The Plant Cell, Vol. 7, 549-559, May 1995 O 1995 American Society of Plant Physiologists
Long-Distance Movement Factor: A Transport Function of
the Potyvirus Helper Component Proteinase
Stephen Cronin, Jeanmarie Verchot, Ruth Haldeman-Cahill, Mary C. Schaad, and James C. Carrington’
Department of Biology, Texas A & M University, College Station, Texas 77843
Transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. Transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process
with requirements differing from those of cell-to-cell movement. Through genetic analysis of tobacco etch virus (TER
potyvirus group), a nove1 long-distance movement factor was identified that facilitates vascular-associated movement
in tobacco. A mutation in the central region of the helper component proteinase (HC-Pro), a TEV-encoded protein with
previously described activities in aphid-mediated transmission and polyprotein processing, inactivated long-distance
movement. This mutant virus exhibited only minor defects in genome amplification and cell-to-cell movement functions.
In situ histochemical analysis revealed that the mutant was capable of infecting mesophyll, bundle sheath, and phloem
cells within inoculated leaves, suggesting that the long-distance movement block was associated with entry into or exit
from sieve elements. The long-distance movement defect was specifically complemented by HC-Pro supplied in trans
by a transgenic host. The data indicate that HC-Pro functions in one or more steps unique to long-distance transport.
INTRODUCTION
Spread of plant viruses from sites of initial infection to dista1
sites can be conceptualized as a multistep process. First, initially infected cells must support viral replication to supply
infectious material for subsequent movement. Second, the virus or viral genome must move from cell to cell through
intercellular connections, the plasmodesmata, within the initially infected leaf. Third, the virus must move through severa1
vasculature-associatedcell types and enter the sieve elements,
where movement occurs passively over long distances within
the same leaf and between organs. Finally, the virus must exit
the sieve elements and reestablish replication and cell-to-cell
movement in tissues distant from the initial infection site.
Cell-to-cell transport requires the activities of movement proteins (MPs) encoded by viruses (Hull, 1991; Maule, 1991; Deom
et al., 1992). It has been proposed that the tobamovirus and
dianthovirus MPs bind directly to their respective genomes and
facilitate passage through plasmodesmata (Citovsky et al.,
1990, 1992; Fujiwara et al., 1993; Giesman-Cookmeyer and
Lommel, 1993; Waigmann et al., 1994). Severa1 viruses, including those in the potexvirus and hordeivirus groups, encode
multiple MPs, among which the required movement activities
are dispersed (Petty et al., 1990; Beck et al., 1991). For some
viruses, such as comoviruses, the MPs form intercellular tubules through which intact virions pass (van Lent et al., 1990;
Kasteel et al., 1993). The bipartite geminiviruses move between
cells through the coordinated activities of two MPs, one that
To whom correspondence should be addressed.
shuttles viral DNA from the nucleus to the cytoplasm and another that facilitates plasmodesmaltransit (Noueiry et al., 1994;
Pascal et al., 1994). The potyviruses, as exemplified by tobacco
etch virus (TEV), differ from each of the aforementionedmodels
in that proteins with dedicated movement functions have not
been identified. The P1 protein has been proposed extensively
in the literature to function as the potyviral MP, but deletions
and modifications of the P1 coding sequence have little direct
effect on movement (Verchot and Carrington, 1995a, 1995b).
On the other hand, the cell-to-cell movement functions have
been hypothesized as being performed by the capsid protein
based on the results of mutational analyses (Dolja et al.,
1994, 1995).
Recent evidence suggests that long-distance transport of
TEV involves viral functions distinct from those necessary for
cell-to-cellmovement. Different domains within the capsid protein (263 amino acid residues) have been shown to play specific
roles in cell-to-cell and long-distance transport (Dolja et al.,
1994, 1995). The core domain, which is required for assembly
of the flexuous rod-shaped virions, is absolutely required for
cell-to-cellmovement. In contrast, the N-terminal(29 residues)
and C-terminal(l8 residues) regions, which comprise surfaceoriented domains not required for assembly, are dispensable
for cell-to-celltransport but necessary for long-distance movement. Mutants that lack either terminal domain move from cell
to cell in primary infection foci but are incapable of vascularassociated movement. These data suggest that the TEV capsid protein interacts with unique cellular and/or viral factors
during cell-to-cell and long-distance transport.
