BACKGROUND
salicylic acid  is a vital signal molecule involved in maintenance and activation of plant defenses.
sa is required for the limitation of pathogen spread during the hypersensitive response , which is a genetically determined resistance mechanism whereby pathogens are restricted to the immediate vicinity of an infection site.
triggering the hr can induce an additional induced resistance mechanism called systemic acquired resistance  that is also sa-dependent.
sar is effective against a very broad spectrum of pathogens, including viruses, oomycetes, fungi and bacteria  <cit> .
sar, induced either as the result of the hr or by application of resistance-inducing chemicals, is associated with dramatic changes in the transcriptome  <cit> .
these changes include increased transcription of genes encoding pathogenesis-related  proteins, several of which contribute to defense against fungi, oomycetes and bacteria  <cit>  but not against viruses .
indeed, induced resistance to viruses remains poorly understood  <cit> .
sa-induced resistance to viruses is not mediated by any of the known pr proteins and is not dependent on the transcriptional activator ‘non-expressor of pr proteins 1’ , which is required for pr gene induction and effective sa-induced resistance and sar against non-viral microbial pathogens  <cit> .
one of the mechanisms that could potentially underlie sa-induced resistance to viruses is rna silencing.
the importance of rna silencing in anti-viral defense may be inferred from the fact that most plant viruses possess counter-defense proteins  that inhibit the activity or stability of one or more components of the host silencing machinery.
the demonstration that the 2b vsr of cucumber mosaic virus , which inhibits rna silencing through binding of small rnas , also inhibited sa-induced resistance to the replication and local movement of this virus  <cit> , indicated a relationship between sa-induced resistance and silencing.
this idea was reinforced by subsequent studies showing that the hc-pro vsrs of potyviruses can have various effects on sa-mediated resistance to virus spread  <cit> .
an independent line of evidence implicating rna silencing in sa-induced resistance to viruses was discovered by xie et al.
<cit> , who found that in tobacco sa increased the accumulation of the transcript encoding rna-dependent rna polymerase   <dig> .
arabidopsis thaliana was also found to possess an sa-inducible rdr <dig> gene  <cit> .
rdrs are host enzymes that can initiate or amplify rna silencing, including antiviral silencing, through synthesis of dsrna molecules that serve as substrates for dicer-like  nucleases .
the products of dcl-mediated cleavage are short-interfering dsrnas that, after further processing to ssrna, direct sequence-specific cleavage or translational arrest of homologous target rna molecules by argonaute  proteins  <cit> .
plants possess a family of rdr paralogs, with six members occurring in arabidopsis  <cit> .
rdr <dig> and rdr <dig>  have demonstrable roles in limiting virus infection or decreasing virus titre in arabidopsis and nicotiana species .
interestingly, the gene encoding ago <dig>  one of the ago proteins with a known antiviral role , was rendered more sensitive to induction by sa in the presence of a transgene expressing the 2b vsr  <cit> .
this further supports a connection between antiviral silencing and sa-induced resistance.
although ample evidence suggests a role for silencing in sa-induced resistance, rna silencing is not absolutely required for successful sa-induced resistance to viruses.
it was shown in arabidopsis that dcls  <dig>   <dig>  and  <dig> are dispensable for sa-induced resistance to tmv and cmv  <cit> .
thus, it appears that sa induces multiple antiviral systems that include not only rna silencing but also other mechanisms; for example, the sa-triggered inhibition of virus replication and movement induced via mitochondria-based signaling processes  <cit> .
meanwhile, the role of rdr <dig> in virus resistance remains incompletely understood.
transgenic tobacco expressing an anti-sense construct for ntrdr <dig> were not compromised in their ability to exhibit sa-induced resistance to tobacco mosaic virus  and potato virus x  .
more recently, it was shown that rdr <dig> expression is inducible not only by sa but also by a wide range of other phytohormones, including jasmonic acid, and that rdr <dig> regulates insect resistance in nicotiana attenuata .
the nicotiana benthamiana accession commonly used for research is a natural rdr <dig> mutant that expresses a non-functional form of the enzyme , which explains why this plant is hypersusceptible to tmv  <cit> .
consistent with an important role for rdr <dig> in virus resistance, tmv-induced disease was ameliorated in transgenic n. benthamiana plants constitutively expressing mtrdr <dig> from medicago truncatula  <cit> .
however, ying and colleagues  <cit>  presented data showing that transgenic expression of tobacco ntrdr <dig> in n. benthamiana enhanced plant susceptibility to the potyvirus plum pox virus.
this was surprising in the light of previous work showing that down-regulation of ntrdr <dig> expression in tobacco plants increased susceptibility to another potyvirus, potato virus y  <cit> .
to better understand the role of rdr <dig> in sa-mediated antiviral defense, we explored the effects of sa on virus accumulation and movement and the effects of sa on the gene expression and activity of rdr <dig> in n. benthamiana.
RESULTS
constitutive mtrdr <dig> expression and sa treatment inhibit spread of tmv in the vicinity of the meristem in n. benthamiana
using tmv engineered to express the green fluorescent protein  we investigated the effect of sa on tmv movement in mtrdr1-transgenic n. benthamiana plants.
five-to-six week old mtrdr1-transgenic and empty vector - control plants  were sprayed on the leaves with either 1 mm sa or a control solution once daily for four consecutive days prior to inoculation with tmv.gfp.
plants were monitored daily for the appearance of gfp fluorescence in the upper, non-inoculated leaves.
in six independent experiments it was noted that sa treatment consistently resulted in a delay of  <dig> to 2 days in the first appearance of tmv.gfp in the upper, non-inoculated leaves.
however, there was no apparent difference between control and mtrdr1-transgenic plants in the timing or patterning of tmv.gfp spread into the upper non-inoculated leaves , though mtrdr1-transgenic plants were less severely stunted than the control plants .
western blot analysis of tmv coat protein accumulation in the systemically infected leaves at 14 days post-inoculation  showed that viral coat protein was present in the upper leaves of both groups of plants .
when the plants were studied under uv illumination to assess tmv.gfp fluorescence in non-inoculated tissue, there was no marked difference in the timing of first appearance or the apparent extent of gfp fluorescence between control and mtrdr1-transgenic n. benthamiana plants in the upper leaves .fig.
