virus resistence

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SUMMARY

Plants have developed numerous approaches to resistinfection by viruses. On the other hand, in many in-stances viruses have evolved to overcome these variousresistance responses and barriers. The extent to whichviruses can overcome some or all of these responses and

barriers determines the extent to which they are able tocolonize plants of a given genotype or species. Resis-tance against plant viruses occurs at different levels andby various mechanisms, most of which largely remainuncharacterized. Here, we will consider these variouslevels of resistance, as well as the types of resistance re-sponses, the isolated genes involved in resistance, thesignalling pathways that have been described, and thevarious resistance factors that have been implicated inresistance to specific viruses.

INTRODUCTION

Francis Holmes (1946) made some poignant observa-tions concerning the types of resistance plants have toviruses, when he examined the host range of Tobaccoetch virus (TEV) and Tobacco mosaic virus (TMV) andshowed that (besides whether or not the plants showedlocal or systemic symptoms) the plants responses couldbe divided into four categories (Table 1). He found thatmany plant species did not show detectable virus multi-plication in the inoculated leaves, while other speciesdid; however, there was also differentiation amongst thelatter group as to whether the plant species become in-fected systemically or not. The proportion of plantspecies in each category differed between the two virus-

es. Four decades later, Zaitlin and Hull (1987) de-scribed resistance operating at four levels: inhibition ofreplication, cell-to-cell movement, and systemic infec-tion (long-distance movement), as well as defense re-sponses restricting infection to a limited number of

Corresponding author: P. PalukaitisFax: +44. 1382.568523E-mail: [email protected]

cells. With one exception, these are still the major formsof resistance that occur against virus infection (rev. byBruening, 2006). Besides these various levels describedbelow, resistance also occurs via the surveillance mecha-nism designated RNA silencing or RNA inhibition(RNAi). We will not consider RNAi in detail here, as ithas been covered in many reviews in recent years

(Brodersen and Voinnet, 2006; Burgyn, 2006; Li andDing, 2006; Vaucheret, 2006; Ding and Voinnet, 2007).Nevertheless, the effects of RNAi may also be a con-tributing factor to either the various types of inducedresistance or the barriers to infection described below.

TYPES OF RESISTANCE

Complete resistance in a plant to virus infection is re-ferred to as immunity (reviewed by Bruening, 2006). Inplants that convey immunity to viruses in one genotypeof a species but not another, the immunity is usuallymanifested in preventing virus replication. Usually, thisis assessed in isolated, single cells (protoplasts), or multi-ple leaf cells co-infected by agroinfiltration of DNA ex-pressing viral genomes. If immunity occurs against allbiotypes of a pathogen and in all cultivars or accessionsof a particular plant species the situation is referred to asnon-host resistance. For viruses, this is a largely an un-explored area, although some strides are being made inunderstanding non-host resistance operating against fun-gi (reviewed by Ellis, 2006; Bittel and Robatzek, 2007;Hckelhoven, 2007; Wise et al., 2007; Hadwiger, 2008).

Some resistance genes have been described as con-veying extreme resistance (ER). In some cases thesemay actually convey immunity, in that no virus multipli-

cation could be detected (Barker and Harrison, 1984;Watanabe et al., 1987) while in the case of Potato virus

X (PVX), replication occurred to a limited extent andthen an induced resistance occurred, leading to preven-tion of further replication (Khm et al., 1993). In cow-pea (Vigna unguiculata) cv. Arlington, where a dominantresistance limiting accumulation ofCowpea mosaic virus(CPMV) in protoplasts was described, the resistance

was due to inhibition of the polyprotein processing ofthis virus (Ponz et al., 1988).

Journal of Plant Pathology (2008), 90 (2), 153-171 Edizioni ETS Pisa, 2008 153

INVITED REVIEW

PLANT RESISTANCE RESPONSES TO VIRUSES

P. Palukaitis1 and J.P. Carr2

1

Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK2Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, UK

001_JPP_Review_153 21-07-2008 9:57 Pagina 153


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In those instances where replication of viruses oc-curred at normal levels, but the virus was preventedfrom moving outside of the inoculated cells, a sublimi-nal infection was said to occur (Sulzinski and Zaitlin,1982) and further infection was thought to be inhibitedby either a dysfunctional viral-encoded movement pro-

tein for that plant genotype or some barrier in that plantgenotype that prevented the normal function of the viralmovement protein (reviewed by Waigmann et al., 2004;Ueki and Citovsky, 2006). In some cases, specific resist-ance genes of the plant have been identified in blockingor preventing viral cell-to-cell movement, with corre-sponding changes occurring in mutants of those virusesthat can overcome the resistance gene (reviewed byKang et al., 2005a; Bruening, 2006).

In some plant genotypes the virus can move cell-to-cellto a limited extent, before a multicellular hypersensitiveresponse (HR) occurs, which first activates defense re-sponses preventing the infection from spreading further

and then kills the cells within the infected zone (reviewedby Gilliland et al., 2006a; Loebenstein and Akad, 2006).The ER and HR are referred to as types of innate resist-ance. Both are associated with dominant resistance genes(reviewed by Bruening, 2006; Maule et al., 2007). Plantsundergoing an HR also induce a state of pathogen-non-specific resistance called systemic acquired resistance(SAR) (reviewed by Gilliland et al., 2006a). SAR is acti-vated by defense signalling responses and will be consid-ered below. If no ER or HR occurs, then the virus maycontinue to spread cell-to-cell throughout the leaf, as wellas into and within the vasculature.

If the virus cannot spread to upper leaves of theplant, then it is likely that there is a barrier to infectionpreventing the virus from ingressing into the sieve ele-ments of the phloem (Waigmann et al., 2004; Bruening,2006; Ueki and Citovsky, 2006). This barrier may pre-vent movement into one or more of the cell types withinthe vasculature: bundle sheath cells, vascular or phloemparenchyma cells, and/or companion cells. Ingress into

the sieve element from the companion cells also may beblocked, as might egress from the sieve elements tocompanion cells, and then to other vascular cells, al-though data from grafting experiments tend to suggestthat ingress rather than egress is the major barrier (Bru-ening, 2006).

The above observations are consistent with the workof Holmes (1946) on the extent of virus infection in 310species of plants infected by TMV vs. TEV (Table 1). Heshowed that infectivity could not be recovered from theinoculated leaves by back inoculation to a susceptiblehost from 111 of 310 plant species inoculated with TMVand 227 of 310 plant species inoculated with TEV; i.e.,they were immune or possibly had a subliminal infection.On the other hand, infectivity could be recovered fromthe inoculated leaves, but not from the upper leaves of127 (TMV) and 22 (TEV) inoculated plant species, whileinfectivity could be recovered from all leaves of 72(TMV) and 61 (TEV) inoculated plant species.

Infection of upper leaves may be limited to a fewleaves, before the plant recovers. This may be a manifes-tation of age-related resistance, which appears to be asalicylic acid (SA)-mediated form of innate resistance(Garcia-Ruiz and Murphy, 2001), or may be due to theinability of the virus to completely block RNAi (Ji andDing, 2001). Most viruses probably encode RNA silenc-ing suppressors, which prevent one or more steps asso-ciated with RNAi (Li and Ding, 2006). In plants show-ing recovery from infection by nepoviruses and to-braviruses, recovery is due to a failure of the virus toprevent RNAi from being established ahead of the virusinfection in upper, newly formed leaves (Ratcliffet al.,1997, 1999). This recovery from infection also occurs insome hosts after infection by Cauliflower mosaic virus(CaMV) (Covey et al., 1997).

