Characterization of the recombinant forms arising from a Potato virus X chimeric virus infection under RNA silencing pressure

Recombination is a frequent phenomenon in RNA viruses whose net result is largely influenced by selective pressures. RNA silencing in plants acts as a defense mechanism against viruses and can be used to engineer virus resistance. Here, we have investigated the influence of RNA silencing as a selective pressure to favor recombinants of PVX-HCT, a chimeric Potato virus X (PVX) vector carrying the helper-component proteinase (HC-Pro) gene from Plum pox virus (PPV). All the plants from two lines expressing a silenced HC-Pro transgene were completely resistant to PPV. However a significant proportion became infected with PVX-HCT. Analysis of viral RNAs accumulating in silenced plants revealed that PVX-HCT escaped silencing-based resistance by removal of the HC-Pro sequences that represented preferential targets for transgene-promoted silencing. The virus vector also tended to lose the HC-Pro insert when infecting transgenic plants containing a nonsilenced HC-Pro transgene or wild-type (wt) Nicotiana benthamiana plants. Nevertheless, loss of HC-Pro sequences was faster in nonsilenced transgenic plants than in wt plants, suggesting the transgene plays a role in promoting a higher selective pressure in favor of recombinant virus versions. These results indicate that the outcome of recombination processes depends on the strength of selection pressures applied to the virus.     

Evidence that RNA silencing-mediated resistance to beet necrotic yellow vein virus is less effective in roots than in leaves

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.     

Recovery of Nicotiana benthamiana plants from a necrotic response induced by a nepovirus is associated with RNA silencing but not with reduced virus titer

Recovery of plants from virus-induced symptoms is often described as a consequence of RNA silencing, an antiviral defense mechanism. For example, recovery of Nicotiana clevelandii from a nepovirus (tomato black ring virus) is associated with a decreased viral RNA concentration and sequence-specific resistance to further virus infection. In this study, we have characterized the interaction of another nepovirus, tomato ringspot virus (ToRSV), with host defense responses during symptom induction and subsequent recovery. Early in infection, ToRSV induced a necrotic phenotype in Nicotiana benthamiana that showed characteristics typical of a hypersensitive response. RNA silencing was also activated during ToRSV infection, as evidenced by the presence of ToRSV-derived small interfering RNAs (siRNAs) that could direct degradation of ToRSV sequences introduced into sensor constructs. Surprisingly, disappearance of symptoms was not accompanied by a commensurate reduction in viral RNA levels. The stability of ToRSV RNA after recovery was also observed in N. clevelandii and Cucumis sativus and in N. benthamiana plants carrying a functional RNA-dependent RNA polymerase 1 ortholog from Medicago truncatula. In experiments with a reporter transgene (green fluorescent protein), ToRSV did not suppress the initiation or maintenance of transgene silencing, although the movement of the silencing signal was partially hindered. Our results demonstrate that although RNA silencing is active during recovery, reduction of virus titer is not required for the initiation of this phenotype. This scenario adds an unforeseen layer of complexity to the interaction of nepoviruses with the host RNA silencing machinery. The possibility that viral proteins, viral RNAs, and/or virus-derived siRNAs inactivate host defense responses is discussed.     

Resistance phenotypes of transgenic tobacco plants expressing different cucumber mosaic virus (CMV) coat protein genes

Tobacco plants expressing a transgene encoding the coat protein (CP) of a subgroup I strain of cucumber mosaic cucumovirus (CMV), I17F, were not resistant to strains of either subgroup I or II. In contrast, the expression of the CP of a subgroup II strain, R, conferred substantial resistance, but only towards strains of the same subgroup. When protection was observed, the levels of resistance were similar when plants were inoculated with either virions or viral RNA, but resistance was more effective when plants were inoculated with viruliferous aphids. Resistance was not dependent on inoculum strength and was expressed as a recovery phenotype not yet described for plants expressing a CMV CP gene. Recovery could be observed either early in infection (less than one week after inoculation) or later (4 to 5 weeks after inoculation). In plants showing early recovery, mild symptoms were observed on the inoculated leaves, and in some cases symptoms developed on certain lower systemically infected leaves, but the upper leaves were symptomless and virus-free. Late recovery corresponded to the absence of both symptoms and virus in the upper leaves of plants that were previously fully infected. Northern blot analyses of resistant plants suggested that a gene silencing mechanism was not involved in the resistance observed.     

