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Plant Cell. Oct 2000; 12(10): 1975–1986.
PMCID: PMC149134

Complex Spatial Responses to Cucumber Mosaic Virus Infection in Susceptible Cucurbita pepo Cotyledons

Abstract

Cucumber mosaic virus infection of its susceptible host Cucurbita pepo results in a program of biochemical changes after virus infection. Applying a spatial analysis to expanding infected lesions, we investigated the relationship between the changes in enzyme activity and gene expression. Patterns of altered expression were seen that could not be detected by RNA gel blot analysis. For all the host genes studied, there was a downregulation (shutoff) of expression within the lesion. In addition, two distinct types of upregulation were observed. The expression of heat shock protein 70 (HSP70) and NADP+-dependent malic enzyme (NADP-ME) showed induction in apparently uninfected cells ahead of the infection. This response was more localized than the upregulation exhibited by catalase expression, which occurred throughout the uninfected regions of the tissue. The experiments showed that virus infection induced immediate and subsequent changes in gene expression by the host and that the infection has the potential to give advance signaling of the imminent infection.

INTRODUCTION

In a compatible interaction between a virus and its host, there must be substantial diversion of metabolites into the accumulation of virus-specific proteins and nucleic acids. In most susceptible plants, this interaction is not accompanied by cell death, and infected cells continue to function despite the presence of massive quantities of virus-specific products. This diversion of resources is evident in the whole plant as changes in plant physiology, growth, and development, that is, symptom formation. How plant cells accommodate virus synthesis while still sustaining some capacity in the plant for further growth is relatively poorly understood. We would expect the exploitation of host metabolism to be transitory, however, thus reducing the immediate damage to the host to a window of time sufficient for the exponential accumulation of progeny virus to a maximally tolerated amount. Because plant viruses invade host tissues progressively, such transitory effects would be spatially restricted and not necessarily detectable by gross analysis of the infected tissues.

Strong support for the transitory nature of responses to virus infection came from a unique study that applied a spatial analysis to changes in host physiology after infection of squash/marrow (Cucurbita pepo) cotyledons with Cucumber mosaic virus (CMV) (Técsi et al., 1996). The study showed that relative to an advancing infection front, changes in primary and secondary metabolism occurred at specific times after the onset of virus replication and virus gene expression. Hence, the center of the lesion had an increase in glycolysis and respiration and a decrease in the Benson–Calvin cycle. Conversely, at the extreme edge of the lesion, the enzymes involved in mobilizing carbon for macromolecular synthesis (anaplerotic reactions) had a transient increase in activity. For these enzymes, the earliest change was seen for the NADP+-dependent malic enzyme (NADP+-ME) (Técsi et al., 1996). There was also a corresponding increase of other anaplerotic activities (e.g., glucose-6-phosphate dehydrogenase [G6P-DH]) of the oxidative pentose phosphate pathway, which persisted into the center of the lesion. This led to the proposition that virus infection triggered a program of physiological responses that resulted in symptom expression.

Transient responses have also been observed in pea tissues infected with Pea seed–borne mosaic virus (potyvirus), Pea early browning virus (tobravirus), White clover mosaic virus (potexvirus), or Beet curly top virus (geminivirus) (Wang and Maule, 1995; Aranda et al., 1996; Escaler et al., 2000a), although these cases assessed changes in host gene expression rather than in host physiology. In infected pea tissues, three patterns of gene expression relative to an advancing infection front were observed. Many host gene transcripts were depleted in a zone occupying 10 to 12 cells behind the infection front, a phenomenon akin to the shutoff of host genes observed for many animal virus infections (Aranda and Maule, 1998). Some other host genes—heat shock protein 70 (HSP70), polyubiquitin, and gor2 (Aranda et al., 1996; Escaler et al., 2000b)—showed a marked but more transient upregulation at the infection front. A third class of genes, comprising actin, tubulin, and heat shock factor, showed no change in the amounts of mRNA after infection (Aranda et al., 1999; Escaler et al., 2000b).

