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336
Viral invasion and host defense: strategies and
counter-strategies
James C Carrington∗ and Steven A Whitham
The outcome of infection of plants by viruses is determined
by the net effects of compatibility functions and defense
responses. Recent advances reveal that viruses have the
capacity to modulate host compatibility and defense functions
by a variety of mechanisms.
Addresses
Institute of Biological Chemistry, Washington State University,
Pullman, WA 99164-6340, USA
∗e-mail: carrington@wsu.edu
Current Opinion in Plant Biology 1998, 1:336–341
http://biomednet.com/elecref/1369526600100336
Current Biology Ltd ISSN 1369-5266
Abbreviations
BMV
brome mosaic virus
CaMV
cauliflower mosaic virus
ER
endoplasmic reticulum
HC-Pro helper component-proteinase
HR
hypersensitive response
mab
maintenance of BMV functions
MP
movement protein
PSBMV pea seedborne mosaic potyvirus
PVX
potato virus X
TBRV
tomato black ring nepovirus
TEV
tobacco etch virus
TMV
tobacco mosaic virus
VIGS
viral induced gene silencing
Introduction
When discussing susceptibility or resistance of plants to
viruses, it is convenient to consider ‘susceptibility factors’
and ‘resistance factors’ as separate entities governing
infection. It can be argued, however, that the distinction
between susceptibility and resistance to viruses may not
be so straightforward. We are particularly intrigued by
the possibility that viruses can be active promoters of
susceptibility or suppressors of defense. In this review,
we will consider selected aspects of susceptibility, such
as virus–host interactions that govern virus genome
replication and intercellular movement, and a limited
discussion of defense responses. We will also summarize
recent findings, as well as our views, on how viruses
may modulate host functions to ensure their own efficient
replication and movement.
Virus–host interactions: susceptibility factors
A plant host is considered fully susceptible to a given virus
if the virus can successfully complete three processes:
genome replication, cell-to-cell movement (local) and
long-distance (vasculature-dependent) movement [1]. For
most RNA-genome viruses, RNA replication is catalyzed
by a core set of virus-encoded enzymes with RNA-de-
pendent RNA polymerase activity and other associated
activities (e.g. helicase, capping, priming) in close association with cytoplasmic surfaces of membranes [2]. The
docking of replication complexes to membranes appears
to be a point of intimate interaction between virus and
host cell. In several cases, complexes associate specifically
with endoplasmic reticulum (ER)-derived membranes
or vesicles through specific membrane-binding functions
of replication proteins [3,4]. The basis for membrane
specificity, however, remains obscure. The participation of
cellular factors, such as host proteins or protein complexes,
has been a point of speculation for many years, with
only limited direct evidence to support functional roles
of host factors [2]. This may change in the near future
through the clever use of yeast as an adopted host for
brome mosaic virus (BMV) to analyze RNA replication;
Paul Ahlquist and colleagues have developed selectable
and counter-selectable BMV RNA-based replicons for
yeast [5•,6,7]. For example, BMV replicons expressing
URA3 allow ura3 yeast to grow in the absence of uracil
and thus provide a positive selection for cells with
active BMV replication. Conversely, URA3 confers a lethal
phenotype in the presence of 5-fluoroortic acid, and thus
provides a selection for yeast cells that lose the ability
to support BMV replication. The characteristics of BMV
replicon amplification are remarkably similar in yeast
cells and plant cells, providing a rationale for applying
yeast genetics to identify host factors that influence
replication. The MAB1, MAB2 and MAB3 (Maintenance of
BMV functions) loci contribute distinct functions to BMV
genomic RNA replication, subgenomic RNA synthesis
and accumulation of BMV-encoded replication proteins
[8••]. Recessive mutations at each locus condition a
temperature-sensitive yeast growth phenotype, suggesting
that the wild-type gene products are necessary for the
normal cellular function. Dissection of the roles of the
MAB gene products in viral RNA replication will represent
a significant milestone in understanding compatibility
during virus–host interactions.
DNA-containing viruses, such as the geminiviruses, replicate in the host plant nucleus where the cellular DNA
replication apparatus is employed. The geminiviruses
use host DNA polymerase and associated factors in
conjunction with a virus-encoded replication protein
(termed RepA or AL1). The RepA/AL1 protein provides
several key activities that integrate viral and host functions
controlling early gene expression, late gene expression and
DNA replication [9,10]. This protein binds to the stem
structure at the replication origin and nicks the DNA prior
to initiation of rolling-circle DNA replication [9,11–13].
Like many animal-infecting DNA viruses that use the
Viral invasion and host defense: strategies and counter-strategies Carrington and Whitham
cellular DNA replication apparatus, the geminiviruses face
a serious problem; they infect terminally differentiated,
resting state (G0) cells, but these cells lack factors required
for DNA replication ([14], and references therein). As
detailed below, geminiviruses likely solve this problem
through modulating factors that normally control the cell
cycle.
