High-level expression of alternative oxidase protein sequences enhances the spread of viral vectors in resistant and susceptible plants

Alex M. Murphy, Androulla Gilliland, Caroline J. York, Belinda Hyman and John P. Carr

Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK

Correspondence
Alex M. Murphy
amm1013{at}hermes.cam.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The alternative oxidase (AOX) is the terminal oxidase of the cyanide-resistant alternative respiratory pathway in plants and has been implicated in resistance to viruses. When tobacco mosaic virus (TMV) vectors were used to drive very high levels of expression of either AOX or AOX mutated in its active site (AOX-E), virus spread was enhanced. This was visualized as the induction of larger hypersensitive-response lesions after inoculation onto NN-genotype tobacco than those produced by vectors bearing sequences of comparable length [the green fluorescent protein (gfp) gene sequence or antisense aox] or the ‘empty’ viral vector. Also, in the highly susceptible host Nicotiana benthamiana, systemic movement of TMV vectors expressing AOX or AOX-E was faster than that of TMV constructs bearing gfp or antisense aox sequences. Notably, in N. benthamiana, TMV.AOX and TMV.AOX-E induced symptoms that were severe and ultimately included cell death, whereas the empty vector, TMV.GFP and the TMV vector expressing antisense aox sequences never induced necrosis. The results show that, if expressed at sufficiently high levels, active and inactive AOX proteins can affect virus spread and symptomology in plants.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The precise details of salicylic acid (SA)-induced plant antiviral mechanisms have not yet been fully elucidated, but they can affect virus replication (Chivasa et al., 1997; Naylor et al., 1998; Wong et al., 2002), cell-to-cell movement (Murphy & Carr, 2002) and long-distance movement from inoculated leaves to other parts of the plant (Naylor et al., 1998). SA is an essential component of the signal-transduction pathway(s) that establish systemic acquired resistance (SAR) (Dempsey et al., 1999). It accumulates in plant tissues following a hypersensitive response (HR), a resistance response that is normally characterized by a zone of programmed cell death (PCD) around the point of entry of an avirulent pathogen (Heath, 2000). SA induces the expression of many defence-related genes, most notably the pathogenesis-related (PR) proteins (van Loon & van Strien, 1999). Some of these have been shown to possess antimicrobial activity in vitro and in planta (Schlumbaum et al., 1986; Mauch et al., 1988; Alexander et al., 1993). However, constitutive expression of several types of PR protein did not elevate resistance towards viruses (Cutt et al., 1989; Linthorst et al., 1990). Indeed, the plant defensive signal-transduction pathway has been shown to branch downstream of SA (Murphy et al., 1999). One branch is dependent on NPR1 activity, which drives PR-protein expression and provides resistance against cellular microbes (Cao et al., 1997). The other branch is independent of NPR1, can be induced with non-lethal concentrations of respiratory inhibitors (Kachroo et al., 2000; Wong et al., 2002) and brings about enhanced resistance to viruses (Chivasa et al., 1997; Chivasa & Carr, 1998; Wong et al., 2002).

The alternative oxidase (AOX) is encoded by a small family of nuclear genes (Vanlerberghe & McIntosh, 1997) and is the terminal oxidase of the cyanide-resistant alternative respiratory pathway (AP; Affourtit et al., 2001, 2002). The AP branches from the main cytochrome pathway (CYT) at the ubiquinone pool, where electrons pass directly to the AOX, bypassing proton pumping by complexes III and IV and thus reducing the proton motive force that is available to drive ATP production. The AP can thus act as a safety valve, siphoning off electrons that could otherwise cause overreduction and subsequent damage of the inner mitochondrial membrane through the accumulation of reactive oxygen species (ROS; Yip & Vanlerberghe, 2001). In fact, the AP has been shown to play a role in the protection of both the mitochondrion and, ultimately, the whole cell (Maxwell et al., 1999).

ROS are produced in plants at all times as by-products of normal metabolic activity (Noctor & Foyer, 1998). To ensure that ROS concentrations do not reach toxic levels, there needs to be a balance between production and detoxification of ROS to maintain ‘redox homeostasis' of the cell and its subcompartments (Noctor & Foyer, 1998; Dutilleul et al., 2003). ROS function as signalling molecules during defence against pathogens, e.g. during the oxidative burst that accompanies the HR (Heath, 2000). Furthermore, changes in cell redox status control SA-induced PR-protein expression by redox-sensitive posttranslational modification of NPR1 and a TGA/OBF transcription factor with which it interacts (Després et al., 2003; Mou et al., 2003). In this way, cellular mechanisms that maintain redox homeostasis can also play a role in defensive signalling.

