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Viroids and Viroid-Host Interactions

February 2005
Annual Review of Phytopathology 43(1):117-39

February 2005
43(1):117-39

DOI:10.1146/annurev.phyto.43.040204.140243

Source
PubMed

Authors:

Raül Flores

University of Barcelona

Carmen Hernández

Universitat Politècnica de València

Ángel Emilio Martínez de Alba

Universidad de Extremadura

José-Antonio Daròs

Instituto de Biología Molecular y Celular de Plantas (IBMCP)

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Although they induce symptoms in plants similar to those accompanying virus infections, viroids have unique structural, functional, and evolutionary characteristics. They are composed of a small, nonprotein-coding, single-stranded, circular RNA, with autonomous replication. Viroid species are clustered into the families Pospiviroidae and Avsunviroidae, whose members replicate (and accumulate) in the nucleus and chloroplast, respectively. Viroids replicate in three steps through an RNA-based rolling-circle mechanism: synthesis of longer-than-unit strands catalyzed by host RNA polymerases; processing to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes; and circularization. Within the initially infected cells, viroid RNA must move to its replication organelle, with the resulting progeny then invading adjacent cells through plasmodesmata and reaching distal parts via the vasculature. To carry out these movements, viroids must interact with host factors. The mature viroid RNA could be the primary pathogenic effector or, alternatively, viroids could exert their pathogenic effects via RNA silencing.

Structural features of viroids. (a) Rod-like secondary structure of members of the family Pospiviroidae. Domains C (central), P (pathogenic), V (variable), and T L and T R (terminal left and right, respectively) are indicated, as well as the CCR (central conserved region, here displayed for the genus Pospiviroid), TCR (terminal conserved region, present in the genera Pospi-and Apscaviroid, and in the two largest members of the genus Coleviroid) and TCH (terminal conserved hairpin, present in the genera Hostu-and Cocadviroid). Arrows indicate flanking sequences that together with the upper CCR strand form a hairpin, and the S-shaped line connects the residues linked after UV irradiation as a consequence of forming part of the loop E. (b) Quasi-rod-like and branched secondary structures of ASBVd and PLMVd, respectively (family Avsunviroidae). Sequences conserved in most natural hammerhead structures are shown within boxes with blue and white backgrounds for () and () polarities, respectively. Broken oval in PLMVd denotes a kissing-loop interaction. (Inset) PLMVd () hammerhead structure represented according to the original scheme (left) and to X-ray crystallography data obtained with artificial hammerhead structures (right), in which a proposed tertiary interaction between loops 1 and 2 enhancing the catalytic activity is indicated. Nucleotides conserved in most natural hammerhead structures are depicted as above. Arrows mark the self-cleavage site, and continuous and broken lines denote Watson-Crick and noncanonical pairs, respectively.

…

Alterations in different organs accompanying infections by some representative viroids. (a) Symptoms of PSTVd on potato tubers (left) and healthy controls (right) (courtesy of T.O. Diener). (b) Symptoms of CEVd on a trifoliate orange rootstock (courtesy of N. Duran-Vila and P. Moreno). (c) Symptoms of PBCVd on pear A20 (courtesy of J.C. Desvignes). (d ) Flower symptoms of CSVd on chrysanthemum (bottom) and a healthy control (top) (courtesy of J.M. Bové). (e) Leaf symptoms of CSVd on chrysanthemum (right) and a healthy control (left). ( f ) Leaf symptoms of CChMVd on chrysanthemum (right) and a healthy control (left). (g) Leaf symptoms of CCCVd on coconut (left) and a healthy control (right). (h) Extreme leaf chlorosis (peach calico) induced by PLMVd. (i) Internode shortening induced by HSVd on cucumber (right) and a healthy control (left). ( j) Symptoms of HSVd on cucumber fruits (left) and a healthy control (right) (courtesy of H.L. Sänger). (k) Fruit symptoms (dapple apple) induced by ASSVd (courtesy of J.C. Desvignes). (l) Fruit symptoms of ASBVd on avocado (courtesy of P.R. Desjardins).

…

Rolling-circle mechanism proposed for viroid replication. The asymmetric pathway, with one rolling circle, is followed by members of the family Pospiviroidae and takes place in the nucleus. The symmetric pathway, with two rolling circles, is followed by members of the family Avsunviroidae and takes place in the chloroplast. White and yellow lines indicate plus () and minus () strands, respectively, and cleavage sites are marked by arrowheads. Self-cleavage mediated by hammerhead ribozymes (Rz) leads to linear monomeric RNAs with 5-hydroxyl and 2-3-cyclic phosphodiester termini; cleavage catalyzed by a host protein (HP) probably generates the same termini. Pol II refers to RNA polymerase II and NEP to nuclear-encoded RNA polymerase. PD and NP are abbreviations for plasmodesmata and nuclear pores, respectively.

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10.1146/annurev.phyto.43.040204.140243

Annu. Rev. Phytopathol. 2005. 43:117–39

doi: 10.1146/annurev.phyto.43.040204.140243

First published online as a Review in Advance on February 7, 2005

VIROIDS AND VIROID-HOST INTERACTIONS

Ricardo Flores,1Carmen Hern´

andez,1

A. Emilio Mart´

ınez de Alba,1Jos ´

e-Antonio Dar `

os,1

and Francesco Di Serio2

1Instituto de Biolog´

ıa Molecular y Celular de Plantas (UPV-CSIC), Universidad

Polit´

ecnica de Valencia, Valencia 46022, Spain; email: rﬂores@ibmcp.upv.es,

cahernan@ibmcp.upv.es, aemarti@ibmcp.upv.es, jadaros@ibmcp.upv.es

2Istituto di Virologia Vegetale (CNR), Dipartimento di Protezione delle Piante e

Microbiologia Applicata, Universit`

adegli Studi di Bari, Bari 70126, Italy;

email: f.diserio@ba.ivv.cnr.it

KeyWords rolling-circle replication, catalytic RNAs, hammerhead ribozymes,

RNA silencing, cross-protection

■Abstract Although they induce symptoms in plants similar to those accompa-

nying virus infections, viroids have unique structural, functional, and evolutionary

characteristics. They are composed of a small, nonprotein-coding, single-stranded, cir-

cular RNA, with autonomous replication. Viroid species are clustered into the families

Pospiviroidae and Avsunviroidae, whose members replicate (and accumulate) in the

nucleus and chloroplast, respectively. Viroids replicate in three steps through an RNA-

based rolling-circle mechanism: synthesis of longer-than-unit strands catalyzed by host

RNA polymerases; processing to unit-length, which in the family Avsunviroidae is me-

diated by hammerhead ribozymes; and circularization. Within the initially infected

cells, viroid RNA must move to its replication organelle, with the resulting progeny

then invading adjacent cells through plasmodesmata and reaching distal parts via the

vasculature. To carry out these movements, viroids must interact with host factors. The

mature viroid RNA could be the primary pathogenic effector or, alternatively, viroids

could exert their pathogenic effects via RNA silencing.

INTRODUCTION

The closest reference term to viroid is virus, reﬂecting the intimate historical links

between viruses, discovered at the end of the nineteenth century in plants, and vi-

roids, also discovered in plants some 70 years later. The ﬁrst viroid, Potato spindle

tuber viroid (PSTVd) (25, 27), was identiﬁed during an attempt to characterize the

virus presumed to cause a potato disease. These and subsequent results obtained

for the same disease (97) as well as for another disease that was also presumed

to have a virus etiology but which turned out to be induced by Citrus exocor-

tis viroid (CEVd) (86, 96), established the viroid concept on solid experimental

ground. Viroids and viruses cannot, in principle, be discriminated according to the

0066-4286/05/0908-0117$20.00 117

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118 FLORES ET AL.

phenotypic effects that they incite in their hosts, which range from severe symptoms

to latent infections. However, both entities differ radically in structure, function,

and evolutionary origin, and if they are collected together in taxonomic schemes

like the one endorsed by the International Committee for Taxonomy of Viruses

(ICTV) (36), it is for historical and practical reasons. (Prions are also included in

this scheme, even though there is an even more drastic difference between them

and viruses). In this review we focus on the interactions of viroids with their host

plants but ﬁrst we brieﬂy describe the properties of these unique pathogens and

their classiﬁcation (for a detailed coverage of different aspects of viroids, see 42).

TAXONOMY OF THE VIROIDAE:

FAMILIES, GENERA, SPECIES

Figure 1 summarizes the structural characteristics of viroids. Because their mech-

anisms of replication and pathogenesis are discussed in detail below, here we

only highlight other properties that distinguish them from viruses. Viroids are

tiny single-stranded RNAs (246–401 nt), approximately tenfold smaller than the

genome of the smallest RNA viruses; they have a circular structure and high de-

gree of self-complementarity that promotes compact folding (87). As a functional

reﬂection of these singular features, viroids, in contrast to viruses, do not appear

to code for speciﬁc proteins (reviewed in 26), hence they must rely almost entirely

on host factors to complete their infectious cycle. This means that the parasitism

developed by viroids and viruses also differs: Viruses and viroids can essentially

be regarded as parasites of the translation and transcriptional apparatus of their

hosts, respectively. However, some viroids are catalytic RNAs and “code” for

hammerhead ribozymes that mediate the self-cleavage of the multimeric RNAs

generated in their replication through a rolling-circle mechanism. Apart from cer-

tain plant satellite RNAs and the RNA of human hepatitis delta virus (reviewed

in 35, 101), which resemble viroids in their circular structure and rolling-circle

replication mode mediated by ribozymes, no other virus-related RNAs have been

characterized as catalytic RNAs. This property constitutes the strongest argument

in support of the idea that viroids may have an ancient evolutionary origin inde-

pendent of viruses, going back to the RNA world postulated to have preceded the

present world on Earth based on DNA and proteins (reviewed in 26).

