Identification of pathogenicity determinants involved in the adaptation of Plum pox virus strain C to Prunus avium and herbaceous hosts

Facultad de Ciencias

Departamento de Biología Molecular

Iden%fica%on
of
pathogenicity
determinants
 involved
in
the
adapta%on
of
Plum
pox


virus
strain
C
to
Prunus
avium
and
 herbaceous
hosts

Memoria
presentada
por
María
Calvo
del
Cas%llo
 para
optar
al
Título
de
Doctor

Esta tesis se ha realizado en el Departamento de Genética Molecular de Plantas del Centro Nacional de Biotecnología (CNB-CSIC) bajo la dirección del Dr. Juan Antonio García.

El trabajo presentado en esta memoria ha sido posible gracias al disfrute de una beca predoctoral I3P del Consejo Superior de Investigaciones Científicas cofinanciada por el

Fondo Social Europeo (CSIC-FSE)


Al
doctor
Juan
Antonio
García
por
dirigir
esta
tesis.


A
la
doctora
Gabriela
Dujovny
por
enseñarme
en
los
comienzos.


A
mis
compañeros
del
laboratorio
313.


A
todas
las
personas
del
CNB
que
me
han
ayudado.

2

ABBREVIATIONS...
 7

VIRUS
CITED ...
 9

RESUMEN
EN
ESPAÑOL...
 10

I.
INTRODUCTION ...
 17

I.1.
General
introduc%on
to
plant
virology ...
 17

... I.1.1.
Disease
symptoms
induced
in
viral
infec;ons
 19 ... I.1.2.
Transmission
of
plant
viruses
 21 ... I.1.3.
Host
range
of
viruses
 23 ... I.1.4.
Dominant
and
recessive
resistance
mechanisms
against
viral
infec;ons.
 24 ... I.1.5.
Viral
evolu;on
and
resistance
breakdown.
 27 I.2.
General
features
of
+ssRNA
plant
viruses...
 30

... 1.2.1.
Genome
transla;on
and
replica;on
of
+ssRNA
plant
viruses.
 31 ... I.2.2.Cell‐to‐cell
and
systemic
movement
of
+ssRNA
viruses
 33 I.3.
The
family
Potyviridae:

Genome
structure
and
genera...
 34

I.4.
Plum
pox
virus
and
sharka
disease...
 35

I.4.1.
Genome
and
expression
of
Plum
pox
virus ....
 36

... 1.4.2.
Sharka
disease.
 41 I.5.
Objec%ves...
 45

II.
MATERIALS
AND
METHODS ...
 49

II.1.
Viruses...
 49

II.2.
Plant
hosts. ...
 49

II.3.
Fluorescence
imaging ...
 50

II.4.
Immunocapture
RT‐PCR
(IC‐RT‐PCR)...
 50

II.5.
Construc%on
of
full‐length
cDNA
infec%ous
clones ...
 50

... II.5.1.
Construc;on
of
pICPPV‐SwCM
 51
 ... II.5.2.
Construc;on
of
clone
pICPPV‐CaBY101n
 52 ... II.5.3.
Construc;on
of
clones
pICPPV‐CBY101‐1,
‐CBY101‐2
and
‐CBY101‐6
 53 ... II.5.4.
Construc;on
of
pICPPV‐CBY101‐2‐I
and
BY101‐2‐II
 53 II.5.5.
Construc;on
of
pICPPV‐SwCM
P1164,
N1169,
N1171,
R1054N1171
and
R1054
mutants
...53

...

II.5.6.
Construc;on
of
P1HCSwCM‐R
 54

...

II.5.7.
Construc;on
of
P1HCR‐SwCM
 55

...

II.5.8.
Construc;on
of
P3+6K1SwCM‐R
 55

...

II.5.9.
Construc;on
of
P3+6K1R‐SwCM
 56

...

II.5.10.
Construc;on
of
CISwCM‐R
 56

...

II.5.11.
Construc;on
of
CIR‐SwCM
 57

...

II.5.12.
Construc;on
of
6K2+NIaSwCM‐R
 57

...

II.5.13.
Construc;on
of
6K2+NIaR‐SwCM
 58

...

II.5.14.
Construc;on
of
NIbSwCM‐R
 58

...

II.5.15.
Construc;on
of
NIbR‐SwCM
 59

...

II.5.16.
Construc;on
of
CPSwCM‐R
 59

...

II.5.17.
Construc;on
of
CPR‐SwCM
 60

...

II.5.18.
Construc;on
of
6K2SwCM‐R
 60


...

II.5.19.
Construc;on
of
VPgSwC‐R
 61

...

II.5.20.
Construc;on
of
ProSwC‐R
 61

...

II.5.21.
Construc;on
of
6K2+VPgSwC‐R
 61

...

II.5.22.
Construc;on
of
VPg+ProSwC‐R
 62

...

II.5.23.
Construc;on
of
P1HC+6K2+NIaR‐SwC
 62

...

II.5.24.
Construc;on
of
P3+6K1+6k2+NIaR‐SwC
 62

...

II.5.25.
Construc;on
of
CI+6K2+NIaR‐SwC
 62

...

II.5.26.
Construc;on
of
6K2+NIa+NIbR‐SwC
 63

...

II.5.27.
Construc;on
of
6K2+NIa+CPR‐SwC
 63

...

II.5.28.
DNA
shuffling
and
construc;on
of
cDNA
full‐length
pICPPV
libraries
 63

II.6.
Biolis%c
inocula%on...
 65

II.7.
Mechanical
hand
inocula%on ...
 65

II.8.
Immunoblot/Western
blot
analysis ...
 66

III.
RESULTS ...
 73

III.1.
The
op%miza%on
of
the
DNA
shuffling
technique
allows
the
genera%on
of
a
cDNA
shuffled
pICPPV‐ NK‐IGFP
library
to
be
used
as
a
tool
for
forced
evolu%on
experiments...
 73

... III.1.1.
Construc;on
of
recombinant
full‐length
cDNA
clones
with
a
shuffled
P1‐HCpro
region.
 74 ... III.1.2.
Sequence
analyses
of
individual
shuffled
clones
 76 III.1.3.
Infec;vity
of
individual
clones
in
N.
benthamiana ....
 80

III.1.4.
Compe;;on
experiment:
Inocula;on
of
a
pool
of
19
shuffled
clones
in
N.
tabacum
(1031‐IXB
X
 NahG)
and
N.
benthamiana ....
 82

III.2.
Gene%c
determinants
involved
in
the
alterna%ve
adapta%on
of
PPV‐C
isolates
to
Nico1ana
 benthamiana
and
Prunus
avium ...
 92

III.2.1.
Construc;on
of
a
full‐length
cDNA
infec;ous
clone
from
a
PPV‐C
isolate
propagated
in
Nico6ana
 clevelandii ....
 92

III.2.2.
The
biological
proper;es
of
the
progeny
of
pICPPV‐SwCM
resemble
those
previously
described
for
 SwC
or
SoC
in
herbaceous
hosts
and
Prunus
persica,
but
the
cloned
isolate
has
a
low
infec;vity
in
Prunus
 avium,
its
natural
host.
...93

III.2.3.
PPV‐C
isolates
propagated
in
P.
avium
cause
delayed
and
mild
infec;on
or
do
not
infect
Nico6ana
 ... plants.
 97 III.2.4.
A
full‐length
cDNA
clone
of
PPV‐C
BY101
is
highly
infec;ous
in
P.
avium
but
only
poorly
infects
 other
Prunus
species.
...99

