Peach latent mosaic viroid: not so latent

MOLECULAR PLANT PATHOLOGY

(2006)

7

(4 ) , 209–221 DOI : 10 .1111/ J .1364-3703.2006.00332.X

© 2006 BLACKWELL PUBL ISH ING LTD

209

Blackwell Publishing Ltd

Pathogen profile

Peach latent mosaic viroid

: not so latent

R ICARDO FLORES

1,

* , SON IA DELGADO

1

, MAR ÍA -ELENA ROD IO

2

, S I LV IA AMBRÓS

3

, CARMEN HERNÁNDEZ

1

AND FRANCESCO D I SER IO

2

1

Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain

2

Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, 70126 Bari, Italy

3

Instituto Valenciano de Investigaciones Agrarias, Moncada 46113, Valencia, Spain

SUMMARY

Taxonomy:


(PLMVd) is the typespecies of the genus

Pelamoviroid

within the family

Avsunviroidae

of chloroplastic viroids with hammerhead ribozymes.

Physical properties:

A small circular RNA of 336–351 nt(differences in size result from the absence or presence of certaininsertions) adopting a branched conformation stabilized by apseudoknot between two kissing loops. This particular conforma-tion is most likely responsible for the insolubility of PLMVd inhighly saline conditions (in which other viroids adopting a rod-like conformation are soluble). Both polarity strands are able toform hammerhead structures and to self-cleave during replica-tion as predicted by these ribozymes.

Biological properties:

Although most infections occurwithout conspicuous symptoms, certain PLMVd isolates induceleaf mosaics, blotches and in the most extreme cases albinism(peach calico, PC), flower streaking, delays in foliation, floweringand ripening, deformations and decolorations of fruits, whichusually present cracked sutures and enlarged roundish stones,bud necrosis, stem pitting and premature ageing of the trees,which also adopt a characteristic growing pattern (open habit).The molecular determinant for PC has been mapped at a 12–14-nt insertion that folds into a hairpin capped by a U-rich looppresent only in certain variants. PLMVd is horizontally transmit-ted by the propagation of infected buds and to a lesser extent bypruning tools and aphids, but not by pollen; the viroid is notvertically transmitted through seed.

Interesting features:

This provides a suitable system forstudying how a minimal non-protein-coding catalytic RNAreplicates (subverting a DNA-dependent RNA polymerase to

transcribe an RNA template), moves, interferes with the metab-olism of its host (inciting specific symptoms and a defensive RNAsilencing response) and evolves following a quasi-species model

characterized by a complex spectrum of variants.

INTRODUCTION

Viroids are a unique class of infectious agents restricted so far tothe plant kingdom. They are solely composed by a non-protein-coding, small, single-stranded, circular RNA able to replicateautonomously in certain hosts (Diener, 2003; Flores

et al

., 2005a;Tabler and Tsagris, 2004). Viroids therefore do not need a helpervirus to complete their infectious cycle as satellite RNAs do. As aside-effect of their replication and accumulation, viroids incitespecific diseases in most of the hosts that they infect. This is thecase, despite its name, of


(PLMVd)(Hernández and Flores, 1992).

The approximately 30 known viroids are classified into twofamilies, each containing several genera (Flores

et al

., 2005b).Members of the family

Pospiviroidae

, type species

Potato spindletuber viroid

(PSTVd) (Diener, 1972; Gross

et al

., 1978), shareconserved motifs including a central conserved region (CCR),replicate and accumulate in the nucleus through an asymmetricrolling-circle mechanism, and their replicative intermediates donot self-cleave through hammerhead ribozymes. By contrast,members of the family

Avsunviroidae,

type specie

s Avocadosunblotch viroid

(ASBVd) (Hutchins

et al

., 1986; Symons, 1981)do not have a CCR, replicate and accumulate in the chloroplastthrough a symmetric rolling-circle mechanism, and their replica-tive intermediates of both polarities self-cleave through hammer-head ribozymes (Flores

et al

., 2000). These properties, initiallyestablished for the type species of both families (Bonfiglioli

et al

.,1994; Branch and Robertson, 1984; Branch

et al

., 1988; Daròs

et al

., 1994; Harders

et al

., 1989; Hutchins

et al

., 1985; Lima

*

Correspondence

: Tel.: +34 96 3877861; Fax: +34 96 3877859; E-mail: [email protected]

210

R. FLORES

et al.


(2006)

7

(4 ) , 209–221 © 2006 BLACKWELL PUBL ISH ING LTD

et al

., 1994; Qi and Ding, 2003), have been extended to othermembers including PLMVd (Bussière

et al

., 1999; Hernández andFlores, 1992) and are presumably shared by the rest. PLMVdbelongs to the family

Avsunviroidae

and is the type species of,and gives the name to, the genus

Pelamoviroid,

the other mem-ber of which is

Chrysanthemum chlorotic mottle viroid

(CChMVd)(Navarro and Flores, 1997).

PLMVd can induce in peach a broad variety of symptoms thatinclude some that are very conspicuous. The term latent in thename of this pathogen and in that of the corresponding disease,peach latent mosaic (PLM) (Desvignes, 1976, 1980), derives fromthe observation that the vast majority of the natural infectionsoccur without leaf symptoms and, when observed, they are notstable and frequently disappear with time. The term latent alsorefers to the prolonged time required for the onset of symptoms.Under field conditions they take at least 2 years to develop, evenafter planting with infected material, and consist of: mosaics orblotches and in the most dramatic cases an albino phenotypethat completely covers the leaf area (peach calico, PC), flowerstreaking, delays in foliation, flowering and ripening, deforma-tions and decolorations of fruits, which usually present crackedsutures and enlarged roundish stones, bud necrosis, stem pittingand premature ageing of the trees, which also adopt a character-istic growing pattern termed open habit (Figs 1 and 2) (Des-vignes, 1986). Under greenhouse conditions, PLMVd isolates areconsidered severe or latent strains depending on whether or notthey induce leaf symptoms on seedlings of the peach indicator GF-305 (Desvignes, 1976, 1980). The economic importance of PLMdisease derives from the fruit alterations, reduced tree longevity,and increased susceptibility to other biotic and abiotic stresses.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF PLMVd: A SECOND HAMMERHEAD VIROID

PLM disease was initially described in France, although it mightcorrespond to disorders reported previously in the US and Japanwith other names (Blodgett, 1944; Kishi

et al

., 1973; Willison,1946), and was assumed to have a viral aethiology because noother cellular pathogen could be isolated from infected tissues(Desvignes, 1976, 1980). However, because efforts aimed at puri-fying and identifying the presumed viral particles were unsuc-cessful and the agent presented a relatively high heat resistance,

Fig. 1 Symptoms in different peach organs induced by PLMVd. (A) Typical leaf mosaic. (B) Pink broken lines on flowers (upper rows) and healthy controls (lower row). (C) Discoloured fruits (lower row) and healthy controls (upper row). (D) Roundish stones (left) and healthy elongated stones (right). Photographs courtesy of J.C. Desvignes.

Fig. 2 Defoliation and characteristic growing pattern (open habit) induced by PLMVd. Photograph courtesy of J.C. Desvignes.


