The determinant of potyvirus ability to overcome the RTM resistance of Arabidopsis thaliana maps to the N-terminal region of the coat protein.
ABSTRACT In Arabidopsis thaliana Columbia (Col-0) plants, the restriction of Tobacco etch virus (TEV) long-distance movement involves at least three dominant RTM (restricted TEV movement) genes named RTM1, RTM2, and RTM3. Previous work has established that, while the RTM-mediated resistance is also effective against other potyviruses, such as Plum pox virus (PPV) and Lettuce mosaic virus (LMV), some isolates of these viruses are able to overcome the RTM mechanism. In order to identify the viral determinant of this RTM-resistance breaking, the biological properties of recombinants between PPV-R, which systemically infects Col-0, and PPV-PSes, restricted by the RTM resistance, were evaluated. Recombinants that contain the PPV-R coat protein (CP) sequence in an RTM-restricted background are able to systemically infect Col-0. The use of recombinants carrying chimeric CP genes indicated that one or more PPV resistance-breaking determinants map to the 5' half of the CP gene. In the case of LMV, sequencing of independent RTM-breaking variants recovered after serial passages of the LMV AF199 isolate on Col-0 plants revealed, in each case, amino acid changes in the CP N-terminal region, close to the DAG motif. Taken together, these findings demonstrate that the potyvirus CP N-terminal region determines the outcome of the interaction with the RTM-mediated resistance.
- Molecular Plant Pathology 08/2014; · 4.49 Impact Factor
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ABSTRACT: To establish a successful infection plant viruses have to overcome a defense system composed of several layers. This review will overview the various strategies plants employ to combat viral infections with main emphasis on the current status of single dominant resistance (R) genes identified against plant viruses and the corresponding avirulence (Avr) genes identified so far. The most common models to explain the mode of action of dominant R genes will be presented. Finally, in brief the hypersensitive response (HR) and extreme resistance (ER), and the functional and structural similarity of R genes to sensors of innate immunity in mammalian cell systems will be described.Frontiers in Plant Science 06/2014; 5:307. · 3.64 Impact Factor
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ABSTRACT: Complete genome sequence of nine isolates of canna yellow streak virus reveals its relationship to the sugarcane mosaic virus (SCMV) subgroup of potyviruses Abstract Complete genome sequences were obtained from nine isolates of canna yellow streak virus (CaYSV). CaYSV belongs to the sugarcane mosaic virus (SCMV) subgroup of potyviruses with johnsongrass mosaic virus (JGMV) as its closest relative. Multiple sequence align-ments showed a pattern of amino acid substitutions in the CP sequences, which enabled us to relate these isolates to South East Asian or European isolates. Biological charac-terization of CaYSV identified Nicotiana benthamiana, Chenopodium quinoa and Phaseolus vulgaris as experi-mental hosts. Given the popularity and global trade of cannas, a clear picture of the genetic diversity of CaYSV is critical to disease management. The family Potyviridae comprises more than 30 % of known plant virus species and the genus Potyvirus contains the largest collection of species within this family [10, 11, 22]. Within the genus Potyvirus, Gibbs and Ohshima  identified eleven potyvirus groups, nine of which reside within two divergent supergroups referred to as the potato virus Y (PVY) supergroup and the bean common mosaic virus (BCMV) supergroup [10, 11]. Canna yellow streak virus (CaYSV) is a major concern in the canna industry and was identified as the cause of severe streaking symptoms in European-grown canna varieties [16, 17]. The first full genome sequence of CaYSV was reported in 2010 . Cannas belong to the plant family Cannaceae  and are a commercial crop producing edible rhizomes that are harvested in South America and South East Asia as a starch food for making chips or boiled to make noodles. Ornamental hybrids are grown worldwide for their colorful foliage and flowers. Cannas are produced and distributed through vegetative propagation, which makes the rhizomes a major source of virus spread through trade among growers. Canna rhizomes from the USA suspected of being infected with CaYSV were collected and grown in the greenhouse. Leaf extracts were taken from plants that showed obvious mosaic symptoms (Fig. S1a), tested by RT-PCR for CaYSV , and examined by electron microscopy to identify flexuous filamentous potyvirus particles (Fig. S1b). Leaf extracts were also subjected to 12.5 % SDS-PAGE and immunoblot analysis using a Pierce Fast Western Immunoblot Kit (Thermo Fisher Sci-entific). As an external control, leaf extracts from plants infected with bean yellow mosaic virus (BYMV) (another potyvirus that infects canna) were included. A commercial potyvirus group monoclonal (MAb) antiserum that detects most potyvirus coat proteins (CPs) (AC Diagnostics) detected both the 30.8-kDa BYMV CP and the 32.4-kDa CaYSV CP (Fig. S1c) but had greater reactivity with the BYMV CP than CaYSV CP . The entire genome of a single isolate of CaYSV (CaYSV-OK) was amplified using total RNA extracted from a six-week-old canna 'Wyoming' plant that was grown in the greenhouse as described previously [4, 20]. RT-PCR was carried out using a One Step Superscript IIArchives of Virology 01/2015; · 2.28 Impact Factor
1302 / Molecular Plant-Microbe Interactions
MPMI Vol. 22, No. 10, 2009, pp. 1302–1311. doi:10.1094/MPMI-22-10-1302. © 2009 The American Phytopathological Society
The Determinant of Potyvirus Ability to Overcome
the RTM Resistance of Arabidopsis thaliana Maps
to the N-Terminal Region of the Coat Protein
V. Decroocq,1 B. Salvador,2 O. Sicard,1 M. Glasa,3 P. Cosson,1 L. Svanella-Dumas,1 F. Revers,1
J. A. García,2 and T. Candresse1
1UMR GDPP, INRA Université Bordeaux II, IBVM, Centre INRA de Bordeaux, BP 81, 33883 Villenave d’Ornon Cedex;
France; 2Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain;
3Institute of Virology, Department of Plant Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 84505 Bratislava,
Submitted 9 March 2009. Accepted 28 May 2009.
