In sexually propagating organisms, genetic, and epigenetic mutations are evolutionarily relevant only if they occur in the germline and are hence transmitted to the next generation. In contrast to most animals, plants are considered to lack an early segregating germline, implying that somatic cells can contribute genetic information to progeny. Here we demonstrate that 2 ARGONAUTE proteins, AGO5 and AGO9, mark cells associated with sexual reproduction in Arabidopsis (Arabidopsis thaliana) throughout development. Both AGOs are loaded with dynamically changing small RNA populations derived from highly methylated, pericentromeric, long transposons. Sequencing of single stem cell nuclei revealed that many of these transposons are co-expressed within an AGO5/9 expression domain in the shoot apical meristem (SAM). Co-occurrence of transposon expression and specific ARGONAUTE (AGO) expression in the SAM is reminiscent of germline features in animals and supports the existence of an early segregating germline in plants. Our results open the path to investigating transposon biology and epigenome dynamics at cellular resolution in the SAM stem cell niche.

Stem cells are vital for plant development and reproduction. The stem cells within shoot apical meristems are known to possess exceptionally effective antiviral defenses against pathogenic viruses which preclude their infection, yet how this is achieved remains poorly understood and scarcely investigated. In this Tansley Insight, we connect very recent experimental results with previous work to summarize the known molecular mechanisms determining stem cell antiviral immunity. More broadly, we attempt to define the viral features triggering immunity and the global consequences of virus infection in these essential cells. This brief article will highlight how these phenomena are fascinating, complex and often crucial for virus-host interactions, while emphasizing the potential for discovery in their investigation.

Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi). In plants, a small group of stem cells harbored within the shoot apical meristem generate all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood. Here, we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

ABSTRACT Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi) ( 1, 2 ). In plants, a small group of stem cells harbored within the shoot apical meristem (SAM) generates all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood ( 3, 4 ). Here we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

Summary paragraphIn sexually propagating organisms, genetic and epigenetic mutations are evolutionarily relevant only if they occur in the germline and provide inherited information to the next generation. In contrast to most animals, plants are thought to lack an early segregating germline, implying that somatic cells can contribute genetic information to the progeny. Here we demonstrate that two ARGONAUTE proteins, AGO5 and AGO9, mark an early-segregating germline. Both AGOs are loaded with dynamically changing populations of small RNAs derived from highly methylated, pericentromeric, long transposons. Sequencing single nuclei revealed that many of these transposons are co-expressed within an AGO5/9 expression domain of the shoot apical meristem (SAM). This indicates a host-parasite tug of war and specific silencing pathways along the plant germline throughout development. Our results open the path to investigate transposon biology and epigenome dynamics at cellular resolution in the SAM stem cell niche.

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 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.

In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection.

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.

