Virus Genes DOI 10.1007/s11262-014-1110-8

What has been happening with viroids? Peter Palukaitis

Received: 8 April 2014 / Accepted: 18 August 2014 Ó Springer Science+Business Media New York 2014

Abstract Viroids are naked nucleic acids that do not code for any proteins and yet are able to be replicated, processed, moved cell-to-cell and systemically through their host plants, as well as resist plant defense response and be transmitted from plant to plant, without a protective coat. All of the information specifying these functions lies within their nucleotide sequence and the RNA structures they form. This review examines what information about these processes has been acquired since 2008. Sequences involved in viroid replication and movement within the plant have been identified, in particular for the nuclearassociated (Pospiviroidae) viroids, as have sequences of one chloroplast-associated viroid (Avsunviroidae) involved in chloroplast uptake. The enzymes involved in ligation of viroids of either of the above two types also have been identified. Viroid sequences that are involved in pathogenicity through the RNA silencing system and the target of their viroid-specific small RNAs also have been identified. Effects of viroid infection on plant gene expression have been assessed for several viroids, and further specific interactions between viroids and host proteins have been identified. The variation in sequence of natural or passaged populations of viroids in various host species has been examined, and the effects of the host have been evaluated. New approaches to obtaining resistance to viroid infection have been examined or implemented, as have combinations of approaches to control viroid infection, and to better understand how viroids are transmitted. Finally, new viroids have also been discovered and characterized.

P. Palukaitis (&) Department of Horticultural Sciences, Seoul Women’s University, Seoul 139-774, Republic of Korea e-mail: [email protected]

Keywords Viroid replication  Viroid movement  Viroid pathogenicity  Viroid transmission  Viroid sequence variation  Viroid resistance

Introduction Viroids are still the smallest pathogen known, consisting of naked, single-stranded, closed circular RNAs, with a high degree of secondary structure, which do not code for any known proteins or peptides [1, 2]. Therefore, their sequence and structure must contain all of the information required for their replication, processing, various movements (to and from their sites of replication within a cell, between cells and systemically throughout the entire plant), as well as their ability to resist defense mechanisms and to induce pathogenic responses in some host plant species. Viroids are still only known from higher plant species, although small circular RNAs have now been detected in archaea [3] as well as various animals [4, 5]. These other circular RNAs can be formed from spliced introns or spliced exons [3–5] and in the latter case, they appear to be molecular sponges of microRNAs (miRNAs) [4, 5]. However, these molecules do not have the secondary structures characteristic of viroids, and as far as is known, they are not infectious. I have chosen a 6-year period to examine since the last comprehensive review was published in 2009 [2] and may not have included some work published in 2008. There were 652 publications listed for the word ‘‘viroid’’ in the Web of Knowledge database from 2008 through 2013, compared with the previous 6 years, for which there were 544 publications. Of these, 190 publications dealt with one or more of the following topics concerning viroid biology, effects on the host, or control. Thus, this review can provide only a snapshot of progress in viroid research.