Virus-encoded susceptibility factors controlling cell-tocell and long-distance movement are called ‘movement
proteins’ (MPs) and have been reviewed extensively
in recent years [1,15,16]. Movement proteins facilitate
movement through interactions with the viral genome or
assembled virus particle, intracellular transport pathways
and structures, and plasmodesmata. For geminiviruses,
two MPs are required for cell-to-cell movement. One
(BR1) is necessary to transport viral ssDNA genomes
between the nucleus and the cytoplasm [17,18]. The
BR1 protein, therefore, is a nuclear shuttle protein with
nuclear import and export signals. The other (BL1)
interacts with ER-derived membranes and plasmodesmata
to facilitate transit of viral DNA to adjacent plant cells,
possibly by modifying the plasmodesmata structure and
transport properties [19,20]. Tobacco mosaic virus (TMV)
MP interacts with viral RNA and modifies plasmodesmata
[21–23]. The TMV MP was shown to interact with
microtubules in infected or transfected cells [24,25], and it
was proposed that this might enable intracellular transport
of movement complexes from sites of genome replication
to plasmodesmata. Recent data, however, suggest that
the MP–microtubule association may occur relatively late
in the infection process, possibly as part of a pathway
to dissipate high local concentrations or to degrade MP
[26]. Early during the infection process, TMV MP, like
the geminivirus BL1 [19], associates with ER-derived
membranes and plasmodesmata [26]. Might the association
between the ER and diverse MPs from different viruses
be telling us something important? Recall that plasmodesmata contain an extension of the ER, the desmotubule,
which is threaded through the central channel [16,27].
Quite possibly, transport complex movement through
plasmodesmata requires specific interactions first with
cortical ER and then with the desmotubule component in
the channel.
Induction of host compatibility factors by
viruses
Do viruses have the ability to promote their own infections
by stimulation of host susceptibility factors or structures
needed for genome replication, genome expression or
movement? Given that the number of cellular factors
shown to play definitive roles in promoting virus infection
is small, there are relatively few examples to ponder. The
geminiviruses, however, provide an excellent case to argue
that viruses have the potential to modulate susceptibility.
As mentioned above, geminiviruses are dependent on
the host DNA replication apparatus for replication, and,
337
therefore, must overcome the lack of DNA replication
factors in G0 cells. Two significant breakthroughs have
shed light on possible mechanisms whereby geminiviruses
solve this problem. First, geminiviruses induce expression
of at least some of the components necessary for DNA
synthesis upon infection. Proliferating cell nuclear antigen
(PCNA), a DNA polymerase accessory factor normally
found only in S-phase cells, is induced in quiescent
mesophyll cells by infection with tomato golden mosaic
geminivirus (TGMV) [14]. Using AL1 transgenic plants,
induction of PCNA was shown to require only the TGMV
AL1 gene. Second, the AL1/RepA protein physically
interacts with host-encoded retinoblastoma-like tumor
suppressor proteins (pRbs) [28••,29–31]. Wheat dwarf
geminivirus mutants with substitutions affecting the probable pRb-like protein binding site of Rep exhibit defects
in viral DNA synthesis [29], supporting the concept
that this interaction is necessary. The well-characterized
mammalian pRB protein serves as key G1 checkpoint
regulator, preventing completion of G1 and entry into
S-phase [32]. Phosphorylation by cyclin-dependent kinases relieves the cell cycle-inhibitory activity of pRb and
allows progression into S-phase [32]. Plants, like mammals,
likely use one or more pRb-like proteins as checkpoint
inhibitors of the cell cycle. It is reasonable to suggest,
therefore, that geminivirus Rep protein interacts with,
and inactivates (or at least diverts), pRb-like protein in
infected cells. This would relieve the transcriptional block
to production of S-phase-specific mRNAs and provide
a pool of enzymes and factors necessary for viral DNA
replication. Thus, plant geminiviruses join the ranks
of animal DNA tumor-inducing viruses, such as SV40
and adenovirus, which encode proteins that affect the
cell cycling apparatus through interactions with tumor
suppressor proteins [33].
A novel defense response against viruses
Defense responses involving dominant resistance (R)
genes, hypersensitive cell death and induction of systemic
acquired resistance have been reviewed nicely by others
[34•,35•,36,37] and will not be covered here. Rather, we
will focus on a novel defense response — post-transcriptional gene silencing — that has only recently been linked
to natural forms of virus resistance.