Several lines of evidence implicate the AOX in signalling during defence against viruses (Singh et al., 2004). SA induces increased aox expression (Raskin et al., 1987; Rhoads & McIntosh, 1992). Treatment of plants with salicylhydroxamic acid (SHAM), an inhibitor of AOX activity, antagonizes SA-induced resistance to viruses, at least in tobacco (Chivasa et al., 1997; Naylor et al., 1998; Wong et al., 2002). Equally, treatment of plants with non-lethal concentrations of inhibitors of the CYT that engage the AP (antimycin A and cyanide) also promotes resistance to viruses (Chivasa & Carr, 1998; Wong et al., 2002; Gilliland et al., 2003) and restores localization of tobacco mosaic virus (TMV) in salicylate hydroxylase-expressing, transgenic, NN-genotype tobacco (Chivasa & Carr, 1998). However, constitutive over- or underexpression of aox in nn-genotype tobacco did not have any gross effect on the timing or appearance of symptoms after TMV inoculation (Ordog et al., 2002; Gilliland et al., 2003). Nevertheless, subsequent detailed examination revealed that overexpression of AOX did antagonize antimycin A-induced resistance to TMV, whereas a reduction in AP capacity transiently enhanced both SA- and antimycin A-induced resistance in directly inoculated tissue (Gilliland et al., 2003). We have proposed that AOX may function as a regulator of ROS-mediated signalling in the mitochondrion and that this potentially AOX-regulated signalling mechanism appears to be involved in the activation of a subset of SA-inducible antiviral defences (Gilliland et al., 2003; Singh et al., 2004).

Here, we set out to further examine the role of AOX in resistance to TMV by transiently overexpressing AOX from a TMV vector in non-transformed NN-genotype tobacco and the highly susceptible host Nicotiana benthamiana.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plant growth conditions.
Seeds of tobacco (Nicotiana tabacum L.) cultivar Xanthi-nc and of N. benthamiana were germinated in soil and maintained under greenhouse conditions with supplementary lighting in winter. Virus-inoculated plants were also maintained under greenhouse conditions.

AP capacity measurements.
The AP capacity of infected plants was measured by obtaining intact cells from leaf strips using 0·5 % (w/v) macerase (Macerozyme R-10; Yakult Pharmaceutical) in 0·7 M mannitol, as described previously (Gilliland et al., 2003). Measurements of oxygen consumption were performed by using a Clarke-type oxygen electrode (Digital model 10; Rank Brothers) in the absence or presence of: 20 µM antimycin A or 1 mM cyanide (CYT inhibitors); 2 mM SHAM (AOX inhibitor); both types of inhibitor; or with the uncoupler p-trifluoromethoxyphenylhydrazone (FCCP; 0·2 µM). For each vector/host combination examined, at least three sets of triplicate measurements were carried out, in order to have statistically valid comparisons of AP capacity.

Plasmid constructions, in vitro transcription and plant inoculation.
Constructs pTMV.AOX and pTMV.asAOX were generated by insertion of sequences encoding AOX1a from ‘Bright Yellow’ tobacco in the sense or antisense orientations, respectively, between the PacI and XhoI sites of the modified TMV cDNA in p30B (Lacomme & Santa Cruz, 1999). The aox fragment was prepared by PCR amplification of the AOX1a cDNA using specific oligonucleotide primers that incorporated restriction-enzyme recognition sites at the 5' (PacI) and 3' (XhoI) termini of the amplified sequence. The antisense aox fragment was prepared in a similar way, except that the primers incorporated an XhoI site at the 5' terminus and a PacI sute at the 3' terminus of the amplified fragment. The construct pTMV.AOX-E was prepared by site-directed mutagenesis of pTMV.AOX, using the oligonucleotides 5'-GAAGCTGAAAATGCCAGGATGCACCTCATGAC-3' and 5'-GTCATGAGGTGCATCCTGGCATTTTCAGCTTC-3' to change glutamate 221 to alanine in the active site of tobacco AOX1a, following the manufacturer's instructions (Quikchange XL site-directed mutagenesis kit; Stratagene). This glutamate residue is the equivalent of the glutamate 270 residue in the Sauromatum guttatum AOX and is one of two that are essential for S. guttatum AOX enzyme activity (Albury et al., 1998; Affourtit et al., 2002).

For infection of plants with viral vectors, infectious RNA transcripts were synthesized from plasmids linearized with KpnI by using a T7 transcription kit (Ambion) and inoculated directly onto three lower leaves (with Carborundum as an abrasive) of 4-week-old N. benthamiana. Infectious sap was collected from N. benthamiana leaves 7 days after inoculation and sap inoculations were carried out on leaves of tobacco, as described previously (Murphy & Carr, 2002). The TMV U1 inoculum consisted of purified virions at a concentration of 1 µg ml–1.

Detection of AOX, TMV CP, PR1 and cytochrome c.
At various times following inoculation, samples were collected by harvesting inoculated or upper non-inoculated leaves. For each viral construct, five plants were inoculated. TMV coat protein (CP), PR1 protein and AOX levels in plant tissue were assessed by Western immunoblot analysis, using previously described methods (Chivasa et al., 1997; Gilliland et al., 2003). Cytochrome c leakage from mitochondria was assessed by comparison of cytochrome c levels in mitochondria versus the cytoplasm, following the method described by Hansen (2000). Experiments were carried out at least four times.

