Chapter

Viroid Elimination by Thermotherapy, Cold Therapy, Tissue Culture, In Vitro Micrografting, or Cryotherapy

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Abstract

Viroids cannot be controlled by therapeutic treatments in fields, orchards, vineyards, or palm plantations. Hence, the elimination of viroids from infected plants has been a challenging issue and different approaches have been studied to produce viroid-free planting material. This chapter describes viroid elimination by thermotherapy, cold therapy, tissue culture, in vitro micrografting, or cryotherapy to obtain viroid-free materials. Elevated temperatures do not inactivate or eliminate most viroids from their host plants, whereas cold therapy can be effective depending on the host-viroid system. Meristem culture and shoot-tip micrografting are good methods to obtain viroid-free plants: the eradication rate depends on the size of the shoot, the viroid or its variant, and the host and its variety. The combination of thermotherapy plus shoot-tip culture is more effective for viroid elimination. Cryotherapy of shoot tips, an innovative technique for pathogen eradication based on cryopreservation, did not result in viroid elimination. Further experiments on other host-viroid systems and on combination of cold therapy and cryotherapy are under evaluation.

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... Often, there are no natural resistance sources and chemical controls against these pathogens are difficult (Kokane, Lawrence, et al., 2021). Therefore, the use of pathogen-free planting materials is pivotal for efficient control of these diseases and the global exchange of genetic resources (Barba et al., 2017;Ghosh et al., 2021Ghosh et al., , 2022Laimer & Barba, 2011;Wang, Cui, et al., 2018). Often, the most straightforward method for obtaining clean plants is by germinating seeds and growing seedlings, because most pathogens are not transmitted through seeds, although there are some exceptions (Bradamante et al., 2021;Ma et al., 2021). ...
... Pathogen eradication can be quite challenging, in part because pathogen-host interactions vary across pathogen and plant types, and the presence of pathogens within plants varies among cell and tissue types (Zhang et al., 2015;Zhao et al., 2018). thermotherapy, cold therapy or chemotherapy with shoot tip culture (Barba et al., 2017;Chilukamarri et al., 2021;Laimer & Barba, 2011;Panattoni et al., 2013;Wang, Cui, et al., 2018). A combination of thermotherapy with shoot tip culture significantly improved virus eradication . ...
... A combination of thermotherapy with shoot tip culture significantly improved virus eradication . The combination of cold therapy with shoot tip culture is usually used for viroid eradication (Barba et al., 2017). In some cases, available pathogen eradication methods are insufficient, and thus new technologies are needed for the production of pathogen-free plants to improve agricultural production. ...
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Diseases caused by plant pathogens such as viruses, viroids and phytoplasmas cause huge economical losses of agricultural production and limit the safe movement of plant materials across borders. The use of pathogen‐free planting materials provides a strategy for efficient management of these diseases and facilitates the global exchange of genetic resources. Shoot tip cryotherapy is a novel biotechnology method that uses cryogenic procedures to eradicate plant pathogens from the diseased plants. Combining thermotherapy or chemotherapy with shoot tip cryotherapy has further enhanced pathogen eradication efficiency. This review provides updated and comprehensive information on shoot tip cryotherapy and the combination of thermotherapy or chemotherapy with shoot tip cryotherapy for pathogen eradication. Prospects are proposed for future studies.
... Micrografting has now been widely adopted as a technique used to eliminate viruses, viroids, and phytoplasmas from fruit trees including citrus, apple, and sweet cherry as well as ornamentals such as chrysanthemum (Barba et al. 2017;Chilukamarri et al. 2021;Jonard 1986;Laimer and Barba 2011;Monteuuis 2012). It has also been used to i) micropropagate plants including fruit trees, ornamentals, vegetables, and forest species (Chilukamarri et al. 2021;Monteuuis 2012); ii) rejuvenate mature plants, particularly perennial woody species (Monteuuis 2012); iii) improve shoot tip regeneration after cryopreservation and genetic transformation in plants that are difficult to regenerate, such as Citrus (Volk et al. 2012;Wu et al. 2019), cotton (Gossypium hirsutum) (Jin et al. 2006), and jujube (Ziziphus jujube) ; and iv) study graft compatibility (Chilukamarri et al. 2021;Goldschmidt 2014;Monteuuis 2012). ...
