“Viroids: filling the lower size niche for RNA genomes”

Ricardo Flores

Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC)Valencia

“DNA habitats and its RNA inhabitants”, Salzburg, 2014

Viroid diseases and viroid characterization

Flores et al., Annu. Rev. Phytopatol. 2005

Sweet pepper ‘Jaguar’ (Capsicum anuum)

Verhoeven et al., Virus Res. 2009

A novel viroid from dahlia with intriguing biologicaland molecular features

Verhoeven et al., J. Gen. Virol. 2013

Analysis by sucrose gradient centrifugation of PSTVd andtwo typical plant viruses and their corresponding RNAs

Diener, Sci. Amer. 1981

UV-absorption profiles of RNA preparations fromhealthy and PSTVd-infected tomato tissue

Diener, Virology 1972

Electronic micrograph of amixture of purified PSTVd RNAand phage T7 RNA

Sogo et al., Virology 1973

Theodor Otto Diener

Slab PAGE analysis of healthy andCEVd-infected Gynura aurantiaca

Semancik, CMI/AAB Descrip. Plant Viruses 1980

Joseph S. Semancik

Gross et al., Nature 1978

Heinz Ludwig Sänger

Triticum aestivum16.000.000.000

Homo sapiens2.900.000.000

Arabidopsis thaliana115.000.000

Saccharomyces cerevisiae12.500.000

Escherichia coli4.720.000

Mycoplasma genitalum580.073

Poxvirus350.000

Adenovirus35.937

Phage MS23.569

Prion gene762

Viroid246

HDV RNA1.678

Replication

Autonomous replication through arolling-circle mechanism

They must replicate by a hostRNA polymerase

Lack of mRNA activity

Structure

High content insecondary structure

Small single-strandedcircular RNAs

Some exhibitribozyme activity

Viroids

Pathogenesis

Subviral pathogens of higher plants

Induce disease by direct interactionwith a cellular factor(s)

VIROID CLASSIFICATION

FAMILY GENUS SPECIES

POSPIVIROIDAE

AVSUNVIROIDAE

POSPIVIROID

COCADVIROID

APSCAVIROID

COLEVIROID

AVSUNVIROID

PELAMOVIROID

HOSTUVIROID

PSTVd (potato spindle tuber)

TCDVd (tomato chlorotic dwarf)

MPVd (mexican papita)

TPMVd (tomato planta macho)

CSVd (chrysanthemum stunt)

CEVd (citrus exocortis)

TASVd (tomato apical stunt)

IrVd 1 (iresine 1)

CLVd (columnea latent)

ASSVd (apple scar skin)

CDVd (citrus dwarfing)

ADFVd (apple dimple fruit)

GYSVd 1 (grapevine yellow speckle 1)

CBLVd (citrus bent leaf)

PBCVd (pear blister canker)

AGVd (australian grapevine)

GYSVd 2 (grapevine yellow speckle 2)

CbVd 1 (coleus blumei 1)

CbVd 2 (coleus blumei 2)

CbVd 3 (coleus blumei 3)

ASBVd (avocado sunblotch)

PLMVd (peach latent mosaic)

CChMVd (chrysanthemum chlorotic mottle)

HSVd (hop stunt)

CCCVd (coconut cadang -cadang)

CTiVd (coconut tinangaja)

HLVd (hop latent)

CBCVd (citrus bark cracking)

ELVd (eggplant latent)ELAVIROIDFlores et al., 8th ICTV Rep. 2005(with modifications)

Viroides

Presencia de una región central

conservada (CCR)

With a Central ConservedRegion (CCR)

Without a Central ConservedRegion (CCR)

Con ribozimas de cabeza de

martillo

With hammerhead ribozymesSin ribozimas de cabeza de Without hammerhead ribozymes

martillo

Viroid classification

(POtato SPIndle tuberVIRoid, PSTVd)

Family Pospiviroidae(AVocado SUNblotch VIRoid,

ASBVd)

Family Avsunviroidae

Sin ribozimas de cabeza de Nuclear replication

martillo

Sin ribozimas de cabeza de Chloroplastic replication

martillo

Confocal micrograph of in situ hybridization showing CEVdaccumulation in tomato cell nucleus (Bonfiglioli et al., Plant J. 1996)

CCRTCRTCH

PSTVd HSVd CCCVd ASSVd

C G CC GG CC GG CC GG CC GG

C G.