These studies point to the elegant control of host gene expression and host physiology after the initiation of virus replication, but separately, they do not show how the processes are related. The work presented here addresses this by applying a spatial analysis of host gene expression to CMV-infected squash/marrow cotyledonary tissue. The results not only show correspondence (gene induction and shutoff) between pea and marrow in the response to virus replication but also indicate that tissues can show two further kinds of responses in uninfected cells ahead of the infectious front. Such responses suggest that host cells have the capacity to respond in advance to signals of the impending infection. The nature of these potential signals is investigated and discussed.

RESULTS

RNA Gel Blot Analysis of Host mRNAs

Three days after manual inoculation of marrow cotyledons with CMV, the infection was visible as small expanding chlorotic lesions. Under our standard experimental conditions, 40 to 50 lesions/cm2 formed on each cotyledon, occupying ~20% of the total leaf area. To obtain a preliminary assessment of changes in the expression of genes encoding some of these key enzymes, we analyzed steady state concentrations of mRNA in whole cotyledonary tissue extracts. For this, partial cDNA clones were isolated, sequenced, and used to prepare probes for RNA gel blot analysis. A list of the clones isolated is given in Table 1. HSP70 cDNA was also included because we had shown previously that this gene was strongly upregulated in response to the replication of a wide range of viruses in pea tissue (Aranda et al., 1996; Escaler et al., 2000a).

Table 1.
Cloned cDNAs from Squash/Marrow

All of the genes showed expression after mock inoculation of the tissues with buffer (Figure 1, lanes 2), although the basal expression of G6P-DH was very low. Of the six genes examined, three showed some increase and three were almost unchanged in steady state mRNA concentrations as a result of virus infection (Figure 1, lanes 1). Notable increases were seen for HSP70, NAD+-dependent malic en-zyme (NAD+-ME), and G6P-DH mRNAs. The mRNAs showing no or little change were those expressed by NADP-ME and by the A and B subunit genes of NADP-dependent glyc-eraldehyde-3-phosphate dehydrogenase (GAP-DH-A and -B).

Figure 1.
Gel Blot Analysis of Host mRNAs from CMV-Infected and Mock-Inoculated Tissues.

These data were surprising because they showed little correspondence with overall activity measurements or with predictions that were based on localized changes relative to the infection front. For example, in the case of NADP-ME, which had previously shown locally increased activity in response to infection (Técsi et al., 1996), the opposite change in mRNA contents was observed. The increase in the expression of HSP70 was in line with our previous observations in pea, although the RNA gel blot analysis did not distinguish between an overall increase in mRNA content and localized changes at the infection front.

Spatially Defined Changes in Host Gene Expression

To resolve the spatial changes in host gene expression, pieces of infected or mock-inoculated tissues were subjected to in situ hybridization to detect host mRNAs; immunohistochemistry was used to detect virus coat protein. We had previously shown a correspondence between the location of CMV coat protein (CP) and CMV RNA and hence the extent of the virus-infected lesion (Técsi et al., 1996).

Because of the complexity of the cotyledonary tissue (i.e., diverse cell types, highly vacuolated cells, and large intercellular spaces), clear changes in host gene expression could be seen only when any upregulation was localized, or when downregulation occurred against a background of high basal expression (i.e., in mock-inoculated tissues). Of the host genes initially selected (Table 1), G6P-DH and NAD-ME mRNAs produced hybridization patterns that were too weak or too dispersed to provide useful information. We also tried a cDNA for fumarate hydratase (an enzyme of the Krebs cycle, involved in respiratory metabolism in the mitochondrion), which also showed increased enzyme activity in the center of the lesion (Técsi et al., 1996), but it too was not useful (data not shown).

Three consecutive sections of tissue from either infected or mock-inoculated tissue were subjected, respectively, to hybridization with negative-sense probes to detect host mRNA, immunohistochemistry to detect CP (and the area of infection) or to hybridization with positive-sense probes as a control. In all the hybridization experiments, an apparently strong reaction was detected in some spongy mesophyll cells within the infected lesion (e.g., Figure 2C, arrow). Because this was seen with the positive-sense control probes, however, we attributed it to a sporadic change in the reaction of the infected cells to the fixation conditions rather than to a specific reaction to virus infection.