Most of the nearly 30 viroid species known (36) (Table 1) belong to the family

Pospiviroidae, type species PSTVd (25, 41), and adopt in vitro a rod-like or quasi-

rod-like secondary structure of minimal free energy (29, 87) with ﬁve structural-

functional domains (52, 89) (Figure 1a). The central conserved region (CCR),

within the C domain, is formed by two stretches of conserved nucleotides, in

which those of the upper strand are ﬂanked by an inverted repeat. Depending of

the nature of the CCR, and on the presence or absence of a terminal conserved

region (TCR) and a terminal conserved hairpin (TCH), members of this family are

allocated to ﬁve genera (36) (Table 1). The other four viroids, Avocado sunblotch

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VIROIDS AND VIROID-HOST INTERACTIONS 119

TABLE 1 Viroid species with their abbreviations, accession numbers of typical sequence

variants, sizes, and genus and family to which they belong

Viroid species Abbreviation Accession Size (nt) Genus Family

Potato spindle tuber PSTVd V01465 359 Pospiviroid Pospiviroidae

Tomato chlorotic

dwarf

TCDVd AF162131 360 Pospiviroid Pospiviroidae

Mexican papita MPVd L78454 360 Pospiviroid Pospiviroidae

Tomato planta

macho

TPMVd K00817 360 Pospiviroid Pospiviroidae

Citrus exocortis CEVd M34917 371 Pospiviroid Pospiviroidae

Chrysanthemum

stunt

CSVd V01107 356 Pospiviroid Pospiviroidae

Tomato apical stunt TASVd K00818 360 Pospiviroid Pospiviroidae

Iresine 1 IrVd-1 X95734 370 Pospiviroid Pospiviroidae

Columnea latent CLVd X15663 370 Pospiviroid Pospiviroidae

Hop stunt HSVd X00009 297 Hostuviroid Pospiviroidae

Coconut

cadang-cadang

CCCVd J02049 246 Cocadviroid Pospiviroidae

Coconut tinangaja CTiVd M20731 254 Cocadviroid Pospiviroidae

Hop latent HLVd X07397 256 Cocadviroid Pospiviroidae

Citrus IV CVd-IV X14638 284 Cocadviroid Pospiviroidae

Apple scar skin ASSVd M36646 329 Apscaviroid Pospiviroidae

Citrus III CVd-III AF184147 294 Apscaviroid Pospiviroidae

Apple dimple fruit ADFVd X99487 306 Apscaviroid Pospiviroidae

Grapevine yellow

speckle 1

GVYSd-1 X06904 367 Apscaviroid Pospiviroidae

Grapevine yellow

speckle 2

GVYSd-2 J04348 363 Apscaviroid Pospiviroidae

Citrus bent leaf CBLVd M74065 318 Apscaviroid Pospiviroidae

Pear blister canker PBCVd D12823 315 Apscaviroid Pospiviroidae

Australian

grapevine

AGVd X17101 369 Apscaviroid Pospiviroidae

Coleus blumei 1 CbVd-1 X52960 248 Coleviroid Pospiviroidae

Coleus blumei 2 CbVd-2 X95365 301 Coleviroid Pospiviroidae

Coleus blumei 3 CbVd-3 X95364 361 Coleviroid Pospiviroidae

Avocado sunblotch ASBVd J02020 247 Avsunviroid Avsunviroidae

Peach latent mosaic PLMVd M83545 337 Pelamoviroid Avsunviroidae

Chrysanthemum

chlorotic mottle

CChMVd Y14700 399 Pelamoviroid Avsunviroidae

Eggplant latent∗ELVd AJ536613 333 Elaviroid Avsunviroidae

∗Pending ICTV approval; whether Apple fruit crinkle and Citrus viroid original source should be considered as new viroid

species of genus Apscaviroid is also pending.

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120 FLORES ET AL.

viroid (ASBVd) (47), Peach latent mosaic viroid (PLMVd) (45), Chrysanthemum

chlorotic mottle viroid (CChMVd) (65), and Eggplant latent viroid (ELVd) (30),

do not have the conserved CCR, TCR, and TCH motifs but, remarkably, both their

polarity strands self-cleave through hammerhead ribozymes; they form the second

family, Avsunviroidae (reviewed in 33), whose type species is ASBVd (formal

inclusion of ELVd in this family is pending ICTV approval) (Figure 1b). Apart

from the core nucleotides conserved in their hammerhead structures, no extensive

sequence similarities exist between them, but PLMVd and CChMVd are grouped

in one genus because of their branched secondary structure (21, 45, 65), which is

stabilized by a pseudoknot (10; S. Gago, M. De la Pe˜na & R. Flores, unpublished

results) (Figure 1b), and their insolubility in2MLiCl (65). ASBVd, the only

viroid with a high A +U content (62%) (47), forms a monospeciﬁc genus, and

ELVd, whose properties fall between those of the members of the other two genera,

has been proposed to constitute its own genus (30). This classiﬁcation scheme is

further supported by phylogenetic reconstructions with entire viroid sequences

(36) and by the different subcellular replication (and accumulation) sites of the

type members of both families, with available data indicating that in this respect

other viroids behave like their corresponding type species. Within each genus, the

criteria to demarcate viroid species are an arbitrary level of below 90% sequence

similarity and distinct biological properties. Viroids, like viruses, propagate in their

hosts as populations of closely related sequence variants (quasi-species), although

one or more may predominate in the population. Heat stress may signiﬁcantly alter

the structure of viroid quasi-species (62). Some viroid variants with minor changes

affecting certain regions are directly related to speciﬁc diseases (57, 71, 83) or to

dramatic alterations in symptom severity (21, 93, 103).

BIOLOGY

Host Range and Host Speciﬁcity

Viroids are the etiologic agents of a number of diseases affecting economically

important herbaceous and ligneous plants including potato, tomato, cucumber,

hop, coconut, grapevine, several subtropical and temperate fruit trees (avocado,

peach, apple, pear, citrus, and plum), and some ornamentals (chrysanthemum and

coleus). Coconut cadang-cadang viroid (CCCVd) and Coconut tinangaja viroid

(CTiVd) infect monocotyledons, whereas the others infect dicotyledons. Some

viroids, among which the most instructive example is Hop stunt viroid (HSVd),

have wide host ranges but others, exempliﬁed by those forming the family Avsun-

viroidae, are mainly restricted to their natural hosts. A single nucleotide substi-

tution converts PSTVd from noninfectious to infectious for Nicotiana tabacum

(108).

Although most viroids are transmitted mechanically and some through seed

or pollen, with only Tomato planta macho viroid (TPMVd) known to be aphid-

transmissible under speciﬁc ecological conditions, the most efﬁcient transmission

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VIROIDS AND VIROID-HOST INTERACTIONS 121

route for viroids is vegetative propagation of infected material. This explains why

certain grapevine and, speciﬁcally, citrus cultivars propagated on infected cultivars

or rootstocks contain complex mixtures of different viroids.

Symptoms and Ultrastructural Effects

Some viroids have destructive consequences, as illustrated by CCCVd that has

killed millions of coconut trees in the Philippines, whereas others affect leaves

(chlorosis in spots or covering the whole blade, epinasty, rugosity, and necro-

sis), stems (pitting, internode shortening, and dwarﬁng), bark (scaling, cracking,

cankers), ﬂowers (size reduction, broken lines on petals), fruits (discolorations and

skin deformations, suture cracking), seeds (enlarged stones), and reserve organs

(malformations), as well as less conspicuous effects including delays in foliation,

ﬂowering and ripening, and growing pattern (open habit) of mature trees (Figure 2).

Certain viroids only induce symptoms in a particular organ (bark or fruit), whereas

others have more general effects. Infections caused by a few viroids result in very

mild or no symptoms. Absence of symptoms is common in naturally infected wild

plants, which can act as reservoirs. Symptom expression is generally favored by

high light intensity and, particularly, high temperature. Viruses, by contrast, have a

broader range of environmental conditions for optimal symptom expression. These

differences may explain why viroids mainly affect crops grown in tropical or sub-

tropical areas (and in greenhouses), and also why curing some viroid infections is

recalcitrant to thermotherapy.

Ultrastructural studies on the cytopathic effects induced by members of the

family Pospiviroidae have shown paramural bodies known as plasmalemmasomes

and aberrations of the thylakoid membranes of chloroplasts (94). Parallel stud-

ies with members of the family Avsunviroidae have revealed grossly disorganized

chloroplasts and membranous bodies in the yellow regions of ASBVd-infected av-

ocado leaves, while in completely chlorotic (“bleached”) leaves some chloroplasts

looked similar to proplastids (23). This latter observation has been reproduced in

PLMVd-infected leaves displaying extensive chlorosis (peach calico), which in

the most dramatic instances covers most of the leaf area (Figure 2h) (M.E. Rodio,

A. Destradis, R. Flores & F. Di Serio, unpublished results).

Cross-Protection

The phenomenon of cross-protection refers to observations that the ability of mem-

bers of both families to infect a host may be inﬂuenced by previous infections by

other strains of the same or closely related viroid (53, 68). More speciﬁcally, when

a plant pre-infected with a mild viroid strain is challenge-inoculated with a se-

vere strain of the same viroid, the typical symptoms of the second strain and the

accumulation level of its RNA are blocked or attenuated for a certain time. On

this basis, even before PSTVd, PLMVd, and CChMVd were identiﬁed as viroids,

the existence of mild or nonsymptomatic strains thereof was postulated and used

to develop cross-protection bioassays. It was also suggested that the nature of

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122 FLORES ET AL.

CChMVd was uncharacteristic (68). The mechanisms underlying cross-protection

have not been determined but may be related to RNA silencing (see below).

REPLICATION

Circular and Oligomeric RNA Templates: Support

foraRolling-Circle Mechanism

On ﬁnding that in PSTVd-infected tomato the most abundant viroid circular RNA,

arbitrarily considered as having (+) polarity, is accompanied by oligomeric (−)

RNAs, it was proposed that the latter were replicative intermediates resulting from

reiterative transcription of the former (6). The existence of CEVd (−)RNAse-

quences in infected Gynura aurantiaca (40) had already indicated that viroid

replication was an RNA-based process and undermined the idea of participa-

tion of DNA intermediates. Moreover, differential centrifugation studies showing

that PSTVd (24) and its complementary strands (99) accumulate in the nucleus

strongly suggested the involvement of a nuclear RNA polymerase in replication.