In Arabidopsis thaliana Columbia (Col-0) plants, the restric-
tion of Tobacco etch virus (TEV) long-distance movement
involves at least three dominant RTM (restricted TEV
movement) genes named RTM1, RTM2, and RTM3. Previ-
ous work has established that, while the RTM-mediated
resistance is also effective against other potyviruses, such
as Plum pox virus (PPV) and Lettuce mosaic virus (LMV),
some isolates of these viruses are able to overcome the
RTM mechanism. In order to identify the viral determi-
nant of this RTM-resistance breaking, the biological prop-
erties of recombinants between PPV-R, which systemically
infects Col-0, and PPV-PSes, restricted by the RTM resis-
tance, were evaluated. Recombinants that contain the PPV-
R coat protein (CP) sequence in an RTM-restricted back-
ground are able to systemically infect Col-0. The use of
recombinants carrying chimeric CP genes indicated that
one or more PPV resistance-breaking determinants map to
the 5′ ′ half of the CP gene. In the case of LMV, sequencing
of independent RTM-breaking variants recovered after
serial passages of the LMV AF199 isolate on Col-0 plants
revealed, in each case, amino acid changes in the CP N-ter-
minal region, close to the DAG motif. Taken together, these
findings demonstrate that the potyvirus CP N-terminal
region determines the outcome of the interaction with the
Plant viruses are systemic pathogens that infect a suscepti-
ble host in a multistep process. Consecutive to entry in host
cells, replication of the viral genome is followed by virus
movement to neighboring cells through the plasmodesmata
(also called cell-to-cell movement), presumably utilizing the
cellular pathways of plasmodesmal transport (Waigmann et al.
2004), and over longer distances through the plant vascular
bundles (the long-distance movement), leading to systemic
infection (Gilbertson and Lucas 1996; Wang et al. 1999). Long-
distance movement occurs through the phloem for most viruses,
following the photoassimilate path from source to sink organs.
Thus, in order to successfully infect distant tissues, viruses must
enter the vascular system, access, move through, and exit from
sieve tubes, exit the vascular system, and infect nonvascular
cells of sink tissues. Depending on the virus, viral genomic
nucleic acids are transported as either virions or nucleoprotein
complexes (Gilbertson and Lucas 1996).
Our understanding of the mechanisms controlling long-dis-
tance movement remains limited, and the role of phloem sap
proteins in long-distance movement of virus particles has yet
to be defined. It has been suggested that plant viruses are pas-
sively transported along with the solutes through the phloem
or that they undergo selective unloading at specific cell boun-
daries (Lucas 2006). These last observations support the notion
that the long-distance movement of viruses in the sieve-tube
system may not simply follow the stream of assimilates and
that phloem exit in sink tissues is highly controlled. Moreover,
viral destination appears to be regulated by protein-protein or
protein-RNA interactions, or both, within phloem tissues (Kehr
and Buhtz 2008; Lough and Lucas 2006). Numerous viral deter-
minants involved in potyvirus infection were identified, among
which three potyviral proteins seem to be involved in long-dis-
tance movement, e.g., the coat protein (CP), the helper compo-
nent-proteinase (HC-Pro), and the genome-linked protein
(VPg) (Revers et al. 1999). Conversely, nearly nothing is
known about host factors involved in the long-distance process
Genetic and functional genomic studies in Arabidopsis have
revealed the importance of specific host proteins in controlling
the vascular movement of Tobacco etch virus (TEV) (Whitham
et al. 1999). During infection with this potyvirus, at least three
RTM (restricted TEV movement) genes, most probably acting
in synergy, prevent systemic but not local TEV infection in
Col-0 and WS wild-type plants (Mahajan et al. 1998). In com-
parison, some other Arabidopsis accessions, i.e., Ler (Lands-
berg erecta) and C24, allow TEV long-distance movement
from inoculated rosette leaves to noninoculated inflorescence
tissues. The RTM1 and RTM2 genes have been identified by
map-based cloning (Chisholm et al. 2000; Whitham et al.
2000). RTM1 encodes a jacalin-like lectin protein, while
RTM2 is related to the multigene family of small heat-shock
proteins. The RTM1 and RTM2 proteins are expressed mostly
or exclusively in the phloem (Chisholm et al. 2001). In fact,
these proteins contribute to a dominant blockage of systemic
infection of Col-0 plants by at least three distinct potyviruses,
namely, TEV, Plum pox virus (PPV), and Lettuce mosaic virus
(LMV) (Decroocq et al. 2006; Mahajan et al. 1998). The three
controlled potyviruses are able to establish a systemic infec-
Corresponding author: V. Decroocq; E-mail: firstname.lastname@example.org
*The e-Xtra logo stands for “electronic extra” and indicates that three
supplemental tables are published online.
Vol. 22, No. 10, 2009 / 1303
tion in Col-0 plants only when at least one of the RTM genes is
mutated (Decroocq et al. 2006; Mahajan et al. 1998). One fas-
cinating challenge with the RTM resistance is the understand-
ing of the mechanism or mechanisms by which the RTM gene
products operate. One way to address this issue is to gain knowl-
edge on one or more viral determinants interacting with the
We have previously reported that, whereas the long distance
movement of PPV isolates is restricted by the RTM resistance,
one PPV isolate, PPV-Rankovic (PPV-R), is able to overcome
the RTM resistance in the Arabidopsis Col-0 accession
(Decroocq et al. 2006). In the present work, the use of a
reverse genetics approach involving the construction of recom-
binants between PPV-R (RTM-breaking isolate) and PPV-PS
(RTM-restricted isolate) allowed the mapping of one or more
PPV RTM-breaking determinants to the N-terminal region of
the viral CP. This result is further confirmed by the analysis of
the biological properties of recombinants involving other PPV
isolates as parents and by the sequencing of RTM-breaking
variants selected for another potyvirus controlled by the RTM
system, LMV. In the case of LMV, the overcoming of the RTM
resistance was found to be associated with point mutations
affecting amino acids of the LMV CP N-terminal region.
These results are further discussed in terms of the manner in
which the RTM complex and viral CP might interplay to
restrict the virus phloem long distance transport.
Restriction of systemic infection of some PPV isolates
in the Col-0 ecotype of Arabidopsis is caused
by the RTM resistance.
We previously showed that, while the PPV-EA (PPV-El-
Amar) long-distance movement is restricted in the Col-0 ac-
cession, several other PPV isolates including PPV-R and PPV-
NAT (non–aphid transmissible) (two members of the PPV-D
strain) are able to invade and accumulate in noninoculated tis-
sues (Decroocq et al. 2006). To examine the ability of other
PPV isolates or strains to systemically invade Col-0, the accu-
mulation of PPV-PSes and PPV-SK68 (both belonging to the
PPV-M strain) was scored by enzyme-linked immunosorbent
assay (ELISA) at 9, 15, 21, and 28 days postinoculation (dpi)
in mechanically inoculated and noninoculated Col-0 tissues.