Tolerance is a manifestation of resistance in whichthe plants may show only mild or no symptoms as afunction of infection (Bruening, 2006). In the experi-ments described by Holmes (1946), virus also could be

154 Resistance to viruses Journal of Plant Pathology (2008), 90 (2), 153-171

Table 1. Susceptibility types to Tobacco mosaic virus (TMV) and Tobacco etch virus (TEV) in 310 plantspecies tested.

Plant responseInoculation with TMV

No. of speciesInoculation with TEV

No. of species

No symptomsNo recovered virus 111 227

No symptomsVirus in inoculated leaves only

100 15

Local symptomsVirus in inoculated leaves only

27 7

No symptomsVirus in upper leaves

15 8

Systemic symptomsVirus in inoculated leaves

57 53



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recovered from various leaves of different plant species,whether they showed symptoms or not (Table 1). Wheregenes conferring tolerance have been delimited, multi-ple, recessive genes are involved (reviewed by Fraser,1990, 1992; Kang et al., 2005a; Bruening, 2006; Mauleet al., 2007). In addition, in most cases examined, toler-

ance is usually associated with a reduced titer of virus inthe plants. The relationship between recovery and toler-ance has not been investigated in detail, although in oneinstance, tolerance specified by two or three specificgenes against Cucumber mosaic virus (CMV) in cucum-ber could be overcome by co-infection with Zucchini

yellow mosaic virus (ZYMV), which increased CMVreplication in the upper leaves (Wang et al., 2004). Thissuggests that tolerance may be a manifestation of RNAioperating against a specific virus.

RESISTANCE GENES

Many resistance genes have been described operatingagainst specific viruses in particular crop species (re-viewed by Fraser, 1990, 1992; Kang et al., 2005a; Bruen-ing, 2006). Most of these resistance genes have not beenisolated and so remain uncharacterized. Many more re-sistance genes operate as quantitative traits loci and thusfunction additively in conferring resistance (Maule etal., 2007). Nevertheless, in the past 15 years, more than20 resistance genes have been isolated and characterized(Maule et al., 2007); most of them fall into two maingroups in terms of common features, while the resist-ance genes that do not, appear quite different from therest in form and presumably function. There is a generalassumption that a dominant resistance gene reflects anencoded positive effect limiting or blocking a virus,

while the allele for susceptibility does not have such arole. By contrast, a recessive resistance gene is thoughtto be one where the encoded protein is unable to func-tion to promote virus infection, while its dominant alleleconveying susceptibility can do so.

Dominant resistance. Most of the resistance genesisolated are dominant genetically and are involved in re-sistances manifested by an HR or an ER (Maule et al.,2007). The proteins encoded by these resistance genesshow considerable similarity in their organization, in

which three functional domains have been defined: anN-terminal domain similar to either a Toll/interleukin-1receptor (TIR) domain or a coiled-coiled (CC) proteindomain; a nucleotide binding domain (NB); and aleucine-rich repeat (LRR) domain. These proteins aresimilar to those associated with resistance to bacteria,fungi, insects and nematodes (Jones and Dangl, 2001,2006). They are all considered to be involved in activat-ing defense signalling responses. A list of the virus re-sistance genes that encode such proteins is shown in

Table 2. These include several resistances operatingagainst specific tobamoviruses, for which resistance-breaking strains have been isolated (Pelham, 1972; Hall,1980; Tbis et al, 1982; Watanabe et al., 1987; Padgettand Beachy, 1993). Resistance-breaking strains have alsobeen identified for the Rx gene operating against PVX

(Moreira et al., 1980) and the Sw-5 gene conferring re-sistance to Tomato spotted wilt virus (TSWV) (Arambu-ru and Mart, 2003; Ciuffo et al., 2005).

The dominant resistance genes that do not encodesuch defense signal response activation proteins includethe Tm-1 gene from tomato and the genes associated

with resistance to systemic infection by TEV in Ara-bidopsis thaliana. The Tm-1 gene product was found toblock virus replication of both TMV and Tomato mosaicvirus (ToMV) in protoplasts (Motoyoshi and Oshima,1977; Fraser and Loughlin, 1980; Watanabe et al., 1987;Meshi et al., 1988; Ishibashi et al., 2007). The encodedprotein, designated p80GCR237, is unrelated to any pro-

tein with a known function, but contains a TIM barrelstructure identified in a wide variety of enzymes(Ishibashi et al., 2007). The resistance of some ecotypesofArabidopsis thaliana to the systemic movement ofTEV requires three genes designatedRTM1,RTM2 and

RTM3 (Chisholm et al., 2000), the last of which was notcharacterized. The RTM1 gene was shown to encode aprotein similar to the lectin jacalin (Chisholm et al.,2000), while the RTM2 gene was shown to encode a 41kDa protein similar to small heat-shock proteins, al-though its expression was not stimulated by heat nordid it function in thermotolerance (Whitham et al.,2000). Both theRTM1 andRTM2 genes were expressedexclusively in phloem-associated cells and the corre-sponding proteins localized to sieve elements (Chisholmet al., 2001). Therefore, these proteins presumably func-tion in preventing long-distance movement of TEV in

A. thaliana, by a yet unknown mechanism.

Recessive resistance. Using a candidate gene ap-proach, several laboratories have isolated recessive resist-ance genes (Table 2) conferring resistance against particu-lar potyviruses (Ruffel et al., 2002, 2005, 2006; Nicaise etal., 2003; Gao et al., 2004; Kang et al., 2005b; Bruun-Ras-mussen et al., 2007), the carmovirus Melon necrotic spotvirus (MNSV) (Nieto et al., 2006), the bymoviruses Bar-ley mild mosaic virus (BMMV) and Barley yellow mosaic

virus (BYMV) (Kanyuka et al., 2005; Stein et al., 2005),and the sobemovirus Rice yellow mottle virus (RYMV)(Albar et al., 2006). These resistance genes all encodetranslation factors involved in the formation of the 40S ri-bosome complex required to initiate translation of RNAs(reviewed by Robaglia and Caranta, 2006). The resistanceto various potyviruses is associated with the translationinitiation factors eIF4E and eIF(iso)4E, while resistancesto the bymoviruses and MNSV are associated with eIF4Eonly, and resistance to RYMV is associated with the trans-

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lation initiation factor eIF(iso)4G. The eIF4E translationinitiation factor has been shown to interact with the VPgof several potyviruses viruses while the allelic mutantsconveying resistance usually, but not always, failed to in-teract (Kang et al., 2005b; Michon et al., 2006; Beau-chemin et al., 2007; Yeam et al., 2007; Charron et al.,

2008). A number ofA. thaliana mutants that showed re-sistance to potyviruses also were associated with changesto eIF4E and eIF(iso)4E, while mutants conveying reces-sive resistance to CMV in A. thalianawere identified asbeing due to changes in eIF4E and eIF4G (reviewed byRobaglia and Caranta, 2006).


Table 2. Cloned genes conferring resistance to viruses.