Hydrogen peroxide detection.
Leaf tissue was immersed in freshly prepared 3,3'-diaminobenzidine (DAB)/HCl stain (1 mg ml–1) for 1 h (Thordal-Christensen et al., 1997). Tissue was cleared in boiling ethanol and mounted in 30 % (v/v) lactic acid. In the presence of hydrogen peroxide and peroxidase, DAB polymerizes instantly to a brown precipitate.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression of active and inactive AOX from a TMV vector enhances virus spread in resistant and susceptible tobacco and N. tabacum plants
Wild-type TMV strain U1 and constructs derived from the TMV vector TMV.30B (Lacomme & Santa Cruz, 1999; Shivprasad et al., 1999) were inoculated in pairs onto opposite halves of NN-genotype tobacco leaves (cultivar Xanthi-nc). The TMV.30B-derived constructs were designed to express gene sequences of tobacco Aox1a (TMV.AOX), Aox1a in the antisense orientation (TMV.asAOX), Aox1a mutated in its predicted active site (Albury et al., 1998; TMV.AOX-E) and the green fluorescent protein (TMV.GFP). Inoculation of two different constructs onto opposite halves of the same leaf is the best method of comparison of HR-lesion size, as the developmental stage of the leaf has a profound effect on the size of the HR.

The HR lesions induced by TMV U1 were significantly larger than those induced by TMV.30B (Fig. 1a). This indicates that the engineered ‘empty’ vector, TMV.30B, is disabled relative to the ‘wild-type’ TMV (TMV U1) from which it is partly derived (i.e. TMV U1 can move more quickly from the point of inoculation prior to onset of the HR, compared to TMV.30B). Addition of the gfp or antisense aox sequence into TMV.30B further disabled the viral vector in terms of its ability to spread. Shivprasad et al. (1999) have previously reported that when the 30B vector carries ORFs the size of gfp or larger, it can no longer move systemically in TMV-susceptible tobacco, although systemic movement occurs in N. benthamiana. The very small HR lesions induced by TMV.GFP and TMV.asAOX were barely visible without magnification (Fig. 1b and data not shown). This makes the HR lesions that are induced by TMV.AOX and TMV.AOX-E all the more remarkable, as these HRs are similar in size to those produced in response to the unmodified, wild-type virus, TMV U1. This shows that insertion of the AOX- and AOX-E-coding sequences into TMV.30B actually enhances viral spread through plant tissue.



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Fig. 1. Wild-type and mutant aox sequences enhance the spread of a viral vector in a resistant host. (a) Diameter of HR lesions on leaves of Xanthi-nc (NN-genotype) tobacco, 3 days after inoculation on opposite leaf halves with combinations of TMV strain U1 (U1), the ‘empty’ viral vector (30B) and viral vectors TMV.AOX expressing sense aox transcripts (T.AOX) or TMV.AOX-E expressing aox transcripts mutated in the putative active site (T.AOX-E). These data are from one experiment and approximately 50 HR lesions developed on each leaf half. The experiment was repeated with similar results. Error bars indicate SEM. HR-lesion diameters that are significantly different by Student's t-test are marked with an asterisk. (b) Appearance of Xanthi-nc tobacco leaf at 6 days after inoculation with TMV.30B (upper leaf panel) or TMV.GFP (lower leaf panel). TMV.GFP induces microscopic HR lesions (arrowed). Bar, 5 mm.

 
Characterization of alternative respiratory capacity in the susceptible host N. benthamiana infected with TMV vectors expressing aox gene sequences
To further analyse the effects of these viral constructs on virus spread and cell physiology, we used a susceptible host that does not normally undergo rapid HR cell death after infection. This makes it easier to examine virus-encoded protein expression and AOX-mediated oxygen consumption, as larger amounts of infected plant tissue can be examined in the absence of the oxidation reactions associated with the HR. We used N. benthamiana because it permits replication of TMV-derived vectors to high levels and, unlike N. tabacum, allows their systemic movement.

N. benthamiana plants were inoculated with TMV.30B, TMV.AOX, TMV.AOX-E or TMV.asAOX. By 10 days post-inoculation (d.p.i.), the virus had moved out from the leaves that were inoculated to the non-inoculated parts of the plants. Samples from the upper non-inoculated leaves were taken to assess the expression levels of AOX in systemically infected tissue. As we had supposed, the level of AOX protein expression that was achievable by using the viral vector was greater than that obtained in a stably transformed plant (Fig. 2a).



