No abstract

Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.

Grapevine (Vitis vinifera) is routinely grafted, and rootstocks inducing drought tolerance represent a source for adapting vineyards to climate change in temperate areas. Our goal was to investigate drought stress effects on microRNA (miRNA) abundance in a drought-resistant grapevine rootstock, M4 (Vitis vinifera 3 Vitis berlandieri), compared with a commercial cultivar, Cabernet Sauvignon, using their autografts and reciprocal grafts. RNA extracted from roots and leaves of droughted and irrigated plants of different graft combinations was used to prepare cDNA libraries for small RNA sequencing and to analyze miRNAs by quantitative real-time polymerase chain reaction (RT-qPCR). Measurements of leaf water potential, leaf gas exchange, and root hydraulic conductance attested that, under irrigation, M4 reduced water loss in comparison with cultivar Cabernet Sauvignon mostly through nonhydraulic, root-specific mechanisms. Under drought, stomatal conductance decreased at similar levels in the two genotypes. Small RNA sequencing allowed the identification of 70 conserved miRNAs and the prediction of 28 novel miRNAs. Different accumulation trends of miRNAs, observed upon drought and in different genotypes and organs, were confirmed by RT-qPCR. Corresponding target transcripts, predicted in silico and validated by RT-qPCR, often showed opposite expression profiles than the related miRNAs. Drought effects on miRNA abundance differed between the two genotypes. Furthermore, the concentration of drought-responsive miRNAs in each genotype was affected by reciprocal grafting, suggesting either the movement of signals inducing miRNA expression in the graft partner or, possibly, miRNA transport between scion and rootstock. These results open new perspectives in the selection of rootstocks for improving grapevine adaptation to drought.

In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles. In plants, RNA interference (RNAi) mediated by small interfering (si)RNA is the main antiviral defence mechanism. To protect themselves against the cell- and non-cell autonomous effects of RNAi, viruses produce viral suppressors of RNA silencing (VSR), whose study is crucial to properly understand the biological cycle of plant viruses and potentially find new solutions to control these pathogens. By combining biochemical approaches, cell-specific inhibition of RNAi movement and peroxisome isolation, we show here that one such VSR, the peanut clump virus (PCV)-encoded P15, isolates siRNA from the symplasm by delivering them into the peroxisomal matrix. Infection with PCV lacking this ability reveals that piggybacking of these VSR-bound nucleic acids into peroxisomes potentiates viral systemic movement by preventing the spread of antiviral siRNA. Collectively, these results highlight organellar confinement of antiviral molecules as a novel pathogenic strategy that may have its direct counterpart in other plant and animal viruses.

In the arms race between plants and viruses, two frontiers have been utilized for decades to combat viral infections in agriculture. First, many pathogenic viruses are excluded from plant meristems, which allows the regeneration of virus-free plant material by tissue culture. Second, vertical transmission of viruses to the host progeny is often inefficient, thereby reducing the danger of viral transmission through seeds. Numerous reports point to the existence of tightly linked meristematic and transgenerational antiviral barriers that remain poorly understood. In this review, we summarize the current understanding of the molecular mechanisms that exclude viruses from plant stem cells and progeny. We also discuss the evidence connecting viral invasion of meristematic cells and the ability of plants to recover from acute infections. Research spanning decades and performed on a variety of virus/host combinations has made clear that, beside morphological barriers, RNA interference (RNAi) plays a crucial role in preventing – or allowing – meristem invasion and vertical transmission. How a virus interacts with plant RNAi pathways in the meristem has profound effects on its symptomatology, persistence, replication rates, and, ultimately, entry into the host progeny.

27 Plant RNA viruses form highly organized membrane-bound virus replication complexes 28 (VRCs) to replicate their genome and multiply. VRCs contain both virus-and host-encoded 29 proteins, and have been investigated through a number of experimental approaches, including 30 genetic screens on surrogate model experimental hosts and pull-down of viral replicase 31 proteins. Double-stranded RNA (dsRNA) is an obligate intermediate of the replication 32 process of all RNA viruses and is the trigger of potent antiviral defenses in all eukaryotes. In 33 this work, we describe the use of a GFP-tagged dsRNA-binding protein (B2:GFP) to pull 34 down viral replicating RNA and associated proteins in planta. Following infection of A.35 thaliana constitutively expressing B2:GFP with tobacco rattle virus (TRV), we were able 36 through GFP immunoprecipitation to pull down dsRNA. Mass spectrometry analysis of the 37 dsRNA-B2:GFP-bound proteins from TRV-infected plants revealed the presence of (i) viral 38 proteins such as the replicase, which attested to the successful isolation of VRCs, and (ii) a 39 number of host proteins, some of which have previously been involved in virus infection. To 40 verify whether the identified host proteins localized at TRV replication complexes, we 41 selected nine candidates, generated genetic fusions with RFP, and transiently expressed them 42 in healthy and TRV-infected N. benthamiana stably expressing B2:GFP. Confocal 43 microscopy analysis revealed that eight out of nine candidates showed dramatic re-44 localization upon infection, and seven of these co-localized with B2-labeled TRV replication 45 complexes, providing ample validation for the immunoprecipitation results. We therefore 46 propose B2:GFP-mediated pull down of dsRNA to be a novel and robust method to explore 47 the proteome of VRCs in planta. 48 49 50 51 AUTHOR SUMMARY 52 Viruses are an important class of pathogens that represent a major problem for human, animal 53 and plant health. They hijack the molecular machinery of host cells to complete their 54 replication cycle, a process frequently associated with the production of double-stranded 55 RNA (dsRNA) that is regarded as a universal hallmark of infection by RNA viruses. Here we 56 exploited the capacity of a GFP-tagged dsRNA-binding protein stably expressed in transgenic 57 Arabidopsis to pull down dsRNA and associated proteins upon virus infection. In this manner 58 we specifically captured short and long dsRNA from tobacco rattle virus (TRV) infected 59 plants, and successfully isolated viral proteins such as the replicase, which attested to the 60 successful isolation of virus replication complexes (VRCs). More excitingly, a number of 61 host proteins, some of which have previously been involved in virus infection, were also 62 captured. Remarkably, among a set of nine host candidates that were analyzed, eight showed 63 dramatic re-localization to viral factories upon infection, and seven of these co-localized 64 dsRNA-labeled VRCs. Being compatible with any plant virus as ...

Peroxisomes are organelles that play key roles in eukaryotic metabolism. Their protein complement is entirely imported from the cytoplasm thanks to a unique pathway that is able to translocate folded proteins and protein complexes across the peroxisomal membrane. The import of molecules bound to a protein targeted to peroxisomes is an active process known as ‘piggybacking’ and we have recently shown that P15, a virus-encoded protein possessing a peroxisomal targeting sequence, is able to piggyback siRNAs into peroxisomes. Here, we extend this observation by analyzing the small RNA repertoire found in peroxisomes of P15-expressing plants. A direct comparison with the P15-associated small RNA retrieved during immunoprecipitation (IP) experiments, revealed that in vivo piggybacking coupled to peroxisome isolation could be a more sensitive means to determine the various small RNA species bound by a given protein. This increased sensitivity of peroxisome isolation as opposed to IP experiments was also striking when we analyzed the small RNA population bound by the Tomato bushy stunt virus-encoded P19, one of the best characterized viral suppressors of RNA silencing (VSR), artificially targeted to peroxisomes. These results support that peroxisomal targeting should be considered as a novel/alternative experimental approach to assess in vivo interactions that allows detection of labile binding events. The advantages and limitations of this approach are discussed.

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.