Resistance genes Plant species Virus targets Reference

Dominant genes

N N. tabacum TMV, ToMV a Whitham et al., 1994

Rx1 S. tuberosum PVX Bendahmane et al., 1999

Rx2 S. tuberosum PVX Bendahmane et al., 2000

Sw-5 S. lycopersicum TSWV, TCSV, GRSV Brommonschenkel et al., 2000

HRT A. thaliana TCV Cooley et al., 2000

RCY1 A. thaliana CMV Takahashi et al., 2002

Y-1 S. tuberosum PVY Vidal et al., 2002

Tm-22 S. lycopersicum ToMV, TMV Lanfermeijer et al., 2003

Rsv1 G. max SMV Hayes et al., 2004

Tm-2 S. lycopersicum ToMV, TMV Lasfermeijer et al., 2005

RT4-4 P. vulgaris CMV Seo et al., 2006

RTM1 A. thaliana TEV Chisholm et al., 2000

RTM2 A. thaliana TEV Whitham et al., 2000

Tm-1 S. lycopersicum TMV, ToMV Ishibashi et al., 2007

PvVTT1 P. vulgaris BDMV Seo et al., 2007

Recessive genes

pvr1/pvr2 C. annuum PVY, TEV Ruffel et al., 2002;Kang et al., 2005b

mol1/mol2 L. sativa LMV Nicaise et al., 2003

sbm1 P. sativum PSbMV, BYMV Gao et al., 2004;Bruun-Rasmussen et al., 2007

rym4/5 H. vulgare BaMMV, BaYMV Kanyuka et al., 2005;Stein et al., 2005

pot-1 S. lycopersicum PVY, TEV Ruffel et al., 2005

rymv1 O. sativa RYMV Albar et al., 2006

nsv C. melo MNSV Nieto et al., 2006

pvr2 + pvr6 C. annuum PVY, TEV Ruffel et al., 2006

(a) Virus names: Barley mild mosaic virus (BaMMV); Barley yellow mosaic virus (BaYMV); Bean dwarfmosaic virus (BDMV); Bean yellow mosaic virus (BYMV); Cucumber mosaic virus (CMV); Groundnutringspot virus (GRSV); Lettuce mosaic virus (LMV); Melon necrotic spot virus (MNSV); Pea seed-bornemosaic virus (PSbMV); Pepper veinal mottlevirus (PVMV); Potato virus X(PVX); Potato virus Y(PVY);Rice yellow mottle virus (RYMV);Soybean mosaic virus (SMV); Tobacco etch virus (TEV); Tobacco mosaicvirus (TMV); Tomato chlorotic spot virus (TCSV); Tomato mosaic virus (ToMV); Tomato spottedwilt virus

(TSWV); Turnip crinkle virus (TCV).



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DEFENSIVE SIGNALLING PATHWAYS

IN RESISTANCE

The occurrence of salicylic acid and its derivatives,during incompatible and compatible interactions withviruses. SA (2-hydroxybenzoic acid) plays a central role

in the signal transduction pathway that results in SARand it is required for localization of viral and otherpathogens during the HR (reviewed by Alvarez, 2000).SA is required for the expression of a group of proteinsthat collectively are referred to as pathogenesis-related(PR) proteins (reviewed by Murphy et al., 1999; vanLoon and van Strien, 1999). The ones described from to-bacco do not appear to have any role in resistance toviruses, but are often used as markers of SAR. Duringvirus-induced HR lesion development, SA biosynthesisoccurs initially in and around the developing lesions, butlater throughout the entire plant (Malamy et al., 1990;Mtraux et al., 1990; Huang et al., 2006; Nobuta et al.,

2007; Strawn et al., 2007). Most of the SA produced isconverted to a glucose conjugate (SA-2-O--D-gluco-side; SAG) that accumulates within cell vacuoles (Enyediet al., 1992; Hennig et al., 1993; Dean and Mills, 2004).It has been suggested that in SAR-expressing tissues,SAG represents a storage form of SA from which free,biologically active SA can be released in response to fur-ther infection (Hennig et al., 1993).

The induction of SAR and, to some extent, mainte-nance of basal resistance to pathogen infection dependson SA-mediated signalling. Consequently, if SA accu-mulation is inhibited by engineering plants to expressthe salicylate-degrading enzyme SA hydroxylase (nahG-transgenic plants; Gaffney et al., 1993; Mur et al., 1997),

or if SA production is decreased by mutation of SAbiosynthetic genes (Nawrath and Mtraux, 1999; Wil-dermuth et al., 2001), plants are not able to expressSAR and are super-susceptible to both virulent and avir-ulent pathogens.

SA can be detected in phloem sap (Mtraux et al.,1990; Rasmussen et al., 1995), consistent with a role inthe spread of SAR induction to all parts of the plant.However, results, sometimes conflicting with each other,obtained from grafting experiments with differentnahG-transgenic tobacco lines (Vernooij et al., 1994;Darby et al., 2000) cast doubt on the idea that SA isnecessarily an essential translocated signal in SAR in-

duction. Subsequently, the isolation of the A. thalianadir1 mutant, defective in a gene encoding a lipid trans-fer protein, suggested that a lipid or lipid-derived sub-stance, possibly jasmonic acid (JA), could be thetranslocated SAR-inducing signal (Maldonado et al.,2002; Truman et al., 2007). Recently, work from theKlessig laboratory showed that transgenic plants si-lenced for the gene encoding a SA-binding (SABP2)

were compromised in gene expression of PR proteinsand SAR induction (Park et al., 2007). SABP2 is an es-

terase that can convert methyl salicylic acid (Me-SA) in-to SA (Kumar et al., 2006). Me-SA is produced in tis-sues undergoing an HR (Shulaev et al., 1997). It is avolatile chemical that can diffuse into the air and eveninduce SAR in nearby, non-infected plants (Shulaev etal., 1997). Taken together these studies provided evi-

dence suggesting that Me-SA is an important long-dis-tance signal in the induction of SAR following a HR.

Although SA is needed for the maintenance of basalresistance, successful pathogen localization during theHR and the establishment of SAR, an increase in itsbiosynthesis can occur during a compatible interaction

with certain virulent pathogens. With respect to virus in-fection, systemic, non-necrotizing infections do not nor-mally trigger biosynthesis of SA and the consequent in-duction of SA-responsive genes (PR genes etc.) (Malamyet al., 1990; Whitham et al., 2003). However, SA-respon-sive gene expression has been observed in A. thaliana(Whitham et al., 2003; Huang et al., 2005) and increased

SA accumulation was observed in potato (Krecic-Stres etal., 2005) and tobacco (A.M. Murphy and J.P. Carr, un-published data), during systemic infections with CMVand potyviruses [Turnip mosaic virus (TuMV) in A.thaliana and Potato virus Y(PVY) in potato].

These results appear to be paradoxical. For example,in the case of CMV, elevation in SA levels in the host,due to treatment with exogenous SA or induction of en-dogenous SA biosynthesis by an avirulent pathogen pri-or to infection with CMV, will inhibit the systemicmovement of the virus (Naylor et al., 1998; Ji and Ding2001; Mayers et al., 2005) (see below). However, elicita-tion of SA biosynthesis by CMV infection is a slowprocess that is only apparent after systemic movementof the virus to all parts of the plant has already occurred(Ji and Ding 2001; Whitham et al., 2003). Because ofthe counter-defensive action of the CMV 2b protein,continued replication or local movement of CMV in sys-temically infected tissues of the plant will be unaffectedby increased SA levels (Naylor et al., 1998; Ji and Ding2001; Murphy and Carr 2002). It remains to be seenhow the induction of what is nominally a defensive re-sponse can benefit CMV.

