Subviral Plant Pathogens Research Articles

Viroids, infectious long non-coding RNAs with autonomous replication

Transcriptome deep-sequencing studies performed during the last years confirmed that the vast majority of the RNAs transcribed in higher organisms correspond to several types of non-coding RNAs including long non-coding RNAs (lncRNAs). The study of lncRNAs and the identification of their functions, is still an emerging field in plants but the characterization of some of them indicate that they play an important role in crucial regulatory processes like flowering regulation, and responses to abiotic stress and plant hormones. A second group of lncRNAs present in plants is formed by viroids, exogenous infectious subviral plant pathogens well known since many years. Viroids are composed of circular RNA genomes without protein-coding capacity and subvert enzymatic activities of their hosts to complete its own biological cycle. Different aspects of viroid biology and viroid-host interactions have been elucidated in the last years and some of them are the main topic of this review together with the analysis of the state-of-the-art about the growing field of endogenous lncRNAs in plants.

Intronic hammerhead ribozymes in mRNA biogenesis

Small self-cleaving ribozymes are a group of natural RNAs that are capable of catalyzing their own and sequence-specific endonucleolytic cleavage. One of the most studied members is the hammerhead ribozyme (HHR), a catalytic RNA originally discovered in subviral plant pathogens but recently shown to reside in a myriad of genomes along the tree of life. In eukaryotes, most of the genomic HHRs seem to be related to short interspersed retroelements, with the main exception of a group of strikingly conserved ribozymes found in the genomes of all amniotes (reptiles, birds and mammals). These amniota HHRs occur in the introns of a few specific genes, and clearly point to a preserved biological role during pre-mRNA biosynthesis. More specifically, bioinformatic analysis suggests that these intronic ribozymes could offer a new form of splicing regulation of the mRNA of higher vertebrates. We review here the latest advances in the discovery and biological characterization of intronic HHRs of vertebrates, including new conserved examples in the genomes of the primitive turtle and coelacanth fish.

Genomisch codierte Hammerhead-Ribozyme

The hammerhead ribozyme is a small RNA motif capable to cleave RNA endonucleolytically. Originally discovered in sub-viral plant pathogens, recent studies have uncovered the presence of this ribozyme in an unprecedented number of bacterial and eukaryal genomes. These findings have evolutionary implications on the origin of this ribozyme and pose the question of the biological function of the uncovered genomic motifs.

The ubiquitous hammerhead ribozyme

The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs.

From alpaca to zebrafish: Hammerhead ribozymes wherever you look

The hammerhead ribozyme was originally discovered in subviral plant pathogens and was subsequently also found in a few other genomic locations. Using a secondary structure-based descriptor, we have searched publicly accessible sequence databases for new examples of type III hammerhead ribozymes. The more than 60,000 entries fulfilling the descriptor were filtered with respect to folding and stability parameters that were experimentally validated. This resulted in a set of 284 unique motifs, of which 124 represent database entries of known hammerhead ribozymes from subviral plant pathogens and A. thaliana. The remainder are 160 novel ribozyme candidates in 50 different eukaryotic genomes. With a few exceptions, the ribozymes were found either in repetitive DNA sequences or in introns of protein coding genes. Our data, which is complementary to a study by De la Peña and García-Robles in 2010, indicate that the hammerhead is the most abundant small endonucleolytic ribozyme, which, in view of no sequence conservation beyond the essential nucleotides, likely has evolved independently in different organisms.

Analysis of symptoms developed in Nicotiana benthamiana plants expressing dimeric forms of Hop stunt viroid.

Viroids are sub-viral plant pathogens consisting of covalently closed circular RNAs that infect herbaceous and woody hosts. They do not code for any peptide or protein and therefore are fully dependent on the host machinery for most biological processes, including pathogenesis. Certain viroid-host combinations show severe symptoms but the biochemical bases of this process are currently unknown. In this work, we have characterized the symptoms induced by Hop stunt viroid (HSVd) when provided as a transgene in Nicotiana benthamiana plants, a natural non-host that is able to process and translocate HSVd. The presence of HSVd in this transgenic plant causes severe stunting and decrease in the leaf size, followed by strong alteration of flower development and reduced fertility. These symptoms are similar to those induced by HSVd in natural infections, indicating that in N. benthamiana HSVd expression mimics the metabolic disorders induced in natural hosts. Therefore, these transgenic plants may be useful to study the viroid-host interactions leading to pathogenesis.

Idiosyncratic cleavage and ligation activity of individual hammerhead ribozymes and core sequence variants thereof

The hammerhead ribozyme is a small RNA endonuclease found in sub-viral plant pathogens, in transcripts from certain animal satellite DNAs and encoded at distinct loci of Arabidopsis thaliana. Kinetic analyses of tertiary stabilised ribozymes from peach latent mosaic viroid (PLMVd), Schistosoma mansoni and A. thaliana revealed a ten-fold difference in cleavage rates. Core nucleotide variations affected cleavage reactions least in the A. thaliana ribozyme, and most in the S. mansoni ribozyme. The reverse ligation reaction was catalysed efficiently by the PLMVd and A. thaliana ribozymes. The different behaviour of the individual hammerhead ribozymes is discussed in terms of structure and function.

