A conserved RNA structure is essential for a satellite RNA-mediated inhibition of helper virus accumulation.

As a class of parasitic, non-coding RNAs, satellite RNAs (satRNAs) have to compete with their helper virus for limited amounts of viral and/or host resources for efficient replication, by which they usually reduce viral accumulation and symptom expression. Here, we report a cucumber mosaic virus (CMV)-associated satRNA (sat-T1) that ameliorated CMV-induced symptoms, accompanied with a significant reduction in the accumulation of viral genomic RNAs 1 and 2, which encode components of the viral replicase. Intrans replication assays suggest that the reduced accumulation is the outcome of replication competition. The structural basis of sat-T1 responsible for the inhibition of viral RNA accumulation was determined to be a three-way branched secondary structure that contains two biologically important hairpins. One is indispensable for the helper virus inhibition, and the other engages in formation of a tertiary pseudoknot structure that is essential for sat-T1 survival. The secondary structure containing the pseudoknot is the first RNA element with a biological phenotype experimentally identified in CMV satRNAs, and it is structurally conserved in most CMV satRNAs. Thus, this may be a generic method for CMV satRNAs to inhibit the accumulation of the helper virus via the newly-identified RNA structure.

Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a ß-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

Cucumber mosaic virus Isolates from Greek Legumes are Associated with Satellite RNAs that are Necrogenic for Tomato.

Worldwide, Cucumber mosaic virus (CMV) is the causal agent of many economically important diseases. Based on immunological or molecular analysis, three distinct subgroups of CMV isolates can be identified (IA, IB, and II). In addition, some CMV isolates are associated with satellite RNAs (satRNAs), a type of noncoding transcript that may alter the symptoms of CMV infections. This study presents an analysis of CMV isolates occurring in legumes in Greece in respect to their genetic diversity, and the presence and diversity of their satRNA. Phylogenetic analysis of the CMV coat protein sequence of 18 legume and 5 tomato CMV isolates collected throughout Greece classified them within subgroups IA and IB, with a limited genetic diversity. The CMV satRNAs found in nine field legumes exhibiting mild symptoms and in one tomato with a necrotic syndrome contained a functional necrogenic motif; therefore, they were grouped within the necrogenic group of CMV-satRNAs. The necrotic phenotype was expressed in all legume CMV isolates containing necrogenic satRNAs when mechanically inoculated onto tomato plants. To our knowledge, this is the first observation that legumes host necrogenic CMV-satRNAs. The possible role of legumes in the epidemiology of CMV and necrogenic satRNA complex is discussed.

Satellite RNAs and Satellite Viruses.

Satellite RNAs and satellite viruses are extraviral components that can affect either the pathogenicity, the accumulation, or both of their associated viruses while themselves being dependent on the associated viruses as helper viruses for their infection. Most of these satellite RNAs are noncoding RNAs, and in many cases, have been shown to alter the interaction of their helper viruses with their hosts. In only a few cases have the functions of these satellite RNAs in such interactions been studied in detail. In particular, work on the satellite RNAs of Cucumber mosaic virus and Turnip crinkle virus have provided novel insights into RNAs functioning as noncoding RNAs. These effects are described and potential roles for satellite RNAs in the processes involved in symptom intensification or attenuation are discussed. In most cases, models describing these roles involve some aspect of RNA silencing or its suppression, either directly or indirectly involving the particular satellite RNA.

A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery.

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5'-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.

Genetic loci controlling lethal cell death in tomato caused by viral satellite RNA infection.

Cucumber mosaic virus (CMV) associated with D satellite RNA (satRNA) causes lethal systemic necrosis (LSN) in tomato (Solanum lycopersicum), which involves programmed cell death. No resistance to this disease has been found in tomato. We obtained a line of wild tomato, S. habrochaitis, with a homogeneous non-lethal response (NLR) to the infection. This line of S. habrochaitis was crossed with tomato to generate F1 plants that survived the infection with NLR, indicating that NLR is a dominant trait. The NLR trait was successfully passed on to the next generation. The phenotype and genotype segregation was analyzed in the first backcross population. The analyses indicate that the NLR trait is determined by quantitative trait loci (QTL). Major QTL associated with the NLR trait were mapped to chromosomes 5 and 12. Results from Northern blot and in situ hybridization analyses revealed that the F1 and S. habrochaitis plants accumulated minus-strand satRNA more slowly than tomato, and fewer vascular cells were infected. In addition, D satRNA-induced LSN in tomato is correlated with higher accumulation of the minus-strand satRNA compared with the accumulation of the minus strand of a non-necrogenic mutant D satRNA.