<dig> tmv.gfp accumulation in upper, non-inoculated leaves in n. benthamiana plants is not prevented by expression of an mtrdr1-transgene or by treatment with salicylic acid.
five-week-old mtrdr1-transgenic plants , or plants transformed with an ‘empty vector’ transformation vector , were pre-treated with a solution of 1 mm sa or a control solution  prior to inoculation with gfp-tagged tmv.
a the movement of tmv.gfp from directly-inoculated leaves into non-inoculated leaves was monitored daily using a hand-held uv lamp and the first appearance of gfp fluorescence in non-inoculated leaves recorded.
there was no apparent difference in the timing of appearance of tmv.gfp in non-inoculated leaves between the two groups of plants although systemic infection with tmv.gfp was delayed by sa treatment in both types of plant.
there were  <dig> plants in each treatment group.
b western blot analysis of tmv.gfp accumulation in systemically-infected leaf tissue of water-  and sa-  treated plants using anti-tmv coat protein .
in each case leaf tissue samples were harvested at 14 days post-inoculation from the uppermost three leaves above the inoculated leaf.
three samples were taken from each treatment group .
equal loading of gel lanes with protein is shown by accumulation of ribulose- <dig>  5-bisphosphate carboxylase/oxygenase large subunit  revealed by ponceau s staining of the western blot membrane.
c the extent of gfp fluorescence in the upper leaves of water-treated tmv.gfp-infected empty vector control  and mtrdr1-transgenic plants  appeared similar when visualised using a hand-held uv lamp and photographed at 14 days post-inoculation.
gfp fluorescence was less extensive in sa-treated plants of both groups.
the data and photographs above are from one experiment, out of a total of four independent experiments.
scale bar = 8 cm
however, closer examination of the uppermost regions of the stems of sa-treated and untreated control and mtrdr1-expressing plants revealed some striking differences in the extent of tmv.gfp spread .
microscopic observation of fluorescence indicated that sa treatment inhibited the extent of spread of tmv.gfp into the upper stem regions of non-transgenic and ev-control plants .
tmv.gfp was excluded entirely from the vicinity of the meristem and much of the upper stems of transgenic plants expressing mtrdr <dig> .
further examination showed that sa treatment enlarged the zone from which tmv.gfp was excluded in mtrdr1-transgenic n. benthamiana plants .fig.
<dig> sa treatment enhances rdr1-mediated exclusion of tmv from tissue adjacent to the apical meristem.
empty vector control , non-transformed and mtrdr1-transgenic plants were treated with 1 mm sa in  <dig>  %  ethanol  or a control solution of  <dig>  %  ethanol  prior to inoculation with tmv.gfp.
by 14 days post-inoculation the upper leaves of all plants with and without sa treatment showed tmv.gfp fluorescence in the upper leaves.
however, when leaves were removed from the stem it was apparent under epifluorescence microscopy  that spread of tmv.gfp into tissue adjacent to meristems of non-transgenic and control transgenic plants was inhibited by sa treatment and that in mtrdr1-transgenic plants, the virus did not enter stem tissue adjacent to the meristem .
stems  of tmv.gfp infected plants were observed under a uv lamp and photographed with a digital camera, revealing that in mtrdr1-transgenic plants the exclusion of tmv.gfp from stem tissue proximal to the meristem was enhanced in sa-treated plants.
photographs in  and  are from two independent experiments
recovery of mtrdr1-transgenic n. benthamiana plants from severe tmv disease was enhanced in sa-treated plants
neither tmv.gfp, nor the tmv.30b vector from which it was derived, induce strong disease symptoms in n. benthamiana  <cit> .
however, infection of this host by the u <dig> strain of tmv causes strong symptoms culminating in systemic necrosis and death of infected plants.
interestingly, tmv-infected mtrdr1-expressing n. benthamiana plants developed milder symptoms and the plants did not die  <cit> .
here we found that sa treatment slowed, but did not prevent, tmv-induced death of ev-control transgenic plants and that all plants  had died by  <dig> dpi .
by this time point no mtrdr1-transgenic plants had died although they did exhibit tmv u1-induced leaf necrosis, preceded by other symptoms  in many leaves, with the onset of necrosis being slower in sa-treated plants.
necrosis did not become sufficiently extensive to entirely kill mtrdr1-transgenic plants.
the progress of disease development on mtrdr1-transgenic plants was monitored until  <dig> dpi and it was noted that on all of these plants the spread of necrosis abated and that newly emerging leaves were green and that growth of the plants resumed .
this apparent recovery from disease was further enhanced on mtrdr1-transgenic plants that had been treated with sa prior to inoculation with tmv.
sa-treated mtrdr1-transgenic plants had grown markedly taller than untreated plants by  <dig> dpi .
western blot analysis using anti-coat protein serum showed that tmv was present in the new leaf tissue produced by the plants  indicating that although the mtrdr1-expressing transgenic plants recovered from tmv-induced disease and that this recovery was enhanced in sa-treated plants, they did not develop true resistance to the virus.fig.
<dig> the effects of mtrdr <dig> expression and sa treatment on the recovery of n. benthamiana plants from tmv-induced plant death.
a five-week-old transgenic control  and mtrdr1-transgenic  plants were pre-treated with a solution of 1 mm sa in  <dig>  %  ethanol  or a  <dig>  %  ethanol control solution prior to inoculation with tmv u <dig>  plants were monitored over a 5-week period and the number of plants killed by the virus recorded.
with the transgenic control plant group, tmv u <dig> had killed all plants within 31 days of inoculation.
pre-treatment with 1 mm sa delayed plant death by a few days but did not prevent it.
tmv u1-induced plant death was suppressed in all the mtrdr1-expressing n. benthamiana plants, regardless of whether they were treated with water or with sa.
b symptoms at 75 days post-inoculation with tmv u <dig> in transgenic control and mtrdr1-transgenic n. benthamiana plants.
mtrdr1-transgenic plants pre-treated with sa  or treated with control solution  recovered but sa-treated plant growth was more vigorous .
data and photographs are from one experiment, out of a total of four independent experiments.
c western blot analysis of tmv using anti-tmv coat protein  serum shows that tmv was present in the newly emerged, green leaf tissue in recovering mtrdr1-transgenic plants treated with sa  or control solution .
leaf samples taken at 75 days post-inoculation or mock-inoculation .
equal loading was confirmed by ponceau s staining of the western blot membrane with the position of the ribulose  <dig> -bisphosphate carboxylase/oxygenase large subunit  indicated
sa treatment increased extractable rdr activity in mtrdr1-transgenic n. benthamiana plants but did not alter mtrdr <dig> transcript accumulation
sa had a long-lasting effect on mtrdr1-transgenic n. benthamiana plants that resulted in an improvement in recovery from tmv-induced disease and resumption of growth .
this suggested that sa enhances or primes rdr1-mediated defense against tmv.
however, mtrdr <dig> transgene expression in these plants is under the control of the cauliflower mosaic virus 35s promoter  <cit>  that is, despite containing an as- <dig> element shared with many sa-responsive plant promoters , not activated by this phytohormone  <cit> .
to confirm that the 35s promoter used in the construction of the mtrdr1-transgenic plants was behaving as expected, i.e.
that it was not responding to sa, we extracted total rna from leaves of control-transgenic and mtrdr1-transgenic n. benthamiana plants 72 h after they were infiltrated with either 1 mm sa or a control solution.
semi-quantitative reverse transcription-polymerase chain reaction  assays showed that sa, but not the control solution, induced expression of the pr1a gene, indicating that this sa concentration was effective for the induction of sa-responsive gene expression .
rt-pcr also indicated that whereas the transcript encoding the native, non-functional rdr <dig> of n. benthamiana  was induced by sa, the transgene-encoded mtrdr <dig> transcript remained at a similar level in mtrdr1-transgenic plants in the presence or absence of sa treatment .
analysis by rt-quantitative pcr  confirmed that steady state levels of mtrdr <dig> transcript were not affected by sa treatment in the mtrdr1-transgenic n. benthamiana plants .
thus, the 35s promoter used in the construction of the mtrdr1-transgenic plants is unaffected by sa, meaning that the observed enhancement of rdr1-mediated antiviral action by sa  must be regulated at a post-transcriptional level.fig.