Figure 2.
Spatial Analysis of the Expression of HSP70 Associated with CMV Infection.

HSP70

Basal expression of HSP70 in control tissue (Figure 2F) could not be resolved above the background staining (Figure 2H). However, infected tissue had a marked accumulation of HSP70 mRNA at the edge of the lesion, that is, in the boundary zone between infected and uninfected regions, which is most obvious in the palisade mesophyll layer (Figure 2A, arrowheads). Aligning the consecutive sections (Figures 2A to 2C, dotted lines, magnified in Figures 2D and 2E) indicated that the upregulation of HSP70 spanned the infection front, resulting in detectable HSP70 mRNA as far as five cells ahead of the last infected cell. In the central portion of the lesion, HSP70 mRNA returned to a value indistinguishable from the background (Figures 2A and 2C).

NADP+-GAP-DH

NADP-GAP-DH, a multisubunit enzyme complex required for photosynthesis, is located in the chloroplast. The enzyme is nuclear-encoded by two highly related genes (for subunits A and B; see Table 1) (Brinkmann et al., 1989). After virus infection, the enzyme activity decreased progressively from the outside to the center of the lesion (Técsi et al., 1996; and illustrated in Figures 3I and and4I4I).

Figure 3.
Spatial Analysis of the Expression of GAP-DH-A Associated with CMV Infection.
Figure 4.
Spatial Analysis of the Expression of GAP-DH-B Associated with CMV Infection.

In situ hybridization showed that in mock-inoculated tissues, genes for both subunit A (Figure 3F) and subunit B (Figure 4F) were highly expressed in the palisade mesophyll cells but very little in the spongy mesophyll cells. The most striking effect of infection on these genes was the sharp depletion of transcript accumulation inside the periphery of the lesion (Figures 3D, ,3E,3E, ,4D,4D, and and4E).4E). Apparently, the removal of GAP-DH-A mRNA was more abrupt after the onset of infection than that seen for subunit B mRNA (cf. Figures 3D, ,3E,3E, ,4D,4D, and and4E).4E). In both cases, residual mRNA contents remained low through the central part of the lesion.

NADP+-ME

In the previous study (Técsi et al., 1996), a change in NADP-ME activity was the earliest specific response to virus invasion at the edge of the expanding lesion. The activity increased and then rapidly declined to the center of the lesion (Figure 5I). In situ hybridization showed that in mock-inoculated tissue, NADP-ME mRNA expression was most abundant in the vascular tissues and the upper cells of the spongy mesophyll layer (Figure 5F). In contrast with the data from the RNA gel blot analysis (Figure 1), which showed an overall slight decrease in NADP+-ME mRNA, in situ hybridization of sections of infected tissue showed an upregulation of expression. This was seen as increased mRNA accumulation outside of the lesion (i.e., in uninfected cells), extending for a considerable distance (~0.5 mm or 20 to 30 palisade mesophyll cells) from the outer edge of the lesion (Figure 5A, arrowheads). The range of this increase extended farther than that seen for HSP70 mRNA (Figure 2A). The increase was greatest close to the infected area and declined with distance from the infection front. The increase did not, however, extend to all the uninfected regions of the leaf. In the example shown in Figure 5A, the mRNA content in the central tissue region between two lesions (arrow) is similar to that in mock-inoculated tissue (Figure 5F). Similarly, the outer edge of the tissue section away from the lesion (Figure 5A, left) shows only a little expression.

Figure 5.
Spatial Analysis of the Expression of NADP+-ME Associated with CMV Infection.

Inside the infected area, the accumulation of NADP+-ME mRNA declined abruptly to less than that seen in mock-inoculated tissue (cf. Figures 5A and 5D with 5F). The exception here appears to be the mRNA within vascular tissues. In Figure 5A, a large vein (probably class III; white arrow) within the infected region retained the mRNA, despite its depletion from the surrounding cells. This was also observed for vascular tissues within other lesions (data not shown).

