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(2003)

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Blackwell Publishing Ltd.

Pathogen profile

Turnip vein-clearing virus

,

from pathogen to host expression profile

ULR ICH MELCHER

Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA

SUMMARY

Taxonomy:

Turnip vein-clearing virus

(TVCV) is a member ofsubgroup 3 of the

Tobamovirus

genus and is thus a member ofthe alphavirus-like supergroup of positive sense RNA-containingviruses.

Physical properties:

Virions, typical of tobamoviruses, arerod-shaped and consist of a single species of four-helix bundlecapsid proteins of 17 kDa helically arranged around a 6.3 kntRNA which accounts for 5% of the virion mass. Virions are stablefor years.

Hosts:

Members of the crucifer family are excellent hosts.Particularly noteworthy is that hosts include the model plant formolecular genetics,

Arabidopsis thaliana.

No non-mechanical

vectors of transmission are known.

INTRODUCTION

That

Turnip vein-clearing virus

(TVCV) shares multiple character-istics with

Tobacco mosaic virus

(TMV), the type member of the

Tobamovirus

genus, identifies TVCV as a

Tobamovirus

(Lartey

et al

., 1994). Viral particles are rod-shaped. The single type ofcapsid protein has a molecular weight near 17 kDa. The particlesencapsidate a single RNA of positive sense. The 6.3 knt RNA isslightly shorter than TMV RNA, but encodes a set of proteins sim-ilar to those encoded by TMV RNA. As with TMV, non-mechanicaltransmission is not known to occur. The virus, principally becauseit infects the model plant

Arabidopsis thaliana

, has been the sub-ject of considerable work, summarized in this profile.

At the time that TVCV was first being characterized in Oklahoma(Lartey

et al

., 1993), three related viral isolates were beingcharacterized elsewhere. One, designated Cr-TMV (for cruciferTMV) by the isolators (Dorokhov

et al

., 1993, 1994a,b), differsonly 6.5% in nucleotide sequence from the TVCV isolate (Lartey

et al

., 1996). Since the name Cr-TMV misleadingly implies thatthe isolate is a strain of TMV, Cr-TMV and the Oklahoma isolateshould be regarded as isolates of the virus TVCV.

Oilseed rapemosaic virus

(ORMV) encompasses the viruses isolated asTMV-C (Oshima

et al

., 1962), passaged through garlic as TMV-Cg(Yamanaka

et al

., 1998),

Chinese rape mosaic virus

(Aguilar

et al

., 1996) and

Youcai mosaic virus

(Pei, 1962). A third virus,Holmes’

ribgrass mosaic virus

(HRMV), the first of the TVCV-relatedviruses to be described (Holmes, 1941; Wetter, 1986), includesisolates characterized at the capsid protein sequence level inJapan (Funatsu and Funatsu, 1968) and in Germany (Jauregui-Adell

et al

., 1969; Wittmann

et al

., 1969) and partially at the nucleotidesequence level (Wang

et al

., 1997). The complete sequenceof a Shanghai isolate of

Ribgrass mosaic virus

has been deter-mined (Zhu

et al

., 2001), but it appears to be more closelyrelated to ORMV than to HRMV. The wasabi strain of TMV, nowdesignated the wasabi strain of crucifer

Tobamovirus

(CTMV-W),is the closest relative of TVCV, ORMV and HRMV. It is about as dif-ferent from the others as the two TVCV strains are from the twoCRMV strains (Shimamoto

et al

., 1998). Other possible relatives,as yet characterized only by the amino acid composition of viri-ons, are

Ullucus mild mottle

and

tomato stripe necrosis

viruses(Gibbs, 1986). It has been suggested (Shimamoto

et al

., 1998)that TVCV, Cr-TMV, ORMV, TMV-Cg and CTMV-W be consideredthe same virus, with HRMV possibly alone being considered a dis-tinct virus. This suggestion is consistent with levels of sequencesimilarity used to make distinctions among other members ofthe

Tobamovirus

genus. Obviously, sequence characterization ofpotential members of the subgroup is needed, as is completionof the nucleotide sequence of HRMV RNA. If HRMV is a strain ofthe species that includes TVCV, precedence requires the speciesbe called HRMV.