The Plant Cell
550
Here, we identify the helper component proteinase(HC-Pro)
of TEV as a trans-actingfactor that plays a specific role in longdistance transport. Potyvirus HC-Pro is a multifunctional protein that has been characterized as a helper factor for
acquisition and transmission of virus by aphids (Figure 1;
reviewed by Riechmann et al., 1992; Shukla et al., 1994). The
helper component domain encompasses at least the N-terminal
and central regions, which also contain determinants involved
in genome amplification and pathogenicity (Thornbury et al.,
1990; Atreya et al., 1992; Atreya and Pirone, 1993; Dolja et al.,
1993; Klein et al., 1994). The C-terminal one-third of HC-Pro
comprises a cysteine-type proteinase domain that catalyzes
autoproteolytic cleavage between itself and the neighboring
P3 protein during maturation of the viral polyprotein (Carrington
et ai., 1989a, 1989b). A model in which HC-Pro provides specific movement functions associated with phloem transport of
virus is proposed.
A
1 kb
w
ur.
sp6
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GUS
B
Nonessential
for replic. or
movement
N
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6 NIa
NIb
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HC-Pro (459 aa)
Cys
'FRN' 'IGN' 'CCCE' 345
L
I
______
His
418
,
Proteinase domain
C
Aphid transmission
Figure 1. Diagrammatic Representation of Relevant Portions of the
TEV-GUS Genome and HC-Pro.
(A) Complementary DNA representing the TEV-GUS genome was
cloned downstream from a bacteriophage SP6 promoter, forming
pTEV7DAN-GLH.The 5'and S'noncoding sequences and polyprotein
coding region (openboxes) and GUS coding region (hatched box) are
shown. The vertical lines throughout the coding region indicate sequences coding for polyprotein cleavage sites. Proteins encoded by
the TEV-GUS genome are indicatedabove the map. Some of the known
or proposedfunctions of the viral proteins are as follows: P1, proteinase;
HC-Pro, helper component for aphid transmission and proteinase; P3,
function unknown; CI, ATPase/helicase;6, membrane binding protein;
Nla, genome-linkedprotein (VPg) and proteinase; Nlb, RNA-dependent
RNA polymerase; CP, capsid protein. Synthetic transcripts were produced using SP6 polymerase after linearization of the DNA with Bglll
(W.
(E) The HC-Proprotein is represented as a black bar. The proteinase
domain within HC-Pro is shown as a thick bar; indicated are the relative positions of Cys-345 and His-418 at the active site. The domain
necessary for helper activity during aphid-mediatedtransmission is
indicated near the N terminus. The positions altered by the FRN, IGN,
and CCCE mutations are also indicated. C, C terminus; N, N terminus; aa, amino acid; replic., replication.
RESULTS
Construction of TEV-GUS Mutants with Alterations
in HC-Pro
Three different mutations, termed FRN, IGN, and CCCE, were
introduced into the HC-Pro sequence of PTEV~DAN-GJH,
a
plasmid from which synthetic RNA transcripts representingthe
TEV-GUS genome are derived (Dolja et al., 1992; Carrington
et al., 1993; Figure 1). Each mutation resulted in substitution
of Arg-Pro-Ala for a tripeptide within the central region of HCPro. The tripeptides selected for substitution were among the
most highly conserved sequences within the central region.
The resulting viruses encoded the P-glucuronidase (GUS)
reporter protein between P1 and HC-Pro. Cleavage sites recognized by P1 and Nla proteinases were engineered at the
sequences encoding the N and C termini of GUS, respectively,
to ensure proteolytic remova1 of GUS from both P1 and HCPro (Dolja et ai., 1992; Carrington et al., 1993). The addition
of the GUS coding sequence to the viral genome was shown
to have relatively minor effects on the systemic infection capabilities of TEV (Dolja et al., 1992).
To determine the effects of the mutations on HC-Pro autoproteolytic function, the sequences encoding a 130-kD
GUSIHC-Prolpartial P3 polyprotein were excised from each
of the parental and mutant full-length plasmids and subcloned
into the vector pTL7SN.3 for in vitro transcription and translation. In addition to subclones containing the three mutations
in the central region of HC-Pro,a plasmid was constructed that
contained a substitution of a Ser codon for the active site His418 codon within the proteolytic domain (termed the H722S
mutation), a modification that inactivates HC-Pro processing
activity (Oh and Carrington, 1989). Transcripts from each plasmid were translated in a rabbit reticulocyte lysate system. The
polyprotein containing the wild-type HC-Pro sequence underwent autoproteolysis to yield a 120-kD GUSIHC-Pro product
(Figure 2A, lane l), whereas the H722S mutant 130-kD polyprotein was stable (lane 2). The polyproteins harboring the
CCCE, FRN, and IGN substitutions were processed to yield
4 2 0 - k D products that comigrated with the wild-type product
(Figure 2A, lanes 3 to 5), indicating that these mutations had
little or no effect on HC-Pro proteolytic activity.