Jasmonic acid and ethylene. The relationship be-tween SA and signalling mediated by JA and its volatileester methyljasmonic acid is intriguing. JA is an oxy-

genated fatty acid (oxylipin) that is a signal in resistanceto certain bacterial and fungal pathogens and against in-sect pests (Reymond and Farmer, 1998; Thaler et al.,2004). Together with the gaseous plant hormone ethyl-ene (ethene), JA also regulates a systemic resistancepathway inducible by non-pathogenic microbes [in-duced systemic resistance (ISR)] that primes resistanceto fungi and bacteria in A. thaliana (Ton et al., 2002).However, based on studies with Turnip crinkle virus(TCV), it appears that ISR is not effective against virus




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infection (Ton et al., 2002).The interplay, or cross-talk, between the pathways

and the choice made between expression of SA-in-duced, anti-microbial gene products (e.g., PR proteins)versus JA-induced antimicrobial gene products (e.g.,defensins) is regulated at several levels. In A. thaliana,

the products of the PAD4 and EDS1 genes promote SA-mediated gene induction and repress gene expressioninduced by JA. These regulatory proteins are them-selves negatively regulated through phosphorylationcatalyzed by a protein kinase (Brodersen et al., 2006)(see next section).

Additional points for cross-talk between SA- and JA-mediated signalling pathways are provided by antago-nistic effects on the re-localization of WRKY transcrip-tion factors from the cytoplasm to the nucleus (Balbiand Devoto, 2007; Miao and Zentgraf, 2007). Variousmembers of this large family of transcription factors(characterized by the amino acid sequence motif

WRKYGQ/KK) are up-regulated during defense re-sponses mediated by both SA and JA and the promotersof many defense-related inducible genes (including PRgenes) contain W-boxes for WRKY binding (Du andChen, 2000; Balbi and Devoto, 2007; van Verk et al.,2008). In A. thaliana, two members of this family,WRKY53 and WRKY70 appear to act as crucial nodesin a network that maintains the equilibrium betweenSA- with JA-mediated signalling and that regulate theresponses of these pathways to either pathogen attackor the developmental and environmental cues that trig-ger senescence (Balbi and Devoto, 2007; Li et al., 2004).

Although the JA and SA pathways are viewed pre-dominantly as being mutually antagonistic, pioneeringtranscript profiling work revealed significant positive as

well as negative cross-talk between the two pathways(Schenk et al., 2000). Other work has shown that JA issynthesized transiently in the earliest stages of a TMV-induced HR and may play a minor role in localization(Kenton et al., 1999; Liu et al., 2004), and a recent studysuggests that JA plays a role in SAR induction, whichuntil recently was considered a JA-independent process(Truman et al., 2007).

Ethylene is produced copiously during the HR and isa strong inducer of certain PR genes. Ethylene-depend-ent signalling is indispensable for the maintenance ofbasal resistance to fungi and bacteria (van Loon and van

Strien, 1999; Geraats et al., 2007). However, ethylenedoes not appear to be essential for the induction of re-sistance to viruses, although since its biosynthesis from1-aminocyclopropane-1-carboxylic acid results in therelease of cyanide (Siegien and Bogatek, 2006), it is con-ceivable that this may contribute to the induction of re-sistance to viruses via the mitochondrial signalling path-

way (se below). It is also possible that ethylene plays arole as a negative regulator of SA-mediated resistance toviruses. It has been proposed that this may explain a de-

crease in susceptibility to infection with CaMV ob-served by Milner and colleagues in the ethylene sig-nalling mutant etr1 inA. thaliana (Love et al., 2007).

Reactive oxygen species, calcium signaling, nitric ox-ide and protein kinase activation. Reactive oxygen

species (ROS) have long been recognized as signals indefense, most notably during the oxidative burst orbursts that occur very early in the HR during a gene-for-gene response (Heath, 2000) or during recognition ofmicrobe- or pathogen-associated molecular patterns(MAMPS or PAMPS, respectively) in basal resistance(see Mackey and McFall, 2006 and references therein).MAMPS have been investigated most extensively instudies of basal resistance to bacterial pathogens, lead-ing to the identification of factors such as bacterial fla-gellin, which trigger basal or innate immunity (Gomez-Gomez and Boller, 2000). However, the triggering oftransient or even sustained ROS and ethylene produc-

tion by TMV coat protein (Allan et al., 2001), aminoacid sequences within the coat proteins of potexviruses(Baurs et al., 2008), or by CaMV gene products (Loveet al., 2005) in susceptible hosts, suggests that certain vi-ral gene products can function as MAMPS.

The oxidative burst, which occurs in cells in the im-mediate vicinity of the infection site, is due predomi-nantly to the activation of NADPH oxidase associated

with the plasma membrane. In a gene-for-gene interac-tion, the burst is biphasic, with an initial small burst(probably wound- or MAMP-induced), followed laterby a sustained burst that is often associated with the on-set of host cell death (reviewed by Torres and Dangl,2005). In A. thaliana, there are ten NADPH genes, of

which two (AtrbohD and F) encode enzymes that func-tion during the HR; the rest play roles in other plantfunctions such as development (Foreman et al., 2003).NADPH oxidase catalyses the formation of superoxide(O2-) anions that are readily converted to other ROS, in-cluding the perhydroxyl radical (.HO2) and hydrogenperoxide (H2O2), via non-enzymatic and enzymaticmechanisms (Lamb and Dixon, 1997).

ROS have several roles during the HR, but from thesignalling point of view, perhaps two are the most im-portant. Firstly, the oxidative burst activates Ca2+ ioninflux across the plasma membrane via cyclic nu-cleotide-gated channels, in addition to mobilization of

Ca2+ ions from intracellular stores (Torres and Dangl,2005; Ma and Berkowitz, 2007). The cytoplasmic do-mains of the NADPH oxidase proteins have EF-handmotifs, characteristic of proteins regulated by Ca2+ ionlevels (Keller et al., 1998). This permits changes in Ca2+

flux to function both upstream and downstream of ROSproduction, resulting in a positive feedback on ROSproduction and, in concert with nitric oxide (NO),helping to drive cell death in the HR (see below).

A second effect of alterations in Ca2+ ion concentra-




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tions in the cytoplasm is triggering of the activity of cal-cium-dependent protein kinases, as well as highly com-plex mitogen-activated protein kinase (MAPK) cascades.MAPKs are activated through phosphorylation byMAPK kinases (MAPKKs) that are in turn activated byupstream protein kinases, MAPKK kinases (MAP-

KKKs), and so on (Ma and Berkowitz, 2006). MAPKcascades function in plant defense responses and proba-bly the best studied of these are the wound- and SA-in-duced protein kinases (WIPK and SIPK; Zhang andKlessig, 1998). From studies in a variety of plant species,it is clear that MAPK cascades play roles in tissues with-in and beyond the infection site and participate in thecontrol of cell death, regulation of ROS production, PRgene induction and establishment of induced resistance(Pedley and Martin, 2006). Nevertheless, althoughMAPK cascades have been studied intensively, it is notalways clear how they control defense, especially with re-spect to defense against viral pathogens. However, there

are a few examples in which their roles have been re-vealed. InA. thaliana, MAPK4 is an important regulatorof the antagonism between SA- and JA-mediated de-fensive signalling (see above). Thus, knockout of thegene for MAPK4 results in constitutive expression ofgenes controlled by the SA-regulated pathway andknockout of responses to treatment with JA (Brodersenet al., 2006). The identities and functions of the proteinsubstrates for defense-related MAPKs remain elusive.Although proteomic studies have revealed likely sub-strates for MAPKs (Merkouropoulos et al., 2008), onlyone in vivo substrate with a clear role in defense hasbeen identified: the ethylene biosynthetic enzyme 1-aminocyclopropane-1-carboxylate synthase, which in A.thaliana is the substrate for MAPK6, the equivalent (or-thologue) to the tobacco SIPK (Liu and Zhang, 2004).