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Replication It is difficult to separate effects on viroid replication from other related aspects such as nuclear import, processing of viroid multimers to monomers and ligation to circles. Nevertheless, a mutagenesis survey of the 2 loops and 25 bulges in the potato spindle tuber viroid (PSTVd) (Fig. 1) showed that alteration in all but bulges 7 and 14 resulted in reduced accumulation of PSTVd in protoplasts [6]. The greatest reduction in accumulation (90–95 %) was caused by mutation of bulges 13 and 15 (the latter is also called loop E) in the central-conserved region (CCR), followed by alteration of loop 1 and bulges 2–4 in the left terminal domain (LTD; 82–86 % reduction), bulges 11, 12, and 16 in the CCR (70–79 % reduction), bulges 5 and 6 in the LTD (65–70 % reduction), and bulges 8–10 in the pathogenic domain (64–75 % reduction), with the least reduction caused by mutation of bulges 18–21 in the variable domain (20–52 % reduction) and bulge 17 of the pathogenic domain (48 % reduction), followed by bulge 22 in the variable domain (58 % reduction) and bulges 23–26 and loop 27 in the right terminal domain (RTD; 45–68 % reduction) [6]. The 70-nt LTD of PSTVd, encompassing a linearized loop 1 and bulges 2–5, also was shown to bind to tomato DNA-dependent RNA polymerase (DdRp) II in vitro, while elimination of any of the bulges in this region inhibited the interaction [7]. The basepaired regions of the 70-nt LTD were required, but the basepaired sequences in these domains could be reversed between upper and lower strands, while the bulge sequences could not be interchanged between upper and lower strands [7]. In the case of citrus exocortis viroid (CEVd), a variant population generated in vivo in Amblycarpa mandarin protoplasts included three mutants that enhanced CEVd accumulation either slightly (A265G, in loop E) or highly (at 62A?, in a polypurine tract within the pathogenic domain, equivalent to PSTVd bulge 9, and at U278A, in the CCR at a position equivalent to PSTVd bulge 14, respectively) [8]. Thus, mutation at the position equivalent to bulge 14 of PSTVd enhanced accumulation of both PSTVd and CEVd, while mutation in the sequences corresponding to PSTVd bulge 15 (loop E) had opposite effects in PSTVd and CEVd [6, 8]. Although viroids have not been found in other kingdoms of the domain Eukaryota, avocado sunblotch viroid (ASBVd) has been shown to be able to replicate in yeast cells, where either (?) RNA or (-) ASBVd multimeric RNA transcripts were expressed from a yeast plasmid [9]. The nature of the polymerase responsible for replication of the viroid sequences was not identified, but it was suggested that the nuclear RNA polymerase II might be responsible [9]. However, as ASBVd, and other viroids in the family Avsunviroidae, replicate in chloroplasts [10–12], which

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have prokaryotic-like polymerases, and Escherichia coli RNA polymerase has been shown to have some copying activity for peach latent mosaic viroid (PLMVd) [13, 14], also a member of the Avsunviroidae, it would seem more likely that a prokaryote-like RNA polymerase from the mitochondria would be the enzyme responsible for replication of ASBVd in yeast. In addition to the multimeric RNAs generated in the yeast cells, monomer linear and circular (?) ASBVd forms, as well as circular (-) ASBVd forms, also were generated [9]. The study could not discriminate between whether the ligation in yeast was due to the ribozymes of ASBVd, or a host RNA ligase. However, recently, a chloroplast isoform of the tRNA ligase from eggplant was shown to ligate self-cleaved, monomeric linear (?) and (-) forms of the four viroids in the family Avsunviroidae, while silencing of the Nicotiana benthamiana chloroplast tRNA ligase reduced the circularization of transiently expressed dimeric (?) RNAs of eggplant latent viroid (ELVd) [15]. Thus, while monomer linear viroids in the family Avsunviroidae may be able to selfligate to form circular molecules in vitro and in vivo, the process is enhanced by chloroplast tRNA ligase. By contrast, viroids such as PSTVd and CEVd, in the family Pospiviroidae, could be circularized by tomato DNA ligase 1, which had been subverted to use an RNA template and the linear, monomer viroid RNAs, but only those which had been linearized from multimers at specific sequences (G95 and G96 of PSTVd) in the stem between bulges 14 and 15 (the latter being the so-called loop E) [16]. Moreover, reducing the expression of DNA ligase 1 in N. benthamiana led to a reduction in the ratio of circular to total PSTVd generated from expressed multimeric RNAs, indicating that DNA ligase 1 is a major, if not the sole ligase involved in circularizing the linear viroid molecules [16]. The template specificity of tomato DNA ligase 1 also required RNA with 50 monophosphate and 30 hydroxyl termini [16], both of which are present in linear, monomer CEVd molecules generated in vivo from multimers [17].

Movement A mutational map of the loops and bulges in PSTVd (Fig. 1) showed that mutations in bulges 6, 7, 10, 12, 17–20, and 24–26 prevented systemic infection, while mutations in bulges 5, 8, 9, 14, 16, and 21–23, as well as loop 27 all led to systemic infection of only a low number of plants, with either reversion of the mutation or additional mutation occurring in the progeny viroid [6]. At least 20 % replication efficiency was required to detect systemic infection, and thus the effects of mutations that also reduced replication by more than 80 % could not be unambiguously evaluated for effects on systemic infection.

