The Plant Cell, Vol. 12, 1975–1985, October 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

Complex Spatial Responses to Cucumber Mosaic Virus Infection in Susceptible

Cucurbita

pepo

Cotyledons

Zoltan Havelda and Andrew J. Maule

1

John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom

Cucumber mosaic virus infection of its susceptible host

Cucurbita pepo

results in a program of biochemical changesafter virus infection. Applying a spatial analysis to expanding infected lesions, we investigated the relationship betweenthe changes in enzyme activity and gene expression. Patterns of altered expression were seen that could not be de-tected by RNA gel blot analysis. For all the host genes studied, there was a downregulation (shutoff) of expressionwithin the lesion. In addition, two distinct types of upregulation were observed. The expression of

heat shock protein 70

(

HSP70

) and

NADP

1

-dependent malic enzyme

(

NADP-ME

) showed induction in apparently uninfected cells ahead of theinfection. This response was more localized than the upregulation exhibited by

catalase

expression, which occurredthroughout the uninfected regions of the tissue. The experiments showed that virus infection induced immediate andsubsequent changes in gene expression by the host and that the infection has the potential to give advance signalingof the imminent infection.

INTRODUCTION

In a compatible interaction between a virus and its host,there must be substantial diversion of metabolites into theaccumulation of virus-specific proteins and nucleic acids.In most susceptible plants, this interaction is not accompa-nied by cell death, and infected cells continue to functiondespite the presence of massive quantities of virus-specificproducts. This diversion of resources is evident in thewhole plant as changes in plant physiology, growth, anddevelopment, that is, symptom formation. How plant cellsaccommodate virus synthesis while still sustaining somecapacity in the plant for further growth is relatively poorlyunderstood. We would expect the exploitation of host me-tabolism to be transitory, however, thus reducing the imme-diate damage to the host to a window of time sufficient forthe exponential accumulation of progeny virus to a maxi-mally tolerated amount. Because plant viruses invade hosttissues progressively, such transitory effects would be spa-tially restricted and not necessarily detectable by grossanalysis of the infected tissues.

Strong support for the transitory nature of responses tovirus infection came from a unique study that applied a spa-tial analysis to changes in host physiology after infection ofsquash/marrow (

Cucurbita pepo

) cotyledons with

Cucumbermosaic virus

(CMV) (Técsi et al., 1996). The study showedthat relative to an advancing infection front, changes in pri-

mary and secondary metabolism occurred at specific timesafter the onset of virus replication and virus gene expres-sion. Hence, the center of the lesion had an increase in gly-colysis and respiration and a decrease in the Benson–Calvincycle. Conversely, at the extreme edge of the lesion, the en-zymes involved in mobilizing carbon for macromolecularsynthesis (anaplerotic reactions) had a transient increase inactivity. For these enzymes, the earliest change was seenfor the NADP

1

-dependent malic enzyme (NADP

1

-ME) (Técsiet al., 1996). There was also a corresponding increase ofother anaplerotic activities (e.g., glucose-6-phosphate de-hydrogenase [G6P-DH]) of the oxidative pentose phosphatepathway, which persisted into the center of the lesion. Thisled to the proposition that virus infection triggered a pro-gram of physiological responses that resulted in symptomexpression.

Transient responses have also been observed in pea tis-sues infected with

Pea seed–borne mosaic virus

(potyvi-rus),

Pea early browning virus

(tobravirus),

White clovermosaic virus

(potexvirus), or

Beet curly top virus

(geminivi-rus) (Wang and Maule, 1995; Aranda et al., 1996; Escaleret al., 2000a), although these cases assessed changes inhost gene expression rather than in host physiology. In in-fected pea tissues, three patterns of gene expression rela-tive to an advancing infection front were observed. Manyhost gene transcripts were depleted in a zone occupying10 to 12 cells behind the infection front, a phenomenonakin to the shutoff of host genes observed for many animalvirus infections (Aranda and Maule, 1998). Some otherhost genes—

heat shock protein 70

(

HSP70

),

polyubiquitin

,

1

To whom correspondence should be addressed. E-mail andy.maule@bbsrc.ac.uk; fax 44-1603-450045.

1976 The Plant Cell

and

gor2

(Aranda et al., 1996; Escaler et al., 2000b)—showeda marked but more transient upregulation at the infectionfront. A third class of genes, comprising

actin

,

tubulin

, and

heat shock factor

, showed no change in the amounts ofmRNA after infection (Aranda et al., 1999; Escaler et al.,2000b).

These studies point to the elegant control of host geneexpression and host physiology after the initiation of virusreplication, but separately, they do not show how the pro-cesses are related. The work presented here addresses thisby applying a spatial analysis of host gene expression toCMV-infected squash/marrow cotyledonary tissue. The re-sults not only show correspondence (gene induction andshutoff) between pea and marrow in the response to virusreplication but also indicate that tissues can show two fur-ther kinds of responses in uninfected cells ahead of the in-fectious front. Such responses suggest that host cells havethe capacity to respond in advance to signals of the im-pending infection. The nature of these potential signals isinvestigated and discussed.