With respect to a role in resistance to viruses forMAPK cascades, no specific substrates have been iden-tified. Nevertheless, several studies using over-expres-sion or virus-induced gene silencing of candidate genesin either tobacco or N. benthamiana plants expressing atransgene for the Nresistance gene indicate that MAPKactivity is required for TMV localization in the HR (re-viewed by Caplan and Dinesh-Kumar, 2006). Thus,WIPK, SIPK and an upstream MAPKK are needed forfull Ngene-mediated resistance to TMV. SIPK, but notWIPK, is required for cell death induction during the

HR, indicating a branch in the MAPK-mediated sig-nalling pathway (Yang et al., 2001; Jin et al., 2003; Liuet al., 2004). In many cases, the phosphorylation targetsfor defense-related MAPKs are likely to be transcriptionfactors. In tobacco and pepper, increased expression ofgenes for TGA, Myb and WRKY transcription factorsoccurred after a TMV-induced HR or following treat-ment with SA (Yang and Klessig, 1996; Chen and Chen,2000; Liu et al., 2004; Park et al., 2006; van Verk et al.,2008). The connection between these factors and virus

resistance is unclear. Although members of the tran-scription factor families are known to be involved in theinduction of PR gene expression, there is no evidencethat the PR proteins are involved in virus resistance (re-viewed by Murphy et al., 1999). Nevertheless, silencingof expression of specific WRKY and Myb factors in to-

bacco did decrease the efficiency of N gene mediatedresistance to TMV, suggesting that new classes of de-fense gene await identification (Liu et al., 2004).

Calcium flux regulates biosynthesis of NO in animalcells and is thought likely to be a strong influence onNO production during plant defense responses. NO isan important defensive signal and probably exerts apositive feedback on Ca2+ flux by enhancing calcium re-lease from endomembrane stores (Ma and Berkowitz,2007; Hong et al., 2008). However, at present, the avail-able data on the mechanism(s) of NO production inplants are confusing (reviewed by Hong et al., 2008).The relative tissue levels of NO and H2O2 appear to

regulate programmed cell death during an HR (Delle-done et al., 2001). NO regulates defense gene expres-sion both at the point of infection and in distal tissues,in part by inducing the biosynthesis of SA (Song andGoodman, 2001). NO may exert some of its effects viamodulation of cyclic nucleotide-based signalling (Wen-dehenne et al., 2004) and by the S-nitrosylation of pro-tein targets (Hong et al., 2008). However, NO also maystimulate changes in nuclear gene expression and defen-sive signalling indirectly through inhibition of cy-tochrome oxidase (Huang et al., 2002). This process canlead to changes in mitochondrial redox and the induc-tion of specific sets of defense related genes (Maxwell etal., 2002). This mode of action may prove to be moreimportant directly in resistance to viruses (see below).

Cell death and the hypersensitive response. Pro-grammed host cell death (PCD) accompanies manygene-for-gene interactions and the term HR is used of-ten, though inaccurately, to describe this correlative fea-ture of resistance. Although hypersensitive cell death hasbeen studied scientifically for almost a century, we stilldo not understand its significance in resistance particu-larly with respect to viruses (Mur et al., 2008). There alsoexists uncertainty about the nature of the PCD that oc-curs during the HR. The process has sometimes been de-scribed as a variant of apoptosis. For example, caspases

are aspartate-specific cysteine proteases that are impor-tant effectors of apoptosis in animal cells and severalgroups have reported increased cysteine protease activityduring the HR (reviewed by Birch et al., 2000; Lam anddel Pozo, 2000). Furthermore, Bax, an animal PCD ef-fector protein, induced PCD when expressed from a vi-ral vector (Lacomme and Santa Cruz, 1999). However,there is no evidence that plant genomes encode eitherorthologues of animal caspases or Bax-type proteins,

while other characteristics of apoptosis, such as DNA




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laddering, are not clearly apparent during the HR (Muret al., 2008). Additionally, plant metacaspases, which aresimilar in several respects to animal caspases, do notcleave caspase substrates and it is not thought that theyare involved in the HR (Bonneau et al., 2008). Perhapsthe nearest functional analog of a caspase found to be as-

sociated with the HR was discovered by Chichkova andcolleagues (2004). This enzyme cleaves only a singlebond in a model substrate for caspase (the virD2 proteinof Agrobacterium tumefaciens) while human caspasecleaves at two sites, suggesting a greater degree of speci-ficity than its mammalian equivalents. An inhibitory pep-tide synthesized to mimic this cleavage site retarded theappearance of HR-associated cell death in the TMV:Ngene interaction (Chichkova et al., 2004).

Recent work with the Ngene-mediated HR in tobac-co suggests that defensive PCD is a form of autophagy(Liu et al., 2005; Mur et al., 2008). In other words, celldeath in the HR involves breakdown and re-cycling of

cellular materials, rather than their outright destruction.In other systems autophagy is used to remove defectivestructures or as a means of rapidly providing intermedi-ates for energy generation (reviewed by Bassham, 2007).The process of autophagy involves the compartmental-ization of cytoplasmic material into double-membranebound vesicles (autophagosomes) that fuse with eitherthe vacuole (in A. thaliana) or small lysosomes (in to-bacco), where degradation occurs (Bassham, 2007). Sig-nificantly, given the likely role of the vacuole and relatedstructures in the autophagy, vacuolar processing en-zymes (VPEs) with caspase-like activity have been re-ported in Nicotiana spp. andA. thaliana, and appear tocontribute both to cell death during the HR and to inhi-bition of pathogen spread (Hatsugai et al., 2004; Rojo etal., 2004). Work with a cysteine protease from tomato,

with 94 % similarity to the tobacco VPE, suggested thatthese proteins may have the capability of acting on thepromoter of the gene for the ethylene biosynthetic en-zyme 1-aminocyclopropane-1-carboxylic acid synthase(Matarasso et al., 2005). How an apparently vacuolar lo-calized protein influences the activity of nuclear DNA ispuzzling. Matarasso and colleagues (2005) proposedthat a posttranslational modification of tomato VPE al-lowed a proportion of the protein synthesized to mi-grate to the nucleus.

Interestingly, from the point of view of plant-virus in-

teractions, expression of the A. thaliana VPE (VPE)was enhanced during a compatible infection with TuMVin which no HR-type necrosis occurred (Rojo et al.,2004). Taken together with the observations of Whithamet al. (2003), who showed increased expression of SA-in-ducible genes by this virus, this suggests that VPE ex-pression is stimulated by SA and may be part of a de-layed resistance response. Consistent with this idea,knockout of VPE gene expression causes a modest in-crease in the accumulation of TuMV (Rojo et al., 2004).