211



(2006)

7

(4 ) , 209–221

the alternative possibility that it could be a viroid was considered.In line with this hypothesis, fractionation studies showed thatthe infectivity was associated with low-molecular-weight nucleicacids (Monsion

et al

., 1988), and analyses with an electrophoreticapproach specifically designed for detecting small circular RNAsrevealed the presence of a viroid-like RNA in extracts frominfected samples that was absent in healthy controls (Flores andLlácer, 1988). Further studies showed the same viroid-like RNA indifferent peach cultivars naturally infected by the PLM agent, butnot in the same cultivars cured by thermotherapy (Flores

et al

.,1990). Koch’s postulates on the viroid nature of this agent werefulfilled by inoculating GF-305 peach seedlings with an electro-phoretically purified preparation of the viroid-like circular RNAfrom a severe PLM isolate: some of the plants developed thecharacteristic symptoms of the disease and an RNA with identicalphysical properties was recovered from them (Flores

et al

., 1990).Molecular characterization of PLMVd involved direct sequenc-

ing with base-specific RNases of a fragment of the viroid RNA,which due to its relatively low accumulation

in vivo

required

considerable purification efforts

,

and then use of this informationto design a primer for the synthesis of the first-strand cDNA thatwas subsequently converted into the double-stranded form andcloned (Hernández and Flores, 1992). When the sequence ofPLMVd (337 nt) was compared with those of typical viroids andsmall satellite RNAs, the similarities found were limited and didnot include the characteristic CCRs of the viroid subgroups thenknown. However, plus and minus PLMVd RNAs presented theconserved sequences of the hammerhead structures mediatingthe self-cleavage of ASBVd and some satellite RNAs (Fig. 3). Bothhammerhead structures of PLMVd have very stable stems III andshort loops closing stems I and II (Fig. 4), resembling in these twoaspects the hammerhead structures of certain satellite RNAsmore than those of ASBVd. Plus and minus, and partial and full-length RNA transcripts of PLMVd containing the hammerheadstructures displayed self-cleavage

in vitro

during transcriptionand after purification as predicted by these structures (Hernándezand Flores, 1992). Together, these data were consistent with theself-cleavage reactions of PLMVd being most likely mediated by

Fig. 3 Primary and predicted secondary structure of lowest free energy of the reference variant of PLMVd RNA (Ambrós et al., 1998; Hernández and Flores, 1992). Plus and minus self-cleavage domains are indicated by flags, nucleotides conserved in most natural hammerhead structures by bars, and the self-cleavage sites by arrows. Black and white symbols refer to plus and minus polarities, respectively. Nucleotides involved in a pseudoknot between positions 178–181 and 211–214, proposed by in vitro assays with nucleases and binding with oligonucleotides (Bussière et al., 2000), are indicated by broken lines. (Inset) Alternative structure of the hammerhead arm similar to the cruciform structure adopted by CChMVd RNA. Adapted from Hernández and Flores (1992), Ambrós et al. (1998) and Bussière et al. (2000), with modifications.

212

R. FLORES

et al.


(2006)

7


single hammerhead structures as opposed to those of ASBVd,which operate through double hammerhead structures (Forster

et al

., 1988), a conjecture later confirmed experimentally (Beaudry

et al

., 1995). Moreover, there is sound evidence supporting thatthe hammerhead structures of PLMVd are also functional

in vivo

(see below).The finding of a second viroid RNA with hammerhead struc-

tures dissipated the doubts that ASBVd could in fact be a satelliteRNA (because avocado trees are often infected by what appearedto be one or more seed-transmissible viruses that could poten-tially act as helper viruses), and established on firm experimentalgrounds what is presently known as the family

Avsunviroidae.

Moreover, molecular characterization of PLMVd (Hernández andFlores, 1992) opened the door to the development of detectionprocedures based on nucleic acid hybridization (Ambrós

et al

.,1995; Loreti

et al

., 1995) and RT-PCR (Shamloul

et al

., 1995),which proved to be considerably more rapid than the existingbiological test (see below).

PLMVd THREE-DIMENSIONAL CONFORMATION: A COMPLEX BRANCHED SECONDARY STRUCTURE STABILIZED BY A PSEUDOKNOT

The initial search for the computer-predicted secondary structureof minimal free energy of PLMVd led to an unexpected branchedconformation very different from the rod-like or quasi-rod-likestructure of other known typical viroids (Hernández and Flores,

1992). Further analyses with multiple sequence variants ofPLMVd supported slightly modified versions of this branchedconformation with a long almost perfect double-stranded stem(the so-called hammerhead arm, capped by the loop A), in whichthe nucleotides forming both hammerhead structures, and theirself-cleavage sites, occur in opposite positions (Ambrós

et al

.,1998, 1999; Pelchat

et al

., 2000) (Fig. 3). This is due to the strongsequence and morphological similarities existing between theplus and minus hammerhead structures of PLMVd, which aremore closely related to each other than to any of the other ham-merhead structures. Consequently, a similar hammerhead arm ispredicted in the most stable conformation of both polaritystrands of PLMVd, an aspect relevant in replication (see below).The peculiar conformation of PLMVd received further supportwhen CChMVd was subsequently cloned and sequenced,because its predicted most stable folding is also a branched con-formation resembling that of PLMVd (Navarro and Flores, 1997).Moreover, the hammerhead arm of PLMVd can alternatively foldin a cruciform structure (Fig. 3) similar to that adopted by CCh-MVd (Ambrós

et al

., 1998). The insolubility of PLMVd andCChMVd in 2

M

LiCl is consistent with a folding different from therod-like or quasi-rod-like structure proposed for most viroids,which are soluble at this salinity (Navarro and Flores, 1997).

Additional data supporting the proposed PLMVd secondarystructure have been obtained by

in vitro

nuclease mapping andoligonucleotide binding shift assays, which also indicate thelikely existence of a pseudoknot-like interaction between twohairpin loops of the branched conformation. Moreover, from a

Fig. 4 (A) Schematic representation of the PLMVd plus (left) and minus (right) hammerhead ribozymes. Nucleotides conserved in most natural hammerhead ribozymes are on a black background, with those forming the catalytic pocket highlighted in yellow, and the self-cleavage sites are denoted by an arrow. A circle delimits the presumed tertiary interaction between loops 1 and 2 (in magenta) enhancing the catalytic activity. (B) Three-dimensional model of the same ribozymes. Helices I, II and III are in blue, loops 1 and 2 in magenta, and the catalytic pocket in yellow. The self-cleavage site is denoted by an arrow. Adapted from De la Peña et al. (2003), with modifications.


213



(2006)

7

(4 ) , 209–221

comparative analysis of PLMVd and CChMVd structures, the pos-sibility of a similar interaction in CChMVd was advanced (Bus-sière

et al

., 2000). The existence of such a kissing-loop interactionhas been proved for CChMVd in a recent study based on thestructural analysis of numerous natural and artificial CChMVdvariants, on bioassays in chrysanthemum to assess their bio-logical properties and on the genetic stability of the resultingprogenies (Gago

et al

., 2005). Two important properties of thekissing-loop interaction are that it is critical for the

in vivo

viabil-ity of CChMVd, probably facilitating the adoption of proper fold-ing, and that apparently it only exists in the plus polarity strand.These data indirectly support that the PLMVd interactionreported

in vitro

between loops GCGG178–181 and CCGC211–214 (Bussière

et al

., 2000) (Fig. 3) is also biologically relevantand, consistent with this notion, the only natural mutation affect-ing the nucleotides involved (C214

→

U) does not disrupt theinteraction (Gago

et al

., 2005). Because evolutionary conserva-tion of a structural RNA motif usually predicts conservation ofbiological function, the conservation in PLMVd and CChMVd,two viroids with an overall low sequence similarity (Hernándezand Flores, 1992; Navarro and Flores, 1997), of a similar kissing-loop interaction suggests that the additional stability it providesis needed in viroids with branched secondary structures fordelimiting the conformation space, avoiding kinetic traps andfacilitating the rapid adoption of the biologically relevant RNAfolding.