Virus Genes HPI

1 R

R

R

R

1

2

3

4

5

T

T

6

7

T

8

9

10

11

T

R

12

13

HPII

359

LTD

HPI

90 R

R

14

15

16

T

17

18

T

T

19

20

21

22 23

T

T

24

25

T

26

CCR

Variable

27

180

HPII

270

Pathogenic

T

RTD

Fig. 1 Map of potato spindle tuber viroid secondary structure showing structural motifs, as well as loops (L1 and L27) and bulges (L2–L26) essential for replication (R) or trafficking (T). The structural motifs indicated are the left terminal domain (LTD), the pathogenic

domain, the central conserved region (CCR), the variable domain, and the right terminal domain (RTD). In addition, sequences that basepair to form metastable hairpin structures I and II (HPI and HPII) are indicated

These included mutations in loop 1 and bulges 2–4, 13, and 15. Therefore, excepting this last group and bulge 11, which reduced replication to *21 %, all of the other bulges and the loop in the RTD affected systemic infection [6]. Bulge 7 had previously been shown specially to affect the movement of PSTVd from bundle sheath cells into phloem cells in N. benthamiana, which is required for systemic infection [18], and a bipartite motif requiring U201 and U309 plus U47/A313 affected the movement of PSTVd in Nicotiana tabacum from bundle sheath to mesophyll cells [19], while bulge 6 contains a novel 3-D structure that is involved in the movement of PSTVd from palisade mesophyll to spongy mesophyll cells in N. benthamiana [20]. In the case of CEVd, mutation at U129A (at the end of a polypurine tract within the variable domain, corresponding to PSTVd bulge 19) enhanced systemic accumulation in seedlings of ‘Etrog’ citron, but did not significantly affect viroid replication, suggesting that this mutation also affected the efficiency of systemic movement [8]. The PSTVd sequences involved in movement of the viroid into the nucleus were delimited using as assay involving potato virus X (PVX) expressing the green fluorescent protein (GFP), showing that the sequences encompassing hairpin I in the upper strand (nucleotides 78–129) were sufficient to promote entry into the nucleus (Fig. 1), as were nucleotides 1–87 and 93–100 [21]. The formation of hairpin I was not essential for the transport of the viroid, which may indicate that more than one sequence in the upper strand can function in nuclear trafficking [21]. When a similar PVX–GFP assay was done using ELVd to delimit sequences involved in movement of this viroid between the cytoplasm and the nucleus, ELVd was shown to transport fused RNAs into the nucleus, and the stem– loop structure encompassing the left half of the ELVd molecule (nucleotides 15–181) was sufficient to carry out this transport function [22]. In addition, ELVd sequences involved in transport into the chloroplast were delimited by using a transient expression vector with viroid sequences as

a 50 untranslated region upstream of a GFP gene [22–24]. These experiments showed that the entire ELVd molecule also could facilitate the transport of ELVd fused to GFP mRNA into the chloroplast for translation there and that nucleotides 52–150, also forming stems and the two loops in the left half of the viroid molecule, were sufficient to promote chloroplast uptake of RNA, at least partially [22– 24]. Thus, the authors have suggested that ELVd, and other viroids in the family Avsunviroidae, upon entry into the cell, would be transported first to the nucleus, and then utilizing a nucleus–chloroplast transport mechanisms, would enter the chloroplast for replication [22]. Subsequently, it is possible that the replicated viroid molecules use the retrograde signaling transduction pathway between chloroplasts and the nucleus [25] to re-enter the nucleus and from there to the cytoplasm for subsequent cell-to-cell movement.

Pathogenicity The main focus in research on pathogenicity has been on identifying sequences of viroids that affect pathogenicity, and assessing the role of RNA silencing in viroid pathogenicity. In the former case, several sequences specifying pathogenicity of various viroids have been described: the single-nucleotide alteration in hop stunt viroid (HSVd) responsible for the cachexia disease of citrus [26]; two adjacent nucleotide alterations in CEVd affecting pathogenicity in a host-specific manner [27]; sequences responsible for either an albinism called peach calico [28] or yellowing and chlorosis-edge [29] caused by different isolates of PLMVd; and a single-nucleotide change in coconut cadang–cadang viroid correlating with an orange spot disease in oil palm [30]. With regard to RNA silencing and the role of viroid-specific small interfering RNAs (vdsiRNAs) in targeting host mRNAs resulting in disease phenotypes, until recently there was no unequivocal evidence for this model. One study showed that RNA-