Post-transcriptional gene silencing is a remarkable process that has been reviewed extensively [38–41]. The
hallmarks of post-transcriptional silencing of nuclear-derived sequences include, among other features, high
transcription levels of the silenced gene but relatively low
steady-state cytoplasmic levels of the transcript. Silencing
occurs through a homology-dependent mechanism and
functions in trans to suppress identical or closely related
sequences. Evidence suggests that silencing is due
to activation of a sequence-specific RNA degradation
apparatus, perhaps by a mechanism involving synthesis of
complementary nucleic acids. Silencing of a nuclear-derived sequence can be triggered by either nuclear genes
338
Plant–microbe interactions
or by cytoplasmic, replicating nucleic acids containing
homologous sequences. Furthermore, a silenced state in
a plant can be generated systemically through transport of
a mobile signal [42••,43••].
Whereas it has been known for some time that viral
sequences can induce silencing of an endogenous nuclear
gene or a transgene [44,45], it was only recently demonstrated that viruses can trigger silencing in the apparent
absence of homologous nuclear sequences. Infection
of Nicotiana clevelandii by tomato blackring nepovirus
(TBRV) strain W22 results in an initial symptomatic phase
in which the virus moves systemically, followed by a recovery state in which new tissue developing post-inoculation
is asymptomatic and largely devoid of virus [46••]. Further,
silencing of the viral sequence is active in the recovered
tissue, conferring effective resistance against W22 and a
closely related strain. Prolonged infection of Brassica napus
by cauliflower mosaic virus (CaMV), a pararetrovirus, also
induces post-transcriptional gene silencing independent of
homologous transgenes [47••]. These findings are highly
significant as they indicate that homology-dependent gene
silencing may be a general antiviral response. The logic
for such a response is impeccable: extreme resistance can
be elicited after infection by a broad range of infecting
viruses using a general, adaptive silencing apparatus. An
important unresolved issue, however, is the extent to
which viruses, other than those mentioned, elicit silencing
upon infection. Most compatible virus–host combinations
do not show the recovery phenotypes as do TBRV and
CaMV. It is possible, however, that silencing is triggered
transiently to limit the extent of virus replication in
infected tissue, thus lessening the extent of disease,
but not in time to limit spread systemically. Silencing
as a general antivirus response in both compatible and
incompatible virus–host combinations deserves careful
attention.
Do viruses fight back?
If one examines the way animal viruses deal with innate
and adaptive immune system defenses, it becomes clear
that these agents are masters of the counter-defensive
strategy. For example, poxviruses encode several types
of cytokine receptor mimic proteins [48]. These attenuate the inflammatory and immune responses normally
triggered by virus-infected cells. Also, numerous animal
viruses encode inhibitors of apoptosis proteins (IAPs),
preventing normal immune signaling that occurs through
activation of cell death pathways [49].
Do plant viruses deploy counter-defensive strategies?
Recent findings offer suggestions that certain plant viruses
are able to interdict defense mechanisms or alter host
cell metabolism for their own benefit. In a series of
elegant experiments examining host cell transcript and
protein levels in situ in tissue across an advancing
infection front, Andy Maule and colleagues demonstrated
that pea seedborne mosaic potyvirus (PSBMV) induces
transient depletion of many, but not all, classes of host
cell mRNAs in pea embryos [50,51]. The host mRNA
modulation occurs specifically in cells supporting virus
replication, and exhibits some of the characteristics of the
heat shock response, such as induction of HSP70 and
polyubiquitin mRNA expression. Although the bases for
suppression and subsequent recovery of transcript levels
are not clear, the consequences of inhibition of host cell
mRNA accumulation on induction of defense responses
are potentially far-reaching. As most types of known
induced defense responses require host gene expression
[34•], inhibition of mRNA accumulation or translation may
severely limit the extent or timing of a defense program.
Additional lines of evidence suggest that potyviruses can
modulate host defense responses, and that modulation
can condition enhanced accumulation and movement of
potyviruses as well as heterologous viruses. Typically,
in single-infections by a non-potyvirus, virus genome
amplification at the single-cell level shuts off after an
exponential amplification phase within 12–24 hours of
inoculation [52]. Tobacco etch virus (TEV), in contrast,
accumulates at a slower initial rate but for considerably
longer periods of time (e.g., 72 hours post-inoculation;
[53]). TEV mutants with defects in helper componentproteinase (HC-Pro) exhibit a premature amplification
shut-off phenotype by 24 hours post-inoculation and
are restricted in long-distance movement [54•]. These
observations suggest that wild-type HC-Pro may function
to suppress one or more induced defense responses.
