060516_umh_ac

Nuevas herramientas para el control de la expresión génica en plantas basadas

en pequeños RNAs artificialesAlberto Carbonell

www.slideshare.net/AlbertoCarbonell1

[email protected]

@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



Gene Silencing controls developmental timing



Gene Silencing defends plants against diseases



Plant resistant to virus

Plant susceptible to virus

Classes of Gene Silencing

Transcriptional Gene Silencing

(TGS)

An

*

X

Gene

AnmRNA

Protein

NoGene 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

Translationalrepression

.............

RNA-dependent RNA polymerase

target RNARDR

Small RNA (sRNA) Silencing Pathways

Artificial sRNA Silencing Pathways

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/

2. Cloning: -Long and slow (multi-step)-Non cost-effective-Non-high throughput capability-Lack of convenient syn-tasiRNAcloning 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 9amiRNAs

-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-SAMShttp://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 EudicotsA

B

Carbonell et al. Plant Physiology (2014)

Osa-MIR390 For MonocotsA

B

Carbonell et al. Plant Journal (2015)

Oligonucleotide Designfor Direct AmiRNA Cloning in MIR390-Based Vectors


AmiRNA Cloning in B/c Vectors


Plant expression vectors

AmiRNA B/c Vectors


GATEWAY-compatible entry vectors


Functionality Of AmiRNA Vectors For Eudicots


- amiRNA

amiR-CH42- +

- U6

amiR-CH42

Severe

Inter-mediate

Weak

No phenotype

Silencing of CHLORINA 42 (CH42)

RN

A ac

cum

ulat

ion

amiR-CH42- +

62/101

25/101

10/101

vector

48/48

- amiRNA

- +- U6

amiR-Trich

amiR-Trichvector

RN

A ac

cum

ulat

ion

amiR-Trich

- + - + - +

52/5333/33

Silencing of TRICH (TRY, CPC, ETC2)

35S:OsMIR390-AtL-Bri1

16/20

35S:OsMIR390-Bri1vector

0/7 7/11

Silencing of BRASSINOSTEROID-INSENSITIVE 1 (BRI1)


Functionality Of AmiRNA Vectors For Monocots

0

0.2

0.4

0.6

0.8

1

1.2

amiR-Bri1- +

BRI1 RNA

-

35S:OsMIR390

35S:OsMIR390-AtL

+

vect

or 35S:OsMIR390-Bri1 AtL-Bri1

21 -

24 -

- amiRNA

- U6

amiR-Spl11

35S:OsMIR390-

Spl11

35S:OsMIR390-AtL-

Spl11vector

0/33 8/8 23/23

vect

or 35S:OsMIR390-Spl11 AtL-Spl11

Silencing of SPOTTED LEAF 11 (SPL11)

- + +

SPL11 RNA

21 -

24 -- amiRNA

- U6

vector

35S:OsMIR390

35S:OsMIR390-AtL


Precursor Selection For Syn-tasiRNA Vectors

Oligonucleotide Design for Cloning in AtTAS1c-based Vectors

Syn-tasiRNA Cloning in B/c Vectors


pMDC123SB-AtTAS1c-B/c

Gateway-compatible entry clonePlant expression vectors

pMDC32B-AtTAS1c-B/c pENTR-TAS1c-B/c

ccdB

BsaI

BsaIAtTAS1c

5’ 3’KanRattL2attL

1ccdB

BsaI

BsaIAtTAS1c

5’ 3’HygRNosR

BLB2x35S

BsaI

KanR

ccdB

BsaI

BsaIAtTAS1c

5’ 3’BastaRNosR

BLB2x35S

BsaI

KanR

Syn-tasiRNA B/c Vectors


Functionality of Syn-tasiRNA Vectors

vector syntasiRNA-TrichsyntasiRNA-Ft


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

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

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320320

340340

360360

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C C A U U U CGAGAUGG. . . . . . . . . . . . . . . .

300300

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GAA

GG

. .

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A G

CA

A. C

GUUG

G A A UUCU

CCUCGG

260

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A 220

AU G

G

GGGU

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CCCUGAUGAGA

CCGAAAGGU

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GGGUC

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CGUCCU

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ACG U UCCUCC A AGAGUCCCUUCCCCA

AACC

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GUACACAGGG

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ACCGAAAGG

UCGG

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AA

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AACC

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CCGUUUCGUCCUUUCGGACUCAUCAGGGAAA

