Renou JP, Navarro L, Berthomé R, Aubourg S, Taconnat L, Jay F, Bitton F, Elftieh S, Yu A & Voinnet O

URGV INRA/CNRS/UEVE, Evry, FranceIBMP CNRS, Strasbourg

A Genomic Approach to study the pri-MIR & Si regulation in

Arabidopsis

CATMA is a Eugene-based chip

(Crowe et al. NAR, 2003; Hilson et al. Genome Res. 2004)

(Foissac et al. NAR, 2003)

At4g31850

GST have been chosen in specific regions : example of a gene from the PPR/PCMP family with conserved motifs (Aubourg et al. 2000; Small and Peeters 2000)

PPR motif

Eugène annotation

TAIR annotation

GST (amplicons) designed by SPADS (Thareau et al. Bioinformatics, 2003)

A Collection of Gene specific tags (GST) covering all the predicted transcripts(CATMA, coord. P. Hilson) , a RNAi resource (AGRIKOLA, coord I. Small)

CATMA (V2.1) : 24576 GSTs

CATMA (V4) : + 6000 GSTs + 1200 GFTs = 32000 (full

genome)(Sclep et al. BMC Bioinf. 2007)

CATMA (V2.2) : + Chl & Mt probes

CATMA (V2.3) : + 1200 miRNA probes

(Navarro et al. in prep)

L. Taconnat, F. Bitton

Data analysis: a normalized protocol M.L. Martin-Magniette, coll. OMIP INA-PG

(Lurin et al. Plant Cell, 2004)

CATMA advances

2003/2007: 130

COLLABORATIONS 19 ANR Programs (GnP & Blan)

Many INRA (36), CNRS, CEA and Univ. labsEU Program: (Compedium of Arabidopsis Gene Expression)

Foreign collaborations (Brazil, Singapour, Czech rep., Germany, Belgium, UK, Spain, Sweden)

> 2900 hybridizations

A huge data-set covering multiple developmental conditions, biotic and abiotic stresses and

mutants

21 publicationsMore to come…

(18 in prep or submitted)

CATdb: a public access to Arabidopsis transcriptome data from the URGV-CATMA

platform(Gagnot et al. NAR 2007, Oct.)

Automatic export and link

Mining the data: FLAGdb++,an integrative database

coding gene additionalannotation

taggedmutant

GSTprobes

repeatelement

protein motifAffymetrix probesMPSS tag

RNA gene

curatedannotation

genefamily/GO

http://urgv.evry.inra.fr/FLAGdb

Links to…GENBANK – PDB – TIGR - PFAM

MIPS – TAIR – ARAMEMNONSWISSPROT – GENEVESTIGATOR

eFP BROWSER - CATdb

Samson et al. NAR, 2004

697 GSTs mapped outside the genes present in the last annotation release (R6.0 from TAIR).

29 GSTs allow to precise gene extremities

Analysis of CATMA data and identification of hundreds of novel genes (Aubourg et al. 2007.BMC Genom.)

GSTs can also be used to detect erroneous gene merging

Affymetrixprimer set

-2,40 2,61

-1,26 0,90

0,60 -0,54

Ratios in 3 independent experiments

cul3 mutant / wt

Nitrogen starv. / control

tor mutant / wt

469 GSTs tag novel genes

100

16 1510

29 30

4246

16

60

16

58

0

20

40

60

80

100

498with

signal

CH3 CH3

CH3

TGS

Chromatin modification

60S40S

n(A)

Translational inhibition

eiF4G

RNA silencing core mechanism

Sense transgenes Endogenous lociInvert-repeat transgenes

Phytoviruses

RNA cleavage

(A)n

PTGS

(A)n

Amplification

RdRP

small RNA duplex 19-25bp (si/miRNA)

RdRP

DicerDicer

RISC assembly (AGO)AGO

DCL1: 20-25nt miRNA -Development -many other processes

DCL2: 22nt nat-siRNA-viral-derived siRNAs

DCL3: 24nt IR-siRNA-DNA methylation-Heretochromatin -TGS

DCL4: 21nt siRNA-viral-derived siRNAs-TasiRNAs- ‘Young’ miRNAs

Four Dicers in Arabidopsis thaliana

>50,000 (200,000)

~100 (conserved miRNAs)

2

5

TE

Nucl.

Cyto.

miR/miR*CH3

CH3

HEN1HYL1

RNA Pol II(A)nDCL1

pri-miRNA

AGO1miR

(A)n

(A)n(U)<10

40S

60SAGO1miR

(A)n

DegradationTranslation inhibition

Predominant in animals Predominant in plants

DCL1

pre-miRNA

HASTY

CH3

CH3

The Arabidopsis miRNA pathway

How to identify mi/siRNAs precursors transcripts in Arabidopsis on CATMA ?