The previously tested PPV-EA and PPV-R were used as con-
trols. Failure to accumulate, as judged by ELISA, was con-
firmed by reverse transcription-polymerase chain reaction (RT-
All tested PPV isolates were detected in inoculated leaves at
9 and 15 dpi. In noninoculated inflorescence tissues (leaves
and flowers), PPV-Pses, and PPV-SK68, the two isolates of the
PPV-M strain behaved as PPV-EA and could not be detected at
any timepoint. On the contrary, accumulation of PPV-NAT and
PPV-R, both belonging to the PPV-D strain, was readily de-
tected at 21 dpi in noninoculated tissues (Supplementary Table
S2). The inability of PPV-PSes and PPV-SK68 isolates to estab-
lish a systemic infection was confirmed even upon direct bio-
listic inoculation of Col-0 plants, and similar results were
again obtained using the green fluorescent protein (GFP)-
tagged version of PPV-PSes. Infectivity of the PPV-PSes and
PPV-SK68 cDNA clones was determined, in parallel, by direct
biolistic inoculation of Ler plants and of rtm mutants (data not
shown), which bear point-mutated, homozygous alleles of
either the RTM1, RTM2, or RTM3 genes in a Col-0 back-
ground (Whitham et al. 1999). All Arabidopsis Ler and rtm
plants inoculated with PPV-PSes and PPV-SK68 were tested
positive for infection of both inoculated and noninoculated
leaves at 9 and 21 dpi, respectively.
Overall, the results obtained demonstrate that similar to PPV-
EA, the PPV-PSes and PPV-SK68 isolates are unable to sys-
temically invade the Col-0 Arabidopsis accession, because their
long-distance movement is restricted by the action of the RTM
resistance system. On the other hand, the ability of both PPV-
NAT and PPV-R to systemically invade Col-0 indicates that they
are RTM-breaking isolates, opening the way to the identification
of the region of the viral genome contributing this property,
using chimeras between the PPV-R and PPV-PSes genomes.
The GFP-tagged version of PPV-R (pICPPVnk-GFP) displays
properties similar to those of its untagged parental isolate.
Figure 1 shows that PPVnk-GFP, progeny of the inoculated
pICPPVnk-GFP, is able to establish a systemic infection in the
wild-type Col-0 by 21 dpi, thereby breaking the RTM resis-
tance (Fig. 1A and D). On the contrary, the P/R(GFP) 7677-
8576 viral clone, corresponding to the PPV-PSes isolate that
contains one single 101-bp PPV-R fragment from position
7677 to 8576 (Fig. 2) is restricted to the initially inoculated
Col-0 leaves. Its behavior is similar to that of the PPV-PSes
isolate, it can move from cell to cell (Fig. 1B and 1C) but it
cannot infect inflorescence tissues (Fig. 1E). Besides, a single
mutation in either the RTM1, RTM2, or RTM3 genes is suffi-
cient to alleviate the restriction of P/R(GFP) 7677-8576 long-
distance movement and systemic infection of Col-0 plants,
since GFP expression was detected at 21 dpi in upper nonin-
oculated leaves of the three mutants (Fig. 1G, H, and I).
The PPV-R–derived RTM-breaking determinants map
in the 5′ ′ region of the CP gene.
Because of the differential behavior of PPV-PSes and PPV-R
towards the RTM resistance, the RTM-breaking properties of
seven available recombinant constructs between these two iso-
lates, including the two GFP-tagged ones, P/R(GFP) 7677-
8576 and P/R(GFP) 7677-9020, were initially assessed (Fig.
2). Six to twelve Col-0 plants were thus mechanically inocu-
lated, using recombinant virus isolates first propagated in
Nicotiana benthamiana. Infection of Col-0 plants was moni-
tored at 9 dpi in inoculated leaves and at 21 dpi in noninocu-
lated inflorescence tissues (leaves and flower buds) by fluores-
cence for P/R(GFP) 7677-9020 and by ELISA and RT-PCR for
the other five recombinants. The experiment was repeated
three times, with similar results. Although not all symmetrical
recombinant pairs are available, the results with the five re-
combinants that are not GFP-tagged are consistent with a role
of the 3′ region of the genome, corresponding to half of the
NIb gene, the CP gene, and the 3′ noncoding region. This is
indicated in particular by the fact that PPV R/P 7677 is not
able to systemically infect Col-0 inflorescences while PPV R/P
2212-7677 develops a systemic infection in this host.
In addition, while P/R(GFP) 7677-8576 long-distance move-
ment is restricted in Col-0 plants (Figs. 1E and 2), the P/R(GFP)
7677-9020 construct is able to infect Col-0 inflorescences
(Figs. 1F and 2). The only difference between these two PPV-
PS constructs is the presence in P/R(GFP) 7677-9020 of the 5′
half of the CP gene (from the first nucleotide to the SacI restric-
tion site) of the PPV-R isolate, which suggests that the CP or,
more precisely, the first 146 amino acids of the N-terminal CP
region is the viral factor involved in the overcoming of the
RTM resistance by PPV-R.
As a control that the restriction of the systemic movement of
the recombinants was indeed caused by the RTM resistance,
all recombinants were also inoculated on rosettes of the Ler
accession and of the various rtm mutants. Viral infection was
assessed by ELISA or, if possible, GFP observation in the
uninoculated upper leaves at 21 dpi. As expected, all recombi-
nants, irrespective of their behavior in Col-0, were readily de-
tected (data not shown) in uninoculated upper leaves of Ler
1304 / Molecular Plant-Microbe Interactions
plants or of the various rtm mutants. The identity of the vari-
ous PS/R recombinants used in those experiments was verified
by partial resequencing of the PPV genome in regions encom-
passing the recombination sites. These control experiments
demonstrate that all tested PPV constructs are fully infectious
in Arabidopsis and that the impaired systemic accumulation
observed in Col-0 for some of them is not due to a nonspecific
loss of infectivity but, rather, to the RTM-mediated resistance
mechanism active in this host.
The first 146 amino acids of the CP control the behavior
of PPV isolates towards the RTM resistance.