<dig> rdr activity, but not mtrdr <dig> transcript accumulation, is induced by sa treatment in mtrdr1-transgenic n. benthamiana plants.
semi-quantitative rt-pcr analysis of pr <dig> transcript accumulation in leaves of  transgenic  control and  mtrdr1-transgenic plants infiltrated with a control solution of  <dig>  %  ethanol or a solution of 1 mm sa in  <dig>  %  ethanol.
infiltrated leaf tissue samples were harvested for rna extraction at 72 h post-infiltration.
pr <dig> transcript accumulation levels were determined by rt-pcr after 40 cycles of pcr and compared relative to the accumulation levels of the elongation factor  <dig> alpha  transcript.
increased pr <dig> accumulation confirmed that sa was taken up by the tissues and was effective in inducing transcriptional changes.
c semi-quantitative rt-pcr analysis showed that there was little difference in mtrdr <dig> transcript accumulation in mtrdr1-transgenic plants infiltrated with control solution or 1 mm sa.
nbrdr1m transcript accumulation was up-regulated in both transgenic control and mtrdr1-transgenic n. benthamiana plants after sa treatment, although the nbrdr1m protein itself is non-functional.
mtrdr <dig> and nbrdr1m transcript accumulation levels after  <dig> and 35 cycles, respectively, were compared relative to the accumulation levels of ef1α.
infiltrated tissue samples were harvested at 72 h post-infiltration.
d rt-qpcr analysis of mtrdr <dig> transcript levels in leaves of empty vector control and mtrdr1-transgenic plants infiltrated with water control or 1 mm sa solution.
mtrdr <dig> was not detected in empty vector control plants.
mean values for relative mtrdr <dig> levels  obtained from three plants  have been given for each treatment group.
error bars represent standard errors of the mean for the three samples.
relative transcript levels of mtrdr <dig> were calculated using the 2-ΔΔc method  <cit>  using ef1α as an internal reference.
e enhancement of rdr activity by sa in mtrdr1-transgenic plants.
leaves from tobacco  and transgenic empty vector control and mtrdr1-transgenic n. benthamiana plants were infiltrated with water  containing  <dig>  %  ethanol or  <dig>  mm sa in  <dig>  %  ethanol  and harvested after 48 h for preparation of rdr1-enriched extracts.
the proxy for rdr <dig> activity was the incorporation of α- ctp into nascent rna analysed by liquid scintillation counting of radioactivity incorporated  into trichloroacetic acid-precipitable material.
error bars are standard errors for the mean for three technical replicates  per sample
in order to determine if sa treatment affected rdr activity levels, extracts enriched in rdr activity  <cit>  were prepared from non-infected control transgenic and mtrdr1-transgenic n. benthamiana leaves infiltrated with sa or control solution 48 h prior to tissue harvesting .
n. tabacum  possesses the sa-inducible gene ntrdr <dig>  which encodes a functional rdr <dig>  <cit> , and so samples from non-transgenic n. tabacum were included in the assays as positive controls for sa-inducible rdr activity.
control-treated ev n. benthamiana plants contained active rdr not attributable to rdr <dig> and as expected, mtrdr1-transgenic n. benthamiana plants contained a higher level of extractable rdr activity than that detected in the ev control plants .
although treatment with sa did not increase the amount of active rdr extractable from ev-control plants , sa treatment did increase rdr activity in mtrdr1-transgenic plants.
this effect was only observed when sa was applied to intact plant tissue, and not when rdr extracts from untreated n. tabacum or mtrdr1-expressing n. benthamiana plants were incubated in vitro with sa or with a biologically inactive isomer of sa .
mtrdr <dig> protein is detectable in rdr extracts from sa-treated mtrdr1-transgenic n. benthamiana plants
since the in vitro rdr activity of extracts from mtrdr1-transgenic plants was increased approximately  <dig>  fold when the plants were pre-treated with sa, we investigated whether there was any change in the accumulation of mtrdr <dig> protein in these extracts.
a polyclonal anti-mtrdr <dig> rabbit serum was prepared using a maltose binding protein  mtrdr <dig> fusion protein made in escherichia coli .
this was used for western blot analysis of rdr-enriched extracts .
as the anti-mtrdr <dig> serum detected a number of background plant protein bands, western blot analysis with preimmune serum was also carried out .
the only polypeptide specifically detected by the anti-mtrdr <dig> serum was of c.131 kda, the mass predicted for mtrdr <dig> , and this was present only in rdr-enriched extracts from sa-treated, mtrdr1-transgenic plants .
ubiquitination and multiple phosphorylation sites are predicted to occur in the mtrdr <dig> protein sequence .
this raises the possibility that sa treatment stabilizes the protein  or, alternatively, that sa triggers recruitment of mtrdr <dig> into an active complex.fig.
<dig> western blot analysis for mtrdr <dig> in rdr1-enriched protein extracts.
leaves of empty vector control transgenic or mtrdr1-transgenic n. benthamiana plants were infiltrated with a control solution of water containing  <dig>  %  ethanol  or  <dig>  mm sa .
rdr1-enriched protein extracts were prepared 48 h later and subjected to western blot analysis with either preimmune rabbit serum  or polyclonal rabbit anti-mtrdr <dig> serum .
the positions of protein molecular mass markers are indicated .
a protein  corresponding to the predicted size of 131 kda for mtrdr <dig> was specifically detected in sa-treated mtrdr1-transgenic plants with the anti-mtrdr <dig> serum but not with the preimmune serum.
equal loading of gel lanes with protein, based on the bradford  <cit>  assay was confirmed by ponceau s staining of the western blot membrane
discussion
we studied the effects of rdr <dig> on the long-distance movement and spread of tmv.
expression of mtrdr <dig> in n. benthamiana did not prevent tmv.gfp movement into non-inoculated leaf tissue or enhance the inhibitory effect of sa on virus movement into these tissues.
however, mtrdr <dig> expression inhibited the extent of spread into the region proximal to the apical meristem and this movement restriction became more pronounced following treatment of mtrdr1-transgenic plants with sa.
schwach and colleagues  <cit>  showed that knock-down of nbrdr <dig> gene expression in the same host  allowed entry of pvx into the meristem and that this resulted in an exacerbation of pvx-induced disease symptoms throughout the plant.
in line with previous work  <cit>  we found that in n. benthamiana plants, tmv.gfp enters tissue adjacent to the meristem, although it does not appear to enter the meristem itself.
in mtrdr1-transgenic plants, however, the ability of tmv.gfp to approach the meristem is drastically curtailed.
furthermore, in the mtrdr1-transgenic plants the normally lethal effect of tmvu <dig> is ameliorated and recovery from disease occurs.
our results with constitutive expression of mtrdr <dig> complement and extend the results and conclusions of schwach and colleagues  <cit> ; indicating that rdr <dig>  as well as rdr <dig>  can function to inhibit virus entry to tissue adjacent to the meristem.