THE INFECTIOUS CYCLE: REPLICATION, MOVEMENT AND TRANSMISSION

Because the general aspects of viroid replication have beenrecently reviewed (Flores

et al

., 2005a; Tabler and Tsagris, 2004),we will focus here on those more specifically related to PLMVd.As already stated, PLMVd replicates through a symmetricalrolling circle mechanism. The prevalent infectious monomericcircular RNA, arbitrarily assigned as having the (+) polarity, isreiteratively transcribed by a host RNA polymerase into oligo-meric (–) RNAs, which, through the hammerhead-embeddedribozymes, self-cleave to unit-length strands that are ligated tothe monomeric (–) circular RNA. This latter species, which servesas the initial template for the second (symmetrical) half of thereplication cycle, has been detected in infected tissue at a con-centration not very different from that of the monomeric (+) cir-cular form (Bussière

et al

., 1999; Delgado

et al

., 2005), whereasthe ratio of (+) to (–) strands is considerably higher in other mem-bers of the family

Avsunviroidae

(Daròs

et al

., 1994; Fadda

et al

.,2003; Hutchins

et al

., 1985; Navarro and Flores, 1997). Alsopeculiar to PLMVd (and CChMVd) is that the monomeric linearRNAs are more abundant than their circular counterparts, andthat longer-than-unit viroid strands are absent in PLMVd-infectedpeach or accumulate at very low concentrations (Bussière

et al

.,

1999; Delgado

et al

., 2005); these differences may result fromthe high catalytic efficiency of PLMVd ribozymes and from theirspecial location within viroid strands (see below).

Early data with ASBVd, obtained by labelling the free 5

′

-triphosphate group characteristic of chloroplastic primarytranscripts with [

α

-

32

P]GTP and guanylyltransferase (

in vitro

capping), together with RNase protection assays, mapped theinitiation sites of ASBVd (+) and (–) RNAs isolated from infectedavocado at similar A+U-rich terminal loops in their predictedquasi-rod-like secondary structures (Navarro and Flores, 2000).By contrast, results from primer-extension analysis of the 5

′

ter-mini of certain PLMVd subgenomic RNAs isolated from infectedtissue, presumed to be replication by-products, and from

in vitro

transcriptions with truncated PLMVd RNAs and the RNA poly-merase of

Escherichia coli

, identified the initiation sites of thisviroid at the loop A capping the hammerhead arm (Fig. 3) (Pelchat

et al

., 2001). However, the presence of free 5

′

-triphosphategroups in the subgenomic RNAs was not examined, and theinitiation complex reconstituted

in vitro

might not reproducethe

in vivo

situation because the specificity of the RNA polymer-ase from

E. coli

may be very different from that of its homologouschloroplastic RNA polymerase (PEP, from plastid encodedpolymerase) (Sugiura, 1992). Moreover, a second RNA polymer-ase class exists in chloroplasts (NEP, from nuclear encodedpolymerase) (Allison

et al

., 1996), with the available evidencesuggesting that the enzyme involved in ASBVd replication has aNEP-like activity (Navarro

et al

., 2000). Attempts to extend theapproach used for mapping the ASBVd initiation sites to PLMVdfailed because the RNAs of this viroid accumulate

in vivo

to con-siderably lower levels than do those of ASBVd. Re-examination ofthis question with a more sensitive methodology, comprising acombination of

in vitro

capping and an RNA ligase-mediatedrapid amplification of cDNA ends developed for identifying thegenuine capped 5

′

termini of eukaryotic messenger RNAs, hasmapped the PLMVd (+) and (–) initiation sites at a similar double-stranded motif of 6–7 bp that also contains the conserved GUCtriplet preceding the self-cleavage site in both polarity strands;this motif is located at the base of the hammerhead arm (Fig. 3),which presumably includes the promoters for a chloroplastic RNApolymerase (Delgado

et al

., 2005). Regarding the nature of thisRNA polymerase, recent data indicate that synthesis (and accu-mulation) of PLMVd strands is very active in leaf areas displayingthe PC symptomatology, in which development of proplastidsinto chloroplasts and processing of certain plastid rRNA precur-sors (and, consequently, translation of plastid-encoded proteins)are impaired (M. E. Rodio

et al.

, manuscript in preparation). Theseobservations are consistent with the involvement in PLMVd rep-lication of a NEP-like enzyme, which could be the same as pro-posed to mediate ASBVd replication on the basis of its sensitivityto the inhibitor tagetitoxin (Navarro

et al

., 2000), or anotherNEP-like enzyme because more than one RNA polymerase of this

214

R. FLORES

et al.


(2006)

7


class has been reported (Bligny

et al

., 2000). The transcriptiontemplates could be the circular PLMVd RNAs or their most abun-dant linear counterparts assuming the involvement of an RNApolymerase that is able to jump over template discontinuities.

The self-cleavage

in vivo

of the PLMVd oligomeric strands isalmost certainly mediated by the hammerhead ribozymesembedded in both polarity strands. First, comparison of the twoinitial PLMVd-cDNA clones (Hernández and Flores, 1992)revealed that the substitutions in the hammerhead structures didnot affect their stability, either because they were found in loopsor, when located in the stems, they were covariations; this was afirst indication, corroborated by the subsequent sequencing ofmany more variants (Ambrós

et al

., 1998, 1999; Malfitano

et al

.,2003; Pelchat

et al

., 2000; Rodio

et al

., 2006), that the hammer-head structures were physiologically significant. Second, primerextension experiments of the PLMVd monomeric linear RNAsextracted from infected tissue have revealed that their mostabundant 5

′

termini are those predicted by the hammerheadstructures (Delgado

et al

., 2005). And third, a correlation hasbeen found between the infectivity of certain PLMVd variants andthe extent of their self-cleavage during

in vitro

transcription(Ambrós

et al., 1998, 1999). Moreover, the high thermodynamicstability of both PLMVd hammerhead structures (Hernández andFlores, 1992), and the specific location of both PLMVd initiationsites at the base of long stable hairpins—entailing that thesequences of the hammerhead structure of the nascent strandare synthesized before those forming the hammerhead structurein the complementary polarity—facilitates self-cleave of nascent(+) and (–) strands during transcription (Delgado et al., 2005),thus explaining the low or undetectable levels of the oligomeric(+) and (–) in infected peach. On the other hand, PLMVd hammer-head structures have been instrumental in uncovering thattertiary interactions between loops 1 and 2 in natural hammerheadribozymes (Fig. 4) play an essential function in self-cleavage atthe low magnesium concentrations existing in vivo (De la Peñaet al., 2003; Khvorova et al., 2003).

Ligation of the PLMVd monomeric linear strands resultingfrom the hammerhead-mediated self-cleavage has been pro-posed to occur non-enzymatically (self-ligation), giving rise invitro (Côté and Perreault, 1997) and in vivo (Côté et al., 2001)to a 2′,5′-phosphodiester bond at the ligation site. However, theligation site of the termini resulting from the hammerhead-mediated self-cleavage of one viroid-like satellite RNA containsa 2′-phosphomonoester, 3′−5′-phosphodiester bond, consistentwith the involvement of an RNA ligase (Kiberstis et al., 1985).Arguments in favour and against the two alternatives have beendiscussed elsewhere (Flores et al., 2005a). If non-enzymaticself-ligation, like self-cleavage, is indeed functional in PLMVd rep-lication, then this process would be largely an RNA-based mech-anism only requiring a host RNA polymerase (Côté et al., 2001).If circularization is catalysed by an RNA ligase, this enzyme

should most likely be nuclear-encoded because PLMVd circularforms have been detected in tissues showing PC symptoms,in which translation of plastid-encoded proteins is severelyaffected (see above). Regardless, ligation (or self-ligation) ofPLMVd linear strands appears to be facilitated by a conformation,like the predicted branched conformation (Fig. 3), in which thetermini to be joined are in close physical proximity and adoptionof the hammerhead structure is impeded; the ribozyme wouldonly form transiently during transcription, when the most stablebranched conformation has not yet formed. Therefore, a con-formational switch is most likely involved in the transitionbetween the two processing steps (cleavage and ligation) of thereplication cycle.