Re-examination of this question with PSTVd and other members of its family using

ﬁner approaches (in situ hybridization and confocal laser scanning and transmis-

sion electron microscopy) conﬁrmed (5, 44) and extended these observations (81).

In contrast, parallel experiments ﬁrst with ASBVd (4, 56, 63) and then with

PLMVd (9) showed the preferential accumulation of their (+) and (−) strands in the

chloroplast, indicating involvement of the enzymatic machinery of this organelle

in the replication of members of the family Avsunviroidae.

Rolling-Circle Mechanism: Asymmetric and

Symmetric Pathways

Figure 3 displays the two alternative pathways: the asymmetric, ﬁrst proposed to

account for replication of PSTVd (6, 7, 31) and HSVd (48), and the symmetric,

initially advanced on theoretical grounds (6) and then experimentally supported

for ASBVd (18, 46) and PLMVd (9). Other members of both families appear to

replicate following the same pathway as their type species. The difference discrim-

inating both pathways is the (−) template: The monomeric (−) circular RNA has

been detected in ASBVd-infected avocado (18, 46) and PLMVd-infected peach

(9), but not in PSTVd-infected tissues (6, 7, 31), or in electroporated protoplasts

(79) in which oligomeric (−)strands accumulate. Thus, cleavage and ligation oc-

cur in (+) and (−) strands in the symmetric pathway with two rolling circles, but

only in (+) strands in the asymmetric pathway with a single rolling circle.

The three catalytic activities required—RNA polymerase, RNase, and RNA

ligase (Figure 3)—were initially presumed to reside in host proteins. However,

in the family Avsunviroidae, the second activity is now known to be mediated

by hammerhead ribozymes embedded in both polarity strands, a ﬁnding with far-

reaching implications. In the context of the ﬁrst activity, the question of how

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VIROIDS AND VIROID-HOST INTERACTIONS 123

viroids redirect the template speciﬁcity of certain host DNA-dependent RNA

polymerases to transcribe RNA is one of the most interesting challenges

unresolved.

RNA Polymerization, Cleavage, and Ligation:

Role of Enzymes and Ribozymes

In light of recent reviews (34, 100), here we summarize only the main features,

novel data, and some unresolved issues regarding RNA polymerization, cleav-

age, and ligation. In vivo and in vitro transcription analyses in the presence of

α-amanitin indicate that RNA strand elongation in representative members of the

family Pospiviroidae is abolished by nanomolar concentrations of this fungal toxin,

which typically inhibit the nucleoplasmic RNA polymerase II (32, 64, 91). In line

with this view, a chromatin-enriched fraction from CEVd-infected tomato, puri-

ﬁed by afﬁnity with a monoclonal antibody to the carboxy-terminal domain of

the largest subunit of RNA polymerase II, has been shown to contain the viroid

(+) and (−) strands (106). Similar analyses in the presence of tagetitoxin support

the role of a nuclear-encoded chloroplastic RNA polymerase (NEP), or another

polymerase resistant to this bacterial inhibitor, in ASBVd strand elongation (67).

In vitro studies with PLMVd and RNA polymerase of Escherichia coli suggest

the participation of the eubacterial-like plastid-encoded polymerase (PEP) (75),

although this is a nonphysiological system. Moreover, synthesis (and accumula-

tion) of PLMVd is particularly active in areas exhibiting peach calico, in which

development of proplastids into chloroplasts and processing of chloroplastic rRNA

precursors (and, consequently, translation of plastid-encoded proteins) appear to

be impaired (M.E. Rodio, R. Flores & F. Di Serio, unpublished results). These lat-

ter observations are more consistent with the involvement of a NEP-like enzyme

in PLMVd replication and suggest that other chloroplastic factors mediating this

process (see below) are also nuclear-encoded.

To determine whether initiation of viroid RNAs is site-speciﬁc (promoter-

driven) or occurs at random (also allowing complete transcription of the circular

template), data obtained by in vitro capping with [α32P]GTP and guanylyltrans-

ferase, which speciﬁcally labels the free 5-triphosphate group characteristic of

chloroplastic primary transcripts, and RNase protection assays have mapped the

initiation of ASBVd (+) and (−)RNAs isolated from infected avocado at similar

A+U-rich terminal loops in their predicted quasi-rod-like secondary structures

(66). Primer-extension analysis of the 5termini of PLMVd subgenomic RNAs

from infected peach, presumed to be replication by-products, and in vitro tran-

scription studies with truncated PLMVd RNAs and RNA polymerase of E. coli

suggest that the initiation sites of this viroid also map at terminal loops (75). How-

ever, in vitro capping studies to map the 5-triphosphate termini of PLMVd (+)

and (−) linear RNAs isolated from infected tissue indicate alternative initiation

sites (S. Delgado, A.E. Mart´ınez de Alba, C. Hern´andez & R. Flores, unpublished

results). Still controversial are the initiation sites in the family of nuclear viroids

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124 FLORES ET AL.

(Pospiviroidae), an issue that has been addressed by in vitro transcription of the

PSTVd monomeric (+) circular RNA with a nuclear extract from potato or with

puriﬁed RNA polymerase II from wheat germ and tomato (100).

Correct processing to the monomeric (+) circular forms has been demon-

strated through assays in vitro with potato nuclear extracts and longer-than-unit

(+) PSTVd RNAs (2, 102) and in vivo with Arabidopsis thaliana transformed

with cDNAs expressing dimeric (+) transcripts of ﬁve representative members

of the family Pospiviroidae (17). These results indicate that the RNase activity

catalyzing cleavage of longer-than-unit (+) strands is a host enzyme(s) whose

site-speciﬁcity is determined by a particular RNA folding. Speciﬁcally, cleavage

of PSTVd (+) strands is proposed to be driven by a branched structure with a

GNRA tetraloop, which subsequently switches to an extended conformation with

an E loop promoting ligation (2). However, this mechanism may not apply to other

members of the family Pospiviroidae that are unable to form the GNRA tetraloop

and the loop E. On the other hand, it has been found that a dimeric (−) transcript

of HSVd expressed transgenically in A. thaliana fails to be processed (17) and

that in infected cultured cells and plants PSTVd (−) strands accumulate in the

nucleoplasm, whereas the (+) strands are localized in the nucleolus as well as in

nucleoplasm (Figure 3) (see below). Such ﬁndings suggest that viroid (+) strands

are processed in the nucleolus where processing of the rRNA and tRNA precursors

also occurs. Which factors determine this differential trafﬁc of viroid (+) and (−)

strands remain an intriguing issue.

In members of the family Avsunviroidae,however, the RNase activity is due not

to an enzyme but to a hammerhead ribozyme, a small RNA motif embedded in both

polarity strands that, through a transesteriﬁcation in the presence of Mg2+, self-

cleaves at a speciﬁc phosphodiester bond producing 5-OH and 2,3-cyclic phos-

phodiester termini (47, 78; reviewed in 35). Hammerhead ribozymes are formed

by a central core of conserved sequences ﬂanked by three double-stranded regions

with loose sequence requirements that are capped by loops. X-ray crystallography

has revealed a complex array of non-canonical interactions between the nucleotides

of the central core, explaining why they are strictly conserved in natural ribozymes

and illustrating that the actual shape does not resemble a hammerhead but rather

an inverted Y in which the stems III and II are almost co-linear (Figure 1b, inset).

There is ﬁrm evidence supporting the proposal that hammerheads mediate the in

vivo self-cleavage of the multimeric viroid RNAs wherein they are inserted, and

that the reaction is under strict control through a conformational switch between

the active ribozyme, which is transiently formed during replication, and an alter-

native folding that promotes ligation (34). In contrast to the accepted view, recent

data show that modiﬁcations of loops 1 and 2 of natural hammerheads induce a

severe reduction in their catalytic activity, indicating that these peripheral regions

play a critical role in catalysis through tertiary interactions between some of their

nucleotides that may favor the active site at the low magnesium concentration

existing in vivo (20, 54) (Figure 1b, inset). These interactions could be stabilized

by chloroplastic proteins behaving as RNA chaperones, which would explain why

they facilitate the hammerhead-mediated self-cleavage of a viroid RNA (16).

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VIROIDS AND VIROID-HOST INTERACTIONS 125

Data on the third replication step are limited, but in PSTVd and nuclear viroids

the reaction is presumably catalyzed by a host enzyme similar to the wheat germ

RNA ligase. Support for this hypothesis comes from data in vitro showing that this

enzyme mediates circularization of the monomeric linear PSTVd RNA isolated

from infected tissue (8) and that a potato nuclear extract can process longer-

than-unit PSTVd RNAs to the monomeric circular forms (2, 102), and in vivo

indicating that A. thaliana seems to have a similar enzyme able to circularize the

monomeric linear forms of representative members of the family Pospiviroidae

(17). Still unclear is whether a chloroplastic RNA ligase exists for members of

the family Avsunviroidae or, alternatively, the reaction is autocatalytic. Support

for this latter view is based on the in vitro self-ligation of the monomeric linear

PLMVd RNAs resulting from hammerhead-mediated self-cleavage, which mostly

leads to 2,5- instead of the conventional 3,5-phosphodiester bonds (14) and the

proposal that these atypical bonds are present in circular PLMVd RNAs isolated

from infected tissue and impede their in vitro self-cleavage (15). In line with these

results, early in vitro studies with RNA of human hepatitis delta virus (HDV)

which, like viroids, also replicates through a rolling-circle mechanism mediated

by ribozymes (albeit of a non-hammerhead class), showed ligation of ribozyme-

cleaved sequences in protein-free conditions (reviewed in 101). However, these

conditions were far from physiological, and more recent studies have shown that a

host-speciﬁc function is needed for ligation of the RNAs resulting from cleavage

by a wide variety of ribozymes, which include hammerhead and HDV ribozymes

(84). Furthermore, the hammerhead-generated 5-OH and 2,3-cyclic phospho-

diester termini are those typically required by a wheat germ-like RNA ligase, a

feasible alternative to self-ligation and, in contrast to a previous report (15), the

circular PLMVd RNAs isolated from infected tissue appear to self-cleave in vitro

(S. Delgado, C. Hern´andez & R. Flores, unpublished results).