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dependent RNA polymerase 6 (RDR6) from N. benthamiana was involved in symptom development by HSVd in this host [31], suggesting a role for RNA silencing in viroid pathogenicity, while another study showed RDR6 was essential for movement of PSTVd into the floral and vegetative meristems, but not for pathogenicity in N. benthamiana [32]. Similarly, vd-siRNAs could be detected in various infected plants [32–53], and some viroids could act as substrates for Dicer-like (DCL) enzymes in vitro or in vivo [39, 54, 55]. However, subsequently it has been shown that PLMVd siRNAs can function in the cleavage of a host mRNA to incite the peach calico pathogenicity, with the target mRNA encoding the chloroplast heat-shock protein 90 [28]. Nevertheless, as those authors also have pointed out, this does not mean that all viroid-induced diseases are necessarily associated with vd-siRNAs [56]. For example, ethylene may be involved in symptom development in tomato [57]. Other studies have indicated that viroid infection can lead to induction of a serine– threonine protein kinase gene [58], overexpression of which resulted in the production of PSTVd-induced symptoms, while application of gibberellic acid (GA) reversed these effects [59]. Either overexpression or inhibition of the expression of this protein kinase affected the transcript levels of GA biosynthesis genes [59]. Similarly, a microarray and deep sequencing analysis of small RNA from PSTVd-infected tomato plants showed that pathogenicity was related to alterations in the biosynthesis of GA and brassinosteroids [60]. On the other hand, in a related study, genes involved in GA and jasmonic acid biosynthesis, which were down-regulated in PSTVd-infected tomato, contained binding sites for PSTVd small RNAs [50], while infection of cucumber by HSVd reactivated rRNA transcription as a result of the demethylation of transcriptionally silenced rRNA gene promoters [61]. In another study, HSVd infection of hops was shown to reduce the accumulation of chalcone synthase H1 (chs_H1; involved in the phenylpropanoid pathway) and also a transcription factor (HlbHLH1) required for synthesis of chs_H1, which also occurred after agroinfiltration of Galinsoga ciliata leaves with transiently expressed HSVd, but not if the tomato bushy stunt virus P19 RNA silencing suppressor also was expressed [62]. Recently, a study was published showing that transgenic expression of an artificial miRNA corresponding to the pathogenicity-modulating domain of PSTVd in N. tabacum or N. benthamiana resulted in the development of phenotypes very similar to those caused by PSTVd infection [63]. The artificial miRNA target shown to be down-regulated during PSTVd infection encodes a soluble inorganic pyrophosphatase, although this gene was not down-regulated in tomato [63], indicating that the hunt is still on. Thus, although it is still too early to draw any general conclusions, perhaps in the

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end, all roads to viroid pathogenicity may ultimately start from some aspect of RNA silencing.

Effects on gene expression Several studies have examined the effects of viroid infection on the pattern of gene expression in their hosts. These include PSTVd infection in tomato [50, 60], CEVd infection in Etrog citron [64], and PLMVd infection in peach [65]. Viroid infection obviously perturbed the gene expression of its tested hosts, with numerous genes upregulated or down-regulated in expression, although very few genes were found altered in expression of PLMVd infection of peach (16 of 4,261), compared with the numbers found altered for CEVd in Etrog citron (132 of 21,081), or especially PSTVd in tomato (5,354 of 10,000). The genes altered in expression in Etrog citron and tomato encoded protein of various functional classes, but also included genes encoding miRNAs in tomato [49, 60]. In a proteomic study involving CEVd and tomato, of 409 proteins showing significant alterations in expression, 45 proteins were identified, with some changes in expression related to transcriptional changes and others caused by post-transcriptional alterations in protein expression [66]. The first group consisted largely of a number of defense proteins (endochitinase, b-glucanase, pathogenesis-related proteins), while the second group included ribosomal proteins and translation factors [including elongation factor 1 (eEF1A) and 2, and initiation factor 5-alpha]. The various defense proteins also could be induced by gentisic acid [66], which previously had been shown to be induced by CEVd infection of tomato [67]. Overall, the various microarray, deep sequencing, and proteomic approaches provide many candidates to investigate as being involved in pathogenicity, although some additional screening will be needed to reduce the list of suspects.