Significantly, when heterologous viruses are co-inoculated
with TEV or other potyviruses, or when heterologous
viruses infect plants or cells in the presence of functional
HC-Pro, the heterologous viruses exhibit an enhanced
genome accumulation phenotype [55,56,57•]. In the case
of infections of potato virus X (PVX) expressing HC-Pro
or an HC-Pro polyprotein precursor, the enhancement
of PVX is actually the result of continued amplification
beyond the point at which amplification normally shuts off
[57•]. A plausible explanation for all of these results is that
potyviruses, through a key activity of HC-Pro, suppress
induction of host defense responses that would normally
result in early amplification shut-off and restricted accumulation (Figure 1). The antiviral defense responses
could have broad-spectrum activity against a range of
diverse viruses, or could be induced to have specificity
against many different viruses. The mechanistic details
of this system are not yet resolved, although recently
obtained evidence is consistent with HC-Pro mediating a
suppression of homology-dependent gene silencing (KD
Kasschau and JC Carrington, unpublished data).
Finally, the potential influence of viruses on signal
transduction pathways controlling a wide range of cellular
activities is just now being realized. Some of the best
ongoing work in this area deals with the virulence-attenuating hypovirus of the chestnut blight fungus, Cryphonectria
parasitica. Donald Nuss and co-workers have shown that
Viral invasion and host defense: strategies and counter-strategies Carrington and Whitham
339
Figure 1
HC-Pro
ER
Local VIGS or
other induced
defense responses
Accumulation of
viral RNAs
Viral RNA
replication complex
Amplification shut-off
or viral RNA degradation
Systemic acquired
silencing or other
systemic responses?
Current Opinion in Plant Biology
Modulation of antiviral defense responses by potyvirus TEV HC-Pro. The effects of HC-Pro on accumulation and movement of TEV and
heterologous viruses [54•,55,56,57•] suggest that HC-Pro protein may suppress local and systemic defense responses. The defense responses
affected by HC-Pro may include virus-induced gene silencing (VIGS), induced (non-HR) reactions, systemic acquired silencing or other systemic
responses. The dotted line indicates that there is currently no direct experimental evidence suggesting that HC-Pro does interfere with a
systemic host defense response. The viral replication complex is comprised of virus-encoded and possibly host-encoded proteins (represented
by gray balls), which synthesize viral RNAs in association with endoplasmic reticulum (ER) derived membranes.
hypovirus infection suppresses a G-protein signaling cascade through down-regulation of the G-protein a-subunit,
CPG-1 [58–60]. This G-protein signaling pathway negatively regulates cAMP levels. Significantly, virus infection,
transgenic co-suppression of CPG-1, and drug-induced
elevation of cAMP levels each result in effects consistently
associated with hypovirulence of the fungus, including
reduced asexual sporulation, reduced pigmentation and
altered colony morphology. The ramifications of induction
of a hypovirulent state by hypoviruses are not immediately
clear, although it is possible that an ecological advantage
is afforded. As sexual reproduction of hypovirus-containing
C. parasitica strains is suppressed, genetic recombination in
a hypovirulent population should be limited. This would
promote proliferation of uniform anastomosis compatibility
groups, which in turn would promote distribution of the
virus by its normal transmission route — hyphal fusion.
While the influence of hypovirus infection on signaling in
C. parasitica is now becoming clear, the effects of plant
viruses on G-protein or other signaling cascades has yet to
be established. The tools are available, however, to address
this issue in detail.
Conclusions and future prospects
Although we have witnessed some significant developments recently, we suggest that the most exciting days in
the quest to understand compatibility and incompatibility
functions governing virus infection are still ahead of us.
A major limitation at this point is the paucity of useful
host mutants that exhibit altered susceptibility phenotypes
and that can serve as starting points for isolation of
relevant genes. Two types of host mutants should
be pursued aggressively. First, gain-of-susceptibility, or
loss-of-resistance, mutants using normally incompatible
(HR- and non-HR-dependent) virus–host combinations
will reveal loci necessary for recognition and response
signaling during defense. Clearly, the successes in recovery
of highly informative mutants with defects in the signaling pathways for defense against bacterial and fungal
pathogens sets an encouraging precedence [61]. Second,
loss-of-susceptibility mutants using normally compatible
virus–host combinations will reveal loci necessary for
genome replication and movement. Besides the yeast mab
mutants with defects in supporting BMV RNA replication,
an Arabidopsis mutant (tom1) has been recovered with
a defect in supporting TMV replication [62]. Recovery
of large numbers of susceptibility mutants, however,
may require particular finesse, as the cellular factors
and structure needed to support virus infection almost
certainly are critical for normal cellular function. We
believe that the necessary mutants are attainable, however,
through development of novel virus–host genetic systems
that are amenable to high-throughput selections and
screens using mass inoculation procedures and selectable
or screenable viruses.
Acknowledgements
Work in the author’s laboratory was supported by grants from the National
Institute of Health (GM18529 to SA Whitham; AI27832 to JC Carrington)
and the United States Department of Agriculture (95-37303-1867).