... Obligate pathogeninduced diseases cause significant losses in agricultural production (Hadidi and Barba 2011). On the other hand, planting pathogen-free stock reduces disease pressure and increases quality and yield (Barba et al. 2017;Laimer and Barba 2011;Wang et al. 2018a). Micrografting has been widely used for pathogen eradication for many plants, particularly woody fruit trees (Fig. 1). ...
... Viroids can usually infect meristematic cells, so eradication of viroids from infected plants has been a challenge (Barba et al. 2017). Micrografting of leaf primordium (LP)-free shoot apical meristems (LP-free SAMs) eradicated chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) from chrysanthemum (Hosokawa 2008). ...
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Micrografting, which was developed almost 50 years ago, has long been used for virus eradication, micropropagation, regeneration, rejuvenation and graft compatibility. Recently, micrografting has been used for studies of long-distance trafficking and signaling of molecules between scions and rootstocks. The graft transmissiveness of obligate plant pathogens, such as viruses, viroids and phytoplasmas, facilitated the use of micrografting to study biological indexing and pathogen transmission, pathogen-induced graft incompatibility, and screening for the pathogen resistance during the past 20 years. The present study provides comprehensive information on the latter subjects. Finally, prospects are proposed to direct further studies.
... Different approaches have been utilized over the years for viroid elimination from infected plants [114][115][116]. Recently, Barba et al. [117] described the possible viroid elimination from infected plant tissue by thermotherapy, cold therapy, tissue culture, in vitro micrografting, or cryotherapy. Among the viroids that were eliminated from infected plants by one or more of these techniques were ASSVd from apple and pear, HLVd and HSVd from hop, CSVd, CChMVd, and HSVd from chrysanthemum, HSVd from peach and pear, PSTVd from potato and tomato, PLMVd from peach, CEVd and HSVd from citrus, and CEVd and TCDVd from tomato [117]. ...
... Recently, Barba et al. [117] described the possible viroid elimination from infected plant tissue by thermotherapy, cold therapy, tissue culture, in vitro micrografting, or cryotherapy. Among the viroids that were eliminated from infected plants by one or more of these techniques were ASSVd from apple and pear, HLVd and HSVd from hop, CSVd, CChMVd, and HSVd from chrysanthemum, HSVd from peach and pear, PSTVd from potato and tomato, PLMVd from peach, CEVd and HSVd from citrus, and CEVd and TCDVd from tomato [117]. Viroid-elimination frequency varied with viroid-host combinations. ...
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... Seker et al. (2015) observed a 10% of surviving plants after cryotherapy treatments. Barba et al. (2017) also evidenced that thermo-and cold-therapy treatments do no inactivate or eliminate most viroids in fruit tree species. In this sense, taking into account the extended presence of HSVd in the cultivated apricot trees (Rubio et al., 2013) these preliminary results indicated that the assayed cold-and thermo-therapy treatments are not suitable to obtain virus and viroid free plants. ...
... Other proposal to eliminate HSVd is the combination of cold-therapy and chemotherapy successfully assayed in peach (El-Dougdoug et al., 2010). On the other hand, Barba et al. (2017) recommended the use of micro-grafting combined with cold-therapy as the most suitable method for viroid elimination in fruit trees. ...
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Chapter
The chapter deals with the history of the discovery of viruses in the late 19 century as well as the progress made over the last 120 years in advancing the science of plant virology. These include but are not limited to biology of virus-or viroid-infected plants, vector and non-vector transmission, the rise of molecular and biophysical virology, replication of RNA and DNA viruses as well as viroids, and many methods used in plant virology. These methods include serology, electron microscopy, confocal microscopy, analytical and preparative ultracentrifugation, density gradient ultracentrifugation, gel electrophoresis, hybridization. polymerase chain reaction, microarrays, genetic engineering, first and next-generation nucleotide sequencing, CRISPR-Cas system editing and several others. Finally, control of viruses and viroids by exclusion was also discussed.