Some aspects of PLMVd movement were studied evenbefore it was identified as a viroid. Long-distance movement wasapproached under field conditions by inoculating trees at onespecific branch and then following, by the cross-protection bio-assay (see below), how infection progressed with time: the viroidwas first detected below the inoculation point, requiring 3–4years to invade completely all tree branches (Desvignes, 1981).Recently, the trafficking pattern of PLMVd in peach has been ana-lysed by in situ hybridization (M. E. Rodio et al., manuscript inpreparation). In contrast to previous observations showing thatPSTVd is absent from the shoot apex (Zhu et al., 2001), PLMVdcan move into the shoot apex, thus establishing an additionaldemarcating difference in the behaviour of two representativemembers of both viroid families. Moreover, because the shootapex is the site of chloroplast biogenesis from proplastids, andbecause proplastids are altered in the albino shoots infected bycertain PLMVd variants inducing PC, this albino phenotype couldresult from the interference with an early step of the evolution ofproplastids into chloroplasts that would also impair the plastid-to-nucleus signalling involved in leaf differentiation. Withininfected trees, PLMVd is well distributed in shoots, leaves, bark,roots and fruits (Flores et al., 1992), although its titre is relativelylow compared with that of most other viroids (with the exceptionof CChMVd). Young expanding fruits appear the best source forobtaining PLMVd-enriched preparations (Delgado et al., 2005).

Horizontal transmission of PLMVd occurs mainly by thepropagation of infected buds; this has been the route for theworldwide spread of PLMVd, particularly considering that thispathogen was not included in most certification schemes untilrecently. PLMVd can also be locally dispersed plant to plant viapruning tools, because the viroid has been experimentally trans-mitted with contaminated blades (Hadidi et al., 1997), and witha low efficiency by aphids (Desvignes, 1986; Flores et al., 1992).However, PLMVd is neither horizontally transmitted throughpollen (Desvignes, 1981) nor vertically transmitted through seed(Desvignes, 1986) and, in agreement with this observation,attempts to detect the viroid in this latter organ have been unsuc-cessful (Flores et al., 1992).

Peach latent mosaic viroid 215

© 2006 BLACKWELL PUBL ISH ING LTD MOLECULAR PLANT PATHOLOGY (2006) 7 (4 ) , 209–221

BIOLOGICAL PROPERTIES: HOST RANGE, STRAINS AND CROSS-PROTECTION

Whether PLMVd is restricted to peach (and peach hybrids) orinfects a wide range of Prunus and non-Prunus species has beendiscussed previously (Flores et al., 2003; Hadidi et al., 2003).Regardless, the available evidence indicates that PLMVd onlyinduces symptoms in peach. These symptoms are very variable. Asindicated above, most of the PLMVd strains are latent when inoc-ulated to the peach indicator GF-305 grown in the greenhouse,and those that induce the typical mosaic are only partially stable(plants revert with time to a symptomless condition) and have tobe maintained by inoculating periodically new indicator plantswith symptomatic tissue (Desvignes, 1976, 1980). Perhaps theonly exceptions are the strains that induce the albino phenotypeknown as PC, which usually remain stable with time. These PCstrains are also peculiar in their sudden emergence in trees thathad previously grown without symptoms, and in the uneven dis-tribution of their symptoms, which are usually only expressed inone or few branches of the affected trees (Malfitano et al., 2003;Rodio et al., 2006).

Under field conditions, symptoms, except the foliar mosaic andPC, are generally elusive and can be confused with physiologicaldisorders or cultivar characteristics. Two examples illustrate thispoint. First, a broad survey of commercial peach and nectarinecultivars collected from five states of the USA showed an unex-pectedly high incidence: approximately 50% from a total of morethat 1000 trees were PLMVd-infected (Skrezeczkowski et al.,1996). And second, certain delays in flowering and fruit ripeninginitially regarded as resulting from agronomic practices or cultivartraits were later found to be caused by PLMVd infection (Gibsonet al., 2001; J.C. Desvignes, personal communication).

Cross-protection phenomena have been known for a longtime in different plant viruses (for a review see Hull, 2002).Briefly, a plant infected with a latent or non-symptomatic strainof a virus becomes protected (it does not develop the character-istic symptoms) against the ulterior inoculation by a severe strainof the same or a closely related virus. This helps in establishingproximity relationships between viruses and has practical impli-cations in those areas in which eradication of a virus appearsto be difficult if not impossible. More recently, interest inphenomena of this kind has been sparked by results showingthat protection against a virus can be elicited by transgenicallyexpressing in plants intact or truncated viral proteins, or non-protein-coding RNA sequences from the viral genome (for areview see Hull, 2002). This so-called pathogen-derived resist-ance has some mechanistic relationships with cross-protectionphenomena, and efforts are being currently dedicated to theirdetailed elucidation. Specifically, RNA-mediated cross-protection has been shown to be functionally equivalent topost-transcriptional gene silencing (Ratcliff et al., 1999).

Before the advent of the viroid concept, and due to the simi-larity of the phenotypic effects incited by viruses and viroids intheir host plants, some diseases that are now known to have aviroid ethiology were presumed to be virus-induced, and thecorresponding cross-protection phenomena were regarded asadditional examples of typical cross-protection. Thus, cross-protection in viroids was reported before their radical differenceswith viruses were uncovered. In the family Pospiviroidae, cross-protection was observed between strains of PSTVd (Fernow, 1967)and between closely related viroids (Niblett et al., 1978). In ret-rospect, it is interesting to note that the lack of cross-protectionbetween the agent of CChM disease and PSTVd (and two othermembers of its family) established a first demarcating criterionbetween what today are recognized as members of the two viroidfamilies. Cross-protection was also observed in PLMVd before itwas characterized as a viroid, and used for setting up the firstdetection protocol for controlling the diffusion of this viroid (Des-vignes, 1976, 1986). In brief, seedlings of the peach indicator GF-305 grown in the greenhouse were first graft-inoculated with apiece of material from the trees under examination (which evenif infected do not usually display conspicuous alterations), andapproximately 2 months later they were challenge-inoculatedwith a severe PLMVd strain. GF-305 seedlings pre-infected witha latent strain of PLMVd do not develop the leaf symptomsinduced by the severe strain, whereas those non-infected initiallydo develop symptoms approximately 1 month after the challengeinoculation.

Although the very different structural, biological and biochem-ical features of the members of both viroid families suggestedthat the mechanisms underlying cross-protection (and pathogen-esis) should be also different, this may not be finally the case anda unifying RNA silencing mechanism might operate in both situ-ations (see below).

MOLECULAR VARIABILITY

The broad symptom variability that characterizes differentPLMVd strains was suspected to result from a parallel variabilityof the viroid RNA. Indeed, the two PLMVd-cDNA clones (hereaf-ter referred to as variants) first reported showed nucleotide changesin 15 positions (Hernández and Flores, 1992). Subsequent char-acterization of many more variants of different origins confirmedthe high variability of the PLMVd RNA, which appears, however,to be restricted by some constraints with regard to the preserva-tion of the branched conformation, the hammerhead structures(Ambrós et al., 1998; Malfitano et al., 2003; Pelchat et al., 2000;Rodio et al., 2006) and the proposed kissing-loop interaction(Bussière et al., 2000). Another pseudoknot involving loops A andB, proposed on the basis of co-variations observed in certainPLMVd variants (Ambrós et al., 1998), may not form in all variants,particularly in those containing an insertion at loop A (see below).

216 R. FLORES et al.

MOLECULAR PLANT PATHOLOGY (2006) 7 (4 ) , 209–221 © 2006 BLACKWELL PUBL ISH ING LTD

Two main factors could determine the high sequence hetero-geneity observed in PLMVd variants from naturally infected peachtrees: repeated inoculations of the same individual field trees,which have a long productive life, or the intrinsic property ofPLMVd RNA to evolve rapidly. Insight into their relative influencewas obtained when the sequence of 36 progeny variants evolvedfrom inoculations of GF-305 peach seedlings with four individualPLMVd variants differing in their pathogenicity was analysed 2–3 months after inoculation, the time required by the pathogenicvariants to induce the onset of symptoms. The wide separation inthe sequence space between the parental and some of the prog-eny variants, most of which had unique sequences, showed thevery dynamic nature of the viroid populations and indicated thatthe extremely heterogeneous character of PLMVd natural isolatesmost probably results from the inherent ability of this RNA toaccumulate changes (Ambrós et al., 1999). These results alsorevealed that PLMVd fits the quasi-species model proposed fordifferent RNA replicons (Domingo and Holland, 1994) includingPSTVd (Góra-Sochacka et al., 1997). This latter study showed therapid generation of new quasi-species of PSTVd evolving fromsingle natural variants inoculated in the experimental host tomato,but the number of nucleotide changes accumulated with respectto the parental variants, which prevailed as main components inthe progenies, was considerably lower than that found for PLMVd,in which the parental variant was retained as a minor componentin only one of the four progenies studied (Ambrós et al., 1999).