TVCV, ORMV, HRMV and CTMV-W can all infect crucifers. Theclustering of protein sequences of the first three separately fromsubgroup 1 and subgroup 2 tobamoviruses in phylogeneticanalysis led to their placement in a new subgroup, subgroup 3(Aguilar

et al

., 1996; Lartey

et al

., 1996).

Odontoglossum ringspotvirus

(ORSV) has an uncertain position relative to these virusesbecause its RNA appears to be a recombinant between a virus ofsubgroup 1 and subgroup 3 (Gibbs

et al

., 2000; Lartey

et al

.,

Correspondence

: Department of Biochemistry and Molecular Biology, 246 NRC,Oklahoma State University, Stillwater OK 74078, USA. E-mail: u-melcher-4@alumni.uchicago.edu

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1996). Since the larger part of the genome is like subgroup 3sequences it is often considered part of subgroup 3, although theimmunodominant capsid protein is encoded by the subgroup 1part of the RNA.

INFECTION OF PLANTS BY TVCV

TVCV and its relatives have mostly been isolated from crucifers,though HRMV was isolated from a non-crucifer, ribgrass (

Plan-tago lanceolata

). In addition to the

Brassicaceae

, at least the

Scrophulariaceae

,

Caryophyllaceae

,

Boraginacae

,

Primulaceae

and

Plantaginaceae

have been observed to harbour TVCV orrelated viruses (Oshima, 1986). Most of these viruses will alsoinfect tobacco and thus have often been misleadingly given TMVdesignations. While some true TMVs do infect crucifers such as

A.thaliana

(Dardick

et al

., 2000; Hughes

et al

., 1995; Rezende

et al

.,1992) they do so less efficiently than do subgroup 3

Tobamovirus

species (Lartey

et al

., 1997). The infectivity of TVCV to

A. thaliana

has made TVCV a favourite subject for the investigation of plant–virus interactions.

An association between placement of a

Tobamovirus

on a phy-logenetic tree and a particular taxonomic family of the originalhost plant has often been noted (Fraile

et al

., 1995; Gibbs, 1999).Thus, subgroup 1 viruses were preferentially isolated fromsolanaceous species, subgroup 3 from crucifers and subgroup 2,the most diverse, from a variety of dicotyledonous plant families(

Cucurbitaceae

,

Leguminosae

and

Malvaceae

). However, mem-bers of each subgroup have also been isolated from speciesoutside the apparently preferred host families. A phylogeneticanalysis (Lartey

et al

., 1996) suggested that

Tobamovirus

specieswere present in the earliest angiosperms and that extant line-ages of subgroups 1 and 2 have diverged from each other sincethe separation of the host families. The most likely position ofthe root of the

Tobamovirus

tree (Fig. 1) is near the junction of the

Hibiscus mosaic virus

,

Sunn-hemp mosaic virus

and

Frangipanimosaic virus

branches. However, this analysis (Lartey

et al

., 1996)also suggested that subgroup 3 arose from a subgroup 1 virusthat infected a cruciferous plant perhaps displacing whatever

Tobamovirus

species may have previously infected that family.TVCV was so named (Lartey

et al

., 1993) because the onlysymptom of infection it produced on turnips was a clearing of thegreen pigment around veins in leaves emerging several weeksafter inoculation. Vein-clearing is also a symptom noted in theTMV-C infection of several

Brassicacae

(Oshima

et al

., 1974).Consistent with the late appearance of symptoms on turnips,TMV-C was reported to produce symptomless infections inseveral

Brassicacae

(Oshima

et al

., 1962; Oshima

et al

., 1974).Symptoms of infection in

A. thaliana

are diverse and dependenton the landrace of

A. thaliana

used (Martín Martín

et al

., 1997).Classification of landraces according to type of symptomsproduced by ORMV correlated with times to flowering, thus

supporting the suggestion that symptom types depend on thedevelopmental stage at which the plant is first infected (Melcher,1989). On other naturally infected plants, a variety of mottles andmosaics is seen (Oshima, 1986). Symptoms on tobacco closelyresemble those caused by TMV, although differences in accumu-lated virus levels occur (discussed below). The ORMV isolate(through its RdRP domain) causes necrotic spots on tobaccoleaves (Mansilla, Sanchez, Brumfield, Padgett, Pogue, Young andPonz, personal communication).