GUACACACUUUCCGACGGUGGGUUCGUCG

ACACCUC

UCC C

CCUCC

CAGG

UACUAUCC

CCUU UC

AA

GGAUG

UGUUC

CCUA

GGAGGG

UGGGUGU A

CCUCUUUUGGAU UGC UC

CGGCCUUCCAGGAGAGA

UAGAGGACGACCUCUCC

CC

A1204060

80

100120

140

160 180 200220

240

260

280

300320

CUUUCCGACGGUGGGUUCGUCG

ACACCUC

UCC C

CCUCC

CAGG

UACUAUCC

CCUU UC

AA

GGAUG

UGUUC

CCUA

GGAGGG

UGGGUGU A

CCUCUUUUGGAU UGC UC

CGGCCUUCCAGGAGAGA

UAGAGGACGACCUCUCC

CCAUAUA1

406080

100120

140

160 180 200220

240

260

280

300320

GGGU

GGUGUGUGCCAC

CCCUGAUGAGA

CCGAAAGGU

CGGGGU

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GGGUC

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AUUC

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AAC

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GGUUUCGCC

AUGGG

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AA

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AAC

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AA

AUUC

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CGUCCU

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AAC

CU

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AAGUGUGGUUCGGCGAA

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GUACACAGGG

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UCG

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AUGGG

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AA

AUUC

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AAC

CU

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AAGUGUGGUUCGGCGAA

UGUA

CCGU GUCCUUU ACUCAUCAGGGAAA

GUACAC CUUUCCGACGGUGGGUUCGUCG

ACACCUC

UCC C

CCUCC

CAGG

UACUAUCC

CCUU UC

AA

GGAUG

UGUUC

CCUA

GGAGGG

UGGGUGU A

CCUCUUUUGGAU UGC UC

CGGCCUUCCAGGAGAGA

UAGAGGACGACCUCUCC

CC

A14060

80

100120

140

160 180 200220

240

260

280

300320

UUUCCGACGGUGGGUUCGUCG

CACCUC

UCC C

CCUCC

CAGG

UACUAUCC

CCUU UC

AA

GGAUG

UGUUC

CCUA

GGAGGG

UGGGUGU A

CCUC UUUGGAU UGC UC

CGGCCUUCCAGGAGAGA

UAGAGGACGACCUCUCC

CCAUAUA1

406080

100120

140

160 180 200220

240

260

280

300320

GGGU

GGUGUGUGCCAC

CCCUGAUGAGA

CCGAAAGU

CGGGU

UUCGCCAU

GGGUC

GGGACUUUAA

AUUC

GGAGGAUU

CGUCCU

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AAC

CU

UACUUUGU

AAGUGUGGUUCGGCGAA

UGUA

CCGUGGG

UGGUGUGUGC

CACCCCGAUG

ACCGAAAG

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GGUUUCGCC

AUGGG

UCGGGACUUU

AA

AUUC

GGAGGAUU

CGUCCU

UUA A


AAC

UUACUUUGU

AAGUGUGGUUCGGCGAA

UGUA

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

CGGA CUAA

AUU C ACACCU GACCUCCUGAGCAGAAAAGAA

AAAAGAAGGCGG CUCGGAGGA

GC

UCCCGAGAA

CCGCUUUUUC

UC

UA

UCUUACUGCUUC

GGGGC

GA

GGGUGUUU

AGCCC

UU

GGAACCGCAGUUGGUUCCU

GCUUCAGG

GA

UCC CG

UGGAA

AC

AA

CUGAAGCCGGG

GAA

AC

CUGGAGCGAACUGGCAAA

GCGCUGUCGCUUCGG

CU

ACU

ACCCGAAAGGAC

CCCUUU

GGUGGGGAGUGCACCCCUCGCC

C

AC

CCAGCGGCCGCGCCCGCAGG

AC

CG

AGGAGUUCCU

UA

CC

AUUCCCGCGGGUGU

CC

UU

GAAA C AGGGUUUU

CACCCUU

CC

UUUC

20 40 60 80

100120 140 160

180200220240260

280300320340

UA C

CGUGGUUCCUG

UGGU

1

CGGA CUAA

AUU C ACACCU GACCUCCUGAGCAGAAAAGAA

AAAAGAAGGCGG CUCGGAGGA

GC

UCCCGAGAA

CCGCUUUUUC

UC

UA

UCUUACUGCUUC

GGGGC

GA

GGGUGUUU

AGCCC

UU

GGAACCGCAGUUGGUUCCU

GCUUCAGG

GA

UCC CG

UGGAA

AC

AA

CUGAAGCCGGG

GAA

AC

CUGGAGCGAACUGGCAAA

GCGCUGUCGCUUCGG

CU

ACU

ACCCGAAAGGAC

CCCUUU

GGUGGGGAGUGCACCCCUCGCC

C

AC

CCAGCGGCCGCGCCCGCAGG

AC

CG

AGGAGUUCCU

UA

CC

AUUCCCGCGGGUGU

CC

UU

GAAA C AGGGUUUU

CACCCUU

CC

UUUC

20 40 60 80

100120 140 160

180200220240260

280300320340

UA C

CGUGGUUCCUG

UGGU

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

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

Location of PSTVd-amiRNA Target Sites

Screening of AmiRNA Anti-PSTVd ActivityNicotiana 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

PSTVdsyn-tasiRNAs

miR173target site

miR173

DCL4

RDR6

AGO1

RDR6

DCL4

AGO1

AGO1 AGO1

AGO1

AGO1

syn-tasiRNA construct

PSTVd

Acknowledgements

Mockler labTodd MocklerSkyler Mitchell

Kevin CoxKevin Reilly

DDPSC Bioinformatics

Noah FahlgrenSteven Hill

Carrington labJim CarringtonAtsushi TakedaJosh T. Cuperus Daròs lab

José Antonio DaròsTeresa Cordero

Verónica Aragonés

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