Functional Hammerhead Ribozymes Naturally Encoded in the Genome of Arabidopsis thaliana

The hammerhead ribozyme (HHRz) is an autocatalytic RNA motif found in subviral plant pathogens and transcripts of repetitive DNA sequences in animals. Here, we report the discovery and characterization of unique HHRzs encoded in a plant genome. Two novel sequences were identified on chromosome IV of Arabidopsis thaliana in a database search, which took into account recently defined structural requirements. The HHRzs are expressed in several tissues and coexist in vivo as both cleaved and noncleaved species. In vitro, both sequences cleave efficiently at physiological Mg(2+) concentrations, indicative of functional loop-loop interactions. Kinetic analysis of loop nucleotide variants was used to determine a three-dimensional model of these tertiary interactions. Based on these results, on the lack of infectivity of hammerhead-carrying viroids in Arabidopsis, and on extensive sequence comparisons, we propose that the ribozyme sequences did not invade this plant by horizontal transfer but have evolved independently to perform a specific, yet unidentified, biological function.

A database search for hammerhead ribozyme motifs

The hammerhead ribozyme is the smallest naturally occurring RNA endonuclease. It is found in subviral plant pathogens and transcripts of satellite DNA from a limited number of organisms. We have performed a database search for novel examples of this catalytic RNA, taking into consideration the recently defined structural requirements for an efficient cleavage under physiological magnesium ion concentrations. In this search, we find, apart from the known examples, several hundreds of motifs in organisms of all kingdoms of life. In a first set of experiments, we analysed hammerhead ribozymes from Arabidopsis thaliana. We found that these sequences are tissue-specifically expressed and that they undergo self-cleavage in planta. Furthermore, their activity under physiological magnesium ion concentrations depends on functional loop-loop interactions, as shown by the lack of activity of appropriate mutants.

A naked plant-specific RNA ten-fold smaller than the smallest known viral RNA: the viroid

Viroids are subviral plant pathogens at the frontier of life. They are solely composed by a single-stranded circular RNA of 246–401 nt with a compact secondary structure. Viroids replicate autonomously when inoculated into their host plants and incite, in most of them, economically important diseases. In contrast to viruses, viroids do not code for any protein and depend on host enzymes for their replication, which in some viroids occurs in the nucleus and in others in the chloroplast, through a rolling-circle mechanism with three catalytic steps. Quite remarkably, however, one of the steps, cleavage of the oligomeric head-to-tail replicative intermediates to unit-length strands, is mediated in certain viroids by hammerhead ribozymes that can be formed by their strands of both polarities. Viroids induce disease by direct interaction with host factors, the nature of which is presently unknown. Some properties of viroids, particularly the presence of ribozymes, suggest that they might have appeared very early in evolution and could represent ˈliving fossilsˈ of the precellular RNA world that presumably preceded our current world based on DNA and proteins.

Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group.

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.

Diagnosis and typing of plant viruses by immunocapture RT-PCR coupled to a fluorogenic exonuclease assay

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been succesfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.

A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been succesfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.

Subviral pathogens of plants: viroids and viroidlike satellite RNAs

Contrary to earlier beliefs, viruses are not the smallest causative agents of infectious diseases. Single-stranded RNAs as small as 246 nucleotides exist in certain higher plants and cause more than a dozen crop diseases. These RNAs have been termed viroids. Despite their extremely limited information content, viroids replicate autonomously in susceptible cells--that is, they do not require helper functions from simultaneously replicating conventional viruses. Viroids are covalently closed circular molecules with a characteristic rodlike secondary structure in which short helical regions are interrupted by internal and bulge loops. Viroids are not translated; they are replicated by a host enzyme (or enzymes) (probably RNA polymerase II) via oligomeric RNA intermediates by a rolling circle mechanism. Viroidlike satellite RNAs resemble viroids in size and molecular structure, but are found within the capsids of specific helper viruses on which they depend for their own replication. These RNAs are of great interest to molecular biology for at least two reasons: 1) they are the smallest and simplest replicating molecules known, and 2) they may represent living fossils of precellular evolution in a hypothetical RNA world.

Book reviews

Books Reviewed in this Article: Ecology and Management of Soilborne Plant Pathogens, edited by C. A. Parker. AD. Rovira, K. J. Moore, P. T. W. Wong and J. F. Kollmorgen. Subviral Pathogens of Plants and Animals: Viroids and Prions, edited by K. Maramorosch and J. J. McKelvey Jr. The Biology and Technology of the Cultivated Mushroom, edited by P. B. Flegg. D. M. Spencer and D. A. Wood.