Long-Range Advance Signaling in Infected Cotyledons

The data from the in situ hybridization analysis for HSP70 and, more particularly, NADP+-ME mRNAs suggest that responses are induced in cells ahead of the advancing infection front. From studies in other systems, we propose some potential candidates for this activity. First, the CMV movement protein (MP) 3a has been shown to move from cell to cell in the absence of infection (Ding et al., 1995). This protein is believed to modify plasmodesmata to facilitate its own trafficking. Second, low molecular weight compounds (e.g., sugars, hormones) can diffuse rapidly through tissues to give distant and even systemic responses.

To investigate the potential for CMV 3a to mediate such signaling, consecutive sections of infected tissue were subjected to inmunohistochemistry with antibodies specific for CMV CP or 3a proteins (Figure 6). Alignment of the sections showed that the 3a protein accumulated throughout the infected area (cf. Figures 6A and 6B), although the concentration of 3a was probably greater at the edge of the lesion (Figures 6A and 6C). However, close examination of the front of infection (Figures 6C and 6D) showed that the accumulation of 3a protein occurred one to two cells ahead of the accumulation of CP, at most.

Figure 6.
Colocalization of the CMV CP and MP in Infected Tissues.

Low molecular weight compounds have been shown to be effector molecules for changing expression of a range of genes in plant–pathogen interactions. For example, peroxidase (Mayda et al., 2000), invertase (Herbers et al., 2000), PR genes (Van Loon, 1997), and catalase (Niebel et al., 1995) have all been shown to exhibit induced changes in expression away from the site of infection. To test whether the upregulation of NADP-ME could be linked to an equivalent signaling process, we wanted to compare the expression of NADP-ME with the pattern of expression of another distantly regulated gene. Unfortunately, for reasons outlined earlier, not all of these genes were suitable for comparative analysis by in situ hybridization. In the case of peroxidase, the enzyme is encoded by a large gene family, not all members of which need be coordinately regulated. In the case of invertase, basal expression in squash cotyledons was very low, and there was no detectable increase in infected tissues (Z. Havelda, unpublished data). PR genes are induced most commonly in response to a hypersensitive response; because no hypersensitive response is associated with the CMV–squash interactions, however, testing PR genes was not appropriate. Catalase, however, is induced in compatible plant–pathogen (bacteria and nematode) interactions (Niebel et al., 1995) and decreases in an incompatible interaction (tobacco mosaic virus infection of resistant tobacco; Yi et al., 1999).

A cDNA for squash catalase was cloned and sequenced (Table 1) and used to prepare probes for in situ hybridization. In mock-inoculated tissue, catalase mRNA accumulated predominantly in and around the vascular tissues (Figure 7F). In infected tissue, catalase mRNA showed increased accumulation outside the vascular tissue and mostly within the spongy mesophyll, particularly its upper cell layers (Figure 7A). This increase was also seen as an overall increase in mRNA, as detected by RNA gel blot analysis of total tissue RNA extracts (Figure 7I). In contrast to the pattern of increased expression seen for NADP-ME, catalase was increased almost uniformly throughout the uninfected regions of the cotyledon (cf. Figures 5A and and7A).7A). As with most of the other host genes examined, the accumulation of catalase mRNA was markedly depleted within the lesion (cf. Figures 7A and 7B) that coincided spatially with the first infected cells (cf. Figures 7D and 7E).

Figure 7.
Spatial Analysis of the Expression of Catalase Associated with CMV Infection.

