Genome structure and sequence are notably conserved between members of the familyPotyviridae. However, some genomic regions of these viruses, such as that encoding the P1 protein, show strikingly high variability. In this study, some partially conserved motifs were identified upstream of the quite well-conserved protease domain located near the P1 C terminus. The irregular distribution of these motifs suggests that the potyviral P1 proteins have undergone complex evolutionary diversification. Evidence was found of recombination events in the P1 N-terminal region, similar to those reported in potyviruses of the bean common mosaic virus subgroup, but also affecting other potyviruses. Moreover, intergeneric recombination events affecting potyviruses and ipomoviruses were also observed. Evidence that these recombination events could be linked to host adaptation is provided. Specific sequence features and differences in net charge help to classify the P1 proteins of members of the familyPotyviridaeinto two groups: those from potyviruses and rymoviruses and those from tritimoviruses. The ipomovirusCucumber vein yellowing virushas two P1 copies arranged in tandem, the most N-terminal one being of the potyvirus type and the other being of the tritimovirus type. These findings suggest that both recombination and gene duplication have contributed to P1 evolution and helped to facilitate successful adaptation of members of the familyPotyviridaeto a wide range of host species.

RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21–24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession. Significance Small RNAs (sRNAs) of 24 nt are associated with transcriptional gene silencing by targeting DNA methylation to complementary sequences. We demonstrated previously that sRNAs move from shoot to root, where they regulate DNA methylation of three endogenous transposable elements (TEs). However, the full extent of root DNA methylation dependent on mobile sRNAs was unknown. We demonstrate that DNA methylation at thousands of sites depends upon mobile sRNAs. These sites are associated with TE superfamilies found in gene-rich regions of the genome, which lose methylation selectively in an sRNA-deficient mutant. If the TEs were able to reactivate, they could cause genome instability and altered gene expression patterns, with negative effects on the plant. Consequently, mobile sRNAs may defend against these TEs.

Summary: 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.

Modifications of RNA sequences by nucleotide insertions, deletions or substitutions can result in expression of multiple proteins in overlapping open reading frames (ORF).…

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.