Plant-viroid interactions As mentioned above, the tomato DdRp II was shown to interact in vitro with the LTD of PSTVd [7]. Other interactions that can be inferred are by a chloroplast tRNA ligase in the circularization of ELVd [15] and by a (nuclear) DNA ligase 1 in circularization of PSTVd [16]. We also can expect interactions between either viroids or their replicative forms and those enzymes that generate the various siRNAs found in deep sequencing [45, 46] or other studies [39, 54, 55], such as DCL1, 2, 3, and 4, the corresponding Argonaute proteins, DRB4, HEN1 and RDR6, the last of which affects movement of a viroid into meristematic tissues [32]. The demethylation of rRNA genes by

Virus Genes

HSVd infection suggests yet further interactions by viroid siRNAs [61]. PSTVd also was shown to interact in vitro with two Arabidopsis proteins that both bind to 5S rRNA: transcription factor IIIA, which is required for transcription of 5S rRNA, and ribosomal protein L5, which is involved in the nucleocytoplasmic transport of 5S rRNA. This suggests that these proteins may facilitate the replication and intracellular movement of nuclear-replicating viroids [68]. In addition, PSTVd has been found to interact with the Nt4/1 protein, a nuclear-cytoplasm shuttle protein, which also can move between cells, and is found primarily in the vasculature [69, 70]. Its role in PSTVd movement is not clear, although Nt-4/1 was found to be associated with living tissues of the veins and silencing the expression of its homolog in N. benthamiana enhanced PSTVd systemic movement and accumulation [70]. Six proteins were found associated with PLMVd in extracts from peach leaves [71]. These proteins were identified as a 30 kDa b-1,3-glucanase, a 45 kDa aminomethyltransferase, the 50 kDa translation factor eEF1A, a putative chaperone, a dynamin-like GTP-binding protein, and ribosomal protein L5 [71]. The interaction of eEF1A with PLMVd was studied in detail further and also was shown to occur with PSTVd [71], as well as between the tomato eEF1A and CEVd [66]; however, is not clear whether the interactions of PLMVd with the other four proteins have any biological significance.

Sequence variation Several studies have examined the sequence variation that occurs among viroid populations, whether within the same sample, various samples of the same species (locally or globally), or different species (usually after passage). These studies were done with CEVd, HSVd, PLMVd and CSVd, and to a lesser extent with other viroids. The genetic diversity of CEVd populations from graft-inoculated trifoliate orange and sour orange after 10 years of evolution were compared with the original isolate maintained in its host, Etrog citron. While the population diversity after propagation in both trifoliate orange and sour orange changed relative to that in Etrog citron, the population composition in the original Etrog citron population and inoculated trifoliate orange population contained a single predominant sequence (haplotype) making up 50–63 % of the various populations. By contrast, the population composition in sour orange did not have a single predominant haplotype, with the most frequent haplotype found at 15–20 % of the populations [72]. Passage of both CEVd orange populations back to Etrog citron showed reversion of the population composition to that seen in the original Etrog citron plant, indicating that the composition of the viroid populations are influenced by the host [72], similar to results described earlier [73].

In the case of HSVd, several analyses of population diversity have been done, including among isolates from various stone fruit trees in Turkey [74], among isolates from plum in China [75], and among isolates from hops and grapevine in Japan and China [76]. The first study indicated that the sequence variability was more associated with the geographical origin of the isolates than the nature of the hosts [74]. The second study identified a novel variant in one sample with five clones contained a 15-nt insert in the lower central region, between nucleotides 244 and 245. After inoculation of the original extract to cucumber, only variants that did not contain the 15-nt insertion could be detected [75]. This supports the conclusion that the host heavily influences the population structure. The third analysis of HSVd from hop plants in Japan and China, and in grapevines from Japan, identified hot spots for sequence variation that were common and unique due to host or geographic location [76]. This study also examined the sequence variation after a 15 year passage experiment in hops of HSVd from different source plants (citrus, plum, grapevines, and hops). The authors concluded that there was convergent evolution of HSVd isolates during passage, especially for hop and grapevine isolates. Furthermore, HSVd RNA transcribed from a cDNA clone of HSVd-grapevine and passaged for 10 years in hops showed that the sequence variants were generated de novo and were not minor variants that were selected for from a pre-existing population [76]. A separate study of viroids infecting grapevines in China and Japan also concluded that HSVd sequence variation showed viroid species dependency in both countries [77]. Grapevines in China infected by grapevine yellow speckle viroid 2 (GYSVd-2) showed some variation in population structure depending on the grapevine cultivar, with the only cultivar containing a major haplotype of more than 50 % of the population represented by plant predicted to be over 150 years old and containing a viroid population less diverse, but with greater sequence variation from the other populations [78]. Analysis of sequence variation in six pathogenic isolates of PLMVd from China showed that each isolate contained a major haplotype, occurring as 32–57 % of the population of that isolate, and non-predominant haplotypes, with one to four nucleotide sequence differences from the predominant haplotype, each occurring as 4–9 % of the population. The predominant haplotypes varied in sequence from each other by 0.6–3.6 %, and from the consensus sequence by 0–1.2 % [79]. An analysis of the sequences of clones from 11 isolates of PLMVd from peach and nectarine trees in Turkey showed sequence identities from 84–100 % with each other and sequence identities to other known isolates of 96–99 % [80]. The most variable (peach) isolate was only 2 % different in sequence from one described

