Viroid Infection Research Articles

Deep sequencing reveals the first fabavirus infecting peach

A disease causing smaller and cracked fruit affects peach [Prunus persica (L.) Batsch], resulting in significant decreases in yield and quality. In this study, peach tree leaves showing typical symptoms were subjected to deep sequencing of small RNAs for a complete survey of presumed causal viral pathogens. The results revealed two known viroids (Hop stunt viroid and Peach latent mosaic viroid), two known viruses (Apple chlorotic leaf spot trichovirus and Plum bark necrosis stem pitting-associated virus) and a novel virus provisionally named Peach leaf pitting-associated virus (PLPaV). Phylogenetic analysis based on RNA-dependent RNA polymerase placed PLPaV into a separate cluster under the genus Fabavirus in the family Secoviridae. The genome consists of two positive-sense single-stranded RNAs, i.e., RNA1 [6,357 nt, with a 48-nt poly(A) tail] and RNA2 [3,862 nt, with a 25-nt poly(A) containing two cytosines]. Biological tests of GF305 peach indicator seedlings indicated a leaf-pitting symptom rather than the smaller and cracked fruit symptoms related to virus and viroid infection. To our knowledge, this is the first report of a fabavirus infecting peach. PLPaV presents several new molecular and biological features that are absent in other fabaviruses, contributing to an overall better understanding of fabaviruses.

The effect of viroid infection of citrus trees on the amoebicidal activity of ‘Maltese half-blood’ (Citrus sinensis) against trophozoite stage of Acanthamoeba castellanii Neff

In order to promote a local Tunisian product, this study was designed to examine, for the first time, the anti-Acanthamoeba activity (Acanthamoeba castellanii Neff) of the essential oils of Tunisian Citrus sinensis peels (Maltese half-blood) and the effect of viroid plant infection on this activity. To do so, three samples of peels’ essential oils were studied: from a healthy plant (Control), a plant inoculated with Citrus exocortis viroid (CEVd) and one inoculated with hot stunt cachexia viroid (HSVd). The samples were extracted by hydrodistillation from dried peels and characterized by GC–MS. Limonene was the major component with a percentage ranging from 90.76 to 93.34% for (CEVd) sample and (Control), respectively. Anti-Acanthamoeba activity of the tested oils was determined by the Alamar Blue® assay. Primary results showed a strong potential anti-Acanthamoeba activity with an IC50 ranging from 36.6 to 54.58 μg/ml for (HSVd) and (CEVd) samples, respectively.In terms of the effect of viroid infection, a strong positive correlation was observed between different chemical classes and anti-Acanthamoeba activity.

Hop stunt viroid: Effect on Host (Humulus lupulus) Transcriptome and Its Interactions With Hop Powdery Mildew (Podospheara macularis).

Viroids are the smallest known plant pathogens that exploit host systems for their replication and cause diseases in many hosts. In this study, the host response of hop plants to Hop stunt viroid (HSVd) infection was studied through transcriptome analysis. RNA sequence analysis of hop leaves infected with HSVd revealed dynamic changes in hop gene expression. Defense-related genes and genes involved in lipid and terpenoid metabolism are the major categories that showed differential expression due to HSVd infection. Additionally, the effect of HSVd on development of hop powdery mildew (Podospheara macularis) (HPM) was studied. Transcriptome analysis followed by quantitative reverse transcription-polymerase chain reaction analysis showed that transcript levels of pathogenesis-related (PR) genes such as PR protein 1, chitinase, and thaumatin-like protein genes are induced in leaves infected with HPM alone. The response in these genes to HPM is significantly down-regulated in leaves with HSVd-HPM mixed infection. These results confirm that HSVd alters host metabolism, physiology, and plant defense responses. Nevertheless, in detached leaf assays, HPM consistently expanded faster on HSVd-negative leaves relative to HSVd-positive leaves. Although HSVd infection suppresses elements associated with the host immunity response, infection by HSVd is antagonistic to HPM infection of hops.

Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants

It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (−) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3′ UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection.

Comprehensive Transcriptome Analyses Reveal that Potato Spindle Tuber Viroid Triggers Genome-Wide Changes in Alternative Splicing, Inducible trans-Acting Activity of Phased Secondary Small Interfering RNAs, and Immune Responses.

Many pathogens express noncoding RNAs (ncRNAs) during infection processes. In the most extreme case, pathogenic ncRNAs alone (such as viroids) can infect eukaryotic organisms, leading to diseases. While a few pathogenic ncRNAs have been implicated in regulating gene expression, the functions of most pathogenic ncRNAs in host-pathogen interactions remain unclear. Here, we employ potato spindle tuber viroid (PSTVd) infecting tomato as a system to dissect host interactions with pathogenic ncRNAs, using comprehensive transcriptome analyses. We uncover various new activities in regulating gene expression during PSTVd infection, such as genome-wide alteration in alternative splicing of host protein-coding genes, enhanced guided-cleavage activities of a host microRNA, and induction of the trans-acting function of phased secondary small interfering RNAs. Furthermore, we reveal that PSTVd infection massively activates genes involved in plant immune responses, mainly those in the calcium-dependent protein kinase and mitogen-activated protein kinase cascades, as well as prominent genes involved in hypersensitive responses, cell wall fortification, and hormone signaling. Intriguingly, our data support a notion that plant immune systems can respond to pathogenic ncRNAs, which has broad implications for providing new opportunities for understanding the complexity of immune systems in differentiating "self" and "nonself," as well as lay the foundation for resolving the long-standing question regarding the pathogenesis mechanisms of viroids and perhaps other infectious RNAs.IMPORTANCE Numerous pathogens, including viruses, express pathogenic noncoding transcripts during infection. In the most extreme case, pathogenic noncoding RNAs alone (i.e., viroids) can cause disease in plants. While some work has demonstrated that pathogenic noncoding RNAs interact with host factors for function, the biological significance of pathogenic noncoding RNAs in host-pathogen interactions remains largely unclear. Here, we apply comprehensive genome-wide analyses of plant-viroid interactions and discover several novel molecular activities underlying nuclear-replicating viroid infection processes in plants, including effects on the expression and function of host noncoding transcripts, as well as the alternative splicing of host protein-coding genes. Importantly, we show that plant immunity is activated upon infection of a nuclear-replicating viroid, which is a new concept that helps to understand viroid-based pathogenesis. Our finding has broad implications for understanding the complexity of host immune systems and the diverse functions of noncoding RNAs.

Propagation and some physiological effects of Citrus bark cracking viroid and Apple fruit crinkle viroid in multiple infected hop (Humulus lupulus L.)

The hop metabolome important for the brewing industry and for medical purposes is endangered worldwide due to multiple viroid infections affecting hop physiology. Combinatorial biolistic hop inoculation with Citrus bark cracking viroid (CBCVd), Apple fruit crinkle viroid (AFCVd), Hop latent viroid, and Hop stunt viroid (HSVd) showed a low CBCVd compatibility with HSVd, while all other viroid combinations were highly compatible. Unlike to other viroids, single CBCVd propagation showed a significant excess of (−) over (+) strands in hop, tomato, and Nicotiana benthamiana, but not in citruses. Inoculation of hop with all viroids led to multiple infections with unstable viroid levels in individual plants in the pre- and post-dormancy periods, and to high plant mortality and morphological disorders. Hop isolates of CBCVd and AFCVd were highly stable, only minor quasispecies were detected. CBCVd caused a strong suppression of some crucial mRNAs related to the hop prenylflavonoid biosynthesis pathway, while AFCVd-caused effects were moderate. According to mRNA degradome analysis, this suppression was not caused by a direct viroid-specific small RNA-mediated degradation. CBCVd infection led to a strong induction of two hop transcription factors from WRKY family and to a disbalance of WRKY/WDR1 complexes important for activation of lupulin genes.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs.

