Eggplant latent viroid is located in the chloroplasts and nuclei of eggplant infected cells.

Viroids that belong to genera Avsunviroid and Pelamovirod (family Avsunviroidae) replicate and accumulate in the chloroplasts of infected cells. In this report, we confirmed by RNA in situ hybridization using digoxigenin-UTP-labelled riboprobes that the positive strands of eggplant latent viroid (ELVd), the only member of genus Elaviroid within the family Avsunviroidae, also accumulate in the chloroplasts of infected cells. However, comparison of ELVd in situ hybridization signals with those from bona fide chloroplastic and nuclear non-coding RNAs, such as chloroplast 5S rRNA and U1 small nuclear RNA, supports the notion that this viroid is also present in the nuclei of infected cells. These results suggest that the subcellular localization of viroids within the family Avsunviroidae may be more complex than previously assumed with dynamic presence in several compartments during the infectious cycle.

High-Throughput Sequencing Reveals New Viroid Species in Opuntia in Mexico.

In the main cactus pear (Opuntia ficus-indica)-producing region in the State of Mexico, fruit production occupies the largest cultivated area with 15,800 ha, while 900 ha are cultivated for edible young Opuntia pads ("nopalitos") which are consumed as vegetables. Two composite samples consisting of cladodes of plants for fruit production (n = 6) and another of "nopalitos" (n = 6) showing virus-like symptoms were collected. Both sample sets were subjected to high-throughput sequencing (HTS) to identify the viruses and viroids. The HTS results were verified using RT-PCR and Sanger sequencing. Subsequently, 86 samples including cladodes from "nopalitos", plants for fruit production, xoconostles, and some wild Opuntia were analyzed via RT-PCR with specific primers for the viruses and viroids previously detected via HTS. Three viruses were discovered [Opuntia virus 2 (OV2), cactus carlavirus 1 (CCV-1), and Opuntia potexvirus A (OPV-A)], along with a previously reported viroid [Opuntia viroid 1 (OVd-1)]. Additionally, two new viroids were identified, provisionally named the Mexican opuntia viroid (MOVd, genus Pospiviroid) and Opuntia viroid 2 (OVd-2, genus Apscaviroid). A phylogenetic analysis, pairwise identity comparison, and conserved structural elements analysis confirmed the classification of these two viroids as new species within the Pospiviroidae family. This is the first report of a pospiviroid and two apscaviroids infecting cactus pears in the world. Overall, this study enhances our understanding of the virome associated with cactus pears in Mexico.

Application of Biotechniques for In Vitro Virus and Viroid Elimination in Pome Fruit Crops.

Sustainable production of pome fruit crops is dependent upon having virus-free planting materials. The production and distribution of plants derived from virus- and viroid-negative sources is necessary not only to control pome fruit viral diseases but also for sustainable breeding activities, as well as the safe movement of plant materials across borders. With variable success rates, different in vitro-based techniques, including shoot tip culture, micrografting, thermotherapy, chemotherapy, and shoot tip cryotherapy, have been employed to eliminate viruses from pome fruits. Higher pathogen eradication efficiencies have been achieved by combining two or more of these techniques. An accurate diagnosis that confirms complete viral elimination is crucial for developing effective management strategies. In recent years, considerable efforts have resulted in new reliable and efficient virus detection methods. This comprehensive review documents the development and recent advances in biotechnological methods that produce healthy pome fruit plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Bacteriophage P22 Capsid as a Pluripotent Nanotechnology Tool.