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Fig. 2. AOX protein accumulation and targeting in leaves of N. benthamiana systemically infected with viral vectors expressing aox sequences. (a) Comparison of AOX protein levels (arrowed) in N. benthamiana leaves systemically infected with TMV.AOX (T.AOX) or TMV.30B (30B) with a non-transgenic and a stably transformed Xanthi-nc (XNN) tobacco plant (Sn6) expressing Aox1a under the CaMV 35S promoter. (b) Immunoblot analysis of AOX protein accumulation in the membrane fraction (proteins released by Triton X-100) of leaves infected with TMV.AOX (expressing sense aox) and TMV.AOX-E (expressing a construct mutated in the putative active site of AOX). Arrows indicate position of the reduced form of AOX (with an apparent molecular mass of 35 kDa). Western blotting for TMV CP (not shown) authenticated the presence of virus in these leaves. (c) Non-reducing and reducing SDS-PAGE and immunoblot analysis of AOX in N. benthamiana systemically infected with viral vectors expressing aox sequences. Proteins were extracted directly into non-reducing SDS loading buffer and one aliquot of this sample was loaded directly onto the gel (–DTT). Another aliquot was mixed with an equal volume of SDS loading buffer containing DTT and half of this was loaded on the gel (+DTT). Upper and lower arrows indicate the apparent molecular masses of oxidized and reduced AOX (70 and 35 kDa, respectively). Western blotting for TMV CP (not shown) authenticated the presence of virus in these leaves.

 
Soluble proteins in the cytosolic fraction and proteins that were solubilized from the membrane fraction of the cells with 0·1 % Triton X-100 were examined by Western blotting. AOX was only detectable in the Triton X-100-released membrane proteins from TMV.AOX- and TMV.AOX-E-infected tissues (Fig. 2b). AOX was not detectable in mock-inoculated plants or plants inoculated with TMV.30B or TMV.asAOX, indicating that the AOX accumulating in the membrane fraction of TMV.AOX- and TMV.AOX-E-infected plants was being expressed from the viral vector. Furthermore, very little AOX or AOX-E was detected in the cytosolic fraction, showing that the AOX expressed from the viral vector was being targeted correctly.

We then investigated whether the AOX expressed from the viral vector accumulated as either the non-covalently linked (reduced) or covalently disulphide-linked (oxidized) dimer in the inner mitochondrial membrane (Umbach & Siedow, 1993) (Fig. 2c). The relevance of this is that the reduced dimer is a more active form of AOX and its proportion of the total AOX increases when the AP is engaged (Umbach & Siedow, 1993). The two forms can be distinguished by non-reducing SDS-PAGE and Western blotting, where the oxidized form has twice the apparent molecular mass of the reduced form (Umbach & Siedow, 1993). Approximately half of the AOX expressed from the viral vector was present as the non-covalently linked dimer and the other half was present as the covalently linked dimer in non-reducing conditions. In the presence of dithiothreitol (DTT), all AOX protein was converted to the non-covalently linked form (Fig. 2c). This demonstrates that roughly 50 % of the AOX expressed from the TMV vector was present as the potentially more active, reduced form.

The respiratory characteristics of infected N. benthamiana plants were examined to assess whether expression of aox, aox-E or asAox sequences from the TMV-based vector had any effect on AP capacity (Fig. 3). It was observed that virus infection per se resulted in greater overall oxygen consumption. Thus, cells from mock-inoculated control plants consumed, on average, 5·65 nmol O2 min–1 (106 cells)–1, whereas infected plants had higher rates of respiration of 10·93, 10·34, 9·38 and 6·77 nmol O2 min–1 (106 cells)–1 in plants infected with TMV.30B, TMV.asAOX, TMV.AOX and TMV.AOX-E, respectively. When the relative contributions of AP and CYT to overall O2 consumption were determined, more striking differences were noted between mock-infected and infected samples. In mock-inoculated plants, the AP capacity was 40 % of the total oxygen consumption; this changed to 48 and 38 % for TMV.30B- and TMV.asAOX-infected plants, respectively (Fig. 3b). AP capacity was consistently enhanced (to 60 % of total oxygen consumption) in TMV.AOX-infected plants, whereas it was reduced (to 28 %) in TMV.AOX-E-infected plants.



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Fig. 3. Respiratory characteristics of systemically infected leaves of N. benthamiana inoculated with viral vectors expressing aox-derived sequences. (a) Cells were isolated from leaves of N. benthamiana that had been mock-inoculated (Mock) or had become systemically infected with TMV.30B (30B), TMV.asAOX, TMV.AOX or TMV.AOX-E and used for the measurement of CYT capacity (white bars) and AP capacity (grey bars). Data are expressed as means±SEM (mock, n=12; TMV.30B, n=8; TMV.asAOX, n=6; TMV.AOX, n=7; TMV.AOX-E, n=12). (b) The same data from (a), expressed as the relative proportion of total oxygen consumption (100 %) attributable to the CYT (white bars) versus the AP (grey bars) in mock-inoculated or infected plants.