III.2.5.
A
genomic
region
coding
for
the
C‐terminus
of
P3,
part
of
P3N‐PIPO,
6K1
and
the
N‐terminus
of
CI
 contains
gene;c
determinants
involved
in
the
alterna;ve
adapta;on
of
PPV‐C
to
N.
benthamiana
and
P.
 avium. ...
 102

III.2.6.
Single
amino
acid
changes
within
or
surrounding
the
NIapro
cleavage
site
between
6K1
and
CI
 promote
the
alterna;ve
adapta;on
of
BY101‐2
to
N.
benthamiana
or
P.
avium ....
 104

III.3.
Avirulence
factors
restric%ng
PPV‐C
infec%on
in
A.
thaliana
and
C.
foe1dum ...
 114

III.3.1.
PPV‐SwCM
does
not
infect
A.
thaliana
or
C.
foe6dum ....
 114


III.3.2.
PPV‐SwCM
and
PPV‐R
do
not
infect
the
infec;vity
of
each
other
in

A.
thaliana
or
C.
foe6dum ...
 116

4

preven;ng
its
infec;on
in
A.
thaliana
and
C.
foe6dum ....
 122 III.3.5.
Different
combina;ons
of
the
6K2+NIa
and
other
coding
sequences
of
PPV‐R
are
necessary
for
 PPV‐SwCM
to
gain
infec;vity
in
A.
thaliana
and
C.
foe6dum ....
 126 III.4.
Iden%fica%on
of
viral
determinants
involved
in
the
systemic
necrosis
caused
by
PPV‐SwCM
in
N.


benthamiana...
 130 III.4.1.
N.
benthamiana
wil;ng
response
does
not
seem
to
depend
on
viral
dosage.
...130 III.4.2.
Systemic
necrosis
displayed
by
the
PPV‐SwCM‐infected
N.
benthamiana
plants
does
not
seem
to


...

be
mediated
by
SA
but
can
be
modulated
by
temperature.
 133

III.4.3.
The
exchange
of
the
P3+6K1
coding
sequences
between
PPV‐SwCM
and
PPV‐R
can
modulate
the
 appearance
of
systemic
necrosis
in
N.
benthamiana ....
 135 III.4.4.
A
high
viral
amplifica;on
rate
of
a
PPV‐C
isolate
is
necessary
but
not
sufficient
to
cause
systemic
 necrosis
in
N.
benthamiana. ...
 137 IV.DISCUSSION
...143

IV.1.
Genera%on
of
a
cDNA
library
containing
ar%ficially
evolved
full‐length
infec%ous
clones
causing
 different
infec%on
phenotypes
and
with
a
possible
increased
fitness
in
permissive
and
par%ally
resistant
 hosts ....
 143

...

IV.1.1.
Genera;on
of
recombinants
with
different
pathogenicity
features
 145 ...

IV.1.2.
Genera;on
of
recombinants
with
an
improved
fitness
 146

...

IV.1.3.
Evolu;onary
poten;al
of
shuffled
clones.
 147

IV.2.
Single
amino
acid
changes
in
the
6K1‐CI
region
can
promote
the
alterna%ve
adapta%on
of
Prunus‐


and
Nico1ana‐propagated
PPV
strain
C
isolates
to
either
host ...
 150 ...

IV.2.1.
A
reliable
PPV‐C
infec;ous
cDNA
clone
with
cherry
specificity
 150 IV.2.2.
Gene;c
determinants
involved
in
alterna;ve
adapta;on
to
N.
benthamiana
and
P.
avium
are


...

located
within
the
C‐P3
(PIPO)/6K1/N‐CI
coding
sequences.
 151

...

IV.2.3.
Single
amino
acid
changes
are
enough
to
facilitate
the
adapta;on
of
the
virus
to
a
new
host.
 151 IV.2.4.
Virus
accumula;on
does
not
posi;vely
correlate
with
symptom
severity
in
either
N.
benthamiana
 or
P.
avium ....
 153

. IV.2.5.
The
puta;ve
role
of
the
proteoly;c
cleavage
between
6K1
and
CI
in
host
adapta;on
of
PPV‐C.
 153 IV.3.
The
NIa
protein
or
their
VPg
and
protease
domains
of
Plum
pox
virus
strain
C
is
a
major


pathogenicity
determinant
that
prevents
the
infec%on
of
resistant
herbaceous
hosts...
 155 IV.3.1.
PPV‐SwCM
does
not
seem
to
trigger
an
R‐gene‐mediated
resistance
mechanism
in
A.
thaliana
or
 C.
foe6dum...
 156 IV.3.2.
The
reciprocal
exchange
of
the
6K2+NIa
coding
sequences
of
PPV‐R
and
PPV‐SwCM
does
not
have


...

a
reciprocal
effect.
 158

IV.3.3.
Muta;ons
P114S
(P1968S)
and
F163L
(F2017L)
in
VPg
appear
to
be
necessary
for
the
systemic
 infec;vity
of
SwCM
VPg‐containing
PPV
chimeras
in
A.
thaliana ....
 159 IV.3.4.
Both
VPg
and
Pro
domains
of
NIa
could
be
involved
in
the
puta;ve
interac;on
with
the
host
factor
 limi;ng
PPV‐C
infec;on
in
A.
thaliana
and
C.
foe6dum ....
 161 IV.3.5.
Different
viral
factors,
in
addi;on
to
NIa,
contribute
to
the
inability
of
PPV‐SwCM
to
infect
A.


thaliana
and
C.
foe6dum ....
 162

IV.3.6.
A
possible
resistance
mechanism
based
on
the
lack
of
an
eIF4e/eIF(iso)4E‐VPg
interac;on,
seems
 to
prevent
PPV‐SwCM
infec;on
in
A.
thaliana
and
probably
also
in
C.
foe6dum ....
 163 IV.4.
The
systemic
necrosis
observed
in
PPV‐SwCM‐infected
N.
benthamiana
plants
is
elicited
by
the
 P3‐6K1
coding
sequence
and
does
not
seem
to
be
related
to
R‐gene
resistance
mechanisms.
...163

IV.4.1.
The
systemic
necrosis
phenotype
does
not
seem
to
depend
on
an
ini;al
high‐viral
dosage,
but
on
 ...

an
efficient
ini;al
replica;on
coupled
to
the
presence
of
specific
pathogenicity
determinants.
 164 IV.4.2.
The
PPV‐SwCM
P3/P3+6K1
proteins
are
the
main
pathogenicity
determinants
for
the
systemic
 necrosis
in
N.
benthamiana,
but
the
N‐terminus
of
CI
might
also
contribute
to
the
necro;c
response.
 165. IV.4.3.
Systemic
necrosis
in
N.
benthamiana
infected
with
PPV‐SwCM
does
not
seem
to
be
the
result
of
a


...

salicylic
acid‐mediated
systemic
hypersensi;ve
response
(SHR).
 167

...

V.
CONCLUSIONS/CONCLUSIONES
 171/173

...

VI.
REFERENCES
 179

...


VII.
Appendix
A‐
Supplementary
data
 193

VII.Appendix
B‐
Publica%ons
...221


6

Measure
units:
Interna;onal
units.