The Salmonella enterica bacteriophage P22 is one of the most promising models for the development of virus-like particle (VLP) nanocages. It possesses an icosahedral T = 7 capsid, assembled by the combination of two structural proteins: the coat protein (gp5) and the scaffold protein (gp8). The P22 capsid has the remarkable capability of undergoing structural transition into three morphologies with differing diameters and wall-pore sizes. These varied morphologies can be explored for the design of nanoplatforms, such as for the development of cargo internalization strategies. The capsid proteic nature allows for the extensive modification of its structure, enabling the addition of non-native structures to alter the VLP properties or confer them to diverse ends. Various molecules were added to the P22 VLP through genetic, chemical, and other means to both the capsid and the scaffold protein, permitting the encapsulation or the presentation of cargo. This allows the particle to be exploited for numerous purposes-for example, as a nanocarrier, nanoreactor, and vaccine model, among other applications. Therefore, the present review intends to give an overview of the literature on this amazing particle.

Mexico: A Landscape of Viroid Origin and Epidemiological Relevance of Endemic Species.

Viroids are single-stranded, circular RNA molecules (234-406 nt) that infect a wide range of crop species and cause economic losses in agriculture worldwide. They are characterized by the existence of a population of sequence variants, attributed to the low fidelity of RNA polymerases involved in their transcription, resulting in high mutation rates. Therefore, these biological entities exist as quasispecies. This feature allows them to replicate within a wide range of host plants, both monocots and dicots. Viroid hosts include economically important crops such as tomato, citrus, and fruit trees such as peach and avocado. Given the high risk of introducing viroids to viroid disease-free countries, these pathogens have been quarantined globally. As discussed herein, Mexico represents a geographical landscape of viroids linked to their origin and comprises considerable biodiversity. The biological features of viroid species endemic to Mexico are highlighted in this communication. In addition, we report the phylogenetic relationships among viroid and viroid strains, their economic impact, geographical distribution, and epidemiological features, including a broad host range and possible long-distance, seed, or insect-mediated transmission. In summary, this review could be helpful for a better understanding of the biology of viroid diseases and future programs on control of movement and spread to avoid economic losses in agricultural industries.

Insights into the Transcriptional Reprogramming in Tomato Response to PSTVd Variants Using Network Approaches.

Viroids are the smallest pathogens of angiosperms, consisting of non-coding RNAs that cause severe diseases in agronomic crops. Symptoms associated with viroid infection are linked to developmental alterations due to genetic regulation. To understand the global mechanisms of host viroid response, we implemented network approaches to identify master transcription regulators and their differentially expressed targets in tomato infected with mild and severe variants of PSTVd. Our approach integrates root and leaf transcriptomic data, gene regulatory network analysis, and identification of affected biological processes. Our results reveal that specific bHLH, MYB, and ERF transcription factors regulate genes involved in molecular mechanisms underlying critical signaling pathways. Functional enrichment of regulons shows that bHLH-MTRs are linked to metabolism and plant defense, while MYB-MTRs are involved in signaling and hormone-related processes. Strikingly, a member of the bHLH-TF family has a specific potential role as a microprotein involved in the post-translational regulation of hormone signaling events. We found that ERF-MTRs are characteristic of severe symptoms, while ZNF-TF, tf3a-TF, BZIP-TFs, and NAC-TF act as unique MTRs. Altogether, our results lay a foundation for further research on the PSTVd and host genome interaction, providing evidence for identifying potential key genes that influence symptom development in tomato plants.

Surveys in the Chrysanthemum Production Areas of Brazil and Colombia Reveal That Weeds Are Potential Reservoirs of Chrysanthemum Stunt Viroid.

The stunting disease, incited by chrysanthemum stunt viroid (CSVd), has become a serious problem in chrysanthemum production areas worldwide. Here we identified 46 weed species from chrysanthemum fields in two producing regions of the State of São Paulo, Brazil. The mechanical inoculation of these weeds with a Brazilian CSVd isolate revealed that this viroid was able to infect 17 of these species, in addition to chrysanthemum, tomato and potato. Plants of Oxalis latifolia and chrysanthemum naturally infected with CSVd were found in chrysanthemum fields in Colombia, which is the first CSVd report in that country. Therefore, weeds have the potential to act as reservoirs of CSVd in the field. These results are the first reports of experimental CSVd infection in the following species: Amaranthus viridis, Cardamine bonariensis, Chamaesyce hirta, Conyza bonariensis, Digitaria sanguinalis, Gomphrena globosa, Helianthus annuus, Lupinus polyphyllus, Mirabilis jalapa, Oxalis latifolia, Portulaca oleracea and Catharanthus roseus. The phylogenetic analyses of the CSVd variants identified herein showed three groups with Brazilian CSVd variants distributed in them all, which suggests that Brazilian CSVd isolates may have different origins through successive introductions of infected germplasm of chrysanthemum in Brazil.