In general, virion accumulation is seen to begin 2 to 3 daysafter inoculation of

A. thaliana

, with infection proceeding incauline leaves more rapidly than in rosette leaves (Lartey

et al

.,1997). Maximum accumulation is reached after 6 days, and ismore rapid in uninoculated than in inoculated tissue. All parts ofthe plant become infected, whether revealed by microscopicdetection of characteristic inclusions, further described below, orby leaf skeleton hybridization (Ernwein and Wetter, 1987; Lartey

et al

., 1997), except for the embryos, which are virus-free, despitethe presence of virus in seed coats and siliques (Lartey

et al

.,1997). Within vascular tissue, virions are seen in phloem paren-chyma and companion cells. Inclusions are found in xylem, aswell as phloem (Ernwein and Wetter, 1987). Virions are alsodetected in large aggregates in the cytoplasm and in intercellularspaces, but not in plastids or nuclei. The infection of plants byHRMC and its relatives results in the accumulation of virions ininclusion bodies (Goldin, 1953) that are quite distinct (Ernweinand Wetter, 1987; Milicic

et al

., 1968) from those formed in TMVinfections and are often used to support identification of theinfecting virus. The most frequently observed inclusion bodies arelayered rounded plates (Ernwein and Wetter, 1987; Goldin, 1953;Milicic

et al

., 1968; Resconich, 1961). Some spiral aggregatesoccur in some hosts (Ernwein and Wetter, 1987). X-bodies, oftenseen with TMV infections, are rare (Resconich, 1961).

TVCV infection may interact with infections by other viruses inunexpected ways. Co-infection of turnip plants with TVCV andthe Cabbage S isolate of

Cauliflower mosaic virus

(CaMV) dra-matically exacerbated the symptoms of infection, a phenomenonnot seen using the CM4-184 isolate of CaMV (Hii

et al

., 2002).The phenomenon was not due to changes in the levels of accu-mulation of either virus, probably because the viruses accumu-lated after the event(s) that determined the severity of thesymptoms. Prior ORMV infection can protect tobacco from infec-tion with

Tobacco mild green mosaic virus

(TMGMV), a subgroup1

Tobamovirus

(Aguilar

et al

., 2000). Conversely, TMGMV infec-tion prevents subsequent infection with ORMV. AlthoughTMGMV also cross-protects against ORMV in

A. thaliana

, thereverse could not be demonstrated because TMGMV infects

A.thaliana

poorly. In contrast, the interaction between TVCV andTMV (also subgroup 3 and 1 tobamoviruses, like ORMV andTMGMV) in tobacco does not exhibit features of cross protection(Jiang, Rajakaruna, Friesen and Melcher, unpublished data). Each

Turnip vein-clearing virus

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virus has its own pattern of accumulation, TVCV accumulatingexclusively early during leaf expansion and TMV accumulatingmore slowly but continuously throughout the life of the leaf. Thepatterns of accumulation of one virus are not appreciablyaffected by the presence of the other. However, in tobacco proto-plasts, the rate of accumulation of viral CP for each virus is accel-erated in the presence of both viruses. In a line of

A. thaliana

protoplasts genetically enfeebled in their ability to replicateORMV, co-infection with

Cucumber mosaic virus

further dramat-ically reduced the accumulation of ORMV-related molecules(Ishikawa

et al

., 1993).

TVCV and its relatives have been detected by the commonlyused methods of Western blotting, ELISA and RT-PCR, the latterbeing capable of detecting an infection of inoculated leaves asearly as 1 day post-inoculation (Pereda

et al

., 2000). Becausevery few proteins migrate on SDS-PAGE gels to the position oftobamoviral coat proteins, the intensity of these bands has beenused as an indicator of virus concentration (Hii

et al

., 2002; Lartey

et al

., 1997). Leaf skeleton hybridization (Melcher

et al

., 1981)has also been used to detect the spread of the virus in differenttissues (Lartey

et al

., 1997; Lartey

et al

., 1998). An improvedimmunostaining procedure that is less destructive to tissue detail