6K1:
6
KDa
protein
1 6K2:
6
KDa
protein
2 aa:
amino
acid Avr:
Avirulence bp:
base
pair cDNA:
DNA
copy CI:
Cylindrical
inclusion CP:
Coat
protein

C‐terminal:
Carboxi‐terminal DNA:
Deoxyribonucleic
acid dpi:
days
post
inocula;on dsRNA:
double‐stranded
RNA

EDTA:
Ethilen‐diamin
tetraace;c
acid eIF:
Eukaryo;c
transla;on
ini;a;on
factor ER:
Extreme
resistance

GFP:
Green
fluorescent
protein HCpro:
Helper‐component
proteinase HR:
Hypersensi;ve
response

JA:
Jasmonic
acid KDa:
Kilo
Dalton

LAR:
Local
acquired
resistance

LSHR:
Lethal
systemic
hypersensi;ve
response MeSA:
Methyl
salycilate

mRNA:
messenger
RNA NIa:
Nuclear
inclusion
“a”

NIb:
Nuclear
inclusion
“b”

NBS‐LRR:
Nucleo;de‐binding
site
–
leucine
rich
region NLS:
Nuclear
localiza;on
signal

NO:
Nitric
oxide nt:
nucleo;de.

N‐terminal:
Amino‐terminal P1:
Protein
1

P3:
Protein
3

PABP:
Poly(A)
binding
protein

PCD:
Programmed
cell
death Poly(A):
Polyadenine

PR:
Pathogenesis‐related

RdRp:
RNA‐dependent
RNA
polymerase R‐gene:
Resistance
gene

RNA:
Ribonucleic
acid RNP:
Ribonucleoprotein

RTM:
Restricted
TEV
movement SA:
Salicylic
acid

SAR:
Systemic
acquired
resistance SHR:
Systemic
hypersensi;ve
response

+ssRNA:
single‐stranded
RNA
of
posi;ve
polarity

‐ssRNA:
single‐stranded
RNA
of
nega;ve
polarity UTR:
Untranslated
region

VPg:
Viral
protein
genome‐linked wt:
Wild
type

8

BMV:
Brome
mosaic
virus.

CVYV:
Cucumber
vein
yellowing
virus.

CTV:
Citrus
tristeza
virus.

CMV:
Cucumber
mosaic
virus.

LMV:
LeCuce
mosaic
virus.

Murine
leukemia
virus.

PPV:
Plum
pox
virus.

PPV‐D:
Plum
pox
virus
Dideron
strain.

PPV‐R:
Plum
pox
virus
strain
D,
Rancovic
isolate.

PPV‐M:
Plum
pox
virus
Marcus
strain.

PPV‐PS:
Plum
pox
virus
strain
M,
PS
isolate.

PPV‐Rec:
Plum
pox
virus
Recombinant
strain.

PPV‐EA:
Plum
pox
virus
El
Amar
strain.

PPV‐W:
Plum
pox
virus
Winona
strain.

PPV‐T:
Plum
pox
virus
Turkey
strain.

PPV‐C:
Plum
pox
virus
Cherry
strain.

PPV‐SwC:
Plum
pox
virus
strain
C,
Sweet
cherry
isolate.

PPV‐SoC:
Plum
pox
virus
strain
C,
Sour
cherry
isolate.

PPV‐CR:
Plum
pox
virus
Cherry‐Russia
strain.

PRSV:
Papaya
ringspot
virus.

PVY:
Potato
virus
Y.

PVA:
Potato
virus
A.

PVX:
Potato
virus
X.

SMV:
Soybean
mosaic
virus.

TBSV:
Tomato
bushy
stunt
virus TCV:
Turnip
crinckle
virus.

TEV:
Tobaco
etch
virus.

TMV:
Tobacco
mosaic
virus.

TuMV:
Turnip
mosaic
virus.

TVMV:
Tobacco
vein
moCling
virus

RESUMEN
EN
ESPAÑOL

Gran parte de las infecciones que sufren las plantas están causadas por virus. A pesar de ello, las infecciones virales son más la excepción que la norma, ya que las plantas poseen complejos mecanismos de defensa para luchar contra los virus e incluso pueden llegar a ser totalmente resistentes.

Entre los mecanismos de defensa más estudiados, aparte del silenciamiento de RNA, se encuentran aquellos mediados por genes R y los mecanismos de resistencia recesiva, que suelen ser los más frecuentes contra Potyvirus (el grupo mayoritario de virus de plantas).

Estos virus logran superar las barreras impuestas por los mecanismos de resistencia gracias a su gran capacidad evolutiva. Por ello, conocer los mecanismos de evolución viral puede ser importante para predecir o minimizar los daños ocasionados por virus que han adquirido una ganancia de virulencia en especies previamente resistentes. El desarrollo de clones DNA virales infectivos ha sido un gran avance para la identificación de determinantes de patogenicidad implicados en la adaptación de los virus a distintos huéspedes.

El virus de la sharka (Plum pox virus, PPV) es un importante patógeno que afecta a árboles del género Prunus y ocasiona grandes pérdidas económicas. Entre las distintas cepas que se han descrito, las más prevalentes en Europa son las cepas D, M y Rec, que infectan melocotoneros, ciruelos y albaricoqueros. La cepa C es una de las más divergentes e infrecuentes y además una de las menos estudiadas. Junto con la cepa CR, es la única capaz de infectar cerezos en la naturaleza. Además, posee características que no comparte con otras cepas del PPV, tales como la incapacidad de infectar importantes huéspedes experimentales como Chenopodium foetidum y Arabidopsis thaliana, o provocar una respuesta necrótica en Nicotiana benthamiana. Por ello, la mayor parte de esta tesis se ha centrado en la identificación de determinantes de patogenicidad implicados en la adaptación de esta cepa a distintos huéspedes y el desarrollo previo de herramientas genéticas para abordar dicho estudio.

El primer paso en esta tesis ha sido la optimización de la técnica de recombinación al azar “DNA shuffling”, con el fin de crear una librería de cDNA compuesta por variantes

10

Posteriormente, se han construido clones infectivos basados en distintos aislados de la cepa C adaptados a Nicotiana y a cerezo. Estos clones se han aprovechado para la construcción de virus recombinantes entre sí y entre aislados de las cepas C y D, mediante técnicas de clonaje clásico y mediante la técnica de “DNA shuffling” previamente puesta a punto. Estos clones se han inoculado tanto en cerezo, como en N. clevelandii, N.

benthamiana, A. thaliana y C. foetidum, con el fin de identificar determinantes de patogenicidad implicados en la adaptación de PPV-C a los distintos huéspedes.

Los resultados obtenidos indican que la adaptación de un aislado de PPV-C de Prunus a Nicotiana y viceversa, puede producirse gracias a un único cambio de amino ácido en la región comprendida entre las proteínas 6K1 y CI, dentro del sitio de reconocimiento de la proteasa NIa o en posiciones adyacentes. Esto sugiere que la adaptación a un huésped u otro podría ser el resultado de una diferente regulación del procesamiento proteolítico, mediado por factores específicos de huésped.

En el caso de A. thaliana y C. foetidum, los resultados obtenidos sugieren que la proteína NIa (VPg+Pro) es el mayor determinante de patogenicidad que impide la infección de PPV-C en estos huéspedes, aunque además existen determinantes de patogenicidad secundarios distribuidos en diferentes zonas del genoma. Virus quiméricos con mutaciones introducidas en la secuencia codificante de la proteína VPg han logrado infectar A. thaliana.