The primary cause of the different evolution rates of PSTVd andPLMVd may result from the involvement in their replication of anuclear and a chloroplastic RNA polymerase, respectively, whichin principle should have different mutation rates, particularlywhen forced to transcribe RNA templates instead of their phy-siological DNA templates. These anomalous RNA templates mayalso decrease the processivity of the RNA polymerases, promot-ing their jumping during replication and facilitating the frequentemergence of novel recombinant variants. Supporting a majorrole of recombination in shaping PLMVd evolution, analysis ofthe population dynamics of PLMVd variants associated with PChas shown that an insertion of 12–14 nt in loop A can beacquired or lost during infection (see below). Moreover, PLMVdseems to be endowed with a particularly high flexibility to accom-modate an extensive number of polymorphic positions (Ambróset al., 1999; Rodio et al., 2006), suggesting that different selec-tive constraints operate on this viroid compared with PSTVd,which has a considerably higher genetic stability (Góra-Sochackaet al., 1997). In summary, the rapid generation of genetic diver-sity observed upon propagation of individual PLMVd variants inthe natural host suggests that repeated fluctuations in thesequence spectrum, due to progressive accumulation of pointmutations and most likely recombination events, may determineto a large extent the fluctuating symptomatology characteristicof most of the severe natural isolates of this viroid.

DETERMINANTS OF PATHOGENICITY

In addition to the signal transduction cascade leading from thefirst primary molecular alteration to symptom expression, anessential step for understanding viroid pathogenesis is the iden-tification in viroid RNAs of sequence or structural motifs thatmodulate their virulence. Such pathogenicity determinants havebeen mapped for PSTVd and other representative members of thefamily Pospiviroidae at small motifs within specific domains ofthe rod-like secondary structure proposed for these RNAs (for areview see Flores et al., 2005a). Initial attempts at mapping thepathogenicity determinant(s) of a mosaic-inducing isolate ofPLMVd, by comparing its spectrum of variants with those of twolatent isolates, were not possible owing to the high variabilityobserved (Ambrós et al., 1998). However, Northern blot analysisof RNAs from a PC isolate showed the presence of two popula-tions of PLMVd molecules with apparently different size (Malfit-ano et al., 2003), a rather unexpected finding because mostPLMVd variants characterized so far had a uniform size between335 and 339 nt (Ambrós et al., 1998, 1999; Hernández andFlores, 1992; Pelchat et al., 2000). Cloning and sequencing of 16full-length PLMVd cDNA clones of this PC isolate revealed that itcomprised two groups of variants: nine had size (336–338 nt)similar to that of typical PLMVd variants of latent and mosaic-inducing isolates, whereas the other seven were longer (348–351 nt) due to an insertion of 12–13 nt. The insertion was alwaysfound in the loop A capping the hammerhead arm (Fig. 5A), hadlimited sequence variability, and folded itself into a hairpin.Because these atypical longer variants appeared as good candi-dates for eliciting PC, three dimeric in vitro transcripts of PLMVdvariants, two with and the other without the insertion, weremechanically inoculated to GF-305 peach seedlings: PC symp-toms were only incited by the RNAs containing the insertion,which was retained in the progeny. These data provided ultimateproof for the long-held assumption regarding PLMVd as thecausal agent of PC, and strongly suggested that only those vari-ants containing the 12–13-nt insertion were able to incite thisspecific syndrome. Definitive evidence for a cause–effect rela-tionship between the 12–13-nt insertion and PC was obtainedby removing the insertion of one of the PC-inducing variants bysite-directed mutagenesis: the resulting variant was able to rep-licate without inciting symptoms (Malfitano et al., 2003).PLMVd appeared therefore peculiar with respect to other viroidsin which changes in pathogenicity have been associated withsubstitutions or insertions/deletions of a more reduced numberof nucleotides.

The finding that the sequence heterogeneity found in the 12–13-nt insertion preserved its folding into a hairpin with a stem of4–6 bp capped by a U-rich tetra- or pentaloop, suggested thatadoption of this folding was a structural constraint and raised thequestion of whether the whole hairpin, or a portion thereof, was



needed for inducing PC (Malfitano et al., 2003). Subsequentcharacterization of two other PC isolates has shed light on therequirements of the insertion containing the pathogenicity deter-minant (Rodio et al., 2006). Whereas PLMVd variants with inser-tions similar to those reported previously (type 1) predominatedin one of the isolates (PC-P2), the second (PC-P1), in addition tothese variants, contained others with insertions in the sameposition and of the same size, but with the hairpin capped by aGA-rich loop (type 2). When symptomatic and non-symptomatictissues from both isolates were implanted on to GF-305 peachseedlings, they reproduced the phenotype of the inoculum source,indicating that variants differing in pathogenicity are unevenlydistributed within single plants. Moreover, characterization of the

progenies resulting from inoculations with the PC-P1 sourceshowed that variants with insertions of type 1 and 2 were pre-dominant in the symptomatic and non-symptomatic seedlings,respectively, confirming the association between PC and variantswith type 1 but not type 2 insertions. Inoculations with dimeric invitro transcripts from PLMVd variants with type 1, type 2 and witha chimeric insertion showed that the variant with type 2 insertionwas latent, and established that the U-rich capping loop has amajor role in PC, although the adjacent stem may also have someinfluence (Fig. 5B). Similar results have been obtained with CCh-MVd pathogenic and latent variants, which provide an intriguingparallelism with PLMVd: the major pathogenic determinant ofCChMVd maps at a U-rich tetraloop (UUUC), which in latent

Fig. 5 (A) Schematic representation of PLMVd variants from PC isolates containing at loop A an insertion denoted by a discontinuous line. (B) The specific sequences of the 12–14-nt insertions of variants PC-C40 (inducing PC, left) and PC-P1.142 (non-symptomatic, right) and their phenotypic effects are presented.



variants is replaced by a GAAA tetraloop (De la Peña and Flores,2002; De la Peña et al., 1999). Further molecular dissection of thePLMVd determinant inducing PC should help to unravel thecontributions of its primary and secondary structure. Anotherpertinent question in this context is whether the moleculardeterminant of PLMVd mosaic-inducing variants maps also atthe loop A or at a different region.

An interesting additional result of these experiments is thatinsertions can be acquired and lost during infection (Malfitanoet al., 2003; Rodio et al., 2006), suggesting that PLMVd latentvariants can evolve into pathogenic variants and vice versa. Thecharacteristic sudden appearance of the PC syndrome in peachtrees that had previously grown without symptoms is consistentwith the de novo emergence of PC-inducing PLMVd variants.The molecular mechanism generating these variants remainsunknown but it may be related to the low processivity of the RNApolymerase catalysing the elongation of PLMVd strands.

RNA SILENCING: A UNIFYING PRINCIPLE LINKING DISTINCT ASPECTS OF PLMVd BIOLOGY?