G C

C G

U A

U G

C G

A U

G C

G C

G

A

A

A

U A

C G.

C G

C G

C GG..

U A

G C

A U

G C

A U

A U

A U

G C

C

G

A

A

AG C

CC GG.

C G

C G

CC GG.

G C

C G

U A

U A

G C

A U

G C

G CG

A

A

AU A

C GG..

C G

C G

CC GG.

U A

C G

A U

C G

C G

U C

G C

U G

C G

G A

U A

CC GG.

G C

U A

CC GG.

U A

G C

UG

U A

U A

C G

C G

C

U

G

C

A

AG C

G C

C GG..

G C

A U

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

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.

CbVd1

Family Pospiviroidae

TL

TERMINALLEFT

TR

TERMINALRIGHT

CCENTRAL

PPATOGENIC

VVARIABLE

Keese and Symons, PNAS USA 1985 (with modifications)

In situ hybridization and transmission electron microscopy showingASBVd accumulation in avocado chloroplasts (Lima et al., Arch. Virol. 1994)

ASBVd

PLMVd o CChMVd

Family Avsunviroidae

I

III

II

Secondary Structure

GAAAC GU

AGUC AG

GUUUCAC

GACU CU CAAAG UGAGUCAG

GUUUC ACUCAGUC

Viroid replication

5’ OH

2’

P

3’

5’ OH

2’

P

3’

5’ P

2’ OH

3’ OH

HF HF

RZRZ

Rolling circle mechanism

Symmetric variant (Family Avsunviroidae & HDV)

Asymmetric variant (Family Pospiviroidae)

Branch and Robertson, Science 1984 (with modifications)

1. Which is the RNA polymerase involved?

RZ

Pospiviroidae

Avsunviroidae

Initiation and elongation of viroid RNAs

Nanomolar

RNA polymerases involved in viroidsynthesis

RNA polimerase I

RNA polimerase II

RNA polimerase III

[!-amanitin]

Insensitive

Micromolar

Familiy Pospiviroidae Nuclear replication

Effects of ! -amanitin on CEVdsynthesis in a nuclei-rich system

[!-amanitin] nM

Mühlbach & Sänger, Nature 1979; Flores & Semancik, PNAS USA 1982

First surprise: viroidssubvert templatespecificity of RNApolymerases

Single-subunit structure related with phage RNA polymerases

Plastid Encoded Polymerase(PEP)

Multimeric structure related with eubacterial RNA polymerases

AT-rich short promoters (15 nt) with a conserved YRT motif

Tagetitoxin sensitive Tagetitoxin insensitive

-10/-35 σ eubacterial-like promotors

70

Nuclear Encoded Polymerase(NEP)

Plastid RNA polymerases

A

ASBVd

RNA 4S

RNA 5S

B

Tagetitoxin (µM)

Effect of tagetitoxin on ASBVd transcription in apreparation of cloroplasts from infected tissue

Rel

ativ

e sy

nthes

is (

%)

100

80

60

40

20

ASBVd RNA (-)

CSVd RNA (-)

0 1 3 5 10 25 50 100 0 10 25 50 100

ASBVd RNA (+)

CSVd RNA (-)

100

80

60

40

20

Tagetitoxin (µM)

Navarro et al., Virology 2000

W WG G

C

L

+ -

PLMVd replicates in albino leaf areas in which PEP is essentially absent

Late

nt s

trai

n

Ethidium bromide

Northern-blot hybridizationwith specific riboprobes forboth PLMVd polarity strands

W

G

Late

nt s

trai

n

Viroid polarity Rodio et al., Plant Cell 2007

Which is the initiation site of synthesis?

Any site?

RZ

Pospiviroidae

Avsunviroidae

Specific site(s)?

Initiation and elongation of viroid RNAs

Plus polarity

Minus polarity

UU

CC

AA

AA

GG

UU

AA

GG

UU

UU

CC

AA

..

..

..

..

..

..

UU

AA

AAAAAAUU

UU

AA

UU

UU

AA

AA

AA

UUUU

GG CC..

UU

CC

AA

AA

GG

UU

AA

GG

UU

UU

CC

AA

..

..

..

..

..

..

AA

AA

UUUUUUUU

UU

UU

UU

AA

AA

UU

AA

AAAA

GG CC..