Virus-viroid interactions: Citrus Tristeza Virus enhances the accumulation of Citrus Dwarfing Viroid in Mexican lime via virus-encoded silencing suppressors.

An assay to identify interactions between Citrus Dwarfing Viroid (CDVd) and Citrus Tristeza Virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd.

A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by prunus necrotic ringspot virus and peach latent mosaic viroid.

BACKGROUND: Microarray profiling is a powerful technique to investigate expression changes of large amounts of genes in response to specific environmental conditions. The majority of the studies investigating gene expression changes in virus-infected plants are limited to interactions between a virus and a model host plant, which usually is Arabidopsis thaliana or Nicotiana benthamiana. In the present work, we performed microarray profiling to explore changes in the expression profile of field-grown Prunus persica (peach) originating from Chile upon single and double infection with Prunus necrotic ringspot virus (PNRSV) and Peach latent mosaic viroid (PLMVd), worldwide natural pathogens of peach trees. RESULTS: Upon single PLMVd or PNRSV infection, the number of statistically significant gene expression changes was relatively low. By contrast, doubly-infected fruits presented a high number of differentially regulated genes. Among these, down-regulated genes were prevalent. Functional categorization of the gene expression changes upon double PLMVd and PNRSV infection revealed protein modification and degradation as the functional category with the highest percentage of repressed genes whereas induced genes encoded mainly proteins related to phosphate, C-compound and carbohydrate metabolism and also protein modification. Overrepresentation analysis upon double infection with PLMVd and PNRSV revealed specific functional categories over- and underrepresented among the repressed genes indicating active counter-defense mechanisms of the pathogens during infection. CONCLUSIONS: Our results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits. We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections. Thus, our data demonstrate for the first time that the simultaneous infection of a viroid and a plant virus synergistically affect the host transcriptome in infected peach fruits. These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.

A single polyprobe for detecting simultaneously eight pospiviroids infecting ornamentals and vegetables.

The spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae), recorded recently in ornamentals and vegetables in several European countries, calls for fast, efficient and sensitive detection methods. Based on bioinformatics analyses of sequence identity among all pospiviroids, a digoxigenin-labeled polyprobe (POSPIprobe) was developed that, when tested by dot-blot and Northern-blot hybridization, detected Potato spindle tuber viroid, Citrus exocortis viroid, Columnea latent viroid, Mexican papita viroid, Tomato planta macho viroid, Tomato apical stunt viroid, Pepper chat fruit viroid and Chrysanthemum stunt viroid. The end-point detection limits of the POSPIprobe ranged from 5(-2) to 5(-4), and from 5(-1) to 5(-3) for nucleic acid preparations obtained by phenol extraction and silica-capture, respectively, similar to those of single probes. Based on sequence identity, the POSPIprobe is expected to detect also the two pospiviroid species not tested in this study (Tomato chlorotic dwarf viroid and Iresine viroid-1). Dot-blot assays with the POSPIprobe were validated by testing 68 samples from tomato, chrysanthemum and argyranthemum infected by different pospiviroids as revealed by RT-PCR, thus confirming the potential of this polyprobe for quarantine, certification and survey programs.

Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation.