both studies point to a relationship between the effectiveness of rna silencing-mediated exclusion of virus from meristematic and adjacent tissues with the severity of virus-induced disease symptoms.
this suggests that, with respect to the elaboration of disease symptoms, that there is a critical developmental stage for plant cells and tissues, which corresponds physically to a certain point along the sub-apical zone.
if viral entry into these tissues is delayed, for example by host defense mechanisms, until after this critical developmental stage has been completed, plants will to some extent be able to recover from virus infection.
it has been postulated that the hyper-susceptibility of the laboratory accession of n. benthamiana to a number of tobamoviruses including tmv can be attributed to the absence of an active rdr <dig> in this species  <cit> .
ying and colleagues  <cit>  proposed an alternative hypothesis, suggesting that rdr <dig> can enhance virus spread and accumulation and that the loss of rdr <dig> functionality in n. benthamiana may be due to selective pressure to maintain high levels of rdr6-dependent antiviral defense.
this hypothesis was formulated mainly to explain results after challenge with non-tobamoviruses, as challenge with tmv did not alter symptoms and at best only modestly decreased virus accumulation in systemically-infected tissue of n. benthamiana plants expressing ntrdr <dig>  <cit> .
we have seen no increase in susceptibility to tmv in our study of n. benthamiana plants expressing the transgene derived from the rdr <dig> of m. truncatula.
furthermore, wild accessions of n. benthamiana that contain a non-truncated, functional nbrdr <dig> are naturally protected against severe symptoms from tobamovirus infection and are not more susceptible to poty- or cucumovirus infection than n. benthamiana carrying nbrdr1m  indicating that rdr <dig> does indeed have a particular role in ameliorating tobamovirus infection in n. benthamiana.
a difference in the effects of rdr <dig> and rdr <dig> on virus spread is apparent in the pattern of spread of pvx.gfp into upper uninoculated leaves previously observed in rdr6i plants  <cit>  and in the pattern of systemic tmv.gfp movement in mtrdr1-transgenic plants in the present study .
specifically, knock-down of nbrdr <dig> accelerated entry of virus into well-developed upper leaves, whereas constitutive expression of mtrdr <dig> did not slow the appearance of tmv.gfp in these leaves.
these differences suggest that the roles of rdrs  <dig> and  <dig> do not overlap completely and that each may be effective in protection against different viruses with differing infection and movement strategies, as suggested previously  <cit> .
these results suggest that rdr <dig> may play a role similar to rdr <dig> in protecting the meristem from viral invasion.
the strong atrdr <dig> promoter activity in arabidopsis phloem tissue observed by xu and colleagues  <cit>  may also help restrict the spread of virus into the vasculature and subsequently the meristem, although curiously the atrdr <dig> promoter:β-glucuronidase reporter transgene was found to be poorly active in younger tissues.
however, since rdr <dig> gene expression is induced by chemical signals associated with sar   <cit>  as well as by many other phytohormones  and wounding  <cit> , it may be that the multiplicity of factors affecting rdr <dig> expression, and its therefore complex regulation, allows plants to reinforce meristem protection against invasion in times of stress or perhaps control the accessibility of this tissue to endogenous factors during development.
previous studies showed that plants expressing a functional rdr <dig> are less susceptible to tmv-induced disease, yet rdr <dig> does not seem to be required for successful sa-induced resistance to viruses in directly inoculated leaves  <cit> .
interestingly, we found here not only that expression of mtrdr <dig> enabled n. benthamiana to recover from severe disease induced by tmv strain u <dig>  but also that this recovery was enhanced by sa treatment.
whilst this could have been due to the activity of one or more of the rdr1-independent sa-inducible antiviral defense systems that are known to exist  <cit> , sa treatment of control-transgenic n. benthamiana plants only delayed tmv-induced plant death by a few days suggesting that the protective activity of other sa-induced antiviral defense systems was insufficient in this case to account for the marked enhancement of recovery after sa treatment in the mtrdr1-transgenic n. benthamiana plants.
although sa and mtrdr <dig> worked synergistically in protection against severe tmv-induced disease, it is not possible to say with certainty whether or not these factors were working together or separately.
however, the hypothesis that sa enhanced rdr <dig> activity was favored by the observation that mtrdr1-transgenic plants contained more extractable rdr activity following sa treatment, despite there being no concomitant sa-induced increase in mtrdr <dig> transgene expression.
there was no direct effect of sa on in vitro rdr activity in plant extracts , which indicates that sa indirectly influences rdr activity in planta.
detection of a band cross-reacting with an antibody raised against mtrdr <dig> in rdr1-enriched preparations from sa-treated but not untreated mtrdr1-transgenic plants was consistent with the detection of greater rdr <dig> activity.
it also suggested that rdr <dig> can be regulated by sa through post-translational process either resulting in a more stable mtrdr <dig> or involving recruitment of mtrdr <dig> protein to a complex.
an in silico analysis of the mtrdr <dig> protein sequence revealed a number of candidate ubiquitination and phosphorylation sites .
post-translational protein modification plays an important role in host immunity  <cit> , for example: ubiquitin-dependent proteolysis negatively regulates defense responses in tobacco  <cit> ; npr <dig> and  <dig> are e <dig> ligase adapters that control npr <dig> activity by proteolysis in an sa-dependent manner in arabidopsis  <cit> ; and sumo proteases negatively regulate sa biosynthesis  <cit> .
it may therefore be that sa induces, in some manner, stabilization of the mtrdr <dig> protein against small modifier protein-mediated degradation and/or activation/deactivation of the protein by phosphorylation.
the idea of assembly of a complex, which includes rdr <dig> and forms in response to sa, is made more plausible by recent findings that several rna silencing factors, including rdr <dig>  form complexes with each other and with other cellular components such as membranes  <cit> .
another possibility is that sa enhances translation of the mtrdr <dig> transcript leading to increased synthesis of mtrdr <dig> protein but it is difficult to envisage a likely mechanism for this.
CONCLUSIONS
in conclusion, our results support an important role for the phytohormone-inducible factor rdr <dig> in resistance by limiting viral access to the region adjacent to the meristem and thereby ameliorating the severity of virus-induced disease.
its ability to inhibit access to the meristematic region correlates with amelioration of tmv-induced disease symptoms and is enhanced by sa.
our data suggests that sa may enhance rdr <dig> activity at a post-transcriptional level in addition to the previously documented effects of this phytohormone on transcription of the rdr <dig> gene.