Dado que la posición de estas mutaciones coincide con la de otras descritas en otros Potyvirus, que provocan cambios de amino ácido que restablecen la interacción de VPg con factores de iniciación de la traducción de la planta, parece que la falta de infectividad de PPV- C en A. thaliana (y posiblemente también en C. foetidum), podría deberse a un mecanismo de resistencia derivado de la falta de un factor del huésped necesario para la replicación del virus.

PPV-C causa en N. benthamiana una necrosis sistémica que no se observa con ninguna otra cepa del PPV. La principal región implicada en el desarrollo de estos síntomas parece ser la de las proteínas P3 y 6K1. La proteína CI también podría participar, aunque de manera secundaria. Sorprendentemente, esta respuesta no parece ser un mecanismo de resistencia

mediado por genes R, sino que parece estar mediada por un mecanismo no demasiado conocido, que no está regulado por ácido salicílico sino por oxilipinas.

En conjunto, a lo largo de esta tesis se han desarrollado herramientas para el estudio de la evolución de virus y para la identificación de determinantes de patogenicidad, que en este caso han supuesto un avance en el conocimiento de los mecanismos moleculares subyacentes al particular comportamiento de la cepa C del PPV en huéspedes leñosos y herbáceos.

12

I.
INTRODUCTION

I.1.
General
introduc%on
to
plant
virology


 Plant pathology is the science field that deals with organisms that provoke disease in plants, the mechanisms that govern the interactions between the causing agent and the plant, and the management strategies to control the disease. Crops and wild species are affected by a vast number of pathogens that include viruses, viroids, bacteria, fungi, and parasitic nematodes and plants. Disease is the result of the interaction between the host, the pathogen and the environment, a fundamental concept in plant pathology known as the “disease triangle” (Lucas, 1998; Moury et al., 2006).

While plant infections affect wild as well as cultivated species, it is usually in crops where the most devastating effects are observed (Prendeville et al., 2012; Roossinck, 2012).

This is due to the greater diversity of wild populations regarding genetic composition, age and spatial dispersion; in contrast to the low genetic diversity, similar age and close distribution of crop plants, which make them more susceptible towards pathogen infection. In addition to this, while a long plant-pathogen co-evolution leads to the establishment of an equilibrium that might regulate plant populations, evolutionary recent plant-pathogen interactions, such as those produced by the artificial introduction of a pathogen in an area where is was not previously present, tends to result in much more severe disease outbreaks, some of which can have important social-economical consequences (Lucas, 1998; Strange and Scott, 2005).

The first report unintentionally describing viral infection symptoms in plants dates from the year 752 AD. It is a poem written by the Japanese empress Koken, in which she describes the “autumnal appearance” of eupatorium plants during the summer. This infection phenotype is nowadays known to be caused by a geminivirus and an accompanying satellite (Saunders et al., 2003) (Fig. 1A). Another early observation of viral symptoms was also reported by the botanist Clusius in 1576 and illustrated by painters from the Dutch School of Art in the 17^th century, who showed the flower-breaking appearance of the highly-valuable Rembrandt tulips, which is a phenotype now known to be caused by a potyvirus (Dekker et al., 1993) (Fig. 1B). Thus, while viral symptoms of infection might be well appreciated from an artistic point of view and might have even increased the value of certain species in the past,

in the case of crops, viral infections can have an enormous negative impact on food supply and economy (Strange and Scott, 2005).

Initial
 experiments
 leading
 to
 the
 First
 identiFication
 of
 a
 virus,
 Tobacco
 mosaic
 virus
(TMV),
were
performed
by
Mayer,
Ivanowsky
and
Beijerinck
during
the
late
years
 of
 the
 19^th
 century.
 During
 that
 time,
 the
 term
 “virus”
was
 used
for
 a
 wide
 range
 of
 infectious
agents.
However,
TMV
structure
and
composition
was
not
elucidated
until
the
 late
30s
of
the
20^th
century.
The
development
of
the
electron
microscope
greatly
boosted
 research
on
plant
and
also
animal
viruses
in
the
following
decade
(Stevens,
1983).

The definition of the term “virus” has suffered modifications as knowledge increased.

According to Hull et al. (2002), a virus can be defined as: “A set of one or more nucleic acid template molecules, normally encased in a protective coat or coats of protein or lipoprotein, that is able to organize is own replication only within suitable host cells. It can usually be horizontally transmitted between hosts. Within such cells, virus replication is (1) dependent on the host’s protein-synthesizing machinery, (2) organized from pools of the required materials (…), (3) located at sites that are not separated from the host cell contents by a

!" #"

Figure
1:
Early
 observa;ons
 of
 virus
 infec;ons
from
 the
 ar;s;c
 point
 of
view.
 A:
Poem
 by
 the
 Japanese
 empress
Koken
taken
from
the
Rui‐shu‐ko‐shu
edi;on
of
the
Man’yoshu
edited
by
Atsukata
Fujiwara
(1071–

1120).
The
text
reads:
“Perhaps
it
does
frost/
In
this
village
morn
by
morn/
For
the
grass
I
saw
in
the
field
of
 summer;me/
 Has
 already
 turned
 yellow”
 (Saunders
 et
 al.,
 2003).
 B:
 An
 example
 of
 Rembrandt
 tulips
 phenotype.
 Pain;ng
 by
 Jacob
 Marrel:
 Page
 from
 a
 tulip
 book,
1640.
 Rijksmuseum,
Amsterdam.
 hpp://

www.preview‐art.com/previews/04‐2009/dutchart.html
(visited
on
November
7th,
2013)

Introduction María Calvo del Castillo - PhD thesis

18

lipoprotein bilayer membrane, and (4) continually giving rise to variants through various kinds of change in the viral nucleic acid”.

I.1.1.
Disease
symptoms
induced
in
viral
infec%ons

The economical importance of viruses is based on the symptoms they induce. The previously mentioned case of the Rembrandt tulips can be regarded as one of the few instances in which the appearance of symptoms was regarded as a positive feature. Other examples of positive plant-virus interactions include those in which mutualistic interactions are established and help infected plants survive longer draught periods or cold weather treatments that uninfected plants cannot support (Roossinck, 2011). On the other hand, while some viruses infect plants without causing any symptoms, others can induce the appearance of necrotic symptoms and may lead to plant death. Between these two situations, a wide variety of phenotypes can be found. Since a given virus with a wide host range can infect several different species and symptoms of infection displayed by each host can be completely different, these are considered to be a consequence of host-specific responses (Stevens, 1983).

Nevertheless, there are numerous factors such as age and stage of development of the host, duration of the infection and environmental and climate conditions, among others, that can alter the infection phenotype caused by a single virus in a single host (Stevens, 1983).

I.1.1.1.
Summary
of
most
common
macroscopic
symptoms
induced
by
plant
viruses

At first sight, macroscopic symptoms of infection can be divided into local or systemic. Local symptoms appear on inoculated leaves at viral entry sites and usually appear as local lesions (Loebenstein, 2009). Systemic symptoms appear at sites distant from the entry point of the virus. Although plant growth impairment is the most commonly observed systemic symptom, other concomitant phenotypes can include mosaic, yellowing, leaf rolling, ring spots, necrosis and developmental abnormalities among others (Hull, 2002).