Artificial microRNAs and synthetic trans-acting small interfering RNAs interfere with viroid infection.

SummaryArtificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs) are two classes of artificial small RNAs (sRNAs) engineered to silence endogenous transcripts as well as viral RNAs in plants. Here, we explore the possibility of using amiRNAs and syn‐tasiRNAs to specifically interfere with infections by viroids, small (250–400‐nucleotide) non‐coding circular RNAs with compact secondary structure infecting a wide range of plant species. The combined use of recent high‐throughput methods for artificial sRNA construct generation and the Potato spindle tuber viroid (PSTVd)–Nicotiana benthamiana pathosystem allowed for the simple and time‐effective screening of multiple artificial sRNAs targeting sites distributed along PSTVd RNAs of (+) or (–) polarity. The majority of amiRNAs were highly active in agroinfiltrated leaves when co‐expressed with an infectious PSTVd transcript, as were syn‐tasiRNAs derived from a construct including the five most effective amiRNA sequences. A comparative analysis showed that the effects of the most effective amiRNA and of the syn‐tasiRNAs were similar in agroinfiltrated leaves, as well as in upper non‐agroinfiltrated leaves in which PSTVd accumulation was significantly delayed. These results suggest that amiRNAs and syn‐tasiRNAs can be used effectively to control viroid infections in plants.

Identification and characterization of microRNAs in Humulus lupulus using high-throughput sequencing and their response to Citrus bark cracking viroid (CBCVd) infection.

BackgroundHop (Humulus lupulus L.) plants are grown primarily for the brewing industry and have been used as a traditional medicinal herb for a long time. Severe hop stunt disease caused by the recently discovered Citrus bark cracking viroid (CBCVd) is one of the most devastating diseases among other viroid infections in hop. MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in gene expression regulation. To identify miRNAs in hop and their response to CBCVd-infection, two small RNA (sRNA) libraries were prepared from healthy and CBCVd-infected hop plants and were investigated by high throughput sequencing.ResultsA total of 67 conserved and 49 novel miRNAs were identified. Among them, 36 conserved and 37 novel miRNAs were found to be differentially recovered in response to CBCVd-infection. A total of 311 potential targets was predicted for conserved and novel miRNAs based on a sequence homology search using hop transcriptome data. The majority of predicted targets significantly belonged to transcriptional factors that may regulate hop leaf, root and cone growth and development. In addition, the identified miRNAs might also play an important roles in other cellular and metabolic processes, such as signal transduction, stress response and other physiological processes, including prenylflavonoid biosynthesis pathways. Quantitative real time PCR analysis of selected targets revealed their negative correlation with their corresponding CBCVd-responsive miRNAs.ConclusionsBased on the results, we concluded that CBCVd-responsive miRNAs modulate several hormone pathways and transcriptional factors that play important roles in the regulation of metabolism, growth and development. These results provide a framework for further analysis of regulatory roles of sRNAs in plant defense mechanism including other hop infecting viroids in particular.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3271-4) contains supplementary material, which is available to authorized users.

Attempts to eradicate graft-transmissible infections through somatic embryogenesis in Citrus ssp. and analysis of genetic stability of regenerated plants

Stigma/style somatic embryogenesis (SE) is proved to be effective in the complete elimination of the main Citrus virus and virus-like diseases in 100 % of analysed cases. In the present research SE was applied on 13 genotypes, belonging to the Algerian germplasm collection of two different Citrus species (Citrus limon and C. sinensis) infected by one or more graft-transmissible agents, to evaluate the genetic stability and the sanitation of regenerants. The infected genotypes were regenerated through stigma/style SE and the obtained plants were tested by serological, molecular and biological assays, 6 and 18 months after plantlet grafting, for assessing the elimination of the virus, viroid and virus-like agents present in the mother plants. No evidence of infections of virus and virus–like agents was present in the regenerated plants; whereas, after 18 months up to 24 % of tested plants were found to be infected by viroids, independently from the species and genotypes of the mother plants. Hop stunt viroid (HSVd) proved to be the most infectious viroid; conversely, Citrus exocortis viroid (CEVd) and Citrus viroid III (CVd-III) were present only in ‘Secile’ lemon, as mixed infection with HSVd. SE from stigma/style explants was 100 % effective in the elimination of mixed viroid infections in two lemons (‘Lunario’ and ‘Sans pepins’) and one orange (‘Mitidja navel’). To evaluate genetic stability of regenerants, DNA analyses were performed. No somaclonal variability was observed in lemon regenerants. However, the amplification products of ‘Washington navel 251’ (C. sinensis) revealed genetic instability in some of the regenerated plants.

