ICTV Virus Taxonomy Profile: Pospiviroidae.

Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/pospiviroidae.

Silencing of transcription factor encoding gene StTCP23 by small RNAs derived from the virulence modulating region of potato spindle tuber viroid is associated with symptom development in potato.

Viroids are small, non-protein-coding RNAs which can induce disease symptoms in a variety of plant species. Potato (Solanum tuberosum L.) is the natural host of Potato spindle tuber viroid (PSTVd) where infection results in stunting, distortion of leaves and tubers and yield loss. Replication of PSTVd is accompanied by the accumulation of viroid-derived small RNAs (sRNAs) proposed to play a central role in disease symptom development. Here we report that PSTVd sRNAs direct RNA silencing in potato against StTCP23, a member of the TCP (teosinte branched1/Cycloidea/Proliferating cell factor) transcription factor family genes that play an important role in plant growth and development as well as hormonal regulation, especially in responses to gibberellic acid (GA). The StTCP23 transcript has 21-nucleotide sequence complementarity in its 3' untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE) confirmed cleavage of StTCP23 transcript at the expected sites within the complementarity with VMR-derived sRNAs. Expression of these VMR sRNA sequences as artificial miRNAs (amiRNAs) in transgenic potato plants resulted in phenotypes reminiscent of PSTVd-RG1-infected plants. Furthermore, the severity of the phenotypes displayed was correlated with the level of amiRNA accumulation and the degree of amiRNA-directed down-regulation of StTCP23. In addition, virus-induced gene silencing (VIGS) of StTCP23 in potato also resulted in PSTVd-like phenotypes. Consistent with the function of TCP family genes, amiRNA lines in which StTCP23 expression was silenced showed a decrease in GA levels as well as alterations to the expression of GA biosynthesis and signaling genes previously implicated in tuber development. Application of GA to the amiRNA plants minimized the PSTVd-like phenotypes. Taken together, our results indicate that sRNAs derived from the VMR of PSTVd-RG1 direct silencing of StTCP23 expression, thereby disrupting the signaling pathways regulating GA metabolism and leading to plant stunting and formation of small and spindle-shaped tubers.

Effects of Host-Adaptive Mutations on Hop Stunt Viroid Pathogenicity and Small RNA Biogenesis.

Accidental transmission of hop stunt viroid (HSVd) from grapevine to hop has led to several epidemics of hop stunt disease with convergent evolution of HSVd-g(rape) into HSVd-h(op) containing five mutations. However, the biological function of these five mutations remains unknown. In this study, we compare the biological property of HSVd-g and HSVd-h by bioassay and analyze HSVd-specific small RNA (HSVd-sRNA) using high-throughput sequencing. The bioassay indicated an association of these five mutations with differences in infectivity, replication capacity, and pathogenicity between HSVd-g and HSVd-h, e.g., HSVd-g induced more severe symptoms than HSVd-h in cucumber. Site-directed mutagenesis of HSVd-g showed that the mutation at position 54 increased pathogenicity. HSVd-sRNA analysis of cucumber and hop plants infected with different HSVd variants showed that several sRNA species containing adaptive nucleotides were specifically down-regulated in plants infected with HSVd-h. Several HSVd-sRNAs containing adaptive mutations were predicted to target cucumber genes, but changes in the levels of these genes were not directly correlated with changes in symptom expression. Furthermore, expression levels of two other cucumber genes targeted by HSVd-RNAs, encoding ethylene-responsive transcription factor ERF011, and trihelix transcription factor GTL2, were altered by HSVd infection. The possible relationship between these two genes to HSVd pathogenicity is discussed.

ICTV Virus Taxonomy Profile: Avsunviroidae.

Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA-RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.

Processing of Potato Spindle Tuber Viroid RNAs in Yeast, a Nonconventional Host.