DISCUSSION

The analysis of the intimate relationship between a plant virus and its host is considerably complicated by the progressive nature of the infection. A particular problem is posed by the time frame for replication within single cells relative to the period required for an infection phenotype to become visible. Hence, whereas replication in single cells may take only a few hours (Dolja et al., 1992), a few days may be required for lesions on inoculated leaves to become visible and days to weeks for full systemic symptoms to appear in susceptible hosts. The approach we have taken previously (Maule et al., 2000), and in this work, is to integrate time into the study of host responses by applying a spatial analysis to an advancing infection front. The benefit of this approach is illustrated well by the RNA gel blot analysis of host mRNAs in cotyledons inoculated with CMV (Figure 1). Of the four mRNAs subjected to gel blot and in situ hybridization analysis, only one (HSP70) showed changes at the tissue level that paralleled changes in recently infected cells (i.e., at the infection front). That is, HSP70 showed an average upregulation in whole-tissue extracts. In contrast, the average unchanged (GAP-DH-A and GAP-DH-B) or reduced (NADP+-ME) steady state accumulation of mRNA in tissues after infection masked specific and highly regulated changes at, ahead of, or behind the infection front. Unfortunately, in situ hybridization has technical limitations associated with its sensitivity and therefore cannot be applied to genes that display very weak expression in the tissues.

A fundamental question related to CMV infection of squash cotyledons was whether there was a correspondence between changes in enzyme activity (Técsi et al., 1996) and changes in host gene expression (mRNA accumulation). To address this, we cloned cDNAs for squash mRNAs that encoded enzymes previously found to be upregulated or downregulated in specific areas of the expanding lesions (i.e., at different times relative to virus infection). We also cloned squash HSP70 cDNA, given that this gene in pea had been shown previously to be tightly upregulated in response to many viruses (Escaler et al., 2000a). NADP+-ME, which showed increased enzyme activity at the periphery of the CMV lesion (Técsi et al., 1996), appears to be controlled at the mRNA level in response to CMV infection. Similarly, the reduced activity of GAP-DH, which correlated with reduced photosynthetic activity in the center of the CMV lesion (Técsi et al., 1996), appears to be related to decreased mRNA accumulation (Figure 8). Unfortunately, when used for in situ hybridization, cDNA probes gave inconclusive results for genes for which the products showed different activity profiles (NAD+-ME, G6P-DH, and fumarate hydratase) after infection (Técsi et al., 1996). This was disappointing with regard to NAD+-ME and fumarate hydratase in particular, given their strong increase in activity in the center of the lesion, an area in which many other activities and most of the mRNAs tested showed a marked decline. G6P-DH, which increased in activity immediately behind the infection front, also could not be analyzed by in situ hybridization. Nevertheless, RNA gel blot analysis for both NAD+-ME and G6P-DH suggests that they may be regulated at the transcriptional or mRNA level.

Figure 8.
Summary of Changes in Host Gene Expression in Response to CMV Infection.

In our previous work in pea (Maule et al., 2000), and for many animal virus infections (reviewed in Aranda and Maule, 1998), a common response to virus infection is seen as the downregulation (or shutoff) of host gene expression coincident with virus infection. Shutoff was also observed after CMV infection in squash cotyledons in which all of the host mRNAs examined by in situ hybridization were substantially depleted within the central area of the CMV-infected lesion (Figure 8). The precise timing of this mRNA turnover varied slightly between mRNA species. The factors controlling this are not known. The potential upregulation of activity and mRNA accumulation for NAD+-ME and G6P-DH inside the lesion (discussed above) may be exceptions to the shutoff process. Such exceptions are not without precedent; we have shown elsewhere that the mRNAs for actin and tubulin escape shutoff in pea after pea seed–borne mosaic virus infection (Escaler et al., 2000b). A second response in common with the pea system was the upregulation of HSP70 in response to CMV infection in marrow, although the precise kinetics of the response might differ slightly in the two virus–host interactions.

