Sequence analysis of five new field isolates demonstrates that the chain length of potato spindle tuber viroid (PSTVd) is not strictly conserved but as variable as in other viroids

The sequence analysis of five new field isolates of potato spindle tuber viroid (PSTVd) of different virulence revealed that the lengthof their RNA chain is not strictly conserved to 359 nucleotides (nts), as one could have inferred from the previously sequenced PSTVd strains. It was now found that the chain length is strain-specific like in the case of practically all other viroids, and that it may vary, so far, between 356 and 360 nts. Taking our previously sequenced and least virulent mild strain PSTVd KF6-M as standard, the new mild strains PSTVd WA-M and PSTVd F-M differ from it by one or two nts. The new intermediate-severe strains PSTVd F-IS and PSTV-F 88-IS differ from the standard mild strain by eight and nine nts, respectively, whereas the new severe-lethal strain PSTVd F-SL differes in seven nts. Most of these mutations are located within the virulence-modulating (VM) region and within the variable region (VR), and only in two strains a single mutation is found in the right terminal domain.     

Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA

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.     

Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA

Trans-cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg(2+) concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg(2+). Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons.     

Potato spindle tuber viroid strains of different pathogenicity induces and suppresses expression of common and unique genes in infected tomato

Viroids are the smallest plant pathogens. These RNAs do not encode proteins and are not encapsidated, and yet they can replicate autonomously, move systemically, and cause diseases in infected plants. Notably, strains of a viroid with subtle differences in nucleotide sequences can cause dramatically different symptoms in infected plants. These features make viroids unique probes to investigate the role of a pathogenic RNA genome in triggering host responses. We conducted a comprehensive analysis of the differential gene expression patterns of tomato plants at various stages of infection by a mild and severe strain of Potato spindle tuber viroid (PSTVd). We also compared tomato gene expression altered by the PSTVd strains with that altered by Tobacco mosaic virus (TMV). Our analyses revealed that the two PSTVd strains altered expression of both common and unique tomato genes. These genes encode products involved in defense/stress response, cell wall structure, chloroplast function, protein metabolism, and other diverse functions. Five genes have unknown functions. Four genes are novel. The expression of some but not all of these genes was also altered by TMV infection. Our results indicate that viroids, although structurally simple, can trigger complex host responses. Further characterization of viroid-altered gene expression in a host plant should help understand viroid pathogenicity and, potentially, the mechanisms of RNA-mediated regulation of plant gene expression.     

Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV)

Sequence analysis by primer-extension at the level of their cDNA showed that the RNA genomes of various field isolates of potato spindle tuber viroid (PSTV) of different virulence differ from each other only in a few nucleotides in two distinct regions of the rod-shaped molecule. Despite insertions and deletions the chain length of 359 nucleotides is strictly conserved in all the isolates studied. Thermodynamic calculations revealed that due to the observed sequence differences the region located at the left hand part of the rod-like secondary structure of the PSTV molecule, denoted 'virulence modulating (VM) region', becomes increasingly unstable with the increasing virulence of the corresponding isolate. Based on these data we propose in molecular terms a model for the mechanism of viroid pathogenicity. It implies that the nucleotides of the VM region specify and modulate the binding- and hence the competition-potential of the PSTV RNA molecule for a still unknown host factor(s) and thus determine the virulence of PSTV.     

Analysis of DNA bending by transient electric birefringence

Transient electric birefringence has been used to analyze DNA bending in six restriction fragments containing 171, 174, 207, 263, 289, and 471 bp in three different low ionic strength buffers. The target fragments contain sequences corresponding to the apparent bend centers in pUC19 and Litmus 28, previously identified by the circular permutation assay (Strutz, K.; Stellwagen, N. C. Electrophoresis 1996, 17, 989-995). The target fragments migrate anomalously slowly in polyacrylamide gels and exhibit birefringence relaxation times that are shorter than those of restriction fragments of the same size, taken from nonbent regions of the same plasmids. Apparent bend angles ranging from 30 degrees to 41 degrees were calculated for the target fragments by tau-ratio method. The bend angles of four of the target fragments were independent of temperature from 4 degrees C to 20 degrees C, but decreased when the temperature was increased to 37 degrees C. The bend angles of the other two target fragments were independent of temperature over the entire range examined, 4 degrees -37 degrees C. Hence, the thermal stability of sequence-dependent bends in random-sequence DNA is variable. The bend angles of five of the six target fragments were independent of the presence or absence of Mg2+ ions in the solution, indicating most of the target fragments were stably bent or curved, rather than anisometrically flexible. Restriction fragments containing 219 and 224 bp, with sequences somewhat offset from the sequence of the 207 bp fragment, were also studied. Comparison of the tau-ratios of these overlapping fragments allowed both the bend angle and bend position to be independently determined. These methods should be useful for analyzing sequence-dependent bending in other random-sequence DNAs.     