 salicylic acid is a vital signal molecule involved in maintenance and activation of plant defenses. sa is required for the limitation of pathogen spread during the hypersensitive response, which is a genetically determined resistance mechanism whereby pathogens are restricted to the immediate vicinity of an infection site. triggering the hr can induce an additional induced resistance mechanism called systemic acquired resistance that is also sa-dependent. sar is effective against a very broad spectrum of pathogens, including viruses, oomycetes, fungi and bacteria. sar, induced either as the result of the hr or by application of resistance-inducing chemicals, is associated with dramatic changes in the transcriptome. these changes include increased transcription of genes encoding pathogenesis-related proteins, several of which contribute to defense against fungi, oomycetes and bacteria but not against viruses. indeed, induced resistance to viruses remains poorly understood. sa-induced resistance to viruses is not mediated by any of the known pr proteins and is not dependent on the transcriptional activator ‘non-expressor of pr proteins 1’, which is required for pr gene induction and effective sa-induced resistance and sar against non-viral microbial pathogens. one of the mechanisms that could potentially underlie sa-induced resistance to viruses is rna silencing. the importance of rna silencing in anti-viral defense may be inferred from the fact that most plant viruses possess counter-defense proteins that inhibit the activity or stability of one or more components of the host silencing machinery. the demonstration that the 2b vsr of cucumber mosaic virus, which inhibits rna silencing through binding of small rnas, also inhibited sa-induced resistance to the replication and local movement of this virus, indicated a relationship between sa-induced resistance and silencing. this idea was reinforced by subsequent studies showing that the hc-pro vsrs of potyviruses can have various effects on sa-mediated resistance to virus spread. an independent line of evidence implicating rna silencing in sa-induced resistance to viruses was discovered by xie et al., who found that in tobacco sa increased the accumulation of the transcript encoding rna-dependent rna polymerase. arabidopsis thaliana was also found to possess an sa-inducible rdr gene. rdrs are host enzymes that can initiate or amplify rna silencing, including antiviral silencing, through synthesis of dsrna molecules that serve as substrates for dicer-like nucleases. the products of dcl-mediated cleavage are short-interfering dsrnas that, after further processing to ssrna, direct sequence-specific cleavage or translational arrest of homologous target rna molecules by argonaute proteins. plants possess a family of rdr paralogs, with six members occurring in arabidopsis. rdr and rdr have demonstrable roles in limiting virus infection or decreasing virus titre in arabidopsis and nicotiana species. interestingly, the gene encoding ago one of the ago proteins with a known antiviral role, was rendered more sensitive to induction by sa in the presence of a transgene expressing the 2b vsr. this further supports a connection between antiviral silencing and sa-induced resistance. although ample evidence suggests a role for silencing in sa-induced resistance, rna silencing is not absolutely required for successful sa-induced resistance to viruses. it was shown in arabidopsis that dcls and are dispensable for sa-induced resistance to tmv and cmv. thus, it appears that sa induces multiple antiviral systems that include not only rna silencing but also other mechanisms; for example, the sa-triggered inhibition of virus replication and movement induced via mitochondria-based signaling processes. meanwhile, the role of rdr in virus resistance remains incompletely understood. transgenic tobacco expressing an anti-sense construct for ntrdr were not compromised in their ability to exhibit sa-induced resistance to tobacco mosaic virus and potato virus x. more recently, it was shown that rdr expression is inducible not only by sa but also by a wide range of other phytohormones, including jasmonic acid, and that rdr regulates insect resistance in nicotiana attenuata. the nicotiana benthamiana accession commonly used for research is a natural rdr mutant that expresses a non-functional form of the enzyme, which explains why this plant is hypersusceptible to tmv. consistent with an important role for rdr in virus resistance, tmv-induced disease was ameliorated in transgenic n. benthamiana plants constitutively expressing mtrdr from medicago truncatula. however, ying and colleagues presented data showing that transgenic expression of tobacco ntrdr in n. benthamiana enhanced plant susceptibility to the potyvirus plum pox virus. this was surprising in the light of previous work showing that down-regulation of ntrdr expression in tobacco plants increased susceptibility to another potyvirus, potato virus y. to better understand the role of rdr in sa-mediated antiviral defense, we explored the effects of sa on virus accumulation and movement and the effects of sa on the gene expression and activity of rdr in n. benthamiana.  constitutive mtrdr expression and sa treatment inhibit spread of tmv in the vicinity of the meristem in n. benthamiana using tmv engineered to express the green fluorescent protein we investigated the effect of sa on tmv movement in mtrdr1-transgenic n. benthamiana plants. five-to-six week old mtrdr1-transgenic and empty vector - control plants were sprayed on the leaves with either 1 mm sa or a control solution once daily for four consecutive days prior to inoculation with tmv.gfp. plants were monitored daily for the appearance of gfp fluorescence in the upper, non-inoculated leaves. in six independent experiments it was noted that sa treatment consistently resulted in a delay of to 2 days in the first appearance of tmv.gfp in the upper, non-inoculated leaves. however, there was no apparent difference between control and mtrdr1-transgenic plants in the timing or patterning of tmv.gfp spread into the upper non-inoculated leaves, though mtrdr1-transgenic plants were less severely stunted than the control plants. western blot analysis of tmv coat protein accumulation in the systemically infected leaves at 14 days post-inoculation showed that viral coat protein was present in the upper leaves of both groups of plants. when the plants were studied under uv illumination to assess tmv.gfp fluorescence in non-inoculated tissue, there was no marked difference in the timing of first appearance or the apparent extent of gfp fluorescence between control and mtrdr1-transgenic n. benthamiana plants in the upper leaves.fig. tmv.gfp accumulation in upper, non-inoculated leaves in n. benthamiana plants is not prevented by expression of an mtrdr1-transgene or by treatment with salicylic acid. five-week-old mtrdr1-transgenic plants, or plants transformed with an ‘empty vector’ transformation vector, were pre-treated with a solution of 1 mm sa or a control solution prior to inoculation with gfp-tagged tmv. a the movement of tmv.gfp from directly-inoculated leaves into non-inoculated leaves was monitored daily using a hand-held uv lamp and the first appearance of gfp fluorescence in non-inoculated leaves recorded. there was no apparent difference in the timing of appearance of tmv.gfp in non-inoculated leaves between the two groups of plants although systemic infection with tmv.gfp was delayed by sa treatment in both types of plant. there were plants in each treatment group. b western blot analysis of tmv.gfp accumulation in systemically-infected leaf tissue of water- and sa- treated plants using anti-tmv coat protein. in each case leaf tissue samples were harvested at 14 days post-inoculation from the uppermost three leaves above the inoculated leaf. three samples were taken from each treatment group. equal loading of gel lanes with protein is shown by accumulation of ribulose- 5-bisphosphate carboxylase/oxygenase large subunit revealed by ponceau s staining of the western blot membrane. c the extent of gfp fluorescence in the upper leaves of water-treated tmv.gfp-infected empty vector control and mtrdr1-transgenic plants appeared similar when visualised using a hand-held uv lamp and photographed at 14 days post-inoculation. gfp fluorescence was less extensive in sa-treated plants of both groups. the data and photographs above are from one experiment, out