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
). Furthermore, small RNA-mediated gene silencing plays essential roles in regulating a wide variety of growth and development processes (4
). 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, overexpressed transgenes, and transposons. RNase III dicer or dicer-like (DCL) proteins and their associated factors cleave precursor RNAs to produce duplex miRNAs or siRNAs. One of the strands is incorporated into the RNA-induced silencing complex (RISC) to guide sequence-specific cleavage of a target RNA or inhibition of translation or into an RNA-induced initiator of transcriptional silencing complex to guide DNA methylation (1
It is generally thought that the dsRNAs that form during the replication of plant viral genomic RNAs serve as the substrates of DCL cleavage, and the resulting small RNAs then guide RISC cleavage of viral RNAs. However, critical analyses suggest that the mechanisms are much more complex, and alternative possibilities must be considered (25
). The predominant plus-strand polarity and genomic map locations suggest that small RNAs are derived mostly from structured regions of genomic RNAs of several tested positive-strand RNA viruses (59
) and the structured region of defective interfering (DI) RNAs (82
). It remains to be demonstrated that the structured viral RNAs are direct substrates for DCL activities. Furthermore, it remains to be established that the proposed viral RNA structures indeed exist in vivo, a basic requirement for them to be bona fide substrates for the biogenesis of small RNAs during infection.
As a counterdefense to host gene silencing, many plant and animal viruses studied to date encode silencing suppressors that interfere with distinct steps of RNA silencing pathways (9
). There is evidence that some satellite RNAs (94
) and DI RNAs (82
) are less susceptible to RNA silencing. The specific mechanism remains unclear, as it could be attributed to subcellular localization or special RNA structural features (6
). The finding that the human adenovirus virus-associated RNAs can function as silencing suppressors by interfering with dicer and RISC activities (3
) raises the question of whether some plant viral RNA structures may possess novel silencing suppressor activities. Thus, the specific roles of infectious RNA structures in counterdefense against host silencing remain to be further investigated.
Viroid infection presents a simple system in which to address issues of RNA-based pathogen-host interactions that impact pathogen survival and replication. Without encoding proteins and encapsidation, the small (250- to 400-nt), circular, and noncoding viroid RNAs replicate to high levels in a host cell, move from cell to cell and from organ to organ to establish systemic infection, and can cause devastating diseases (21
). Therefore, these infectious RNAs are exposed to almost every conceivable means of cellular surveillance, detection, and destruction. Indeed, RNA gel blots revealed small viroid-specific RNAs of both positive and negative polarities that are characteristic of RNA silencing in infected plants, suggesting that viroid RNAs can trigger RNA silencing (40
How the viroid small RNAs are produced remains poorly understood. Landry and Perreault (44
) showed that a hairpin structure of Peach latent mosaic viroid
(PLMVd) could be a substrate for dicer-like cleavage in wheat germ extract. Because PLMVd replicates in the chloroplasts that are not known yet to possess RNA silencing machinery, how small RNAs are derived from this viroid in vivo remains an intriguing question to be addressed. The presence of viroid small RNAs suggested activation of host silencing against viroid replication. In general, however, the accumulation of viroid small RNAs does not lead to elimination or even reduced accumulation of PSTVd genomic RNAs. In fact, higher accumulation levels of small RNAs can be associated with higher accumulation of viroid genomic RNAs (40
). The secondary structure of a viroid could play a role in resistance to silencing (94
), but the role of subcellular localization or novel silencing suppressor activities cannot be excluded.
Thus, the biogenesis and function of viroid and viral small RNAs, as well as how viroid and viral RNAs respond to host RNA silencing, remain to be fully understood. To provide further insights into these issues, we use infection of the 359-nt Potato spindle tuber viroid
(PSTVd) as a model system. The secondary structure of PSTVd, the type species of the family Pospiviroidae
, consists of five broad domains: (i) central region, (ii) pathogenicity domain, (iii) variable domain, (iv) left-terminal domain, and (v) right-terminal domain (Fig. [43
]). Importantly, there is evidence to suggest that the secondary structure of PSTVd exists in vivo (95
), making it a compelling model to address the question of whether infectious RNA structures could be substrates for small RNA biogenesis in vivo.
FIG. 1. Genomic map locations of srPSTVds and PSTVd siRNAs. The single-nucleotide difference between PSTVdInt (A) and PSTVdIntU257A (B) is indicated by the arrowhead. The patterns of PSTVd siRNAs produced in vitro are shown in panel C. Blue and red bars represent (more ...)
PSTVd replicates in the nucleus by utilizing the host DNA-dependent RNA polymerase II (75
). During asymmetric rolling-circle replication (10
), the unit-length plus circular strands serve as initial templates for the synthesis of concatemeric linear minus strands. Such minus strands then serve as the replication intermediates to direct synthesis of concatemeric, linear plus strands, which are finally cleaved into monomers and circularized. While the minus strands are anchored in the nucleoplasm, the plus strands traffic into the nucleolus, presumably for processing (65
This study addresses several basic questions concerning the biogenesis and function of viroid small RNAs as well as viroid responses to RNA silencing. (i) Are the viroid small RNAs derived from structured viroid RNAs in vivo? (ii) Are they biologically active in RNA silencing? (iii) How do viroid RNAs evade host RNA silencing-based defense? Here we present molecular and biochemical evidence that small RNAs of PSTVd (srPSTVds) are predominantly produced from the secondary structure of PSTVd RNAs. Our analyses with a reporter system demonstrate that srPSTVds are biologically active guide RNAs in RISC-mediated silencing pathways. Finally, we show that PSTVd replication is resistant to RNA silencing. This resistance can be largely attributed to the resistance of its secondary structure to RISC-mediated cleavage rather than an RNA-based silencing suppressor activity or subcellular localization. We discuss the implications of our findings in studying RNA-based plant-pathogen interactions.