove target sites that contain 15-nt sequence form positions 6-20 (core target pairing sequence) that perfectly match a non-target transcript  Step 3: Target sites are grouped by the core target pairing sequence, only target site groups that contain all input genes are considered further.  Step 4: Grouped sites are scored and ranked based on group-wise similarity and the identity of nucleotides at positions 1, 2, 3 and 21.  Step 5: For each group site, a guide RNA is designed to target all sites with the additional criteria that position 1 and 19 are a U and a C, and that position 21 is mismatched  Step 6: P-SAMS uses TargetFinder to predict target RNAs for each guide RNA. -Optimal Results: include guide RNAs predicted to target exclusively transcripts from input genes -Sub-Optimal Results: guide RNAs predicted to target transcripts from input genes AND from non-input genes
  • 20.
    Precursor Selection ForAmiRNA Vectors Ath-MIR390a For Eudicots A B Carbonell et al. Plant Physiology (2014) Osa-MIR390 For Monocots A B Carbonell et al. Plant Journal (2015)
  • 21.
    Oligonucleotide Design for DirectAmiRNA Cloning in MIR390-Based Vectors Carbonell et al. Plant Physiology (2014)
  • 22.
    AmiRNA Cloning inB/c Vectors Carbonell et al. Plant Physiology (2014)
  • 23.
    Plant expression vectors AmiRNAB/c Vectors Carbonell et al. Plant Physiology (2014) GATEWAY-compatible entry vectors Carbonell et al. Plant Journal (2015)
  • 24.
    Functionality Of AmiRNAVectors For Eudicots Carbonell et al. Plant Physiology (2014) G - U6 - amiRNA amiR- Trich - + amiR- Lfy - + amiR- Ch42 - + amiRNA accumulation in Arabidopsis transgenic lines amiR- Ft - + 21- 24- - amiRNA amiR-CH42 - + - U6 amiR- CH42 Severe Inter- mediate Weak No phenotype C Weak 35S:GUS Intermediate Severe No phenotype Silencing of CHLORINA 42 (CH42) LFY - + amiR-Lfy TargetmRNA relativeexpression 0 0.2 0.4 0.6 0.8 1 1.2 CH42 - + amiR-Ch42 RNAaccumulation amiR-CH42 - + 62/101 25/101 10/101 vector 48/48 35S:GUS 35S:AtMIR390a-Trich G - U6 - amiRNA amiR- Trich - + amiR- Lfy - + amiR- Ch42 - + amiRNA accumulation in Arabidopsis transgenic lines amiR- Ft - + 21- 24- - amiRNA - + - U6 amiR-Trich amiR-Trichvector TRY CPC ETC2 - + - + - + amiR-Trich RNAaccumulation LFY - + amiR-Lfy TargetmRNA relativeexpression 0 0.2 0.4 0.6 0.8 1 1.2 amiR-Trich - + - + - + 52/5333/33 Silencing of TRICH (TRY, CPC, ETC2)
  • 25.
    UC U AG C U A GACAGGCGUAAGAUUGCG CGCAAUCUUC GCC UGCUC A U G C 35S:OsMIR390-AtL- Bri1 16/20 AC U UG C U A GACAGGCGUAAGAUUGCG CGCAAUCUU C GCC UGCUC A U G C 35S:OsMIR390- Bri1vector 0/7 7/11 Silencing of BRASSINOSTEROID- INSENSITIVE 1 (BRI1) Carbonell et al. Plant Journal (2015) Functionality Of AmiRNA Vectors For Monocots 0 0.2 0.4 0.6 0.8 1 1.2 1.4 amiR-Bri1 - + BRI1 RNA - 35S:OsMIR390 35S:OsMIR390- AtL + vector 35S:OsMIR390- Bri1 AtL-Bri1 21 - 24 - - amiRNA - U6 amiR-Spl11 35S: OsMIR390- Spl11 35S: OsMIR390-AtL- Spl11vector 0/33 8/8 23/23 AC U UG C U A UGGCUACUGCUCAGAUCG CGAUCUGAG A GUA GCCCA U A G A C UG C U A UGGCUACUGCUCAGAUCG CGAUCUGAG A GUA GCCCA U A G A U A vector 35S:OsMIR390- Spl11 AtL-Spl11 Silencing of SPOTTED LEAF 11 (SPL11) - + + SPL11 RNA 21 - 24 - - amiRNA - U6 LFY - + amiR-Lfy TargetmRNA relativeexpression 0 0.2 0.4 0.6 0.8 1 1.2 vector 35S:OsMIR390 35S:OsMIR390- AtL
  • 26.