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previously from peach in Tunisia [81]. A comparison of nine Iranian isolates and four Australian isolates of PLMVd showed 94–99 % sequence identity with other reported PLMVd isolates. The Iranian isolates were more variable from each other than the Australian isolates [82]. Inoculation of an infectious DNA of one Australian isolate to peach seedlings showed the rapid accumulation of mutations in the PLMVd population, with the appearance of ten mutations in the progeny viroids from each of two plants within 30 days, including different mutations at six common positions [82]. A different study, using two primer sets and deep sequencing via the 454 pyrosequencing system to examine variation in sequence of PLMVd 6 months after inoculation to a peach tree, showed sequence variation encompassing up to 17 % of the genome, which was detected with 3,939 unique reads among 291,959 total corrected/adjusted reads [83]. Up to 50 % of the genome remained completely conserved, with most sequences containing 4.6–6.4 mutations relative to the starting inoculum. The evolution of the changes that occurred could be discerned using appropriate algorithms [83], indicating the power of this approach. Sequence variation of citrus dwarfing viroid was analyzed in field populations and after propagation in specific hosts. The former study showed that pathogenic variants were present among different isolates and these also had different levels of sequence diversity [84]. The latter study showed specific host effect on either the generation of numerous variants or the selection of specific haplotypes, even to the exclusion of all others [85]. Cloned CSVd also showed host-specific changes in sequence after inoculation to several symptomless hosts, including tomato and subsequently potato [86]. Similarly, PSTVd isolated from ornamental plants showed increased diversity after inoculation to potato [87]. An analysis of pear blister canker viroid sequences indicated that about 31 % of the genome showed sequence variation which was influenced by both geographical origin and host [88]. Analysis of global sequence in CSVd showed that variation occurred in 29 % of the genome among isolates and clones, that sequence variation within a country was as great as sequence variation between countries, and that some isolates showed little or no variation in population composition, while others showed as much variation in composition as between countries [89]. Inoculation of RNA transcribed from dimeric cDNA clones of coleus blumei viroid 1 (CbVd-1) -3, -5 and -6, each to five C. blumei plants derived from mother plants varying in leaf shape and color, resulted in differences in the timing and incidences of infection, as well as sequence variation among the four different coleus viroids [90]. CbVd-5 infected all five variant plants between 45 and 65 days post-inoculation (dpi), while CbVd-1 infected all

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five variant plants between 60 and 80 dpi. CbVd-6 infected only two of the five variant plants at 80 or 110 dpi, and CbVd-3 infected only one plant at 300 dpi. Sequence analyses of the viroids in the variant plant type infected by all four coleus viroids showed differences in the accumulation of the sequence variants, based on sequencing 20 cDNA clones generated from the viroids from each plant: 95 % of the CbVd-5 population was the inoculum haplotype, with 5 % represented by a singleton with a singlenucleotide change; 80 % of the CbVd-1 population was the inoculum haplotype with four singletons showing one-tothree nucleotide differences from the inoculum haplotype; 60 % of the CbVd-6 population consisted of the inoculum haplotype, with 10 % (2 clones) representing a variant with one nucleotide change, and six singletons each with one nucleotide difference from the dominant haplotype; and none of the CbVd-3 haplotypes was representative of the inoculum sequence, but the dominant haplotype, with one nucleotide difference from the inoculum, represented 80 % of the CbVd-3 population, with four singletons showing two-to-three nucleotide differences from the inoculum haplotype. The observations with CbVd-3 suggested that adaptation was required for this viroid to infect coleus varieties in China, as it was rarely detected in China [90]. Sequence variation in infectious RNA populations is determined by the mutation rate and the selection efficiency of various haplotypes of a population. The above results showed that various factors can influence the selection efficiency for particular haplotypes within populations. Since CSVd has much less genetic variability than CChMVd, in general, viroids in the family Pospiviroidae may have much lower mutation rates than viroids in the family Avsunviroidae [91]. While the mutation rates for any members of the family Pospiviroidae are not known, the mutation rate for one member of the family Avsunviroidae, CChMVd, was determined to be one in 400 nt, the highest for any biological organism [92].