Fig. 1 Neighbour joining phylogenetic tree derived from the alignment of movement protein amino acid sequences of tobamoviruses. SG-1 to SG-3 indicate the branches subtending the three major subgroups of tobamoviruses. Neighbour joining was as implemented in CLUSTALX. Abbreviations and accession numbers of virus or virus strains used are: CFMMV, NP_072163, Cucumber fruit mottle mosaic virus; CGMMVC, BAA87613, Cucumber green mottle mosaic virus 1, C; CRMV, Q66221, Chinese rape mosaic virus; Cr-TMV, S48700, Tobacco mosaic virus (strain Cr-TMV); CTMV-W, NP_543051, Crucifer tobamovirus, Watermelon; FPMV, AAD44359, Frangipani mosaic virus; HiMV, AF395899, Hibiscus mosaic virus; KGMMV, CAC44968, Kyuri green mottle mosaic virus; ORSVD, AAB53795, Odontoglossum ringspot virus, Dawson; ORSVNP, NP_056811, Odontoglossum ringspot virus, Ryu & Park; PPMVS, P29097, Pepper mild mottle virus, Spain; RMVS, NP_115482, Ribgrass mosaic virus, Shanghai; SHMV, P03585, Sunn-hemp mosaic virus; TMGMV, NP_062915, Tobacco mild green mosaic virus; TMGMVJ, BAB83988, Tobacco mild green mosaic virus, Japanese; TMGMVU, P18338, Tobacco mild green mosaic virus, U2; TMV2a, BAA04268.1, Tobacco mosaic virus, 2a; TMVCg, conceptually translated from D38444, Tobacco mosaic virus, Cg; TMVCH, JC1339, Tobacco mosaic virus, China; TMVK, AAD44328.1, Tobacco mosaic virus, K; TMVOb, Q83485, Tobacco mosaic virus Ob; TMVR, Q98746, Tobacco mosaic virus, Rakkyo; TMVV, P03583, Tobacco mosaic virus, vulgare; ToMVL, P03584, Tomato mosaic virus L.; TVCV, NP_046153, Turnip vein-clearing virus; ZGMMV, CAC82484, Zucchini green mottle mosaic virus. In a tree showing relationships among coat protein amino acid sequences, the Holmes Ribgrass mosaic virus branch emerges from the branch labelled SG-3.

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than previous methods has been used to locate the virus in elec-tron micrograph images (Ghoshroy and Citovsky, 1998).

TVCV MOLECULAR BIOLOGY

Fibre diffraction studies (Stubbs, 2001) resulted in a detailedthree-dimensional description of the structure of the HRMV cap-sid (Wang et al., 1997). The description was facilitated by theobservation, derived from the TVCV nucleotide sequence, that theHRMV coat protein sequences determined by protein chemicalmeans were likely mistaken in the order of amino acids in a(his,gly) dipeptide (Lartey et al., 1996). Three features not foundin the structures of TMV and Cucumber green mottle mosaic virus(CGMMV) were revealed. The C-terminal-most helix is shorter,giving the monomer a stubbier appearance. The predominantloop on the inner surface has a β-hairpin instead of an irregularstructure. Most interestingly, a patch of negative charge noted forTMV and CGMMV capsid monomers also exists for HRMV, but iscreated by a different set of amino acid residues.

With some notable exceptions, the organization of the TVCVRNA genome is typical of tobamoviral RNAs. TVCV RNA has a 5′-capstructure demonstrated by a newly developed method, calledcap jumping, for identifying such caps (Efimov et al., 2001). Inthis method, an oligonucleotide is covalently attached to thecap’s 2′,3′-cis glycol, after which it can serve as part of the tem-plate for reverse transcriptases since these have a terminal trans-ferase activity that forms a bridge past the triphosphate. Capstructures are also present on RNAs of other Tobamovirus species.Non-translated regions exist at 5′ and 3′ ends (Lartey et al.,1995). That at the 5′ end is G-poor, a feature thought to facilitate5′-to-3′ uncoating of TVCV rods upon infection. The 3′ non-translatedregion can assume secondary and three-dimensional structuresthat for TMV RNA have been demonstrated to be acylatablewith histidine (Mans et al., 1991). The TMV 3′ end is also recog-nized by the tRNA terminal nucleotidyl transferase which can addCCA to the 3′ end of the RNA if it is not present in the transcript(Zhang et al., 1999).