(+) polarityPlus polarity (-) polarityMinus polarity

5’5’ 3’3’ 5’5’ 3’3’

Navarro & Flores, EMBO J. 2000

ASBVd

Cleavage of viroid RNAs

Pospiviroidae

Avsunviroidae

5’ OH

2’

P

3’

RZ

HF HF

HF (host RNase)

RZ

Chloroplastic viroids: self-cleavage mediated by hammerhead ribozymes

5’ 3’

GA

AA

H

AG

UC

GA

5’

3’

17

34

56

1413

12

89

1.1 1.2 1.3 1.4

2.1 2.2 2.3 2.4

11.4 11.3 11.1 11.1

10.4 10.3 10.2 10.1

Helix

Helix

Helix

__

N N N

N N N

__

__

N

N

__

Loop

3’

5’

N N C

N N G

N

N

Loop

Loop

U__

N N__

N N__

NN__

NN__

C G__

N N__

N N__

NN __ NN __

16.1

16.4

16.5

16.2

16.3

16.6

15.1

15.4

15.5

15.2

15.3

15.65’ 3’

GA

AH

AG

UC

NG

A

5’

3’

17

34

56

1413

12

78

9

1.1 1.2 1.3 1.4

2.1 2.2 2.3 2.4

11.4 11.3 11.1 11.1

10.4 10.3 10.2 10.1

N N N

N N N

N

N

__

3’

5’

N N C

N N G

__

__

N

N

__

U__

N N__

N N__

NN__

NN__

C G__

N N__

N N__

NN __ NN __

16.1

16.4

16.5

16.2

16.3

16.6

16.1

16.4

16.5

16.2

16.3

16.6

15.1

15.4

15.5

15.2

15.3

15.6

15.1

15.4

15.5

15.2

15.3

15.6

Self-cleavage site

III

1

III

2

3

Prody et al., Science 1986; Forster & Symons, Cell 1987

C

3’ F +

3’ F -

5’ F -

5’ F +

C

3’ F -

5’ F -

5’ F +

3’ F +

M (+) (-) M (+) (-)T T Sc Sc

587

434

267234213192184

124

104

89

80

nt

Plus

T3 P

BamHI

399

T3 RNA Pol1295

485nt C

294

297nt

188nt 5’F +

Minus

T7 P

Eco RI

T7 RNA Pol

244nt

294

C-

2951 399

223nt

Self-cleavage467nt

3’F+

+

5’F -

3’F-

Self-cleavage

Navarro & Flores, PNAS USA 1997

GTCG*AGA

G

CG*GCA

T

T G C A R T G C A R

DETERMINATION OF THE EXACTSELF-CLEAVAGE SITES OF THE

CChMVd HAMMERHEAD STRUCTURES

UU AA

UU AA

CC GG

UU AA

CC GG

UUAA

AAGGCC

GG

AA

UU

GG

CC

AA

UU

GG

CC

UUAA

AAGG UU AA

GG

AAGG

CCAA

CC

GG

CC AA

UU GG

CC

CC

GG

UU

AA

AA

335335 282282

5´5´3´3´

GG

CC

IIIIII

IIII II

.. .. .. .. ..

..

..

..

..

..

..

.. .. .. ..

UU

AA

UU..

C C .. GG

PLUS

HAMMERHEAD STRUCTURES OF HAMMERHEAD STRUCTURES OF PLMVdPLMVd(Natural (Natural variabilityvariability supportssupports in vivoin vivo functionfunction))

aa uu

cc

gguu

aa

UU AA

AA UU

UU AA

UU AA

CC GG

UUAA

AAGGCC

GG

AA

UU

GG

CC

CC

GG

GG

CC

UUAA

AAGG UU AA

GG

AAGG

CCAA

CC

GG

CC CC

GG GG

CC

CC

GG

UU

AA

AA

22 5757

5´5´3´3´

AA

UU

IIIIII

IIII II

.. .. .. .. ..

..

..

..

..

..

..

. . .. .. .. ..

UU

AA

GG CC..AA UU..