The presence of high-molecular-weight viral RNAs interferes with the detection of viral small RNAs.

Viral small interfering RNA (siRNA) accumulation in plants is reported to exhibit a strong strand polarity bias, with plus (+) strand siRNAs dominating over minus (-) strand populations. This is of particular interest, as siRNAs processed from double-stranded RNA would be expected to accumulate equivalent amounts of both species. Here, we show that, as reported, (-) strand viral siRNAs are detected at much lower levels than (+) strand-derived species using standard Northern hybridization approaches. However, when total RNA is spiked with in vitro-transcribed antisense viral genomic RNA, (-) strand viral siRNAs are detected at increased levels equivalent to those of (+) strand siRNA. Our results suggest that (+) and (-) strand viral siRNAs accumulate to equivalent levels; however, a proportion of the (-) strand siRNAs are sequestered from the total detectable small RNA population during gel electrophoresis by hybridizing to the high-molecular-weight sense strand viral genomic RNA. Our findings provide a plausible explanation for the observed strand bias of viral siRNA accumulation, and could have wider implications in the analysis of both viral and nonviral small RNA accumulation.

Cucumber mosaic virus satellite RNAs that induce similar symptoms in melon plants show large differences in fitness.

Two groups of Cucumber mosaic virus (CMV) satellite RNAs (satRNAs), necrogenic and non-necrogenic, can be differentiated according to the symptoms they cause in tomato plants, a host in which they also differ in fitness. In most other CMV hosts these CMV-satRNA cause similar symptoms. Here, we analyse whether they differ in traits determining their relative fitness in melon plants, in which the two groups of CMV-satRNAs cause similar symptoms. For this, ten necrogenic and ten non-necrogenic field satRNA genotypes were assayed with Fny-CMV as a helper virus. Neither type of CMV-satRNA modified Fny-CMV symptoms, and both types increased Fny-CMV virulence similarly, as measured by decreases in plant biomass and lifespan. Necrogenic and non-necrogenic satRNAs differed in their ability to multiply in melon tissues; necrogenic satRNAs accumulated to higher levels both in single infection and in competition with non-necrogenic satRNAs. Indeed, multiplication of some non-necrogenic satRNAs was undetectable. Transmission between hosts by aphids was less efficient for necrogenic satRNAs as a consequence of a more severe reduction of CMV accumulation in leaves. The effect of CMV accumulation on aphid transmission was not compensated for by differences in satRNA encapsidation efficiency or transmissibility to CMV progeny. Thus, necrogenic and non-necrogenic satRNAs differ in their relative fitness in melon, and trade-offs are apparent between the within-host and between-host components of satRNA fitness. Hence, CMV-satRNAs could have different evolutionary dynamics in CMV host-plant species in which they do not differ in pathogenicity.

De novo emergence of a novel satellite RNA of cucumber mosaic virus following serial passages of the virus derived from RNA transcripts.

Satellite RNA (satRNA) is often associated with cucumber mosaic virus (CMV); however, its origin remains unexplained and a subject for speculation. We passaged progeny of molecularly cloned CMV-Fny and CMV-LS in Nicotiana tabacum cv. Ky 14 under greenhouse conditions. A satRNA emerged after at least eight successive transfers of CMV-Fny, but no satRNA was recovered after eleven serial transfers of CMV-LS under the same conditions. The sequences of the newly emerged satRNA were determined, and an infectious cDNA clone was synthesized. Comparison of the sequences of the newly emerged satRNA with those of known CMV satRNAs showed that it is unique. This observation raises interesting questions regarding the enigmatic nature of the origin of CMV satRNAs.

[Construction of infectious cDNA clone of cucumber mosaic virus satellite RNA XJs1 and preliminary study on its biological function].