The remainder of the TVCV RNA contains ORFs for at least fourpolypeptides (Dorokhov et al., 1994a; Lartey et al., 1995). The5′-most ORF encodes a 125 kDa polypeptide, which is slightlysmaller than the TMV homologue. It contains motifs which arerecognized as characteristic of a guanine methyltransferase andan RNA helicase. The ORF ends at a UAG non-sense codon. Aswith other Tobamovirus and Tobravirus species, this codon isread-through with appreciable frequency to produce an RNA rep-licase domain from the immediately 3′-adjacent sequence. Incor-poration of the sequence surrounding the UAG in TVCV and othersubgroup 3 tobamoviruses into an alignment of sequences sur-rounding read-through stop codons led to the identification ofone of six groups of viral sequences facilitating read-through(Harrell et al., 2002).

The 3′ most ORFs encode a movement protein (MP) and thecoat protein (CP). The MP, a member of the 30 K superfamily ofmovement proteins (Koonin et al., 1991; Melcher, 1990; Melcher,2000), is required for movement of the infection both from cell tocell in turnip and tobacco and over long distances in A. thalianaand other plants (Zhang and Melcher, unpublished data). The ori-gin of virion assembly is probably in the MP ORF (Lartey et al.,1996) as it is in subgroup 1 viruses (Jonard et al., 1977; Zimmern,1977), but not in subgroup 3 viruses (Fukuda et al., 1980; Fukudaet al., 1981), where it is in the CP ORF. There are RNA sequenceand predicted secondary structure similarities between the sub-group 1 and subgroup 2 RNAs in the region of TMV’s origin ofassembly and virus particles of a size expected for the encapsida-tion of subgenomic CP mRNA have not been detected for TVCVor its relatives. A small ORF overlaps the 3′ end of the MP codingregion in a different frame in many tobamoviruses (Morozovet al., 1993). Its predicted product has been suggested to func-tion in infection in Nicotiana benthamiana but not in N. tabacum(P. Paulkaitis, T. Canto, S.A. MacFarlane, unpublished data). How-ever, efforts to establish a role in the infection of other specieshave not been successful, though an interaction with EF-1-αtranslation elongation factor has been demonstrated (Fedorkinet al., 1995). The ORF may be non-functional in TVCV due to theabsence of an in-frame AUG initiation codon, but alternate initi-ation codons are possible.

A distinguishing feature of the genome of TVCV and subgroup3 viruses is that MP and CP ORFs overlap for a much larger dis-tance than in the viral genomes of other subgroups. The initiationcodon of TVCV CP synthesis occurs 75 nt 5′ of the stop codon forthe MP ORF. In this stretch, two different reading frames obtainexpression in protein sequence. Analysis of sequence divergencesin this region suggested that the CP ORF was the ancestral func-tional ORF and that its 5′ region acquired the ability to encode afunctional C-terminal end of the MP (Lartey et al., 1996).

Replication of TVCV-like tobamoviruses is likely to occur incomplexes bound to membranes. Accumulation of the TMV-Cgisolate of ORMV is much reduced in tom1 mutant A. thaliana(Ishikawa et al., 1991). The reduction of TMV-Cg levels reflectsa much diminished ability of the virus to replicate in tom1protoplasts (Ishikawa et al., 1993). TOM1 encodes a six or seventransmembrane-helix protein (Yamanaka et al., 2000) found inplasma and tonoplast (Hagiwara et al., 2003). Its interaction withthe helicase region of the ORMV 130 kDa protein suggests thatit anchors replication complexes to the membrane. Its function inhealthy plants is unclear. A TOM1 homologue, TOM3, has recentlybeen described and accounts for the incomplete reduction ofTMV-Cg levels in tom1 plants (Yamanaka et al., 2002).

