B. Navarro

Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection

The discovery of viroids about 45 years ago heralded a revolution in Biology: small RNAs comprising around 350 nt were found to be able to replicate autonomously-and to incite diseases in certain plants-without encoding proteins, fundamental properties discriminating these infectious agents from viruses. The initial focus on the pathological effects usually accompanying infection by viroids soon shifted to their molecular features-they are circular molecules that fold upon themselves adopting compact secondary conformations-and then to how they manipulate their hosts to be propagated. Replication of viroids-in the nucleus or chloroplasts through a rolling-circle mechanism involving polymerization, cleavage and circularization of RNA strands-dealt three surprises: (i) certain RNA polymerases are redirected to accept RNA instead of their DNA templates, (ii) cleavage in chloroplastic viroids is not mediated by host enzymes but by hammerhead ribozymes, and (iii) circularization in nuclear viroids is catalyzed by a DNA ligase redirected to act upon RNA substrates. These enzymes (and ribozymes) are most probably assisted by host proteins, including transcription factors and RNA chaperones. Movement of viroids, first intracellularly and then to adjacent cells and distal plant parts, has turned out to be a tightly regulated process in which specific RNA structural motifs play a crucial role. More recently, the advent of RNA silencing has brought new views on how viroids may cause disease and on how their hosts react to contain the infection; additionally, viroid infection may be restricted by other mechanisms. Representing the lowest step on the biological size scale, viroids have also attracted considerable interest to get a tentative picture of the essential characteristics of the primitive replicons that populated the postulated RNA world.

FIRST REPORT OF CHRYSANTHEMUM STUNT VIROID IN ARGYRANTHEMUM FRUTESCENS IN ITALY

Chrysanthemum stunt viroid (CSVd) is a quarantine pathogen for chrysanthemum (Dendranthema spp.) in the European countries (Plant Health Directive 2000/29/EC), because this host is severely affected, thus comes down with a disease characterized by stunting, leaf chlorosis and floral disorders. CSVd spread in Europe has efficiently been restrained so far, although several outbreaks were recorded in the past. Here we report the first occurrence of CSVd in Italy, as detected in several symptomless cultivars of Argyranthemum frutescens (marguerite daisy) by RT-PCR with specific primers and by Northern blot hybridization with a specific digoxigenin-labeled riboprobe. Viroid identity was ultimately ascertained by cloning and sequencing cDNA amplicons. Molecular characterization of CSVd isolates from six different A. frutescens cultivars disclosed viroid RNA populations with a prevalent size of 354 nt and sequences 98-100% identical to those of CSVd variants reported previously from D. grandiflora and A. frutescens. These results call for a prompt extension of surveys for assessing the presence of CSVd in symptomless A. frutescens and other ornamentals, which could constitute hidden reservoirs of this pathogen. In view of this, a tissue-printing hybridization method for detecting CSVd in A. frutescens was tested and validated.

Survey on viroids infecting grapevine in Italy: identification and characterization of Australian grapevine viroid and Grapevine yellow speckle viroid 2

Five viroid species have been reported from grapevine. Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1) are distributed worldwide, whereas Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd) and Citrus exocortis viroid (CEVd) are found only sporadically. However, the presence of AGVd and GYSVd-2 in several countries, including China, Turkey and Tunisia, suggests a wider dissemination, possibly also in Europe, where AGVd has never been found and GYSVd-2 has been occasionally identified in Italy. Taking advantage of a multiplex RT-PCR assay recently developed for detecting simultaneously these five viroids, vines growing in Italy in commercial vineyards and germplasm collections were surveyed. Besides confirming the widespread presence of HSVd and GYSVd-1 in the field, GYSVd-2 and/or AGVd were identified in two grapevine table cultivars (Sultanina Bianca and Red Globe) from germplasm collections. Tests extended to vines cultivated in southern Italy confirmed the presence of both viroids, which were further characterized. No major sequence divergences between the AGVd and GYSVd-2 variants from Italy and those previously described from other countries were observed. Phylogenetic analysis supported the close relationships among AGVd variants from Italy, Tunisia and Australia. To our knowledge this is the first report of AGVd in Europe and the first molecular characterization of GYSVd-2 isolates from a European country.