RESULTS

RNA Gel Blot Analysis of Host mRNAs

Three days after manual inoculation of marrow cotyledonswith CMV, the infection was visible as small expanding chlo-rotic lesions. Under our standard experimental conditions,40 to 50 lesions/cm

2

formed on each cotyledon, occupying

z

20% of the total leaf area. To obtain a preliminary assess-

ment of changes in the expression of genes encoding someof these key enzymes, we analyzed steady state concentra-tions of mRNA in whole cotyledonary tissue extracts. Forthis, partial cDNA clones were isolated, sequenced, andused to prepare probes for RNA gel blot analysis. A list ofthe clones isolated is given in Table 1.

HSP70

cDNA wasalso included because we had shown previously that thisgene was strongly upregulated in response to the replicationof a wide range of viruses in pea tissue (Aranda et al., 1996;Escaler et al., 2000a).

All of the genes showed expression after mock inocula-tion of the tissues with buffer (Figure 1, lanes 2), althoughthe basal expression of

G6P-DH

was very low. Of the sixgenes examined, three showed some increase and threewere almost unchanged in steady state mRNA concentra-tions as a result of virus infection (Figure 1, lanes 1). Notableincreases were seen for

HSP70

,

NAD

1

-dependent malic en

-

zyme

(

NAD

1

-ME

), and

G6P-DH

mRNAs. The mRNAs show-ing 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

).These data were surprising because they showed little

correspondence with overall activity measurements orwith predictions that were based on localized changes rel-ative to the infection front. For example, in the case ofNADP-ME, which had previously shown locally increasedactivity in response to infection (Técsi et al., 1996), the op-posite change in mRNA contents was observed. The in-crease in the expression of

HSP70

was in line with ourprevious observations in pea, although the RNA gel blotanalysis did not distinguish between an overall increase inmRNA content and localized changes at the infectionfront.

Table 1.

Cloned cDNAs from Squash/Marrow

Enzyme/Metabolic Area cDNA

a

Size (bp) mRNA

b

Size (nt) EMBL

c

Accession No. Identity (%)

d

Mitochondrial respirationNAD

1

-ME 494

<

2300 AF260732 72–83Photosynthesis

NADP

1

-dependent GAP-DH-ANADP

1

-GAP-DH-B513516

<

1400

<

1500AF260733AF260734

79–8278–80

AnapleroticNADP

1

-MEG6P-DH (plastidic)

531580

<

2100

<

1900AF260735AF260736

76–7978–79

Chaperone HSP70 401

<

2200 AF260731 73–82Detoxification of active oxygen species

Catalase 491

<

1800 AF260737 70–100

a

Length of the cloned and sequenced

C. pepo

cDNA.

b

Expected and recorded size of the

C. pepo

mRNA from comparisons with homologs from other plant species and from gel migration distances.

c

Sequences submitted to the EMBL database.

d

Percentage of identity with homologs from other plant species.

Host Responses to Virus Infection 1977

Spatially Defined Changes in Host Gene Expression

To resolve the spatial changes in host gene expression,pieces of infected or mock-inoculated tissues were sub-jected to in situ hybridization to detect host mRNAs; immu-nohistochemistry was used to detect virus coat protein. Wehad previously shown a correspondence between the loca-tion of CMV coat protein (CP) and CMV RNA and hence theextent 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 inter-cellular spaces), clear changes in host gene expressioncould be seen only when any upregulation was localized, orwhen downregulation occurred against a background ofhigh basal expression (i.e., in mock-inoculated tissues). Ofthe host genes initially selected (Table 1),

G6P-DH

and

NAD-ME

mRNAs produced hybridization patterns that weretoo weak or too dispersed to provide useful information. Wealso tried a cDNA for

fumarate hydratase

(an enzyme of theKrebs cycle, involved in respiratory metabolism in the mito-chondrion), which also showed increased enzyme activity inthe center of the lesion (Técsi et al., 1996), but it too was notuseful (data not shown).

Three consecutive sections of tissue from either infectedor mock-inoculated tissue were subjected, respectively, tohybridization with negative-sense probes to detect hostmRNA, immunohistochemistry to detect CP (and the area ofinfection) or to hybridization with positive-sense probes as acontrol. In all the hybridization experiments, an apparentlystrong reaction was detected in some spongy mesophyllcells within the infected lesion (e.g., Figure 2C, arrow). Be-cause this was seen with the positive-sense control probes,however, we attributed it to a sporadic change in the reac-

tion of the infected cells to the fixation conditions ratherthan to a specific reaction to virus infection.