Resistance and control Resistance research to viroids consists largely of screening germplasm for resistance, developing pathogen-derived resistance, obtaining stock plants free of viroid infection via tissue culture methods, and disinfection of tools. There is limited or no natural resistance to viroids in the major cultivated crops that are infected by viroids. In the case of potato, some resistance has been identified in Solanum acaule, Solanum guerreroense, and Solanum berthaultii [93] and resistance has also been identified in Solanum etuberosum, Solanum sucrense and Solanum chacoense, with Solanum stoloniferum exhibiting tolerance [94]. However, none has yet been transferred to commercial

Virus Genes

potato varieties. In the case of chrysanthemums, one study identified a cultivar of chrysanthemum which showed a reduced rate of CSVd accumulation. Self-pollination of this cultivar (Utage) and grafting to an infection source identified three plants with very low CSVd accumulation [95]. A different screening program identified one resistant cultivar (Okayamaheiwa) showing strong resistance to CSVd accumulation, and crossing this cultivar with two susceptible cultivars showed resistance in either one of eight or 13 of 76 progeny plants, indicating that the resistance was heritable [96]. A third screening program identified two resistant chrysanthemum cultivars from 85 cultivars examined, with one cultivar (Sei no Issei) showing decreased graft-inoculum levels with time and the other cultivar (Mari Kazaguruma) showing a very low CSVd accumulation titer [97]. Pathogen-derived protection against PSTVd, CEVd, and CChMVd was achieved to varying extents in tomato, gynura and chrysanthemum plants using either mechanical coinoculation or co-agroinfiltration. The former was done with the viroids and either viroid dsRNA, or small RNAs generated by RNase III cleavage of dsRNAs as the co-inoculum. The latter was done by co-infiltrating Agrobacterium harboring plasmids expressing infectious dimeric transcripts of PSTVd and hairpin dsRNAs of part the PSTVd genome with an intron loop (hpRNAs). Many or most plants became infected, but viroid accumulation was delayed and symptoms were reduced or absent [98]. Transgenic expression of PSTVd hpRNAs in tomato resulted in the accumulation of vd-siRNAs and led to excellent resistance to infection by PSTVd in some lines [99]. On the other hand, transgenic N. benthamiana expressing the same hpRNA construct from a companion cell-specific promoter showed only a delay in PSTVd accumulation in two of four lines. This delay also occurred when the transgenic plants were used as rootstocks and an inoculated scion was examined for accumulation of PSTVd [100]. Tissue culture methods have been used for some time to obtain viroid-free material [reviewed in 101]. These have involved meristem tip culture, thermotherapy (high temperature treatment), psychrotherapy (low temperature treatment), chemotherapy, or combinations of different approaches. Leaf primordial-free shoot apical meristem culture have been used for CSVd elimination [101]. Rough lemon and trifoliate orange were developed free of CEVd and HSVd by tissue micropropagation [102], while stigma/ style somatic embryogenesis was used in another study to regenerate citrus germplasm apparently free of CEVd and HSVd [103]. The use of meristem tip culture, psychrotherapy and virazole were found to eliminate HSVd from peach and pear [104], while meristem tip culture, psychrotherapy and ribavirin were found to eliminate CSVd from chrysanthemum [105].