    Carbonell et al.Plant Physiology (2014) Precursor Selection For Syn-tasiRNA Vectors Oligonucleotide Design for Cloning in AtTAS1c- based Vectors
  • 27.
    Syn-tasiRNA Cloning inB/c Vectors Carbonell et al. Plant Physiology (2014)
  • 28.
    pMDC123SB-AtTAS1c-B/c Gateway-compatible entry clonePlantexpression vectors pMDC32B-AtTAS1c-B/c pENTR-TAS1c-B/c ccdB BsaI BsaIAtTAS1c 5’ 3’ KanRattL2attL 1 ccdB BsaI BsaIAtTAS1c 5’ 3’ HygRNosR B L B 2x35S BsaI KanR ccdB BsaI BsaIAtTAS1c 5’ 3’ BastaRNosR B L B 2x35S BsaI KanR Syn-tasiRNA B/c Vectors Carbonell et al. Plant Physiology (2014)
  • 29.
    AtTAS1c-D3&D4-Trich target mRNA 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||||||:| 3’AGGGTAAGCTATGACGAGTGA TRY TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||||||:| AGGGTAAGCTATGACGAGTGA 5’ AtTAS1c-D3&D4-Ft target mRNA 5’ TTGGTTATAAAGGAAGAGGCC |||||||||||||||||:||| 3’ AACCAATATTTCCTTCTTCGG TTGGTTATAAAGGAAGAGGCC 3’ |||||||||||||||||:||| AACCAATATTTCCTTCTTCGG 5’ AtTAS1c-D3Trich-D4Ft target mRNA 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||||||:| 3’ AGGGTAAGCTATGACGAGTGA TTGGTTATAAAGGAAGAGGCC 3’ |||||||||||||||||:||| AACCAATATTTCCTTCTTCGG 5’ FT TRY TRY FT FT CPC 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG ETC2 5’ TCCCATTCGATACTGCTCGCC ||||||||||| ||:|||:|| 3’ AGGGTAAGCTACGATGAGTGA CPC TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| AGGGTAAGCTATGATGAGTGG 5’ ETC2 TCCCATTCGATACTGCTCGCC 3’ ||||||||||| ||:|||:|| AGGGTAAGCTACGATGAGTGA 5’ CPC 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG 5’ 2x35S:AtTAS1c miR173 target site miR173 5’ GUGAUUUUUCUCUACAAGCGAA 3’ |||||||:||||| |||||||| 3’ CACUAAAGAGAGACGUUCGCUU 5’ syn-tasiRNAs syn-tasiRNA-1 (3’D3[+]) syn-tasiRNA-2 (3’D4[+]) AtTAS1c-D3&D4-Trich target mRNA 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||||||:| 3’ AGGGTAAGCTATGACGAGTGA TRY TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||||||:| AGGGTAAGCTATGACGAGTGA 5’ AtTAS1c-D3&D4-Ft target mRNA 5’ TTGGTTATAAAGGAAGAGGCC |||||||||||||||||:||| 3’ AACCAATATTTCCTTCTTCGG TTGGTTATAAAGGAAGAGGCC 3’ |||||||||||||||||:||| AACCAATATTTCCTTCTTCGG 5’ AtTAS1c-D3Trich-D4Ft target mRNA 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||||||:| 3’ AGGGTAAGCTATGACGAGTGA TTGGTTATAAAGGAAGAGGCC 3’ |||||||||||||||||:||| AACCAATATTTCCTTCTTCGG 5’ FT AtTAS1c-D3Ft-D4Trich target mRNA 5’ TTGGTTATAAAGGAAGAGGCC |||||||||||||||||:||| 3’ AACCAATATTTCCTTCTTCGG FT TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||||||:| AGGGTAAGCTATGACGAGTGA 5’ TRY TRY TRY FT FT CPC 5’ TCCCATTCGATACTGCTCGCC ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG ETC2 5’ TCCCATTCGATACTGCTCGCC ||||||||||| ||:|||:|| 3’ AGGGTAAGCTACGATGAGTGA CPC TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| AGGGTAAGCTATGATGAGTGG 5’ ETC2 TCCCATTCGATACTGCTCGCC 3’ ||||||||||| ||:|||:|| AGGGTAAGCTACGATGAGTGA 5’ CPC 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG 5’ ETC2 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||| ||:|||:|| 3’ AGGGTAAGCTACGATGAGTGA 5’ CPC 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||||||:|||:|| 3’ AGGGTAAGCTATGATGAGTGG 5’ ETC2 5’ TCCCATTCGATACTGCTCGCC 3’ ||||||||||| ||:|||:|| 3’ AGGGTAAGCTACGATGAGTGA 5’ miR173 5’ GUGAUUUUUCUCUACAAGCGAA 3’ |||||||:||||| |||||||| 3’ CACUAAAGAGAGACGUUCGCUU 5’ syn-tasiRNA-1 (3’D3[+]) syn-tasiRNA-2 (3’D4[+]) Functionality of Syn-tasiRNA Vectors 35S:GUS Trich Ft 35S:AtMIR390- 35S:GUS Trich Ft D3&D4-Trich D3&D4-Ft D3Trich-D4Ft D3Ft-D 35S:AtMIR390- 35S:AtTAS1c- vector syntasiRNA-Trich syntasiRNA-Ft 35S:GUS Trich Ft D3&D4-Trich D3&D4-Ft D3Trich-D4Ft D3Ft-D4Trich 35S:AtMIR390- 35S:AtTAS1c- Carbonell et al. Plant Physiology (2014)
  • 30.
     syn-tasiRNA vectorsfor targeting single or multiple (sequence unrelated) genes -Arabidopsis (and close species) vectors: AtTAS1c-based -In other species if MIR173 is co-expressed  amiRNA vectors for targeting single or multiple (sequence related) genes: -Eudicot vectors: AtMIR390a-based -Monocot vectors: OsMIR390-AtL-based Development of a new platform to design, clone and express plant artificial small RNAs in a simple, fast, cost-effective and effective manner to silence single or multiple genes in plants Summary  P-SAMS webtool with two apps (P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer) for the automated design of amiRNAs and syn-tasiRNAs, respectively AtMIR390a-B/c vector amiRNA insert Oligo annealing BsaI digestion + ligation E. coli transformation Plasmid purification Sequencing amiRNA construct 1 h 5 min Plant transformation Day 6 Agrobacterium liquid culture Day 7 Day 2 amiRNA oligos Agrobacterium transformation Day 4 E. coli liquid culture Day 1 P-SAMS amiRNA Design Oligo ordering Day 3 1-10 min amiRNA sequence
  • 31.
    World-Wide Usage OfP-SAMS Number of Sessions
  • 32.
    B/c Vectors areAvailable @ www.addgene.org
  • 33.
    Some Institutions HavingRequested B/c Vectors
  • 34.