Combined Activity of DCL2 and DCL3 Is Crucial in the Defense against Potato Spindle Tuber Viroid.

Viroids are self replicating non-coding RNAs capable of infecting a wide range of plant hosts. They do not encode any proteins, thus the mechanism by which they escape plant defenses remains unclear. RNAi silencing is a major defense mechanism against virus infections, with the four DCL proteins being principal components of the pathway. We have used Nicotiana benthamiana as a model to study Potato spindle tuber viroid infection. This viroid is a member of the Pospiviroidae family and replicates in the nucleus via an asymmetric rolling circle mechanism. We have created knock-down plants for all four DCL genes and their combinations. Previously, we showed that DCL4 has a positive effect on PSTVd infectivity since viroid levels drop when DCL4 is suppressed. Here, we show that PSTVd levels remain decreased throughout infection in DCL4 knockdown plants, and that simultaneous knockdown of DCL1, DCL2 or DCL3 together with DCL4 cannot reverse this effect. Through infection of plants suppressed for multiple DCLs we further show that a combined suppression of DCL2 and DCL3 has a major effect in succumbing plant antiviral defense. Based on our results, we further suggest that Pospoviroids may have evolved to be primarily processed by DCL4 as it seems to be a DCL protein with less detrimental effects on viroid infectivity. These findings pave the way to delineate the complexity of the relationship between viroids and plant RNA silencing response.

Effects of potato spindle tuber viroid infection on tomato metabolic profile

Viroids are the smallest plant pathogens consisting of a single stranded circular RNA molecule with a strong secondary structure, lacking a coat protein or any other proteins. The mechanism of viroid pathogenicity has remained unclear. Recent advances in instrumentation and data mining have made it possible to study the effects of various stresses on primary and secondary metabolisms. Here, we have utilized metabolic profiling approach to show how PSTVd infection alters tomato metabolic profile and the related pathways. Three terminal leaflets of third true leaf of 20-day-old tolerant tomato cultivar ‘Moneymaker’ were mechanically inoculated by PSTVd intermediate variant cDNAs and samples were taken from eighth leaf, 19days post-inoculation. Metabolites were extracted and analyzed by gas chromatography/mass spectrometry (GC/MS) and subjected to statistical data analysis. Affected pathways were identified by Pathway Tools program and were compared with microarray data previously reported. The study showed that 79 metabolites changed significantly and 23 pathways were identified in relation to these metabolites. Fourteen of these pathways were similar to those reported in other works. The altered pathways in PSTVd infected tomato leaves included, eight cutin and wax biosynthesis, seven pathways that produce defense related compounds, two energy generator pathways, three hormone biosynthesis pathways, two signal transduction pathways, and one nucleotide biosynthesis pathway. Our data on up/down-regulation of pathways supported the data produced on their corresponding gene(s) up/down-regulation.

Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection.

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection.

Molecular diagnostics of potato infections with PVY and PLRV by immunochromatography

An immunochromatography test system has been developed for molecular diagnostics of the potato virus Y and PLRV infection. To increase a low yield of PLRV and raise antibodies against the PLRV antigen, chimerical virus was constructed comprising the PLRV coat protein and recombinant RNA of a tobamovirus, in which capsid protein gene was replaced by the PLRV coat protein gene. Binary vector containing the DNA copy of the recombinant RNA was infectious, and yield of the chimerical virus increased up to 800 times in comparison with the wild type PLRV. On the basis of experience in the development of the diagnostics of viral and viroid infections, rational tactics are proposed for the mass laboratory and field diagnosis of viral infections on the molecular level.