 
These data indicate that AOX expressed by the TMV vector is functional and can enhance the AP, whereas mutant AOX-E expressed from the viral vector can interfere with and decrease the AP capacity of host cells. Thus, we assume that AOX-E is genuinely mutated in its active site and can depress AP capacity by interfering with the formation of active, dimeric AOX, presumably by dimerization with pre-existing wild-type AOX monomers. AP capacity was not affected by infection with TMV.asAOX, indicating that transcription of an antisense version of the aox cDNA did not decrease aox expression, for example by initiating RNA silencing of native aox transcripts. Altogether, these results demonstrate that a TMV vector can mediate delivery of AOX or AOX-derived proteins to the inner mitochondrial membrane in a correctly folded and active state.

Expression of both active and inactive forms of AOX from a TMV vector enhances virus movement and symptom induction, as well as inducing cell death, in the susceptible host N. benthamiana
N. benthamiana infected with TMV.30B, TMV.AOX or TMV.AOX-E showed typical symptoms of vein-clearing and leaf curling in the upper leaves between 5 and 6 d.p.i. Symptoms were most severe in plants inoculated with TMV.AOX and TMV.AOX-E (Fig. 4). TMV.asAOX-infected plants showed symptoms much later (8–12 d.p.i.), whereas TMV.GFP-infected plants typically showed symptoms at 7–10 d.p.i.



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Fig. 4. In N. benthamiana, wild-type and mutant aox sequences induce cell death when expressed from a TMV vector. (a) Leaves 7 days after inoculation with TMV.30B, TMV.AOX or TMV.AOX-E. Arrows indicate chlorosis. (b) Comparison of stunting and deformation in plants infected with TMV.AOX, TMV.AOX-E, TMV.asAOX or TMV.30B. Photograph was taken 15 days after inoculation. (c) Cell death (arrowed) in a leaf where unloading of TMV.AOX occurred during the sink–source transition. Photograph was taken 15 days after inoculation. (d) Overhead view of TMV.AOX-infected N. benthamiana 15 days after inoculation. Arrow indicates necrosis. (e) Detail of cell death (arrowed) in leaves systemically infected with TMV.AOX-E, 15 days after inoculation. Bars, 1 cm.

 
Accumulation of TMV CP in directly inoculated, as well as upper non-inoculated, leaves was assayed at various time points by Western blotting as a measure of viral vector accumulation (Fig. 5a and b). In line with the timing of symptom appearance in systemically infected leaves, TMV CP was detected earliest in plants inoculated with TMV.30B, TMV.AOX or TMV.AOX-E. Insertion of an additional gene sequence (e.g. gfp or antisense aox) into the TMV vector hindered virus spread, but vectors carrying the aox or aox-E gene sequences moved systemically at a rate similar to that of the empty vector, indicating that these sequences enhance the ability of the virus to spread.



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Fig. 5. TMV CP accumulation in N. benthamiana and tobacco plants infected with the viral vector expressing aox sequences. (a) TMV.30B (30B), TMV.AOX, TMV.AOX-E and TMV.asAOX CP accumulation in inoculated and systemically infected N. benthamiana leaves. (b) TMV.GFP, TMV.AOX and TMV.asAOX CP accumulation in inoculated and systemically infected leaves. (c) TMV.GFP and TMV.AOX CP accumulation in directly inoculated Xanthi tobacco leaves (nn genotype) at 10 d.p.i. Proteins were extracted from inoculated or systemically infected upper leaves and separated by SDS-PAGE, immunoblotted and assayed by using polyclonal anti-TMV CP serum.

 
Cell death occurred only in plants inoculated with TMV.AOX or TMV.AOX-E (Fig. 4). In the inoculated leaves, striking chlorosis that was first noticeable at 6 d.p.i. (Fig. 4a) preceded cell death, which was first apparent at the virus infection sites by 9–11 d.p.i. Chlorosis, followed by cell death, did not occur in the directly inoculated leaves of plants inoculated with TMV.30B, TMV.asAOX or TMV.GFP. Cell death also occurred in the systemically infected leaves of TMV.AOX- and TMV.AOX-E-inoculated plants between 10 and 15 d.p.i., usually appearing slightly earlier in the TMV.AOX-E-inoculated plants (Fig. 4b, c, d and e). Virus accumulation and movement were slower in TMV.asAOX-inoculated plants (Fig. 5a and b); this may account for the milder symptoms seen in these plants. However, TMV.30B (the empty vector), TMV.AOX and TMV.AOX-E accumulated to similar levels, demonstrating that the more intense symptoms and cell death seen after TMV.AOX and TMV.AOX-E inoculation were not due to increased virus accumulation in these plants.

