Targeting of genomic and negative-sense strands of viral RNA contributes to antiviral resistance mediated by artificial miRNAs and promotes the emergence of complex viral populations.

Technology based on artificial small RNAs, including artificial microRNAs (amiRNAs), exploits natural RNA silencing mechanisms to achieve silencing of endogenous genes or pathogens. This technology has been successfully employed to generate resistance against different eukaryotic viruses. However, information about viral RNA molecules effectively targeted by these small RNAs is rather conflicting, and factors contributing to the selection of virus mutants escaping the antiviral activity of virus-specific small RNAs have not been studied in detail. In this work, we transformed Nicotiana benthamiana plants with amiRNA constructs designed against the potyvirus plum pox virus (PPV), a positive-sense RNA virus, and obtained lines highly resistant to PPV infection and others showing partial resistance. These lines have allowed us to verify that amiRNA directed against genomic RNA is more efficient than amiRNA targeting its complementary strand. However, we also provide evidence that the negative-sense RNA strand is cleaved by the amiRNA-guided RNA silencing machinery. Our results show that the selection pressure posed by the amiRNA action on both viral RNA strands causes an evolutionary explosion that results in the emergence of a broad range of virus variants, which can further expand in the presence, and even in the absence, of antiviral challenges.

Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones.

An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by Agrobacterium-mediated delivery to host plants. We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development.

Sterol isomerase HYDRA1 interacts with RNA silencing suppressor P1b and restricts potyviral infection.

Plants use RNA silencing as a strong defensive barrier against virus challenges, and viruses counteract this defence by using RNA silencing suppressors (RSSs). With the objective of identifying host factors helping either the plant or the virus in this interaction, we have performed a yeast two-hybrid screen using P1b, the RSS protein of the ipomovirus Cucumber vein yellowing virus (CVYV, family Potyviridae), as a bait. The C-8 sterol isomerase HYDRA1 (HYD1), an enzyme involved in isoprenoid biosynthesis and cell membrane biology, and required for RNA silencing, was isolated in this screen. The interaction between CVYV P1b and HYD1 was confirmed in planta by Bimolecular Fluorescence Complementation assays. We demonstrated that HYD1 negatively impacts the accumulation of CVYV P1b in an agroinfiltration assay. Moreover, expression of HYD1 inhibited the infection of the potyvirus Plum pox virus, especially when antiviral RNA silencing was boosted by high temperature or by coexpression of homologous sequences. Our results reinforce previous evidence highlighting the relevance of particular composition and structure of cellular membranes for RNA silencing and viral infection. We report a new interaction of an RSS protein from the Potyviridae family with a member of the isoprenoid biosynthetic pathway.

Truncation of a P1 leader proteinase facilitates potyvirus replication in a non-permissive host.

The Potyviridae family is a major group of plant viruses that includes c. 200 species, most of which have narrow host ranges. The potyvirid P1 leader proteinase self-cleaves from the remainder of the viral polyprotein and shows large sequence variability linked to host adaptation. P1 proteins can be classified as Type A or Type B on the basis, amongst other things, of their dependence or not on a host factor to develop their protease activity. In this work, we studied Type A proteases from the Potyviridae family, characterizing their host factor requirements. Our in vitro cleavage analyses of potyvirid P1 proteases showed that the N-terminal domain is relevant for host factor interaction and suggested that the C-terminal domain is also involved. In the absence of plant factors, the N-terminal end of Plum pox virus P1 antagonizes protease self-processing. We performed extended deletion mutagenesis analysis to define the N-terminal antagonistic domain of P1. In viral infections, removal of the P1 protease antagonistic domain led to a gain-of-function phenotype, strongly increasing local infection in a non-permissive host. Altogether, our results shed new insights into the adaptation and evolution of potyvirids.

Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors with Compatible Replication Origins.