Replication of the HC-Pro Mutants in Protoplasts
The effects of the FRN, CCCE, and IGN mutations on genome
amplification were measured. Full-lengthtranscripts representing the parental TEV-GUS and mutant genomes were used
to inoculate tobacco protoplasts, and GUS activity was assayed
at time intervals postinoculation (pi). GUS activity, which accumulates to significant levels only in cells supporting virus
replication, has proven to be an effective quantitative marker
for genome amplification (Carrington et al., 1993; Dolja et
al., 1994; Restrepo-Hartwig and Carrington, 1994; Li and
Virus Movement Function
A
130-kD
C
120-kD
C
GUS
-Pro Tl
HC-Pro
TP3
GUS
HC-Pro
XJ
P3
in
cs
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551
Carrington, 1995). The FRN mutant was amplified to levels
comparable to parental TEV-GUS at 48 hr pi, although a modest reduction was observed at 72 hr pi (Figure 3A). Cells
inoculated with the VNN mutant, which contains a replicationdebilitating mutation affecting the Nib polymerase (Carrington
et al., 1993; Li and Carrington, 1995), failed to accumulate
significant GUS activity. The CCCE and IGN mutants were amplified to levels ~25 and 1%, respectively, of that of the parental
virus at 72 hr pi (Figure 3B). Although IGN mutant-induced
GUS activity was low, it was significantly greater (P < 0.03)
than the minimal activity in VNN mutant-inoculated cells at
all time points tested. These results indicate that the FRN and
CCCE mutations had only modest effects on genome amplification, whereas the IGN mutation resulted in a more serious
amplification defect.
Systemic Infection by the HC-Pro Mutants
1
B
2
3
0027-1031
Transgen.
4
5
V
HC-Pro - — •*—
(52-kD)
Figure 2. Synthesis and Processing of HC-Pro in Vitro and in Transgenic Plants.
(A) Synthetic transcripts encoding a wild-type (wt) or mutagenized
GUS/HC-Pro/partial P3 polyprotein (diagram) were produced in vitro
and translated in a rabbit reticulocyte lysate in the presence of
^S-methionine. Radiolabeled proteins were detected after SDS-PAGE
and autoradiography (bottom), The HC-Pro mutations present in the
various transcripts are indicated above each lane. The molecular
masses (in kilodaltons) of the precursor polyprotein (130 kD) and
GUS/HC-Pro processed product (120 kD) are indicated at left.
(B) Transgenic (Transgen.; lanes 1 and 2) or TEV-infected (V; lane 3)
plants were subjected to immunoblot analysis using an anti-HC-Pro
serum. The immunoreaction was detected using a chemiluminescent
second antibody and autoradiography. Ten times more of the transgenic extracts were loaded compared with the virus-infected extract.
Tobacco plants were inoculated with transcripts of TEV-GUS
and mutant genomes, and systemic infection was assayed by
the appearance of symptoms and detection of GUS activity
at 12 days pi. Symptoms of parental TEV-GUS infection were
observed in most plants by 5 days pi (Figure 4A). Plants inoculated with transcripts from the FRN and IGN mutant genomes
exhibited mild symptoms, and these were delayed by at least
2 days, compared with the appearance of parental TEV-GUS
symptoms. No symptoms of infection were detected in CCCE
mutant-inoculated plants. GUS activity was detected in leaves
two positions above the inoculated leaves of plants infected
with parental TEV-GUS and the FRN and IGN mutants, although the levels of activity differed greatly among plants
inoculated with the different viruses (Figure 4B). Accumulation of the FRN and IGN mutants was approximately two and
four orders of magnitude, respectively, lower than that of parental TEV-GUS. The levels of GUS activity among individual
FRN and IGN mutant-infected plants were also quite variable.
Among plants inoculated with the FRN and IGN mutants, at
least one failed to exhibit GUS activity in upper leaves, even
though analysis of inoculated leaves indicated that infection
had occurred. The differences between TEV-GUS and the
FRN and IGN mutants were highly significant (P < 0.0001 in
all comparisons). Because these two mutants were debilitated
in plants to a greater degree than in protoplasts, they appeared
to possess slow or debilitated cell-to-cell and/or long-distance
movement phenotypes.
No GUS activity was detected in upper leaves of CCCE mutant-inoculated plants (Figure 4B), even at 16 days pi (data
not shown). All inoculated leaves had GUS activity, however,
indicating that this virus may have been confined to initial infection sites. Considering both the protoplast and whole-plant
results, the CCCE mutant appeared to display a significant
defect in systemic infectivity. Because of this striking phenotype, the movement characteristics of the CCCE mutant were
analyzed in greater detail.
552
The Plant Cell
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Figure 3. Amplification of Parental and Mutant TEV-GUS Genomes in Protoplasts.