Potato spindle tuber viroid (PSTVd) is a circular, single-stranded, noncoding RNA plant pathogen that is a useful model for studying the processing of noncoding RNA in eukaryotes. Infective PSTVd circles are replicated via an asymmetric rolling circle mechanism to form linear multimeric RNAs. An endonuclease cleaves these into monomers, and a ligase seals these into mature circles. All eukaryotes may have such enzymes for processing noncoding RNA. As a test, we investigated the processing of three PSTVd RNA constructs in the yeast Saccharomyces cerevisiae Of these, only one form, a construct that adopts a previously described tetraloop-containing conformation (TL), produces circles. TL has 16 nucleotides of the 3' end duplicated at the 5' end and a 3' end produced by self-cleavage of a delta ribozyme. The other two constructs, an exact monomer flanked by ribozymes and a trihelix-forming RNA with requisite 5' and 3' duplications, do not produce circles. The TL circles contain nonnative nucleotides resulting from the 3' end created by the ribozyme and the 5' end created from an endolytic cleavage by yeast at a site distinct from where potato enzymes cut these RNAs. RNAs from all three transcripts are cleaved in places not on path for circle formation, likely representing RNA decay. We propose that these constructs fold into distinct RNA structures that interact differently with host cell RNA metabolism enzymes, resulting in various susceptibilities to degradation versus processing. We conclude that PSTVd RNA is opportunistic and may use different processing pathways in different hosts.IMPORTANCE In higher eukaryotes, the majority of transcribed RNAs do not encode proteins. These noncoding RNAs are responsible for mRNA regulation, control of the expression of regulatory microRNAs, sensing of changes in the environment by use of riboswitches (RNAs that change shape in response to environmental signals), catalysis, and more roles that are still being uncovered. Some of these functions may be remnants from the RNA world and, as such, would be part of the evolutionary past of all forms of modern life. Viroids are noncoding RNAs that can cause disease in plants. Since they encode no proteins, they depend on their own RNA and on host proteins for replication and pathogenicity. It is likely that viroids hijack critical host RNA pathways for processing the host's own noncoding RNA. These pathways are still unknown. Elucidating these pathways should reveal new biological functions of noncoding RNA.

Plant-specific 4/1 polypeptide interacts with an endoplasmic reticulum protein related to human BAP31.

MAIN CONCLUSION: The plant-specific 4/1 protein interacts, both in yeast two-hybrid system and in vitro, and co-localizes in plant cells with plant BAP-like protein, the orthologue of human protein BAP31. In yeast two-hybrid system, we identified a number of Nicotiana benthamiana protein interactors of Nt-4/1, the protein known to affect systemic transport of potato spindle tuber viroid. For one of these interactors, an orthologue of human B-cell receptor-associated protein 31 (BAP31) termed plant BAP-like protein (PBL), the ability to interact with Nt-4/1 was studied in greater detail. Analyses of purified proteins expressed in bacterial cells carried out in vitro with the surface plasmon resonance (SPR) spectroscopy revealed that the N. tabacum PBL (NtPBL) was able to interact with Nt-4/1 with high-affinity, and that their complex can form at physiologically relevant concentrations of both proteins. Subcellular localization studies of 4/1-GFP and NtPBL-mRFP transiently co-expressed in plant cells revealed the co-localization of the two fusion proteins in endoplasmic reticulum-associated bodies, suggesting their interaction in vivo. The N-terminal region of the Nt-4/1 protein was found to be required for the specific subcellular targeting of the protein, presumably due to a predicted amphipathic helix mediating association of the Nt-4/1 protein with cell membranes. Additionally, this region was found to contain a trans-activator domain responsible for the Nt-4/1 ability to activate transcription of a reporter gene in yeast.

Viroid quasispecies revealed by deep sequencing.

Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250-400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than [Formula: see text], quite comparable to the value of [Formula: see text] reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.

Discriminative Stimulus Properties of the Endocannabinoid Catabolic Enzyme Inhibitor SA-57 in Mice.