Transmission Transmission of CSVd occurs both from the pollen and ovule to the seed, but to a much greater extent form the ovule than via the pollen, and if both parents are infected the transmission frequency is increased greatly. There is also a seasonal effect on transmission, with the transmission frequency higher in the late spring than in the summer months [106]. The incidence in coleus seeds of CbVd-1 was found to be 0–100 %, indicating that CbVd-1 is seed transmissible [107], while the incidence of seed transmission of CbVd-5 and CbVd-6 from infected plant sources was 48 and 58 %, respectively [90]. Tomato chlorotic dwarf viroid (TCDVd) was found in the ornamental plant Vinca minor, where it could overwinter. TCDVd from V. minor could be transmitted to tomato by mechanical inoculation, and was then found to be seed-borne in tomato and transmitted at a high frequency to seedlings. The viroid could not be removed by soaking the seed in sodium hypochlorite [108]. TCDVd also was found to be transmitted between tomato plants by bumble bees [109]. However, TCDVd was not detectable in parts of the placenta, the ovules or in the integuments of the embryo sac [110]. CEVd was found to be present at a high frequency in symptomless Impatiens and Verbena varieties, and was shown to be seed transmissible in Impatiens walleriana and Verbina x hybrid [111]. PSTVd found in tomato in both Italy and The Netherlands was ascribed to have been transmitted from Solanum jasminoides [112, 113], or Physalis peruviana [113]; both symptomless ornamental host species that were found to be highly infected in surveys done of those plants. No transmission of PSTVd via thrips, honey bees or bumble bees could be demonstrated from ornamental plants to tomato [114]. PSTVd also was found to be transmissible to tomato or potato during hydroponic growth, as well as by watering potato plants with water containing infected leaf extracts [115]. On the other hand, in an earlier study, the latter method did yield infection of tomato plants, although there may have been differences between the conditions used [116].

New viroids A number of new viroids also has been identified during the last 6 years. Citrus viroid V was isolated from Atalantia citroides, a species previously shown to be immune from other citrus viroids. This 293–294 nt RNA contained similar sequences in the CCR and the terminal conserved region (TCR) with other members of the genus Apscaviroid, showing 39.1–73.5 % sequence identity to these viroids [117]. The 396 nt persimmon viroid (also known as persimmon latent viroid), isolated from Japanese persimmon

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and containing less than 70 % sequence identity with any viroid, was placed into the genus Apscaviroid, based on sequence similarities of the CCR and the TCR [118]. Persimmon viroid 2 was isolated from American persimmon and contained 358 nt. This viroid showed *72 % sequence homology to citrus viroid VI (formerly citrus viroid OS) and 52 % sequence homology to persimmon latent viroid, and also was placed in the genus Apscaviroid [119]. Pepper chat fruit viroid was identified in pepper plants in The Netherlands and was shown to be seed transmissible. This 348-nt viroid is most similar to viroids in the genus Pospiviroid [120]. Chinese grapevine viroid (possibly to be designated GYSVd-3) variants of 366-nt were found in seven grapevine samples, with sequence similarities to GYSVd-1 (*88 %) and GYSVd-2 (*77 %) [121]. A 274-nt viroid designated CbVd-5 was found in coleus in China and could be transmitted to coleus. This viroid shared *66 % sequence identity with other members of the genus Coleviroid [122]. A recombinant coleviroid was found in a coleus plant previously shown to be infected with both CbVd-1 and CbVd-5. This 342-nt viroid, designated CbVd-6, contained the left half of CbVd-3 and the right half of CbVd-5, flanking the CCR [123]. The 342-nt dahlia latent viroid also appears to be a recombinant viroid, having a CCR identical to HSVd, but with the TCR found in members of the genus Pospiviroid and absent from HSVd, and lacking the terminal conserved hairpin found in HSVd [124].

Conclusions The above snapshot is not exhaustive of work done in the field of viroid research during the last 6 years, since, among other things, it does not include studies to improve detection or identification of viroids, which of course is made more difficult by the appearance of new viroids and recombinant viroids. Studies related to the epidemiology or incidence of viroid-induced diseases have not been covered. However, it is clear that there are a number of symptomless viroid hosts which may be the source of new infections, including, but not limited to ornamental plants. Nevertheless, the work described above shows that the field of viroid research is thriving and has identified new ways to assess viroid population structures and how viroids are replicated, ligated, transported within the plant and transmitted from plant to plant. As these studies are extended to other viroids, new ways of inhibiting viroid replication or movement, or preventing viroid-induced disease will follow, hopefully eventually putting all viroidologists out of a job. Acknowledgments The author thanks various colleagues for their comments and clarifications. This work was supported by a special grant from Seoul Women’s University in 2013.

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