There is no direct evidence from any system showingthat cell death is an absolute requirement for the limita-tion of virus spread during an HR. Although host celldeath is a prominent feature ofNgene-mediated resist-ance to TMV, several studies indicate that it is dispensa-ble. Weststeijn (1981) exploited the temperature-sensi-

tive nature of necrotic lesion formation and TMV local-ization in tobacco containing the N resistance gene toshow that increased temperature could facilitate the es-cape of virus from lesions for up to 12 days followingthe first appearance of TMV-induced lesions. SantaCruz and colleagues (Wright et al., 2000) used geneti-cally engineered TMV expressing green fluorescent pro-tein to confirm that virus remained in living cells at theperiphery of the HR lesion for several days followingthe appearance of the HR in NNgenotype Nicotiana ed-wardsonii. Mittler et al. (1996) grew NN genotype to-bacco plants in an oxygen depleted atmosphere to in-hibit ROS formation and cell death following inocula-

tion with TMV, and more recently, Kirly et al. (2008)applied antioxidant agents to inhibit TMV-inducedROS generation. In both studies plants were still able toinhibit the spread of the virus, confirming that celldeath induction and virus localization are separateprocesses. Studies in other systems have shown that celldeath is either not required for resistance or can be sep-arated from resistance on a genetic basis (Schoelz et al.,2006). Thus, in potato, the gene-for-gene resistance toPVX, conditioned by the Rx gene and the viral coatprotein, does not normally elicit cell death and appearsto be an extreme form of resistance (ER; see above).Only when the coat protein was expressed using agroin-filtration in large swathes of leaf tissues inRx-containingpotato plants was HR-associated cell death induced(Bendahmane et al., 1999).

Cell death and resistance can be induced by separatedomains within a single viral elicitor molecule. Thus, astudy of HR-type resistance to CMV in cowpea showedthat specific and distinct amino acids within the viralRNA polymerase sequence were responsible for the in-duction of virus localization and the elicitation of celldeath (Kim and Palukaitis, 1997). Furthermore, celldeath and resistance induction triggered by CaMV inNicotiana species are controlled by separate host genes(Cole et al., 2001). Consistent with the idea that celldeath and resistance are separate processes it was ob-

served during the study of a caspase-like activity in to-bacco that while pharmacological inhibition of its activi-ty slowed the cell death process, it did not prevent local-ization of TMV in NNgenotype tobacco (Chichkova etal., 2004). Similarly, silencing of a newly discovered HR-induced, jasmonate-regulated F-box protein in tobaccoinhibited Ngene associated cell death but did not inhib-it N gene mediated restriction of TMV infection (vanden Burg et al., 2008).




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RESISTANCE FACTORS

Achievement of dominant gene-mediated resistanceusually involves factors and components at the begin-ning and at the end of pathways activated by the domi-nant resistance genes. These components include vari-

ous transcription factors (described above) and en-zymes involved in various processes, including, kinases,proteinases, RNA polymerases, RNases, replication in-hibitors, and cellular responses to ROS. Various exam-ples of such factors are described below. In addition tothose listed below, plants have been shown to produceother antiviral factors that either when added to virusinocula or previously applied to plants can inhibit virusinfection to various degrees. These include the poke-

weed antiviral protein, a ribosome-inactivating protein,the action of which has been studied extensively (re-viewed by Nielsen and Boston, 2001; Park et al.,2004a), and others that have been reported but not as-

sessed further.

Alternative Oxidase (AOX) and mitochondrial re-dox signalling. Although, so far, we have consideredROS as products of the oxidative burst, ROS are pro-duced at all times as by-products of normal metabolicactivity (Noctor and Foyer, 1998). Within mitochondriathe respiratory electron transport chain constantly givesrise to ROS as a by-product of its activity (Yip and Van-lerberghe, 2001). Plant mitochondria can minimizeROS generation while maintaining efficient electronflow into and through the respiratory chain using an en-zyme called the alternative oxidase (AOX). AOX is thesole component of a distinct branch of the respiratory

pathway, the so-called alternative or cyanide-resistantpathway, which connects oxidation of the ubiquinol/ubiquinone (UQ) pool directly to the reduction of oxy-gen to water. By engaging AOX, excess electrons flow-ing into the UQ pool are dissipated. Although no ATPis generated by the alternative respiratory pathway, itsactivity ensures that respiratory metabolism can contin-ue under conditions of stress (Maxwell et al., 1999; Af-fourtit et al., 2001, 2002; Yip and Vanlerberghe, 2001;Moore et al., 2002; Pasqualini et al., 2007). In addition,strong evidence exists to indicate that the ability ofAOX to modulate the levels of ROS in the mitochondri-on allows it to regulate ROS-mediated signal transduc-

tion and indirectly, nuclear gene expression (Maxwell etal., 2002). Among the genes affected in this way aresome of those encoding members of the Aox gene fami-ly themselves (Norman et al., 2004).

AOX is synthesized in the cytoplasm and is post-translationally translocated into the mitochondrion(Vanlerberghe and McIntosh, 1997). In all plants exam-ined to date, AOX is encoded by a small family of nu-clear genes, a subset of which are inducible. For exam-ple, A. thaliana has at least four Aox genes, of which

one, Aox 1a, is inducible by chemicals such as cyanideor antimycin A, which inhibit electron flow through thecytochrome pathway, causing an increase in mitochon-drial ROS levels (Wong et al., 2002; Singh et al., 2004).IncreasedAox gene expression and AOX activity is alsotriggered by natural and synthetic inducers of pathogen

resistance such as SA, NO and 2,6-dichloroisonicotinicacid (Rhoads and McIntosh, 1993; Chivasa et al., 1999;Huang et al., 2002). These chemicals probably inducechanges in Aox gene expression by inhibition of elec-tron flow through the cytochrome pathway (Xie andChen, 1999; Huang et al., 2002; Norman et al., 2004).

Aox gene expression also increased in tobacco (Lennonet al., 1997; Chivasa and Carr, 1998) and A. thaliana(Lacomme and Roby, 1999; Simons et al., 1999) in tis-sues undergoing a HR induced by viruses or otherpathogens. TheAox 1a promoter from tobacco has cer-tain sequence motifs in common with the promoters ofgenes encoding PR protein (Rhoads and McIntosh,

1993). Nevertheless, it cannot be regulated by the samesignalling pathway as the PR proteins since its expres-sion is not dependent upon NPR1, the key regulator ofSA-induced PR gene expression (Wong et al., 2002).