In our experience, when wild-type U1 TMV is inoculated into N. benthamiana, symptoms can include necrosis, but this only begins to occur after several weeks (in excess of 30 d.p.i.; data not shown and Yang et al., 2004). This is much later than the extensive cell death that is apparent in plants infected with TMV.AOX or TMV.AOX-E (10–15 d.p.i.). However, it should be noted that some researchers find that wild-type TMV U1 or clones derived from it can cause severe necrosis and death within 1 week of inoculation (e.g. Rabindran & Dawson, 2001). We suspect that the speed of onset of TMV-induced necrosis is dependent on growth conditions.

TMV CP accumulation was also analysed in the susceptible tobacco host Xanthi (nn genotype) inoculated with TMV.GFP or TMV.AOX (Fig. 5c). TMV.AOX CP accumulated to greater levels than TMV.GFP, again indicating that expression of the aox sequence enhances the ability of the virus to spread. Ten days after inoculation, no necrosis was apparent on TMV.AOX-inoculated Xanthi leaves, nor were any symptoms visible on upper non-inoculated leaves (data not shown), indicating that in N. tabacum hosts, expression of AOX does not enable the TMV vector to overcome barriers to systemic movement.

Necrosis induced by TMV.AOX and TMV.AOX-E is not accompanied by induction of markers for PCD or SAR
Markers for PCD were also investigated in infected leaves showing visible necrosis. Cytochrome c leakage from mitochondria was not evident in TMV.AOX- or TMV.AOX-E-infected leaves (Fig. 6a); neither was DNA laddering (data not shown). However, DNA laddering is not always associated with plant PCD (reviewed by Birch et al., 2000).



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Fig. 6. TMV.AOX- and TMV.AOX-E-infected cells that are dying do not leak cytochrome c, but do accumulate hydrogen peroxide. (a) Cytochrome c leakage in leaves infected with TMV.30B (30B), TMV.asAOX, TMV.AOX or TMV.AOX-E (15 d.p.i.). Cytochrome c protein was analysed in the cytosolic (C) and the membrane/organelle (P) fractions from infected leaves. Arrow indicates the apparent molecular mass of 14 kDa that corresponds to cytochrome c. (b) DAB staining for hydrogen peroxide accumulation in TMV.30B-, TMV.AOX- and TMV.AOX-E-infected leaves at 15 d.p.i. Bars, 200 µm.

 
We did find that hydrogen peroxide, an often-used biological indicator for PCD, accumulated in chlorotic areas of leaves prior to cell death in TMV.AOX- or TMV.AOX-E-infected leaves, but did not accumulate in the chlorotic areas of TMV.30B-infected leaves (Fig. 6b). These results show that accumulation of high levels of both AOX and AOX-E brought about an increase of hydrogen peroxide that preceded cell death.

During the HR, cell death is usually accompanied by increased biosynthesis of SA, which leads to induction of PR-protein genes. To further characterize the cell death, we analysed the induction of PR1 protein after inoculation with TMV.30B, TMV.AOX, TMV.AOX-E or TMV.asAOX (Fig. 7). Surprisingly, the strongest induction of PR1 was in TMV.30B-infected N. benthamiana leaves. PR1 was also induced by the other constructs, but more slowly than in TMV.30B-infected tissues (Fig. 7a). By 17 d.p.i., when cell death had been apparent on the upper leaves of TMV.AOX- and TMV.AOX-E-infected leaves for 3 days, PR1 had accumulated to similar levels in TMV.30B-, TMV.AOX- and TMV.AOX-E-infected leaves (Fig. 7b). However, PR1 levels were still relatively low in TMV.asAOX-infected leaves.



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Fig. 7. PR1 protein induction in N. benthamiana leaves by virus vectors. (a) Immunoblot analysis of PR1 accumulation in inoculated and systemically infected upper leaves of plants inoculated with TMV.30B (30B), TMV.AOX, TMV.AOX-E or TMV.asAOX. (b) PR1 and TMV CP accumulation in the upper leaves of TMV.30B-, TMV.AOX-, TMV.AOX-E- and TMV.asAOX-infected plants. For (a) and (b), protein was also extracted from N. benthamiana plants that had been pretreated with 1 mM SA for 3 days to provide an N. benthamiana PR1 size marker.

 
These results show that the degree of PR1 accumulation was not dependent on cell death, so we cannot conclude that the cell death observed in these experiments had exactly the same characteristics as PCD during the HR.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Use of a TMV vector to drive transient, high-level expression of either AOX or AOX-E enhanced virus spread and thereby induced larger HR lesions than those produced by the ‘empty’ viral vector in NN-genotype tobacco. Also, systemic movement of TMV vectors expressing AOX or AOX-E in the susceptible host N. benthamiana progressed at a similar rate to that of the empty vector TMV.30B and faster than that of TMV constructs bearing sequences of comparable length (gfp or antisense aox). Eventually, though, all of the viral constructs reached similar levels in systemically infected leaves. Notably, in N. benthamiana, TMV.AOX and TMV.AOX-E induced symptoms that were severe and ultimately included cell death. In contrast, TMV.30B, TMV.asAOX and TMV.GFP induced milder symptoms and never induced cell death. The results show that, if expressed at sufficiently high levels, active and inactive AOX proteins can affect virus spread and symptomology in plants.