Mosaic symptoms are characterized by the development of a pattern of light and dark green areas in leaves. Although mosaic symptoms are very frequent, they show a great variability depending on the specific plant-virus interaction. One example of this kind of symptoms can be found in Nicotiana tabacum leaves infected with TMV (Fig. 2A). A plant

initially displaying mosaic symptoms can start to develop “dark green islands” that will appear in younger leaves during the course of the infection. Flower breaking can also be classified as a mosaic symptom.

Yellowing symptoms usually begin as a veinal clearing that finally expands through the leaf lamina. One example of this kind of symptom may be that observed in cucumber infected with Cucumber vein yellowing virus (CVYV) (Fig. 2B).

Ring spot symptoms can appear as concentric rings that tend to grow and fuse. They are observed in leaves and sometimes in fruits. Rings are visualized when cells from superficial layers become chlorotic or necrotic. Rings are often observed in papaya leafs and fruits infected with Papaya ringspot virus (PRSV) (Fig. 2C).

Necrotic symptoms can appear as a result of a systemic hypersensitive response and are often accompanied by wilting, which can altogether lead to plant death (see next section). Necrotic symptoms, wilting and plant death can be observed in Nicotiana benthamiana plants infected with PPV isolate SwCM (Fig. 2D) (see section III of this thesis).

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$" %"

Introduction María Calvo del Castillo - PhD thesis

20

Figure
 2:
 Macroscopic
 symptoms
 produced
 by
 plant
 viruses.
 A:
 Example
 of
 mosaic
 symptoms
 produced
 by
 Tobacco
mosaic
virus
(TMV)
in
a
tobacco
leaf.


Source:
 hpp://www.apsnet.org/publica;ons/

apsneqeatures/pages/tmv.aspx
 (visited
 on
 November
 7th,
2013)

B:
 Example
 of
 vein
 yellowing
 symptoms
 produced
 by
 Cucumber
 vein
 yellowing
 virus
 (CVYV)
 in
 a
 cucumber
 leaf.


Source:
 hpp://www.eppo.int/QUARANTINE/virus/

Cucumber_vein_yellowing_virus/CVYV00_images.htm
 (visited
on
November
7th,
2013)

C:
 Example
of
 ringspot
 symptoms
 produced
 by
 Papaya
 ringspot
virus
(PRSV)
in
a
papaya
fruit.

Source:
 hpp://www.apsnet.org/edcenter/intropp/

lessons/viruses/Pages/PapayaRingspotvirus.aspx
 (visited
 on
November
7th,
2013)

D:
Example
of
necro;c
symptoms
(ini;al
veinal
necrosis)
 produced
 by
 Plum
 pox
 virus
 (PPV)
 in
 a
 Nico6ana
 benthamiana
leaf.

I.1.1.2. Summary of microscopic symptoms induced by plant viruses

Microscopic symptoms of infection can be analysed at the tissue or at the cell level.

Tissue alterations include necrosis, hypoplasia and hyperplasia (Hull, 2002).

Cytological alterations affecting organelles can be often observed in the nucleus, mitochondria and chloroplasts (Hull, 2002). Some viruses express proteins that form aggregates in the nucleus and/or nucleolus and also in the cytoplasm, such as the nuclear inclusions a and b (NIa and NIb) and the cylindrical inclusion (CI) of potyviruses (Baunoch et al., 1991; Rajamäki and Valkonen, 2009) (Fig. 3).

I.1.2.
Transmission
of
plant
viruses

Plant viruses are parasites whose spread or transmission from plant to plant is necessary to ensure their survival. Since plants are sessile organisms, viruses often need specific vectors for their natural transmission. Viruses that do not count on vectors are usually spread by means of pollen or seeds. Other artificial methods widely used under experimental conditions, are mechanical and grafting transmission (Stevens, 1983).

Transmission by living vectors may be circulative or non-circulative. In circulative transmission the virus crosses the body barriers of the vector, enters the circulatory system and accumulates inside the body. During this process, the virus might propagate or not. In

Figure
3:
Electron
 microscope
picture
of
a
thin
 sec;on
of
the
cytoplasm
of
a
Nico6ana
 benthamiana
plant
 cell
 infected
with
PPV,
showing
‘pinwheel’
inclusions.
Bar
equals
200
nm.
Courtesy
of
D.
López‐Abella,
CIB‐

CSIC
(Madrid,
Spain).

non-circulative transmission, the virus does not cross vector body barriers (Raccah and Fereres, 2009).

The most common plant vectors are arthropods and they transmit viruses in a “non- persistent”, “persistent” or “semi-persistent” manner. In non-persistent transmission mediated by arthropods, viruses can be readily transmitted by the vector after feeding on the infected plant, while when viruses are trasmitted in the persistent mode, they require an incubation time (> 12 hours) in the vector. Semi-persistent viruses are those that fall between these two categories (Ng and Perry, 2004).

Aphids are the most frequent and efficient plant virus vectors (Brault et al., 2010) and are the only known vectors that transmit viruses in a non-persistent way. Although with this mode of transmission the virus might stay in the vector only for a few minutes, a specific interaction between the virus and the aphid stylet must be established. The coat protein (CP) can be the only viral determinant that participates in this interaction, such as in the case of Cucumber mosaic virus (CMV) (Perry et al., 1998), but usually other viral factors play an essential role as helper-components, as for instance the P2 protein of caulimoviruses (Lutz et al., 2012) or the HCpro protein of potyviruses (Govier and Kassanis, 1974). In this last case, the “bridge hypothesis” proposes the HCpro as the connector or “bridge” between the aphid stylet and the CP from virions (Blanc et al., 1997).

Non-persistent transmission has the advantage of viruses being easily transmitted using mechanical methods, which is an appealing feature for experimental procedures.

Although in this thesis all work has been done with Plum pox virus (PPV), which is transmitted in nature by aphids in a non-persistent mode, no aphid-mediated transmission experiments have been performed. Instead, mechanical transmission using infected extracts was the most convenient method for viral passages (see Materials and Methods section).

I.1.3.
Host
range
of
viruses

According to Dawson (1992) “natural hosts” are plant species that are found to be infected by a virus in wild or agricultural ecosystems. “Experimental hosts” are those in which the virus is able to replicate efficiently under experimental conditions.

Introduction María Calvo del Castillo - PhD thesis

22

Natural infections are considered to be more the exception than the rule. To develop a successful infection, the virus needs to be transmitted into a compatible host by a compatible vector. After getting through the structural barriers of the host, it must establish the necessary interactions with host factors to replicate and move from the first infected cell to neighbouring cells and to distant leaves, circumventing or counteracting defensive responses from the host, such as RNA silencing and those described in the following sections. Therefore, a defective interaction at any infection step may result in resistance and will limit the host range of the virus. For this reason, hosts in which the virus is restrained to the inoculated leaf can be called

“local hosts” and hosts in which the virus can spread systemically to different leaves can be called “systemic hosts” (Hull, 2002).

Some viruses have a relatively reduced host range that include closely related plant species, such as in the case of Citrus tristeza virus (CTV), which only infects plants from the genus Citrus and related species (Dawson et al., 2013). These are considered to be specialist parasites. Generalist parasites are those that have a wide host range. For instance, CMV has the largest host range of any virus known to date, being able to infect thousands of species from different families (Palukaitis et al., 1992; Mochizuki and Ohki, 2012). In some cases, some viral strains of a given virus can have a broader host range than others, as it happens with PPV.