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This study examined the influence of low temperature on the production of reactive oxygen species and antioxidant metabolism in two strawberry (Fragaria ananassa Duch.) cultivars (cv. Zoji and Toyonaka). Low temperature treatment was imposed by maintaining the plants at 0℃ for 2, 4, 6, 8, 12, 24, 48 and 72 h in an artificial intelligent growth chamber. During the period of low temperature treatments, the activities of peroxidative (POD), superoxide disumutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate (DHAR), glutathione reductase (GR), the production of O 2 -and H 2 O 2 , as well as the contents of antioxidant such as dehydroascorbate and reduced glutathione were up-regulated exception of the contents of ascorabte and chlorophyll in compared with the control. Meanwhile, cv. Toyonaka showed more tolerant to low temperatures than cv. Zoji since it showed higher activities of antioxidative enzymes, more contents of osmolytes (proline and soluble sugar) and less lipid peroxidation during the low temperature treatments. The present study suggested that plant had signal molecules sensing the environmental stress, following erecting its defensive system protective them against the damage caused by stress environment. However, if the stress were too powerful, plants could not response to acclimate that stress, the plants would be injured or even be dead finally. In the two cultivars examined, cv. Toyonaka has a more efficient antioxidant system against low temperature than cv. Zoji.
Article
Stunting caused by the Chrysanthemum stunt viroid (CSVd) is one of the most damaging diseases in cultivated chrysanthemum (Chrysanthemum morifolium). Infected seedlings of asymptomatic cultivars may spread CSVd to other susceptible cultivars in nurseries. Thus, breeding of a CSVd-resistant cultivar would represent an important step in developing a protection strategy against CSVd. Our objectives were to (i) find CSVd-resistant cultivars and (ii) determine whether their resistance is heritable. We screened 35 chrysanthemum lines and cultivars, wild chrysanthemum species and interspecific hybrids for resistance to CSVd. Scions of screened cultivars were inoculated with CSVd by grafting them onto CSVd-infected plant roots. Every month after grafting inoculation, we used the reverse-transcription polymerase chain reaction to search for viroids in upper leaves of the scions. Viroids were not detected in ‘Okayamaheiwa’ 210 days after inoculation with CSVd, demonstrating strong resistance in this cultivar. F1 progeny were produced by crossing this resistant cultivar with the susceptible cultivars ‘Sei-elza’ and ‘Anri’. Among the 76 and 8 F1 progeny produced by these two crosses, 13 and 1, respectively, were not infected with CSVd following inoculation. Hence, CSVd resistance was expressed in the first hybrid generation. This is the first report demonstrating that resistance is heritable in crosses between a CSVd-resistant chrysanthemum cultivar and CSVd-susceptible cultivars. Chemical and cultural approaches for control of CSVd epidemics are difficult; therefore, breeding for CSVd resistance provides a promising alternative.
Article
The disease symptoms of chrysanthemum chlorotic mottle viroid (CChMVd) that had not been observed in chrysanthemum (Dendranthema grandiflorum) plants grown in Japan were found recently. In our first experimental test to examine whether the disease symptoms, such as chlorosis of newly expanded leaves in Japanese chrysanthemum, confirmed that the disease was induced by CChMVd. The viroid was detected in cultivars with the disease symptoms by polymerase chain reaction (PCR). The nucleotide sequence of CChMVd extracted from an infected 'Piato' plant, almost completely agreed with that of the non-symptomatic CChMVd strain that was reported previously. In the second experiment from cultivars randomly collected from various areas in Japan, namely, Fukuoka, Hiroshima, Osaka, Shiga, Kyoto and Aichi prefectures, CChMVd was detected in many of the collected cultivars. Moreover, CChMVd was detected in several samples at low concentrations from cut chrysanthemum flowers sold in Kyoto prefecture shipped from various areas in Japan and the Netherlands. Hence, we concluded that CChMVd had already spread in Japan. In the third experiment to develop a CChMVd-free plant through a newly established method, that is, leaf primordia-free shoot apical meristem (LP-free SAM) culture was utilized to eliminate CChMVd from infected cultivars. These excised LP-free SAMs derived from CChMVd-infected 'Piato' and 'Sttetsuman' plants were attached to the root tip of in vitro-sown cabbage (Brassica oleracea) 'Harunami' and regenerated. One out of 29 regenerated 'Piato' plants and two out of 6 regenerated 'Sttetsuman' plants were ascertained to be CChMVd-free by nested PCR.