MOVEMENT

Viroid movement received scant attention in early studies on these peculiar patho-

gens but an increasing number of recent reports are now elucidating the mecha-

nisms and putative host factors involved in this step of the viroid infectious cycle.

This area has been further promoted by the possible relevance of these results to

the general processes of RNA transport in plants, including mRNAs and mobile

silencing signals (see below). In contrast to viruses, which encode their own move-

ment proteins, viroids must interact directly with host factors for mobility. Viroids

therefore offer a unique system to dissect RNA trafﬁc in plants.

Intracellular Movement

After entering a cell, the viroid RNA must move to its replication site, either the

nucleus or the chloroplast, to generate the progeny for release to the cytoplasm

to invade neighboring cells. Import of ﬂuorescein-labeled PSTVd transcripts into

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126 FLORES ET AL.

the nucleus has been studied in permeabilized tobacco cells and shown to be a

cytoskeleton-independent process mediated by a speciﬁc and saturable receptor

(109). This process was demonstrated to be sequence or structure speciﬁc because

no import was observed for mRNA fragments of the same size or for twoviroids that

replicate and accumulate in the chloroplast. These results were validated in planta

with an RNA virus vector expressing a green ﬂuorescent protein (GFP) reporter

gene bearing an intron that was either unmodiﬁed or contained an embedded full-

length PSTVd copy. Inoculation with these cytoplasmic replicating viral constructs

caused ﬂuorescence only with the PSTVd-containing vector, indicating that the

viroid sequence targeted the recombinant RNA to the nucleus where the intron

was removed, with the spliced mRNA returning to the cytoplasm where it was

translated (110).

Overall, these data suggest that nuclear import of PSTVd results from interac-

tion of a viroid sequence or structural motif with cellular factors that may lead to

the formation of a ribonucleoprotein complex, which would shuttle the viroid to the

nucleus. Pertinent to this point is the identiﬁcation of a bromodomain-containing

protein from tomato that binds speciﬁcally PSTVd in vitro and in vivo (61). This

Viroid RNA-binding Protein 1 (VirP1), which was isolated from an RNA-ligand

screening through its ability to interact with PSTVd (+)RNA,isamember of

afamily of transcriptional regulators associated with chromatin remodeling. As

expected, VirP1 contains a nuclear localization signal and is a candidate to mediate

PSTVd transfer (and that of other related viroids) into or out of the nucleus. Further

work has mapped the RNA motif responsible for the speciﬁc interaction between

VirP1 and PSTVd to an asymmetrical internal loop (termed RY motif owing to its

base composition), within the right terminal domain, which is present twice in this

viroid and in most members of the genus Pospiviroid, once in HSVd, and is partially

conserved in the genus Cocadviroid (39, 58). Mutations in any of the two RY motifs

of PSTVd abolished infectivity, a ﬁnding that supports its biological relevance.

The general picture of the intracellular viroid movement becomes more complex

when analyzed at the organelle level. Using improved sample preparation and in

situ hybridization, it has recently been shown that the PSTVd (−) strand localizes

in the nucleoplasm of infected tomato and N. benthamiana plants or cultured cells,

whereas the (+) strand localizes in the nucleolus as well as in the nucleoplasm

(Figure 3), with distinct spatial patterns that may represent successive stages of the

viroid RNA migration or processing (81). These results suggest that after synthesis

of PSTVd (+) and (−)RNAsinthe nucleoplasm, the latter stays anchored to this

compartment while the (+) strand is transported to the nucleolus. This ﬁnding

points to the existence of highly specialized machinery in eukaryotic cells able to

discriminate the opposite strands of an RNA and may have implications in gene

regulation and pathogen infection.

As regards intracellular movement in members of the family Avsunviridae, the

transfer mechanism to the chloroplast has yet to be investigated. Novel transport

pathways may well be discovered in plant cells, given that no other alien or cellular

RNAs have been reported to trafﬁc inside this organelle.

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VIROIDS AND VIROID-HOST INTERACTIONS 127

Cell-to-Cell Movement

Once it has infected the ﬁrst cells, a viroid has to colonize adjacent cells prior to

invading the more distal plant parts. Proteins and nucleic acids, either endogenous

or of viral origin, have been reported to move cell-to-cell via the plasmodesmata

(PD), the plant organella providing cytoplasmic connections between cells. Stud-

ies with PSTVd suggest that viroids follow the same pathway for intercellular

movement (28). PSTVd was retained when microinjected into symplasmically

isolated guard cells of mature tomato and tobacco leaves; however, when injected

into symplasmically connected mesophyll cells, the viroid moved quickly from

cell to cell. Remarkably, the fusion to PSTVd aided the transport through PD of

an otherwise nonmobile RNA, suggesting that the viroid contains a motif for PD

transport.

Long-Distance Movement

Systemic spread of viroids occurs through the vasculature and allows their access

to tissues far away from those initially infected. As in viruses, this transport in-

volves loading the viroid into the phloem and follows the ﬂow of photoassimilates

from the photosynthetic source to sink tissues/organs of the plant (72, 111). Evi-

dence of this movement at the cellular level has been provided for PSTVd by in situ

hybridization, which also showed that trafﬁcking within the phloem is probably

sustained by viroid replication and tightly regulated by plant developmental and

cellular factors (111). The viroid was not detected in shoot apical meristems (SAM)

of infected N. benthamiana or tomato plants, whereas it was present in the vascu-

lar tissues (most probably the procambium and/or protophloem) below the SAM,

suggesting that PD at some cellular boundaries between the SAM and the rest of

plant body restrict PSTVd trafﬁcking into the SAM. Moreover, PSTVd was absent

in developing ﬂowers, whereas mature ﬂowers contained the viroid in parenchyma

cells of sepals but, intriguingly, not in petals, stamens, styles, or ovaries, although

the phloem connections were already established. The absence of PSTVd in some

ﬂoral organs of N. benthamiana may be attributable to restricted trafﬁc in these

organs and not to replication suppression (112). This restriction could be nonop-

erative under certain conditions because PSTVd is seed-transmissible in tomato

and therefore able to infect ovules and/or pollen eventually. In contrast to the

strong vascular tropism that PSTVd shows below the SAM in tomato, PLMVd

appears able to invade cell layers very close to the SAM in peach (M.E. Rodio, S.

Delgado, M.D. G´omez, R. Flores & F. Di Serio, unpublished results), suggesting

that different mechanisms regulate movement in the two viroid families.

Viroid translocation via the phloem is most probably facilitated by host pro-

teins. Evidence has been obtained in vitro and in vivo of the formation of a ri-

bonucleoprotein complex between HSVd and one of the most abundant phloem

polypeptides of cucumber, the dimeric lectin known as Phloem Protein 2 (PP2)

(37, 38, 69). This protein fulﬁls the requirements of an RNA chaperone protein in-

volved in systemic movement of HSVd: RNA-binding activity probably mediated

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128 FLORES ET AL.

by a double-stranded RNA binding motif, ability to interact with PD and increase

their exclusion limit, and competence for long-distance translocation. Speciﬁcally,

this protein is able to move and aid HSVd movement through intergeneric grafts

involving a host rootstock and a nonhost scion, although the viroid remains largely

conﬁned within the vascular tissue of the scion, suggesting that additional host

factors are required for HSVd to leave the phloem efﬁciently (38). Indeed, phloem

entry and exit are probably mediated by different motifs, as revealed by the iden-

tiﬁcation of two PSTVd mutants able to enter and replicate in the phloem of N.

tabacum but unable to leave the vascular tissue (112). The same viroid variants

infected all cells of the upper uninoculated leaves in N. benthamiana, indicating

that the regulation mechanisms of phloem-mediated RNA trafﬁc may differ in

distinct plant species. Moreover, in PSTVd, a motif has recently been identiﬁed

that mediates trafﬁcking from the bundle sheath into the mesophyll but not in the

opposite direction (82).

It has been suggested that VirP1 from tomato, like PP2 from cucumber, is

involved in the systemic spread of PSTVd in this host, given that a PSTVd mutant

defective for VirP1 binding is unable to spread systemically (58). However, in

contrast to PP2, the ability of VirP1 to interact with PD and move long distance

has yet to be proved. In conclusion, viroids must have evolved to exploit existing

transport routes and, given their peculiar characteristics, one may expect these

pathogens to be instrumental in elucidating structural traits, mechanisms, and

cellular factors that mediate RNA trafﬁc in plants.

PATHOGENESIS

Lacking protein-coding capacity, viroids must incite disease by direct interaction

of their genomic RNA or derivatives thereof with host factors (proteins or nucleic

acids). This primary interaction triggers a cascade of events, still poorly understood,

that eventually lead to macroscopic symptoms.

Is the Mature Viroid RNA the Direct Pathogenic Effector?

In PSTVd, mutations of 3–4 nucleotides, with marked effects on symptoms, have

been mapped at a virulence-modulating (VM) region that overlaps a premelting

(PM) region within a domain of the rod-like structure (93). Because a similar

situation was observed in CEVd (103), this domain was termed pathogenic (P)

(52) (Figure 1a). Although an inverse correlation was found between the ther-

modynamic stability of the PM region and virulence in tomato for some PSTVd

strains (93), other data do not support this correlation (70). Alternatively, from

comparisons of the most stable secondary structures of PSTVd variants of differ-

ent pathogenicity and considerations about bending of RNA helices, it has been

advanced that major differences in their VM region geometry and concomitant al-

terations in RNA-protein interactions are the primary cause of viroid pathogenicity

(70, 92). In this context, a correlation between the virulence of PSTVd strains and

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VIROIDS AND VIROID-HOST INTERACTIONS 129

the activation of certain protein kinases has been reported (reviewed in 26). It is also

possible that differential interactions with host proteins involved in viroid replica-

tion, movement, or accumulation could be the initial event in viroid pathogenesis

(Figure 4). Moreover, other data indicate the contribution of additional domains to

symptom expression (89), including the C domain, in which a speciﬁc nucleotide

(A257) in PSTVd induces severe growth stunting and ﬂat top of the tomato shoot

(80). Pathogenicity determinants outside the P domain have also been identiﬁed in

other members of the family Pospiviroidae such as CCCVd (85) and HSVd (71,

83).