Small RNAs (sRNA), including microRNAs (miRNA) and small interfering RNAs (siRNA), are produced abundantly in plants and animals and function in regulating gene expression or in defense against virus or viroid infection. Analysis of siRNA profiles upon virus infection in plant may allow for virus identification, strain differentiation, and de novo assembly of virus genomes. In the present study, four suspected virus-infected tomato samples collected in the U.S. and Mexico were used for sRNA library construction and deep sequencing. Each library generated between 5-7 million sRNA reads, of which more than 90% were from the tomato genome. Upon in-silico subtraction of the tomato sRNAs, the remaining highly enriched, virus-like siRNA pools were assembled with or without reference virus or viroid genomes. A complete genome was assembled for Potato spindle tuber viroid (PSTVd) using siRNA alone. In addition, a near complete virus genome (98%) also was assembled for Pepino mosaic virus (PepMV). A common mixed infection of two strains of PepMV (EU and US1), which shared 82% of genome nucleotide sequence identity, also could be differentially assembled into their respective genomes. Using de novo assembly, a novel potyvirus with less than 60% overall genome nucleotide sequence identity to other known viruses was discovered and its full genome sequence obtained. Taken together, these data suggest that the sRNA deep sequencing technology will likely become an efficient and powerful generic tool for virus identification in plants and animals.

Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus Pospiviroid.

Tomato planta macho viroid (TPMVd) and Mexican papita viroid (MPVd) are two closely related (>90% sequence identity) members of the genus Pospiviroid. Their current status as members of separate species is based upon the reported ability of TPMVd to replicate in Gomphrena globosa and the inability of this viroid to evoke flower break in N. glutinosa. Characterization of a viroid recently isolated from diseased tomato plants grown in Mexico (identical to GenBank accession GQ131573) casts doubt on this earlier report and indicates that these viroids should be classified as members of a single species. Giving priority to the older name, we propose including both of these viroids in the current species Tomato planta macho viroid.

Viroides, pequenos RNAs patogênicos capazes de replicação autônoma: modelos moleculares para o estudo de interações patógeno-hospedeiro e evolução / Viroids, small pathogenic RNAs capable of autonomous replication: molecular models for the study on host-pathogen interactions and evolution

Os viroides, apesar de serem constituídos por um pequeno RNA de fita simples, fortemente estruturado, circular, que não codifica proteínas, são capazes de se replicar de maneira autônoma em plantas superiores e causar doença interagindo diretamente com fatores do hospedeiro. Nesta revisão, serão apresentados e discutidos alguns dos mais recentes trabalhos envolvendo a interação de viroides com fatores do hospedeiro, incluindo aspectos relacionados à replicação, movimento e patogênese, além de suas características evolutivas. Nos últimos anos, alguns grupos de pesquisa têm se aventurado na busca por fatores do hospedeiro e mecanismos moleculares relacionados ao ciclo infeccioso dos viroides, tentando desvendar como esses pequenos RNAs interagem com o hospedeiro induzindo sintomas. Os viroides não codificam proteínas supressoras de silenciamento e, portanto, devem garantir sua existência utilizando estratégias baseadas em sua estrutura secundária, na compartimentalização em organelas, associação com fatores do hospedeiro e eficiência na replicação. A complexidade do ciclo infeccioso desses minúsculos RNAs indica que muitas interações desses patógenos com fatores do hospedeiro ainda devem ser identificadas.

Viroids are small, single-stranded, highly structured, circular RNAs that replicate autonomously in their hosts, without messenger RNA activity. Because they do not encode for proteins, viroids have to interact directly with host factors. This review presents recent progress in understanding the possible role of recently identified viroid-binding host proteins related to replication, trafficking and pathogenesis. It also discusses some aspects on viroid evolution. In recent years, efforts to understand how viroids replicate, cause disease and induce symptoms have prompted details on molecular mechanisms related to the viroid infectious cycle. Inasmuch as viroids lack protein-encoding capacity, including suppressors of gene silencing, their existence could be ensured by their compact conformation, compartimentalization in organelles, association with host factors or by their highly efficient replication. The complexity of the infectious cycle of these tiny pathogenic RNAs indicates that several interactions with host factors remain to be identified.