The (beta)gamma subunits of G proteins gate a K(+) channel by pivoted bending of a transmembrane segment

The molecular mechanism of ion channel gating remains unclear. Using approaches such as proline scanning mutagenesis and homology modeling, we localize the gate of the K(+) channels controlled by the (beta)gamma subunits of G proteins at the pore-lining bundle crossing of the second transmembrane (TM2) helices. We show that the flexibility afforded by a highly conserved glycine residue in the middle of TM2 is crucial for channel gating. In contrast, flexibility introduced immediately below the gate disrupts gating. We propose that the force produced by channel-G(beta)gamma interactions is transduced through the rigid region below the helix bundle crossing to bend TM2 at the glycine that serves as a hinge and open the gate.     

RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct

Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (–)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.     

Nuclear targeting by fragmentation of the potato spindle tuber viroid genome

Transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a Potato virus X-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide Potato spindle tuber viroid (PSTVd) RNA genome. To further delimit the putative nuclear-targeting signal, PSTVd subgenomic fragments were embedded within the intron, and recombinant reporter RNAs were inoculated onto Nicotiana benthamiana plants. Appearance of green fluorescence in leaf tissue inoculated with PSTVd-fragment-containing constructs indicated shuttling of the RNA into the nucleus by fragments as short as 80 nucleotides in length. Plant-to-plant variation in the timing of intron removal and subsequent GFP fluorescence was observed; however, earliest and most abundant GFP expression was obtained with constructs containing the conserved hairpin I palindrome structure and embedded upper central conserved region. Our results suggest that this conserved sequence and/or the stem-loop structure it forms is sufficient for import of PSTVd into the nucleus.     

Dynamics of transfer RNAs analyzed by normal mode calculation.

Normal mode calculation is applied to tRNAPhe and tRNAAsp, and their structural and vibrational aspects are analyzed. Dihedral angles along the phosphate-ribose backbone (alpha, beta, gamma, epsilon, zeta) and dihedral angles of glycosyl bonds (chi) are selected as movable parameters. The calculated displacement of each atom agrees with experimental data. In modes with frequencies higher than 130 cm-1, the motions are localized around each stem and the elbow region of the L-shape. On the other hand, collective motions such as bending or twisting of arms are seen in modes with lower frequencies. Hinge axes and bend angles are calculated without prior knowledge. Movements in modes with very low frequencies are combinations of hinge bending motions with various hinge axes and bend angles. The thermal fluctuations of dihedral angles well reflect the structural characters of transfer RNAs. There are some dihedral angles of nucleotides located around the elbow region of L-shape, which fluctuate about five to six times more than the average value. Nucleotides in the position seem to be influential in the dynamics of the entire structure. The normal mode calculation seems to provide much information for the study of conformational changes of transfer RNAs induced by aminoacyl-tRNA synthetase or codon during molecular recognition.     

Probing Na(+)-induced changes in the HIV-1 TAR conformational dynamics using NMR residual dipolar couplings: new insights into the role of counterions and electrostatic interactions in adaptive recognition

Many regulatory RNAs undergo large changes in structure upon recognition of proteins and ligands, but the mechanism by which this occurs remains poorly understood. Using NMR residual dipolar coupling (RDCs), we characterized Na+-induced changes in the structure and dynamics of the bulge-containing HIV-1 transactivation response element (TAR) RNA that mirrors changes induced by small molecules bearing a different number of cationic groups. Increasing the Na+ concentration from 25 to 320 mM led to a continuous reduction in the average inter-helical bend angle (from 46 degrees to 22 degrees ), inter-helical twist angle (from 66 degrees to -18 degrees ), and inter-helix flexibility (as measured by an increase in the internal generalized degree of order from 0.56 to 0.74). Similar conformational changes were observed with Mg2+, indicating that nonspecific electrostatic interactions drive the conformational transition, although results also suggest that Na+ and Mg2+ may associate with TAR in distinct modes. The transition can be rationalized on the basis of a population-weighted average of two ensembles comprising an electrostatically relaxed bent and flexible TAR conformation that is weakly associated with counterions and a globally rigid coaxial conformation that has stronger electrostatic potential and association with counterions. The TAR inter-helical orientations that are stabilized by small molecules fall around the metal-induced conformational pathway, indicating that counterions may help predispose the TAR conformation for target recognition. Our results underscore the intricate sensitivity of RNA conformational dynamics to environmental conditions and demonstrate the ability to detect subtle conformational changes using NMR RDCs.     