UU

MINUS

gg

cc

aauu

uu aa

uu

aa

uu

uugg

cc

aauu

uu

Hernández & Flores, PNAS USA 1992; Ambrós et al., J. Virol. 1997; Malfitano et al., Virology 2003

b

C

U

C

U

G

G

A

A

A

C

U

C

U

3´

G

A

G

A

C

A

G

U

U

G

A

G

A

5´

G

C

A

C

A

C

C

G

U

G

U

C

AG

U

III

III

U

GA G

U

A

U A

A

Khvorova et al., Nature Struct. Biol. 2003De la Peña et al., EMBO J. 2003Martick & Scott, Cell 2006Chi et al., PLoS Biol. 2008Dufour et al., Nucleic Acids Res. 2009

Secondary structure and tridimensional modelproposed for most natural hammerheads

a

UU AA

CC GG

UU AA

CC GG

UUAA

AAGGCC

GG

AA

UU

GG

CC

AA

UU

GG

CC

UUAA

AAGG UU AA

GGAA

GG

CCAA

CC

GG

CC AA

UU GG

CC

CC

GG

UU

AA

AA

5´5´3´3´

GG

UU

III III

IIII II

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

UU

AA

..

UU

..

..

..

..

..

c

Second surprise: cleavage in thefamily Avsunviroidae is not catalyzedby enzymes, but by ribozymes

5’ OH

2’

P

3’

5’ OH

2’

P

3’

HF HF

RZRZ

Ligation of viroid linear RNAs

Pospiviroidae

Avsunviroidae Mediated by a chloroplastic RNA ligase

Mediated by a nuclear RNA ligase

-First plant RNA ligase: Englert & Beier Nucleic Acids Res. 2005-A second plant RNA ligase most likely exist

5’ P 3’ OH

T4 – 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20

5’ P 3’ OH

mc

ml

KCl

0.1 – 2 M

WG – 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20

KCl

0.1 – 2 M

2’

3’

P5’ OH

mc

ml

Nohales et al., PNAS USA 2012

Biochemical evidence for nuclear DNA ligase 1 mediatingcircularization of PSTVd RNA (family Pospiviroidae)

Third surprise:viroids subvert thesubstrate specificityof DNA ligases

Viroid pathogenesis

Albinism induced bysome PLMVd variants

Malfitano et al., Virology 2003

Rodio et al., J. Gen. Virol. 2006

Rodio et al., Plant Cell 2007

G UCCCAAAC

CCG

CG

AU

UG

CG G

AU CGCA

UGC GA UU GU AA U

CG

U

G

U

G

U

G

C

G

C

G

U

G

U

A

C

G

G

CCUUGG

A

U

GGAACC

AU

G

GC

G

U

A

A

A

U

C

G

C

G

U

A

C

G

G

G

C

AA

CGAA

. . . . .

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.......

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. . . . . . . .

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..

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.

.

U A

C GA G

A

U

A

A

G

C

A

C

U

G

G CC

.

CG .

.

GG

U

AU

A

U

AG

C

A

GU G

.... ..

CA

CUUG

GGC A

UU

UU

. . . .

CU

AA

G UU U CA

U C AA AG

U

AU

CC A

G U

G

C

A

AC A

A

C

UU

G

C

U

A

C

GA

GC

G

A

U

U

A

C

A

. . . .. . . . . . . . . . ..U

AA

U

A

C

C

G

U

A

C

G

U

A

C A

G U

G

C

U

A

G

CA

G

A

GG

C

U

G. . . . . . . . . . . A

UC

U

A

. . .A G

U C U

. .U CCC

A GUG

C

G

U

A

A G C

G

. . . . . . . ..U

A

A

U

AUG

C

G

C

.

G

U

A

U

GUG

C

A

A

C

C

UG

....

A

UG

C

G

CAU

G

CU

C

U

A

A

AA G

A

UG

C G

C GC G

C GU U

C U

C CA C

GCG

C

U

A AG

C AG

C A

A

U

U

G

C

G

C

C

..

...

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.

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.

...

..

.

U

A

U

AA

G

CC C GC G

C G

U A

UGG U

CCCAAAC

CCG

CG

CG

CG

AU

UG

CG G

AU CGCA

UGC GA UU GU AA U

CG

U

G

U

G

U

G

C

G

C

G

U

G

U

A

C

G

G

CCUUGG

A

U

GGAACC

AU

G

GC

G

U

A

A

A

U

C

G

C

G

U

A

C

G

G

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. . . . .

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.......

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A

U

A

A

G

C

A

C

U

G

G CC

.