To experimentally validate the contribution of the first 146
amino acids of the CP to the resistance phenotype in Col-0, a
series of PPV recombinant cDNA clones was designed to ex-
change this N-terminal CP region between various PPV iso-
lates. This region was symmetrically exchanged between PPV-R
and PPV-PSes, yielding the R/P 8576-9020 and P/R 8576-9020
recombinant pair. In addition, the N-terminal region of PPV-EA
was introduced in PPV-R and that of PPV-SK68 in PPV-PSes,
yielding recombinants R/EA 8576-9020 and P/SK68 8576-9020,
The results obtained show that replacing the sequence cod-
ing for N-terminal CP region of PPV-R by a similar KpnI/SacI
fragment issued from PPV-PSes or PPV-EA compromises virus
systemic accumulation in Col-0 plants (Fig. 2) but not in Ler
or rtm mutants (data not shown). Conversely, replacing the
PPV-PSes N-terminal CP region by that of PPV-R (but not that
Fig. 1. Distribution of green fluorescent protein (GFP)-tagged Plum pox virus (PPV) isolates within inoculated rosette leaves and uninoculated inflorescen-
ces of Col-0 wild-type or rtm-mutated plants. Photographs were taken under UV light as described by Decroocq and associates (2006). A, Inoculated rosette
leaves at 9 days postinoculation (dpi) and D, uninoculated inflorescence tissues at 21 dpi of Col-0 plants infected with the restricted TEV movement (RTM)-
breaking isolate, pICPPVnk-GFP (PPV-R). B, Stereomicroscopy images obtained with a B or C, a GFP3 filter at 9 dpi from a rosette leaf of a Col-0 plant
inoculated with P/R(GFP) 7677-8576; E, Images obtained at 21 dpi from uninoculated inflorescences of Col-0 plants infected with P/R(GFP) 7677-8576 or
F, P/R(GFP) 7677-9020. G through I, Uninoculated inflorescence and rosette leaves of Col-0 mutants rtm1-1, rtm1-2, and rtm3-1, respectively, inoculated
with P/R(GFP) 7677-8576. Bar = 2 mm.
Vol. 22, No. 10, 2009 / 1305
of PPV-SK68, a non–resistance breaking isolate) resulted in a
recombinant virus that systemically accumulated in Col-0
plants (Fig. 2), demonstrating that the exchanged CP N-termi-
nal region is sufficient to determine the systemic invasion phe-
notype in Col-0 plants.
Amino acid changes in the N-terminal region
of the LMV CP correlate with the ability
to overcome the RTM resistance.
While the LMV-AF199 isolate is able to systemically infect
numerous Arabidopsis thaliana accessions, it is restricted to
the inoculated leaves in Col-0 (Revers et al. 2003) and this
long-distance movement-restriction phenotype has been shown
to be mediated by the RTM resistance (Decroocq et al. 2006).
However, systemic accumulation of LMV-AF199 in Col-0
plants is occasionally observed in a low percentage of the
inoculated plants (not shown). Serial propagation in Col-0 of
the viral isolate present in these plants results in the selection
of LMV-AF199 variants able to systemically infect 100% of
the inoculated Col-0 plants, as revealed by ELISA and RT-
PCR assays (data not shown). In this way, a total of four RTM-
breaking variants were independently obtained, deriving from
independent inoculation experiments of Col-0 plants. Given
the involvement of the PPV CP N-terminal region in RTM-
breaking, initial characterization was carried out on one of
these variants, LMV-AFVAR1, by sequencing its N-terminal
CP region and comparing it with that of the original LMV-
AF199 inoculum maintained in lettuce. A single G to A muta-
tion at position 9,063 of the genome causing a glycine to
aspartic acid amino acid change at position 10 of the CP was
identified (Fig. 3). Because no infectious cDNA clone of
LMV-AF199 is available, site-directed mutagenesis cannot be
used to validate the effect of this mutation on the phenotype
towards the RTM resistance. As an alternative the full genome
sequence of LMV-AFVAR1 was determined and compared
with its parent LMV-AF199, and three other independent
RTM-breaking variants were characterized.
In total, two additional mutations were identified between
the complete genome of LMV-AFVAR1 and that of its LMV-
AF199 parent, a G to A mutation at position 3,883 of the ge-
nome in the P3 coding region and a C to T mutation at position
8,729 within the NIb coding region. However, these two muta-
tions are synonymous ones, which do not change the sequence
of the viral polyprotein, so that LMV-AFVAR1 differs by a
single amino acid change in the CP N-terminal region from its
LMV-AF199 progenitor (Fig. 3).
Analysis of the three other independently obtained RTM-
breaking variants was carried out by partial sequencing in the
regions surrounding the mutations identified in LMV-AFVAR1.
None of these three variants contained the silent mutations at
Fig. 2. Schematic representation of the Plum pox virus (PPV) genome and of different PPV recombinants tested on the Col-0 accession. The outcome of Col-
0 infection with PPV isolates and recombinants is depicted on the right: + indicates systemic infection at 21 dpi was detected and – that no systemic infection
of uninoculated inflorescences detected at 21 dpi. PPV-PSes and PPV-R sequences are shown as open and filled boxes, respectively. Restriction sites used in
the cloning are indicated on the top. A genetic map of PPV, indicating the positions of the encoded proteins, is shown in the middle. The names of the virus
proteins are indicated in the PPV genome map as detailed by Urcuqui-Inchima and associates (2001). Infectious clones and recombinants are fully described
in this manuscript and by Sáenz and associates (2000). The location of the introduced green fluorescent protein (GFP) open reading frame is indicated under-
neath the corresponding PPV construct.
1306 / Molecular Plant-Microbe Interactions
positions 3,883 or 8,729 (result not shown), but all three of them
contained nonsynonymous mutations in their CP N-terminal
region (Fig. 3). One variant presents the same mutation as
LMV-AFVAR1 at position 10 of the CP, while the other two
show, respectively, a serine to asparagine mutation at position
11 (LMV-AFVAR2) or an aspartate to glycine mutation at
position 14 of the CP (LMV-AFVAR3) (Fig. 3).
Biophysical parameters of the N-terminal CP region
in PPV and LMV.
Previous work has demonstrated the influence of CP phos-
phorylation, O-glycosylation, and N-terminal net charge on
potyvirus infectivity or ability to mount systemic infection in
host plants (Chen et al. 2005; Fernández- Fernández et al. 2002;
Ivanov et al. 2003). In order to evaluate such a possibility in
the case of the RTM resistance, the isoelectric point, net charge,
and total number of predicted potential sites for phosphoryla-
tion or glycosylation (Thr + Ser residues) were compared for
the PPV and LMV CP N-terminal regions of RTM-restricted
and RTM-breaking isolates (Table 1).