of a total of four independent experiments. scale bar = 8 cm however, closer examination of the uppermost regions of the stems of sa-treated and untreated control and mtrdr1-expressing plants revealed some striking differences in the extent of tmv.gfp spread. microscopic observation of fluorescence indicated that sa treatment inhibited the extent of spread of tmv.gfp into the upper stem regions of non-transgenic and ev-control plants. tmv.gfp was excluded entirely from the vicinity of the meristem and much of the upper stems of transgenic plants expressing mtrdr. further examination showed that sa treatment enlarged the zone from which tmv.gfp was excluded in mtrdr1-transgenic n. benthamiana plants.fig. sa treatment enhances rdr1-mediated exclusion of tmv from tissue adjacent to the apical meristem. empty vector control, non-transformed and mtrdr1-transgenic plants were treated with 1 mm sa in  % ethanol or a control solution of  % ethanol prior to inoculation with tmv.gfp. by 14 days post-inoculation the upper leaves of all plants with and without sa treatment showed tmv.gfp fluorescence in the upper leaves. however, when leaves were removed from the stem it was apparent under epifluorescence microscopy that spread of tmv.gfp into tissue adjacent to meristems of non-transgenic and control transgenic plants was inhibited by sa treatment and that in mtrdr1-transgenic plants, the virus did not enter stem tissue adjacent to the meristem. stems of tmv.gfp infected plants were observed under a uv lamp and photographed with a digital camera, revealing that in mtrdr1-transgenic plants the exclusion of tmv.gfp from stem tissue proximal to the meristem was enhanced in sa-treated plants. photographs in and are from two independent experiments recovery of mtrdr1-transgenic n. benthamiana plants from severe tmv disease was enhanced in sa-treated plants neither tmv.gfp, nor the tmv.30b vector from which it was derived, induce strong disease symptoms in n. benthamiana. however, infection of this host by the u strain of tmv causes strong symptoms culminating in systemic necrosis and death of infected plants. interestingly, tmv-infected mtrdr1-expressing n. benthamiana plants developed milder symptoms and the plants did not die. here we found that sa treatment slowed, but did not prevent, tmv-induced death of ev-control transgenic plants and that all plants had died by dpi. by this time point no mtrdr1-transgenic plants had died although they did exhibit tmv u1-induced leaf necrosis, preceded by other symptoms in many leaves, with the onset of necrosis being slower in sa-treated plants. necrosis did not become sufficiently extensive to entirely kill mtrdr1-transgenic plants. the progress of disease development on mtrdr1-transgenic plants was monitored until dpi and it was noted that on all of these plants the spread of necrosis abated and that newly emerging leaves were green and that growth of the plants resumed. this apparent recovery from disease was further enhanced on mtrdr1-transgenic plants that had been treated with sa prior to inoculation with tmv. sa-treated mtrdr1-transgenic plants had grown markedly taller than untreated plants by dpi. western blot analysis using anti-coat protein serum showed that tmv was present in the new leaf tissue produced by the plants indicating that although the mtrdr1-expressing transgenic plants recovered from tmv-induced disease and that this recovery was enhanced in sa-treated plants, they did not develop true resistance to the virus.fig. the effects of mtrdr expression and sa treatment on the recovery of n. benthamiana plants from tmv-induced plant death. a five-week-old transgenic control and mtrdr1-transgenic plants were pre-treated with a solution of 1 mm sa in  % ethanol or a  % ethanol control solution prior to inoculation with tmv u plants were monitored over a 5-week period and the number of plants killed by the virus recorded. with the transgenic control plant group, tmv u had killed all plants within 31 days of inoculation. pre-treatment with 1 mm sa delayed plant death by a few days but did not prevent it. tmv u1-induced plant death was suppressed in all the mtrdr1-expressing n. benthamiana plants, regardless of whether they were treated with water or with sa. b symptoms at 75 days post-inoculation with tmv u in transgenic control and mtrdr1-transgenic n. benthamiana plants. mtrdr1-transgenic plants pre-treated with sa or treated with control solution recovered but sa-treated plant growth was more vigorous. data and photographs are from one experiment, out of a total of four independent experiments. c western blot analysis of tmv using anti-tmv coat protein serum shows that tmv was present in the newly emerged, green leaf tissue in recovering mtrdr1-transgenic plants treated with sa or control solution. leaf samples taken at 75 days post-inoculation or mock-inoculation. equal loading was confirmed by ponceau s staining of the western blot membrane with the position of the ribulose -bisphosphate carboxylase/oxygenase large subunit indicated sa treatment increased extractable rdr activity in mtrdr1-transgenic n. benthamiana plants but did not alter mtrdr transcript accumulation sa had a long-lasting effect on mtrdr1-transgenic n. benthamiana plants that resulted in an improvement in recovery from tmv-induced disease and resumption of growth. this suggested that sa enhances or primes rdr1-mediated defense against tmv. however, mtrdr transgene expression in these plants is under the control of the cauliflower mosaic virus 35s promoter that is, despite containing an as- element shared with many sa-responsive plant promoters, not activated by this phytohormone. to confirm that the 35s promoter used in the construction of the mtrdr1-transgenic plants was behaving as expected, i.e. that it was not responding to sa, we extracted total rna from leaves of control-transgenic and mtrdr1-transgenic n. benthamiana plants 72 h after they were infiltrated with either 1 mm sa or a control solution. semi-quantitative reverse transcription-polymerase chain reaction assays showed that sa, but not the control solution, induced expression of the pr1a gene, indicating that this sa concentration was effective for the induction of sa-responsive gene expression. rt-pcr also indicated that whereas the transcript encoding the native, non-functional rdr of n. benthamiana was induced by sa, the transgene-encoded mtrdr transcript remained at a similar level in mtrdr1-transgenic plants in the presence or absence of sa treatment. analysis by rt-quantitative pcr confirmed that steady state levels of mtrdr transcript were not affected by sa treatment in the mtrdr1-transgenic n. benthamiana plants. thus, the 35s promoter used in the construction of the mtrdr1-transgenic plants is unaffected by sa, meaning that the observed enhancement of rdr1-mediated antiviral action by sa must be regulated at a post-transcriptional level.fig. rdr activity, but not mtrdr transcript accumulation, is induced by sa treatment in mtrdr1-transgenic n. benthamiana plants. semi-quantitative rt-pcr analysis of pr transcript accumulation in leaves of transgenic  control and mtrdr1-transgenic plants infiltrated with a control solution of  % ethanol or a solution of 1 mm sa in  % ethanol. infiltrated leaf tissue samples were harvested for rna extraction at 72 h post-infiltration. pr transcript accumulation levels were determined by rt-pcr after 40 cycles of pcr and compared relative to the accumulation levels of the elongation factor alpha transcript. increased pr accumulation confirmed that sa was taken up by the tissues and was effective in inducing transcriptional changes. c semi-quantitative rt-pcr analysis showed that there was little difference in mtrdr transcript accumulation in mtrdr1-transgenic plants infiltrated with control solution or 1 mm sa. nbrdr1m transcript accumulation was up-regulated in both transgenic control and mtrdr1-transgenic n. benthamiana plants after sa treatment, although the nbrdr1m protein itself is non-functional. mtrdr and nbrdr1m transcript accumulation levels after and 35 cycles, respectively, were compared relative to the accumulation levels of ef1α. infiltrated tissue samples were harvested at 72 h post-infiltration. d rt-qpcr analysis of mtrdr transcript levels in leaves of empty vector control and mtrdr1-transgenic plants infiltrated with water control or 1 mm sa solution. mtrdr was not detected in empty vector control plants. mean values for relative mtrdr