    Viroids  Single-stranded circularRNA (246-401 nt)  High secondary structure content  Do not code for proteins  Need host factors for replication G CUGA 120 C U U C A G G U C U CG A C U G . . . . . . . . . . . . G A 160 180 200 240 380 GG C ACCUGA U G C C U A G C G U C C A A C C C C G G G A G G AAA GGGGUUGGG A C G G G C C A G U C C C A G U C G G U U C G C U C UCG U A G U C A C A G C C A C U G G G GA A CC U A G G C AG A U GGCUGGACG G A U G U C U A G U C C CC G A GC .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 U C C U U U G G A G UA C U C C A G A G G A . . . . . . . . . . . CCGGAA C G UU C CG . . . . . .. U U G G G U U UGA A A C C C C C A A A G G U A A A UA C C U C . . . . . C U C U A A G G G A G . . . . A GG U C G U A A A A C U U C C. . . . . 140 60 100 U C C A U U U C U C A U CA G G A A A C C C A . . . . . .. . . . . . . . .. . . . U G G C U G U G C G . 20 U C GGU G UCUGA A GAAAC CUC U 40 G A U C C A U GA C A G A U C . . . . . 80 U A A A G G A G G C U U U C C C C C U C C A A G G C U U C . . . . . . . . . . . . . . 320 340 360 280 C C A U U U C GAGAUGG .. . . . . . . 300 U C U GA A G G . . U A G CA A . C G U U G G A A U U C U C C UCGG 260 . C U A 220 A U G G G CUGA 120 C U U C A G G U C U CG A C U G . . . . . .. . . . . . . . . . . .. . . . . . G A 160 180 200 240 380 GG C ACCUGA U G C C U A G C G U C C A A C C C C G G G A G G AAA GGGGUUGGG A C G G G C C A G G G C C A G U C C C A G U U C C C A G U C G G U U G G U U C G C U G C U C UCG U A G U C A C A G C C A C U G G G GA A CC U A G G C AG A U GGCUGGAC GGCUGGACG G A U G U C U A G U CG U C U A G U C C CC G A GC .. . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . .. . . . . 1 U C C U U U G G A G U U C C U U U G G A G UA C U C C A G A G G A . . . . . . . . . . . CCGGAA C G UU C CG . . . . . .. U U G G G U U UGA A A C C C C C A A A G G U A A A UA C C U C . . . . . C U C U C U C U A A G G G A G G G A G . . . . . . . . A GG U C G U A A A A C U U C C. . . . . 140 60 100 U C C A U U U C U C A U CA G G A A A C C C A . . . . . .. . . . . ... . . . . .. . . . . . . .. . . .. . .. . . . U G G C U G G G C U G U G C G . 20 U C GGU G UCUGA A GAAAC CUC U 40 G A U C C A U GA C A G A U C . . . . . 80 U A A A G G A G G C G G A G G G G A G G C U U U C C C C C U C C A A G G C U U C C C C U C C A A G G C U U C C C U C C A A G G C U U C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320320 340340 360360 280 C C A U U U C GAGAUGG .. . . . . . ... . . . . . . 300300 U C U GA A G G . . U A G CA A . C G U U G G A A U U C U C C UCGG 260 . C U A 220 A U G G GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CGG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGUUUCG U CCUUUCGG A CUCAUCAGGG AAA GUACACA GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CGG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGUUUCG U CCUUUCGG A CUCAUCAGGG AAA GUACACACU U UCC GAC GG U GGG U UCGUC G A C A C C UC U C C C C C U C C C A G G U A C U A U C C C C U U U C A A G G A U G U G U U C C C U A G G A G G G U G G G U G U A C CUCUUUUGGAU UGC U C C GGCCUUCCAGGAGAG A U AGA G GACGACCUC U