Cucumber mosaic virus (CMV) sugar beet isolate caused yellow mosaic, leaf distortion, crinkle and stunt symptoms on sugar beet in nature. It exhibited some special biological properties with narrower host range and had no symptom on Nicotiana glutinosa L. and Nicotiana tobacum L. cv. NC-89. A new satellite RNA, XJs1 was found to be associated with the helper virus. In order to know the cause of the special pathogenicity of the CMV isolate. Full-length infectious cDNA clone of CMV satellite RNA XJs1, pMSC20, was constructed by reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis showed that the satellite RNA consists of 384 nucleotides (nt) (GenBank accession number: D0070748). Compared the nucleotide sequence of satellite RNA XJsl with those of other representative CMV satellite RNAs displayed that it contains typical necrogenic consensus sequence block from positions 325 to 350, and shared 73.27% - 91.93% nucleotide sequence identity with some published CMV satellite RNAs. By in vitro transcription, satellite RNA XJsl was inoculated on Nicotiana tabacum and Nicotiana glutinosa together with CMV-AH, a CMV isolate without satellite RNA. The results showed that satellite RNA XJsl could attenuate symptoms on Nicotiana tabacum and Nicotiana glutinosa induced by CMV-AH. Detection by RT-PCR and Northern blot hybridization revealed XJs1 obtained replication in the above two host plants, showing the pathogenicity changes of CMV-AH on Nicotiana tabacum and Nicotiana glutinosa were induced by co-infecting with satellite RNA XJsl. These results indicated that XJsl is probably an attenuate satellite RNA. The relationship between helper virus, satellite RNA and host plants is discussed.

A shift in plant proteome profile for a Bromodomain containing RNA binding Protein (BRP1) in plants infected with Cucumber mosaic virus and its satellite RNA.

Host proteins are the integral part of a successful infection caused by a given RNA virus pathogenic to plants. Therefore, identification of crucial host proteins playing an important role in establishing the infection process is likely to help in devising approaches to curbing disease spread. Cucumber mosaic virus (Q-CMV) and its satellite RNA (QsatRNA) are important pathogens of many economically important crop plants worldwide. In a previous study, we demonstrated the biological significance of a Bromodomain containing RNA-binding Protein (BRP1) in the infection cycle of QsatRNA, making BRP1 an important host protein to study. To further shed a light on the mechanistic role of BRP1 in the replication of Q-CMV and QsatRNA, we analyzed the Nicotiana benthamiana host protein interactomes either for BRP1 alone or in the presence of Q-CMV or QsatRNA. Co-immunoprecipitation, followed by LC-MS/MS analysis of BRP1-FLAG on challenging with Q-CMV or QsatRNA has led us to observe a shift in the host protein interactome of BRP1. We discuss the significance of these results in relation to Q-CMV and its QsatRNA infection cycle. BIOLOGICAL SIGNIFICANCE: Host proteins play an important role in replication and infection of eukaryotic cells by a wide-range of RNA viruses pathogenic to humans, animals and plants. Since a given eukaryotic cell typically contains ~30,000 different proteins, recent advances made in proteomics and bioinformatics approaches allowed the identification of host proteins critical for viral replication and pathogenesis. Although Cucumber mosaic virus (CMV) and its satRNA are well characterized at molecular level, information concerning the network of host factors involved in their replication and pathogenesis is still on its infancy. We have recently observed that a Bromodomain containing host protein (BRP1) is obligatory to transport satRNA to the nucleus. Consequently, it is imperative to apply proteomics and bioinformatics approaches in deciphering how host interactome network regulates the replication of CMV and its satRNA. In this study, first we established the importance of BRP1 in CMV replication. Then, application of co-immunoprecipitation in conjunction with LC-MS/MS allowed the identification of a wide range of host proteins that are associated with the replication of CMV and its satRNA. Interestingly, a shift in the plant proteome was observed when plants infected with CMV were challenged with its satRNA.

[Studies on construction of artificial mutants of Cucumber mosaic virus satellite RNA and their biological activity].