HSP70

Basal expression of

HSP70

in control tissue (Figure 2F)could not be resolved above the background staining (Fig-ure 2H). However, infected tissue had a marked accumula-tion of

HSP70

mRNA at the edge of the lesion, that is, inthe boundary zone between infected and uninfected re-gions, which is most obvious in the palisade mesophylllayer (Figure 2A, arrowheads). Aligning the consecutivesections (Figures 2A to 2C, dotted lines, magnified in Fig-ures 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. Inthe central portion of the lesion,

HSP70

mRNA returned toa value indistinguishable from the background (Figures 2Aand 2C).

NADP

1

-GAP-DH

NADP-GAP-DH, a multisubunit enzyme complex requiredfor photosynthesis, is located in the chloroplast. The en-zyme is nuclear-encoded by two highly related genes (forsubunits A and B; see Table 1) (Brinkmann et al., 1989). Af-ter virus infection, the enzyme activity decreased progres-sively from the outside to the center of the lesion (Técsi etal., 1996; and illustrated in Figures 3I and 4I).

In situ hybridization showed that in mock-inoculated tis-sues, genes for both subunit A (Figure 3F) and subunit B(Figure 4F) were highly expressed in the palisade mesophyllcells but very little in the spongy mesophyll cells. The moststriking effect of infection on these genes was the sharp de-pletion of transcript accumulation inside the periphery of thelesion (Figures 3D, 3E, 4D, and 4E). Apparently, the removalof

GAP-DH-A

mRNA was more abrupt after the onset of in-fection than that seen for

subunit B

mRNA (cf. Figures 3D,3E, 4D, and 4E). In both cases, residual mRNA contents re-mained low through the central part of the lesion.

NADP

1

-ME

In the previous study (Técsi et al., 1996), a change in

NADP-ME

activity was the earliest specific response to vi-rus invasion at the edge of the expanding lesion. The activ-ity increased and then rapidly declined to the center of thelesion (Figure 5I). In situ hybridization showed that inmock-inoculated tissue,

NADP-ME

mRNA expression wasmost abundant in the vascular tissues and the upper cellsof the spongy mesophyll layer (Figure 5F). In contrast with

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

Total mRNAs, prepared from CMV-infected (lanes 1) or mock-inocu-lated (lanes 2) tissues, were separated under denaturing conditions,blotted, and hybridized with probes (top) for HSP70, GAP-DH-A,GAP-DH-B, NADP1-ME, NAD1-ME, or G6P-DH mRNAs. Relativegel loadings are shown from ethidium bromide staining of the rRNAs(bottom).

1978 The Plant Cell

the data from the RNA gel blot analysis (Figure 1), whichshowed an overall slight decrease in

NADP

1

-ME

mRNA, insitu

hybridization of sections of infected tissue showed anupregulation of expression. This was seen as increasedmRNA accumulation outside of the lesion (i.e., in uninfectedcells), extending for a considerable distance (

z

0.5 mm or20 to 30 palisade mesophyll cells) from the outer edge ofthe lesion (Figure 5A, arrowheads). The range of this in-crease extended farther than that seen for

HSP70

mRNA(Figure 2A). The increase was greatest close to the infectedarea and declined with distance from the infection front.The increase did not, however, extend to all the uninfectedregions of the leaf. In the example shown in Figure 5A, themRNA content in the central tissue region between two le-sions (arrow) is similar to that in mock-inoculated tissue(Figure 5F). Similarly, the outer edge of the tissue sectionaway from the lesion (Figure 5A, left) shows only a little ex-pression.

Inside the infected area, the accumulation of

NADP

1

-ME

mRNA declined abruptly to less than that seen inmock-inoculated tissue (cf. Figures 5A and 5D with 5F).The exception here appears to be the mRNA within vascu-lar 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 wasalso 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

1

-ME

mRNAs suggest thatresponses are induced in cells ahead of the advancing in-fection front. From studies in other systems, we proposesome potential candidates for this activity. First, the CMVmovement protein (MP) 3a has been shown to move fromcell to cell in the absence of infection (Ding et al., 1995).This protein is believed to modify plasmodesmata to facil-itate its own trafficking. Second, low molecular weight com-pounds (e.g., sugars, hormones) can diffuse rapidly throughtissues to give distant and even systemic responses.

Figure 2.

Spatial Analysis of the Expression of

HSP70

Associatedwith CMV Infection.

(A)

to

(C)

Consecutive sections of CMV-infected tissues analyzed byin situ hybridization using a negative-sense

(A)

or a positive-sense

(C)

RNA probe to detect

HSP70

mRNAs or by immunocytochemis-try using antiserum to CMV CP

(B)

. The alignment is shown relativeto the edge of the CMV lesions (dotted lines). Irrespective of theprobe used, in situ hybridization resulted in the formation of artifac-tual dark spots within the infected areas, as shown by (e.g.) the ar-row in

(C)

.

(D)

and

(E)

Magnification (

3

2.5) of the alignment at one edge of a le-sion, as marked by the triangles in

(A)

and

(B)

. The direction of virusmovement from infected to uninfected tissues is identified (white ar-rowhead) between

(D)

and

(E)

.