CG .

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GG

U

AU

A

U

AG

C

A

GU G

.... ..

CA

CUUG

GGC A

UU

UU

. . . .CUUG

GGC A

UU

UU

. . . CUUG CUUG

GGC A GGC A

UU

UU

. . .UU

UU

. . . .

CU

AA

G UU U CA

U C AA AG

U

AU

CC A

G U

G

C

A

AC A

A

C

UU

G

C

U

A

C

GA

G

A

GC

G

A

U

U

A

C

A

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

Pathogenicitydeterminant

GG

AA AA

AA

Localización de los posibles

determinantes patogénicos del

CChMVd

CC C

C

A

A A AAG G

G G. . . . . . . ..A

A C CU

GG G

. . .

A

20

40

60

80

100

CCA

U

GC

UU

CUAC

C CGG G U

ACC

C

CC

G G G GAU UGU

. . . . . . .. . . . . . .GU

G G

GGG GG GU UAA C CCC C C. . .. . . . ..

G

G

G

A

AAA

C A

CG

AA

GG

G

AA

CC

CU

.100

CCA

U

GC

UU

CUAC

C CGG G U

ACC

C

CC

G G G GAU UGU

. . . . . . .. . . . . . .GU

G G

GGG GG GU UAA C CCC C C. . .. . . . ..

G

G

G

A

AAA

C A

CG

AA

GG

G

AA

CC

CU

..

..

.

U

A

AA

U

U

U

G

G

CU

.

C GA

GG

U.

CU.

.

AA G

C

..

..

.

GA

UC

GG

U

CC

A

A AA

U

1

120

140

160

180

200

A

G

CU

UG

GC

CC

AA

G

..

.

UU

U

CCA

AA G

380

C U

UUU

CC

GG

A

A

A

GG

G

CC

.

.

.

.

.

.

.

.

GA

CU .

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C

CC

C

C

GG

AA

GG

G

CC

UU

..

..

.

..

A

AA

G

C C U

200

220240

UU UU UUU

UU

U

C

C

C C

CC

A A

U

A

A AG

G U GG

G

U

C

C GG. ..

280

.. . . ..

AA

G

G GA. . ..

GGCCG

GC

G

UC

A

.

.

300

320

AUCC

AAGG

.

.

GG

CC

.

.

.

.

AGU

UC

.

.

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GGUU

CGCU

CCGA

A

.

.

.

.CACU

GA

.

.

.C

C

U

UG

AA

G

C

360

340

UGGGG UG C A

G GUU

C U A CUG

A. . . ... ..

..

.

260

G C.C G.

U

UU

.G U U U C

C A A A G. . . .

C

C

.CUAGG

GAUCC

.

.

.

.

G

A A

AC U

C

U

A

C

U A

G CGCU A

GC

.

.

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.

UG C.

G C..

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GA

U

C

C

CU

U

.

.

.

.

.

.

AA

A

CU

U

U

U U

U U UC

C C

C

A

A A AAG G

G G. . . . . . . ..A

A C CU

GG G

. . .

A

20

40

60

80

100

CCA

U

GC

UU

CUAC

C CGG G U

ACC

C

CC

G G G GAU UGU

. . . . . . .. . . . . . .GU

G G

GGG GG GU UAA C CCC C C. . .. . . . ..

G

G

G

A

AAA

C A

CG

AA

GG

G

AA

CC

CU

.100

CCA

U

GC

UU

CUAC

C CGG G U

ACC

C

CC

G G G GAU UGU

. . . . . . .. . . . . . .GU

G G

GGG GG GU UAA C CCC C C. . .. . . . ..

G

G

G

A

AAA

C A

CG

AA

GG

G

AA

CC

CU

..

..

.

U

A

AA

U

U

U

G

G

CU

.

C GA

GG

U.

CU.

.

AA G

C

..

..

.

GA

UC

GG

U

CC

A

A AA

U

1

120

140

160

180

200

A

G

CU

UG

GC

CC

AA

G

..

.

UU

U

CCA

AA G

380

C U

UUU

CC

GG

A

A

A

GG

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.

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.

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CU .

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.

..

A

AA

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C C U

200

220240

UU UU UUU

UU

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CC

A A

U

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G U GG

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280

.. . . ..

AA

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300

320

AUCC

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CC

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340

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A. . . ... ..