RNAi mediated inhibition of viroid infection in transgenic plants expressing viroid-specific small RNAs derived from various functional domains.

Previous attempts to develop RNAi-mediated viroid-resistant transgenic plants using nearly full-length Potato spindle tuber viroid (PSTVd) hairpin RNA (hpRNA) were successful; however unusual phenotypes resembling viroid infection occurred. Therefore, in the present work, transgenic Nicotiana benthamiana lines expressing both partial and truncated versions of PSTVd hpRNA were developed. Specifically, seven partial or truncated versions of PSTVd sequences were selected according to the hotspots of both PSTVd-sRNAs and functional domains of the PSTVd. A total of 21 transgenic lines Nicotiana benthamiana were developed under the control of either the CaMV-35S or the CoYMV promoters. All of the transgenic lines established here were monitored for the induction of phenotypic changes, for PSTVd-sRNA expression and for the resistance against PSTVd infection. Additionally, this study demonstrates the use of inverted repeat construct sequences as short as 26- to -49 nucleotides for both the efficient expression of the PSTVd-sRNA and the inhibition of PSTVd infection.

K. Subramanya Sastry: Plant virus and viroid diseases in the tropics, Volume 1: Introduction of plant viruses and sub-viral agents, classification, assessment of loss, transmission, and diagnosis

Throughout the world, most of the plants are naturally affected by number of virus and viroid diseases, besides fungal and bacterial pathogens. Virus and viroid diseases are economically important because of the losses they cause in productivity and quality of the plant products. The profitable crop production depends on the appropriate diagnosis of the etiological agents and application of management measures. In the first volume, the author Dr. K Subramanya Sastry, has provided up-to-date information on different aspects of plant virus and viroid diseases of crop plants of South East Asia, Africa, South American countries in five chapters, which are interlinked and exhaustive. Detail information on classification, crop yield losses, transmission and diagnosis of virus and viroid diseases at advanced as well as comprehensive level has been described. In the introductory chapter, the author has presented the information on tropical countries, their climate, tropical crops and the prevalent virus and viroid diseases. The second chapter is focused on introduction of virus and virus-like diseases and their classification based on the 9th report of ICTV published by King et al. (2012). In the third chapter, the author has provided the extensive yield losses both quantitatively and qualitatively in large number of crops due to virus and viroid infections. Viruses cause their damage in such diverse ways like reducing the vigor and life span of the infected plants of the orchards, groves and pastures. In the fourth chapter, Dr. Sastry, has discussed the different agents and factors involved in the spread of virus and viroid diseases, which are very much required for framing suitable management measures. The author has also provided exhaustive information on seed transmission of plant virus and viroid diseases which would be quite useful to the personnel involved at quarantine and agriculture. The fifth chapter describes the diagnosis and detection of plant virus and viroid diseases by the application of techniques including the electron microscopy, serological techniques like variants of ELISA, monoclonal antibody techniques, nucleic acid hybridization technologies, PCR/RT-PCR, microarray technologies, biosensors, etc., which are the outcome of number of years of hard work in different research laboratories at global level. The diagnostic techniques which are sensitive, rapid and accurate in virus and viroid diagnosis are provided. This volume 1 will serve as one of the valuable and comprehensive reference book for the basic, practical and applied plant pathologists, scientists, teaching faculty and researchers of plant virology, plant pathology and plant biology. This volume is relevant and timely in view of the rapidly expanding international trade of plant and plant products. The author, Dr. K Subramanya Sastry, has done sincere hard work and major efforts to provide the latest information and it is my pleasure to complement him, for his outstanding and comprehensive volume 1 on plant virus and viroid diseases in the tropics.

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants.

The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.