Improved plants are necessary to meet human needs. Agrobacterium-mediated transformation is the most common method used to rewire plant capabilities. For plant gene delivery, DNA constructs are assembled into binary T-DNA vectors that rely on broad host range origins for bacterial replication. Here we present pLX vectors, a set of mini binary T-DNA plasmids suitable for Type IIS restriction endonuclease- and overlap-based assembly methods. pLX vectors include replicons from compatible broad host range plasmids. Simultaneous usage of pBBR1- and RK2-based pLX vectors in a two-plasmid/one-Agrobacterium strain strategy allowed multigene delivery to plants. Adoption of pLX vectors will facilitate routine plant transformations and targeted mutagenesis, as well as complex part and circuit characterization.

Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance.

The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.

The Potyviridae P1a leader protease contributes to host range specificity.

The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serine proteases present in Potyviridae family members. P1a is located at the N-terminal end of the viral polyprotein, and is closely related to potyviral P1 protease. For its proteolytic activity, P1a requires a still unknown host factor; this might be linked to involvement in host specificity. Here we built a series of constructs and chimeric viruses to help elucidate the role of P1a cleavage in host range definition. We demonstrate that host-dependent separation of P1a from the remainder of the polyprotein is essential for suppressing RNA silencing defenses and for efficient viral infection. These findings support the role of viral proteases as important determinants in host adaptation.

Rapid fluorescent reporter quantification by leaf disc analysis and its application in plant-virus studies.

BACKGROUND: Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent protein genes are among the most commonly used reporters in transient RNA silencing and heterologous protein expression assays. Fluorescence intensity is used to quantify fluorescent protein accumulation by image analysis or spectroscopy of protein extracts; however, these methods might not be suitable for medium- to large-scale comparisons. RESULTS: We report that laser scanners, used routinely in proteomic studies, are suitable for quantitative imaging of plant leaves that express different fluorescent protein pairs. We developed a microtiter plate fluorescence spectroscopy method for direct quantitative comparison of fluorescent protein accumulation in intact leaf discs. We used this technique to measure a fluorescent reporter in a transient RNA silencing suppression assay, and also to monitor early amplification dynamics of a fluorescent protein-labeled potyvirus. CONCLUSIONS: Laser scanners allow dual-color fluorescence imaging of leaf samples, which might not be acquired in standard stereomicroscope devices. Fluorescence microtiter plate analysis of intact leaf discs can be used for rapid, accurate quantitative comparison of fluorescent protein accumulation.

Virus-induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation.

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so-called virus-induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5' end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3' end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3' end, and an overall increase in CG methylation in the 5' end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.

The hypervariable amino-terminus of P1 protease modulates potyviral replication and host defense responses.

The replication of many RNA viruses involves the translation of polyproteins, whose processing by endopeptidases is a critical step for the release of functional subunits. P1 is the first protease encoded in plant potyvirus genomes; once activated by an as-yet-unknown host factor, it acts in cis on its own C-terminal end, hydrolyzing the P1-HCPro junction. Earlier research suggests that P1 cooperates with HCPro to inhibit host RNA silencing defenses. Using Plum pox virus as a model, we show that although P1 does not have a major direct role in RNA silencing suppression, it can indeed modulate HCPro function by its self-cleavage activity. To study P1 protease regulation, we used bioinformatic analysis and in vitro activity experiments to map the core C-terminal catalytic domain. We present evidence that the hypervariable region that precedes the protease domain is predicted as intrinsically disordered, and that it behaves as a negative regulator of P1 proteolytic activity in in vitro cleavage assays. In viral infections, removal of the P1 protease antagonistic regulator is associated with greater symptom severity, induction of salicylate-dependent pathogenesis-related proteins, and reduced viral loads. We suggest that fine modulation of a viral protease activity has evolved to keep viral amplification below host-detrimental levels, and thus to maintain higher long-term replicative capacity.

Diverse amino acid changes at specific positions in the N-terminal region of the coat protein allow Plum pox virus to adapt to new hosts.

Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N terminus of the coat protein (CP) includes host-specific pathogenicity determinants. Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3' region of PPV-D (including the region coding for the CP) in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and N. benthamiana when specific amino acids were modified or deleted in a short 30-amino-acid region of the N terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus spp. although others impaired systemic infection in this host. We propose a model in which the N terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanism in Nicotiana spp. Mutations in this short region allow PPV to overcome the defense response in this host but can compromise the efficiency of PPV systemic movement in other hosts such as Prunus spp.

pFiD188, the linear virulence plasmid of Rhodococcus fascians D188.

Rhodococcus fascians is currently the only phytopathogen of which the virulence genes occur on a linear plasmid. To get insight into the origin of this replicon and into the virulence strategy of this broad-spectrum phytopathogen, the sequence of the linear plasmid of strain D188, pFiD188, was determined. Analysis of the 198,917 bp revealed four syntenic regions with linear plasmids of R. erythropolis, R. jostii, and R. opacus, suggesting a common origin of these replicons. Mutational analysis of pFi_086 and pFi_102, similar to cutinases and type IV peptidases, respectively, showed that conserved region R2 was involved in plasmid dispersal and pointed toward a novel function for actinobacterial cutinases in conjugation. Additionally, pFiD188 had three regions that were unique for R. fascians. Functional analysis of the stk and nrp loci of regions U2 and U3, respectively, indicated that their role in symptom development was limited compared with that of the previously identified fas, att, and hyp virulence loci situated in region U1. Thus, pFiD188 is a typical rhodococcal linear plasmid with a composite structure that encodes core functions involved in plasmid maintenance and accessory functions, some possibly acquired through horizontal gene transfer, implicated in virulence and the interaction with the host.

Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features.

Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host.

Antiviral strategies in plants based on RNA silencing.

One of the challenges being faced in the twenty-first century is the biological control of plant viral infections. Among the different strategies to combat virus infections, those based on pathogen-derived resistance (PDR) are probably the most powerful approaches to confer virus resistance in plants. The application of the PDR concept not only revealed the existence of a previously unknown sequence-specific RNA-degradation mechanism in plants, but has also helped to design antiviral strategies to engineer viral resistant plants in the last 25 years. In this article, we review the different platforms related to RNA silencing that have been developed during this time to obtain plants resistant to viruses and illustrate examples of current applications of RNA silencing to protect crop plants against viral diseases of agronomic relevance. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.

Constraints to virus infection in Nicotiana benthamiana plants transformed with a potyvirus amplicon.

BACKGROUND: Plant genomes have been transformed with full-length cDNA copies of viral genomes, giving rise to what has been called 'amplicon' systems, trying to combine the genetic stability of transgenic plants with the elevated replication rate of plant viruses. However, amplicons' performance has been very variable regardless of the virus on which they are based. This has boosted further interest in understanding the underlying mechanisms that cause this behavior differences, and in developing strategies to control amplicon expression. RESULTS: Nicotiana benthamiana plants were transformed with an amplicon consisting of a full-length cDNA of the potyvirus Plum pox virus (PPV) genome modified to include a GFP reporter gene. Amplicon expression exhibited a great variability among different transgenic lines and even among different plants of the same line. Plants of the line 10.6 initially developed without signs of amplicon expression, but at different times some of them started to display sporadic infection foci in leaves approaching maturity. The infection progressed systemically, but at later times the infected plants recovered and returned to an amplicon-inactive state. The failure to detect virus-specific siRNAs in 10.6 plants before amplicon induction and after recovery suggested that a strong amplicon-specific RNA silencing is not established in these plants. However, the coexpression of extra viral silencing suppressors caused some amplicon activation, suggesting that a low level of RNA silencing could be contributing to maintain amplicon repression in the 10.6 plants. The resistance mechanisms that prevent amplicon-derived virus infection were also active against exogenous PPV introduced by mechanical inoculation or grafting, but did not affect other viruses. Amplicon-derived PPV was able to spread into wild type scions grafted in 10.6 rootstocks that did not display signs of amplicon expression, suggesting that resistance has little effect on virus movement. CONCLUSIONS: Our results suggest that amplicon-derived virus infection is limited in this particular transgenic line by a combination of factors, including the presumed low efficiency of the conversion from the transgene transcript to replicable viral RNA, and also by the activation of RNA silencing and other defensive responses of the plant, which are not completely neutralized by viral suppressors.