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Viral invasion and host defense: strategies and
counter-strategies
James C Carrington∗ and Steven A Whitham
The outcome of infection of plants by viruses is determined
by the net effects of compatibility functions and defense
responses. Recent advances reveal that viruses have the
capacity to modulate host compatibility and defense functions
by a variety of mechanisms.
Addresses
Institute of Biological Chemistry, Washington State University,
Pullman, WA 99164-6340, USA
∗e-mail: carrington@wsu.edu
Current Opinion in Plant Biology 1998, 1:336–341
http://biomednet.com/elecref/1369526600100336
Current Biology Ltd ISSN 1369-5266
Abbreviations
BMV
brome mosaic virus
CaMV
cauliflower mosaic virus
ER
endoplasmic reticulum
HC-Pro helper component-proteinase
HR
hypersensitive response
mab
maintenance of BMV functions
MP
movement protein
PSBMV pea seedborne mosaic potyvirus
PVX
potato virus X
TBRV
tomato black ring nepovirus
TEV
tobacco etch virus
TMV
tobacco mosaic virus
VIGS
viral induced gene silencing
Introduction
When discussing susceptibility or resistance of plants to
viruses, it is convenient to consider ‘susceptibility factors’
and ‘resistance factors’ as separate entities governing
infection. It can be argued, however, that the distinction
between susceptibility and resistance to viruses may not
be so straightforward. We are particularly intrigued by
the possibility that viruses can be active promoters of
susceptibility or suppressors of defense. In this review,
we will consider selected aspects of susceptibility, such
as virus–host interactions that govern virus genome
replication and intercellular movement, and a limited
discussion of defense responses. We will also summarize
recent findings, as well as our views, on how viruses
may modulate host functions to ensure their own efficient
replication and movement.
Virus–host interactions: susceptibility factors
A plant host is considered fully susceptible to a given virus
if the virus can successfully complete three processes:
genome replication, cell-to-cell movement (local) and
long-distance (vasculature-dependent) movement [1]. For
most RNA-genome viruses, RNA replication is catalyzed
by a core set of virus-encoded enzymes with RNA-de-
pendent RNA polymerase activity and other associated
activities (e.g. helicase, capping, priming) in close association with cytoplasmic surfaces of membranes [2]. The
docking of replication complexes to membranes appears
to be a point of intimate interaction between virus and
host cell. In several cases, complexes associate specifically
with endoplasmic reticulum (ER)-derived membranes
or vesicles through specific membrane-binding functions
of replication proteins [3,4]. The basis for membrane
specificity, however, remains obscure. The participation of
cellular factors, such as host proteins or protein complexes,
has been a point of speculation for many years, with
only limited direct evidence to support functional roles
of host factors [2]. This may change in the near future
through the clever use of yeast as an adopted host for
brome mosaic virus (BMV) to analyze RNA replication;
Paul Ahlquist and colleagues have developed selectable
and counter-selectable BMV RNA-based replicons for
yeast [5•,6,7]. For example, BMV replicons expressing
URA3 allow ura3 yeast to grow in the absence of uracil
and thus provide a positive selection for cells with
active BMV replication. Conversely, URA3 confers a lethal
phenotype in the presence of 5-fluoroortic acid, and thus
provides a selection for yeast cells that lose the ability
to support BMV replication. The characteristics of BMV
replicon amplification are remarkably similar in yeast
cells and plant cells, providing a rationale for applying
yeast genetics to identify host factors that influence
replication. The MAB1, MAB2 and MAB3 (Maintenance of
BMV functions) loci contribute distinct functions to BMV
genomic RNA replication, subgenomic RNA synthesis
and accumulation of BMV-encoded replication proteins
[8••]. Recessive mutations at each locus condition a
temperature-sensitive yeast growth phenotype, suggesting
that the wild-type gene products are necessary for the
normal cellular function. Dissection of the roles of the
MAB gene products in viral RNA replication will represent
a significant milestone in understanding compatibility
during virus–host interactions.
DNA-containing viruses, such as the geminiviruses, replicate in the host plant nucleus where the cellular DNA
replication apparatus is employed. The geminiviruses
use host DNA polymerase and associated factors in
conjunction with a virus-encoded replication protein
(termed RepA or AL1). The RepA/AL1 protein provides
several key activities that integrate viral and host functions
controlling early gene expression, late gene expression and
DNA replication [9,10]. This protein binds to the stem
structure at the replication origin and nicks the DNA prior
to initiation of rolling-circle DNA replication [9,11–13].
Like many animal-infecting DNA viruses that use the
Viral invasion and host defense: strategies and counter-strategies Carrington and Whitham
cellular DNA replication apparatus, the geminiviruses face
a serious problem; they infect terminally differentiated,
resting state (G0) cells, but these cells lack factors required
for DNA replication ([14], and references therein). As
detailed below, geminiviruses likely solve this problem
through modulating factors that normally control the cell
cycle.