Within the family Avsunviroidae, motifs involved in pathogenesis have also

been recognized. ASBVd variants slightly diverging in the poly-A right terminal

loop have been associated with different leaf symptoms (95), although difﬁculties

inherent to bioassays in avocado have precluded a direct analysis. In CChMVd,

a tetraloop in the in vivo branched RNA conformation has been mapped as the

major pathogenicity determinant because site-directed mutagenesis and bioassays

have shown that the change from UUUC to GAAA converts a variant from se-

vere into latent without altering the ﬁnal viroid titer (19, 21). A similar method-

ology was used to demonstrate that PLMVd variants containing a 12–13-nt in-

sertion incite peach calico. This insertion is always found in the same position,

has limited sequence variability, and folds itself into a hairpin (57). Moreover,

the pathogenicity determinant might be restricted to the U-rich tetraloop of this

hairpin. Although the precise role of this loop in symptom development is not

known, electron microscopy observations have revealed that chloroplast differ-

entiation is blocked in symptomatic tissues (M.E. Rodio, S. Delgado, R. Flores

&F.DiSerio, unpublished results). The identiﬁcation of pathogenicity determi-

nants with a similar structure (a U-rich tetraloop) in CChMVd and PLMVd is

intriguing.

Instead of proteins as the primary host target, base-pair interactions between

PSTVd and host RNAs, resulting in interference with rRNA maturation, mRNA

splicing, or 7S RNA assembly into the signal recognition particle, were proposed

as possible molecular events initiating pathogenesis (reviewed in 26). Although

these hypotheses fail to explain the differential symptoms induced by the same

viroid variant on closely related species (26), the observation that the PSTVd (+)

strand is able to migrate to the nucleolus and cause a redistribution of the small

nucleolar RNAs has led to the suggestion that PSTVd could incite deleterious

effects on rRNA processing and related events by competing for certain nucleolar

factors (81).

Early studies on the signal-transduction cascade leading to symptom expression

showed that viroids activate a general plant defense system based on pathogenesis-

related (PR) proteins. This system is also induced by other pathogens including

viruses, bacteria and fungi, which modify the concentrations of certain hormones

and metabolites such as polyamines (13). More recently, a macroarray-based ap-

proach has shown that PSTVd infection alters the gene expression pattern in tomato

and that the regulation of speciﬁc genes, which are not involved in the host reaction

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130 FLORES ET AL.

to other pathogens, may be affected in a strain-dependent manner (50). The role

of these genes in pathogenesis is unknown, except in tomato plants infected with

a PSTVd strain with the U257A substitution; here the stunted growth is caused

by restricted cell expansion, but not cell division or differentiation, and has been

correlated with the down-regulated expression of an expansin gene, LeExp2 (80).

Although it is generally accepted that viroid diseases are induced by speciﬁc

interference with regulation of host gene expression, many questions about the

underlying mechanism remain unanswered. The discovery that a group of small

antisense RNAs, namely the small interfering RNAs (siRNAs) and the microRNAs

(miRNAs) (reviewed in 1, 11), mediate a regulatory layer of host gene expression

in eukaryotes has led to new attractive hypotheses for viroid pathogenesis.

Do Viroids Exert their Pathogenic Effects via Speciﬁc siRNAs?

miRNAs (21–24 nt long) negatively regulate the expression of genes involved

mainly in development (11). They are generated by an enzyme of the RNase

III class (Dicer) (3), Dicer-like (DCL) in plants, acting on miRNA precursors,

which are endogenous nonprotein-coding transcripts adopting a hairpin conforma-

tion with partially double-stranded regions. Depending on the degree of sequence

complementarity with their cognate mRNAs, miRNAs guide the RNA-induced

silencing complex (RISC) (43) to degrade the target RNA or, less frequently in

plants, to repress its translation. Plant siRNAs (21–26-nt long) are associated with

both transcriptional gene silencing (TGS) and posttranscriptional gene silencing

(PTGS) (reviewed in 1). TGS controls gene expression negatively by siRNA-

directed DNA methylation of certain promoters, a process that is linked to histone

modiﬁcation in plants. PTGS, a sequence-speciﬁc RNA-degradation mechanism,

was ﬁrst identiﬁed in plants as a defense response against invading RNAs from

transgenes, transposons, and viruses (reviewed in 1, 55). siRNAs play a key role

in this system because, like miRNAs, they are loaded into RISC and guide it to

degrade speciﬁc RNAs. siRNAs also resemble miRNAs in that they are generated

by Dicer, although acting on double-stranded RNA (dsRNA) in this case. Most

transgenic and transposon dsRNAs derive from transcription of inverted repeats in

the nucleus, with other dsRNAs that accumulate in the cytoplasm resulting from

replication of single-stranded RNA (ssRNA) viruses. To counteract this antiviral

defense, plant viruses code for speciﬁc proteins, called silencing suppressors, that

inhibit PTGS (reviewed in 55).

The possibility that viroids may inﬂuence host gene expression at both post-

transcriptional and transcriptional levels and, indirectly, induce symptoms (see

below) is supported by the recent identiﬁcation of viroid-speciﬁc siRNAs in plants

infected by members of the families Pospiviroidae (49, 59, 73) and Avsunviroidae

(59, 60), together with the previous ﬁnding that replicating PSTVd induces de

novo methylation of PSTVd sequences transgenically inserted in the plant genome

(107). Where and how these viroid-speciﬁc siRNAs are generated is unresolved.

Since DCL isoenzymes with nuclear localization have been described (reviewed

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VIROIDS AND VIROID-HOST INTERACTIONS 131

in 1) (Figure 4), a nuclear origin for the siRNAs in the family Pospiviroidae

seems plausible. In contrast, because no chloroplastic DCL has been reported,

siRNA generation in the cytoplasm during viroid cell-to-cell movement seems

more likely in the family Avsunviroidae. This possibility could also apply to nu-

clear viroids, given that PSTVd-derived siRNAs accumulate in the cytoplasm (22)

(Figure 4). Regarding the templates, the primary candidates for siRNA genera-

tion in the family Pospiviroidae are the nuclear dsRNA intermediates of viroid

replication. However, the mature genomic forms (structurally similar to miRNAs

precursors) could also be degraded by DCL-1 in the nucleus or, alternatively,

serve as templates for the synthesis of secondary dsRNAs catalyzed by a cytoplas-

mic RNA-dependent RNA polymerase (RdRp) (90) (Figure 4). Indeed, although

PSTVd and ASBVd monomeric forms are resistant in vitro to human Dicer (12),

they appear to be sensitive to wheat germ DCL (A.E. Mart´ınez de Alba and R.

Flores, unpublished results). In this framework, direct characterization of the small

RNAs derived from replicating PSTVd indicates that they are predominantly of the

(+) polarity resembling, as a population, miRNAs rather than siRNAs (B. Ding,

personal communication).

Are viroid-speciﬁc siRNAs relevant in pathogenesis or are they just by-products

of the RNA silencing machinery? The in vivo concentration of these siRNAs cannot

explain differences in symptom development because their levels are essentially

the same in infections induced by severe, mild, or latent strains of PSTVd and

CChMVd (60, 73). However, an inverse correlation exists in chloroplastic viroids

between the accumulation of genomic viroid forms and their corresponding siR-

NAs: the in vivo PLMVd and CChMVd concentrations are low but their siRNAs

are easily detectable, whereas in tissues accumulating a very high ASBVd con-

centration the corresponding siRNAs are undetectable (60) or accumulate poorly

(59). This ﬁnding is consistent with the siRNA participation in a PTGS defense

response of certain hosts aimed at attenuating the in vivo viroid titer (60); this re-

sponse should take place in the cytoplasm where RISC is assumed to act. Other data

support this view indirectly. First, PSTVd infection of transgenic plants, express-

ing a reporter gene 3fused to partial-length PSTVd sequences, activates degra-

dation of the recombinant transcript (and methylation of the nuclear transgenic

viroid DNA) (104), probably via the siRNAs that result from PSTVd replication.

Second, preliminary observations indicate that the infectivity of some viroids is

reduced when they are coinoculated with an excess of their cognate dsRNAs (A.

Carbonell, S. Gago & R. Flores, unpublished results), suggesting that the latter

are processed by DCL in vivo and the resulting siRNAs target the genomic forms

for RISC-mediated degradation (Figure 4). And third, accumulation of PSTVd-

speciﬁc siRNAs precedes tomato plant recovery from severe symptoms of PSTVd

infection (88). These results argue against the proposal that viroids are resistant

to RNA silencing–mediated degradation and might have evolved their compact

secondary structure to escape this host defense pathway (105). Such folding might

also have appeared to provide resistance against RNases or certain inactivating

proteins that target RNA single-stranded regions (74).

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132 FLORES ET AL.

