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RNA viruses have evolved sophisticated strategies to exploit the limited encoded information within their typically compact genomes. One of them, named transcriptional slippage (TS), is characterized by the appearance of indels in nascent viral RNAs, leading to changes in the open reading frame (ORF). Although members of unrelated viral families express key proteins via TS, the available information about this phenomenon is still limited. In potyvirids (members of the Potyviridae family), TS has been defined by the insertion of an additional A at An motifs (n ≥ 6) in newly synthesized transcripts at a low frequency, modulated by nucleotides flanking the A-rich motif. Here, by using diverse experimental approaches and a collection of plant/virus combinations, we discover cases not following this definition. In summary, we observe (i) a high rate of single-nucleotide deletions at slippage motifs, (ii) overlapping ORFs acceded by slippage at an U8 stretch, and (iii) changes in slippage rates induced by factors not related to cognate viruses. Moreover, a survey of whole-genome sequences from potyvirids shows a widespread occurrence of species-specific An/Un (n ≥ 6) motifs. Even though many of them, but not all, lead to the production of truncated proteins rather than access to overlapping ORFs, these results suggest that slippage motifs appear more frequently than expected and play relevant roles during virus evolution. Considering the potential of this phenomenon to expand the viral proteome by acceding to overlapping ORFs and/or producing truncated proteins, a re-evaluation of TS significance during infections of RNA viruses is required.IMPORTANCETranscriptional slippage (TS) is used by RNA viruses as another strategy to maximize the coding information in their genomes. This phenomenon is based on a peculiar feature of viral replicases: they may produce indels in a small fraction of newly synthesized viral RNAs when transcribing certain motifs and then produce alternative proteins due to a change of the reading frame or truncated products by premature termination. Here, using plant-infecting RNA viruses as models, we discover cases expanding on previously established features of plant virus TS, prompting us to reconsider and redefine this expression strategy. An interesting conclusion from our study is that TS might be more relevant during RNA virus evolution and infection processes than previously assumed.

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P1 is the first protein translated from the genomes of most viruses in the family Potyviridae, and it contains a C-terminal serine-protease domain that cis-cleaves the junction between P1 and HCPro in most cases. Intriguingly, P1 is the most divergent among all mature viral factors, and its roles during viral infection are still far from understood. In this study, we found that telosma mosaic virus (TelMV, genus Potyvirus) in passion fruit, unlike TelMV isolates present in other hosts, has two stretches at the P1 N terminus, named N1 and N2, with N1 harboring a Zn finger motif. Further analysis revealed that at least 14 different potyviruses, mostly belonging to the bean common mosaic virus subgroup, encode a domain equivalent to N1. Using the newly developed TelMV infectious cDNA clones from passion fruit, we demonstrated that N1, but not N2, is crucial for viral infection in both Nicotiana benthamiana and passion fruit. The regulatory effects of N1 domain on P1 cis cleavage, as well as the accumulation and RNA silencing suppression (RSS) activity of its cognate HCPro, were comprehensively investigated. We found that N1 deletion decreases HCPro abundance at the posttranslational level, likely by impairing P1 cis cleavage, thus reducing HCPro-mediated RSS activity. Remarkably, disruption of the Zn finger motif in N1 did not impair P1 cis cleavage and HCPro accumulation but severely debilitated TelMV fitness. Therefore, our results suggest that the Zn finger motif in P1s plays a critical role in viral infection that is independent of P1 protease activity and self-release, as well as HCPro accumulation and silencing suppression. IMPORTANCE Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy. P1, the first protein translated from most potyvirid genomes, is the most variable viral factor and has attracted great scientific interest. Here, we defined a Zn finger motif-encompassing domain (N1) at the N terminus of P1 among diverse potyviruses phylogenetically related to bean common mosaic virus. Using TelMV as a model virus, we demonstrated that the N1 domain is key for viral infection, as it is involved both in regulating the abundance of its cognate HCPro and in an as-yet-undefined key function unrelated to protease processing and RNA silencing suppression. These results advance our knowledge of the hypervariable potyvirid P1s and highlight the importance for infection of a previously unstudied Zn finger domain at the P1 N terminus.

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Eukaryotic genomics frequently revealed historical spontaneous endogenization events of external invading nucleic acids, such as viral elements. In plants, an extensive occurrence of endogenous plant pararetroviruses (EPRVs) is usually believed to endow hosts with an additional layer of internal suppressive weaponry. However, an actual demonstration of this activity remains speculative. We analyzed the EPRV component and accompanying silencing effectors of Solanum lycopersicum, documenting that intronic/intergenic pararetroviral integrations bearing inverted-repeats fuel the plant's RNA-based immune system with suitable transcripts capable of evoking a silencing response. A surprisingly small set of rearrangements explained a substantial fraction of pararetroviral-derived endogenous small-interfering (si)RNAs, enriched in 22-nt forms typically associated with anti-viral post-transcriptional gene silencing. We provide preliminary evidence that such genetic and immunological signals may be found in other species outside the genus Solanum. Based on molecular dating, bioinformatics, and empirical explorations, we propose that homology-dependent silencing emerging from particular immuno-competent rearranged chromosomal areas that constitute an adaptive heritable trans-acting record of past infections, with potential impact against the unlocking of plant latent EPRVs and cognate-free pararetroviruses.

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Cassava brown streak disease (CBSD), dubbed the "Ebola of plants", is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC-MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups.