GRAPEVINE VIROIDS AND GRAPEVINE FANLEAF VIRUS IN NORTH- WEST IRAN

Grapevine-infecting viroids do not induce symptoms, except for Grapevine yellow speckle viroid-1 (GYSVd-1) and Grapevine yellow speckle viroid-2 (GYSVd-2), the agents of yellow speckle (YS), a disease characterized by yellow spots or flecks scattered on the leaf blade. The association of these viroids with Grapevine fanleaf virus (GFLV) is thought to elicit vein banding (VB), a syndrome characterized by chrome yellow flecks localized along the main veins and progressing into the interveinal areas of affected vines. The occurrence of these diseases and their causal agents was investigated in north-west Iran with a survey in which 137 vines were tested by multiplex RT-PCR for the presence of the five known grapevines viroids. GYSVd-1, GYSVd-2, Australian grapevine viroid (AGVd) and Hop stunt viroid (HSVd) were detected in 91%, 64%, 95%, and 100% of the tested samples, respectively, whereas Citrus exocortis viroid (CEVd) was not found. Combinations of three and four different viroids were present in most plants (88%) whereas GFLV was found in 50 samples (37%). The Iranian isolates of GYSVd-1, GYSVd-2, HSVd and AGVd showed minor molecular changes compared with the respective reference strains from grapevine. VB occurred in 22 vines infected by GYSVd-1, GYSVd-2 and GFLV, whereas YS symptoms, which occurred in 10% of the tested plants, were always shown by vines infected by GYSVd-1 and/or GYSVd-2. These findings are in line with the notion that assigns to GYSVd-1 and GYSVd-2 a role in the induction of YS and to both viroids and GFLV the genesis of VB.

Origin and Evolution of Viroids

With their small, circular, nonprotein-coding RNA genome, which may be endowed with catalytic activity, viroids have been proposed to be “fossils” of an RNA world preceding the cellular world based on DNA and proteins. This chapter summarizes the arguments sustaining this proposal and discusses the possible evolutionary scenarios for the adaptation of ancestor viroids to a cellular environment. The implications of the quasispecies nature of viroid populations and the constraints governing their evolution are also addressed, highlighting how a deeper understanding of viroid evolution is closely linked to advances in the molecular mechanisms mediating plant–viroid interactions.

Viroids Infecting the Grapevine

Viroids are nonprotein-coding, small, circular RNAs infecting plants in which they may induce specific symptoms. Five different viroids have been identified in the grapevine in the period elapsed from 1985 to 1990. Since then, no new viroid has been reported from grapevines until the application of next-generation sequencing allowed the discovery of an additional viroid and a new viroid-like RNA. Possibly, new small, circular RNAs will be identified in the future by metagenomic approaches, but bioassays, which are time intensive and require phytopathological expertise, will always be needed for establishing conclusively their true identity as viroids. Although viroids generally do not elicit severe symptoms in grapevines, some of them are the agent of diseases in certain environmental conditions or in combination with certain viruses. Some of grapevine-infecting viroids may cause severe diseases in other crops. This chapter reviews the molecular, biological, and epidemiological features of viroids and viroid-like RNAs infecting grapevines and the methods for their detection and control and discusses the future perspectives of research.

The role of plant viroids in diseases - new developments

Work in B. Navarro’s and F. Di Serio’s laboratory has been supported by a grant from the Italian Ministry of the Agriculture (OIGA-‘PSTVd-free’ 2009–2011) and a dedicated grant from the Italian Ministry of Economy and Finance to the CNR (Legge no. 191/2009), and in R. Flores’s laboratory by the Ministerio de Ciencia e Innovacio´n of Spain (grant nos. BFU2008-03154 and BFU2011-28443).

Chapter 9 – Viroid Pathogenesis

The small size and noncoding nature of viroid RNAs raises intriguing and specific questions about how they induce disease. The absence of viroid-encoded proteins, in sharp contrast to viruses, led originally to the assumption that viroid diseases were the result of direct interaction of the genomic viroid RNA (or its complement) with cellular constituents. Recently, however, the possible involvement of indirect interactions via RNA silencing and other existing regulatory networks has received increasing attention. This chapter summarizes early views and discusses newly available evidence that RNA silencing directed by viroid-derived small RNAs inhibits, at least in some cases, both the spread of viroid infection and the expression of key host genes that ultimately may result in symptom induction. Viroid infection is also accompanied by changes in the levels of certain microRNAs as well as other host-encoded small RNAs derived from ribosomal RNA transcripts. Clearly, viroid pathogenesis engages multiple regulatory networks, thus demanding network-based experimental strategies to complement the current gene-by-gene approaches.

Small Circular Satellite RNAs

Small circular satellite RNAs (sc-satRNAs) assume compact conformations, display catalytic activity mediated by hammerhead or hairpin ribozymes, and, in most cases, do not code for proteins, thus resembling viroids in several structural and functional features. However, in contrast to viroids, and similarly to other viral satellites, sc-satRNAs rely on a helper virus for replication and transmission. sc-satRNAs may modulate helper virus accumulation and symptomatology through unknown molecular mechanisms including RNA silencing.