Host-specific effect of P1 exchange between two potyviruses.

The potyviruses Plum pox virus (PPV) and Tobacco vein mottling virus (TVMV) have distinct host ranges and induce different symptoms in their common herbaceous hosts. To test the relevance of the P1 protein in host compatibility and pathogenicity, hybrid viruses were constructed in which the P1 coding sequence of PPV was completely or partially replaced by the corresponding sequences from TVMV. Infections induced by these chimeric viruses revealed that the TVMV P1 and a PPV/TVMV hybrid P1 proteins are functionally equivalent in herbaceous plants to the P1 protein of a PPV isolate adapted to these hosts, in spite of having high sequence divergence. Moreover, the presence of TVMV P1 sequences enhanced the competence of a low-infectivity PPV-D-derived chimera in Nicotiana clevelandii. Conversely, all PPV/TVMV hybrids were unable to infect Prunus persicae, a specific host for PPV, suggesting that TVMV P1 is not functionally competent in this plant. Together, these data highlight the importance of the P1 protein in defining the virus host range.

MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily evolves to escape this silencing mechanism.

Since the discovery of microRNA (miRNA)-guided processing, a new type of RNA silencing, the possibility that such a mechanism could play a role in virus defense has been proposed. In this work, we have analyzed whether Plum pox virus (PPV) chimeras bearing miRNA target sequences (miR171, miR167, and miR159), which have been reported to be functional in Arabidopsis, were affected by miRNA function in three different host plants. Some of these PPV chimeras had clearly impaired infectivity compared with those carrying nonfunctional miRNA target sequences. The behaviors of PPV chimeras were similar but not identical in all the plants tested, and the deleterious effect on virus infectivity depended on the miRNA sequence cloned and on the site of insertion in the viral genome. The effect of the miRNA target sequence was drastically alleviated in transgenic plants expressing the silencing suppressor P1/HCPro. Furthermore, we show that virus chimeras readily escape RNA silencing interference through mutations within the miRNA target sequence, which mainly affected nucleotides matching the 5'-terminal region of the miRNA.

The phytopathogen Rhodococcus fascians breaks apical dominance and activates axillary meristems by inducing plant genes involved in hormone metabolism.

SUMMARY Rhodococcus fascians is a Gram-positive bacterium that interacts with many plant species and induces multiple shoots through a combination of activation of dormant axillary meristems and de novo meristem formation. Although phenotypic analysis of the symptoms of infected plants clearly demonstrates a disturbance of the phytohormonal balance and an activation of the cell cycle, the actual mechanism of symptom development and the targets of the bacterial signals are unknown. To elucidate the molecular pathways that are responsive to R. fascians infection, differential display was performed on Nicotiana tabacum as a host. Four differentially expressed genes could be identified that putatively encode a senescence-associated protein, a gibberellin 2-oxidase, a P450 monooxygenase and a proline dehydrogenase. The differential expression of the three latter genes was confirmed on infected Arabidopsis thaliana plants by quantitative reverse transcription polymerase chain reactions, supporting their general function in R. fascians-induced symptom development. The role of these genes in hormone metabolism, especially of gibberellin and abscisic acid, in breaking apical dominance and in activating axillary meristems, which are processes associated with symptom development, is discussed.

Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants.

RNA silencing has a well-established function as an antiviral defence mechanism in plants and insects. Using an Agrobacterium-mediated transient assay, we report here that NS1 protein from human influenza A virus suppresses RNA silencing in plants in a manner similar to P1/HC-Pro protein of Tobacco etch potyvirus, a well-characterized plant virus silencing suppressor. Moreover, we have shown that NS1 protein expression strongly enhances the symptoms of Potato virus X in three different plant hosts, suggesting that NS1 protein could be inhibiting defence mechanisms activated in the plant on infection. These data provide further evidence that an RNA silencing pathway could also be activated as a defence response in mammals.