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Capítulo ICapítulo I

·

··

·· Symptomatic

U UU U

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De la Peña et al., PNAS USA 1999; De la Peña & Flores, J. Mol. Biol. 2002

Chrysanthemum chlorotic mottle viroid(CChMVd)

G AAA

Asymptomatic

Virus- (and viroid)-induced RNA silencing

DRB4DCL4/2

HEN1

dsRNA

Virus RNA

Viral

RdRP

am

pli

fica

tion

RDR6SGS3, SDE5

Aberrant viral RNA

Folded viral RNA

CH3

CH3

Translational inhibition?

AGO1RISC

Virus RNA degradation

AGO1

siRNAs

21-22 nt

AGO1

Burgyan, 2008

siRNAs, hallmarks of RNA silencing, accumulate in plantsinfected with nuclear-replicating viroids (PSTVd)

Papaefthimiou et al., Nucleic Acids Res. 2001

Itaya et al., Mol. Plant-Microbe Interact. 2001

Itaya et al., J. Virol. 2007

Martin et al., Virology 2007

minusplus

siRNAs, hallmarks of RNA silencing, accumulate in plantsinfected with chloroplast-replicating viroids (CChMVd)

20

40

60

80

100120

160

180

200

220

240

260

280

300

320

340

360

380

GGC

ACCUGAC

GUC

GGUG

UCCUGAUGAA

G

A

U

C

CA

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C

AG

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A

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CAC

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GGAAGG

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

. .

. .

.

.

.

.

.

. .

. .

.

. . . . . . . . .

.

.

.

.

.

.

.

.

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.

.

. .

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. .

. .

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.

.

.

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.

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.

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.

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.

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.

.

.

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

.

.

.

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.

.

.

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.

.

.

.

.

.

.

.

.

.

.

.

1

140

HealthyCChMVd HealthyCChMVd

1 2 3 4 5 6 1 2 3 4 5 6

siRNAs

Martínez de Alba et al., J. Virol. 2002

Proposed model for pathogenesis of viroids (and satellite RNAs)

Wang et al., PNAS USA 2004; Navarro et al., Plant J. 2012

Viroids and the origin of life

Viroids: the oldest RNA replicons?

-Circular structure: no need of genomic tags for replication

-No protein-coding capacity: they could have emerged before the advent of the ribosome

-Rich in G+C: important for primitive RNA polymerases with low fidelity

-Structural periodicities: they could have been assembled modularly

-Some of them with ribozymes, the signature of the RNA world

-Minimal genomes (250-400 nt)

Diener, PNAS USA 1989; Flores et al., Annu. Rev. Microbiol. 2014

Hammerhead viroids can tolerate this elevated mutation rate becausethey have a very small genome

CChMVd mutation rate is the highest reported for any biological entity

CChMVd

Viroids

CChMVd

Viroids

10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 1010 2

10 – 2

10 – 3

10 – 4

10 – 5

10 – 6

10 – 7

10 – 8

10 – 9

10 – 10

10 – 11

Higher

eukaryotesssDNA

viruses

Prokaryotes

dsDNA

viruses

Lower

eukaryotes

RNA

viruses

Genome size

Muta

tion

rate

10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 1010 2

10 – 2

10 – 3

10 – 4

10 – 5

10 – 6

10 – 7

10 – 8

10 – 9

10 – 10

10 – 11

Higher

eukaryotesssDNA

viruses

Prokaryotes

dsDNA

viruses

Lower

eukaryotes

RNA

viruses

Genome size

Muta

tion

rate

Gago et al., Science, 2009; Flores et al., Annu. Rev. Microbiol., 2014

These results suggest that emergence of replication fidelity mechanisms wascentral to the evolution of complexity in the early history of life

““LL’’importance de limportance de l’’infiniment petit est infiniment grandinfiniment petit est infiniment grand””L. L. PasteurPasteur

“Natura numquam magis est tota quam in minimis”(In no other place is Nature as a whole as in its smallest creatures)

Plinio, dead victim of his own scientific curiosity while observingVesuvius eruption in 79 A.C.

““When we study the smallest components of matter,When we study the smallest components of matter,wewe addressaddress thethe deepestdeepest questionsquestions ofof universeuniverse””

R. R. HeuerHeuer ( CERN general director) ( CERN general director)