Virus-encoded susceptibility factors controlling cell-tocell and long-distance movement are called ‘movement
proteins’ (MPs) and have been reviewed extensively
in recent years [1,15,16]. Movement proteins facilitate
movement through interactions with the viral genome or
assembled virus particle, intracellular transport pathways
and structures, and plasmodesmata. For geminiviruses,
two MPs are required for cell-to-cell movement. One
(BR1) is necessary to transport viral ssDNA genomes
between the nucleus and the cytoplasm [17,18]. The
BR1 protein, therefore, is a nuclear shuttle protein with
nuclear import and export signals. The other (BL1)
interacts with ER-derived membranes and plasmodesmata
to facilitate transit of viral DNA to adjacent plant cells,
possibly by modifying the plasmodesmata structure and
transport properties [19,20]. Tobacco mosaic virus (TMV)
MP interacts with viral RNA and modifies plasmodesmata
[21–23]. The TMV MP was shown to interact with
microtubules in infected or transfected cells [24,25], and it
was proposed that this might enable intracellular transport
of movement complexes from sites of genome replication
to plasmodesmata. Recent data, however, suggest that
the MP–microtubule association may occur relatively late
in the infection process, possibly as part of a pathway
to dissipate high local concentrations or to degrade MP
[26]. Early during the infection process, TMV MP, like
the geminivirus BL1 [19], associates with ER-derived
membranes and plasmodesmata [26]. Might the association
between the ER and diverse MPs from different viruses
be telling us something important? Recall that plasmodesmata contain an extension of the ER, the desmotubule,
which is threaded through the central channel [16,27].
Quite possibly, transport complex movement through
plasmodesmata requires specific interactions first with
cortical ER and then with the desmotubule component in
the channel.
Induction of host compatibility factors by
viruses
Do viruses have the ability to promote their own infections
by stimulation of host susceptibility factors or structures
needed for genome replication, genome expression or
movement? Given that the number of cellular factors
shown to play definitive roles in promoting virus infection
is small, there are relatively few examples to ponder. The
geminiviruses, however, provide an excellent case to argue
that viruses have the potential to modulate susceptibility.
As mentioned above, geminiviruses are dependent on
the host DNA replication apparatus for replication, and,
337
therefore, must overcome the lack of DNA replication
factors in G0 cells. Two significant breakthroughs have
shed light on possible mechanisms whereby geminiviruses
solve this problem. First, geminiviruses induce expression
of at least some of the components necessary for DNA
synthesis upon infection. Proliferating cell nuclear antigen
(PCNA), a DNA polymerase accessory factor normally
found only in S-phase cells, is induced in quiescent
mesophyll cells by infection with tomato golden mosaic
geminivirus (TGMV) [14]. Using AL1 transgenic plants,
induction of PCNA was shown to require only the TGMV
AL1 gene. Second, the AL1/RepA protein physically
interacts with host-encoded retinoblastoma-like tumor
suppressor proteins (pRbs) [28••,29–31]. Wheat dwarf
geminivirus mutants with substitutions affecting the probable pRb-like protein binding site of Rep exhibit defects
in viral DNA synthesis [29], supporting the concept
that this interaction is necessary. The well-characterized
mammalian pRB protein serves as key G1 checkpoint
regulator, preventing completion of G1 and entry into
S-phase [32]. Phosphorylation by cyclin-dependent kinases relieves the cell cycle-inhibitory activity of pRb and
allows progression into S-phase [32]. Plants, like mammals,
likely use one or more pRb-like proteins as checkpoint
inhibitors of the cell cycle. It is reasonable to suggest,
therefore, that geminivirus Rep protein interacts with,
and inactivates (or at least diverts), pRb-like protein in
infected cells. This would relieve the transcriptional block
to production of S-phase-specific mRNAs and provide
a pool of enzymes and factors necessary for viral DNA
replication. Thus, plant geminiviruses join the ranks
of animal DNA tumor-inducing viruses, such as SV40
and adenovirus, which encode proteins that affect the
cell cycling apparatus through interactions with tumor
suppressor proteins [33].
A novel defense response against viruses
Defense responses involving dominant resistance (R)
genes, hypersensitive cell death and induction of systemic
acquired resistance have been reviewed nicely by others
[34•,35•,36,37] and will not be covered here. Rather, we
will focus on a novel defense response — post-transcriptional gene silencing — that has only recently been linked
to natural forms of virus resistance.