GUS activity was measured in protoplasts inoculatedwith TEV-GUS or mutant transcripts at 24,48, and 72 hr pi. The data in (A) and (B) represent
the mean of three concomitant, replicate samples using the same preparation of protoplasts. The experiments in (A) and (B) were conducted
using different protoplast preparations.
(A) Parental TEV-GUS and FRN and VNN mutants.
(B) Parental TEV-GUS and CCCE, IGN, and VNN mutants.
Cell-to-Cell Movement of TEV-GUS and the CCCE
Mutant
The cell-to-cell movement rates of parental TEV-GUS and the
CCCE mutant were measured in inoculated leaves. lnfection
foci were visualized at 24, 48, 72, and 96 hr pi by infiltration
with the GUS histochemical substrate 5-bromo-4-chloro-3indolyl P-D-glucuronide (X-gluc). The diameters of foci were
determined microscopically and expressed in numbers of
epidermal cells. At 24 hr pi, the mean diameters of foci produced by TEV-GUS and the CCCE mutant were similar (P =
0.57), although many foci consisted of single cells in both cases
(Figure 5A). Cell-to-cell movement of the CCCE mutant continued at an average rate of 0.12 cells per hr, which was slightly
slower than the parental TEV-GUS rate of 0.23 cells per hr.
Measurements of TEV-GUS foci were not possible at 96 hr
pi due to extensive secondary movement through the vasculature. These data indicate that the CCCE mutant was capable
of cell-to-cell movement but at a reduced rate compared with
parental TEV-GUS.
Continued incubation of TEV-GUS-infected and CCCE mutant-infected plants to 8 days pi showed that parental virus
spread throughout the inoculated leaves by cell-to-cell and
vascular-associated movement. The CCCE mutant, however,
remained confined to initial infection foci (Figure 56). Culturing of the CCCE mutant-infected plants to 16 days pi revealed
that foci continued to expand via cell-to-cell movement but failed
to exhibit vascular-associatedmovement (data not shown). The
CCCE mutant, therefore, contained a major defect in systemic
or long-distance movement.
Long-distance movement through the vasculature first requires passage through a number of distinct cell types,
including mesophyll, bundle sheath, and phloem companion
cells. The inability to infect vasculature-associatedcells would
account for the CCCE mutant phenotype. The cell types infected by TEV-GUS and the CCCE mutant were examined in
sections of Historesinembedded leaf tissue treated with X-gluc.
Strong histochemical GUS reactions were detected in bundle
sheath and phloem cells of tissue infected by parental (Figures
6A and 66) and CCCE mutant (Figures 6C and 6D) viruses.
The reactions were particularly evident in the cytoplasm-dense
phloem cells, some of which were companion cells.'This staining appeared not to have resulted simply from the diffusion
of the X-gluc reaction product from adjacent infected cells because the intensityof the reaction was often high in the phloem
cells and low in neighboring bundle sheath cells. The pattern
of staining within virtually all positive cells was confined to the
cytoplasm, with little or no leakage of the X-gluc reaction product into the vacuole or the nucleus (Figure 6). These data
suggest that the CCCE mutant was capable of reaching phloem
cells of inoculated leaves, indicating that the long-distance
movement defect was associated with a point later in the movement pathway.
Complementation of the Long-Distance Movement
Defect of the CCCE Mutant in HC-Pro-Expressing
Transgenic Plants
Transgenic plants that expressed the 5' region of the TEV genome, including P1 and HC-Pro sequences, were produced.
Virus Movement Function
A protein with an electrophoretic mobility identical to that of
authentic HC-Pro was detected in transgenic plants using an
anti-HC-Pro serum (Figure 28), indicating accurate processing of the transgenic polyprotein. The bulk level of accumulation
of transgenic HC-Pro was -10% that of HC-Pro in TEVGUS-infected plants.
Protoplasts from transgenic and nontransgenic plants were
inoculated with full-length vira1 RNA transcripts. The relative
levels of GUS activity induced by TEV-GUS and the CCCE
mutant were the same in each protoplast preparation (data not
shown), indicatingthat HC-Prosupplied by the transgenic cells
had little effect on genome amplification of parental or mutant
viruses at the single-cell level.