Experimental hosts like Nicotiana species are used in the laboratory to propagate and maintain viral isolates due to their high susceptibility. Species from the genus Chenopodium have traditionally been used as indicator plants and more recently, Arabidopsis thaliana has enabled the identification of plant factors involved in viral infections thanks to the availability of resources to perform reverse genetic studies (see next sections).

I.1.4.
Dominant
and
recessive
resistance
mechanisms
against
viral
infec%ons


As is has been previously mentioned, plants do not normally get infected by viruses because they are mostly resistant. In general, non-host resistance is the most common type of resistance in plants (Heath, 2000) and it is shown by an entire plant species towards a specific pathogen. On the contrary, host resistance acts specifically against a virus to which the species as a whole tends to be susceptible (Fraser, 1990).

Plants have developed sophisticated mechanisms to counteract viral infections. Apart from RNA silencing (reviewed by Voinnet, 2001), other responses involving dominantly and recessively inherited resistance genes have been described.

I.1.4.1.
Overview
of
resistance
mechanisms
associated
to
dominant
R‐genes

On some occasions, viruses start to replicate on inoculated leaves but do not progress towards upper parts of the plant, thus remaining localized to small areas surrounding local lesions. Hosts that only develop local lesions are often used as indicators in the laboratory (Loebenstein, 2009; Yongqiang et al., 2012), as it is the case of the aforementioned Chenopodium species.

Local lesions are considered to be the result of the activation of a resistance mechanism known as hypersensitive response (HR) (Loebenstein, 2009). HR is typically mediated by resistance (R) genes, which are generally dominantly inherited and code for proteins that contain a highly conserved nucleotide-binging site and leucine-rich region (NBS-LRR) domains, known to be involved in protein-protein interactions (Moffett et al., 2002). It is important to remark that R-gene-mediated resistance sometimes takes place without HR and with undetectable virus accumulation even in the inoculated leaves. This response is named extreme resistance (ER) (Bendahmane et al., 1999). R-genes specifically interact with pathogen elicitors (also known as effectors), which are proteins encoded by avirulence (Avr) genes, in a process known as “gene for gene” response (Flor, 1971). As opposed to R-genes, Avr genes can be very diverse, and almost any viral protein can act as an Avr factor (Harrison and Robinson, 2005). The R-Avr interaction might not always be direct, but might also include a third factor from the plant (Van der Biezen and Jones, 1998; Luderer and Joosten, 2001), although the precise mechanism is still unclear. Some examples of R-Avr interactions involving viruses include the Potato virus X (PVX) CP-Rx in tomato (Bendahmane et al., 1995), Turnip crinkle virus (TCV) CP-HRT in Arabidopsis (Dempsey et al., 1997; Cooley et al., 2000), and TMV replicase-N in tobacco (Culver and Dawson, 1991;

Padgett et al., 1997).

The R-Avr recognition triggers a very complex biochemical reaction cascade that induces the accumulation of compounds such as salicylic acid (SA), nitric oxide (NO) and ethylene (Marco and Levy, 1979; Klessig et al., 2000; Verberne et al., 2003) that lead to the Introduction María Calvo del Castillo - PhD thesis

24

expression of pathogenesis-related (PR) genes (Linthorst and Van Loon, 1991; Grüner et al., 2003). SA accumulation initially takes place during the development of the local lesion in surrounding cells, but is later found in other areas of the same leaves and distant parts of the plant as well. However, methyl salicylate (MeSA) and not SA, is the mobile signal that is necessary for conferring systemic acquired resistance (SAR) (Park et al., 2007), which provides an “antiviral” state and thus prevents the re-infection of the plant by similar or related viruses.

When the R-triggered response is coupled to programmed cell death (PCD) necrotic lesions appear. But PCD is not necessary to restrict viral infection (Bendahmane et al., 1999).

The ER, which is not coupled to PCD, does not cause a visible phenotype but shares biochemical characteristics with the previously described necrotic HR, such as the accumulation of SA and ethylene (Sansregret et al., 2013). The ER takes place when the elicitor dosage is low and the plant is able to counteract viral infection before the local lesion is produced (Bendahmane et al., 1999).

RNA silencing suppressors are among the virus factors able to elicit HR and ER reactions (Sansregret et al., 2013). This interesting crosstalk between R-gene mediated responses and RNA silencing mechanisms suggests that specific defence mechanisms can be triggered to counteract the activity of virus factors able to disturb more general antiviral defenses.

As it was mentioned previously, systemic symptoms of infection may also course with a necrotic phenotype. Since viruses are biotroph pathogens and thus depend on living cells to develop their infections, necrotic systemic symptoms are not the most commonly observed (Xu and Roossinck, 2000). However, despite sharing some biochemical features with local HR (Seo et al., 2006; Komatsu et al., 2010), the mechanisms causing systemic necrosis have been less investigated. In some cases, systemic necrosis is thought to be the result of a weak or incomplete HR that does not restraint virus spread. This response in known as systemic HR (SHR) (Dinesh-Kumar et al., 2000). Other factors, such as temperature shifts, incomplete dominance of R-genes or inhibition of SA accumulation, may cause a HR to develop into a SHR (Hajimorad et al., 2005). Depending on the viral strain, SHR might even lead to plant death. In these cases, the response in known as lethal SHR (LSHR) (Hajimorad et al., 2005).

As in the case of local HR, programmed cell death (PCD) appears to be the mechanism by which systemic necrosis occurs (Xu and Roossinck, 2000; Chichkova et al., 2004; Komatsu et al., 2010). PCD is mediated by SA in R-gene-based resistance mechanisms, but more recently, PCD in systemic necrosis, produced during plant-viral compatible interactions, has been reported to be regulated by oxylipin synthesis (García-Marcos et al., 2013). Although this second mechanism has been much less studied, it is important to take into consideration that PCD can be the result of two different pathways.

I.1.4.2.
Overview
of
recessive
resistance
mechanisms

In contrast to R-genes, many other genes conferring resistance against viral infections are recessively inherited (Fraser, 1990). According to published data, resistance mediated by genes that are recessively inherited (that is, recessive resistance), seems to be much more common against viruses than against other plant pathogens (Truniger and Aranda, 2009).

Recessive resistance tends to be the result of a missing host factor or of a defective interaction between a host and a viral factor, necessary for the early amplification steps of the virus (Fraser, 1990). Thus, recessive resistance usually takes place on inoculated leaves either restraining viral replication or movement (Decroocq et al., 2006) and no visible phenotypes are associated.

Some of the natural recessive resistance genes identified so far include: mo1 in lettuce;

pot-1 in tomato; pvr-1, -2 and -6 in pepper and sbm-1, wlv and cyv2 in pea (Wang and Krishnaswamy, 2012). All of them code for translation initiation factors eIF4E and eIF4G and their isoforms (Truniger and Aranda, 2009; Wang and Krishnaswamy, 2012). This kind of resistance is particularly common against potyviruses, and is thought to derive usually from defects in specific interactions between the potyviral protein genome-linked (VPg) and the translation initiation factor (Léonard et al., 2000). Similar resistance can be artificially engineered in other plant-virus pathosystems. For instance, downregulation of eIF(iso)4E by RNA interference confers resistance against PPV in plum (Wang et al., 2013).

