Manipulation of Plant Host Susceptibility: An Emerging Role for Viral Movement Proteins?

Viruses encode viral suppressors of RNA silencing (VSRs) to counteract RNA silencing, a major antiviral defense response in plants. Recent studies indicate a role of virus-derived siRNAs in manipulating the expression of specific host genes and that certain plant viral movement proteins (MPs) can act as viral enhancers of RNA silencing (VERs) by stimulating the spread of silencing between cells. This suggests that viruses have evolved complex responses capable to efficiently hijack the host RNA silencing machinery to their own advantage. We draw here a dynamic model of the interaction of plant viruses with the silencing machinery during invasion of the host. The model proposes that cells at the spreading front of infection, where infection starts from zero and the VSR levels are supposedly low, represent potential sites for viral manipulation of host gene expression by using virus- and host-derived small RNAs. Viral MPs may facilitate the spread of silencing to produce a wave of small RNA-mediated gene expression changes ahead of the infection to increase host susceptibility. When experimentally ascertained, this hypothetical model will call for re-defining viral movement and the function of viral MPs

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The Nuclear dsRNA Binding Protein HYL1 Is Required for MicroRNA Accumulation and Plant Development, but Not Posttranscriptional Transgene Silencing

AbstractMicroRNAs (miRNAs) are 21–24 nucleotides long molecules processed from imperfect double-stranded RNAs (dsRNAs). They regulate gene expression by targeting complementary mRNA for cleavage or interfering with their translation [1–6]. In Arabidopsis, point mutations in or short truncations of the nuclear DICER-LIKE1 (DCL1) or HEN1 protein reduce miRNA accumulation and increase uncleaved target mRNAs accumulation, resulting in developmental abnormalities [7–12]. Here, we show that miRNA accumulation also depends on the activity of HYL1, a nuclear dsRNA binding protein [13]. hyl1 mutants exhibit developmental defects overlapping with that of dcl1 and hen1 mutants, suggesting that DCL1, HEN1, and HYL1 act together in the nucleus. We validate additional target mRNAs and show that reduced miRNA accumulation in hyl1 correlates with an increased accumulation of uncleaved target mRNAs, including meristem- and auxin-related genes, providing clues for the developmental abnormalities of hyl1 and for the previous identification of hyl1 as a mutant with altered responses to phytohormones [13]. Lastly, we show that posttranscriptional transgene silencing occurs in hyl1, suggesting that HYL1 has specialized function in the plant miRNA pathway, whereas the HYL1-related RDE-4 and R2D2 proteins associate with DICER in the cytoplasm and act in the RNAi pathway in C. elegans and Drosophila, respectively [14–15]

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siRNAs from miRNA sites mediate DNA methylation of target genes

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plant

Specific Impact of Tobamovirus Infection on the Arabidopsis Small RNA Profile

Tobamoviruses encode a silencing suppressor that binds small RNA (sRNA) duplexes in vitro and supposedly in vivo to counteract antiviral silencing. Here, we used sRNA deep-sequencing combined with transcriptome profiling to determine the global impact of tobamovirus infection on Arabidopsis sRNAs and their mRNA targets. We found that infection of Arabidopsis plants with Oilseed rape mosaic tobamovirus causes a global size-specific enrichment of miRNAs, ta-siRNAs, and other phased siRNAs. The observed patterns of sRNA enrichment suggest that in addition to a role of the viral silencing suppressor, the stabilization of sRNAs might also occur through association with unknown host effector complexes induced upon infection. Indeed, sRNA enrichment concerns primarily 21-nucleotide RNAs with a 5′-terminal guanine. Interestingly, ORMV infection also leads to accumulation of novel miRNA-like sRNAs from miRNA precursors. Thus, in addition to canonical miRNAs and miRNA*s, miRNA precursors can encode additional sRNAs that may be functional under specific conditions like pathogen infection. Virus-induced sRNA enrichment does not correlate with defects in miRNA-dependent ta-siRNA biogenesis nor with global changes in the levels of mRNA and ta-siRNA targets suggesting that the enriched sRNAs may not be able to significantly contribute to the normal activity of pre-loaded RISC complexes. We conclude that tobamovirus infection induces the stabilization of a specific sRNA pool by yet unknown effector complexes. These complexes may sequester viral and host sRNAs to engage them in yet unknown mechanisms involved in plant:virus interactions

Listening to the parent voice to inform person-centred neonatal care.

Family integrated care (FIC), where parents are an integral part of their baby’s care and decision-making can enhance parental involvement and empowerment, contributing to decreased parental separation and stress. It follows that parents can also be a central part of neonatal education for staff in the neonatal speciality. This paper focuses on what students and staff can learn from parents about what they feel is important to make their experience better. A narrative, interpretive approach was undertaken to collect and analyse parent interview narratives. A specific question was posed to a purposive sample of parents who have had premature babies about what health professionals can learn from them. Thematic analysis revealed five key themes relating to the importance of: communicating; listening; empathising; acknowledging (the parent’s role); realising (what matters to parents). These elements were incorporated into a framework named by the mnemonic, ‘CLEAR’. This highlights what parents want staff to be cognisant of when caring for them and their babies. Learning from the parents in our care enables a greater understanding of their experiences at difficult and challenging times. Having a deeper understanding of parents’ experiences can contribute to enhanced empathic learning.Peer reviewedFinal Accepted Versio

siRNAs from miRNA sites mediate DNA methylation of target genes

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections

Novel Polyfermentor Intestinal Model (PolyFermS) for Controlled Ecological Studies: Validation and Effect of pH

Peer reviewe