However, some viroid-speciﬁc siRNAs might also direct host DNA methylation

(76) or act like endogenous miRNAs targeting host mRNAs for degradation (59, 73,

105) (Figure 4). Supporting this idea, transgenic tomato plants expressing inverted

repeats of an almost complete (noninfectious) PSTVd sequence showed symptoms

similar to, though milder than, those induced by infectious PSTVd RNA. These

transgenic plants accumulated PSTVd-derived siRNAs, whereas neither siRNAs

nor typical symptoms were detected in plants expressing direct repeats of the same

viroid sequence (105). However, the effects of expressing sequences from a mild

PSTVd strain were not determined, very few small RNAs derived from replicating

PSTVd correspond to domains responsible for symptom expressiod(same)]TJT*[(viroid)-262.5(sequence)-262.4((105).)-262.5(Ho)25(we)25(v)15(e)0(r)40(,)-262.4(the)-262.5(ef)25(fects)-262.5(of)-262.4(e)15(xpressing)-262.5(sequences)-262.4(from)-262.5(a)-262.4(mild)]TJT*[(PSTVd)-201.4(strain)-201.4(were)-201.3(not)-201.4(determined,)-201.2(v)15(ery)-201.4(fe)25(w)-201.4(small)-201.3(RN)35(As)-201.4(deri)25(v)15(e)0(d)-201.4(from)-201.3(replicating)]TJT*[(PSTVd)-204.9(correspond)-204.8(to)-204.9(domains)-204.9(responsible)-204.8(for)-204.9(symptom)-204.9(e)15(xpressiod(same)]TJT*[(sam2TJT*[(PSTVd)-204.9(h24not)-201.h)]xpres19 Tm4.8(orssam2TJTw75.01.4(determined,)-2-r.1 -170 9.9626343)-20athw0.2 ays26343)-2 7116(tar343)2(Inasmuch26343)-2as26343 P43 s43ers43 not ho01.ol(v)15(ercomplete)-TG5219 Tm4.8(orssa259(,)4(der521ader521co)0(drder521defen32)T5219s)-t.1 -17yed fr5215.1(v521T*[(P(v5210.5649 der521dromoder521(ef)25(.8(to)-204.op2)T5TJTw75.0T5TJT9(domains)5TJ3strain)-20?0T5TJTP-201ing)]TJ5TJ3sco)ld0T5TJTb2dordedTJ5TJ3s[(PST5TJTpreirPST5TJT)15( -17.5TJ3sconhe)-han, 000117.933m3 ref245es1-149.6(,f245e204)19(,)utf245es1one v6(,f245e 755.9245e 7b)-2245e20 rtressi ref245et 017.933o.29995.8(i8(ocii ref995.1Tw75.0T995.8()-201.hs.0T995.8(262erni r(e)15(xpree)-65(,f995.1Tpresxpry0T995.8(co)ld0T995.8(me)]TJT*0T995.8(w75.0T995.8(pressi95.1T1.8(acc95.8(meetected)-19bor.3(re2.7)-3ed)e3(re2.7)sy)-2m3(re2.7)because3(re2.7)(endogenre2.7)-s)-201.4enre2.7)has263e2.7)bee4enre2.7)i8(ocii egenre2.7)w75.0T9e2.7)-sild

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VIROIDS AND VIROID-HOST INTERACTIONS 133

of life”). Additionally, it has led to discoveries transcending virology and the

plant world: the hammerhead ribozyme, with deep functional, biotechnological,

and evolutionary repercussions (47, 78; reviewed in 35), and RNA-dependent

DNA methylation (107) that mediates transcriptional silencing. This illustrates,

once again, how unveiling apparently marginal details of a speciﬁc system can

illuminate far-reaching questions. But this may not be the end. Recently, viroids

have become a tool for dissecting aspects of transcriptional and posttranscriptional

gene silencing, as well as for understanding how minimal RNAs can elicit disease

(reviewed in 26, 34, 100). Moreover, viroids are being used as probes to study

the factors governing the intracellular, cell-to-cell, and long-distance movement

of RNAs (81, 109, 111, 112). Within this context, studies on chloroplast viroids

could elucidate how a foreign RNA manages to cross the membrane surrounding

this organelle. Last but not least, whether viroids exist out of the plant kingdom

is still a challenging and exciting question. After all, since viruses were initially

found in plants and subsequently in most types of other organisms, why should

viroids be restricted to the green world?

ACKNOWLEDGMENTS

We apologize to colleagues whose articles were not cited because of space lim-

itations. We thank B. Ding and R.A. Owens for access to unpublished data and

suggestions, respectively. Work in R. Flores and J.A. Dar`os laboratories has been

supported by the Ministerio de Ciencia y Tecnolog´ıa (BMC2002-03,694) and the

Generalidad Valenciana (Spain).

The Annual Review of Phytopathology is online at

http://phyto.annualreviews.org

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110. Zhao Y, Owens RA, Hammond RW.

2001. Use of a vector based on potato virus

Xinawhole plant assay to demonstrate

nuclear targeting of potato spindle tuber

viroid. J. Gen. Virol. 82:1491–97

111. Zhu Y, Green L, Woo Y-M, Owens R,

Ding B. 2001. Cellular basis of potato

spindle tuber viroid systemic movement.

Virology 279:69–77

112. Zhu Y, Qi Y, Xun Y, Owens R, Ding

B. 2002. Movement of potato spindle

tuber viroid reveals regulatory points

of phloem-mediated RNA trafﬁc. Plant

Physiol. 130:138–46

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VIROIDS AND VIROID-HOST INTERACTIONS C-1

Figure 1 Structural features of viroids. (a) Rod-like secondary structure of mem-

bers of the family Pospiviroidae. Domains C (central), P (pathogenic), V (variable),

and TLand TR(terminal left and right,respectively) are indicated, as well as the

CCR (central conserved region,here displayed for the genus Pospiviroid), TCR

(terminal conserved region,present in the genera Pospi- and Apscaviroid,and in the

two largest members of the genus Coleviroid) and TCH (terminal conserved hairpin,

present in the genera Hostu- and Cocadviroid). Arrows indicate flanking sequences

that together with the upper CCR strand form a hairpin, and the S-shaped line con-

nects the residues linked after UV irradiation as a consequence of forming part of the

loop E. (b) Quasi-rod-like and branched secondary structures of ASBVd and

PLMVd, respectively (family Avsunviroidae). Sequences conserved in most natural

hammerhead structures are shown within boxes with blue and white backgrounds for

() and () polarities, respectively. Broken oval in PLMVd denotes a kissing-loop

interaction. (Inset) PLMVd () hammerhead structure represented according to the

original scheme (left) and to X-ray crystallography data obtained with artificial ham-

merhead structures (right), in which a proposed tertiary interaction between loops 1

and 2 enhancing the catalytic activity is indicated. Nucleotides conserved in most

natural hammerhead structures are depicted as above. Arrows mark the self-cleavage

site, and continuous and broken lines denote Watson-Crick and noncanonical pairs,

respectively.

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C-2 FLORES ET AL.

Figure 2 Alterations in different organs accompanying infections by some repre-

sentative viroids. (a) Symptoms of PSTVd on potato tubers (left) and healthy con-

trols (right) (courtesy of T.O. Diener). (b) Symptoms of CEVd on a trifoliate orange

rootstock (courtesy of N. Duran-Vila and P. Moreno). (c) Symptoms of PBCVd on

pear A20 (courtesy of J.C. Desvignes). (d) Flower symptoms of CSVd on chrysan-

themum (bottom) and a healthy control (top) (courtesy of J.M. Bové). (e) Leaf

symptoms of CSVd on chrysanthemum (right) and a healthy control (left). ( f)Leaf

symptoms of CChMVd on chrysanthemum (right) and a healthy control (left).

(g)Leaf symptoms of CCCVd on coconut (left) and a healthy control (right).

(h) Extreme leaf chlorosis (peach calico) induced by PLMVd. (i) Internode shorten-

ing induced by HSVd on cucumber (right) and a healthy control (left). ( j)Symptoms

of HSVd on cucumber fruits (left) and a healthy control (right) (courtesy of H.L.

Sänger). (k) Fruit symptoms (dapple apple) induced by ASSVd (courtesy of J.C.

Desvignes). (l) Fruit symptoms of ASBVd on avocado (courtesy of P.R. Desjardins).

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VIROIDS AND VIROID-HOST INTERACTIONS C-3

Figure 3 Rolling-circle mechanism proposed for viroid replication. The asymme-

tric pathway, with one rolling circle, is followed by members of the family

Pospiviroidae and takes place in the nucleus. The symmetric pathway, with two

rolling circles, is followed by members of the family Avsunviroidae and takes place

in the chloroplast. White and yellow lines indicate plus () and minus () strands,

respectively, and cleavage sites are marked by arrowheads. Self-cleavage mediated

by hammerhead ribozymes (Rz) leads to linear monomeric RNAs with 5-hydroxyl

and 2-3-cyclic phosphodiester termini; cleavage catalyzed by a host protein (HP)

probably generates the same termini. Pol II refers to RNA polymerase II and NEP to

nuclear-encoded RNA polymerase. PD and NP are abbreviations for plasmodesma-

ta and nuclear pores, respectively.

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C-4 FLORES ET AL.

Figure 4 Hypothetical mechanisms of viroid pathogenesis. Symptoms could result

from direct interaction between the genomic viroid RNAs and a host factor (HF, pro-

tein or RNA), either in the organelle where the viroid replicates and accumulates or

in the cytoplasm during viroid movement. Alternatively, RNA silencing could medi-

ate symptom development. In the family Pospiviroidae viroid-specific siRNAs could

accumulate either in the nucleus (from the action of DCL1 on the genomic viroid

RNAs, or of DCL3 and/or DCL4 on the dsRNAs formed during replication), or in

the cytoplasm (from the action of DCL2 on the aberrant RNAs generated by a cyto-

plasmic RdRp, or on the genomic viroid RNAs, not shown). In the family

Avsunviroidae viroid-specific siRNAs should be generated only in the cytoplasm

because no chloroplastic DCL has been characterized. siRNAs would then load

RISC for degradation or translation repression of host mRNAs with complementary

sequences, or for direct methylation of host DNA. siRNAs could also target geno-

mic viroid RNAs and regulate their titer through a feed-back mechanism. The sizes

of the different molecules are not proportional. Both hypothetical mechanisms

should not be necessarily exclusive. PD, plasmodesmata; NP, nuclear pores.