(F)

to

(H)

Consecutive sections of control mock-inoculated tissues

analyzed by in situ hybridization using a negative-sense

(F)

or a pos-itive-sense

(H)

RNA probe to detect

HSP70

mRNAs or by immuno-cytochemistry using antiserum to CMV CP

(G).Comparing (A) with (B) and (F) shows that infection increasedHSP70 mRNA to more than that seen in mock-inoculated tissues.This increase occurred at the edge of the lesion (arrowheads), whichon closer examination ([D] and [E]) was seen to span the edge of theinfected zone to include approximately five cells in the apparentlyuninfected area. PM, palisade mesophyll layer; SM, spongy meso-phyll layer. Bars in (C) and (H) 5 0.5 mm and apply to (A) to (C) and(F) to (H); bar in (E) 5 200 mm and applies to (D) and (E).

Host Responses to Virus Infection 1979

To investigate the potential for CMV 3a to mediate suchsignaling, consecutive sections of infected tissue were sub-jected to inmunohistochemistry with antibodies specific forCMV CP or 3a proteins (Figure 6). Alignment of the sectionsshowed that the 3a protein accumulated throughout the in-fected area (cf. Figures 6A and 6B), although the concentra-tion of 3a was probably greater at the edge of the lesion(Figures 6A and 6C). However, close examination of thefront of infection (Figures 6C and 6D) showed that the accu-mulation of 3a protein occurred one to two cells ahead ofthe accumulation of CP, at most.

Low molecular weight compounds have been shown tobe effector molecules for changing expression of a range ofgenes in plant–pathogen interactions. For example, peroxi-dase (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 ex-pression away from the site of infection. To test whether theupregulation of NADP-ME could be linked to an equivalentsignaling process, we wanted to compare the expression ofNADP-ME with the pattern of expression of another dis-tantly regulated gene. Unfortunately, for reasons outlinedearlier, not all of these genes were suitable for comparativeanalysis by in situ hybridization. In the case of peroxidase,the enzyme is encoded by a large gene family, not all mem-bers of which need be coordinately regulated. In the case ofinvertase, basal expression in squash cotyledons was verylow, and there was no detectable increase in infectedtissues (Z. Havelda, unpublished data). PR genes are in-duced most commonly in response to a hypersensitiveresponse; because no hypersensitive response is associ-ated with the CMV–squash interactions, however, testingPR genes was not appropriate. Catalase, however, is in-duced in compatible plant–pathogen (bacteria and nema-tode) interactions (Niebel et al., 1995) and decreases in anincompatible interaction (tobacco mosaic virus infection ofresistant tobacco; Yi et al., 1999).

A cDNA for squash catalase was cloned and se-quenced (Table 1) and used to prepare probes for in situhybridization. In mock-inoculated tissue, catalase mRNAaccumulated predominantly in and around the vasculartissues (Figure 7F). In infected tissue, catalase mRNAFigure 3. Spatial Analysis of the Expression of GAP-DH-A Associ-

ated with CMV Infection.

(A) to (C) Consecutive sections of CMV-infected tissues analyzed byin situ hybridization using a negative-sense (A) or a positive-sense(C) RNA probe to detect GAP-DH-A mRNAs or by immunocy-tochemistry using antiserum to CMV CP (B). The alignment is shownrelative to the edge of the CMV lesions (dotted lines).(D) and (E) Magnification (32.5) of the alignment at one edge of a le-sion, as marked by the triangles in (A) and (B). The direction of virusmovement from infected to uninfected tissues is identified (white ar-rowhead) between (D) and (E).(F) to (H) Consecutive sections of control mock-inoculated tissuesanalyzed by in situ hybridization using a negative-sense (F) or a pos-itive-sense (H) RNA probe to detect GAP-DH-A mRNAs or by immu-nocytochemistry using antiserum to CMV CP (G).

(I) Relative GAP-DH-A enzyme activity across the lesion (redrawnfrom Técsi et al., 1996).In mock-inoculated tissues, GAP-DH-A was expressed predomi-nantly in the palisade mesophyll layer (F). This was also true in in-fected tissues except within the zone of infection, which wasmarkedly depleted in transcript accumulation, as shown in (A) and(D). This decrease correlates with a decrease in GAP-DH enzymeactivity within the lesion (I). PM, palisade mesophyll layer; SM, spongymesophyll layer. Bars in (C) and (H) 5 0.5 mm and apply to (A) to (C)and (F) to (H); bar in (E) 5 200 mm and applies to (D) and (E).

1980 The Plant Cell

showed increased accumulation outside the vascular tis-sue and mostly within the spongy mesophyll, particularlyits upper cell layers (Figure 7A). This increase was alsoseen as an overall increase in mRNA, as detected by RNAgel blot analysis of total tissue RNA extracts (Figure 7I). Incontrast to the pattern of increased expression seen forNADP-ME, catalase was increased almost uniformlythroughout the uninfected regions of the cotyledon (cf.Figures 5A and 7A). As with most of the other host genesexamined, the accumulation of catalase mRNA was mark-edly depleted within the lesion (cf. Figures 7A and 7B)that coincided spatially with the first infected cells (cf.Figures 7D and 7E).