Based on the full length cDNA clone of a Cucumber mosaic virus satellite RNA, which was 369nt in size, artificial mutants were developed by the method of error-prone PCR and DNA shuffling. The new satellite cDNAs were transcribed in vitro into ssRNA and pseudo-recombined with a helper Cucumber mosaic virus, which contains no satellite RNA. Sequence analysis showed that A to T/G or G to A replacement all the four mutants, named MS1, MS5, MS6 and MS11 respectively, and there is no C to G or G to C replacement, but amongst, only the mutants MS11 could replicated when recombined with the helper virus strain. No satellite RNA could be detected by RT-PCR amplification and double-stranded RNA analysis for those pseudo-recombination constitution of Cucumber mosaic virus strain with mutants MS1, MS5 and MS6.Sequence homological comparison showed that the single replacement of mutants MS1, MS5 and MS6 occurred in the highly conservative regions and the T to A replacement of mutant MS11 was located in the normal-variation region. This is the first artificial mutation of satellite RNA of plant RNA viruses. The results indicated that single base in the region of satellite RNA maybe important to maintaining the biological activity of satellite RNA for its replication and stability. The variation and evolution of satellite RNA could be hopefully studied through combination directed evolution by DNA shuffling with pseudo-recombination in vitro.

Heterologous replicase driven 3' end repair of Cucumber mosaic virus satellite RNA.

To investigate the extent of the 3' end repair in a satellite RNA of Cucumber mosaic virus (CMV) strain Q (Q(sat)) by a heterologous Tomato aspermy virus (TAV), a set of biologically active agrotransformants corresponding to the three genomic RNAs of TAV was developed. Analysis of Nicotiana benthamiana plants agroinfiltrated with TAV and either wild type or each of the six 3' deletion mutants of Q(sat) revealed that (i) heterologous replicase failed to generate Q(sat) multimers, a hallmark feature of homologous replicase dependent replication of Qsat; (ii) manifestation of severe symptom phenotypes and progeny analysis suggested that heterologous replicase was competent to repair Q(sat) deletion mutants lacking up to 3'13 nucleotides (nt) but not beyond and (iii) comparative in silico analysis indicated that the 3' secondary structural features of the repaired Q(sat) progeny from heterologous vs homologous driven replicases are remarkably very similar. The significance of these observations is discussed.

Analysis of mechanisms involved in the Cucumber mosaic virus satellite RNA-mediated transgenic resistance in tomato plants.

Transgenic tomato (Lycopersicon esculentum Mill. cv. UC82) plants expressing a benign variant of Cucumber mosaic virus satellite RNA (CMV Tfn-satRNA) were generated. The transformed plants did not produce symptoms when challenged with a satRNA-free strain of CMV (CMV-FL). The same plant lines initially were susceptible to necrosis elicited by a CMV strain supporting a necrogenic variant of satRNA (CMV-77), but a phenotype of total recovery from the necrosis was observed in the newly developing leaves. The features of the observed resistance were analyzed and are consistent with two different mechanisms of resistance. In transgenic plants inoculated with CMV-FL strain, the symptomless phenotype was correlated to the down-regulation of CMV by Tfn-satRNA, amplified from the transgene transcripts, as the first resistance mechanism. On the other hand, the delayed resistance to CMV-77 in transgenic tomato lines was mediated by a degradation process that targets satRNAs in a sequence-specific manner. Evidence is provided for a correlation between a reduced accumulation level of transgenic messenger Tfn-satRNA, the accumulation of small (approximately 23 nucleotides) RNAs with sequence homology to satRNAs, the progressively reduced accumulation of 77-satRNA in infected tissues, and the transition in infected plants from diseased to healthy. Thus, events leading to the degradation of satRNA sequences indicate a role for RNA silencing as the second mechanism determining resistance of transgenic tomato lines.

Molecular epidemiology of Cucumber mosaic virus and its satellite RNA.