These computations show that the original wild-type LMV
CP and one of its variants (LMV-AFVAR1) contain the same
number of positively and negatively charged amino acids, giv-
ing an equivalent slightly negative net charge (Table 1). Sur-
prisingly, in LMV-AFVAR3, the terminal CP net charge is null,
which is commonly found to be deleterious to potyvirus sys-
temic movement in host plants like zucchini squash, tobacco,
and N. benthamiana (Kimalov et al. 2004; López-Moya and
Pirone 1998). In comparison, the N-terminal CP net charge of
all PPV isolates is significantly negative. However, multiple
amino acid changes between PPV-NAT (an RTM breaking
isolate) and PPV-SK68 (an RTM-restricted isolate) did not
modify the total net charge (–6) of this CP region. Overall, this
analysis does not reveal a clear correlation between the CP N-
terminal net charge and the ability to overcome the RTM
resistance in Col-0. Interestingly, while the LMV wild-type
isolate and two of its variants display similar amounts of Thr
and Ser residues, the PPV RTM-restricted isolates (PPV-PSes,
PPV-SK68, PPV-EA) harbor a significantly higher number of
Thr residues (Table 1) in comparison to the PPV RTM-
breaking isolates (PPV-R and PPV-NAT). However, the situa-
tion is reversed concerning the Ser phosphorylation or gly-
cosylation sites. Moreover, an RTM-breaking isolate, PPV-R,
and the RTM-restricted isolate PPV-SK68 display a similar
proportion of Thr + Ser residues (20%). Therefore, we cannot
conclude on a significant correlation between the number of
Fig. 3. Alignment of the amino acid sequences of A, Plum pox virus (PPV), B, Lettuce mosaic virus (LMV), and C, Tobacco etch virus (TEV) N-terminal
coat protein (CP) regions. Identical amino acids are indicated by horizontal bars. The 15 amino acid ‘NAT’ deletion is represented by dots. The amino acid
highly variable N-terminal sequence used for the analysis of the CP biophysical parameters is underlined with stars. The amino-acids highlighted in gray cor-
respond to the start of the core conserved region of PPV, LMV and TEV CP. Residues in bold are those consistently differing between restricted TEV move-
ment (RTM)-breaking (PPV-R and PPV-NAT) and RTM-restricted (PPV-PSes, PPV-SK68, and PPV-EA) isolates. The corresponding amino acid substitu-
tions are represented above the PPV sequence. The sequences of PPV-R and PPV-PSes used in this alignment correspond to the CP sequences of pICPPVnk
and pICPPVPSes isolates, respectively. The TEV-7DA (named TEV-GUS) N-terminal CP sequence depicted in this figure belongs to the TEV isolate initially
used to identify host genes controlling RTM in Arabidopsis (Mahajan et al. 1998; Whitham et al. 1999).
Vol. 22, No. 10, 2009 / 1307
potentially phosphorylated or glycosylated residues and RTM
Systemic infection of RTM-restricted and RTM-breaking
PPV and LMV isolates in the OGT mutant plants.
To examine the ability of RTM-restricted PPV and LMV iso-
lates to overcome the RTM resistance in OGT (O-linked N-ace-
tylglucosamine transferase)–depleted mutant plants, the accu-
mulation of PPV-PSes, PPV-EA, and LMV-AF199 was scored
by ELISA at 9 and 21 dpi, in mechanically inoculated and non-
inoculated tissues of Arabidopsis knock-out mutants (Supple-
mentary Table S3). The previously tested PPV-R (pICPPVnk-
GFP; Chen et al. 2005) was used as control. In comparison, the
OGT Col-0 mutants (namely, sec-2 and spy-1) were also inocu-
lated with the RTM-breaking LMV variants (LMV-AFVAR 1 to
LMV-AFVAR 3). Failure to accumulate, as judged by ELISA,
was confirmed by RT-PCR analysis. In this study, only the
RTM-breaking PPV isolate and LMV-AFVAR variants were
able to complete a successful systemic infection in the OGT-mu-
tant plants. In consequence, reduction of the activity of one or
the other Arabidopsis OGT did not alleviate the RTM resistance.
In Arabidopsis, the RTM-mediated restriction of potyvirus
long-distance movement involves at least three host proteins,
i.e., RTM1, RTM2, and RTM3 (Chisholm et al. 2001; Mahajan
et al. 1998; Whitham et al. 1999). This resistance machinery is
active against at least three distinct potyviruses, TEV, PPV, and
LMV (Decroocq et al. 2006; Mahajan et al. 1998). Since all
TEV isolates initially tested presented the same restriction
phenotype, no information about one or more viral factors
interacting with the RTM proteins, or at least relevant for the
RTM resistance, could be obtained. We previously reported
that several PPV isolates, PPV-R among them, are able to
overcome the RTM resistance in the Arabidopsis Col-0 acces-
sion (Decroocq et al. 2006). In the present study, we extend the
number of RTM-breaking and RTM-restricted PPV isolates.
Here, we show clearly that, similarly to TEV (Mahajan et al.
1998), the RTM-restricted PPV-PSes isolate is not defective in
accumulation and cell-to-cell movement within the inoculated
leaf but is, rather, deficient in establishing an effective systemic
infection of Col-0 plants (Fig. 1). The two identified RTM-
breaking isolates belong to the PPV-D strain, while the RTM-
restricted isolates belong either to the PPV-M or PPV-EA
strains. The first PPV-PS isolate, initially tested in Col-0
(Decroocq et al. 2006; Nicaise et al. 2007) and which appeared
to break down the RTM resistance, was later on resequenced
and shown to belong to the PPV-D strain and to be a close
variant of PPV-R (V. Decroocq, unpublished data). Whether
the behavior towards the RTM resistance is a homogenous
property shared by all isolates within a given PPV strain will
have to be specifically investigated.
The sequence of the N-terminal region
of the CP determines the breaking
of the RTM-mediated resistance in Col-0 plants.
A reverse-genetics approach was used to map the viral deter-
minants of RTM-breaking by analyzing the infection pheno-
type in Col-0 plants of recombinant constructs between PPV-R
and PPV-PSes (Fig. 2). This allowed the identification of a key
role for the region encoding the first 146 amino acids of the
CP. However, PPV RTM-breaking and RTM-restricted isolates
are substantially divergent within their N-terminal CP region
(Fig. 3), making it difficult to pinpoint single amino acids that
could be relevant for RTM-resistance breaking.
In the case of LMV, complete genome comparison between
RTM-breaking variants and the non–resistance breaking pro-
genitor allowed the identification of three-point mutations, a
single one of which changes the sequence of the viral polypro-
tein. Although a contribution of the two noncoding mutations
to the resistance-breaking phenotype cannot be absolutely
ruled out, the fact that none of these mutations was observed in
the three other independently obtained resistance-breaking
variants strongly argues against such a hypothesis. On the con-
trary, the fact that the glycine to aspartate mutation at position
10 of the CP was observed in two independently obtained vari-
ants is a very strong indication of its implication in the resis-
tance-breaking phenotype. This is further supported by the ob-
servation of coding mutations at closely located positions of
the CP of two other independently obtained resistance-break-
ing variants (Fig. 3). Interestingly, despite the relative ease
with which resistance-breaking variants could be selected for
LMV, it was not possible to select similar variants for PPV, de-
spite extensive efforts involving the inoculation of batches of
24 to 48 Col-0 plants with three different PPV isolates
(pICPPVPSes, PPV-SK68, or PPV-EA).