1217 candidates miRNAs

Collaboration : O. Voinnet, L. Navarro (IBMP Strasbourg) , B. Arnold (JIT Norwich UK)

465 non-redundant miR or Si precursors

60 mers oligoprobes added on the CATMA arrays

ORF1 ORF2

Bioinformatic analysis:

Identification of stable stem-loop structures throughout the Arabidopsis genome

miRspot706=

Hunt-729_777

miRspot285=

hunt-556_605

miRspot730=

Hunt-861_908

miRspot327=

hunt-742_790

miRspot1047=

Hunt-193_245

Ex : 5 new precursors validated by Q-PCR

(Navarro et al. in prep)

hypmiR / miR in FLAGdb++

known miR

% identity with the closest paralogous region

hypmiR

S. AubourgV. BrunaudF. Samson

25 miRNA precursors are more elevated in naïve silencing mutant seedlings

22 out of 25 are specifically more elevated in dcl1-9 mutant seedlings

5 miRNA precursors have not yet been identified (yellow)

Validation of the microarray approach using silencing mutant seedlingsdcl2-1 dcl3-1 dcl4-2 rdr6-1 rdr2-1 dcl1-9 hasty hen1-1

Col0 Col0 Col0 Col0 Col0 Ler Ler LermiRNA precursor miRspot name Rat Rat Rat Rat Rat Rat

At-miR156c mirspot_455 0,15 0,00 -0,12 1,07 -0,05 -0,07At-miR156d mirspot_1098 0,06 -0,06 -0,02 0,99 -0,08 0,14At-miR157a mirspot_165/457 -0,02 0,07 -0,02 1,70 0,05 -0,40At-miR160a mirspot_465/211/1064/466 -0,17 -0,09 -0,38 1,52 0,25 0,18At-miR160c mirspot_110/1002/460/461/464/463/458/462 0,87 0,23 0,25 1,99 0,16 0,65At-miR166a mirspot_858 -0,04 -0,05 -0,06 3,15 -0,06 -0,19At-miR166b mirspot_856/857 -0,12 -0,07 0,18 2,26 -0,02 -0,18At-miR166e mirspot_855 -0,13 0,05 -0,16 0,86 0,13 -0,04At-miR167a mirspot_1180/1181 -0,02 0,15 -0,06 0,94 -0,02 0,01At-miR168a mirspot_141 0,01 -0,14 -0,24 1,03 0,03 0,29At-miR171a mirspot_880/967/881/137/417/882/123/1 0,18 -0,11 0,01 1,86 -0,04 -0,08At-miR171b mirspot_966/475/965/474 -0,03 -0,04 0,09 3,48 -0,09 0,02At-miR171e mirspot_885/883 0,24 -0,18 -0,05 1,39 -0,24 0,47At-miR172a mirspot_477/478/476 -0,19 0,14 0,08 1,36 -0,05 0,03At-miR172b mirspot_930/931/127/571/406/479/572/515/480/516/509/514/512 0,16 -0,05 0,13 2,19 -0,07 -0,24At-miR172e mirspot_512/510/884/405 -0,09 0,17 0,29 1,47 0,03 -0,20At-miR398c mirspot_896/895/4035/402/894/4 0,00 -0,03 0,09 1,81 0,02 0,19At-miR400 mirspot_772/10/771 0,11 0,03 0,00 1,75 0,02 -0,14

hunt-556_605 mirspot_285 -0,07 -0,13 0,08 2,59 -0,12 -0,06Hunt-729_777 mirspot_706 0,56 0,29 0,15 1,21 -0,12 0,01hunt-742_790 mirspot_1157/327/1158 -0,06 -0,41 0,00 1,91 -0,12 -0,09Hunt-861_908 mirspot_730 -0,02 0,09 -0,38 0,94 -0,12 0,41At-miR169h mirspot_1123 -0,13 0,10 -0,03 -0,03 -0,01 1,96At-miR415 mirspot_504 -0,13 -0,13 2,30 -0,03 -0,03 -0,03 -0,03

Hunt-193_245 mirspot_1047 -0,13 -0,13 -0,13 0,74 -0,03 -0,03 -0,03 -0,03

(Navarro et al. in prep)

Small RNAs in PAMP-triggered immunity

Bacterial cell

Yonekura et al., 2003

Flg22 is a “Pathogen associated molecular patterns” that triggers an innate immune response in plants (Felix et al.,

1998)

TLR5

FLS2

- Highly conserved- Present in different organisms - Play a pivotal role for the pathogen

21 out of 24 flg22-induced pri-miRNA/siRNA are novel

14 are hyper-induced in dcl4 treated seedlings and are likelyearly evolving pri-miRNAs or siRNA precursors

