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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., (© 2022 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Proteins of the alkylation B (AlkB) superfamily show RNA demethylase activity removing methyl adducts from N 6 -methyladenosine (m 6 A). m 6 A is a reversible epigenetic mark of RNA that regulates human virus replication but has unclear roles in plant virus infection. We focused on Potyvirus-the largest genus of plant RNA viruses-and report here the identification of AlkB domains within P1 of endive necrotic mosaic virus (ENMV) and an additional virus of a putative novel species within Potyvirus. We show that Nicotiana benthamiana m 6 A levels are reduced by infection of plum pox virus (PPV) and potato virus Y (PVY). The two potyviruses lack AlkB and the results suggest a general involvement of RNA methylation in potyvirus infection and evolution. Methylated RNA immunoprecipitation sequencing of virus-infected samples showed that m 6 A peaks are enriched in plant transcript 3' untranslated regions and in discrete internal and 3' terminal regions of PPV and PVY genomes. Down-regulation of N. benthamiana AlkB homologues of the plant-specific ALKBH9 clade caused a significant decrease in PPV and PVY accumulation. In summary, our study provides evolutionary and experimental evidence that supports the m 6 A implication and the proviral roles of AlkB homologues in Potyvirus infection., (© 2022 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

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.

Understanding biological mechanisms that regulate emergence of viral diseases, in particular those events engaging cross-species pathogens spillover, is becoming increasingly important in virology. Species barrier jumping has been extensively studied in animal viruses, and the critical role of a suitable intermediate host in animal viruses-generated human pandemics is highly topical. However, studies on host jumping involving plant viruses have been focused on shifting intra-species, leaving aside the putative role of "bridge hosts" in facilitating interspecies crossing. Here, we take advantage of several VPg mutants, derived from a chimeric construct of the potyvirus Plum pox virus (PPV), analyzing its differential behaviour in three herbaceous species. Our results showed that two VPg mutations in a Nicotiana clevelandii-adapted virus, emerged during adaptation to the bridge-host Arabidopsis thaliana, drastically prompted partial adaptation to Chenopodium foetidum. Although both changes are expected to facilitate productive interactions with eIF(iso)4E, polymorphims detected in PPV VPg and the three eIF(iso)4E studied, extrapolated to a recent VPg:eIF4E structural model, suggested that two adaptation ways can be operating. Remarkably, we found that VPg mutations driving host-range expansion in two non-related species, not only are not associated with cost trade-off constraints in the original host, but also improve fitness on it. [ABSTRACT FROM AUTHOR]