levels obtained from three plants have been given for each treatment group. error bars represent standard errors of the mean for the three samples. relative transcript levels of mtrdr were calculated using the 2-ΔΔc method using ef1α as an internal reference. e enhancement of rdr activity by sa in mtrdr1-transgenic plants. leaves from tobacco and transgenic empty vector control and mtrdr1-transgenic n. benthamiana plants were infiltrated with water containing  % ethanol or  mm sa in  % ethanol and harvested after 48 h for preparation of rdr1-enriched extracts. the proxy for rdr activity was the incorporation of α- ctp into nascent rna analysed by liquid scintillation counting of radioactivity incorporated into trichloroacetic acid-precipitable material. error bars are standard errors for the mean for three technical replicates per sample in order to determine if sa treatment affected rdr activity levels, extracts enriched in rdr activity were prepared from non-infected control transgenic and mtrdr1-transgenic n. benthamiana leaves infiltrated with sa or control solution 48 h prior to tissue harvesting. n. tabacum possesses the sa-inducible gene ntrdr which encodes a functional rdr, and so samples from non-transgenic n. tabacum were included in the assays as positive controls for sa-inducible rdr activity. control-treated ev n. benthamiana plants contained active rdr not attributable to rdr and as expected, mtrdr1-transgenic n. benthamiana plants contained a higher level of extractable rdr activity than that detected in the ev control plants. although treatment with sa did not increase the amount of active rdr extractable from ev-control plants, sa treatment did increase rdr activity in mtrdr1-transgenic plants. this effect was only observed when sa was applied to intact plant tissue, and not when rdr extracts from untreated n. tabacum or mtrdr1-expressing n. benthamiana plants were incubated in vitro with sa or with a biologically inactive isomer of sa. mtrdr protein is detectable in rdr extracts from sa-treated mtrdr1-transgenic n. benthamiana plants since the in vitro rdr activity of extracts from mtrdr1-transgenic plants was increased approximately fold when the plants were pre-treated with sa, we investigated whether there was any change in the accumulation of mtrdr protein in these extracts. a polyclonal anti-mtrdr rabbit serum was prepared using a maltose binding protein mtrdr fusion protein made in escherichia coli. this was used for western blot analysis of rdr-enriched extracts. as the anti-mtrdr serum detected a number of background plant protein bands, western blot analysis with preimmune serum was also carried out. the only polypeptide specifically detected by the anti-mtrdr serum was of c.131 kda, the mass predicted for mtrdr, and this was present only in rdr-enriched extracts from sa-treated, mtrdr1-transgenic plants. ubiquitination and multiple phosphorylation sites are predicted to occur in the mtrdr protein sequence. this raises the possibility that sa treatment stabilizes the protein or, alternatively, that sa triggers recruitment of mtrdr into an active complex.fig. western blot analysis for mtrdr in rdr1-enriched protein extracts. leaves of empty vector control transgenic or mtrdr1-transgenic n. benthamiana plants were infiltrated with a control solution of water containing  % ethanol or  mm sa. rdr1-enriched protein extracts were prepared 48 h later and subjected to western blot analysis with either preimmune rabbit serum or polyclonal rabbit anti-mtrdr serum. the positions of protein molecular mass markers are indicated. a protein corresponding to the predicted size of 131 kda for mtrdr was specifically detected in sa-treated mtrdr1-transgenic plants with the anti-mtrdr serum but not with the preimmune serum. equal loading of gel lanes with protein, based on the bradford assay was confirmed by ponceau s staining of the western blot membrane discussion we studied the effects of rdr on the long-distance movement and spread of tmv. expression of mtrdr in n. benthamiana did not prevent tmv.gfp movement into non-inoculated leaf tissue or enhance the inhibitory effect of sa on virus movement into these tissues. however, mtrdr expression inhibited the extent of spread into the region proximal to the apical meristem and this movement restriction became more pronounced following treatment of mtrdr1-transgenic plants with sa. schwach and colleagues showed that knock-down of nbrdr gene expression in the same host allowed entry of pvx into the meristem and that this resulted in an exacerbation of pvx-induced disease symptoms throughout the plant. in line with previous work we found that in n. benthamiana plants, tmv.gfp enters tissue adjacent to the meristem, although it does not appear to enter the meristem itself. in mtrdr1-transgenic plants, however, the ability of tmv.gfp to approach the meristem is drastically curtailed. furthermore, in the mtrdr1-transgenic plants the normally lethal effect of tmvu is ameliorated and recovery from disease occurs. our results with constitutive expression of mtrdr complement and extend the results and conclusions of schwach and colleagues; indicating that rdr as well as rdr can function to inhibit virus entry to tissue adjacent to the meristem. both studies point to a relationship between the effectiveness of rna silencing-mediated exclusion of virus from meristematic and adjacent tissues with the severity of virus-induced disease symptoms. this suggests that, with respect to the elaboration of disease symptoms, that there is a critical developmental stage for plant cells and tissues, which corresponds physically to a certain point along the sub-apical zone. if viral entry into these tissues is delayed, for example by host defense mechanisms, until after this critical developmental stage has been completed, plants will to some extent be able to recover from virus infection. it has been postulated that the hyper-susceptibility of the laboratory accession of n. benthamiana to a number of tobamoviruses including tmv can be attributed to the absence of an active rdr in this species. ying and colleagues proposed an alternative hypothesis, suggesting that rdr can enhance virus spread and accumulation and that the loss of rdr functionality in n. benthamiana may be due to selective pressure to maintain high levels of rdr6-dependent antiviral defense. this hypothesis was formulated mainly to explain results after challenge with non-tobamoviruses, as challenge with tmv did not alter symptoms and at best only modestly decreased virus accumulation in systemically-infected tissue of n. benthamiana plants expressing ntrdr. we have seen no increase in susceptibility to tmv in our study of n. benthamiana plants expressing the transgene derived from the rdr of m. truncatula. furthermore, wild accessions of n. benthamiana that contain a non-truncated, functional nbrdr are naturally protected against severe symptoms from tobamovirus infection and are not more susceptible to poty- or cucumovirus infection than n. benthamiana carrying nbrdr1m indicating that rdr does indeed have a particular role in ameliorating tobamovirus infection in n. benthamiana. a difference in the effects of rdr and rdr on virus spread is apparent in the pattern of spread of pvx.gfp into upper uninoculated leaves previously observed in rdr6i plants and in the pattern of systemic tmv.gfp movement in mtrdr1-transgenic plants in the present study. specifically, knock-down of nbrdr accelerated entry of virus into well-developed upper leaves, whereas constitutive expression of mtrdr did not slow the appearance of tmv.gfp in these leaves. these differences suggest that the roles of rdrs and do not overlap completely and that each may be effective in protection against different viruses with differing infection and movement strategies, as suggested previously. these results suggest that rdr may play a role similar to rdr in protecting the meristem from viral invasion. the strong atrdr promoter activity in arabidopsis phloem tissue observed by xu and colleagues may also help restrict the spread of virus into the vasculature and subsequently the meristem, although curiously the atrdr promoter:β-glucuronidase reporter transgene was found to be poorly active in younger tissues. however, since rdr gene expression is induced by chemical signals associated with sar as well as by many other phytohormones and wounding, it may be that the multiplicity of factors affecting rdr expression, and