Although the molecular mechanisms through which viroidsinduce their pathogenic effects remain largely unknown, it is gen-erally accepted that viroids modify host gene expression interfer-ing with the normal developmental pathways. Until recently, themature viroid RNA or some of its replicative intermediates havebeen regarded as the primary pathogenic effectors interactingwith a host protein or RNA (Diener, 2001). This first interactionshould be in principle diverse for members of the two families,Pospiviroidae and Avsunviroidae, because they differ in struc-tural, biological and biochemical features (Flores et al., 2005b).However, in the last few years, a new paradigm has emerged pro-posing that viroid symptoms may be elicited by the small inter-fering RNAs (siRNAs) resulting from the host RNA silencingdefensive response triggered by double- or quasi-double-stranded RNAs. This paradigm is based on the recent discovery ofviroid-specific siRNAs of both polarities corresponding to differ-ent genomic regions, the hallmarks of RNA silencing, in plantsinfected by members of both families: PSTVd (Itaya et al., 2001;Papaefthimiou et al., 2001) and CEVd (Markarian et al., 2004), aswell as PLMVd (Martínez de Alba et al., 2002), and later con-firmed (Landry et al., 2004), CChMVd (Martínez de Alba et al.,2002) and ASBVd (Markarian et al., 2004). Whereas the genera-tion of the PSTVd-specific siRNAs may take place in the nucleus,where this viroid replicates and some Dicer-like (DCL) isoforms—an RNase III-like enzyme catalysing the process—are located (fora review see Baulcombe, 2004), synthesis of the siRNAs derivedfrom PLMVd should occur in the cytoplasm during cell-to-cellviroid movement because RNA silencing has not been reported inthe chloroplast (Martínez de Alba et al., 2002). For chloroplastic

viroids, the mature genomic RNA appears as the most likely sub-strate for generating the primary siRNAs and, consistent with thisview, they are also produced in vitro by the DCL activities fromwheat-germ acting on a highly structured PLMVd fragment (thehammerhead arm) (Landry and Perreault, 2005), and on theASBVd circular forms (A.E. Martínez de Alba and R. Flores,unpublished data). By binding to mature viroid RNAs, the primarysiRNAs could prime a cytoplasmic RNA-dependent RNA polymer-ase (RdRp) (Schiebel et al., 1998), giving rise to dsRNAs andthrough the subsequent DCL action to secondary siRNAs, thusresulting in a signal amplification cascade. PLMV-specific siRNAsof different sizes have been detected in vitro (Landry and Per-reault, 2005), and in vivo data (Landry et al., 2004; Martínez deAlba et al., 2002) also suggest the existence of more than oneclass of siRNAs that could result from the participation of morethan one DCL isoform in their genesis.

Therefore, viroids and particularly PLMVd induce an RNAsilencing response in their hosts and most likely they are also tar-gets of this response. By loading the second key RNase of theRNA silencing pathway, forming part of RISC (from RNA inducedsilencing complex) (for a review see Baulcombe, 2004), theviroid-specific siRNAs could guide this complex against theircognate genomic RNAs. The inverse correlation observed in threechloroplastic viroids (PLMVd, CChMVd and ASBVd) between theaccumulation levels of the mature viroid forms and their corre-sponding siRNAs (Martínez de Alba et al., 2002) is consistentwith this idea: viroid titre, and the associated damaging effects,would thus be attenuated by the integrated action of DCL andRISC. In this same context, the cross-protection phenomenareported in both viroid families may also be explained if the siR-NAs derived from the pre-inoculated mild strain target for degra-dation the RNA of the challenging severe strain. The compactsecondary structure of viroids may have evolved to provide someresistance to RISC (Wang et al., 2004).

In addition, it has been proposed that certain viroid-specificsiRNAs could by chance guide RISC against certain host messen-ger RNAs, thereby down-regulating the expression of specifichost genes and eventually leading to symptom development(Papaefthimiou et al., 2001; Wang et al., 2004). This hypothesisis intriguing because, as in the case of cross-protection, the samemechanism could mediate pathogenesis in both viroid familiesand even in certain satellite RNAs (Wang et al., 2004), butconclusive evidence is lacking (Flores et al., 2005a). The recentidentification in PLMVd of a small hairpin as the pathogenicitydeterminant of PC (Malfitano et al., 2003; Rodio et al., 2006)(and the similar situation reported previously in CChMVd, seeabove) offers an opportunity for testing this hypothesis by clon-ing the viroid-specific siRNAs from PC and latent isolates: thepresence of siRNAs derived from the PC pathogenicity determi-nant in the severe isolate would be consistent with the hypothe-sis whereas their absence would argue against.



CONCLUDING REMARKS

For almost 20 years since its discovery, PLMVd has providedmany insights into fundamental questions regarding how a min-imal non-protein-coding RNA replicates, moves, incites specificpeach symptoms and evolves. Moreover, recent data have estab-lished a link between certain macroscopic symptoms (the albinophenotype induced by some PLMVd variants) and the underlyingcytopathic and molecular alterations (malformed proplastids andimpairment of their evolution into chloroplasts most likely result-ing from the inefficient translation of plastid-encoded proteins)(M.E. Rodio et al., manuscript in preparation). These observationspave the way for an integrated and comprehensive understand-ing of the whole pathogenic process.

ACKNOWLEDGEMENTS

Work in R.F.’s laboratory has been partially supported by theMinisterio de Ciencia y Tecnología (MCyT) (BMC2002-03694),the Ministerio de Educación y Ciencia (BFU2005-06808/BMC)and the Generalidad Valenciana (GV) (Grupos 03/064) of Spain.The laboratories of F.D.S and R.F. have been jointly supported bythe CNR-CSIC project 2004IT0028. During this course of thisstudy, S.D. received postdoctoral fellowships from the MCyT andthe GV, and M.E.R. a predoctoral fellowship from the Universitàdegli Studi di Bari.

REFERENCES

Allison, L.A., Simon, L.D. and Maliga, P. (1996) Deletion of rpoB revealsa second distinct transcription system in plastids of higher plants. EMBOJ. 15, 2802–2809.

Ambrós, S., Desvignes, J.C., Llácer, G. and Flores, R. (1995) Peachlatent mosaic and pear blister canker viroids: detection by molecularhybridization and relationships with specific maladies affecting peachand pear trees. Acta Hortic. 386, 515–521.

Ambrós, S., Hernández, C., Desvignes, J.C. and Flores, R. (1998)Genomic structure of three phenotypically different isolates of peachlatent mosaic viroid: implications of the existence of constraints limitingthe heterogeneity of viroid quasi-species. J. Virol. 72, 7397–7406.

Ambrós, S., Hernández, C. and Flores, R. (1999) Rapid generationof genetic heterogeneity in progenies from individual cDNA clones ofpeach latent mosaic viroid in its natural host. J. Gen. Virol. 80, 2239–2252.

Baulcombe, D. (2004) RNA silencing in plants. Nature, 431, 356–363.Beaudry, D., Bussiere, F., Laureau, F., Lessard, C. and Perrault, J.P. (1995)

The RNA of both polarities of the peach latent mosaic viroid self-cleavagein vitro solely by single hammerhead structures. Nucleic Acids Res. 23,745–752.

Bligny, M., Courtois, F., Thaminy, S., Chang, C.C., Lagrange, T.,Baruah-Wolff, J., Stern, D. and Lerbs-Mache, S. (2000) Regulation ofplastid rDNA transcription by interaction of CDF2 with two different RNApolymerases. EMBO J. 19, 1851–1860.

Blodgett, E.C. (1944) Peach calico. Phytopathology, 34, 650–657.

Bonfiglioli, R.G., McFadden, G.I. and Symons, R.H. (1994) In situhybridization localizes avocado sunblotch viroid on chloroplastthylakoid membranes and coconut cadang cadang viroid in the nucleus.Plant J. 6, 99–103.

Branch, A.D., Benenfeld, B.J. and Robertson, H.D. (1988) Evidence fora single rolling circle in the replication of potato spindle tuber viroid.Proc. Natl Acad. Sci. USA, 85, 9128–9132.

Branch, A.D. and Robertson, H.D. (1984) A replication cycle for viroidsand other small infectious RNAs. Science, 223, 450–454.