DISCUSSION

The analysis of the intimate relationship between a plant vi-rus and its host is considerably complicated by the progres-sive nature of the infection. A particular problem is posed bythe time frame for replication within single cells relative tothe period required for an infection phenotype to becomevisible. Hence, whereas replication in single cells may takeonly a few hours (Dolja et al., 1992), a few days may be re-quired for lesions on inoculated leaves to become visibleand days to weeks for full systemic symptoms to appear insusceptible hosts. The approach we have taken previously(Maule et al., 2000), and in this work, is to integrate time intothe study of host responses by applying a spatial analysis toan advancing infection front. The benefit of this approach isillustrated well by the RNA gel blot analysis of host mRNAsin cotyledons inoculated with CMV (Figure 1). Of the fourmRNAs subjected to gel blot and in situ hybridization analy-sis, only one (HSP70) showed changes at the tissue levelthat paralleled changes in recently infected cells (i.e., at theinfection front). That is, HSP70 showed an average upregu-lation in whole-tissue extracts. In contrast, the average un-changed (GAP-DH-A and GAP-DH-B) or reduced (NADP1-ME) steady state accumulation of mRNA in tissues after in-fection masked specific and highly regulated changes at,

Figure 4. Spatial Analysis of the Expression of GAP-DH-B Associ-ated with CMV Infection.

(A) to (C) Consecutive sections of CMV-infected tissues analyzed byin situ hybridization using a negative-sense (A) or a positive-sense(C) RNA probe to detect GAP-DH-B mRNAs or by immunocy-tochemistry using antiserum to CMV CP (B). The alignment is shownrelative to the edge of the CMV lesions (dotted lines).(D) and (E) Magnification (32.5) of the alignment at one edge of a le-sion, as marked by the triangles in (A) and (B). The direction of virusmovement from infected to uninfected tissues is identified (white ar-rowhead) between (D) and (E).(F) to (H) Consecutive sections of control mock-inoculated tissuesanalyzed by in situ hybridization using a negative-sense (F) or a pos-itive-sense (H) RNA probe to detect GAP-DH-B mRNAs or by immu-nocytochemistry using antiserum to CMV CP (G).

(I) Relative GAP-DH-B enzyme activity across the lesion (redrawnfrom Técsi et al., 1996).In mock-inoculated tissues, GAP-DH-B was expressed predomi-nantly in the palisade mesophyll layer (F). This was also true in in-fected tissues except within the zone of infection; see (A) and (D).As with GAP-DH-A, there was a marked depletion in transcript ac-cumulation although with some delay, as seen by comparing (D)and (E) with Figures 3D and 3E. Again, this decrease correlateswith a decrease in GAP-DH activity within the lesion (I). PM, pali-sade mesophyll layer; SM, spongy mesophyll layer. Bars in (C) and(H) 5 0.5 mm and apply to (A) to (C) and (F) to (H); bar in (E) 5 200mm and applies to (D) and (E).

Host Responses to Virus Infection 1981

ahead of, or behind the infection front. Unfortunately, in situhybridization has technical limitations associated with itssensitivity and therefore cannot be applied to genes thatdisplay very weak expression in the tissues.

A fundamental question related to CMV infection ofsquash cotyledons was whether there was a correspon-dence between changes in enzyme activity (Técsi et al.,1996) and changes in host gene expression (mRNA accu-mulation). To address this, we cloned cDNAs for squashmRNAs that encoded enzymes previously found to be up-regulated or downregulated in specific areas of the expand-ing lesions (i.e., at different times relative to virus infection).We also cloned squash HSP70 cDNA, given that this gene inpea had been shown previously to be tightly upregulated inresponse to many viruses (Escaler et al., 2000a). NADP1-ME, which showed increased enzyme activity at the periph-ery of the CMV lesion (Técsi et al., 1996), appears to be con-trolled at the mRNA level in response to CMV infection.Similarly, the reduced activity of GAP-DH, which correlatedwith reduced photosynthetic activity in the center of theCMV lesion (Técsi et al., 1996), appears to be related todecreased mRNA accumulation (Figure 8). Unfortunately,when used for in situ hybridization, cDNA probes gave in-conclusive results for genes for which the products showeddifferent activity profiles (NAD1-ME, G6P-DH, and fumaratehydratase) after infection (Técsi et al., 1996). This was disap-pointing with regard to NAD1-ME and fumarate hydratase inparticular, given their strong increase in activity in the centerof the lesion, an area in which many other activities andmost of the mRNAs tested showed a marked decline. G6P-DH, which increased in activity immediately behind the in-fection front, also could not be analyzed by in situ hybridiza-tion. Nevertheless, RNA gel blot analysis for both NAD1-MEand G6P-DH suggests that they may be regulated at thetranscriptional or mRNA level.