Post-transcriptional gene silencing is a remarkable process that has been reviewed extensively [38–41]. The
hallmarks of post-transcriptional silencing of nuclear-derived sequences include, among other features, high
transcription levels of the silenced gene but relatively low
steady-state cytoplasmic levels of the transcript. Silencing
occurs through a homology-dependent mechanism and
functions in trans to suppress identical or closely related
sequences. Evidence suggests that silencing is due
to activation of a sequence-specific RNA degradation
apparatus, perhaps by a mechanism involving synthesis of
complementary nucleic acids. Silencing of a nuclear-derived sequence can be triggered by either nuclear genes
338
Plant–microbe interactions
or by cytoplasmic, replicating nucleic acids containing
homologous sequences. Furthermore, a silenced state in
a plant can be generated systemically through transport of
a mobile signal [42••,43••].
Whereas it has been known for some time that viral
sequences can induce silencing of an endogenous nuclear
gene or a transgene [44,45], it was only recently demonstrated that viruses can trigger silencing in the apparent
absence of homologous nuclear sequences. Infection
of Nicotiana clevelandii by tomato blackring nepovirus
(TBRV) strain W22 results in an initial symptomatic phase
in which the virus moves systemically, followed by a recovery state in which new tissue developing post-inoculation
is asymptomatic and largely devoid of virus [46••]. Further,
silencing of the viral sequence is active in the recovered
tissue, conferring effective resistance against W22 and a
closely related strain. Prolonged infection of Brassica napus
by cauliflower mosaic virus (CaMV), a pararetrovirus, also
induces post-transcriptional gene silencing independent of
homologous transgenes [47••]. These findings are highly
significant as they indicate that homology-dependent gene
silencing may be a general antiviral response. The logic
for such a response is impeccable: extreme resistance can
be elicited after infection by a broad range of infecting
viruses using a general, adaptive silencing apparatus. An
important unresolved issue, however, is the extent to
which viruses, other than those mentioned, elicit silencing
upon infection. Most compatible virus–host combinations
do not show the recovery phenotypes as do TBRV and
CaMV. It is possible, however, that silencing is triggered
transiently to limit the extent of virus replication in
infected tissue, thus lessening the extent of disease,
but not in time to limit spread systemically. Silencing
as a general antivirus response in both compatible and
incompatible virus–host combinations deserves careful
attention.
Do viruses fight back?
If one examines the way animal viruses deal with innate
and adaptive immune system defenses, it becomes clear
that these agents are masters of the counter-defensive
strategy. For example, poxviruses encode several types
of cytokine receptor mimic proteins [48]. These attenuate the inflammatory and immune responses normally
triggered by virus-infected cells. Also, numerous animal
viruses encode inhibitors of apoptosis proteins (IAPs),
preventing normal immune signaling that occurs through
activation of cell death pathways [49].
Do plant viruses deploy counter-defensive strategies?
Recent findings offer suggestions that certain plant viruses
are able to interdict defense mechanisms or alter host
cell metabolism for their own benefit. In a series of
elegant experiments examining host cell transcript and
protein levels in situ in tissue across an advancing
infection front, Andy Maule and colleagues demonstrated
that pea seedborne mosaic potyvirus (PSBMV) induces
transient depletion of many, but not all, classes of host
cell mRNAs in pea embryos [50,51]. The host mRNA
modulation occurs specifically in cells supporting virus
replication, and exhibits some of the characteristics of the
heat shock response, such as induction of HSP70 and
polyubiquitin mRNA expression. Although the bases for
suppression and subsequent recovery of transcript levels
are not clear, the consequences of inhibition of host cell
mRNA accumulation on induction of defense responses
are potentially far-reaching. As most types of known
induced defense responses require host gene expression
[34•], inhibition of mRNA accumulation or translation may
severely limit the extent or timing of a defense program.
Additional lines of evidence suggest that potyviruses can
modulate host defense responses, and that modulation
can condition enhanced accumulation and movement of
potyviruses as well as heterologous viruses. Typically,
in single-infections by a non-potyvirus, virus genome
amplification at the single-cell level shuts off after an
exponential amplification phase within 12–24 hours of
inoculation [52]. Tobacco etch virus (TEV), in contrast,
accumulates at a slower initial rate but for considerably
longer periods of time (e.g., 72 hours post-inoculation;
[53]). TEV mutants with defects in helper componentproteinase (HC-Pro) exhibit a premature amplification
shut-off phenotype by 24 hours post-inoculation and
are restricted in long-distance movement [54•]. These
observations suggest that wild-type HC-Pro may function
to suppress one or more induced defense responses.