Transgenic and nontransgenic plants were inoculated with
TEV-GUS and the CCCE mutant, and systemic infectivitywas
monitored by symptom production and GUS activity. As previously described, TEV-GUS induced systemic symptoms by
5 days pi in nontransgenic plants, whereas infection by the
CCCE mutant failed to result in symptoms. In contrast, both
TEV-GUS and the CCCE mutant induced systemic symptoms
in 1000/0 of transgenic plants, although symptoms caused by
the mutant were delayed by 2 to 3 days (Figure 7A). Indistinguishable levels of GUS activity were detected in transgenic
and nontransgenic plants infected by TEV-GUS. GUS activity
induced by the CCCE mutant was detected only in transgenic
plants (Figure 78) and at a level reduced considerably relative to that in TEV-GUS-infected plants. The same results
A
100 7
553
were obtained using each of two independent transgenic lines
(data not shown). Systemic infection by the CCCE mutant was
dueto complementation rather than to recombinationbetween
the mutant genome and the transgene because inoculation
of nontransgenic plants with the virus recovered from transgenic plants resulted in an infection phenotype identical to that
of the original CCCE virus derived from transcripts.
To determine the effect of transgenic HC-Pro on cell-to-cell
movement of the CCCE mutant in inoculated leaves, the in
situ histochemical assay was conducted at time intervals
through 72 hr pi. lnfection of transgenic plants with the CCCE
mutant resulted in only modest stimulation of cell-to-cell
movement (Figure 8A). Interestingly, cell-to-cell movement
of TEV-GUS was suppressed partially in the transgenic plants,
resulting in infection foci of sizes comparable to the CCCE
mutant at each time point.
Secondary, vascular-associated movement was analyzed
in situ in inoculated and noninoculated leaves of transgenic
and nontransgenic plants at 13 days pi. Extensive secondary
movement was detected in transgenic leaves inoculated with
the CCCE mutant, whereas only individual, primary infection
foci were visible in nontransgenic plants (Figure 88). Upper
noninoculatedleaves also exhibited GUS activity radiating from
primary veins (Figure 8C), clearly showing vascular-associated
movement to systemic tissues. These data indicate that HCPro supplied in frans complemented, at least partially, the longdistance movement defect of the CCCE mutant.
o TEV-GUS
O
o 1 2 3 4 5 6 7 8 9 1 0
Days Post-Inoculation
TEV- FRN IGNCCCE
GUS
Figure 4. Systemic lnfection of Plants by Parenta1 TEV-GUS and Mutants.
(A) Vira1 symptoms in upper leaves were scored at daily intervals after inoculation with parental TEV-GUS or mutants containing the FRN, IGN,
or CCCE mutations. The percentage of plants exhibiting symptoms is plotted.
(B) GUS activity was quantified in extracts from leaves two positions above the inoculated leaves at 12 days pi. Each bar represents the mean
from seven to 10 plants. Activity values from individual plants are indicated.
The Plant Cell
554
A
DISCUSSION
» TEV-GUS
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15 25 35 45 55 65 75 85 95 105
Hours Post-Inoculation
B
TEV-GUS
CCCE
(Inoculated Xanthi leaves)
Figure 5. Cell-to-Cell Movement of Parental TEV-GUS and the CCCE
Mutant in Inoculated Leaves.
(A) Diameters of infection foci, expressed in numbers of epidermal
cells, in inoculated tobacco leaves were measured microscopically at
daily intervals postinoculation. Each point represents the mean from
at least 39 foci.
(B) Infection of inoculated leaves by parental TEV-GUS or the CCCE
mutant was visualized by infiltration with X-gluc at 8 days pi. Note that
parental virus spread throughout the leaf, whereas the CCCE mutant
was confined to individual infection foci.
Previous investigations revealed that cell-to-cell movement and
long-distance transport of TEV are distinct processes with
different requirements (Dolja et al., 1994,1995). In fact, it has
been suggested that these two modes of movement are unique
processes in other viral systems as well (e.g., Dawson, 1992;
Taliansky and Garcia-Arenal, 1995). Here, we demonstrated
that TEV encodes a nonstructural protein, HC-Pro, which provides a specific frans-active function in vascular-associated
transport.
The collective analyses of three mutants indicated that the
HC-Pro long-distance movement activity is distinct from the
role HC-Pro may play in genome amplification (Table 1). First,
the long-distance movement-defective CCCE mutant could
amplify in protoplasts to 25% of the level of parental TEV-GUS.
Based on results with other TEV-GUS mutants, such as those
with defects in the P1 protein (Verchot and Carrington, 1995b),
this level of replication within single cells is clearly not debilitating to cell-to-cell or long-distance movement. Second, the IGN
mutant encoded a debilitated HC-Pro that resulted in drastically reduced genome amplification levels (<1% compared
with parental virus), yet this mutant was capable of limited
long-distance movement. The limited systemic infection capability of the IGN mutant may have resulted from decreases in
both genome amplification and long-distance movement rates,
although it could have been due solely to indirect effects of
limited amplification. Because the defect produced by the
CCCE mutation had only a minor effect on genome amplification but a completely debilitating effect on long-distance
movement (Table 1), these two functions are genetically separable. Finally, the fact that HC-Pro supplied in transgenic cells
could restore long-distance movement activity to the CCCE
mutant but could not stimulate genome amplification of the
CCCE mutant at the single-cell level in protoplasts implies
that the movement and replication functions of HC-Pro are
distinct.