In our previous work in pea (Maule et al., 2000), and formany animal virus infections (reviewed in Aranda and Maule,1998), a common response to virus infection is seen as the

Figure 5. Spatial Analysis of the Expression of NADP1-ME Associ-ated with CMV Infection.

(A) to (C) Consecutive sections of CMV-infected tissues analyzed byin situ hybridization using a negative-sense (A) or a positive-sense(C) RNA probe to detect NADP1-ME mRNAs or by immunocy-tochemistry using antiserum to CMV CP (B). The alignment is shownrelative to the edge of the CMV lesions (dotted lines).(D) and (E) Magnification (32.5) of the alignment at one edge of a le-sion, as marked by the triangles in (A) and (B). The direction of virusmovement from infected to uninfected tissues is identified (white ar-rowhead) between (D) and (E).(F) to (H) Shown are consecutive sections of control mock-inocu-lated tissues analyzed by in situ hybridization using a negative-sense(F) or a positive-sense (H) RNA probe to detect NADP1-ME mRNAsor by immunocytochemistry using antiserum to CMV CP (G).

(I) Relative NADP1-ME enzyme activity across the lesion (redrawnfrom Técsi et al., 1996).In mock-inoculated tissues (F), NADP1-ME was expressed predom-inantly in the vascular tissues and in the palisade and the upperspongy mesophyll layers. In infected tissues (A), a complex patternof expression was seen. Within the lesion, NADP1-ME transcriptswere markedly depleted from the mesophyll layers but not from thevascular tissues (white arrow). Outside the lesion, an increase inNADP1-ME expression (arrowheads) extended for z0.5 mm fromthe edge of the lesion; beyond this point, expression was equivalentto that seen in uninfected tissues (black arrow). This expression pro-file correlated with the activity profile across the lesion (I). PM, pali-sade mesophyll layer; SM, spongy mesophyll layer; V, vasculartissues. Bars in (C) and (H) 5 0.5 mm and apply to (A) to (C) and (F)to (H); bar in (E) 5 200 mm and applies to (D) and (E).

1982 The Plant Cell

downregulation (or shutoff) of host gene expression coinci-dent with virus infection. Shutoff was also observed afterCMV infection in squash cotyledons in which all of the hostmRNAs examined by in situ hybridization were substantiallydepleted within the central area of the CMV-infected lesion(Figure 8). The precise timing of this mRNA turnover variedslightly between mRNA species. The factors controlling thisare not known. The potential upregulation of activity andmRNA accumulation for NAD1-ME and G6P-DH inside thelesion (discussed above) may be exceptions to the shutoffprocess. Such exceptions are not without precedent; wehave shown elsewhere that the mRNAs for actin and tubulinescape shutoff in pea after pea seed–borne mosaic virus in-fection (Escaler et al., 2000b). A second response in com-mon with the pea system was the upregulation of HSP70 inresponse 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 CMVinfection was a spatially restricted increase in gene expres-sion ahead of the advancing infection front (Figure 8). Thiswas observed for HSP70 (Figure 2) and more notably forNADP1-ME (Figure 5). For the latter, increased mRNA accu-mulation was observed z0.5 mm (z20 to 30 palisade meso-phyll cells) beyond the outer edge of the lesion. Thetriggering of remote responses to infection is common, par-ticularly for the hypersensitive response reactions, in whichthe induction of systemic acquired resistance is accompa-nied by the upregulation of many genes (reviewed in Maleckand Dietrich, 1999). Similarly, local stresses may result in animbalance of hormones or other low molecular weight com-pounds and may alter the expression of genes at remotesites. Generally, we would expect these responses to beless restricted than those seen for NADP1-ME and HSP70.For example, we examined the expression of catalase, forwhich the activity of the enzyme it encodes is known to in-crease systemically in susceptible potato in response tonematode and bacterial infection (Niebel et al., 1995). Wefound that in contrast to the pattern of expression seen forNADP1-ME, catalase mRNA increased uniformly in the up-per spongy mesophyll layer of the cotyledon outside of theinfected lesions (Figure 7).

If the increases in NADP1-ME and HSP70 mRNAs outsideof the lesion do not represent a general stress response,then we should perhaps consider virus-associated factorsas forward signals for the upregulation of these genes. Ge-neric effects associated with virus multiplication, such as analteration in cell-to-cell communication (Balachandran et al.,1995; Olesinski et al., 1995) or the diversion of metabolisminto virus synthesis, could potentially act as indirect sourcesof remote signaling events. In the latter case, virus replica-tion conceivably could serve as a sink for the products ofprimary metabolism drawn from the neighboring uninfectedtissues. This would be equivalent to the “green island” ef-fects observed for some fungal infections (see Goodman etal., 1986). Alternatively, two virus-derived products might beconsidered as more direct candidates for signaling mole-cules. While modifying plasmodesmal function, virus MPscan move between cells (Ding et al., 1995) and could providea forward signaling function. However, within the limits ofsensitivity of the CMV 3a MP antibody, we were unable to de-tect the MP at more than one to two cells beyond the zone ofvirus infection. The second candidate, which has not beentested, is the population of small 25-nucleotide RNA frag-ments that accompany virus infection and may be the prod-uct of a post-transcriptional gene silencing–based defenseagainst the invading virus (Hamilton and Baulcombe, 1999).