Significantly, when heterologous viruses are co-inoculated
with TEV or other potyviruses, or when heterologous
viruses infect plants or cells in the presence of functional
HC-Pro, the heterologous viruses exhibit an enhanced
genome accumulation phenotype [55,56,57•]. In the case
of infections of potato virus X (PVX) expressing HC-Pro
or an HC-Pro polyprotein precursor, the enhancement
of PVX is actually the result of continued amplification
beyond the point at which amplification normally shuts off
[57•]. A plausible explanation for all of these results is that
potyviruses, through a key activity of HC-Pro, suppress
induction of host defense responses that would normally
result in early amplification shut-off and restricted accumulation (Figure 1). The antiviral defense responses
could have broad-spectrum activity against a range of
diverse viruses, or could be induced to have specificity
against many different viruses. The mechanistic details
of this system are not yet resolved, although recently
obtained evidence is consistent with HC-Pro mediating a
suppression of homology-dependent gene silencing (KD
Kasschau and JC Carrington, unpublished data).
Finally, the potential influence of viruses on signal
transduction pathways controlling a wide range of cellular
activities is just now being realized. Some of the best
ongoing work in this area deals with the virulence-attenuating hypovirus of the chestnut blight fungus, Cryphonectria
parasitica. Donald Nuss and co-workers have shown that
Viral invasion and host defense: strategies and counter-strategies Carrington and Whitham
339
Figure 1
HC-Pro
ER
Local VIGS or
other induced
defense responses
Accumulation of
viral RNAs
Viral RNA
replication complex
Amplification shut-off
or viral RNA degradation
Systemic acquired
silencing or other
systemic responses?
Current Opinion in Plant Biology
Modulation of antiviral defense responses by potyvirus TEV HC-Pro. The effects of HC-Pro on accumulation and movement of TEV and
heterologous viruses [54•,55,56,57•] suggest that HC-Pro protein may suppress local and systemic defense responses. The defense responses
affected by HC-Pro may include virus-induced gene silencing (VIGS), induced (non-HR) reactions, systemic acquired silencing or other systemic
responses. The dotted line indicates that there is currently no direct experimental evidence suggesting that HC-Pro does interfere with a
systemic host defense response. The viral replication complex is comprised of virus-encoded and possibly host-encoded proteins (represented
by gray balls), which synthesize viral RNAs in association with endoplasmic reticulum (ER) derived membranes.
hypovirus infection suppresses a G-protein signaling cascade through down-regulation of the G-protein a-subunit,
CPG-1 [58–60]. This G-protein signaling pathway negatively regulates cAMP levels. Significantly, virus infection,
transgenic co-suppression of CPG-1, and drug-induced
elevation of cAMP levels each result in effects consistently
associated with hypovirulence of the fungus, including
reduced asexual sporulation, reduced pigmentation and
altered colony morphology. The ramifications of induction
of a hypovirulent state by hypoviruses are not immediately
clear, although it is possible that an ecological advantage
is afforded. As sexual reproduction of hypovirus-containing
C. parasitica strains is suppressed, genetic recombination in
a hypovirulent population should be limited. This would
promote proliferation of uniform anastomosis compatibility
groups, which in turn would promote distribution of the
virus by its normal transmission route — hyphal fusion.
While the influence of hypovirus infection on signaling in
C. parasitica is now becoming clear, the effects of plant
viruses on G-protein or other signaling cascades has yet to
be established. The tools are available, however, to address
this issue in detail.
Conclusions and future prospects
Although we have witnessed some significant developments recently, we suggest that the most exciting days in
the quest to understand compatibility and incompatibility
functions governing virus infection are still ahead of us.
A major limitation at this point is the paucity of useful
host mutants that exhibit altered susceptibility phenotypes
and that can serve as starting points for isolation of
relevant genes. Two types of host mutants should
be pursued aggressively. First, gain-of-susceptibility, or
loss-of-resistance, mutants using normally incompatible
(HR- and non-HR-dependent) virus–host combinations
will reveal loci necessary for recognition and response
signaling during defense. Clearly, the successes in recovery
of highly informative mutants with defects in the signaling pathways for defense against bacterial and fungal
pathogens sets an encouraging precedence [61]. Second,
loss-of-susceptibility mutants using normally compatible
virus–host combinations will reveal loci necessary for
genome replication and movement. Besides the yeast mab
mutants with defects in supporting BMV RNA replication,
an Arabidopsis mutant (tom1) has been recovered with
a defect in supporting TMV replication [62]. Recovery
of large numbers of susceptibility mutants, however,
may require particular finesse, as the cellular factors
and structure needed to support virus infection almost
certainly are critical for normal cellular function. We
believe that the necessary mutants are attainable, however,
through development of novel virus–host genetic systems
that are amenable to high-throughput selections and
screens using mass inoculation procedures and selectable
or screenable viruses.
Acknowledgements
Work in the author’s laboratory was supported by grants from the National
Institute of Health (GM18529 to SA Whitham; AI27832 to JC Carrington)
and the United States Department of Agriculture (95-37303-1867).
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