Molecular analysis of viral isolates can yield information that facilitates an understanding of virus epidemiology and has been termed molecular epidemiology. This approach has only recently been applied to plant viruses. Results on the molecular epidemiology of Cucumber mosaic virus (CMV) and its satellite RNA (satRNA) in Spain, where CMV is endemic in vegetable crops are presented here. To characterise the genetic structure of CMV populations, c. 300 isolates, representing 17 outbreaks (i.e. sub-populations) in different crops, regions and years, were compared. Genetic analyses of CMV isolates were done by ribonuclease protection assay of cRNA probes representing RNA1, RNA2 and the two open reading frames in RNA3. All isolates belonged to one of three genetic types: Sub-group II and two types of Sub-group I. The genetic structure of the 17 sub-populations varied randomly, without correlation with location, year, or host plant species. Thus, CMV in Spain shows a metapopulation structure with local extinction and random recolonisation from local or distant virus reservoirs. The frequency of mixed infections and of new genetic types generated by reassortment of genomic segments or by recombination was also estimated. Results indicate that heterologous genetic combinations are not favoured. About 30% of CMV isolates were supporting a satRNA. The frequency of CMV isolates with a satRNA differed for each sub-population, being c. 1 in eastern Spain in 1990 and decreasing to c. 0 in distant regions and in subsequent years. Molecular analyses of CMV-satRNA isolates show high genetic diversity, due both to the accumulation of point mutations and to recombination. The CMV-satRNA population is a single, unstructured one. Thus, the CMV-satRNA population has a genetic structure and dynamics different from those of its helper virus. This indicates that CMV-satRNA has spread epidemically on the extant virus population from an original reservoir in eastern Spain. The relevance of these results for the control of CMV infections is discussed.

Some Indian strains of cucumber mosaic virus (CMV) lacking satellite RNA.

Virus strains isolated from Ocimum sanctum and Zinnia elegans were identified as cucumber mosaic virus (CMV) strains on the basis of non-persistant aphid transmission, 28 nm particles, 26 kDa coat protein subunits and serological relationships with CMV and chrysanthemum aspermy virus. The strains showed some biological, serological and satellite RNA based differentiation with other CMV strains isolated earlier from chrysanthemum, petunia and tobacco.

Role of satellite RNA of an Indian isolate of cucumber mosaic virus in inducing lethal necrosis of tobacco plants.

Lethal necrosis or systemic stem necrosis followed by death of Nicotiana benthamiana, severe leaf deformations of N. tabacum cv. white burley and blister formations on N. tabacum cv. samsun NN symptoms were induced by experimental inoculations of CMV RNA preparations containing satellite RNA (sat-RNA). Inoculations of RNA preparations without sat-RNA did not induce that severe symptoms on these plants, only late mild mosaic was observed. It is suggested that sat-RNA of CMV isolate has a certain role for enhancing severity of symptoms in tobacco plants. Local and systemic lethal necrosis of N. benthamiana is due to sat-RNA present with genome of CMV isolate. It is the first report of lethal necrosis induced in N. benthamiana by CMV satellite.

Emergence of a new satellite RNA from cucumber mosaic virus isolate P1.

The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species. After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars and tomato. A specific band of approximately 0.3 kb in length was amplified by RT-PCR with primers synthesized based on reported CMV satellite RNA (satRNA) sequences. Sequence analysis showed there were two satRNAs (Sat-P1-1 and Sat-P1-2). Sat-P1-1 contained 335 nucleotides, and Sat-P1-2 contained 394 nucleotides. These two satRNAs shared 64% overall nucleotide sequence homology, and differences between the two satRNAs included mutations as well as deletions. Sat-P1-1 was identical to a satRNA (Z96099) reported in 1995 in CMV P1. Based on differences in the sequence and secondary structure between these two satRNAs, we conclude that Sat-P1-2 represents the emergence of a new satellite (necrotic satellite) from attenuated satRNA populations. The possible effect of the emergence of this new satRNA is discussed.

Repair of the 3' proximal and internal deletions of a satellite RNA associated with Cucumber mosaic virus is directed toward restoring structural integrity.

The phenomenon of rapid turnover of 3' proximal nucleotides (nt) lost by the action of nuclease in RNA viruses is integral to replication. Here, a set of six deletions encompassing the 3' 23 nt region of a satellite RNA (satRNA) of Cucumber mosaic virus (CMV) strain Q (Q-sat), were engineered. Repair of the 3' end was not observed in the absence of CMV. However, co-expression with CMV in planta revealed that Q-sat mutants lacking the 3' 18 nt but not the 3' 23 nt are repaired and the progeny accumulation was inversely proportional to the extent of the deletion. Progeny of the 3'Δ3 mutant were repaired to wild type (wt) while those from the remaining four mutants were heterogeneous, exhibiting a wt secondary structure. Analysis of additional 3' internal deletions mutants revealed that progeny with a repaired sequence reminiscent of wt secondary structure were competent for replication and systemic spread.