Silencing by RNAi of the gene for Pns12, a viroplasm matrix protein of Rice dwarf virus, results in strong resistance of transgenic rice plants to the virus

The non-structural protein Pns12 of Rice dwarf virus is one of the early proteins expressed in cultured insect cells, and it is one of 12 proteins that initiate the formation of the viroplasm, the putative site of viral replication. Pns4 is also a non-structural protein, visible as minitubules after nucleation of the viroplasm. We introduced Pns12- and Pns4-specific RNA interference (RNAi) constructs into rice plants. The resultant transgenic plants accumulated short interfering RNAs specific to the constructs. The progeny of rice plants with Pns12-specific RNAi constructs, after self-fertilization, were strongly resistant to viral infection. By contrast, resistance was less apparent in the case of rice plants with Pns4-specific RNAi constructs, and delayed symptoms appeared in some plants of each line. Our results suggest that interference with the expression of a protein that is critical for viral replication, such as the viroplasm matrix protein Pns12, might be a practical and effective way to control viral infection in crop plants.     

Molecular Evolution of a Viral Non-Coding Sequence under the Selective Pressure of amiRNA-Mediated Silencing

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.     

Symptom attenuation by a normally virulent satellite RNA of turnip crinkle virus is associated with the coat protein open reading frame.

Many satellite RNAs (sat-RNAs) can attenuate or intensify the symptoms produced by their helper virus. Sat-RNA C, associated with turnip crinkle virus (TCV), was previously found to intensify the symptoms of TCV on all plants in which TCV produced visible symptoms. However, when the coat protein open reading frame (ORF) of TCV was precisely exchanged with that of cardamine chlorotic fleck virus, sat-RNA C attenuated the moderate symptoms of the chimeric virus when Arabidopsis plants were coinoculated with the chimeric virus. Symptom attenuation was correlated with a reduction in viral RNA levels in inoculated and uninoculated leaves. In protoplasts, the presence of sat-RNA C resulted in a reduction of approximately 70% in the chimeric viral genomic RNA at 44 hr postinoculation, whereas the sat-RNA wa consistently amplified to higher levels by the chimeric virus than by wild-type TCV. TCV with a deletion of the coat protein ORF also resulted in a similar increase in sat-RNA C levels in protoplasts, indicating that the TVC coat protein, or its ORF, downregulates the synthesis of sat-RNA C. These results suggest that the coat protein or its ORF is a viral determinant for symptom modulation by sat-RNA C, and symptom attenuation is at least partly due to inhibition of virus accumulation.     

Beet curly top virus DI DNA-mediated resistance is linked to its size

Beet curly top virus (BCTV) infection is associated with the de novo synthesis of a heterogeneous population of subgenomic viral DNAs. Nicotiana benthamiana plants transformed with a partial repeat of one such subgenomic DNA remained susceptible to infection but produced ameliorated symptoms when agroinoculated with BCTV. Symptom amelioration is associated with the mobilization of subgenomic DNA from the integrated copy. In an attempt to improve the resistance, N. benthamiana has been transformed with a partial repeat of a much smaller subgenomic DNA. However, transgenic plants showed almost no resistance although subgenomic DNA was mobilised from the host genome. To further understand the molecular basis of the interference phenomenon, we compared the ability of BCTV to replicate and accumulate in leaf discs derived from resistant and non-resistant transgenic plants. Both subgenomic DNAs were able to interfere with virus replication but only in case of resistant plants the DI DNA efficiently suppressed viral accumulation.     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

Genetically engineered transgenic plants with the domain 1 sequence of tobacco mosaic virus 126 kDa protein gene are completely resistant to viral infection

In many plant RNA viruses, Domains 1, 2 and 3 are conserved in replicase proteins. In order to examine the interference of viral replication by the Domain 1 sequence, we generated transgenic plants transformed with DNA corresponding to the Domain 1 sequence of the TMV 126 kDa protein. This DNA sequence includes the TMV RNA from nucleotides 1 to 2,149, which comprises both the 5'-untranslated and methyl transferase region. The transgenic plants obtained showed complete resistance to TMV infection. The presence of the Domain 1 sequence in the plants completely prevented local necrosis in Nicotiana tabacum cv. Xanthi nc, and any systemic development of symptoms in Nicotiana tabacum Xanthi upon TMV inoculation. Most transgenic plants sustained the conferred resistance even under TMV inoculum concentrations up to as high as 1,000 microg/ml. To detect any accumulation of TMV coat protein or viral RNA in infected transgenic plants, immunochemical tests and Northern blot analyses were carried out. Neither viral RNA or coat protein was detectable in the systemic leaves of the completely resistant transgenic plants, whereas they were accumulated in large quantities in all of the control plants. Because of the conservation of Domain 1 in many plant RNA viruses, the acquisition of resistance to virus infection using the Domain 1 sequence appears to be a very effective strategy for breeding of viral resistant plants.     

Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants

Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5' end of AC1 and the embedded AC4, the intergenic region and 5' end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoV-specific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene-produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.