Host genes involved in recessive resistance against plant viruses have been also identified in natural Arabidopsis accessions (Revers et al., 2003; Decroocq et al., 2006), and new recessive resistance sources have been first generated by random mutagenesis and then characterized by appropriate mutant screenings (Hagiwara et al., 2003). Host factors whose Introduction María Calvo del Castillo - PhD thesis

26

depletion could be used to generate recessive resistance are also being characterized in heterologous genetic systems, thanks to the ability of some plant viruses to replicate in yeast cells (Kushner et al., 2003; Panavas et al., 2005).

However, viruses can overcome recessive resistance by introducing mutations in viral factors that facilitate the productive interaction with the defective host factor or even allow the virus to escape from the dependence on this host factor. Thus, potyviruses break eIF4E- mediated resistance by introducing adaptive mutations mainly in the VPg coding sequence (Moury et al., 2004; Charron et al., 2008), showing that a functional interaction between VPg and eIF4E or eIF(iso)4E is needed to establish an efficient viral infection. However, also mutations in other potyviral proteins, such as CI (Hjulsager et al., 2006; Abdul-Razzak et al., 2009) or P1 (Nakahara et al., 2010), allow potyviruses to escape from eIF4E-related resistance, which indicates the involvement of these viral proteins at the viral infection step in which eIF4E plays a role. Moreover, HCPro is an additional interaction partner of eIF4E (Ala-Poikela et al., 2011), further illustrating the complexity of the interactions in which this host factor is involved .

I.1.5.
Viral
evolu%on
and
resistance
breakdown

Adaptation to a new host involves resistance breaking (Janzac et al., 2010).

Resistance-breaking viruses can have one or more mutations in their genomes that circumvent the host’s resistance either at the replication, cell-to-cell or systemic movement steps, and thus confer a gain of function that is translated into the gain of infectivity. This is due to the establishment of compatible interaction/s between host and viral factor/s that was/were previously defective, or hiding viral elicitors from recognition by host resistance mechanisms.

The emergence of resistance-breaking viruses should be analysed in the viral population context.

The main processes leading to genetic variation within a viral population are mutation and recombination. Genetic variation is generated by errors occurring during the replication process (García-Arenal et al., 2001). RNA-dependent RNA polymerases from viruses with single-stranded RNA genomes of positive polarity (+ssRNA), which are the majority of the plant viruses, have an estimated error rate of around 10^-4 mutations per nucleotide per

replication round. This high error rate is the consequence of lacking a proofreading activity, which gives rise to a wide variety of virus variants within a single population that accumulate random mutations in their genomes.

Recombination is the process by which genomic fragments of RNA or DNA are exchanged between different viral genetic variants (García-Arenal et al., 2001), and is a main contributor to the evolution of plant viruses. Recombination events have been particularly frequently detected in natural populations of viruses from the family Potyviridae, which is one of the largest family of plant viruses (see next sections) (Bujarski, 2013). Recombination, which has a main role in helping to conserve the genomic integrity, has also been postulated to be an important process for the emergence of resistance-breaking viruses (Moury et al., 2011).

Therefore, the population of an infecting RNA virus is not formed by a single viral genotype, but by a great number of individual genotypes, differing in a very reduced number of nucleotides, thus conforming mutant clouds known as quasiespecies (Domingo et al., 2012). This heterogeneity is not normally observed when the consensus sequence of the entire population is analysed. This structure of the virus population makes easier the emergence of viruses breaking resistance barriers.

Basically, the evolution of viral populations is driven by natural selection and genetic drift. Selection is a deterministic process that can be either positive or negative. While the most fitted virus variants will increase their frequency in favourable environmental conditions (positive selection), less fitted variants will decrease their frequency (negative or purifying selection). The effects of selection on virus variants differing only in one or two point mutations might be very strong (Fabre et al., 2012). Genetic drift is a stochastic process that depends on the effective population size. The effective population size is usually much smaller than the total population (Zwart et al., 2011). Thus, populations encounter narrow bottlenecks that limit the fixation of advantageous mutations and allow the appearance of deleterious mutations that might even lead to the extinction of the population if the effective population size is too small (a process known as Müller’s ratchet) (Sacristán et al., 2003; de la Iglesia and Elena, 2007; Monsion et al., 2008; Domingo et al., 2012).

Introduction María Calvo del Castillo - PhD thesis

28

Therefore, since viral populations that start colonizing a new host tend to be rather small, resistance-breaking viruses might be present in the original population, rather than appear through de novo mutations in the new host. However, the generation of potentially resistance-breaking virus variants in a population does not necessary imply that these will out- compete the other avirulent variants (Acosta-Leal and Xiong, 2013). For resistance breakdown to occur, three steps must be accomplished: 1) appearance of an adapted mutant in the pathogen population, 2) co-existence with other viral genotypes present in the same population and substantial accumulation, and 3) efficient transmission to the resistant host with the result of resistance breakdown (Quenouille et al., 2013).

The adaptation to various hosts is supposed to be constrained by the fact that mutations that provide a fitness advantage in one environment can be disadvantageous in another environment. Such effects are known as adaptation trade-offs, and must be taken into consideration when artificially inducing the adaptation of viruses from natural hosts to experimental hosts, since the adaptation of a virus to an experimental host can have negative consequences regarding viral fitness in the natural host (Wallis et al., 2007).

I.1.5.1.
 Gene;c
 engineering
 for
 the 
iden;fica;on
 of
 viral 
determinants
 involved
 in
 host
 adapta;on
and
ar;ficial
evolu;on
of
viral
vectors

Genetic determinants involved in host adaptation (or resistance breakdown) can be identified by examining and comparing naturally occurring variants or through experimental evolution assays, such as serial passages in the laboratory (Hajimorad et al., 2003; Hajimorad et al., 2011). However, genetic engineering techniques have enabled the development of cDNA infectious clones, which allow to start a viral infection with well-defined uniform genotypes. This has been an important technical advance that has greatly facilitated the identification of genetic determinants involved in host adaptation of viruses. Nowadays, the generation of full-length cDNA infectious clones is widely used for reverse genetic study of viral genes. The first cDNA clone of a plant virus was that of Brome mosaic virus (BMV) (Ahlquist and Janda, 1984), after which cDNA clones of many other viruses were constructed.

The development of in vitro evolution techniques, such as DNA shuffling, has allowed the optimization and selection of desired properties of viruses. The DNA shuffling procedure was developed in order to mimic the outcome of a natural evolution process by means of

random recombination between homologous sequences and random introduction of point mutations (Stemmer, 1994). Although DNA shuffling has been mostly used for the optimization of enzyme activities, this technique has also proven to be useful for viral studies.

Soong et al. (2000) were the first to use this technique to generate an evolved retrovirus library of Murine leukemia virus, from which recombinant viruses with improved cell tropism were selected to be used in gene therapy. Another example of virus engineering was the creation of an evolved cardiotropic adeno-associated virus vector through DNA shuffling, also to be used in gene therapy (Yang and Xiao, 2013). In the case of plant viruses, DNA shuffling of the TMV movement protein coding region has proven to be a powerful technique to generate clones with increased cell-to-cell and systemic movement capacities in restrictive hosts (Toth et al., 2002).

