RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21-to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.A major focus of current biology is to understand how a pathogen has evolved mechanisms to achieve a balance among several interrelated activities that are crucial to establish a full infection: evading or suppressing host defense, minimizing destructive interference of host metabolism, and maximizing utilization of host factors to support replication and systemic spread. A full understanding of these mechanisms is not only necessary to build a foundation for developing technologies to combat pathogen diseases but also can provide fundamental mechanistic insights into the regulation of basic cellular processes.Recent studies have discovered small RNA-mediated gene silencing as a powerful antiviral mechanism in plants and animals (6,22,25,47,49,50,72,77,84,(87)(88)(89)98). Furthermore, small RNA-mediated gene silencing plays essential roles in regulating a wide variety of growth and development processes (4-6, 11, 13, 17, 23, 28, 42). A key mediator of RNA silencing is several classes of 21-to 24-nucleotide (nt) small RNAs.MicroRNAs (miRNAs) are produced by cleavage of hairpin RNA precursors encoded by the genome of an organism. Short interfering RNAs (siRNAs) are generated by cleavage of double-stranded RNAs (dsRNAs) that may originate from several sources, including cellular genomes, viral replication intermediates, aberrant cellular RNAs, overexpresse...
Potato spindle tuber viroid (PSTVd), an RNA plant pathogen encoding no known proteins, induces systemic symptoms on tomato plants. We report detection of small RNAs of approximately 25 nucleotides with sequence specificity to PSTVd in infected plants: an indication of the presence of RNA silencing. RNA silencing, however, did not appear to be responsible for the differing symptoms induced by a mild and a severe strain of PSTVd. The unique structural and biological features of viroids make them attractive experimental tools to investigate mechanisms of RNA silencing and pathogen counterdefense.
In higher plants, O-phosphohomoserine (OPH) represents a branch point between the methionine (Met) and threonine (Thr) biosynthetic pathways. It is believed that the enzymes Thr synthase (TS) and cystathionine ␥-synthase (CGS) actively compete for the OPH substrate for Thr and Met biosynthesis, respectively. We have isolated a mutant of Arabidopsis, designated mto2-1, that over-accumulates soluble Met 22-fold and contains markedly reduced levels of soluble Thr in young rosettes. The mto2-1 mutant carries a single base pair mutation within the gene encoding TS, resulting in a leucine-204 to arginine change. Accumulation of TS mRNA and protein was normal in young rosettes of mto2-1, whereas functional complementation analysis of an Escherichia coli thrC mutation suggested that the ability of mto2-1 TS to synthesize Thr is impaired. We concluded that the mutation within the TS gene is responsible for the mto2-1 phenotype, resulting in decreased Thr biosynthesis and a channeling of OPH to Met biosynthesis in young rosettes. Analysis of the mto2-1 mutant suggested that, in vivo, the feedback regulation of CGS is not sufficient alone for the control of Met biosynthesis in young rosettes and is dependent on TS activity. In addition, developmental analysis of soluble Met and Thr concentrations indicated that the accumulation of these amino acids is regulated in a temporal and spatial manner.
The plasmodesmata and phloem form a symplasmic network that mediates direct cell-cell communication and transport throughout a plant. Selected endogenous RNAs, viral RNAs, and viroids traffic between specific cells or organs via this network. Whether an RNA itself has structural motifs to potentiate trafficking is not well understood. We have used mutational analysis to identify a motif that the noncoding Potato spindle tuber viroid RNA evolved to potentiate its efficient trafficking from the bundle sheath into mesophyll that is vital to establishing systemic infection in tobacco (Nicotiana tabacum). Surprisingly, this motif is not necessary for trafficking in the reverse direction (i.e., from the mesophyll to bundle sheath). It is not required for trafficking between other cell types either. We also found that the requirement for this motif to mediate bundle sheath-to-mesophyll trafficking is dependent on leaf developmental stages. Our results provide genetic evidence that (1) RNA structural motifs can play a direct role in mediating trafficking across a cellular boundary in a defined direction, (2) the bundle sheath-mesophyll boundary serves as a novel regulatory point for RNA trafficking between the phloem and nonvascular tissues, and (3) the symplasmic network remodels its capacity to traffic RNAs during plant development. These findings may help further studies to elucidate the interactions between RNA motifs and cellular factors that potentiate directional trafficking across specific cellular boundaries.
RNA-templated RNA replication is essential for viral or viroid infectionAccording to the "RNA world" scenario, the appearance of RNA molecules simultaneously capable of self-replication and information storage signaled a major milestone in the evolution of life (14,24,25). In modern biology, RNA replication is central to viral or viroid infection, as well as to the regulation of cellular gene expression. Virus-encoded RNA-dependent RNA polymerases play a major role in the replication of RNA viruses (10, 23). Cellular RNA-dependent RNA polymerases generate double-stranded RNAs as triggers for RNA silencing (1). Intriguingly, the DNA-dependent cellular RNA polymerases can also transcribe at least two types of RNA templates: viroid RNAs (11,15,59) and the human hepatitis delta virus (HDV) RNA (28, 60). The replication of viroid and HDV RNAs raises the question of whether the DNA-templated transcription machinery also replicates other cellular RNAs yet to be identified. Elucidating the replication mechanisms of these infectious RNAs should help address this question of profound biological interest.Viroids are the smallest known nucleic acid-based infectious agents and self-replicating genetic units. Their "genomes" consist of single-stranded, circular RNAs ranging in size from 250 to 400 nucleotides (23). Viroids can replicate and spread throughout an infected plant, although they do not encode proteins, do not have encapsidation mechanisms, and do not require helper viruses. Furthermore, they cause devastating diseases by altering host gene expression and developmental processes (11,59). Evidently, viroid RNA genomes contain all of the sequence and structural information needed to mediate or trigger the various functions associated with infection. Potato spindle tuber viroid (PSTVd) is the type member of the family Pospiviroidae (8, 12). The PSTVd genome consists of 359 nucleotides and assumes a rod-shaped secondary structure in the native state (48) with five structural domains, as shown in Fig. 1A (26). This secondary structure, which is typical of viroids in the family Pospiviroidae, comprises many loops and bulges flanked by short Watson-Crick helices. Formation of this secondary structure is necessary for infection (62). During asymmetric rolling-circle replication of PSTVd (5), the plus circular strands serve as templates for the synthesis of concatemeric, linear minus strands, which then function as the replication intermediates for the synthesis of concatemeric, linear plus strands. These are subsequently cleaved into monomers and ligated into circular molecules (Fig. 1B). Without encoding proteins, PSTVd replicates in the nucleus of a host cell and
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