Whereas the CCCE mutant was complemented in HCPro-expressing transgenic plants, it was not restored to the
level of the parental virus. This could be due to one or more
reasons. Immunoblot analysis indicated that the transgenic
plants accumulated HC-Pro to levels 10% of those detected
in TEV-GUS —infected plants. The relatively low level of functional HC-Pro may have had a quantitative effect on the
long-distance movement function. Alternatively, the CCCE mutant HC-Pro accumulating in infected cells may have been
frans-inhibitory to the complementing activity of the transgenic
protein. If the movement function of HC-Pro requires multiple
domains or binding sites and the CCCE mutation affects only
one of these putative domains, then the mutant protein may
have partially interfered with the movement process. Such
frans-dominant interference was proposed to explain the lack
of transgenic complementation of long-distance movement of
a TEV mutant lacking the C-terminal domain of capsid protein
(Dolja et al., 1995).
Virus Movement Function
A
555
B
V
,
.V;..
Figure 6. In Situ Histochemical Analysis of Cell Types Infected by Parental TEV-GUS and the CCCE Mutant.
Inoculated leaves were infiltrated with X-gluc at 7 days pi. Tissue near the edge of an infection focus was embedded in Historesin, sectioned
(6 urn), and viewed by phase-contrast light microscopy. Cross-sections of minor veins are present near the center of (A), (B), (C), and (D). The
cells staining most intensely are phloem companion or phloem parenchyma cells. The same magnification was used for each photomicrograph.
(A) Parental TEV-GUS-infected leaf.
(B) Parental TEV-GUS-infected leaf.
(C) CCCE mutant-infected leaf.
(D) CCCE mutant-infected leaf. Bar = 30 nm.
How might HC-Pro participate specifically in vascularassociated long-distance movement? It is proposed that longdistance movement of TEV involves an interaction between
HC-Pro and capsid protein (or virions). If this were the case,
similar movement phenotypes would be predicted if mutations
were introduced into either HC-Pro or the capsid protein. Indeed, the CCCE HC-Pro mutant and the mutants lacking the
terminal domains of the capsid protein each have the same
phenotype: they can amplify and move from cell to cell, but
they fail to move long distances. Furthermore, mutants that
lack the ability to be transmitted by aphids may possess defects
in either the N-terminal or central region of HC-Pro or the N
terminus of capsid protein (Atreya et al., 1990, 1991, 1992;
Thornbury et al., 1990; Atreya and Pirone, 1993; Dolja et al.,
1993). Thus, two different processes have similar requirements
for both HC-Pro and the capsid protein. However, the aphid
transmission-defective mutants referenced previously still possess long-distance movement capability. These data suggest
556
The Plant Cell
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A
O
2
4
6
8
1 0 1 2
Days Post-Inoculation
TEV- mv- ccc
GUS GUS
ccc
Xan. Tran. Xan.Tran.
Figure 7. Complementation of the CCCE Mutant in Transgenic Plants.
Nontransgenic (Xanthior Xan.)or HC-Pro-expressingtransgenic (Transgen.or Tran.) plants were inoculatedwith parenta1TEV-GUS or the CCCE
(CCC) mutant.
(A) Vira1 symptoms in upper leaves were scored at daily intervals after inoculation.The percentage of plants exhibiting symptoms is plotted.
(e) GUS activity was quantified in extracts from leaves two positions above the inoculated leaves 12 days pi. Each bar represem the mean
from five to seven plants. Activity values from individual plants are indicated.
that HC-Pro and the capsid protein interact to form active complexes that facilitate insect transmission and long-distance
movement but that the nature of the interaction required for
each process may differ.
It is further proposed that the putative HC-Pro-capsid protein interaction provides a unique function in movement through
the companion celllsieve element boundary. The histochemical analysis indicated that the CCCE mutant could reach at
least as far as the phloem companion cells of inoculated leaves.
Therefore, the mutant may have been blocked at the point of
entry into sieve elements or at the point of exit from sieve elements back into companion cells at sites dista1 to the-initial
infection. The idea that transport across the companion celll
sieve element boundary requires a set of unique movement
functions is attractive because evidence suggests that plasmodesmata at this junction are quite different from plasmodesmata
between mesophyll cells (Kempers et al., 1993). The plasmodesmata between mesophyll cells and between phloem
cells were shown, in dye injection experiments, to differ functionally (Ding et al., 1992). In addition, the density of companion
celllsieve element plasmodesmata per unit area is relatively
low in tobacco (Van Bel and Gamalei, 1992). HC-Pro mayfacilitate transport through these specialized plasmodesmata. We
do not know whether HC-Pro also enters the sieve element
along with the virion or transport complex. Entry of HC-Pro
into sieve elements is clearly plausible, given that MPs from
severa1 diverse virus groups are able to move themselves
through plasmodesmata (Fujiwara et al., 1993; Noueiry et al.,
1994; Waigmann et al., 1994).