I.2.
General
features
of
+ssRNA
plant
viruses

Plant viruses can be classified according to different criteria, such as the structure of the viral particle, serological relationships, physicochemical properties, etc, although nowadays the most common classification is based on the properties of the genomic nucleic acids. The organization and strategy of viral genomes place plant viruses into different families and genera.

Double-stranded DNA (dsDNA) plant viruses are circumscribed to the family Caulimoviridae. Single-stranded DNA (ssDNA) plant viruses are classified in two families, Geminiviridae and Circoviridae. Double-stranded RNA (dsRNA) plant viruses are distributed in the family Reoviridae and the genus Varicosavirus. There are single-stranded RNA plant viruses of negative polarity (–ssRNA) in the family Rhabdoviridae and plant viruses with ambisense RNA genomes in the family Bunyaviridae. However, the vast majority of plant viruses have a single-stranded RNA genome with positive polarity (+ssRNA). These viruses are classified in the order Tymovirales and Picornavirales, and in the families Bromoviridae, Closteroviridae, Luteoviridae, Potyviridae and Sequiviridae and in some genera that have still not been included in taxonomic entities of higher order (http://ictvonline.org/

virusTaxonomy.asp, viewed on 15-10-13). Potyviridae is one of the largest families.

Introduction María Calvo del Castillo - PhD thesis

30

Based on similarities of the genome structures and of some protein sequences, the family Potyviridae, has been included, together with the family Secoviridae and other families of animal viruses of the order Picornavirales in the proposed supergroup of picorna-like viruses (Goldbach et al., 1994).

I.2.1.
Genome
transla%on
and
replica%on
of
+ssRNA
plant
viruses

The genome of +ssRNA viruses can be mono- or multipartite. Positive sense genomic RNAs can function as mRNAs to be directly translated by the host translation machinery to express the necessary products for the viral infection cycle. In addition, viral genomes also contain regulatory sequences, located both in untranslated regions (UTRs) and inside coding sequences, which control the expression of the proteins and are involved in viral replication (Hull, 2002).

There are general aspects regarding the replication of +ssRNA viruses and also particular aspects specifically related to each group. One general characteristic is that after entering the cell and uncoating (a mechanism which is largely unknown but might be favoured by the interaction with ribosomes and the translation machinery), the genome of +ssRNA viruses is translated and some of the resulting proteins, like the RNA-dependent RNA polymerase (RdRp), are directly recruited for replication. Because this polymerase is not encapsidated, RNA translation must precede RNA replication, although these are tightly linked processes.

Since viruses do not code for their own ribosomes, they depend entirely on the host translation machinery for the expression of viral products. Unlike mRNAs from the plant, some +ssRNA genomes lack a cap structure at the 5’-end. In some cases, the presence of a 5’- end internal ribosomic entry site (IRES) has been shown to enhance the translation of viral products. In the case of potyviruses both IRES-dependent (Zeenko and Gallie, 2005) and leaky scanning (Simón-Buela et al., 1997) mechanisms have been proposed to operate for translation initiation.

Instead of the cap structure at the 5’ end, picorna-like +ssRNA viruses may possess a covalently attached viral protein genome-linked (VPg). The potyviral VPg interacts with the

translation initiation machinery binding the eIF4E/eIF(iso)4E factor, which suggests a role in viral genome translation. However the mRNA of animal picornaviruses loses its VPg before translation (Nomoto et al., 1977), and the VPg of the potyvirus PPV is not needed, or at least it is not indispensable, for the first translation event (Riechmann et al., 1990). Interestingly, some results indicate that the VPg-eIF4E interaction downregulates host gene mRNA expression and thus, the translation of viral products is favoured (Eskelin et al., 2011). The VPg also interacts with the poly(A) binding protein (PABP), which causes the genome circularization and enhances the translation efficiency. Therefore, the VPg could be used for the recruitment of host factors necessary for translation initiation (Léonard et al., 2004).

Viral factors involved in replication interact with host factors and form the viral replication complexes (VRCs) in modified membranous compartments (Miller and Krijnse- Locker, 2008; den Boon and Ahlquist, 2010; Nagy and Pogany, 2012). The origin of these membranous structures (whether they are derived from the endoplasmic reticulum, mitochondria, chloroplasts, etc…) can be different for each virus. The formation of vesicles is thought to be useful not only for the recruitment and concentration of factors required for the replication but also to protect the RNA from degradation and to avoid induction of host defences. The +ssRNA is used in the VRCs for the generation of a negative polarity single- stranded RNA (–ssRNA), which is then used to synthesize more +ssRNA viral molecules in the 3’–5’ direction, which will be used in another replication cycle or will be translated or encapsidated (Buck, 1999). Since the number of +ssRNA molecules generated exceeds that of –ssRNA molecules, replication is said to be asymmetric. The viral RNA not only codes for viral replication proteins, but also contains nucleotide sequences known as cis-active elements that are essential by their own in the replication process (Mahajan et al., 1996; Steil and Barton, 2009).

I.2.2.Cell‐to‐cell
and
systemic
movement
of
+ssRNA
viruses

After translation and replication, viruses move through the plant. Viruses must move from cell to cell of the initially infected leaf to reach the vascular tissue (cell-to-cell movement), move to upper leaves, mainly trough the phloem sieve tubes (long-distance or systemic movement) and move again from cell-to-cell in younger leaves. Thus, to cause a Introduction María Calvo del Castillo - PhD thesis

32

systemic infection, viruses must overcome the barriers imposed at each of the three steps (Hull, 2002).

1.2.2.1
Cell‐to‐cell
movement

Cell-to-cell movement occurs through plasmodesmata, which are structures that physically communicate neighbour cells in the epidermis or mesophyll. Basically, these structures are formed by two concentric membrane cylinders, the plasma membrane and the endoplasmic reticulum (desmotubule), both associated to plasmodesmatal proteins, which traverse the cellulose walls between adjacent cells. (Hull, 2002).

The first viral movement protein (MP) was identified in TMV. It was shown to bind single stranded RNA to form a ribonucleoprotein (RNP) complex, interact with the host machinery to increase the size exclusion limit of the plasmodesmata, localize in plasmodesmatal structures and mediate its own trafficking as well as that of the viral RNA progeny from one cell to another. Depending on the genera, viruses use different cell-to-cell movement strategies. While some viruses like TMV possess a specific MP for cell-to-cell movement, others use a set of two or three different proteins to develop this function. The interaction between viral MPs and plasmodesmata increases the exclusion size limit of the pore to allow the trafficking of the virus as a virion or RNP complex. Moreover, in some cases, the CP also plays a relevant role in cell-to-cell movement (Lucas, 2006).

1.2.2.2.
Long‐distance
movement

For long-distance movement, viruses have to be translocated from mesophil cells to sieve elements of the phloem, crossing the bundle sheath, vascular parenchima and companion cells. Once in the sieve element, viruses are transported following the photoassimilate route (from source to sink tissues), until they exit the phloem and continue propagating in the new infection site (Citovsky and Zambryski, 2000; Liu et al., 2005).

Although most of the +ssRNA viruses appear to move though the phloem, virus transport though the xylem has also been reported (Verchot et al., 2001; Moreno et al., 2004; Betti et al., 2012)

In any case, replication and movement appear not to be independent processes. MPs associate to viral replication complexes, which traffic intracellularly through the