Whereas the inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the respective major hydrolytic enzymes of N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), elicits no or partial substitution for Δ(9)-tetrahydrocannabinol (THC) in drug-discrimination procedures, combined inhibition of both enzymes fully substitutes for THC, as well as produces a constellation of cannabimimetic effects. The present study tested whether C57BL/6J mice would learn to discriminate the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) from vehicle in the drug-discrimination paradigm. In initial experiments, 10 mg/kg SA-57 fully substituted for CP55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a high-efficacy CB1 receptor agonist in C57BL/6J mice and for AEA in FAAH (-/-) mice. Most (i.e., 23 of 24) subjects achieved criteria for discriminating SA-57 (10 mg/kg) from vehicle within 40 sessions, with full generalization occurring 1 to 2 hours postinjection. CP55,940, the dual FAAH-MAGL inhibitor JZL195 (4-​nitrophenyl 4-​(3-​phenoxybenzyl)piperazine-​1-​carboxylate), and the MAGL inhibitors MJN110 (2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate) and JZL184 (4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) fully substituted for SA-57. Although the FAAH inhibitors PF-3845 ((N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) and URB597 (cyclohexylcarbamic acid 3'-(aminocarbonyl)-[1,1'-biphenyl]-3-yl ester) did not substitute for SA-57, PF-3845 produced a 2-fold leftward shift in the MJN110 substitution dose-response curve. In addition, the CB1 receptor antagonist rimonabant blocked the generalization of SA-57, as well as substitution of CP55,940, JZL195, MJN110, and JZL184. These findings suggest that MAGL inhibition plays a major role in the CB1 receptor-mediated SA-57 training dose, which is further augmented by FAAH inhibition.

Δ9-tetrahydrocannabinol and endocannabinoid degradative enzyme inhibitors attenuate intracranial self-stimulation in mice.

A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and 2-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type 2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on ICSS. Thus, THC, MAGL inhibition, and dual FAAH-MAGL inhibition not only reduce ICSS, but also decrease other reinforced and nonreinforced behaviors.

The Remarkable Legacy of Theodor O. Diener (1921-2023): Preeminent Plant Pathologist and the Discoverer of Viroids.

Theodor ("Ted") Otto Diener, the discoverer of viroids, died on 28 March 2023 at his home in Beltsville, Maryland, USA [...].

Role of RNA silencing in plant-viroid interactions and in viroid pathogenesis.

Viroids are small, single-stranded, non-protein coding and circular RNAs able to infect host plants in the absence of any helper virus. They may elicit symptoms in their hosts, but the underlying molecular pathways are only partially known. Here we address the role of post-transcriptional RNA silencing in plant-viroid-interplay, with major emphasis on the involvement of this sequence-specific RNA degradation mechanism in both plant antiviroid defence and viroid pathogenesis. This review is a tribute to the memory of Dr. Ricardo Flores, who largely contributed to elucidate this and other molecular mechanisms involved in plant-viroid interactions.

Global analysis of tomato gene expression during Potato spindle tuber viroid infection reveals a complex array of changes affecting hormone signaling.

Viroids like Potato spindle tuber viroid (PSTVd) are the smallest known agents of infectious disease-small, highly structured, circular RNA molecules that lack detectable messenger RNA activity, yet are able to replicate autonomously in susceptible plant species. To better understand the possible role of RNA silencing in disease induction, a combination of microarray analysis and large-scale RNA sequence analysis was used to compare changes in tomato gene expression and microRNA levels associated with PSTVd infection in two tomato cultivars plus a third transformed line expressing small PSTVd small interfering RNAs in the absence of viroid replication. Changes in messenger (m)RNA levels for the sensitive cultivar 'Rutgers' were extensive, involving more than half of the approximately 10,000 genes present on the array. Chloroplast biogenesis was down-regulated in both sensitive and tolerant cultivars, and effects on mRNAs encoding enzymes involved in the biosynthesis of gibberellin and other hormones were accompanied by numerous changes affecting their respective signaling pathways. In the dwarf cultivar 'MicroTom', a marked upregulation of genes involved in response to stress and other stimuli was observed only when exogenous brassinosteroid was applied to infected plants, thereby providing the first evidence for the involvement of brassinosteroid-mediated signaling in viroid disease induction.

Lolium latent virus (Alphaflexiviridae) coat proteins: expression and functions in infected plant tissue.

The genome of Lolium latent virus (LoLV; genus Lolavirus, family Alphaflexiviridae) is encapsidated by two carboxy-coterminal coat protein (CP) variants (about 28 and 33 kDa), in equimolar proportions. The CP ORF contains two 5'-proximal AUGs encoding Met 1 and Met 49, respectively promoting translation of the 33 and 28 kDa CP variants. The 33 kDa CP N-terminal domain includes a 42 aa sequence encoding a putative chloroplast transit peptide, leading to protein cleavage and alternative derivation of the approximately 28 kDa CP. Mutational analysis of the two in-frame start codons and of the putative proteolytic-cleavage site showed that the N-terminal sequence is crucial for efficient cell-to-cell movement, functional systemic movement, homologous CP interactions and particle formation, but is not required for virus replication. Blocking production of the 28 kDa CP by internal initiation shows no major outcome, whereas additional mutation to prevent proteolytic cleavage at the chloroplast membrane has a dramatic effect on virus infection.