METHODS
Strains and Plants
A modified form of the highly aphid-transmissible strain of tobacco
etch virus (TEV), the genome of which is represented as cDNA in
pTEV7DA (Dolja et al., 1992), was used in this study. Nicotiana tabacum cv Xanthi-nc was used as the host in whole-plant experiments
and as the source of protoplasts. All cloning of DNA was conducted
in fscherichia coli HBlO1.
Two independenttransgenic tobacco lines, termed 0027-1031-19x8
and 0027-1031-1XB,that expressed the P1-helpercomponent proteinase
(HC-Pro)region of the TEV genome were produced using the leaf disc
method (Horsch et al., 1985; Carrington and Freed, 1990). Progeny
from the RI or R2 generationswere analyzed for expression of HC-Pro
by immunoblot analysis using an anti-HC-Pro serum (Carrington et
al., 1990) and a chemiluminescence detection system (Amersham).
Site-Directed Mutagenesis of the HC-Pro Sequence
Three substitution mutations(FRN, IGN, and CCCE)were introduced
into the HC-Pro coding sequence within the plasmid pTL7SN-
Virus Movement Function
0823GJH, which contains cDNA corresponding to TEV nucleotides
849 to 2332 of the TEV-GUS genome between two Xbal sites. This
plasmid also contains the 3-glucuronidase (GUS) coding sequence
between the P1 and HC-Pro regions as well as codons for an Nla proteinase cleavage site between GUS and HC-Pro (Carrington et al.,
1993). For each mutant, three codons were replaced by the sequence
AGGCCTGCT, which contains a Stul site and codes for the tripeptide
Arg-Pro-Ala. The mutations affected Phe-182-Arg-183-Asn-184 (FRN),
lle-251-Gly-252-Asn-253 (IGN), and Cys-293-Cys-294-Cys-295
(CCCE). The CCCE mutant also contained a substitution of Asp for
» TEV-GUSXan.
o TEV-GUS Transgen.
• CCCEXan.
V CCCE Transgen.
E =
a _ 1073 8'a.
u
d
c
557
6-
Glu-299. Mutagenesis was conducted by the oligonucleotide-directed
4-
15
25
35
45
55
65
Hours Post-Inoculation
75
method (Kunkel et al., 1987) and verified by nucleotide sequence
analysis.
The mutations were introduced into a GUS-containing, full-length
cDNA of the TEV genome by transfer of the Xbal-Xbal fragment from
pTL7SN-0823GiH into pTEV7DAN. The resulting plasmidswere named
pTEV7DAN-GiH/FRN, pTEV7DAN-GiH/IGN, and DTEV7DAN-GIH/
CCCE. These plasmids contain a bacteriophage SP6 promoter adjacent to the TEV 5' sequence and a Bglll site adjacent to the 3' poly(A)
region.
The region encoding GUS, mutagenized HC-Pro, and a 10-kD segment of the P3 protein was subcloned from the full-length plasmids
described previously into the vector pTL7SN.3 (Carrington and Freed,
1990). These plasmidswere termed pTL7SN-1027GIH/FRN, pTL7SN1027GiH/IGN, and DTL7SN-1027G1H/CCCE.
In Vitro Transcription and Translation
Xanthi
Transgenic
CCCE-Inoculated Leaves
TEV-GUS
CCCE
Systemically Infected Transgenic Leaves
Full-length TEV transcripts were generated using SP6 RNA polymerase and Bglll-linearized DMA as described previously (Dolja et al., 1992).
These transcripts were used for inoculation of plants and protoplasts.
Transcripts were also produced from the pTL7SN-1027GiH-based
plasmids using SP6 RNA polymerase. These transcripts were translated in a rabbit reticulocyte lysate (Promega) in the presence of
35
S-methionine (Du Pont-New England Nuclear) for 1 hr. The radiolabeled products were subjected to electrophoresis through an
SDS-10% polyacrylamide gel and detected by autoradiography.
Table 1. Properties of the FRN, IGN, and CCCE Mutants in
Nontransformed Plants and Protoplasts
Relative
Genome
Amplification3
Figure 8. Movement of Parental TEV-GUS and the CCCE Mutant in
Inoculated Systemically Infected Leaves of Transgenic Plants.