A novel and surprising feature of the responses to CMV infection was a spatially restricted increase in gene expression ahead of the advancing infection front (Figure 8). This was observed for HSP70 (Figure 2) and more notably for NADP+-ME (Figure 5). For the latter, increased mRNA accumulation was observed ~0.5 mm (~20 to 30 palisade mesophyll cells) beyond the outer edge of the lesion. The triggering of remote responses to infection is common, particularly for the hypersensitive response reactions, in which the induction of systemic acquired resistance is accompanied by the upregulation of many genes (reviewed in Maleck and Dietrich, 1999). Similarly, local stresses may result in an imbalance of hormones or other low molecular weight compounds and may alter the expression of genes at remote sites. Generally, we would expect these responses to be less restricted than those seen for NADP+-ME and HSP70. For example, we examined the expression of catalase, for which the activity of the enzyme it encodes is known to increase systemically in susceptible potato in response to nematode and bacterial infection (Niebel et al., 1995). We found that in contrast to the pattern of expression seen for NADP+-ME, catalase mRNA increased uniformly in the upper spongy mesophyll layer of the cotyledon outside of the infected lesions (Figure 7).

If the increases in NADP+-ME and HSP70 mRNAs outside of the lesion do not represent a general stress response, then we should perhaps consider virus-associated factors as forward signals for the upregulation of these genes. Generic effects associated with virus multiplication, such as an alteration in cell-to-cell communication (Balachandran et al., 1995; Olesinski et al., 1995) or the diversion of metabolism into virus synthesis, could potentially act as indirect sources of remote signaling events. In the latter case, virus replication conceivably could serve as a sink for the products of primary metabolism drawn from the neighboring uninfected tissues. This would be equivalent to the “green island” effects observed for some fungal infections (see Goodman et al., 1986). Alternatively, two virus-derived products might be considered as more direct candidates for signaling molecules. While modifying plasmodesmal function, virus MPs can move between cells (Ding et al., 1995) and could provide a forward signaling function. However, within the limits of sensitivity of the CMV 3a MP antibody, we were unable to detect the MP at more than one to two cells beyond the zone of virus infection. The second candidate, which has not been tested, is the population of small 25-nucleotide RNA fragments that accompany virus infection and may be the product of a post-transcriptional gene silencing–based defense against the invading virus (Hamilton and Baulcombe, 1999).

Although not tested, the invocation of a defense reaction to provide an advance signal for an anaplerotic activity (NADP+-ME) to the advantage of the virus raises an important conceptual point about the interpretation of host responses. It is tempting to view the highly regulated changes in host expression and biochemistry as events directed either by—and to the advantage of—the virus or, reciprocally, by and to the advantage of the host. The reality is more likely a mutual balance in which active (defense) responses by the host provide a selection pressure on the virus to adapt in ways that exploit or facilitate tolerance of the change. Nevertheless, whether these effects are interpreted as cause or consequence in virus replication, their biological significance will be appreciated only by studying them in the context of a temporal sequence of induced events.

METHODS

Plant Material

Squash (or marrow) plants (Cucurbita pepo cv Green Bush) were grown under greenhouse conditions at 20 to 22°C with a 16-hr supplemented photoperiod. Eight-day-old green cotyledons were inoculated with cucumber mosaic virus (CMV).

Virus Material and Inoculation

Plants were inoculated with the Kin strain of CMV exactly as described by Técsi et al. (1994); control plants were inoculated with a buffer homogenate prepared from noninfected marrow plants. Tissues were sampled for RNA extraction 3 days after inoculation and were processed for in situ hybridization and immunocytochemistry.

RNA Gel Blot Analysis

Total RNA was isolated from infected and mock-inoculated cotyledons by using RNA Isolator (Genosys Biotechnologies, Pampisford, UK); 15 μg of this was used for RNA gel blot analysis. Denaturing RNA gels were run as described by Aranda et al. (1999), followed by capillary transfer of the RNA to Hybond NX nylon membranes (Amersham Life Sciences, UK) for hybridization with radioactive probes, as described by Feinberg and Vogelstein (1983).

Sequence Analysis

Plasmid DNA was used as a template for automated sequencing with the Thermo Sequenase Dye Terminator Cycle Sequencing kit (Amersham Life Sciences). Forward and reverse M13 primers and oligonucleotides derived from internal sequences were used to sequence DNA templates. DNA sequence data were analyzed with the Genetics Computer Group (Madison, WI) package.