Sequence analysis of three citrus viroids infecting a single Tunisian citrus tree (Citrus, reticulata, Clementine)

We report the nucleotide sequences of three citrus viroids belonging to three different genera: Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus viroid-III (CVd-III) isolated from a single natural infected Citrus reticulata var. Clementine tree growing in a tree nursery in Manouba (near Tunis Capital). We describe the sequence variability of these viroids from their natural host without using an alternative passage by an indicator host or an artificial inoculation. This work confirms that naturally occurring viroid infections contain a mixture of sequence variants. These are the first sequences of citrus viroids from Africa.

Occurence of citrus viroids in Costa Rica

A survey for citrus viroids was conducted in the major citrus commercial growing areas in Costa Rica. Screening of 36 sweet orange and 12 lemon trees resulted in the detection of members of four of the five citrus viroid groups as determined by nucleic acid hybridization using specific RNA probes and polymerase chain reaction (PCR) using specific oligonucleotide primers. CEVd, CVd-IIa, CVD-IIb and CVd-III viroids were found to be widespread in the three main regions of commercial citrus production. CVd-Ib was only found in lemon in Nicoya

Evidence for heterologous encapsidation of potato spindle tuber viroid in particles of potato leafroll virus.

The aphid Myzus persicae (Sulz.) was shown to transmit potato spindle tuber viroid (PSTVd) to potato clone DTO-33 from source plants doubly infected with potato leafroll virus (PLRV) and PSTVd. Transmission was of the persistent type and did not occur when the insects were allowed to feed on singly infected plants. Only low levels of PSTVd were associated with purified PLRV virions, but its resistance to digestion with micrococcal nuclease indicates that the viroid RNA is encapsidated within the PLRV particles. Epidemiological surveys carried out at three locations in China revealed a strong correlation between PSTVd infection and the presence of PLRV, suggesting that PLRV can facilitate PSTVd spread under field conditions.

Occurrence of citrus viroids in Costa Rica.

A survey for citrus viroids was conducted in the major citrus commercial growing areas in Costa Rica. Screening of 36 sweet orange and 12 lemon trees resulted in the detection of members of four of the five citrus viroid groups as determined by nucleic acid hybridization using specific RNA probes and polymerase chain reaction (PCR) using specific oligonucleotide primers. CEVd, CVd-IIa, CVD-IIb and CVd-III viroids were found to be widespread in the three main regions of commercial citrus production. CVd-Ib was only found in lemon in Nicoya.

Mexican papita viroid: putative ancestor of crop viroids.

The potato spindle tuber disease was first observed early in the 20th century in the northeastern United States and shown, in 1971, to be incited by a viroid, potato spindle tuber viroid (PSTVd). No wild-plant PSTVd reservoirs have been identified; thus, the initial source of PSTVd infecting potatoes has remained a mystery. Several variants of a novel viroid, designated Mexican papita viroid (MPVd), have now been isolated from Solanum cardiophyllum Lindl. (papita güera, cimantli) plants growing wild in the Mexican state of Aguascalientes. MPVd's nucleotide sequence is most closely related to those of the tomato planta macho viroid (TPMVd) and PSTVd. From TPMVd, MPVd may be distinguished on the basis of biological properties, such as replication and symptom formation in certain differential hosts. Phylogenetic and ecological data indicate that MPVd and certain viroids now affecting crop plants, such as TPMVd, PSTVd, and possibly others, have a common ancestor. We hypothesize that commercial potatoes grown in the United States have become viroid-infected by chance transfer of MPVd or a similar viroid from endemically infected wild solanaceous plants imported from Mexico as germplasm, conceivably from plants known to have been introduced from Mexico to the United States late in the 19th century in efforts to identify genetic resistance to the potato late blight fungus, Phytophthora infestans.

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.