Viroids and most viral satellites have small, noncoding, and highly structured RNA genomes. How they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. Recent studies have shown that both viroids and satellites are capable of inducing RNA silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral RNAs. Here we show that preventing RNA silencing in tobacco, using a silencing suppressor, greatly reduces the symptoms caused by the Y satellite of cucumber mosaic virus. Furthermore, tomato plants expressing hairpin RNA, derived from potato spindle tuber viroid, developed symptoms similar to those of potato spindle tuber viroid infection. These results provide evidence suggesting that viroids and satellites cause disease symptoms by directing RNA silencing against physiologically important host genes. We also show that viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens.V iroids and most viral satellites, which are the smallest known infectious agents in plants, have single-stranded RNA genomes of 200-400 nt and do not encode proteins (1-3). Whereas viroids replicate autonomously by using host-encoded RNA polymerase, satellite RNAs multiply only in the presence of a helper virus that provides the appropriate RNA-dependent RNA polymerase (2, 4). Intriguingly, some viroids and satellites can induce unique, highly host species-specific disease symptoms despite their exceedingly small size and lack of mRNA activity. Previous studies have shown that one, or a few, nucleotide changes in their RNA genomes can dramatically alter the virulence of these subviral RNAs or the host-plant specificity of the disease symptoms (5-7). Despite intensive investigation, major questions remain as to how these minor sequence variations modulate viroid and satellite pathology and how host plants develop symptoms in response to specific sequences. A striking similarity among viroids and small satellites is that they tend to form characteristic secondary structures due to intramolecular base-pairing. These structures are clearly important, because the evolution of these small RNAs appears to be constrained by the need to preserve their distinct structural features. However, the host factor(s) that imposes this evolutionary pressure has yet to be identified.RNA silencing is a sequence-specific RNA degradation process directed by double-stranded RNA (dsRNA) or selfcomplementary hairpin RNA (hpRNA). This dsRNA or hpRNA is cleaved by an RNase III-like enzyme known as Dicer to generate small (21-to 25-nt) RNAs, termed small interfering RNAs (siRNAs), which are used to guide siRNAribonuclease complexes [known as RNA-induced silencing complexes (RISC)] to degrade cognate single-stranded RNA (8). Recent studies have shown that plants infected with pota...

RNA silencing is a sequence-specific mechanism regulating gene expression and has been used successfully for antiviral defense against RNA viruses. Similar strategies to develop resistance against DNA containing Tomato leaf curl virus (TLCV) and some other geminiviruses have been unsuccessful. To analyze this silencing escape, we transformed tomato plants with a hairpin construct from the TLCV C2 open reading frame (ORF). The transgenic plants showed a strong RNA silencing response, and following TLCV inoculation, their infection was delayed. However, the viral infection was not prevented and TLCV DNA accumulated to the levels found in nontransgenic plants. To determine the fate of a transgene carrying homology to the virus, we used transgenic plants carrying the TLCV C4 gene, which induces a distinct phenotype. Upon TLCV infection, the phenotype was abolished and C4 transcript disappeared. Concurrently, TLCV-specific small interfering RNAs were produced. In situ hybridization showed abundant levels of TLCV DNA in phloem cells of TLCV-infected C4 transgenic plants. However, the C4 transcripts were no longer detectable in nonvascular cells. Analysis of the transgene by methylation sequencing revealed a high level of de novo methylation of asymmetric cytosines in both the C4 ORF and its 35S promoter. A high level of methylation also was found at both symmetric and asymmetric cytosines of the complementary-sense strand of TLCV double-stranded DNA. Given the previous finding that methylated geminiviral DNA is not competent for replication, we provide a model whereby TLCV evades host defense through a population of de novo synthesized unmethylated DNA.

Begomoviruses (the family Geminiviridae) are transmitted by the whitefly Bemisia tabaci and contain monopartite or bipartite circular single-stranded (ss)DNA genomes. They have emerged as severe problems in the production of agricultural and horticultural crops worldwide. Here, we report the identification of a tomato breeding line, FLA653, that confers a high level of resistance to Tomato leaf curl virus (TLCV, monopartite). Genetic analysis indicated that the resistance is controlled by a single recessive allele named tgr-1, which is in contrast to previous reports that multiple genetic factors are involved in tomato resistance to begomoviruses. Particle bombardment of an infectious TLCV DNA construct into the detached leaves of FLA653 resulted in the viral replication, but the viral ssDNA accumulated at a much lower level than that in susceptible controls. In situ localization of TLCV in the bombarded leaves suggests that tgr-1 impaired TLCV movement, raising the possibility that it may specify a host factor essential for viral systematic infection. This makes tgr-1 a strong candidate for developing resistance in major crops carrying the gene homologue.