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Small (s)RNAs play crucial roles in the regulation of gene expression and genome stability across eukaryotes where they direct epigenetic modifications, post-transcriptional gene silencing, and defense against both endogenous and exogenous viruses. It is known that Chlamydomonas reinhardtii, a well-studied unicellular green algae species, possesses sRNA-based mechanisms that are distinct from those of land plants. However, definition of sRNA loci and further systematic classification is not yet available for this or any other algae. Here, using data-driven machine learning approaches including Multiple Correspondence Analysis (MCA) and clustering, we have generated a comprehensively annotated and classified sRNA locus map for C. reinhardtii. This map shows some common characteristics with higher plants and animals, but it also reveals distinct features. These results are consistent with the idea that there was diversification in sRNA mechanisms after the evolutionary divergence of algae from higher plant lineages.

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Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability.IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

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The presence of CpG and UpA dinucleotides is restricted in the genomes of animal RNA viruses to avoid specific host defenses. We wondered whether a similar phenomenon exists in nonanimal RNA viruses. Here, we show that these two dinucleotides, especially UpA, are underrepresented in the family Potyviridae, the most important group of plant RNA viruses. Using plum pox virus (PPV; Potyviridae family) as a model, we show that an increase in UpA frequency strongly diminishes virus accumulation. Remarkably, unlike previous observations in animal viruses, PPV variants harboring CpG-rich fragments display just faint (or no) attenuation. The anticorrelation between UpA frequency and viral fitness additionally demonstrates the relevance of this particular dinucleotide: UpA-high mutants are attenuated in a dose-dependent manner, whereas a UpA-low variant displays better fitness than its parental control. Using high-throughput sequencing, we also show that UpA-rich PPV variants are genetically stable, without apparent changes in sequence that revert and/or compensate for the dinucleotide modification despite its attenuation. In addition, we also demonstrate here that the PPV restriction of UpA-rich variants works independently of the classical RNA silencing pathway. Finally, we show that the anticorrelation between UpA frequency and RNA accumulation applies to mRNA-like fragments produced by the host RNA polymerase II. Together, our results inform us about a dinucleotide-based system in plant cells that controls diverse RNAs, including RNA viruses.IMPORTANCE Dinucleotides (combinations of two consecutive nucleotides) are not randomly present in RNA viruses; in fact, the presence of CpG and UpA is significantly repressed in their genomes. Although the meaning of this phenomenon remains obscure, recent studies with animal-infecting viruses have revealed that their low CpG/UpA frequency prevents virus restriction via a host antiviral system that recognizes, and promotes the degradation of, CpG/UpA-rich RNAs. Whether similar systems act in organisms from other life kingdoms has been unknown. To fill this gap in our knowledge, we built several synthetic variants of a plant RNA virus with deoptimized dinucleotide frequencies and analyzed their viral fitness and genome adaptation. In brief, our results inform us for the first time about an effective dinucleotide-based system that acts in plants against viruses. Remarkably, this viral restriction in plants is reminiscent of, but not identical to, the equivalent antiviral response in animals.

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Nucleotide binding site leucine-rich repeat (NLR) proteins of the plant innate immune system are negatively regulated by the miR482/2118 family miRNAs that are in a distinct 22-nt class of miRNAs with a double mode of action. First, they cleave the target RNA, as with the canonical 21-nt miRNAs, and second, they trigger secondary siRNA production using the target RNA as a template. Here, we address the extent to which the miR482/2118 family affects expression of NLR mRNAs and disease resistance. We show that structural differences of miR482/2118 family members in tomato (Solanum lycopersicum) are functionally significant. The predicted target of the miR482 subfamily is a conserved motif in multiple NLR mRNAs, whereas for miR2118b, it is a noncoding RNA target formed by rearrangement of several different NLR genes. From RNA sequencing and degradome data in lines expressing short tandem target mimic (STTM) RNAs of miR482/2118, we confirm the different targets of these miRNAs. The effect on NLR mRNA accumulation is slight, but nevertheless, the tomato STTM lines display enhanced resistance to infection with the oomycete and bacterial pathogens. These data implicate an RNA cascade of miRNAs and secondary siRNAs in the regulation of NLR RNAs and show that the encoded NLR proteins have a role in quantitative disease resistance in addition to dominant gene resistance that has been well characterized elsewhere. We also illustrate the use of STTM RNA in a biotechnological approach for enhancing quantitative disease resistance in highly bred cultivars.

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RNA viruses have very compact genomes and so provide a unique opportunity to study how evolution works to optimize the use of very limited genomic information. A widespread viral strategy to solve this issue concerning the coding space relies on the expression of proteins with multiple functions. Members of the family Potyviridae, the most abundant group of RNA viruses in plants, offer several attractive examples of viral factors which play roles in diverse infection-related pathways. The Helper Component Proteinase (HCPro) is an essential and well-characterized multitasking protein for which at least three independent functions have been described: (i) viral plant-to-plant transmission; (ii) polyprotein maturation; and (iii) RNA silencing suppression. Moreover, multitudes of host factors have been found to interact with HCPro. Intriguingly, most of these partners have not been ascribed to any of the HCPro roles during the infectious cycle, supporting the idea that this protein might play even more roles than those already established. In this comprehensive review, we attempt to summarize our current knowledge about HCPro and its already attributed and putative novel roles, and to discuss the similarities and differences regarding this factor in members of this important viral family.

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We describe here a forward genetic screen to investigate the biogenesis, mode of action, and biological function of miRNA-mediated RNA silencing in the model algal species,Chlamydomonas reinhardtii Among the mutants from this screen, there were three at Dicer-like 3 that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon- and repeat-derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild-type and dcl3 mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant in dcl3 mutant strains than in wild-type cells were not due to sRNA-targeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. Our analysis reveals that the miRNA-mediated RNA silencing in C. reinhardtii differs from that of higher plants and informs about the evolution and function of this pathway in eukaryotes.

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