Col-0 Flg22 30' dcl2-1 Flg22 30' dcl3-1 Flg22 30' dcl4-1 Flg22 30' Ler Flg22 30' Ler Flg22 60' dcl1-9 Flg22 30' dcl1-9 Flg22 60'Col-0 Flg22Agro dcl2-1 Flg22Agro dcl3-1 Flg22Agro dcl4-1 Flg22Agro Ler Flg22Agro Ler Flg22Agro dcl1-9 Flg22Agro dcl1-9 Flg22Agro

miRNA precursors miRspot name Rat Rat Rat Rat Rat Rat Rat Rat

Hunt-76_102 mirspot_997/107/998 0,80 2,17 -0,68 0,84 1,40 1,40

Hunt-134_176 mirspot_156 -0,04 0,37 0,10 0,30 1,12 -0,70

Hunt-729_777 mirspot_701/703 0,19 -0,94 1,44 0,16 1,06 1,03

At5g36280_5_3 mirspot_521 -0,06 2,41 -1,11 0,95 1,02 1,78

At-miR398c mirspot_4/895/402/896/894 0,08 3,16 -3,55 1,46 0,98 0,71

Hunt-729_777 mirspot_326/715 0,12 -1,18 1,29 0,09 0,75 0,63

Hunt-128_165 mirspot_142/1016/1018 -0,11 2,59 -1,46 0,59 0,53 1,35

At-miR405 mirspot_35/23 0,89 0,85 -0,13 0,02 0,28 0,90

At-miR405 mirspot_292 -0,09 1,10 -0,08 0,39 -0,60 0,62

Hunt-831_879 mirspot_1174/337 -0,31 -1,46 1,20 0,05 0,21 0,13

At-miR160c mirspot_110/1002/460 -0,12 -2,35 3,31 0,22 0,16 0,12

Hunt-636_684 mirspot_300 0,20 2,09 -1,76 -0,30 0,10 -0,12

At-miR396b mirspot_495/496/497 0,01 1,52 -1,49 -0,33 -0,07 -0,18

Hunt-729_777 mirspot_711/712/713/325 0,12 -1,28 1,56 0,12 0,05 0,52

Hunt-729_777 mirspot_705/704 0,05 -1,22 1,60 0,27 0,03 0,57

At-miR395e mirspot_182 -0,07 -1,17 0,67 0,33 0,26 -0,07

Hunt-30_37 mirspot_73/384 0,14 1,03 -0,61 -0,11 0,34 -0,05

Hunt-1037_1083 mirspot_375 0,02 0,97 -0,38 0,29 0,45 0,64

Hunt-1064_1110 mirspot_1213 -0,38 0,88 -1,49 0,23 -0,25 -0,38

At-miR419 mirspot_383 -0,02 0,88 -0,14 0,08 -0,44 0,21

Hunt-729_777 mirspot_709/324 0,38 -0,85 1,04 0,28 -0,05 0,27

Hunt-193_245 mirspot_194 -0,10 0,83 -0,88 0,49 -0,35 0,16

Hunt-507_556 mirspot_658/275 0,01 0,77 -0,33 0,25 -0,13 0,66

Hunt-444_494 mirspot_258 -0,09 0,75 -0,26 0,28 0,26 0,56

Hunt-322_375 mirspot_108 0,23 -0,33 1,24 0,15 0,00 0,30

Hunt-432_484 mirspot_255/1102 -0,20 0,67 1,62 -0,07 0,23 0,19

Hunt-683_731 mirspot_1147/1148 0,33 -0,27 1,02 0,59 0,50 0,64

Hunt-26_30 mirspot_64 -0,28 -0,67 0,83 0,25 -0,09 0,10

(Navarro et al. in prep)

Small RNAs in PAMP-triggered immunity

miRspot730miRspot292 miRspot300 miRspot495 miRspot1213

AuxRE motif ARF TFs

RY motifB3-type TFs

Novel motif

Present in:

14 promoters

14 promoters

13 promoters

These cis-acting elements are not over-represented in a promoter setof 29 non Flg22-induced miRNA precursors

(Navarro et al. in prep)

Over-representation of regulatory cis-acting elements in Flg22 induced pri-miR promoters

Collaborators

The ADT membersR. Berthomé (CR2), J. Yansouni (IE GnP), S. Balzergue (IE) , F. Bitton (IE), S.

Mainguet (PhD St), L. Taconnat (AI), A. Yu (IE GnP), S. Elftieh (IE GnP)S. Pelletier (TR) & D. Glissant (Post-Doc)

The Bioinformatics groupA. Lecharny, V. Brunaud, F. Samson & M.L. Martin-Magniette (Stats)