CC C C A1 204060 80 100 120 140 160 180 200 220 240 260 280 300320 CU U UCC GAC GG U GGG U UCGUC G A C A C C UC U C C C C C U C C C A G G U A C U A U C C C C U U U C A A G G A U G U G U U C C C U A G G A G G G U G G G U G U A C CUCUUUUGGAU UGC U C C GGCCUUCCAGGAGAG A U AGA G GACGACCUC U CC C CAUAU A1 4060 80 100 120 140 160 180 200 220 240 260 280 300320 GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CGG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGU GGG U GGUGUGUGC CAC CCCGAUG A CCGAAAGG U CAAAU GAAAU GG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGU GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGUUUCG U CCUUUCG A CUCAUCAGGG AAA GUACACA GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAGG U CG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGU G U CCUUU A CUCAUCAGGG AAA GUACAC CU U UCC GAC GG U GGG U UCGUC G A C A C C UC U C C C C C U C C C A G G U A C U A U C C C C U U U C A A G G A U G U G U U C C C U A G G A G G G U G G G U G U A C CUCUUUUGGAU UGC U C C GGCCUUCCAGGAGAG A U AGA G GACGACCUC U CC C C A1 4060 80 100 120 140 160 180 200 220 240 260 280 300320 U U UCC GAC GG U GGG U UCGUC G C A C C UC U C C C C C U C C C A G G U A C U A U C C C C U U U C A A G G A U G U G U U C C C U A G G A G G G U G G G U G U A C CUC UUUGGAU UGC U C C GGCCUUCCAGGAGAG A U AGA G GACGACCUC U CC C CAUAU A1 4060 80 100 120 140 160 180 200 220 240 260 280 300320 GGG U GGUGUGUGC CAC CCCUGAUGAG A CCGAAAG U CG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CCGU GGG U GGUGUGUGC CAC CCCGAUG A CCGAAAG U CAAA GAGG GGU UUCGCC AU GGG UC GGGACUUU A A AU U C GGAGGA UU CGU CC U U UA A ACG U UCCUCC A AGAGUCCC UUCCC C A A A C U U A C U U U G U A A G U G U G G U U C GGCGAA U GU A CC  Plant pathogens Trifoliate orange Semancik and Weathers Virology (1972) Gross et al. Europ. J. Biochem. (1982) Tomato Chrysanthemum
  • 35.
    Potato Tuber SpindleViroid (PSTVd) Potato Tomato 1 C G GA CUAA A UU C ACACCU GA CCUC CU GAGCAG AA AAGA A AA A AGAAGGCGG CUCGG A GG A G C UCCCGAG AA CCGCUUUUU C U C U A UCUU AC UGCUU C GGGG C G A GGGUGU UU AGCC C U U GGAACCGCAGUUGGUUC C U GCUUCAG G G A UCC C G U GGA A A C A A CUGAAGC CGGG G A A A C CUGGAGCGA A C U GGC A A A GC GCUGUCGCUUCGG C U AC U ACCCG AAAGG AC C CCUUU GGUGGGGAGUG CACCCCUCGCC C AC CCAGCGGCCG CGCCCGCAGG AC CG AGGAG UUCCU UA CC AUUCCCG CGGGUGU CC UU GAAA C AGGGU U U U C ACCCU U C C UUUC 20 40 60 80 100 120 140 160 180200220240 260 280300320340 U A C C GUGGUUCC U G U GGU 1 C G GA CUAA A UU C ACACCU GA CCUC CU GAGCAG AA AAGA A AA A AGAAGGCGG CUCGG A GG A G C UCCCGAG AA CCGCUUUUU C U C U A UCUU AC UGCUU C GGGG C G A GGGUGU UU AGCC C U U GGAACCGCAGUUGGUUC C U GCUUCAG G G A UCC C G U GGA A A C A A CUGAAGC CGGG G A A A C CUGGAGCGA A C U GGC A A A GC GCUGUCGCUUCGG C U AC U ACCCG AAAGG AC C CCUUU GGUGGGGAGUG CACCCCUCGCC C AC CCAGCGGCCG CGCCCGCAGG AC CG AGGAG UUCCU UA CC AUUCCCG CGGGUGU CC UU GAAA C AGGGU U U U C ACCCU U C C UUUC 20 40 60 80 100 120 140 160 180200220240 260 280300320340 U A C C GUGGUUCC U G U GGU N. benthamiana Gross et al. Nature (1978) Diener Virology (1971)
  • 36.