A chimeric satellite transgene sequence is inefficiently targeted by viroid-induced DNA methylation in tobacco

In plants, transgenes containing Potato spindle tuber viroid (PSTVd) cDNA sequences were efficient targets of PSTVd infection-mediated RNA-directed DNA methylation. Here, we demonstrate that in PSTVd-infected tobacco plants, a 134 bp PSTVd fragment (PSTVd-134) did not become densely methylated when it was inserted into a chimeric Satellite tobacco mosaic virus (STMV) construct. Only about 4–5% of all cytosines (Cs) of the PSTVd-134 were methylated when flanked by satellite sequences. In the same plants, C methylation was approximately 92% when the PSTVd-134 was in a PSTVd full length sequence context and roughly 33% when flanked at its 3′ end by a 19 bp PSTVd and at its 5′ end by a short viroid-unrelated sequence. In addition, PSTVd small interfering RNAs (siRNAs) produced from the replicating viroid failed to target PSTVd-134-containing chimeric STMV RNA for degradation. Satellite RNAs appear to have adopted secondary structures that protect them against RNA interference (RNAi)—mediated degradation. Protection can be extended to short non-satellite sequences residing in satellite RNAs, rendering them poor targets for nuclear and cytoplasmic RNAi induced in trans.     

Conformational effects of Gly-X-Gly interruptions in the collagen triple helix

The collagen model peptide with sequence (Pro-Hyp-Gly)4-Pro-Gly-(Pro-Hyp-Gly)5 contains a central Gly-Pro-Gly interruption in the consensus collagen sequence. Its high-resolution crystal structure defines the molecular consequences of such an interruption for the collagen triple-helical conformation, and provides insight into possible structural and biological roles of similar interruptions in the -Gly-X-Y- repeating pattern found in non-fibrillar collagens. The peptide (denoted as the Hyp minus peptide or Hyp-) forms a rod-like triple helix structure without any bend or kink, and crystallizes in a quasi-hexagonal lattice. The two Pro-Hyp-Gly zones adopt the typical triple-helical collagen conformation with standard Rich and Crick II hydrogen bonding topology. Notably, the central zone containing the Gly-Pro-Gly interruption deviates from the standard structure in terms of hydrogen bonding topology, torsion angles, helical, and superhelical parameters. These deviations are highly localized, such that the standard features are regained within one to two residues on either side. Conformational variations and high temperature factors seen for the six chains of the asymmetric unit in the zone around the interruption point to the presence of a local region of considerable plasticity and flexibility embedded within two highly rigid and ordered standard triple-helical segments. The structure suggests a role for Gly-X-Gly interruptions as defining regions of flexibility and molecular recognition in the otherwise relatively uniform repeating collagen conformation.     

Biological and molecular analysis of the pathogenic variant C3 of potato spindle tuber viroid (PSTVd) evolved during adaptation to chamomile (Matricaria chamomilla)

Abstract Viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small RNAs (vsRNAs). We describe a new PSTVd variant C3, evolved through sequence adaptation to the host chamomile (Matricaria chamomilla) after biolistic inoculation with PSTVd-KF440-2, which causes extraordinary strong ('lethal') symptoms. The deletion of a single adenine A in the oligoA stretch of the pathogenicity (P) domain appears characteristic of PSTVd-C3. The pathogenicity and the vsRNA pool of PSTVd-C3 were compared to those of lethal variant PSTVd-AS1, from which PSTVd-C3 differs by five mutations located in the P domain. Both lethal viroid variants showed higher stability and lower variation in analyzed vsRNA pools than the mild PSTVd-QFA. PSTVd-C3 and -AS1 caused similar symptoms on chamomile, tomato, and Nicotiana benthamiana, and exhibited similar but species-specific distributions of selected vsRNAs as quantified using TaqMan probes. Both lethal PSTVd variants block biosynthesis of lignin in roots of cultured chamomile and tomato. Four 'expression markers' (TCP3, CIPK, VSF-1, and VPE) were selected from a tomato EST library to quantify their expression upon viroid infection; these markers were strongly downregulated in tomato leaf blades infected by PSTVd-C3- and -AS1 but not by PSTVd-QFA.     

The genetic stability of potato spindle tuber viroid (PSTVd) molecular variants.

RNA viruses propagate as a population of genetically related entities composing a quasi-species. Specific representatives are the result of both a high mutation rate during replication and competition between the continuously arising sequence variants. Similar to other RNA pathogens, potato spindle tuber viroid (PSTVd) propagates as a population of similar but nonidentical sequences. The sequence of progeny molecules derived from cloned molecular variants of PSTVd were studied after one and six consecutive plant passages. Although the severe parental sequence S23 was found to be genetically stable, all five other parental sequences analyzed, irrespective of their pathogenicity, led to the appearance of complex populations. Divergence of the progeny was observed at the sequence level, but also, more surprisingly, at the level of the pathogenicity of individual progeny molecules. In two cases, the parental sequence was retained in the progeny population. In the other cases, it was completely out-competed and eliminated, sometimes in as little as one plant passage. Although it has been observed previously that artificially mutated PSTVd molecules may revert rapidly to the wild-type sequence, this study presents direct evidence for the rapid evolution of naturally occurring PSTVd sequence variants.