The paradigm for the translation of tobamoviral MP and CPORF’s is the production of subgenomic RNAs whose 5′ ends are justupstream of the start codons for MP or CP synthesis. Though suchRNAs are produced during TVCV infection, evidence suggests

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that translation can also initiate from genomic RNA by ribosomesbinding directly to an internal ribosome entry site (IRES) (Ivanovet al., 1997). In vitro studies using dicistronic constructs revealedthat an IRES exists in TVCV just upstream of both the CP (Ivanovet al., 1997) and MP genes (Skulachev et al., 1999). Only the MPproximal IRES seems to be present in TMV. What role, if any, inter-nal ribosome entry plays in natural TVCV infection is unclear.However, an examination of the Cr-TMV CP IRES sequence led tothe discovery that (GA2–5)n behaves as a strong IRES in plants,mammals and yeast (Dorokhov et al., 2002). The simplicity of thissignal allows an identification of IRES sequences in the 5′ non-translated regions of mRNAs by computational searches and mayalter our concept of the initiation of protein synthesis.

PLANT–VIRUS INTERACTIONS

The ability of TVCV to infect A. thaliana led to its use in the inves-tigation of virus–plant interactions, including the production ofsymptoms of infection, movement of the infection through thehost, resistance to infection, and suppression of gene silencing.Interaction of viral components with host molecules must occurthroughout the infection process. On entry of the virus into anuninfected cell, viral RNA requires host components for replica-tion. The need for TOM1 (Yamanaka et al., 2000) and TOM3(Yamanaka et al., 2002) proteins for organizing replication com-plexes has already been mentioned. A mutation in the TOM2region also reduces ORMV (Ohshima et al., 1998) accumulationin protoplasts. The tom2 mutation has recently been shown to bea deletion that includes two genes, TOM2A and TOM2B, that arerequired for the efficient replication of ORMV (Tsujimoto et al.,2003). TOM2A encodes a membrane protein whose principallocation, along with that of TOM1, appears to be in tonoplastmembranes (Hagiwara et al., 2003). Tonoplast membranes ofORMV-infected plants contain complexes competent in viral RNAreplication.

Certain physiological changes can make plant cells inhospitableto viral replication. Salicylic acid induces resistance to TVCVinfection, as do the alternate oxidase targeting chemicals cyanideand antimycin A (Wong et al., 2002). The latter do not induce PRproteins and an active NPR1 gene (active in PR protein inductiondownstream of salicylic acid) is not required for resistance.

The MP, an early viral product, has reported interactions withRNA and four host proteins. TVCV MP binding to RNA (Ivanovet al., 1994) inhibits the translatability and infectivity to proto-plasts of the RNA (Karpova et al., 1997). TVCV MP is a proteinkinase substrate. Phosphorylation of the TMV MP by proteinkinase prevented the inhibition of translation and infection(Karpova et al., 1999) due to binding to RNA. The RNA binding islikely to be not RNA-sequence specific since co-bombardment ofthe MP gene with a MP-defective Potato virus X (PVX) genomerestored the ability of PVX to move from cell to cell (Morozov

et al., 1997). A screen of a cDNA expression library by the farWestern method using TMV MP as probe identified an orthologueof the KELP transcription activator as a protein that binds theCTMV-W MP (Matsushita et al., 2001). The same method alsoidentified a transcriptional co-activator, multiprotein bridge fac-tor 1 (MBF1), that binds the MP of CTMV-W (Matsushita et al.,2002). A tobacco protein that interacts with the TVCV MP wasidentified as a pectin methylesterase (Chen et al., 2000). The sig-nificance for the infection process of each of these interactions isas yet unclear.

As the virus becomes established in the initially infected cell,it begins to spread to neighbouring cells. TMV is restricted in itsmovement on the inoculated leaves of turnip plants, while TVCVmoves efficiently locally and systemically. Gene exchangeexperiments demonstrated that the TMV MP is not responsiblefor the restriction of TMV local movement (Zhang et al., 1999).Similar gene exchange experiments between TMV-Cg and TMVrevealed that the TMV MP is also not responsible for the slownessof TMV spread in A. thaliana (Arce-Johnson, Medina, Padgett,Huanca and Espinoza, manuscript in preparation). Thus, someother tobamoviral products must be responsible for theseactivities.