Different models for the RTM-mediated restriction
of viral long-distance movement.
The CP of plant viruses is multifunctional (Urcuqui-Inchima
et al. 2001). In addition to its obvious implication in the encap-
Table 1. Biophysical parameters characterizing Plum pox virus (PPV), Lettuce mosaic virus (LMV), and Tobacco etch virus (TEV) N-terminal coat protein
% Target residues for
12.2% (T) 7.8% (S)
11.8% (T) 7.9% (S)
15.6% (T) 5.6% (S)
14.4% (T) 5.6% (S)
27.7% (T) 3.2% (S)
5.3% (T) 10.5% (S)
5.3% (T) 7.9% (S)
5.3% (T) 10.5% (S)
5.3% (T) 10.5% (S)
3.8% (T) 7.7% (S)
5 5 0
a The predicted isolelectric point (pI) and the net charge were determined from the nonconserved amino-acid region of the PPV and LMV CP, using the
ExPASy ProtParam tool.
b Total number of negatively charged residues (Asp + Glu).
c Total number of positively charged residues (Arg + Lys).
d Net charge calculated as follows: (Arg + Lys) - (Asp + Glu).
1308 / Molecular Plant-Microbe Interactions
sidation of the genomic RNA, the potyviral CP has been shown
to play roles in vector transmission (Atreya et al. 1990; Blanc
et al. 1997), plasmodesmatal gating (Rojas et al. 1997), and
cell-to-cell (Dolja et al. 1995) and long distance (Andersen
and Johansen 1998) movement, and the CP of one potyvirus
has been shown to display NTPase activity (Rakitina et al.
2005). In PPV, the N-terminal region of the CP was linked to
aphid transmission (Maiss et al. 1989) and to virus spread and
accumulation (Chen et al. 2005). Dolja and associates (1995)
have shown that the N- and C- terminal regions of TEV CP are
necessary for long-distance movement. The N-terminal region
of the CP is broadly variable in length and sequence between
potyviruses and, at a lower level, within potyvirus species, so
that its sequence is frequently used to distinguish strains
within the same virus species (Shukla and Ward 1989). Various
authors have proposed that this variability could represent a
way for the virus to modulate its interaction with specific host
factors involved in cell-to-cell or long-distance movement. In
this concept, amino acid variation presumably modifies the
ability of the virus to interact with one or more host compo-
nents of a movement complex and, in consequence, alters the
capacity of the virus for systemic movement (López-Moya and
Pirone 1998). The first hypothesis concerning the mechanism
underlying the RTM resistance is that the N-terminal region of
CP interacts with host factors to potentiate virus movement
and that the RTM resistance corresponds to an interference
with these systemic movement-promoting interactions. It is
conceivable that the RTM system could prevent virus move-
ment by sequestering the viral CP. An alternative possibility is
that the same N-terminal region of CP could independently
interact with movement-promoting host factors and with the
RTM proteins. In this scenario, the same CP region would be
related to virus restriction through its interaction with the RTM
complex and, related to virus long-distance movement,
through its interaction with other host factors. The RTM pro-
teins and other host factors would thus compete for interaction
with the CP sequence. It is worth noting that, while the PPV
and LMV CP N-terminal regions are rather distinct (Fig. 3),
both sequences are presumably able to interact with the same
RTM complex. The fact that the RTM resistance is dominant is
consistent with a direct or indirect interaction of the RTM pro-
teins with viral movement proteins such as the CP.
In both scenarios, if one of the RTM genes is mutated, as in
the rtm mutants, the virus determinant would be free to interact
with other host factors promoting movement. Similarly, amino
acid modifications in the CP N-terminal region impeding the
direct or indirect interaction of the capsid with the RTM com-
plex would liberate the sequences involved in CP–host factor
interactions, thus promoting systemic movement. Of course, we
cannot rule out the possibility that independent mechanisms of
RTM restriction and support of virus movement coexist, involv-
ing distinct CP determinants and possibly other viral proteins
and finely tuning the systemic invasion of the host plant.
The sole CP primary sequence is not sufficient
to explain the occurrence or not of RTM resistance.
It is intriguing to note that all three LMV variant positions
are located close to the DAG triplet in the N-terminal CP se-
quence (Fig. 3). The importance of this observation for the
breaking of the RTM-mediated resistance is not yet clear.
However, in PPV, the presence (in PPV-R) or the deletion (in
PPV-NAT) of the 15 amino acids immediately downstream to
the DAG (Fig. 3) does not seem to affect resistance-breaking
ability, since both PPV isolates are able to mount a systemic
infection in Col-0. In addition, introducing this 15–amino acid
deletion in the N-terminal CP region of pICPPVPSes did not
alleviate its RTM-mediated restriction (data not shown). An-
other intriguing remark is that the N-terminal region upstream
of the core conserved domain of the CP is significantly differ-
ent between RTM-restricted PPV, LMV, and TEV isolates,
both in length and in amino acid sequence (Fig. 3). However,
all three viral proteins are presumably able to interact with the
RTM proteins and, in doing so, to determine the outcome of
the potyvirus infection. In consequence, except by postulating
a role for shared posttranslational modifications, it is rather
difficult to relate the CP-RTM interaction and specific amino
Several lines of evidence have suggested posttranslational
modifications in the CP N-terminal region of potyviruses, either
in the amino acid content, net charge, or phosphorylation or gly-
cosylation state, affecting the ability of a virus to spread in its
host plant (Chen et al. 2005; Fernández- Fernández et al. 2002;
Ivanov et al. 2003). We initially hypothesized that they could
affect not only the compatible interactions needed for systemic
movement but also interactions with the potentially competing
RTM system. Indeed, the CP of two potyviruses, Potato virus A
(Ivanov et al. 2001) and PPV (Fernández- Fernández et al.