    Goal: To induce PSTVdresistance in tomato plants using efficient plant artificial sRNAs: -amiRNAs -syn-tasiRNAs
  • 37.
    |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| A Modified TA primary tran syn-tasiRNAs TAS1c- tasiRNAs (…) miR173 targetsite miR173 dsR DCL4 Virus A An RDR6 AGO1 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||RDR6 ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| DCL4 AGO1 AGO1 AGO1 AGO1 AGO1 TAS1c-bas syn-tasiRN B. B. P-SAMS design of multiple amiRNAs against a given plant virus (repeat this process for each target virus) amiRNA constructs A. syn-tasiRNA construct Cloning in B/c amiRNA vector Plant transformation Rapid amiRNA screening (e.g. agroinfiltration in N. benthamiana) Selected sRNAs Cloning in B/c syn-tasiRNA vector Transgenic plant expressing antiviral syn-tasiRNAs Analysis of the plant multiviral resistance Transgenic plant long lasting resistant to multiple viruses sRNA sequences Carbonell et al. RNA&Disease (2016) Methodology
  • 38.
    P-SAMS-Based amiRNA Design 164optimal results PSTVd(+)-amiRNAs 6 amiRNAs selected* PSTVd(-)-amiRNAs GUS-amiRNAs 2 amiRNAs selected 148 optimal results 3 optimal results 6 amiRNAs selected* *Criteria: -Low score -Target different genomic locations -With non-overlapping target sites 1 C G G A A C U A AAC U C G U G G U U C C U G U G G U U C A C A C C U G A C C U C C U G A C A A G A A A A G A A A A A A G A A G G C G G C U C G G A G G A G C UCCCGAG AA CCGCUUUUU C U C U A UCUU A CUG C UC C GGGG C G A GGGUGU UU AGCC C U U GGAACCGCAGUUGGUUC C U G C U U C A G G G A U C C C G U GGA A A C A A CUGAAGC C G G G G A A A C C U G G A G C G A A C U G G C A A A GC GCUGUCGCUUCGG C U AC U A CCCG A A A G G A C C CCUUU G G U G G G G A G U G CACCCCUCGCC C AC C C A G C G G C C G CGCCCGCAGG A C CG A G G A G UUCCU U A CC A U U C C C G CGGGUGU C C UU G A A A C A G G G U U U U C ACCCU U C C UUUC 20 40 60 80 100 120 140 160 180200220 260 280300320 340 U G U U C U C A A AA AU U A U U TCR (11-26) CCR (81-108;258-283) (+)-4 (+)-1 (+)-2(+)-3(-)-1 (-)-3 (-)-6 (-)-4 (-)-5 (+)-5 (+)-6 (-)-2 Location of PSTVd-amiRNA Target Sites
  • 39.
    0 0.5 1 1.5 2 1 2 34 5 6 7 8 9 10 amiR-PSTVd(+) 1 2 3 4 5 6- 1 GUS 2 amiR-GUS PSTVd * * * - PSTVd 0 0.5 1 1.5 1 2 3 4 5 6 7 8 9 10 amiR-PSTVd(-) 1 2 3 4 5 6- 1 GUS 2 amiR-GUS PSTVd * * * - PSTVd Screening of AmiRNA Anti-PSTVd Activity Nicotiana benthamiana transient assays amiRNA 2x35S T PSTVd 2x35S T -Collect leaves 2 dpi -RNA extraction -Northern-blot analysis From Wikipedia.org amiRNAs against PSTVd(-)amiRNAs against PSTVd(+)
  • 40.
    Syn-tasiRNAs Against PSTVd |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||| An PSTVd syn-tasiRNAs miR173 targetsite miR173 DCL4 RDR6 AGO1 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||RDR6 ||||||||||||||||||| ||||||||||||||||||| ||||||||||||||||||| DCL4 AGO1 AGO1 AGO1 AGO1 AGO1 syn-tasiRNA construct PSTVd
  • 41.
    Acknowledgements Mockler lab Todd Mockler SkylerMitchell Kevin Cox Kevin Reilly DDPSC Bioinformatics Noah Fahlgren Steven Hill Carrington lab Jim Carrington Atsushi Takeda Josh T. Cuperus Daròs lab José Antonio Daròs Teresa Cordero Verónica Aragonés