Bussière, F., Lehoux, J., Thompson, D.A., Skrzeczkowski, L.J. andPerreault, J.P. (1999) Subcellular localization and rolling circle replicationof peach latent mosaic viroid: hallmarks of group A viroids. J. Virol. 73,6353–6360.

Bussière, F., Ouellet, J., Côté, F., Lévesque, D. and Perreault, J.P. (2000)Mapping in solution shows the peach latent mosaic viroid to possess anew pseudoknot in a complex, branched secondary structure. J. Virol. 74,2647–2654.

Côté, F., Lévesque, D. and Perreault, J.P. (2001) Natural 2′,5′-phosphodiester bonds found at the ligation sites of peach latentmosaic viroid. J. Virol. 75, 19–25.

Côté, F. and Perreault, J.P. (1997) Peach latent mosaic viroid is locked bya 2′,5′-phosphodiester bond produced by in vitro self-ligation. J. Mol.Biol. 273, 533–543.

Daròs, J.A., Marcos, J.F., Hernández, C. and Flores, R. (1994) Replica-tion of avocado sunblotch viroid: evidence for a symmetric pathway withtwo rolling circles and hammerhead ribozyme processing. Proc. NatlAcad. Sci. USA, 91, 12813–12817.

De la Peña, M. and Flores, R. (2002) Chrysanthemum chlorotic mottleviroid RNA: dissection of the pathogenicity determinant and comparativefitness of symptomatic and non-symptomatic variants. J. Mol. Biol. 321,411–421.

De la Peña, M., Gago, S. and Flores, R. (2003) Peripheral regions of nat-ural hammerhead ribozymes greatly increase their self-cleavage activity.EMBO J. 22, 5561–5570.

De la Peña, M., Navarro, B. and Flores, R. (1999) Mapping the moleculardeterminat of pathogenicity in a hammerhead viroid: a tetraloop withinthe in vivo branched RNA conformation. Proc. Natl Acad. Sci. USA, 96,9960–9965.

Delgado, S., Martínez de Alba, E., Hernández, C. and Flores, R. (2005)A short double-stranded RNA motif of peach latent mosaic viroid con-tains the initiation and the self-cleavage sites of both polarity strands.J. Virol. 79, 12934–12943.

Desvignes, J.C. (1976) The virus diseases detected in greenhouse and fieldby the peach seedlings GF-305 indicator. Acta Hortic. 67, 315–323.

Desvignes, J.C. (1980) Different symptoms of the peach latent mosaic.Acta Phytopath. Acad. Sci. Hung. 15, 183–190.

Desvignes, J.C. (1981) Epidemiologie de la mosaïque latent du pêcher(PLMV). 1er Colloque sur les Rechereches Fruitières Bordeuax, 263–276.

Desvignes, J.C. (1986) Peach latent mosaic and its relation to peachmosaic and peach yellow mosaic virus diseases. Acta Hortic. 193, 51–57.

Diener, T.O. (1972) Potato spindle tuber viroid VIII. Correlation of infectivitywith a UV-absorbing component and thermal denaturation properties ofthe RNA. Virology, 50, 606–609.

Diener, T.O. (2001) The viroid: biological oddity or evolutionary fossil? Adv.Virus Res. 57, 137–184.

Diener, T.O. (2003) Discovering viroids—a personal perspective. NatureRev. Microbiol. 1, 75–80.



Domingo, E. and Holland, J.J. (1994) Mutation rates and rapid evolutionof RNA viruses. In: The Evolutionary Biology of Viruses (Morse, S.S., ed.),pp. 161–184. New York: Raven Press.

Fadda, Z., Daròs, J.A., Fagoaga, C., Flores, R. and Duran-Vila, N.(2003) Eggplant latent viroid (ELVd): candidate type species for a newgenus within family Avsunviroidae (hammerhead viroids). J. Virol. 77,6528–6532.

Fernow, K.H. (1967) Tomato as a test plant for detecting mild strains ofpotato spindle tuber virus. Phytopathology, 57, 1347–1352.

Flores, R., Daròs, J.A. and Hernández, C. (2000) The Avsunviroidae fam-ily: viroids with hammerhead ribozymes. Adv. Virus Res. 55, 271–323.

Flores, R., Hernández, C., Avinent, L., Hermoso, A., Llácer, G., Juárez, J.,Arregui, J.M., Navarro, L. and Desvignes, J.C. (1992) Studies on thedetection, transmission and distribution of peach latent mosaic viroid inpeach trees. Acta Hortic. 309, 325–330.

Flores, R., Hernández, C., Desvignes, J.C. and Llácer, G. (1990) Someproperties of the viroid inducing the peach latent mosaic disease. Res.Virol. 141, 109–118.

Flores, R., Hernández, C., Llácer, G., Shamloul, A.M., Giunchedi, L.and Hadidi, A. (2003) Peach latent mosaic viroid in peach. In: Viroids(Hadidi, A., Flores, R., Randles, J.W. and Semancik, J.S., eds), pp. 156–160. Collingwood, Australia: CSIRO Publishing.

Flores, R., Hernández, C., Martínez de Alba, E., Daròs, J.A. andDi Serio, F. (2005a) Viroids and viroid–host interactions. Ann. Rev.Phytopathol. 43, 117–139.

Flores, R. and Llácer, G. (1988) Isolation of a viroid-like RNA associatedwith peach latent mosaic disease. Acta Hortic. 235, 325–332.

Flores, R., Randles, J.W., Owens, R.A., Bar-Joseph, M. and Diener, T.O.(2005b) Viroids. In: Virus Taxonomy. Eighth Report of the InternationalCommittee on Taxonomy of Viruses (Fauquet, C.M., Mayo, M.A.,Maniloff, J., Desselberger, U. and Ball, A.L., eds), pp. 1145–1159. Lon-don: Elsevier/Academic Press.

Forster, A.C., Davies, C., Sheldon, C.C., Jeffries, A.C. and Symons,R.H. (1988) Self-cleaving viroid and newt RNAs may only be active asdimers. Nature, 334, 265–267.

Gago, S., De la Peña, M. and Flores, R. (2005) A kissing-loop interactionin a hammerhead viroid RNA critical for its in vitro folding and in vivoviability. RNA, 11, 1073–1083.

Gibson, P.G., Reighard, G.L., Scott, S.W. and Zimmerman, M.T. (2001)Identification of graft-transmissible agents from ‘Ta-Tao 5’ peach andtheir effect on ‘Coronet’ peach. Acta Hortic. 550, 309–314.

Góra-Sochacka, A., Kierzek, A., Candresse, T. and Zagórski, W. (1997)The genetic stability of potato spindle tuber viroid (PSTVd) molecularvariants. RNA, 3, 68–74.

Gross, H.J., Domdey, H., Lossow, C., Jank, P., Raba, M., Alberty, H.and Sänger, H.L. (1978) Nucleotide sequence and secondary structureof potato spindle tuber viroid. Nature, 273, 203–208.

Hadidi, A., Giunchedi, L., Osaki, H., Shamloul, A.M., Crescenzi, A.,Gentit, P., Nemchinov, L., Piazzola, P. and Kyriakopoulou, P.E.(2003) Peach latent mosaic viroid in temperate fruit hosts. In: Viroids(Hadidi, A., Flores, R., Randles, J.W. and Semancik, J.S., eds), pp. 161–164. Collingwood, Australia: CSIRO Publishing.

Hadidi, A., Giunchedi, L., Shamloul, A.M., Poggi-Pollini, C. and Amer,M.A. (1997) Occurrence of peach latent mosaic viroid in stone fruits andits transmission with contaminated blades. Plant Dis. 81, 154–158.

Harders, J., Lukacs, N., Robert-Nicoud, M., Jovin, J.M. and Riesner, D. (1989)Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridiza-tion and confocal laser scanning microscopy. EMBO J. 8, 3941–3949.