Transient expression of aox-derived sequences from a viral vector enhances vector movement in resistant and susceptible host plants
Transient overexpression of both AOX and mutant AOX-E protein from the TMV vector enhanced virus spread in the TMV-resistant (NN-genotype) tobacco cultivar Xanthi-nc and the susceptible host N. benthamiana. In Xanthi-nc tobacco, this was seen as an increase in HR-lesion size, relative to that induced by infection with TMV.asAOX or TMV.GFP. Thus, although TMV.AOX and TMV.AOX-E, just like wild-type TMV U1 or TMV.30B, are prevented from spreading systemically by the N gene-mediated HR, they are able to spread more rapidly than TMV.asAOX or TMV.GFP before the onset of the HR. In N. benthamiana, the enhanced speed of movement of TMV.AOX and TMV.AOX-E relative to that of TMV.asAOX or TMV.GFP resulted in more rapid kinetics of viral protein accumulation in non-inoculated leaves and the earlier onset of symptoms. Although TMV.AOX caused necrosis in N. benthamiana, it did not in Xanthi (nn-genotype) tobacco. Thus, we hypothesize that the TMV.AOX-induced HR lesions on tobacco Xanthi-nc (nearly isogenic to the susceptible cultivar Xanthi) are a genuine measure of virus spread prior to the onset of cell death, rather than a combination of HR cell death and necrosis such as that seen in N. benthamiana.

These results suggest that expression of either AOX or AOX-E is allowing the viral vector to overcome, at least to some extent, a pre-existing or basal resistance to the spread of TMV. This contrasts with some of our own earlier results and conclusions (Gilliland et al., 2003), in which we found that although altering aox gene expression in stably transformed tobacco could affect certain aspects of induced resistance, it did not affect basal resistance or susceptibility to infection with TMV. We can only suggest that the far higher levels of aox expression (achievable with the viral vectors) are negatively affecting the operation of some form of basal resistance to the spread of TMV in Xanthi-nc tobacco and N. benthamiana. We have argued previously that mitochondrial ROS are signals that are involved in the regulation of a subset of antiviral-resistance responses (Gilliland et al., 2003; Singh et al., 2004). If this is true, any perturbation of mitochondrial redox could compromise the ability of the host to limit the spread of virus. The enhanced spread of viral vectors carrying AOX-derived proteins is consistent with this idea, as expression of either the functional (AOX) or non-functional (AOX-E) protein in plants did affect the operation of the AP. Possible mechanisms of action of the two proteins are discussed in the next section.

Interestingly, we recently found that although TMV.AOX can readily spread and form lesions in NN-genotype Xanthi-nc tobacco, it does not form HR lesions on plants of another NN-genotype cultivar, Samsun NN, unless these are transgenic for the bacterial salicylate hydroxylase-encoding gene, nahG (B. Hyman & J. P. Carr, unpublished data). This suggests that Samsun NN tobacco possesses a basal resistance to the spread of TMV that is stronger than that of Xanthi-nc. Although this basal resistance must be dependent in part on SA, it cannot be overcome by high-level expression of aox sequences from the virus. The result suggests that tobacco varieties vary widely in their ability to limit virus infection, independently of whether they possess a major single-gene resistance, such as that controlled by the N gene. In addition, the inability of TMV.AOX and TMV.AOX-E to induce HR lesions on Samsun NN indicates that these constructs are less fit than the wild-type TMV (which induces normal HR lesions).

Severe symptoms and cell death are induced by TMV-derived vectors expressing AOX and AOX-E in the susceptible host N. benthamiana
TMV.AOX and TMV.AOX-E caused more severe symptoms, including cell death, in the susceptible host than did the ‘empty’ TMV-vector, TMV.30B, or the vectors expressing gfp or the antisense aox sequence. This result was surprising because it did not appear to correlate either with the similar titres that were eventually attained by the various viral constructs, or in a straightforward way with the physiological effects of TMV.AOX and TMV.AOX-E.

We had seen that virus-mediated overexpression of active AOX or inactive AOX-E caused the predicted physiological effects of respectively enhancing or lowering the AP capacity (Fig. 3). However, as one function of the AOX is to lower mitochondrial (and ultimately cellular) ROS (Maxwell et al., 1999), we anticipated that TMV.AOX infection would decrease cellular ROS levels, whereas TMV.AOX-E infection would elevate them. However, increased amounts of hydrogen peroxide, a marker for ROS production, accumulated not only in TMV.AOX-E-infected cells, but also in those infected with TMV.AOX (Fig. 6b), and both constructs eventually caused cell death in their N. benthamiana hosts.