In some plant–virus interactions, infection is not successfuldue to the triggering of a defence mechanism that targets RNAfor destruction. TVCV has been used in unravelling the geneticrequirements for post-transcriptional gene silencing (PTGS)(Vaucheret et al., 2001). Infection by the TVCV of an A. thalianaplant that has a β-glucuronidase (GUS) transgene silenced byPTGS resulted in an alleviation of the silencing, suggesting thatTVCV encodes a suppressor of silencing (Mourrain et al., 2000).Indeed, levels of TVCV RNA accumulation in these plants and inplants that can not carry out silencing due to mutations in SDE1/SGS2 and SGS3 genes were comparable. A similar result wasobtained with A. thaliana plants with mutations in an RNA-dependent RNA polymerase-encoding SDE1/SGS2 gene (Dalmayet al., 2000). The sde1 mutation severely reduces the ability of theplant to silence the expression of a GFP transgene. However, theresult was interpreted to suggest that virus induced gene silenc-ing was not dependent on SDE1/SGS2 function rather than thata suppression of PTGS occurred. The effect of TVCV infection ontransgene-induced GFP silencing was not tested. A role for aTVCV-encoded suppressor of gene silencing is consistent with thefinding that TMV also has a PTGS suppressing gene (Voinnetet al., 1999). However, other interpretations are possible (Dalmayet al., 2001).

The silencing signal and the viral infection must each travelfrom the leaf in which they were first established to other sites inthe plant. Systemic movement requires interactions with addi-tional host products. A recessive mutation, in a VSM-1 gene, con-ditions the resistance of the plant to symptom development(Lartey et al., 1998). In this mutant, systemic, but not local,

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movement of TVCV and Tomato mosaic virus was impaired,confirming that specific protein products are required for sys-temic movement. The systemic movement of TVCV in A. thalianais inhibited by concentrations of cadmium ion that are not toxicto the plants (Ghoshroy et al., 1998). Inhibition is not dependenton the salicylic acid pathway and the inhibition is specific, in thatmovement of the unrelated Tobacco etch virus was not affected(Citovsky et al., 1998). Such cadmium ion concentrations alsoinhibit post-transcriptional gene silencing (Ueki and Citovsky,2001), suggesting a pathway shared, at least in part, by thespread of virus infection and the spread of the silencing signal.Effective cadmium ion concentrations up-regulate the synthesisof a glycine rich protein that appears to play a role in callosedeposition at the exit site of the phloem (Ueki and Citovsky,2002).

The description of the interactions between A. thaliana withviruses is just beginning, with many more interesting interactionslikely to be uncovered. For example, a screen of ethylmethanesulphonate-mutagenized A. thaliana produced a mutant thatreacted more severely than the wild-type to TVCV infection(Sheng et al., 1998). The effect of the recessive mutation in a sin-gle gene, VID-1, could be repressed by administration of auxin,indicating complex interactions with host physiology and devel-opment. Another fertile source for discovering how TVCV inter-acts with its A. thaliana host is gene expression profiling. Thelevels of mRNAs for over 400 A. thaliana transcription factorshave been followed for the first 5 days of infection by TVCV anda few selected other viruses (Chen et al., 2002). The data allowcomparisons among viruses and among different kinds of patho-gens and provide a rich source of clues as to how the hostresponds to viral invasion.

ACKNOWLEDGEMENTS

The author thanks colleagues that have shared their resultsprior to publication and those that have read drafts of themanuscript. Work from the author’s laboratory mentioned inthe article was supported by the USDA Biological ImpactAssessment Program, the National Science Foundation EPSCoRprogramme, the Robert J. Sirny Professorship and the OklahomaAgricultural Experiment Station, whose Director has approved itspublication.

NOTE ADDED IN PROOF

A. thaliana heat shock protein genes are among those inducedwithin 1 day of infection with TVCV and ORMV but not with otherviruses [Whitman, S.A., Quan, S., Chang, H.-S., Cooper, B., Etes,B., Zhu, T., Wang, X. and Hou, Y.-M. (2003) Diverse RNA viruseselicit the expression of common sets of genes in susceptibleArabidopsis thaliana plants. Plant J. 33, 271–283.]

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