2002), have been shown to be phosphorylated, whereas the PPV
CP is also modified by O-linked N-acetylglucosamination, pos-
sibly counteracting phosphorylation on Ser + Thr residues
(Lefebvre et al. 2003). O-GlcNAc modification of PPV CP takes
place in Arabidopsis and reduction of O-linked N-OGT activity
in the sec-2 mutants has been shown to partially reduce virus
spread and accumulation (Chen et al. 2005). The hypothesis that
CP O-GlcNAcylation contributes to either PPV movement,
RTM restriction, or both would be consistent with conclusions
obtained recently with pumpkin phloem proteins (Taoka et al.
2007), which showed that protein-protein interactions between
non–cell autonomous proteins (NCAP) and NCAP protein 1 and
their subsequent transport through plasmodesmata and phloem
are dependent on O–glycosylation or phosphorylation posttrans-
lational modifications. However, computer predictions did not
show a significant difference of total Thr and Ser phosphoryla-
tion or glycosylation residues between RTM-breaking and
RTM-restricted PPV isolates. Moreover, this parameter cannot
account for the behavior of the LMV RTM-breaking variants
since only one of them has a mutation affecting a Ser or Thr
residue (Fig. 3). The other two mutated residues (G and D) are
neither phosphorylated nor glycosylated. An alternative explana-
tion would be that O-glycosylation might be influenced by the
residues surrounding the modified amino acids and could be dis-
turbed by changes in these residues in the RTM-breaking PPV
isolates and the LMV-AFVAR variants. However, infection of
sec-2 and spy-1 mutants in a Col-0 background with RTM-
restricted LMV and PPV isolates (LMV-AF199, PPVPSes, and
PPV-EA) did not result in a breakdown of the RTM resistance,
clearly demonstrating that O-GlcNAc modification is not re-
quired for RTM restriction of LMV and PPV systemic infection
in Col-0 plants.
In the present study, we showed that the CP N-terminal
region is the key viral factor that determines the outcome of
the interaction of two potyviruses, PPV and LMV, in Arabi-
dopsis Col-0 plants. However, we were not able to reveal a
clear correlation between the CP N-terminal primary sequence
or its biophysical characteristics and the ability to break down
RTM-mediated resistance. Therefore, the challenge ahead will
be to understand how the RTM host proteins and the viral CP
interplay to restrict viral long-distance movement.
MATERIALS AND METHODS
All plants were grown under greenhouse conditions. Arabi-
dopsis thaliana Col-0 and Ler seeds were initially obtained
Vol. 22, No. 10, 2009 / 1309
from the Nottingham Arabidopsis Stock Centre (accessions
N1092 and N8581, respectively). Chemically induced rtm mu-
tants (Mahajan et al. 1998; Whitham et al. 1999) were pro-
vided by J. Carrington, Oregon State University (Corvallis, OR,
U.S.A.) and OGT mutants in Col-0 (sec-2 and spy-1) (Hartweck
et al. 2002), by N. Oszewski, University of Minnesota (Minne-
Virus isolates and plasmid construction.
PPV isolates were maintained in N. benthamiana plants.
LMV-AF199, described by Krause-Sakate and associates
(2002), was propagated in the lettuce cv. Trocadéro.
Isolates from three PPV strains were used in this study,
namely PPV-R and PPV-NAT, belonging to the D strain (Maiss
et al. 1989; Riechmann et al. 1990), PPV-PSes and PPV-SK68,
belonging to the M strain (Palkovics et al. 1993; Sáenz et al.
2001), and PPV-EA, which typifies the El-Amar strain (Wetzel
et al. 1991).
Construction of the PS/R and R/PS recombinant cDNA
clones (Fig. 2) was described previously (Sáenz et al. 2000).
Construction of pICPPVnk and pICPPVnk-GFP containing the
full-length nucleotide sequence of PPV-R coupled or not with
the GFP gene has also been described (Fernández-Fernández
et al. 2001). pICPPVPSes (Salvador 2007), P/R(GFP) 7677-
8576 and P/R(GFP) 7677-9020, derive from the same
pGPPVPS clone with A1395G and G1764A substitutions in
the HCPro coding sequence (pGPPVPSes) that has been previ-
ously described (Sáenz et al. 2001). P/R(GFP) 7677-8576 and
P/R(GFP) 7677-9020 contain the GFP reporter gene and the 3′
NIb coding sequence [P/R(GFP) 7677-8576] or the 3′ NIb and
5′ CP coding sequence [P/R(GFP) 7677-9020] of pICPPVnk-
GFP replacing the original PPV-PS sequence of pICPPVPSes
and pGPPVPSes, respectively (B. Salvador, P. Sáenz, J. B.
Quiot, C. Simón-Mateo and J. A. García, unpublished results).
In order to refine the position of the viral RTM-breaking deter-
minant, new chimeric viruses were constructed as follows.
R/P 8576-9020 and R/EA 8576-9020. pICPPVnk harbors a
NaeI-KpnI site between the NIb and CP sequences
(Fernández-Fernández et al. 2001), while all PPV isolates,
PPV-R, PPV-PS, PPV-SK68, and PPV-EA included, present a
unique SacI site about 455 bp downstream of the Ala (A)
codon starting the CP coding sequence.
The PPV-PS 5′ CP KpnI/SacI fragment was ligated into the
KpnI and SacI digested and linearized pICPPVnk vector. From
PPV-EA, the 5′ CP gene fragment was amplified by PCR with
the following primers: CP PPV-EA/KpnI sense (GATGGTACC
AATGTAGTTGTCCAT) and CP PPV-EA/SacI antisense (CT
GTGGAGCTCGCGTGTTCGAC). KpnI and SacI restriction
sites are underlined in the sense and antisense primers, respec-
tively. The PPV-EA 5′ CP gene PCR fragment was double-
digested with KpnI and SacI and was substituted for the KpnI-
SacI PPV-R fragment into the full-length pICPPVnk cDNA
infectious clone. The final viral clones were then referred to as
R/P 8576-9020 and R/EA 8576-9020, respectively.
P/R 8576-9020 and P/SK68 8576-9020. The P/R 8576-9020
and P/SK68 8576-9020 recombinants were engineered by PCR
in the pICPPVPSes cDNA infectious clone (Salvador 2007).
Beforehand, the strategy implied the creation, into the
pICPPVPSes clone, of a KpnI restriction site between the NIb
and CP sequences, similarly to the pICPPVnk construct. For
this purpose, we amplified first 1.2 kb of the NIb 3′ terminal
region from pICPPVPSes with the following primers:
NIb(#111)PPV-PSes sense (AAGGTTTTGAAGAATG) and
NIb(#940)PPV-PSes/KpnI antisense (GGTACCAGCCTGGTG
TATAACAATGTTGG). The KpnI restriction site is underlined.