This does not appear to be either a consequence of virus infection per se, as TMV.30B infection did not induce hydrogen peroxide accumulation or cell death. Nor is it due to the functionality or otherwise of the AOX active site, as expression of both the unmodified and mutant aox sequences enhanced ROS production and caused cell death. One possibility is that any interference with the fine control of redox poise can eventually result in a runaway loss of control, leading to an unstoppable increase in ROS that causes cell death. There is at least one previous study that supports this idea. Creissen et al. (1999) made transgenic tobacco plants with increased capacity to synthesize a key antioxidant, glutathione (GSH), in the chloroplasts. Paradoxically, these plants suffered continual oxidative stress and the authors proposed that the high levels of GSH disrupted the redox-sensing process, which, in turn, upset the balance between ROS production and detoxification. Perhaps, in our study, the high-level expression of AOX or AOX-E protein interfered indirectly with the AOX-mediated detoxification of ROS in the mitochondria by upsetting the balance between active and less active dimeric forms of AOX (which itself is redox-controlled). Interestingly, interference with chloroplastic ROS production has also been implicated in basal viral defence, as silencing of a gene encoding a component of the oxygen-evolving complex of photosystem II enhanced virus replication in N. benthamiana (Abbink et al., 2002).

An alternative, but less likely, possibility that could account for increased ROS production and cell death in N. benthamiana plants infected with TMV.AOX or TMV.AOX-E is that extreme overaccumulation of any protein in the mitochondrion can in itself cause mitochondrial dysfunction. However, we found no obvious biochemical evidence of mitochondrial dysfunction prior to cell death, as shown by respiration measurements (Fig. 3), and examination of infected tissues by transmission electron microscopy did not reveal any consistent effect of AOX or AOX-E on mitochondrial ultrastructure (data not shown). Also, the late appearance of cell death in TMV.AOX- or TMV.AOX-E-infected N. benthamiana cells may not be consistent with the idea of protein overaccumulation causing mitochondrial damage.

Regardless of the exact mechanism that underlies cell death induced by high-level expression of AOX or AOX-E, it does not appear to have the characteristics of apoptotic cell death, such as cytochrome c leakage (Balk et al., 1999). Furthermore, cell-death induction does not appear to be simulating the HR-type cell death that occurs in resistant, NN-genotype hosts, as there is no correlation between PR-protein induction and cell death.

On balance, therefore, this necrosis phenomenon does not fulfil the criteria required for HR-related death (Heath, 2000). However, it has been noted that AOX protein does build up in tissues undergoing the HR, with very high levels probably occurring at the lesion centres (Lennon et al., 1997; Chivasa & Carr, 1998). It therefore remains a possibility that although AOX may not be the predominant determinant or trigger of cell death in the HR, it may contribute to cell death in a small number of cells by destabilizing mitochondrial function through one or both of the mechanisms outlined above.

Hypersusceptibility of N. benthamiana to TMV may prime it for induction of necrosis
N. benthamiana is remarkable in that it is generally far more susceptible to systemic infection with RNA viruses than other members of the genus Nicotiana and, for this reason, it has become one of the preferred model hosts for studies of resistance to viruses (Yang et al., 2004). It was recently shown that this hypersusceptibility to certain viruses, including TMV, results from possession of a RNA-dependent RNA polymerase 1 (RdRP1) gene encoding a non-functional product.

Host-encoded RdRPs can participate in the induction of RNAi, a homology-based destruction mechanism that can be targeted against foreign, overexpressed or aberrant RNA molecules in a sequence-specific manner (Grant, 1999; Baulcombe, 2001; Moissiard & Voinnet, 2004). In plants and other eukaryotes, RNAi has been shown to act as an antiviral mechanism (Moissiard & Voinnet, 2004). In tobacco and Arabidopsis thaliana, RdRP1 is known to be important in limiting viral titre through mediating the turnover of viral RNA, but is not required for the complete silencing of viral RNA (Xie et al., 2001; Yu et al., 2003). RdRP1 is also inducible by SA, but is not required for the induction of SA-induced resistance to viruses (Xie et al., 2001), although we have suggested that it may constitute an important component of SA-induced resistance to viruses (Gilliland et al., 2003). Could the responses of N. benthamiana to infection with TMV.AOX or TMV.AOX-E be explained by the defective nature of the RdRP1 gene in this host? The fact that TMV.AOX spread more rapidly than TMV.GFP in the susceptible tobacco variety Xanthi, but did not induce necrosis, indicates that cell-death induction by AOX is a process that requires a level of protein expression from the viral vector that can only be reached in the hypersusceptible host N. benthamiana or, we hypothesize, in the cells at the centre of an HR lesion.


   ACKNOWLEDGEMENTS
 
This work was funded with support from the Biotechnology and Biological Sciences Research Council (to J. P. C.) and the University of Cambridge Broodbank Trust (to A. M. M.). We thank Simon Santa Cruz for p30B and pTMV.GFP, Tom Elthon for anti-AOX mAb and Mike Wilson for anti-TMV CP serum.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 21 June 2004; accepted 18 August 2004.



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