its therefore complex regulation, allows plants to reinforce meristem protection against invasion in times of stress or perhaps control the accessibility of this tissue to endogenous factors during development. previous studies showed that plants expressing a functional rdr are less susceptible to tmv-induced disease, yet rdr does not seem to be required for successful sa-induced resistance to viruses in directly inoculated leaves. interestingly, we found here not only that expression of mtrdr enabled n. benthamiana to recover from severe disease induced by tmv strain u but also that this recovery was enhanced by sa treatment. whilst this could have been due to the activity of one or more of the rdr1-independent sa-inducible antiviral defense systems that are known to exist, sa treatment of control-transgenic n. benthamiana plants only delayed tmv-induced plant death by a few days suggesting that the protective activity of other sa-induced antiviral defense systems was insufficient in this case to account for the marked enhancement of recovery after sa treatment in the mtrdr1-transgenic n. benthamiana plants. although sa and mtrdr worked synergistically in protection against severe tmv-induced disease, it is not possible to say with certainty whether or not these factors were working together or separately. however, the hypothesis that sa enhanced rdr activity was favored by the observation that mtrdr1-transgenic plants contained more extractable rdr activity following sa treatment, despite there being no concomitant sa-induced increase in mtrdr transgene expression. there was no direct effect of sa on in vitro rdr activity in plant extracts, which indicates that sa indirectly influences rdr activity in planta. detection of a band cross-reacting with an antibody raised against mtrdr in rdr1-enriched preparations from sa-treated but not untreated mtrdr1-transgenic plants was consistent with the detection of greater rdr activity. it also suggested that rdr can be regulated by sa through post-translational process either resulting in a more stable mtrdr or involving recruitment of mtrdr protein to a complex. an in silico analysis of the mtrdr protein sequence revealed a number of candidate ubiquitination and phosphorylation sites. post-translational protein modification plays an important role in host immunity, for example: ubiquitin-dependent proteolysis negatively regulates defense responses in tobacco; npr and are e ligase adapters that control npr activity by proteolysis in an sa-dependent manner in arabidopsis; and sumo proteases negatively regulate sa biosynthesis. it may therefore be that sa induces, in some manner, stabilization of the mtrdr protein against small modifier protein-mediated degradation and/or activation/deactivation of the protein by phosphorylation. the idea of assembly of a complex, which includes rdr and forms in response to sa, is made more plausible by recent findings that several rna silencing factors, including rdr form complexes with each other and with other cellular components such as membranes. another possibility is that sa enhances translation of the mtrdr transcript leading to increased synthesis of mtrdr protein but it is difficult to envisage a likely mechanism for this.  in conclusion, our results support an important role for the phytohormone-inducible factor rdr in resistance by limiting viral access to the region adjacent to the meristem and thereby ameliorating the severity of virus-induced disease. its ability to inhibit access to the meristematic region correlates with amelioration of tmv-induced disease symptoms and is enhanced by sa. our data suggests that sa may enhance rdr activity at a post-transcriptional level in addition to the previously documented effects of this phytohormone on transcription of the rdr gene. 
but although it contributes to basal resistance to tobacco mosaic virus it is dispensable for sa-induced resistance in inoculated leaves. rdr is constitutively expressed and was previously shown to limit invasion of nicotiana benthamiana meristem tissue by potato virus x and thereby inhibit disease development. thus, rdr may act in a similar fashion to rdr mtrdr and sa acted additively to further promote recovery from disease symptoms in mtrdr1-transgenic plants. in mtrdr1-transgenic n. benthamiana plants the spread of tmv expressing the green fluorescent protein into upper, non-inoculated, leaves was not inhibited. following sa treatment of mtrdr1-transgenic plants, extractable rdr enzyme activity was increased and western blot analysis of rdr extracts revealed a band cross-reacting with an antibody raised against mtrdr expression of mtrdr in the transgenic n. benthamiana plants was driven by a constitutive 35s promoter derived from cauliflower mosaic virus, confirmed to be non-responsive to sa. however, in these plants exclusion of tmv.gfp from the apical meristem and adjacent stem tissue was greater than in control plants and this exclusion effect was enhanced by sa. recovery from disease was markedly enhanced by sa treatment in mtrdr1-transgenic plants whereas in control plants sa delayed but did not prevent systemic necrosis and death. the laboratory accession of n. benthamiana is a natural rdr mutant and highly susceptible to tmv. tmv normally kills n. benthamiana plants but although mtrdr1-transgenic plants initially displayed virus-induced necrosis they subsequently recovered. this suggests that the effects of sa on mtrdr are exerted at a post-transcriptional level. however, tmv-induced symptoms are ameliorated in transgenic plants expressing medicago truncatula rdr rdr is inducible by salicylic acid and several other phytohormones.
 host rna-dependent rna polymerases and contribute to antiviral rna silencing in plants. rdr is constitutively expressed and was previously shown to limit invasion of nicotiana benthamiana meristem tissue by potato virus x and thereby inhibit disease development. rdr is inducible by salicylic acid and several other phytohormones. but although it contributes to basal resistance to tobacco mosaic virus it is dispensable for sa-induced resistance in inoculated leaves. the laboratory accession of n. benthamiana is a natural rdr mutant and highly susceptible to tmv. however, tmv-induced symptoms are ameliorated in transgenic plants expressing medicago truncatula rdr  in mtrdr1-transgenic n. benthamiana plants the spread of tmv expressing the green fluorescent protein into upper, non-inoculated, leaves was not inhibited. however, in these plants exclusion of tmv.gfp from the apical meristem and adjacent stem tissue was greater than in control plants and this exclusion effect was enhanced by sa. tmv normally kills n. benthamiana plants but although mtrdr1-transgenic plants initially displayed virus-induced necrosis they subsequently recovered. recovery from disease was markedly enhanced by sa treatment in mtrdr1-transgenic plants whereas in control plants sa delayed but did not prevent systemic necrosis and death. following sa treatment of mtrdr1-transgenic plants, extractable rdr enzyme activity was increased and western blot analysis of rdr extracts revealed a band cross-reacting with an antibody raised against mtrdr expression of mtrdr in the transgenic n. benthamiana plants was driven by a constitutive 35s promoter derived from cauliflower mosaic virus, confirmed to be non-responsive to sa. this suggests that the effects of sa on mtrdr are exerted at a post-transcriptional level.  mtrdr inhibits severe symptom development by limiting spread of virus into the growing tips of infected plants. thus, rdr may act in a similar fashion to rdr mtrdr and sa acted additively to further promote recovery from disease symptoms in mtrdr1-transgenic plants. thus it is possible that sa promotes mtrdr activity and/or stability through post-transcriptional effects. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords systemic acquired resistancehypersensitive responsevirus movementrnaipost-transcriptional gene silencingeffector-triggered immunitydefensive signal transductionhttp://dx.doi.org/http://dx.doi.org/ /501100003341cambridge overseas trustministry of education of taiwanhttp://dx.doi.org/ /501100000268biotechnology and biological sciences research councilbb/d014376/1bb/j011762/1the leverhulme trustf/09741/frpg-20120667the cambridge university isaac newton trusthttp://dx.doi.org/http://dx.doi.org/ /100000944samuel roberts noble foundationissue-copyright-statement© the author 2016 