Article
Studies conducted over the last 10 years have revealed that the disease caused by the apple scar skin viroid (ASSVd) is extremely rare in Europe. ASSVd was detected by molecular hybridization and indexing in field plots on the apple indicators Starkrimson and Indo, which showed symptoms of dapple apple disease within 2 years, and rough scarred skin within 3 years, respectively. Results from both approaches were in agreement. In an attempt to improve the biological detection of ASSVd, the Japanese PK13 isolate was inoculated to 4 Prunus, 13 Malus, 17 Pyrus, and 17 other pomaceous species. All the species tested of the Malus, Pyrus, Sorbus, Chaenomeles, Cydonia, and Pyronia genera were susceptible to ASSVd based upon back indexing and hybridization, but none developed leaf or bark symptoms during a 2-year period. The viroid was not detected in the tested members of genera Amelanchier, Aronia, Cotoneaster, Crataegus, Prunus, and Pyracantha. Symptoms on fruit of 42 commercial apple cultivars experimentally inoculated with ASSVd fell into five groups ranging from inconspicuous spots to severely scarred skin and cracking. ASSVd was eliminated from most of the infected apple plants when they were subjected to a dormant stage followed by thermotherapy and shoot tip grafting. Analysis of more than 400 apple seedlings, originated from Starkrimson and Indo fruits with typical ASSVd symptoms, showed that there is little or no seed transmission of this viroid. However, ASSVd was transmitted at a low rate under field conditions to adjacent trees.
Article
Chrysanthemum stunt viroid (CSVd) is one of the problematic pathogens known to infect chrysanthemum (Dendranthema grandiflorum) plants and causes various symptoms, such as a reduction in plant height, which is a serious problem in cut flower production. No natural sources with resistance to CSVd have been reported. By quantifying the CSVd titer within the plant, we identified the cultivar 'Utage' as a plant in which the increase of the CSVd titer was slowest among 6 cultivars tested. 'Utage' self-pollinated, and 67 resulting seedlings were screened for CSVd resistance using reverse transcription polymerase chain reaction (RT-PCR), nested-PCR, micro-tissue (MT) direct RT-PCR, and real-time RT-PCR. Of these 67 seedlings, 9 plants lacked the obvious CSVd band detected by RT-PCR. Five months after grafting to CSVd-infected plants, the CSVd titers of 3 plants (C7, A30, and A27) were about 1/240,1/41000, and 1/125000 compared to that of 'Utage', respectively. These 3 plants were comfirmed to have strong resistance to CSVd. In C7, local distribution of CSVd was observed in the youngest expanded leaf by micro-tissue direct RT-PCR and in situ hybridization. For A30 and A27, CSVd was hardly detected in the whole plant. These 3 plants will contribute to the elucidation of CSVd resistance mechanisms. JSHS
Article
Two aspects of hop latent viroid (HLVd) relevant to control were examined: the production of viroid-free plants from infected material and transmission of HLVd in the field. Plants free from HLVd were obtained by a combination of storing infected source plants at low temperature (2–4oC in the dark) for several months followed by meristem culture using small explants. A total of 77 plants of six cultivars and male pollinator clones were grown from meristems and 28 of these were free from HLVd. Tests showed that the cutting of stems (mimicking the use of tools) was more effective than abrasion (mimicking natural plant to plant contact) for the mechanical transmission of HLVd between hop plants. When field-grown test plants were inoculated, infection occurred more commonly in May before plants had grown large enough for significant contact between neighbouring plants than later in the season. The aphid Phorodon humuli could not be shown to transmit HLVd. These results indicate that all hop varieties and pollinator clones can be made available to the industry free from HLVd and that the chances of infection can be reduced by avoiding early-season cultural operations that cut into hop shoots.
Article
SUMMARY‘Mistletoe’ chrysanthemums infected with stunt were grown at 35°C for 14–37 weeks, and meristem-tips cultured from them at intervals. Of 337 meristems, seventy-two survived to plants but their development took 50% longer than did that of stunt-free meristem-tips. All plants were symptomless for at least 5 weeks after planting in 75 mm pots of soil-mix, and only three showed symptoms after 9 weeks. No more plants developed symptoms during the next 5 (winter) months, but between mid-March and late May sixty-seven did so. Only two plants were freed from stunt. ‘Mini-cuttings’ rooted from shoot tips of infected chrysanthemums grown at 35°C for 37 weeks all developed stunt symptoms within 27 weeks.