Studies of the potential role of AOX in pathogen re-sistance led to the discovery that SA-induced resistanceto viruses is mediated in part by a pathway that appearsto involve signals transduced through changes in redoxor ROS in the mitochondria. Moreover, this pathway isseparate from the NPR1-dependent pathway requiredfor SA-induced PR gene expression and resistance tofungi and bacteria (Chivasa et al., 1997; Murphy et al.,1999; Wong et al., 2002; Singh et al., 2004). Initial evi-dence for this included observations that resistance toreplication and/or movement of CMV, PVX and TMVin tobacco, as well as of Turnip vein clearing virus inA.thaliana, can be induced with non-toxic levels of an-timycin A or cyanide (Chivasa and Carr 1998; Wong etal., 2002). Similar findings with respect to the DNAvirus CaMV inA. thaliana have been reported (Love etal., 2005, 2007; Gilliland et al., 2006b). Evidence for theexistence of a mitochondrial signalling pathway regulat-ed by AOX came from studies using transgenic plantsor viral vectors to alter alternative pathway capacity(Gilliland et al., 2003; Murphy et al., 2004). Increasingthe capacity of the alternative pathway to scavenge mi-tochondrial ROS compromised resistance to TMV in-

duced by antimycin A, and resistance induction by SAor antimycin A was enhanced in transgenic tobaccoplants with decreased alternative pathway capacity. Thissuggested that AOX acts as a negative regulator of in-duced resistance to viruses (Gilliland et al., 2003). How-ever the work also indicated that SA can activate addi-tional AOX-independent resistance mechanisms againstTMV, including RNAi mediated by RDR1 (Gilliland etal., 2003). The effects of altering alternative pathway ca-pacity on chemically-induced resistance to TMV were




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subtle. In contrast, high-level expression of wild-typeand mutant AOX proteins from a TMV-derived vectorgreatly enhanced the susceptibility ofN. benthamiana tovirus infection (Murphy et al., 2004). However, in morerecent work it has been found that even the relativelysmall changes in alternative pathway capacity, which can

be produced by constitutive expression ofAox-derivedtransgenes in tobacco (Gilliland et al., 2003; Pasqualiniet al., 2007), can have dramatic effects on the ability ofplants to resist viruses other than TMV (R. Fu, J. Ver-chot-Lubicz, W.S. Lee and J.P. Carr, unpublished data).Other recent experiments with Aox-transgenic tobaccoindicate that, whereas it was thought that NO inducedresistance to TMV via an SA-dependent mechanism(Song and Goodman, 2001), it now appears that NOcan induce resistance rapidly via an additional, AOX-regulated, SA-independent mechanism (W.S. Liang and

J.P. Carr, unpublished data).

Tobacco ethylene response factor 5 (NtERF5). ERF5is a tobacco transcription factor, so named because it re-sembled ERF1 from A. thaliana in sequence and there

were four other tobacco ERFs previously identified,even though NtERF5 gene expression did not respondto ethylene (Fischer and Drge-Laser, 2004). NtERF5gene expression was enhanced by infection with TMV,but its natural expression was temperature sensitive,

with little expression at 32C. Transgenic over-expres-sion of NtERF5 was found to reduce TMV accumula-tion in the inoculated leaves, and to prevent systemic in-fection of TMV in tobacco plants expressing the Ngeneat 32C, when the N gene, SA-mediated defense re-sponse is inactive against TMV. NtERF5 is also not acti-vated by either SA or JA (Fischer and Drge-Laser,2004), and therefore represents an independent defenseresponse against TMV from that activated by the SA-dependent defense pathway.

RNA-dependent RNA polymerase (RdRp1). Infec-tion of tobacco and numerous other species by viruseshad been shown to induce an RNA-dependent RNApolymerase (RdRp) (reviewed by Fraenkel-Conrat,1986) now designated RdRp1 (reviewed by Wasseneg-ger and Krczal, 2006). Its exact role in virus infection

was not known, although at one time there was specula-tion that it might be involved in virus infection

(Fraenkel-Conrat, 1983). A cDNA clone of a gene tran-script encoding an RdRp (RDR) was isolated fromtomato (Schiebel et al., 1998). Work on defining genesinvolved in RNAi in Neurospora crassa and A. thalianaidentified a gene in each case resembling the tomato

RDR gene (Cogoni and Macino, 1999; Dalmay et al.,2000; Mourrain et al., 2000). Interrogation of the A.thaliana genome identified six genes with similarities tothe tomatoRDR gene (Yu et al., 2003). Thus, based onphylogenetic analysis, the tomato gene was designated

RDR1 and the A. thaliana RDR gene shown to be in-volved in RNAi was designated RDR6. Silencing the

RDR1 gene was shown to affect infection by TMV andPVX in N. tabacum (Xie et al., 2001), and by Tobaccorattle virus (TRV) and TMV-Cg inA. thaliana (Yu et al.,2003).RDR1 gene expression was stimulated in both to-

bacco andA. thaliana by SA (Xie et al., 2001; Yu et al.,2003) and silencing RDR1 gene expression in A.thaliana did not affect RNAi (Yu et al., 2003), indicatingthat the RdRp1 was not required for RNAi, at least notagainst the viruses tested. In tobacco, the stimulation of

RDR1 gene expression by SA, independent of the AOXpathway (Gilliland et al., 2003), and silencing of tobac-co RDR1 did not prevent the SA-mediated resistancefrom being induced (Xie et al., 2001), indicating thatRdRp1 was one of several components acting againstTMV during the SA-mediated defense response.

N. benthamianawas shown to express a translational-ly defective RDR1 gene, which was thought to explain

the susceptibility of this host to many viruses (Yang etal., 2004). However, constitutive expression of aMedica-

go truncatula RDR1 orthologue of NtRDR1 in trans-genic N. benthamiana altered the disease resistance pro-file after infection by several tobamoviruses tested, butnot against CMV or PVX (Yang et al., 2004). Thus, therole of RdRp1 in virus resistance appears to be limitedto specific viruses. Whether or not RdRp1 functionsthough a separate RNAi pathway is not clear. RdRp1does not require primers for complementary RNA syn-thesis (Fraenkel-Conrat, 1983, 1986) and may functionby making regions of single-stranded viral RNA tem-plates double-stranded, thus masking regulatory signalsand preventing them from functioning in translation orreplication.

The inhibitor of TMV replication, p80GCR237. TheTm-1 gene encodes an 80-kDa protein, designatedp80GCR237, that binds to the 126 and 183 kDa proteins ofboth TMV and ToMV and prevents them from assem-bling into the active replicase complex. However, oncethe replicase complex has been formed, the 126 and 183kDa proteins cannot bind to p80GCR237 (Ishibashi et al.,2007). These data also suggest that p80GCR237 functionsstoichiometrically and not catalytically and therefore canbe saturated or lead to resistance breakdown allowingdelayed infection to occur with time, which in fact was

described (Fraser and Loughlin, 1980). Nevertheless, inother instances, the Tm-1 gene has been shown to pro-vide resistance in the field, although resistance-breakingstrains have been identified (Pelham, 1972; Hall, 1980;Watanabe et al., 1987), with mutations in the viral-en-coded 126 kDa protein (Meshi et al., 1988; Hamamotoet al. 1997). The success of this resistance gene may re-flect the fact that the 126 kDa protein is also the RNA si-lencing suppressor of ToMV and TMV and is involvedin virus movement and activation of defense responses




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(Kubota et al., 2003; Ding et al., 2004). Thus, a reducedavailability of the 126 kDa protein may have more thanone effect on virus accumulation.