Hernández, C. and Flores, R. (1992) Plus and minus RNAs of peach latentmosaic viroid self-cleave in vitro via hammerhead structures. Proc. NatlAcad. Sci. USA, 89, 3711–3715.

Hull, R. (2002) Matthews’ Plant Virology, 4th edn. London: AcademicPress.

Hutchins, C.J., Keese, P., Visvader, J.E., Rathjen, P.D., McInnes, J.L. andSymons, R.H. (1985) Comparison of multimeric plus and minus forms ofviroids and virusoids. Plant Mol. Biol. 4, 293–304.

Hutchins, C., Rathjen, P.D., Forster, A.C. and Symons, R.H. (1986) Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid.Nucleic Acids Res. 14, 3627–3640.

Itaya, A., Folimonov, A., Matsuda, Y., Nelson, R.S. and Ding, B. (2001)Potato spindle tuber viroid as inducer of RNA silencing in infectedtomato. Mol. Plant–Microbe Interact. 14, 1332–1334.

Khvorova, A., Lescoute, A., Westhof, E. and Jayasena, S.D. (2003)Sequence elements outside the hammerhead ribozyme catalytic coreenable intracellular activity. Nature Struct. Biol. 10, 708–712.

Kiberstis, P.A., Haseloff, J. and Zimmern, D. (1985) 2′ Phosphomo-noester, 3′-5 phosphodiester bond at a unique site in a circular viral RNA.EMBO J. 4, 817–822.

Kishi, K., Takanashi, K. and Abiko, K. (1973) New virus diseases ofpeach, yellow mosaic, oil blotch and star mosaic. Bull. Hort. Res. Sta.Japan, Series A, 12, 197–208.

Landry, P. and Perreault, J.P. (2005) Identification of a peach latent mosaicviroid hairpin able to act as a dicer-like substrate. J. Virol. 79, 6540–6543.

Landry, P., Thompson, D. and Perreault, J.P. (2004) The role of viroids ingene silencing: the model case of peach latent mosaic viroid. Can. J.Plant Pathol. 26, 31–38.

Lima, M.I., Fonseca, M.E.N., Flores, R. and Kitajima, E.W. (1994) Detec-tion of avocado sunblotch viroid in chloroplasts of avocado leaves by insitu hybridization. Arch. Virol. 138, 385–390.

Loreti, S., Faggioli, F. and Barba, M. (1995) A rapid extraction methodto detect peach latent mosaic viroid by molecular hybridization. ActaHortic. 386, 560–564.

Malfitano, M., Di Serio, F., Covelli, L., Ragozzino, A., Hernández, C.and Flores, R. (2003) Peach latent mosaic viroid variants inducing peachcalico contain a characteristic insertion that is responsible for this symp-tomatology. Virology, 313, 492–501.

Markarian, N., Li, H.W., Ding, S.W. and Semancik, J.S. (2004) RNAsilencing as related to viroid induced symptom expression. Arch. Virol.149, 397–406.

Martínez de Alba, A.E., Flores, R. and Hernández, C. (2002) Two chlo-roplastic viroids induce the accumulation of the small RNAs associatedwith post-transcriptional gene silencing. J. Virol. 76, 3094–3096.

Monsion, M., Bachelier, J.C., Candresse, T., Desvignes, J.C., Mac-quaire, G. and Dunez, J. (1988) Investigations on the infectious agentresponsible for peach latent mosaic disease. Acta Hortic. 235, 247–255.

Navarro, B. and Flores, R. (1997) Chrysanthemum chlorotic mottle viroid:unusual structural properties of a subgroup of viroids with hammerheadribozymes. Proc. Natl Acad. Sci. USA, 94, 11262–11267.

Navarro, J.A. and Flores, R. (2000) Characterization of the initiation sitesof both polarity strands of a viroid RNA reveals a motif conserved insequence and structure. EMBO J. 19, 2662–2670.

Navarro, J.A., Vera, A. and Flores, R. (2000) A chloroplastic RNApolymerase resistant to tagetitoxin is involved in replication of avocadosunblotch viroid. Virology, 268, 218–225.

Niblett, C.L., Dickson, E., Fernow, K.H., Horst, R.K. and Zaitlin, M.(1978) Cross-protection among four viroids. Virology, 91, 198–203.



Papaefthimiou, I., Hamilton, A.J., Denti, M.A., Baulcombe, D.C.,Tsagris, M. and Tabler, M. (2001) Replicating potato spindle tuberviroid RNA is accompanied by short RNA fragments that are characteristicof post-transcriptional gene silencing. Nucleic Acids Res. 29, 2395–2400.

Pelchat, M., Côté, F. and Perreault, J.P. (2001) Study of the polymeriza-tion step of the rolling circle replication of peach latent mosaic viroid.Arch. Virol. 146, 1753–1763.

Pelchat, M., Levesque, D., Ouellet, J., Laurendeau, S., Levesque, S.,Lehoux, J., Thompson, D.A., Eastwell, K.C., Skrzeczkowski, L.J. andPerreault, J.P. (2000) Sequencing of peach latent mosaic viroid variantsfrom nine North American peach cultivars shows that this RNA folds intoa complex secondary structure. Virology, 271, 37–45.

Qi, Y. and Ding, B. (2003) Differential subnuclear localization of RNAstrands of opposite polarity derived from an autonomously replicatingviroid. Plant Cell, 15, 2566–2577.

Ratcliff, F., MacFarlane, S. and Baulcombe, D.C. (1999) Gene silencingwithout DNA. RNA-mediated cross-protection between viruses. PlantCell, 11, 1207–1216.

Rodio, M.E., Delgado, S., Flores, R. and Di Serio, F. (2006) Variants ofpeach latent mosaic viroid inducing peach calico: uneven distribution ininfected plants and requirements of the insertion containing the patho-genicity determinant. J. Gen. Virol. 87, 231–240.

Schiebel, W., Pelissier, T., Riedel, L., Thalmeir, S., Schiebel, R., Kempe, D.,Lottspeich, F., Sänger, H.L. and Wassenegger, M. (1998) Isolation of

an RNA-directed RNA polymerase-specific cDNA clone from tomato.Plant Cell, 10, 2087–2101.

Shamloul, A.M., Minafra, A., Hadidi, A., Waterworth, H.E., Giunchedi, L.and Allam, E.K. (1995) Peach latent mosaic viroid: nucleotide sequenceof an Italian isolate, sensitive detection using RT-PCR and geographic dis-tribution. Acta Hortic. 386, 522–530.

Skrezeczkowski, L.J., Howell, W.E. and Mink, G.I. (1996) Occurrence ofpeach latent mosaic viroid in commercial peach and nectarine cultivarsin the US. Plant Dis. 80, 823.

Sugiura, M. (1992) The chloroplast genome. Plant Mol. Biol. 19, 149–168.Symons, R.H. (1981) Avocado sunblotch viroid: primary sequence

and proposed secondary structure. Nucleic Acids Res. 9, 6527–6537.

Tabler, M. and Tsagris, M. (2004) Viroids: petite RNA pathogens with dis-tinguished talents. Trends Plant Sci. 9, 339–348.

Wang, M.B., Bian, X.Y., Wu, L.M., Liu, L.X., Smith, N.A., Isenegger, D.,Wu, R.M., Masuta, C., Vance, V.B., Watson, J.M., Rezaian, A., Den-nis, E.S. and Waterhouse, P.M. (2004) On the role of RNA silencing inthe pathogenicity and evolution of viroids and viral satellites. Proc. NatlAcad. Sci. USA, 101, 3275–3280.

Willison, R.S. (1946) Peach blotch. Phytopathology, 36, 273–276.Zhu, Y., Green, L., Woo, Y.-M., Owens, R. and Ding, B. (2001) Cellular

basis of potato spindle tuber viroid systemic movement. Virology, 279,69–77.

Documents

Peach latent mosaic viroid: not so latent