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“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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.
U
A
U
AA
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CC C GC G
C G
U A
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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
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G
CCUUGG
A
U
GGAACC
AU
G
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G
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A
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. . . . .
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CG .
.
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
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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
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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
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AAA
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G
AA
CC
CU
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CU.
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..
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GA
UC
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1
120
140
160
180
200
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GC
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380
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..
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AA
G
C C U
200
220240
UU UU UUU
UU
U
C
C
C C
CC
A A
U
A
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G U GG
G
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280
.. . . ..
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300
320
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A
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GA
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C
U
UG
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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
.
.
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G
A A
AC U
C
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U A
G CGCU A
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C C
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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
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.
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 .
.U
GCA ..
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
.
.
.C
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
Capítulo ICapítulo I
·
··
·· Symptomatic
U UU U
80
GU
GGGAGU
AAA
GC
U CGCU
CUCUCC
.
.
.
.
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
UGA
C
AG
G
A
U
C
GAAACCU
C
U
U
CC A
G U UUC
GG
C
UU
G
UG
UG
G
G
A
GUA
AA
GC
U
U U
C
GC
U
C
U
C
U
C
CA
C
A
G
CC
UC A UCA GGAAACC
CAC
UU CAGGU
CUCG ACU
GGAAGG
UC
G
UU AA
A
CUUCCCC
U
C
U
A
A G
C
G
G
A
GU AGAGGU
A AA
UACCUCC
G
UC CA ACC CCGG G
A
G
GAA
AGGGGUUGGG
A
CCCGGAAC A
G
AU
CCA
GUUCCGGU
C
C
U
UU
G
G
A
G
U
CC AU UU C U C UCG U U
GGAU A
U
UCUCC
UC
G
GAGAAGG
GAGAUGG
G
G
C
C
A
G
U
C
C
CA
GUC
G
G
U
U
C
GCU
C
UC
G
U
AGUCACA
G
C
C
AC
UG
G
G
GAACC
UAG
G
CAGAUGGCUGGACG
G
A GUCUU AGUC
C A
C
U
C
C
AG
A
G
GA
CCUUGGG
U
UUG
AA
ACC C
CCAAG
A
GGUC
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. .
. .
.
.
.
.
.
. .
. .
.
. . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
. .
. .
. .
. .
. .
. .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. . . . . . . . . . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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)
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