Tobacco inhibitor of virus replication (IVR). A pro-tein induced by TMV infection in Ngene tobacco was

isolated and was found to inhibit the accumulation ofTMV (reviewed by Loebenstein and Akad, 2006). Al-though IVR was induced by the N gene-mediated re-sponse against TMV, the IVR was not target-specific inits interference; IVR applied to leaf discs or leaves couldinhibit accumulation of CMV, PVX or PVY (Gera andLoebenstein, 1983). Constitutive expression of IVR, ei-ther in transgenic tobacco (Akad et al., 2005) or natural-ly in a hybrid Nicotiana derived from a cross between N.

glutinosa x N. debneyi (Loebenstein et al., 1990), gaveenhanced resistance to TMV. The mode of action ofIVR is not clear, but as it can be applied to protoplastsinfected with TMV 4 to 18 h after inoculation and still

reduce virus accumulation, it apparently interferes withvirus replication (Loebenstein and Gera, 1981).Whether it also can interfere with subsequent steps invirus infection is not know. IVR does not have single-stranded RNase activity (Gera and Loebenstein, 1983),although whether it has double-stranded RNase activityhas not been evaluated. Since it accumulates in the in-tercellular spaces (Spiegel et al., 1989), IVR probably isnot a transcription factor. It is not known by what path-

way transcription of the tobacco IVR gene is induced,although it was not stimulated by exogenously appliedSA (M. Takeshita and P. Palukaitis, unpublished).

Tobacco antiviral factor (AVF). AVF is a family ofphosphorylated glycoproteins stimulated in TMV-in-fected N-gene tobacco that when mixed with TMV, pri-or to inoculation, led to inhibition of virus accumulation(Sela and Appelbaum, 1962; Sela, 1981). Both AVF andhuman -interferon stimulated plants to produce nu-cleotides with antiviral activity (Reichman et al., 1983).AVF was purified using antibodies to human -interfer-on; however, the purified proteins did not resemble in-terferon in sequence (Edelbaum et al., 1990, 1991). Thetwo purified glycoproteins, pg35 and gp22, appeared tobe a -1,3-glucanase and an isoform of PR-5 (Edelbaumet al., 1991).

Resistance factors induced in Capsicum annuum. InCapsicum spp., resistance to several tobamoviruses iscontrolled by the allelic genes L1-L4 (reviewed byGrube et al., 2000; Sawada et al., 2004). Pathotypes ofthe tobamoviruses TMV and Pepper mild mottle virus(PMMV) can break resistance conferred by specific Lgenes (Alonso et al., 1991; Tsuda et al., 1998; Grube etal., 2000; Hamada et al., 2002; Genda et al., 2007). Inaddition, several factors have been identified that mayplay key roles in resistance to tobamoviruses in Cap-

sicum spp. These include PR-10 and the Tin2 geneproduct, both of which were found to be induced dur-ing an incompatible reaction between TMV-P0 and C.annuum cv. Bugang carrying the L2 resistance gene(Shin et al., 2003; Park et al., 2004b). Other peppergenes that appear to be involved in the defense response

were shown to be induced during this incompatible re-sponse, such as genes encoding a lipid transfer protein(CaLTP1) (Park et al., 2002) and an alanine aminotrans-ferase (CaAlaAT1) (Kim et al., 2005), but their roles inthe defense against virus infection are not known.

Transcription of the gene encoding PR-10 (CaPR-10)was induced in leaves during the incompatible responseelicited by TMV-P0, but not during a compatible reactionbetween TMV-P1,2 and hot pepper carrying theL

2 resist-ance gene. By contrast, CaPR-10 was expressed constitu-tively in roots and was not upregulated by infection withTMV0. CaPR-10 was shown to be an 18 kDa RNase withno apparent sequence specificity, capable of degrading

both TMV RNA and plant RNA. During induction ofCaPR-10 in leaves, the encoded protein was phosphory-lated, which increased its specific activity; the RNase PR-10 present in roots was also a mixture of phosphorylatedand non-phosphorylated protein. CaPR-10 transcription

was induced by SA, JA, ethylene, sodium chloride andthe herbicide methyl viologen (MV; also known asparaquat), which generates superoxide radicals (Park etal., 2004b).

The C. annuum, TMV-induced gene (CaTin2) encodesa mature 23 kDa protein that contains little sequencesimilarity to other cell wall proteins, except for the 26-amino acid signal peptide. However, fluorescently-taggedCaTin2 appeared to localize to the cell wall in cells tran-siently expressing this fusion protein, and in common

with other cell wall proteins, CaTin2was expressed pref-erentially in leaves and roots, as well as to a lesser extentin flowers, but not detectably in stems or fruit. Interest-ingly, constitutive expression ofCaTin2 in transgenic to-bacco resulted in some resistance against TMV or CMV,between one and two weeks after inoculation, althoughthe resistance was lost by one month after inoculation.This suggested that the expression of the CaTin2 cell wallprotein probably was limiting virus movement and accu-mulation. CaTin2 was induced during the incompatibleresponse elicited by TMV-P0, but not by the compatibleresponse elicited by TMV-P1,2. CaTin2 also was induced

by treatment with SA, ethylene, JA, and more slowly byABA, sodium chloride, and MV (Shin et al., 2003).

CONCLUDING REMARKS

Since the initial experiments done by Holmes (1946)demonstrating different levels of resistance to two virus-es, considerable progress has been made in understand-ing the nature of resistance and in isolating resistance




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genes. Over the last 15 years there has been immenseprogress in understanding defensive signalling in resist-ance to viruses and other pathogens, although much stillremains to be determined. We still do not fully under-stand the exact pathway of resistance, or the mechanismby which any resistance gene inhibits virus accumula-

tion or spread. However, in some cases, we know howthe resistance gene is triggered to activate the signallingpathway and some components of the signalling path-

way have been identified. Some aspects of the interac-tion of a resistance gene products and a viral-encodedprotein also have been identified. This is particularly thecase for recessive resistance genes operating against po-tyviruses, although the exact mechanism by which virusinfection is inhibited is still not clear.

Compared to these areas, relatively little progress hasbeen made in understanding how resistance responsesinhibit virus replication, cell-to-cell and long-distancemovement (see Zaitlin and Hull, 1987). In many cases,

these resistance responses have not been correlated withthe presence of specific genes, while in other cases wherespecific genes have been identified, the mechanisms by

which they affect the above process has not been deter-mined. This is particularly true with respect to specificblocks in long-distance movement. In some cases thesemay be due to RNAi, although these then would have tobe aspects of RNA silencing that occur in one genotypeof a species but not another. Although we know thatRNAi plays a part in induced and basal resistance andthat many aspects of the mechanism of RNAi have beenelucidated, other mechanisms remain elusive.

With the tools now available to isolate genes, interro-gate plant genome sequences, analyse changes in the ex-pression of multiple genes or their encoded proteins,and functional genomics, we can expect to see progressin gaining a better understanding of the identity of spe-cific resistance factors and the mechanisms by whichthey confer resistance to the infection process by specif-ic viruses. It is also hoped that some progress will bepossible in the largely unexplored area of non-host re-sistance, which may provide new sources of potentiallybroad-spectrum, durable resistance that can be exploit-ed. As we become more familiar with the effects of cli-mate change on the durability of the current array of de-ployed natural resistant genes, new sources of resistancethat are thermotolerant may also be required. Thus, un-

derstanding how resistance mechanisms operate toblock virus infection at the different levels is critical tosafeguarding the food supply against the effects of infec-tion by viral pathogens.

ACKNOWLEDGEMENTS

Work on virus resistance mechanisms in the authorslaboratories was supported by Workpackage 1.5 from

the Rural and Environment Research and Analysis Di-rectorate (RERAD) (PP), and by grants from theBiotechnology and Biological Sciences Research Coun-cil (BBSRC) and the Cambridge University NewtonTrust (JPC).

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