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P1: KUV

July 14, 2005 11:17 Annual Reviews AR250-FM

Annual Review of Phytopathology

Volume 43, 2005

CONTENTS

FRONTISPIECE,Robert K. Webster xii

BEING AT THE RIGHT PLACE,AT THE RIGHT TIME,FOR THE RIGHT

REASONS—PLANT PATHOLOGY,Robert K. Webster 1

FRONTISPIECE,Kenneth Frank Baker

KENNETH FRANK BAKER—PIONEER LEADER IN PLANT PATHOLOGY,

R. James Cook 25

REPLICATION OF ALFAMO-AND ILARVIRUSES:ROLE OF THE COAT PROTEIN,

John F. Bol 39

RESISTANCE OF COTTON TOWARDS XANTHOMONAS CAMPESTRIS pv.

MALVACEARUM,E. Delannoy, B.R. Lyon, P. Marmey, A. Jalloul, J.F. Daniel,

J.L. Montillet, M. Essenberg, and M. Nicole 63

PLANT DISEASE:ATHREAT TO GLOBAL FOOD SECURITY,Richard N. Strange

and Peter R. Scott 83

VIROIDS AND VIROID-HOST INTERACTIONS,Ricardo Flores,

Carmen Hern´

andez, A. Emilio Mart´

ınez de Alba, Jos´

e-Antonio Dar`

os,

and Francesco Di Serio 117

PRINCIPLES OF PLANT HEALTH MANAGEMENT FOR ORNAMENTAL PLANTS,

Margery L. Daughtrey and D. Michael Benson 141

THE BIOLOGY OF PHYTOPHTHORA INFESTANS AT ITS CENTER OF ORIGIN,

Niklaus J. Gr¨

unwald and Wilbert G. Flier 171

PLANT PATHOLOGY AND RNAi: A BRIEF HISTORY,John A. Lindbo

and William G. Doughtery 191

CONTRASTING MECHANISMS OF DEFENSE AGAINST BIOTROPHIC AND

NECROTROPHIC PATHOGENS,Jane Glazebrook 205

LIPIDS,LIPASES,AND LIPID-MODIFYING ENZYMES IN PLANT DISEASE

RESISTANCE,Jyoti Shah 229

PATHOGEN TESTING AND CERTIFICATION OF VITIS AND PRUNUS SPECIES,

Adib Rowhani, Jerry K. Uyemoto, Deborah A. Golino,

and Giovanni P. Martelli 261

MECHANISMS OF FUNGAL SPECIATION,Linda M. Kohn 279

vii

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P1: KUV

July 14, 2005 11:17 Annual Reviews AR250-FM

viii CONTENTS

PHYTOPHTHORA RAMORUM:INTEGRATIVE RESEARCH AND MANAGEMENT

OF AN EMERGING PATHOGEN IN CALIFORNIA AND OREGON FORESTS,

David M. Rizzo, Matteo Garbelotto, and Everett M. Hansen 309

COMMERCIALIZATION AND IMPLEMENTATION OF BIOCONTROL,D.R. Fravel 337

EXPLOITING CHINKS IN THE PLANT’SARMOR:EVOLUTION AND EMERGENCE

OF GEMINIVIRUSES,Maria R. Rojas, Charles Hagen, William J. Lucas,

and Robert L. Gilbertson 361

MOLECULAR INTERACTIONS BETWEEN TOMATO AND THE LEAF MOLD

PATHOGEN CLADOSPORIUM FULVUM,Susana Rivas

and Colwyn M. Thomas 395

REGULATION OF SECONDARY METABOLISM IN FILAMENTOUS FUNGI,

Jae-Hyuk Yu and Nancy Keller 437

TOSPOVIRUS-THRIPS INTERACTIONS,Anna E. Whitﬁeld, Diane E. Ullman,

and Thomas L. German 459

HEMIPTERANS AS PLANT PATHOGENS,Isgouhi Kaloshian

and Linda L. Walling 491

RNA SILENCING IN PRODUCTIVE VIRUS INFECTIONS,Robin MacDiarmid 523

SIGNAL CROSSTALK AND INDUCED RESISTANCE:STRADDLING THE LINE

BETWEEN COST AND BENEFIT,Richard M. Bostock 545

GENETICS OF PLANT VIRUS RESISTANCE,Byoung-Cheorl Kang, Inhwa Yeam,

and Molly M. Jahn 581

BIOLOGY OF PLANT RHABDOVIRUSES,Andrew O. Jackson, Ralf G. Dietzgen,

Michael M. Goodin, Jennifer N. Bragg, and Min Deng 623

INDEX

Subject Index 661

ERRATA

An online log of corrections to Annual Review of Phytopathology chapters

may be found at http://phyto.annualreviews.org/

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Understanding Citrus Viroid Interactions: Experience and Prospects

Article

Full-text available

Apr 2024

Citrus is the natural host of at least eight viroid species, providing a natural platform for studying interactions among viroids. The latter manifests as antagonistic or synergistic phenomena. The antagonistic effect among citrus viroids intuitively leads to reduced symptoms caused by citrus viroids, while the synergistic effect leads to an increase in symptom severity. The interaction phenomenon is complex and interesting, and a deep understanding of the underlying mechanisms induced during this viroid interaction is of great significance for the prevention and control of viroid diseases. This paper summarizes the research progress of citrus viroids in recent years, focusing on the interaction phenomenon and analyzing their interaction mechanisms. It points out the core role of the host RNA silencing mechanism and viroid-derived siRNA (vd-siRNA), and provides suggestions for future research directions.

Hop stunt viroid infection alters host heterochromatin

Preprint

Full-text available

Dec 2023

Viroids are pathogenic non-coding RNAs that completely rely on their host molecular machinery to accomplish their life cycle. Several interactions between viroids and their host molecular machinery have been identified, including an interference with epigenetic mechanisms such as DNA methylation. Despite this, whether viroids influence changes in other epigenetic marks such as histone modifications remained unknown. Epigenetic regulation is particularly important during pathogenesis processes because it might be a key regulator of the dynamism of the defense response. Here we have analyzed the changes taking place in Cucumis sativus facultative and constitutive heterochromatin during hop stunt viroid (HSVd) infection using chromatin immunoprecipitation (ChIP) of the two main heterochromatic marks: H3K9me2 and H3K27me3. We find that HSVd infection is associated with changes in both H3K27me3 and H3K9me2, with a tendency to decrease the levels of repressive epigenetic marks through infection progression. These epigenetic changes are connected to the transcriptional regulation of their expected targets, genes and transposable elements. Indeed, several genes related to the defense response are targets of both epigenetic marks. Our results highlight another host regulatory mechanism affected by viroid infection, providing further information about the complexity of the multiple layers of interactions between pathogens/viroids and hosts/plants.

The Secondary Structure of Potato Spindle Tuber Viroid Determines Its Infectivity in Nicotiana benthamiana

Article

Full-text available

Nov 2023

The function of RNAs is determined by their structure. However, studying the relationship between RNA structure and function often requires altering RNA sequences to modify the structures, which leads to the neglect of the importance of RNA sequences themselves. In our research, we utilized potato spindle tuber viroid (PSTVd), a circular-form non-coding infectious RNA, as a model with which to investigate the role of a specific rod-like structure in RNA function. By generating linear RNA transcripts with different start sites, we established 12 PSTVd forms with different secondary structures while maintaining the same sequence. The RNA secondary structures were predicted using the mfold tool and validated through native PAGE gel electrophoresis after in vitro RNA folding. Analysis using plant infection assays revealed that the formation of a correct rod-like structure is crucial for the successful infection of PSTVd. Interestingly, the inability of PSTVd forms with non-rod-like structures to infect plants could be partially compensated by increasing the amount of linear viroid RNA transcripts, suggesting the existence of additional RNA secondary structures, such as the correct rod-like structure, alongside the dominant structure in the RNA inoculum of these forms. Our study demonstrates the critical role of RNA secondary structures in determining the function of infectious RNAs.

Xanthohumol and echinocystic acid induces PSTVd tolerance in tomato

Article

Full-text available

Jun 2024

Tomato is a popular vegetable worldwide; its production is highly threatened by infection with the potato spindle tuber viroid (PSTVd). We obtained the full‐length genome sequence of previously conserved PSTVd and inoculated it on four genotypes of semi‐cultivated tomatoes selected from a local tomato germplasm resource. SC‐5, which is a PSTVd‐resistant genotype, and SC‐96, which is a PSTVd‐sensitive genotype, were identified by detecting the fruit yield, plant growth, biomass accumulation, physiological indices, and PSTVd genome titer after PSTVd inoculation. A non‐target metabolomics study was conducted on PSTVd‐infected and control SC‐5 to identify potential anti‐PSTVd metabolites. The platform of liquid chromatography‐mass spectrometry detected 158 or 123 differential regulated metabolites in modes of positive ion or negative ion. Principal component analysis revealed a clear separation of the global metabolite profile between PSTVd‐infected leaves and control regardless of the detection mode. The potential anti‐PSTVd compounds, xanthohumol, oxalicine B, indole‐3‐carbinol, and rosmarinic acid were significantly upregulated in positive ion mode, whereas echinocystic acid, chlorogenic acid, and 5‐acetylsalicylic acid were upregulated in negative ion mode. Xanthohumol and echinocystic acid were detected as the most upregulated metabolites and were exogenously applied on PSTVd‐diseased SC‐96 seedlings. Both xanthohumol and echinocystic acid had instant and long‐term inhibition effect on PSTVd titer. The highest reduction of disease symptom was induced by 2.6 mg/L of xanthohumol and 2.0 mg/L of echinocystic acid after 10 days of leaf spraying, respectively. A superior effect was seen on echinocystic acid than on xanthohumol. Our study provides a statistical basis for breeding anti‐viroid tomato genotypes and creating plant‐originating chemical preparations to prevent viroid disease.