(A) Diameters of infection foci, expressed in numbers of epidermal
cells, in inoculated nontransgenic (Xanthi [Xan.]) or HC-Prc-expressing
transgenic (Transgen.) tobacco leaves were measured microscopically
at daily intervals postinoculation. Each point represents the mean from
at least 10 foci.
(B) Infection of inoculated leaves of nontransgenic (Xanthi) and HCPro-expressing transgenic leaves by the CCCE mutant was visualized by infiltration with X-gluc at 13 days pi. Note that the secondary
movement defect of the CCCE mutant was rescued in the transgenic
plants.
(C) Leaves Systemically infected by parental TEV-GUS and the CCCE
mutant in HC-Pro-expressing transgenic plants are shown. Sites of
infection were visualized as given in (B).
Virus
(O/o)
TEV-GUS
FRN mutant
IGN mutant
CCCE mutant
100
62
0.6
25
a
Relative Systemic
GUS Activity"
(%)
Cell-to-Cell
Movement
Rate
100
0.23 cell/hr
NDC
NDC
0.1 2 cell/hr
1.6
0.04
0.00
Based on GUS activity in protoplasts at 72 hr pi as shown in Figure
3, using the TEV-GUS value as the 100% standard.
b
Based on GUS activity in upper, noninoculated leaves at 12 days
pi as shown in Figure 4, using the TEV-GUS value as the 100%
standard.
c
Not determined.
558
The Plant Cell
lnoculation of Plants and Protoplasts
Carborundum-dustedplants were inoculated mechanicallyusing RNA
transcripts ( 4 0 wg) or inoculum prepared by grinding 1 g of virusinfected leaves in 2 mL of TE buffer (10 mM Tris-HCI, 1 mM EDTA,
pH 7.6).
Protoplastswere prepared from greenhouse-grown plants and inoculated (7.5 x 105cells) with SP6 transcripts using the polyethylene
glycol method described previously (Negrutiu et al., 1987; Carrington
and Freed, 1990). lnoculations were conducted in triplicate, using a
single preparationof protoplastsfor each experiment. Transcripts derived from pTEV7DAN-GJH (the plasmid yielding TEV-GUS) and
pTEV7DAN-G.1HlVNN served as the positive and negative amplification controls, respectively.The latter contained a mutation within the
Nlb polymerase sequence, resulting in an amplification-defective
phenotype (Carrington et al., 1993; Li and Carrington, 1995).
GUS Activity Assays
Protoplasts (2.5 x 105)were harvestedat 24, 48, and 72 hr postinoculation (pi), frozen at -80°C, and resuspended in GUS lysis buffer (40
mM sodium phosphate, 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium
lauryl sarcosine,0.07°/o p-mercaptoethanol,pH 7.0), and GUS activity
was measured using the fluorometric substrate 4-methylumbelliferyl
glucuronide (Jefferson, 1987). GUS activity was also measured using
the fluorometric assay in extracts from virus-infected leaves. Leaf tissue was ground in 5 volumes of GUS lysis buffer and clarified by
centrifugation at 13,OOOg for 10 min.
GUS activity was visualized in virus-infected leaves after vacuum
infiltration of 5-bromo-4-chloro-3-indolyl p-D-glucuronide (X-gluc), a
colorimetric GUS substrate(Dolja et al., 1992). In some experiments,
infectionfocus diameter (expressedin number of epidermal cells) was
measured (Dolja et al., 1994). Statisticalanalysis of data was performed
using the StatView4.0 program(Abacus Concepts, Inc., Berkeley,CA).
Histochemistry and Microscopy
lnoculatedleaves of nontransgenictobacco plantswere infiltratedwith
X-gluc at 7 days pi. Tissue near the edge of an infection focus was
excisedwith a razor blade, fixed in 50 mM Pipes, pH 7.4,4% paraformaldehyde(Electron MicroscopySciences, Fort Washington, PA), 05%
glutaraldehyde(Sigma),dehydrated through a graded acetone series,
and embedded in Historesin(Reichert-Jung, Heidelberg, Germany).
Sections (6-bm) were cut with glass knives using an Ultracut microtome and mountedin Aqua-polymount(Polysciences, Inc., Warrington,
PA) on glass slides. Sections were viewed with an Olympus 6x50
microscope (Tokyo, Japan) using phase-contrast optics.
Agriculture(No. 91-373036435),the NationalScience Foundation(No.
lBN-9158559),andthe National lnstitutesof Health (No. Al27832). M.C.S.
was supported by a National Research Service Award from the National lnstitutes of Health (No. A109121).
Received January 23, 1995; accepted March 16, 1995.
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Plant Cell 1995;7;549-559
DOI 10.1105/tpc.7.5.549
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