Blockade of endocannabinoid hydrolytic enzymes attenuates precipitated opioid withdrawal symptoms in mice.

Δ(9)-Tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB(1) and CB(2) cannabinoid receptors, the role these receptors play in reducing the variety of opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this study was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces naloxone-precipitated morphine withdrawal symptoms in in vivo and in vitro models of opioid dependence. Morphine-dependent mice challenged with naloxone reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, diarrhea, and weight loss. THC and the MAGL inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) dose dependently reduced the intensity of most measures through the activation of CB(1) receptors. JZL184 also attenuated spontaneous withdrawal signs in morphine-dependent mice. The FAAH inhibitor N-(pyridin-3-yl)-4-(3-(5-(trifluoromethyl)pyridin-2-yloxy)benzyl)-piperdine-1-carboxamide (PF-3845) reduced the intensity of naloxone-precipitated jumps and paw flutters through the activation of CB(1) receptors but did not ameliorate incidence of diarrhea or weight loss. In the final series of experiments, we investigated whether JZL184 or PF-3845 would attenuate naloxone-precipitated contractions in morphine-dependent ilea. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, although this model does not account mechanistically for the autonomic withdrawal responses (i.e., diarrhea) observed in vivo. These results indicate that endocannabinoid catabolic enzymes are promising targets to treat opioid dependence.

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement.

Cell-to-cell movement of potexviruses requires coordinated action of the coat protein and triple gene block (TGB) proteins. The structural properties of Alternanthera mosaic virus (AltMV) TGB3 were examined by methods differentiating between signal peptides and transmembrane domains, and its subcellular localization was studied by Agrobacterium-mediated transient expression and confocal microscopy. Unlike potato virus X (PVX) TGB3, AltMV TGB3 was not associated with the endoplasmic reticulum, and accumulated preferentially in mesophyll cells. Deletion and site-specific mutagenesis revealed an internal signal VL(17,18) of TGB3 essential for chloroplast localization, and either deletion of the TGB3 start codon or alteration of the chloroplast-localization signal limited cell-to-cell movement to the epidermis, yielding a virus that was unable to move into the mesophyll layer. Overexpression of AltMV TGB3 from either AltMV or PVX infectious clones resulted in veinal necrosis and vesiculation at the chloroplast membrane, a cytopathology not observed in wild-type infections. The distinctive mesophyll and chloroplast localization of AltMV TGB3 highlights the critical role played by mesophyll targeting in virus long-distance movement within plants.

Russian Isolates of Potato spindle tuber viroid Exhibit Low Sequence Diversity.

Potato spindle tuber viroid (PSTVd) is currently widespread in seed potatoes grown in Russia. Characterization of 39 PSTVd isolates collected over a 15-year period from widely separated areas in Russia revealed the presence of 17 different sequence variants, all but one of which were previously unknown. Most variants were recovered only once, but two were more widely distributed; one of these was a mild variant previously isolated in Germany, the second was a novel variant inducing symptoms similar to those of the type strain in tomato. Despite this apparent lack of population diversity, several informative PSTVd variants were recovered. Sequence changes in the pathogenicity and variable domains were particularly common, but previously unknown changes were also detected within the loop E motif in the central domain, a structural motif known to play a key role in PSTVd replication and host range determination.

Inhibition of the endocannabinoid-regulating enzyme monoacylglycerol lipase elicits a CB1 receptor-mediated discriminative stimulus in mice.