Symptoms and disease virulence assessment in commercial pepper cultivars caused by Pepper chat fruit viroid

Article

May 2024

The symptoms caused by viroids differ, ranging from asymptomatic to mild-or-severe symptoms. Pepper plant symptoms caused by the Pepper chat fruit viroid (PCFVd) are mild compared to those affecting tomato plants; however, there is not much more known of the symptomatology on pepper plants. Symptoms that could be used for disease virulence assessment in pepper plants were elucidated from 31 commercial pepper cultivars belonging to Capsicum annuum and C. frutescens that had been purchased from agricultural shops. The plants were mechanically sap-inoculated at the seedling growth stage and observed weekly for symptom development, with disease virulence evaluations performed at 4, 8, and 12 weeks post inoculation. Infection of all plants was verified based on reverse transcription-polymerase chain reaction, along with visual evidence of growth reduction, including leaf rugosity and leaf size reduction, a narrow canopy of the blocky and elongated fruit shapes for C. annuum and explicit apical stunting with small apical leaves of the elongated fruit type for C. frutescens. The disease virulence assessment was designed based on these symptoms to produce a score with 0–10 disease virulence levels (DVLs). The results showed that the pepper cultivars displayed responses to PCFVd with DVL scores of 1.00–8.00, with no PCFVd transmission being recorded from seeds to seedlings for the 3 test cultivars. This finding indicated that the genetic resources of pepper cultivars against PCFVd were as low as 1.00 DVL. However, the low DVL pepper cultivars could provide an inoculum source to other susceptible plants via mechanical transmission.

Molecular, genetic, and morphological interactions of viruses, viroids, bacteria, insects, and nematodes on pepper

Chapter

Jan 2024

Viroid-induced RNA silencing and its secondary effect on the host transcriptome

Chapter

Jan 2024

Molecular detection of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in Palestine

Article

Full-text available

Jan 2024

Citrus hosts various phytopathogens that have impacted productivity, including viroids. Missing data on the status of viroids in citrus in Palestine were not reported. This study was aimed to detect any of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in the Palestinian National Agricultural Research Center (NARC) germplasm collection Field inspections found symptoms such as leaf epinasty; vein discoloration, and bark cracking on various citrus varieties. RT-PCR revealed a significant prevalence of CVd-IV; CEVd and CVd-III (47%, 31%, and 22%; respectively). CVd-III variants with 91.3% nucleic acid sequence homology have been reported. The sequence of each viroid were deposited in GenBank as (OP925746 for CEVd, OP902248 and OP902249 for CVd-III-PS-1 and -PS-2 isolates, and OP902247 for CVd-IV). This was the first to report three of citrus viroids in Palestine, appealing to apply of phytosanitary measures to disseminate healthy propagating materials free from viroids.

Challenges to Cannabis sativa Production from Pathogens and Microbes—The Role of Molecular Diagnostics and Bioinformatics

Article

Full-text available

Dec 2023
INT J MOL SCI

The increased cultivation of Cannabis sativa L. in North America, represented by high Δ9-tetrahydrocannabinol-containing (high-THC) cannabis genotypes and low-THC-containing hemp genotypes, has been impacted by an increasing number of plant pathogens. These include fungi which destroy roots, stems, and leaves, in some cases causing a build-up of populations and mycotoxins in the inflorescences that can negatively impact quality. Viroids and viruses have also increased in prevalence and severity and can reduce plant growth and product quality. Rapid diagnosis of the occurrence and spread of these pathogens is critical. Techniques in the area of molecular diagnostics have been applied to study these pathogens in both cannabis and hemp. These include polymerase chain reaction (PCR)-based technologies, including RT-PCR, multiplex RT-PCR, RT-qPCR, and ddPCR, as well as whole-genome sequencing (NGS) and bioinformatics. In this study, examples of how these technologies have enhanced the rapidity and sensitivity of pathogen diagnosis on cannabis and hemp will be illustrated. These molecular tools have also enabled studies on the diversity and origins of specific pathogens, specifically viruses and viroids, and these will be illustrated. Comparative studies on the genomics and metabolomics of healthy and diseased plants are urgently needed to provide insight into their impact on the quality and composition of cannabis and hemp-derived products. Management of these pathogens will require monitoring of their spread and survival using the appropriate technologies to allow accurate detection, followed by appropriate implementation of disease control measures.

Symptomology, prevalence, and impact of Hop latent viroid on greenhouse-grown cannabis ( Cannabis sativa L.) plants in Canada

Article

Nov 2023

Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV)

Article

Sep 1985

Sequence analysis by primer‐extension at the level of their cDNA showed that the RNA genomes of various field isolates of potato spindle tuber viroid (PSTV) of different virulence differ from each other only in a few nucleotides in two distinct regions of the rod‐shaped molecule. Despite insertions and deletions the chain length of 359 nucleotides is strictly conserved in all the isolates studied. Thermodynamic calculations revealed that due to the observed sequence differences the region located at the left hand part of the rod‐like secondary structure of the PSTV molecule, denoted ‘virulence modulating (VM) region’, becomes increasingly unstable with the increasing virulence of the corresponding isolate. Based on these data we propose in molecular terms a model for the mechanism of viroid pathogenicity. It implies that the nucleotides of the VM region specify and modulate the binding‐ and hence the competition‐potential of the PSTV RNA molecule for a still unknown host factor(s) and thus determine the virulence of PSTV.

Concentration and distribution of mild and severe strains of potato spindle tuber viroid in cross-protected tomato plants

Article

Jan 1988
PHYTOPATHOLOGY

Viroidae

Article

Jan 2005

An Infectious and Replicating RNA of Low Molecular Weight: The Agent of the Exocortis Disease of Citrus

Article

Dec 1972
Adv Biosci

Heinz Ludwig Sänger

The transmissible agent of the exocortis disease of citrus (ExC) exhibits properties of an unusual free infectious RNA. It can be isolated from its herbaceous host Gynura aurantiaca DC by any method designed to extract and to concentrate undegraded host or viral RNA. The infectivity of ExC is found in nuclei and in association with the chromatin of the host cell. The agent of ExC is insensitive to different organic solvents, to proteolytic enzymes, and to DNase, but it is inactivated by RNase and by formaldehyde. In rate sedimentation experiments, it sedimented with about 6 to 8 s. Electrophoretic analysis in appropriate polyacrylamide gels revealed that infectivity of ExC is consistently associated with a monodisperse fraction of RNA with a molecular weight of ca. 50–60.000 daltons which would comprise molecules with a chain length of ca. 150–200 nucleotides.

Induced tolerance to mal secco disease in Etrog citron and Rangpur lime by infection with the citrus exocortis viroid

Article

Jan 1995
PLANT DIS

Avocado Sunblotch

Chapter

Jan 1987

Paul R. Desjardins

The sunblotch disease of avocado has been known for over 50 years. It was first described as a physiological (Coit, 1928) or genetic (Home, 1929) disorder. Shortly thereafter, Horne and Parker (1931) transmitted the causal agent from diseased scions to healthy rootstocks. By 1941, graft transmissibility of the agent was well established (Horne et al., 1941). Hence, the causal agent was considered to be a virus, and like some of the other plant viroids was studied as such for many years (Whitsell, 1952; Wallace, 1958; Wallace and Drake, 1962; Desjardins et al., 1980).

Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato

Article

Dec 1998

Winfried Schiebel

A 3600-bp RNA-directed RNA polymerase (RdRP)–specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)–, and poly(U)–Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.

Induced Tolerance to Mal Secco Disease in Etrog Citron and Rangpur Lime by Infection with the Citrus Exocortis Viroid

Article

Jan 1995
PLANT DIS

Z. Sole

Etrog citron and Rangpur lime plants were each inoculated with one of four isolates of the citrus exocortis viroid (CEVd) and incubated for 1 yr, during which time the viroid spread systemically. CEVd caused severe leaf curling and short internodes in Etrog citron but not in Rangpur lime. After leaf inoculation with Phoma tracheiphila, typical mal secco disease symptoms developed on leaves of both cultivars, regardless of prior infection by CEVd, but growth of the mycelium from the infected leaves into the branches was greatly affected by CEVd infection. In Etrog citron, P. tracheiphila was isolated from only 9.1% of the branches sampled from CEVd-infected plants, compared with 100% from CEVd-free plants. In Rangpur lime, the incidence of branches containing P. tracheiphila varied in CEVd-infected plants from 16.7 to 50% among CEVd isolates, compared with 70% in the viroid-free controls. This is believed to be the first report of systemic resistance induced in a woody plant by prior inoculation with a viroid

Potato spindle tuber “virus”

Article

Aug 1971

Theodor O Diener

Earlier results had indicated that the spindle tuber disease of potato is incited by free RNA, and that neither conventional virions nor proteins that could be construed as viral coat proteins are synthesized in infected plants. By a combination of density-gradient centrifugation and polyacrylamide gel electrophoresis, using internal marker RNAs, it is now shown that the infectious RNA occurs in the form of several species with molecular weights ranging from 2.5 × 104 to 1.1 × 105 daltons. No evidence for the presence in uninoculated plants of a latent helper virus was found. Thus, potato spindle tuber “virus” RNA, which is too small to contain the genetic information necessary for self-replication, must rely for its replication mainly on biosynthetic systems already operative in the uninoculated plant. Several possible mechanisms are discussed. The term “viroid” is proposed to designate potato spindle tuber “virus” RNA and other RNAs with similar properties.

RNA silencing in plants

Article

Jan 2004

D. Baulcombe

In the quest for plant regulatory sequences capable of driving nematode-triggered effector gene expression in feeding structures, we show that promoter tagging is a valuable tool, A large collection of transgenic Arabidopsis plants was generated, They were transformed with a beta-glucuronidase gene functioning as a promoter tag, Three T-DNA constructs, pGV1047, p Delta gusBin19, and pMOG553, were used. Early responses to nematode invasion were of primary interest. Six lines exhibiting beta-glucuronidase activity in syncytia induced by the beet cyst nematode were studied. Reporter gene activation was also identified in galls induced by root knot and ectoparasitic nematodes. Time-course studies revealed that all six tags were differentially activated during the development of the feeding structure. T-DNA-flanking regions responsible for the observed responses after nematode infection were isolated and characterized for promoter activity.

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