Simultaneous detection of Tomato spotted wilt virus, Dahlia mosaic virus and Chrysanthemum stunt viroid by multiplex RT-PCR in dahlias and their distribution in Japanese dahlias.

Tomato spotted wilt virus (TSWV), Dahlia mosaic virus (DMV) and Chrysanthemum stunt viroid (CSVd) are economically important viruses and viroid that infect cultivated dahlias. Prior to this investigation, no multiplex RT-PCR assay for the detection of dahlia virus and viroid infections existed. In this study, we report the development of a multiplex RT-PCR that simultaneously detects TSWV, DMV and CSVd infections in dahlias. In addition, a simple RT-PCR method that does not require RNA extraction, microtissue direct RT-PCR, could be used to prepare samples for analysis by this multiplex RT-PCR. A field survey validated our results, indicating that TSWV was the dominant virus found in the Kansai region, DMV in the Tohoku and Kyushu regions, and CSVd in the Hokkaido region. This method represents a rapid, sensitive and cost effective approach to diagnose viral infections in dahlias. The multiplex RT-PCR assay described in this study is the first report of simultaneous detection of virus and viroid in dahlia. This method represents a rapid, sensitive and cost effective approach to diagnose viral infections in dahlias. A field survey validated our results, indicating that TSWV was the dominant virus found in the Kansai region, DMV in the Tohoku and Kyushu regions and CSVd in the Hokkaido region.

Expression of SANT/HTH Myb mRNA, a plant morphogenesis-regulating transcription factor, changes due to viroid infection

Potato spindle tuber viroid (PSTVd) belongs to plant-pathogenic, circular, non-coding RNAs. Its propagation is accompanied by (mis)regulation of host genes and induction of pathogenesis symptoms including changes of leaf morphogenesis depending on the strength of viroid variant. We found strong genotype-dependent suppression of tomato morphogenesis-regulating transcription factor SANT/HTH-Myb (SlMyb) due to viroid pathogenesis. Its relative mRNA level was found to be significantly decreased in PSTVd-sensitive tomato (cvs Rutgers and Heinz 1706) due to degradation processes, but increased in PSTVd-tolerant (cv. Harzfeuer). In heterologous system of Nicotiana benthamiana, we observed a SlMyb-associated necrotic effect in agroinfiltrated leaf sectors during ectopic overexpression. Leaf sector necroses were accompanied by activation of nucleolytic enzymes but were suppressed by a strongly pathogenic PSTVd variant. Contrary to that, PSTVd's effect was inhibited by the silencing suppressor p19. It was found that in both, Solanum lycopersicum leaves and N. benthamiana leaf sectors, SlMyb mRNA degradation was significantly stronger in viroid-infected tissues. Necroses induction as well as gene silencing experiments using the SANT/HTH-Myb homologues revealed involvement of this Myb in physiological changes like distortions in flower morphogenesis and growth suppression.

λ-Carrageenan Suppresses Tomato Chlorotic Dwarf Viroid (TCDVd) Replication and Symptom Expression in Tomatoes.

The effect of carrageenans on tomato chlorotic dwarf viroid (TCDVd) replication and symptom expression was studied. Three-week-old tomato plants were spray-treated with iota(ɩ)-, lambda(λ)-, and kappa(κ)-carrageenan at 1 g·L−1 and inoculated with TCDVd after 48 h. The λ-carrageenan significantly suppressed viroid symptom expression after eight weeks of inoculation, only 28% plants showed distinctive bunchy-top symptoms as compared to the 82% in the control group. Viroid concentration was reduced in the infected shoot cuttings incubated in λ-carrageenan amended growth medium. Proteome analysis revealed that 16 tomato proteins were differentially expressed in the λ-carrageenan treated plants. Jasmonic acid related genes, allene oxide synthase (AOS) and lipoxygenase (LOX), were up-regulated in λ-carrageenan treatment during viroid infection. Taken together, our results suggest that λ-carrageenan induced tomato defense against TCDVd, which was partly jasmonic acid(JA) dependent, and that it could be explored in plant protection against viroid infection.