Substantial challenges exist for investigating the cannabinoid receptor type 1 (CB1)-mediated discriminative stimulus effects of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA), compared with exogenous CB1 receptor agonists, such as Δ9-tetrahydrocannabinol (THC) and the synthetic cannabinoid CP55,940. Specifically, each endocannabinoid is rapidly degraded by the respective hydrolytic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Whereas MAGL inhibitors partially substitute for THC and fully substitute for CP55,940, FAAH inhibitors do not substitute for either drug. Interestingly, combined FAAH-MAGL inhibition results in full THC substitution, and the dual FAAH-MAGL inhibitor SA-57 serves as its own discriminative training stimulus. Because MAGL inhibitors fully substitute for SA-57, we tested whether the selective MAGL inhibitor MJN110 would serve as a training stimulus. Twelve of 13 C57BL/6J mice learned to discriminate MJN110 from vehicle, and the CB1 receptor antagonist rimonabant dose-dependently blocked its discriminative stimulus. CP55,940, SA-57, and another MAGL inhibitor JZL184, fully substituted for MJN110. In contrast, the FAAH inhibitor PF-3845 failed to substitute for the MJN110 discriminative stimulus, but produced a 1.6 (1.1-2.2; 95% confidence interval) leftward shift in the MJN110 dose-response curve. Inhibitors of other relevant enzymes (i.e., ABHD6, COX-2) and nicotine did not engender substitution. Diazepam partially substituted for MJN110, but rimonabant failed to block this partial effect. These findings suggest that MAGL normally throttles 2-AG stimulation of CB1 receptors to a magnitude insufficient to produce cannabimimetic subjective effects. Accordingly, inhibitors of this enzyme may release this endogenous brake producing effects akin to those produced by exogenously administered cannabinoids.

Phylogenetic and functional analyses of a plant protein related to human B-cell receptor-associated proteins.

Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway.

Pathogenesis by subviral agents: viroids and hepatitis delta virus.

The viroids of plants are the simplest known infectious genetic elements. They have RNA genomes of up to 400 nucleotides in length and no protein encoding capacity. Hepatitis delta virus (HDV), an infectious agent found only in humans co-infected with hepatitis B virus (HBV), is just slightly more complex, with an RNA genome of about 1700 nucleotides, and the ability to express just one small protein. Viroid and HDV RNAs share several features that include circular structure, compact folding, and replication via a rolling-circle mechanism. Both agents were detected because of their obvious pathogenic effects. Their simplicity demands a greater need than conventional RNA or DNA viruses to redirect host components for facilitating their infectious cycle, a need that directly and indirectly incites pathogenic effects. The mechanisms by which these pathogenic effects are produced are the topic of this review. In this context, RNA silencing mediates certain aspects of viroid pathogenesis.

In silico prediction and validation of potential gene targets for pospiviroid-derived small RNAs during tomato infection.

Viroids are small, covalently closed, circular non-coding RNA pathogens of flowering plants. It is proposed that the symptoms of viroid pathogenesis result from a direct interaction between the viroid genomic RNA and unknown host plant factors. Using a comparative genomic approach we took advantage of the detailed annotation of the Arabidopsis thaliana (Arabidopsis) genome to identify sequence homologies between putative viroid-derived small RNAs (vd-sRNAs) and coding regions in the plant genome. A pool of sequence homologies among 29 species of the Pospiviroidae family and the Arabidopsis genome was analyzed. Using this strategy we identified putative host gene targets that may be involved in symptom expression in viroid-infected plants. In this communication, we report the in silico prediction and the experimental validation of pospiviroid-derived sRNAs conserved in the lower strand of the pathogenicity domain of seven viroid species infecting tomato; those vd-sRNAs targeted for cleavage the host mRNA encoding a conserved tomato WD40-repeat protein (SolWD40-repeat; SGN_U563134). Analysis of SolWD40-repeat expression indicated that this gene is down-regulated in tomato plants infected with tomato planta macho viroid (TPMVd). Furthermore, 5' RLM-RACE revealed that the SolWD40-repeat mRNA is cleaved at the predicted target site showing complementarity to a corresponding TPMVd-sRNA identified in silico. Our approach proved to be useful for the identification of natural host genes containing sequence homologies with segments of the Pospiviroidae genome. Using this strategy we identified a functionally conserved gene in Arabidopsis and tomato, whose expression was modified during viroid infection in the host genome; regulation of this gene expression could be guided by vd-sRNA:mRNA complementarity, suggesting that the comparison of the Arabidopsis genome to viroid sequences could lead to the identification of unexpected interactions between viroid RNAs and their host.