Although not tested, the invocation of a defense reactionto provide an advance signal for an anaplerotic activity(NADP1-ME) to the advantage of the virus raises an impor-tant conceptual point about the interpretation of host re-sponses. It is tempting to view the highly regulated changes

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

(A) and (B) Consecutive sections of CMV-infected cotyledonary tis-sues subjected to immunocytochemical analysis with antiserumspecific for the viral movement (A) or CP (B), respectively. The align-ment is shown relative to the edge of the CMV lesion (dotted linesbetween arrowheads).(C) and (D) Magnification (32.5) of the alignment at one edge of a le-sion, as marked by the triangles in (A) and (B). The locations of thetwo proteins correspond across the lesion, although the distributionof the MP (C) extends by one to two cells beyond that of the CP (D).(E) and (F) Consecutive sections of control, mock-inoculated tissuestreated with antisera specific for the MP and CP, respectively.PM, palisade mesophyll layer; SM, spongy mesophyll layer. Bars in(B) and (F) 5 0.5 mm for (A), (B), (E), and (F); bar in (D) 5 200 mm for(C) and (D).

Host Responses to Virus Infection 1983

in host expression and biochemistry as events directed ei-ther by—and to the advantage of—the virus or, reciprocally,by and to the advantage of the host. The reality is morelikely a mutual balance in which active (defense) responsesby the host provide a selection pressure on the virus toadapt in ways that exploit or facilitate tolerance of thechange. Nevertheless, whether these effects are interpretedas cause or consequence in virus replication, their biologicalsignificance will be appreciated only by studying them in thecontext of a temporal sequence of induced events.

METHODS

Plant Material

Squash (or marrow) plants (Cucurbita pepo cv Green Bush) weregrown under greenhouse conditions at 20 to 228C with a 16-hrsupplemented photoperiod. Eight-day-old green cotyledons were in-oculated with cucumber mosaic virus (CMV).

Virus Material and Inoculation

Plants were inoculated with the Kin strain of CMV exactly as de-scribed by Técsi et al. (1994); control plants were inoculated with abuffer homogenate prepared from noninfected marrow plants. Tis-sues were sampled for RNA extraction 3 days after inoculation andwere processed for in situ hybridization and immunocytochemistry.

RNA Gel Blot Analysis

Total RNA was isolated from infected and mock-inoculated cotyle-dons by using RNA Isolator (Genosys Biotechnologies, Pampisford,UK); 15 mg of this was used for RNA gel blot analysis. DenaturingRNA gels were run as described by Aranda et al. (1999), followedby capillary transfer of the RNA to Hybond NX nylon membranes

Figure 7. Spatial Analysis of the Expression of Catalase Associatedwith CMV Infection.

(A) to (C) Consecutive sections of CMV-infected tissues analyzed byin situ hybridization using a negative-sense (A) or a positive-sense(C) RNA probe to detect catalase mRNA or by immunocytochemis-try using antiserum to CMV CP (B). The alignment is shown relativeto the edge of the CMV lesions (dotted lines).(D) and (E) Magnification (32.5) of the alignment at one edge of a le-sion, as marked by the triangles in (A) and (B). The direction of virusmovement from infected to uninfected tissues is identified (white ar-rowhead) between (D) and (E).(F) to (H) Consecutive sections of control mock-inoculated tissuesanalyzed by in situ hybridization using a negative-sense (F) or a pos-itive-sense (H) RNA probe to detect catalase mRNAs or by immuno-cytochemistry using antiserum to CMV CP (G).

(I) Gel blot analysis of catalase mRNA (top) extracted from infected(lanes 1) or mock-inoculated (lanes 2) cotyledons. Relative gelloadings are shown from ethidium bromide staining of the rRNAs(bottom).In mock-inoculated tissues, catalase was expressed predominantlyin the vascular tissues (F). In infected tissues, expression extendedto the upper layers of the spongy mesophyll (A), where it accumu-lated uniformly in the uninfected areas in the cotyledon lamina. Thecatalase transcripts were markedly depleted within the lesion. Thisdownregulation abruptly coincided with the edge of lesion (i.e., thezone of maximal virus accumulation). Despite the combination ofupregulation and downregulation, an increase in the averagesteady state amounts of catalase mRNA was detected by gel blotanalysis of total RNA (I). PM, palisade mesophyll layer; SM, spongymesophyll layer; V, vascular tissues. Bars in (C) and (H) 5 0.5 mmand apply to (A) to (C) and (F) to (H); bar in (E) 5 200 mm and ap-plies to (D) and (E).