Cloning cDNA Probes for RNA Analysis

cDNA to marrow RNA was obtained by reverse transcription–polymerase chain reaction (RT-PCR). Five micrograms of total RNA from healthy or virus-infected cotyledons was used for RT. The reaction was performed at 37°C for 90 min with oligo(dT) as a primer and M-MLV Reverse Transcriptase (Life Technologies, Paisley, UK) in a 40-μL reaction. Five microliters of this reaction mix was used as template for PCR reactions. cDNA clones for squash genes were obtained by PCR with degenerate oligonucleotides (Genosys Biotechnologies). These were designed to conform to conserved regions in sequences available in the EMBL sequence database. The oligonucleotide sequences were as follows: glucose-6-phosphate dehydrogenase (G6P-DH; EC 1.1.1.49), sense primer 5′-GGACWMGGRTTRTTGTTGARAARCC-3′, antisense primer 5′-GCYTTCAKTANR-AARGGMACACCKTCCC-3′; NAD+-dependent malic enzyme (NAD+-ME; EC 1.1.1.39), sense primer 5′-GATCGTGGRGARATGATG-TCAATG-3′, antisense primer 5′-TCAATCATTGGYCTKCCYTGNGC-3′; NADP+-dependent malic enzyme (NADP+-ME; EC 1.1.1.40), sense primer 5′-GGAGAYYTKGGYTGYCAGGGAATGG-3′, antisense primer 5′-GARTCCACMAGCCAAAYCTTCTTGCG-3′; NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP-DH; EC 1.2.1.13), sense primer 5′-GCAATGCTTCTTGCACCACTAACTG-3′, antisense primer 5′-AACCCCAYTCATTRTCATACCAAGC-3′; heat shock protein 70 (HSP70), sense primer 5′-GTTGGWGGNTCMACKAGRATH-CC-3′, antisense primer 5′-CCYCTBGGDGCWGGWGGDATNCC-3′; and catalase (EC 1.11.1.6), sense primer 5′-TCCAYTGGAARCCNACTTGYGG-3′, antisense primer 5′-CCTCATCTCTRTGCATRA-AGTTC-3′. In these sequences, equation M1, equation M2, equation M3, equation M4, equation M5, equation M6, equation M7, equation M8, and equation M9.

The standard PCR reactions were prepared by using 30 to 60 pmol of each primer and 2 units of Taq DNA polymerase (Promega) in the buffer supplied by the manufacturer at a final volume of 50 μL. The reaction conditions were 94°C for 3 min followed by 40 cycles of 40 to 50°C for 30 sec, 72°C for 1 min, and 94°C for 30 sec. The gel-purified PCR products were ligated into the pGEM-T Easy vector (Promega) according to the manufacturer's instructions.

In Situ Hybridization

Hybridization probes to detect target RNAs by in situ hybridization were prepared as described previously (Wang and Maule, 1995; Aranda et al., 1996). The negative- or positive-sense probes were prepared after linearizing the pGEM-T Easy vectors containing the target sequence with NdeI or NcoI and transcribing with SP6 or T7 RNA polymerase. Digoxigenin-11-UTP–labeled probes (Boehringer Mannheim) were hybridized to tissue sections and detected with alkaline phosphatase–conjugated anti-digoxigenin antibody, as described previously (Wang and Maule, 1995). Polyclonal rabbit antisera against CMV coat protein (CP) (Técsi et al., 1994) and movement protein (MP) (Gal-On et al., 1994) were used at dilutions of 1:500 and 1:250, respectively. The immunohistochemical procedures with anti–CMV CP and anti–CMV MP were as described previously (Wang and Maule, 1994).

Acknowledgments

We thank Margaret Boulton, Carsten Lederer, Carole Thomas, Alison Smith, and Stuart Harrison for comments on the manuscript before submission. We are grateful to Prof. Peter Palukaitis for providing antibody for the CMV MP and to Dr. Laszlo Técsi for continuing interest in the project. Z.H. was in receipt of an EMBO Fellowship. The John Innes Centre receives a grant-in-aid from the Biotechnology and Biological Research Council.

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