The PPV-PSes sequence 487 bp downstream of the hybridiza-
tion site of the NIb(#111)PPV-PSes sense primer displays a
SalI restriction site. The PCR fragment was consequently double
digested with SalI and KpnI and was subcloned in a pCRII-
TOPO vector (Invitrogen, Carlsbad, CA, U.S.A.) upstream of a
KpnI-SacI PPV-R or PPV-SK68 5′ terminal CP gene fragment.
The PPV-R KpnI-SacI 5′ CP gene region was obtained by
double KpnI and SacI digestion of the pICPPVnk clone and
was subcloned in the recipient pCRII-TOPO vector. The PPV-
SK68 KpnI-SacI 5′ CP gene region was amplified by PCR
with the CP PPV-SK68/KpnI sense (CAGGCTGGTACCGAG
GAAGACGAT) and CP PPV-SK68/SacI antisense (TTGTGG
AGCTCGTGTGTTTGACAAG) primers. The fusion frag-
ments obtained between the PPV-PSes 3′ terminal NIb gene
region and the PPV-R or PPV-SK68 5′ terminal CP gene
region were substituted by triple digestion and ligation of SalI-
BglI, BglI-SacI, and SalI-SacI back into the full-length
pICPPVPSes cDNA infectious clone. Clones were then referred
to as P/R 8576-9020 and P/SK68 8576-9020, respectively.
Virus inoculation methods.
Arabidopsis rosette leaves were inoculated mechanically
with extracts of N. benthamiana infected with the various PPV
isolates as described by Decroocq and associates (2006). LMV
inoculation of Arabidopsis plants was as described by Revers
and associates (2003).
Transcripts were obtained for the five PS/R or R/PS recom-
binant constructs as well as for P/R(GFP) 7677-9020 using T7
RNA polymerase. They were then mechanically inoculated to
N. benthamiana before transfer to Arabidopsis plants.
Because the second set of PPV recombinant isolates was
constructed under the Cauliflower mosaic virus 35S promoter,
the infectivity of the newly obtained PPV constructs was as-
sessed first on N. benthamiana and, later, directly on Arabi-
dopsis Ler, Col-0, and rtm1, rtm2, and rtm3 plants by biolistic
inoculation. Direct particle bombardment inoculation was also
performed with the pICPPVnk, pICPPVPSes, PPVSK68,
pICPPVnk-GFP, P/R(GFP) 7677-8576 clones, using a hand-
held device (Bio-Rad, Hercules, CA, U.S.A.) and starting from
0.1 μg of infectious DNA per shooting.
To control the identity of the recombinant PPV clones, RT-
PCR was used to reamplify and sequence short fragments
spanning the recombination points as detailed in Supplemen-
tary Table S1.
Accumulation and spread of viral progenies originating
from inoculating pICPPVnk-GFP, P/R(GFP) 7677-8576, and
P/R(GFP) 7677-9020 clones in the inoculated and noninocu-
lated tissues of Arabidopsis plants was analyzed by monitoring
of GFP fluorescence following the procedure of Decroocq and
Virus accumulation in uninoculated inflorescence tissues
was assayed at 21 dpi by double-antibody sandwich-ELISA
using anti-PPV CP antibodies (M+D antibody, LCA Labora-
tory, Blanquefort, France) and, if needed, confirming by RT-
PCR. Viral RNA was extracted and subjected to RT-PCR as
described previously by Decroocq and associates (2006). LMV
detection in Arabidopsis inflorescence tissues was carried out
using ELISA as described by Revers and associates (2003).
Sequencing of the LMV variant genome.
Total RNA was extracted from LMV-infected Col-0 plants
using the SV total RNA isolation system (Promega, Madison,
WI, U.S.A.). First-strand cDNA synthesis was performed using
the Superscript II reverse transcriptase using the LD-polyT
primer (CACTGGCGGCCGCTCGAGCATGTACT30NN). Five
overlapping PCR fragments spanning the complete LMV ge-
nome were obtained using the Advantage 2 PCR kit (Clontech,
1310 / Molecular Plant-Microbe Interactions
Mountain View, CA, U.S.A.) and the following primer pairs:
0.1cP (AAAATAAAACAACCCAACACAACTC) and 0.2144M
(CATTTGGATTGCGTCTTAGGTG); 0.1870P (GGCACAAG
CATCGAACCATGT) and 0.4415M (G(G/A)CGAGTTGGTT
CGATGAGT); 0.4148P (GAACTTTACTCTTCTCGCGTG)
and 0.6702AM (TTTGG(G/A)ATCAGTGC(A/T)GGAGC);
0.8072M (CAGCAAGCAGAGTGTCGAG); 0.7852P (GGCT
GTTGGTGCACTATATAG) and LD-Prim (CACTGGCGGCC
GCTCGAGCATGTAC). Cycling parameters were 1 min at
95°C followed by 30 cycles (1 min at 95°C, 3 min at 68°C)
and a final step of 3 min at 68°C.
Automated DNA sequencing of the various uncloned PCR
products was performed by Cogenics (Meylan, France). The
sequence was compared with that of the published wild-type
LMV-AF199 (GenBank AJ278854; Krause-Sakate et al. 2002)
using ClustalW. Differences between the sequence of the vari-
ant and either the wild-type LMV-AF199 or other variants
were confirmed by direct sequencing of viral cDNAs obtained
as short PCR products spanning the regions of interest.
Biophysical parameters of the N-terminal CP region
of PPV and LMV isolates.
The predicted isoelectric point and the net charge were de-
termined for the 90 or 39 first amino acids of the CP of various
isolates or variants of PPV and LMV, respectively (Fig. 3),
using the ExPASy ProtParam tool.
We are grateful to L. Palkovics (Plant Pathology Department, Faculty of
Horticultural Science, Corvinus University, Budapest, Hungary) for the PPV-
SK68 cDNA infectious clone. We thank T. Mauduit and M. Roncoroni for
the production and maintenance of the Arabidopsis plants and J.-P.
Eyquard for technical assistance. Experiments were carried out in compli-
ance with the current French and European guidelines concerning quaran-
tine and recombinant pathogens. This work was supported in part by the
bilateral Hubert Curien France-Spain (PICASSO 11023ZA) and France-
Slovakia (STEFANIK 11130TM) funds. This work is currently funded by
the European Union through the FP7 Small Collaborative Project KBBE-
204429 (SHARCO acronym). B. Salvador and J. A. García were also sup-
ported by grant BIO2007-67283 from Spanish Ministerio de Ciencia e
Innovación. M. Glasa was supported by grant APVV-51-0402-07 from
Slovak Research and Development Agency.
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