1984 The Plant Cell

(Amersham Life Sciences, UK) for hybridization with radioactiveprobes, as described by Feinberg and Vogelstein (1983).

Sequence Analysis

Plasmid DNA was used as a template for automated sequencing withthe Thermo Sequenase Dye Terminator Cycle Sequencing kit (Amer-sham Life Sciences). Forward and reverse M13 primers and oligonu-cleotides derived from internal sequences were used to sequenceDNA templates. DNA sequence data were analyzed with the Genet-ics Computer Group (Madison, WI) package.

Cloning cDNA Probes for RNA Analysis

cDNA to marrow RNA was obtained by reverse transcription–poly-merase chain reaction (RT-PCR). Five micrograms of total RNA fromhealthy or virus-infected cotyledons was used for RT. The reactionwas performed at 378C for 90 min with oligo(dT) as a primer andM-MLV Reverse Transcriptase (Life Technologies, Paisley, UK) in a40-mL reaction. Five microliters of this reaction mix was used astemplate for PCR reactions. cDNA clones for squash genes were ob-tained by PCR with degenerate oligonucleotides (Genosys Bio-technologies). These were designed to conform to conservedregions in sequences available in the EMBL sequence database. Theoligonucleotide sequences were as follows: glucose-6-phosphatedehydrogenase (G6P-DH; EC 1.1.1.49), sense primer 59-GGACWM-GGRTTRTTGTTGARAARCC-39, antisense primer 59-GCYTTCAKTANR-AARGGMACACCKTCCC-39; NAD1-dependent malic enzyme (NAD1-

ME; EC 1.1.1.39), sense primer 59-GATCGTGGRGARATGATG-TCAATG-39, antisense primer 59-TCAATCATTGGYCTKCCYTGNGC-39;NADP1-dependent malic enzyme (NADP1-ME; EC 1.1.1.40), senseprimer 59-GGAGAYYTKGGYTGYCAGGGAATGG-39, antisense primer59-GARTCCACMAGCCAAAYCTTCTTGCG-39; NADP1-dependent glyc-eraldehyde-3-phosphate dehydrogenase (GAP-DH; EC 1.2.1.13),sense primer 59-GCAATGCTTCTTGCACCACTAACTG-39, antisenseprimer 59-AACCCCAYTCATTRTCATACCAAGC-39; heat shock pro-tein 70 (HSP70), sense primer 59-GTTGGWGGNTCMACKAGRATH-CC-39, antisense primer 59-CCYCTBGGDGCWGGWGGDATNCC-39;and catalase (EC 1.11.1.6), sense primer 59-TCCAYTGGAARC-CNACTTGYGG-39, antisense primer 59-CCTCATCTCTRTGCATRA-AGTTC-39. In these sequences, R 5 A 1 G, Y 5 C 1 T, M 5 A 1 C,K 5 G 1 T, W 5 A 1 T, H 5 A 1 T 1 C, B 5 G 1 T 1 C, D 5 G 1 A 1T, and N 5 A 1 G 1 C 1 T.

The standard PCR reactions were prepared by using 30 to 60 pmolof each primer and 2 units of Taq DNA polymerase (Promega) in thebuffer supplied by the manufacturer at a final volume of 50 mL. Thereaction conditions were 948C for 3 min followed by 40 cycles of 40to 508C for 30 sec, 728C for 1 min, and 948C 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 hybridizationwere prepared as described previously (Wang and Maule, 1995;Aranda et al., 1996). The negative- or positive-sense probes were pre-pared after linearizing the pGEM-T Easy vectors containing the targetsequence with NdeI or NcoI and transcribing with SP6 or T7 RNA poly-merase. Digoxigenin-11-UTP–labeled probes (Boehringer Mannheim)were hybridized to tissue sections and detected with alkaline phos-phatase–conjugated anti-digoxigenin antibody, as described previ-ously (Wang and Maule, 1995). Polyclonal rabbit antisera against CMVcoat protein (CP) (Técsi et al., 1994) and movement protein (MP) (Gal-Onet 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, AlisonSmith, and Stuart Harrison for comments on the manuscript beforesubmission. We are grateful to Prof. Peter Palukaitis for providing an-tibody for the CMV MP and to Dr. Laszlo Técsi for continuing interestin the project. Z.H. was in receipt of an EMBO Fellowship. The JohnInnes Centre receives a grant-in-aid from the Biotechnology and Bi-ological Research Council.

Received May 31, 2000; accepted August 4, 2000.

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Figure 8. Summary of Changes in Host Gene Expression in Re-sponse to CMV Infection.

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DOI 10.1105/tpc.12.10.1975 2000;12;1975-1985Plant Cell

Zoltan Havelda and Andrew J. MauleCotyledons

pepo CucurbitaComplex Spatial Responses to Cucumber Mosaic Virus Infection in Susceptible

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