Article
Viroid-free potato and chrysanthemum plants were obtained from meristem-tips cut from potato spindle tuber viroid-infected potato plants and from chrysanthemum plants infected with chrysanthemum stunt, chrysanthemum chlorotic mottle or cucumber palefruit viroids after 6 months therapy in a growth chamber at 5 °C and 16 hours daily light of 5.000 lx intensity. Chrysanthemum plants survived quite well the conditions of therapy while potato plants grown from stem cuttings survived these conditions much worse and potato plants grown from tubers did not survive these conditions. PSTV-free plants were obtained from meristem-tips cut from sprouts grown from potato tubers infected with severe (s-PSTV) or mild (m-PSTV) strains of potato spindle tuber viroid after 6 months therapy at 6–7 °C in the dark. The tubers survived these conditions quite well.The 3 months therapy period was found too short for any plant material. The efficiency of 6 months therapy in viroid elimination varied for different viroids and different plant material from 18.5 to 80.0 %.
Article
In situ hybridization was used to analyze the distribution pattern of Tomato chlorotic dwarf viroid (TCDVd) in floral organs of tomato plants. Following TCDVd invasion of floral organs, it became localized only in sepals at an early developmental stage, then reached other floral organs at the flower opening stage, with the exception of part of the placenta and ovules. When distribution of TCDVd was compared with that of Potato spindle tuber viroid (PSTVd), TCDVd was not detected in the outer integument around the embryo sac even though PSTVd was able to invade there, suggesting that such specific distribution might reflect the frequent occurrence of viroid disease on crops caused by PSTVd-seed transmission. KeywordsIn situ hybridization–Ovules–Potato spindle tuber viroid–Seed transmission–Shoot apical meristem–Tomato
Article
Citrus exocortis viroid was eradicated from infected tomato plants (Lycopersicon esculentum Mill. ‘Rutgers’) by in vitro shoot-tip culture of axillary buds excised from greenhouse-grown plants. Plantlets developed from 23 of 67 shoot tips excised. The 6 plantlets that demonstrated normal root development were shown to be free of CEV by polyacrylamide gel electrophoresis and bioassay. Fully developed recovered plants showed the same characteristics as the ‘Rutgers’ seedlings.
Article
Viroids are small, nontranslatable pathogenic RNAs that replicate autonomously and traffic systemically in their host plants. We have used in situ hybridization to analyze the trafficking pattern of Potato spindle tuber viroid (PSTVd) in tomato and Nicotiana benthamiana. When PSTVd was inoculated onto the stem of a plant, it replicated and trafficked to sink, but not source, leaves. PSTVd was absent from shoot apical meristems. In the flowers of infected plants, PSTVd was present in the sepals, but was absent in the petals, stamens, and ovary. The replicative form of PSTVd was detected in the phloem. Our data demonstrate that (i) PSTVd traffics long distance in the phloem and this trafficking is likely sustained by replication of the viroid in the phloem, and (ii) PSTVd trafficking is governed by plant developmental and cellular factors. The dependency of PSTVd and other viroids on cellular mechanisms for RNA trafficking makes them excellent tools to study such mechanisms.
Article
Viroids are small noncoding and infectious RNAs that replicate autonomously and move systemically throughout an infected plant. The RNAs of the family Pospiviroidae contain a central conserved region (CCR) that has long been thought to be involved in replication. Here, we report that the CCR of Potato spindle tuber viroid (PSTVd) also plays a role in pathogenicity. A U257A change in the CCR converted the intermediate strain PSTVd(Int) to a lethal strain that caused severe growth stunting and premature death of infected plants. PSTVd with nucleotide U257 changed to C or G did not cause such symptoms. The pathogenic effect of the U257A substitution was abolished by a C259U substitution in the same RNA. Analyses of the pathogenic effects of the U257A substitution in three other PSTVd variants established A257 as a new pathogenicity determinant that functions independently and synergistically with the classic pathogenicity domain. The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism. The stunted growth of PSTVd(Int)U257A-infected tomato plants resulted from restricted cell expansion but not cell division or differentiation. This was correlated positively with the downregulated expression of an expansin gene, LeExp2. Our results demonstrate that specific nucleotides in a noncoding, pathogenic RNA have a profound effect in altering distinct cellular responses, which then lead to well-defined alterations in plant growth and developmental patterns. The feasibility of correlating viroid RNA sequence/structure with the altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system in which to investigate host factors for symptom expression and perhaps also to characterize the mechanisms of RNA regulation of gene expression in plants.
Article
A new method to regenerate plants from leaf primordia-free shoot apical meristem domes (LP-free SAMs) was developed by establishing the meristem dome on the cut surface of root tips. Ten days after culture, the viable rate of LP-free SAMs of chrysanthemum 'Piato' attached to chrysanthemum root tips was >40%. Shoot regeneration was not observed from LP-free SAMs without the root tips. When LP-free SAMs of chrysanthemum were transferred to root tips of either petunia, cabbage, or carnation, the highest shoot regeneration rate was observed with cabbage root tips. Microscopic observation documented that the LP-free SAM temporarily adhered to the cut surface of the root tip of cabbage.
Article
A direct reverse transcription-polymerase chain reaction (RT-PCR) method for detecting the chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) to screen for a viroid-free chrysanthemum plant at a small plant size was established and named microtissue direct RT-PCR. A razor or syringe needle was used for RNA template preparations. Under a stereoscopic microscope, a razor or syringe needle was used to pierce, a tissue sample to a depth of 0.1-0.2mm, and the sample was directly transferred to the RT mixtures. Methods using razors or needles for the preparation of templates could detect CSVd and CChMVd with a high sensitivity. The most sensitive method used a razor or syringe needle to acquire template from the shoot tips. Using the microtissue direct RT-PCR method, both viroids could be detected from the high- and low-viroid-concentration plants. The microtissue direct RT-PCR method was more sensitive than a conventional template preparation method. Using the microtissue direct RT-PCR method established in this study, the laborious subculture step could be omitted because detecting viroids and screening for viroid-free plants even at a small plant size before the subculture could be possible. In addition, the microtissue direct RT-PCR method could also be a powerful tool for clarifing the viroid distribution among microtissues, such as shoot apical meristems.
Article
In this research we eliminated chrysanthemum stunt viroid (CSVd) from a highly infected chrysanthemum cultivar using a newly established method. 'Piato' is one of the most difficult cultivars in which to obtain CSVd-free plants by conventional methods. Leaf primordium-free shoot apical meristems (LP-free SAMs) of 'Piato' plants were dissected and attached to CSVd-free chrysanthemum or cabbage root tips. As shown by nested-PCR, CSVd was not detected in some shoots regenerated on both types of root tip. The production rates of CSVd-free plants using chrysanthemum and cabbage root tips were 14% and 3%, respectively. Regeneration of plants from LP-free SAMs of chrysanthemum plants by attaching these SAMs to root tips is an efficient method of generating CSVd-free chrysanthemum plants.
Article
Peach latent mosaic viroid (PLMVd) is a chloroplast-replicating RNA that propagates in its natural host, peach (Prunus persica), as a complex mixture of variants, some of which are endowed with specific structural and pathogenic properties. This is the case of variant PC-C40, with an insertion of 12 to 13 nucleotides that folds into a hairpin capped by a U-rich loop, which is responsible for an albino-variegated phenotype known as peach calico (PC). We have applied a combination of ultrastructural, biochemical, and molecular approaches to dissect the pathogenic effects of PC-C40. Albino sectors of leaves infected with variant PC-C40 presented palisade cells that did not completely differentiate into a columnar layer and altered plastids with irregular shape and size and with rudimentary thylakoids, resembling proplastids. Furthermore, impaired processing and accumulation of plastid rRNAs and, consequently, of the plastid translation machinery was observed in the albino sectors of leaves infected with variant PC-C40 but not in the adjacent green areas or in leaves infected by mosaic-inducing or latent variants (including PC-C40Delta, in which the 12- to 13-nucleotide insertion was deleted). Protein gel blot and RT-PCR analyses showed that the altered plastids support the import of nucleus-encoded proteins, including a chloroplast RNA polymerase, the transcripts of which were detected. RNA gel blot and in situ hybridizations revealed that PLMVd replicates in the albino leaf sectors and that it can invade the shoot apical meristem and induce alterations in proplastids, bypassing the RNA surveillance system that restricts the entry of a nucleus-replicating viroid and most RNA viruses. Therefore, a non-protein-coding RNA with a specific structural motif can interfere with an early step of the chloroplast developmental program, leading ultimately to an albino-variegated phenotype resembling that of certain variegated mutants in which plastid rRNA maturation is also impaired. Our results highlight the potential of viroids for further dissection of RNA trafficking and pathogenesis in plants.