The dataset contains all the original raw files sorted by figure and figure panel. NGS data has been deposited on GEO (GSE176378). The content of each file is the following: FIGURE 1: -1A: Northern blots raw TIFF image files for the detection of TuYVs81 RNA on membrane HMW7 and methylene blue staining of the same membrane. -1B: Pictures of Col-0, ago1-57, ago1-27 and ago1-38 infected with TuYVs81 WT and -P0. -1C: Axiozoom pictures of TuMV-GFP AS9 infected plants of Col-0, ago2-1, ago1-57 (sup149.1) and ago2-1/ago1-57. Both raw czi and jpg files are provided. Additional pictures for each genotype are also provided. -1D: Western blot (W579) and Coomassie staining raw image files for the detection of GFP from TuMV-AS9-GFP infected plants. -1E&G: Northern blots raw film scan for the detection of TuYVs81 RNA and TRV-PDS RNS on membrane HMW24 and HMW25 respectively. Methylene blue staining of the same membrane. Total RNA quantification and input volumes on gel (spreadsheet). -1F&H: -1I: Signal quantification of HMW24/25 using imageJ (spreadsheet). -1J: Northern blots raw film scan and TIFF images/raw phoshphoimager .gel files for the detection of TuYVs81 RNA 3’, 5’, TuYV siRNA, mir159 and U6 probe on membrane PPM70. For the detection of PDS, mir408 and U6 on membrane PPM71 Pictures of Col-0, ago2-1, ago1-57 (sup149.1) and ago2-1/ago1-57 infected with TuYVs81 WT and TRV-PDS at 20dpi. Total RNA quantification and input volumes on gel (spreadsheet). -1K: Shortstack quantification of normalized reads per category (see legend). Original bargraph and html document retracing the analysis steps. -1L: Distribution of TuYVs81-derived sRNA reads (20-nt to 25-nt) along the TuYVs81 genome in Col-0 total RNA (library JBT5) and AGO1 IP replicate 1 (library JBT13), with MISIS. Output files from MISIS and final .png image of the distribution. FIGURE 2: -2A&C: Kinetic of systemic TuYVs81 and TuMV-GFP WT infection in Col-0, ago1-57, ago1-27 and ago1-38 represented as the cumulated percentage of infected plants in the inoculated population. Raw spreadsheet with day to day counting. Original graphs. -2B: Northern blots raw TIFF image files for the detection of TuYVs81 RNA on membrane HMW22 and methylene blue staining of the same membrane. -2D: Western blot (W555) and Coomassie staining raw image files for the detection of GFP from TuMV-GFP infected plants. -2E: qPCR data for the quantification of TuYV RNA in inoculated leaves of Col-0, ago1-27 and ago1-57. 2F: Western blot (W779) and Coomassie staining raw image files for the detection of RT viral proteins from TuYVs81 inoculated leaves. 2G: Western blot (W626-W629) and Coomassie staining raw image files for the detection of CFP-AGO1 and P0-myc in N. benthamiana leaves. Northern blot raw TIFF image files for the detection of P0 CABYV, P0 BMYV and P0 PLRV on membrane HMW28 and methylene blue staining of the same membrane. Protein sequence of the P0 constructs used. 2H&I: DAS-ELISA result and fresh weight of Col-0 and ago1-57 plants infected via aphids with WT TuYV. FIGURE 3: -3A: Northern blots TIFF images/raw phoshphoimager .gel files for the detection of TuYVs81 RNA 3’, TuYV siRNA, siR255, IR71, siR1003 and U6 probe on membrane PPM102. Total RNA quantification and input volumes on gel (spreadsheet). -3B: Pictures of Col-0, dcl2-1, dcl4-2, ago1-57, dcl2-1/dcl4-2, dcl2-5/dcl3-1, dcl2-1/ago1-57, dcl4-2/ago1-57, dcl2-1/dcl4-2/ago1-57 infected with TuYVs81 WT at 16dpi. -3C: qPCR data for the quantification of TuYV RNA in TuYVs81 infected Col-0, dcl2-1, dcl4-2, ago1-57, dcl2-1/dcl4-2, dcl2-5/dcl3-1, dcl2-1/ago1-57, dcl4-2/ago1-57, dcl2-1/dcl4-2/ago1-57 leaves. -3D: All raw .lif files and Fiji processed TIFF microscopy images of B2-GFP expressing N. benthamiana plants infiltrated with tRFP, DCL2-tRFP and DCL4-tRFP are in the folder Figure S5 (related to this panel) FIGURE 4: 4A: qPCR data for the quantification of TuYV RNA in TuYVs81 infected leaves and vasculature of Col-0 and dcl2-1 at 17dpi. 4B: Northern blots TIFF images/raw phoshphoimager .gel files for the detection of TuYVs81 RNA 3’, TuYV siRNA, siR255, IR71, miR822, miR168, miR162 and U6 probe on membrane PPM90. Total RNA quantification and input volumes on gel (spreadsheet). 4C: Western blot and Coomassie staining raw image files for the detection of DCL1 (W746) and the RT viral protein (W721) from TuYVs81 infected leaves and vasculatures. 4D: qPCR data for the quantification of DCL2 and DCL4 RNA in whole leaves and vasculatures of TuYVs81 infected Col-0 and dcl2-1 plants. 4E: Northern blots TIFF images/raw phoshphoimager .gel files for the detection of PDS, TRV 3’UTR and U6 (PPM96) and TRV 3’UTR (HMW38) in leaf and vasculature of plants infected with TRV-PDS. Total RNA quantification and input volumes on gel (spreadsheet). 4F: qPCR data for the quantification of TuYV RNA in TuYVs81 WT and P0- infected leaves and vasculature of Col-0 plants. Raw Ct and calculation spreadsheet are with Figure 4A. 4G: Northern blots TIFF images/raw phoshphoimager .gel files for the detection of TuYV RNA 3’, TuYV siRNA and U6 (PPM102) in leaf and vasculature of Col-0 and dcl2-1 plants infected withTuYVs81 WT or P0-. Total RNA quantification and input volumes on gel (spreadsheet). FIGURE 5: -5A: qPCR data for the quantification of TuYV RNA in whole leaves and vasculatures of TuYVs81 infected Col-0 and ago1-57 plants. -5B: Western blot and Coomassie staining raw image files for the detection of AGO1 (W758) and AGO2(W759) from TuYVs81 infected leaves and vasculatures. -5C: AGO1 signal quantification across five biological replicates in Mock and TuYVs81 infected leaves and vasculatures. -5D&E: All raw .lif files and Fiji processed TIFF microscopy images of B2-GFP expressing N. benthamiana plants infiltrated with tRFP-AGO1 and P0-tRFP are in the folder Figure S8 (related to this panel). -5F: qPCR data for the quantification of AGO1 and AGO2 RNA in TuYVs81 infected leaves and vasculature of Col-0 and ago1-57 plants. Raw Ct and calculation spreadsheet are with Figure 5A. -5G: Western blot and Coomassie staining raw image files for the detection of AGO1 and RT viral protein (W809) and AGO2 (W810) from TuYVs81 WT and TuYVs81 P0- infected leaves and vasculatures. -5H: Pictures of SUC-SUL (SS, parental), SS/pCoYMV:P0-HA WT (CWSS6-3) and SS/pCoYMV-P0-HA LP1 (CLSS6-3) adult plants. -5I: Western blot and Coomassie staining raw image files for the detection of AGO1 and P0-HA (W723) from SUC-SUL (SS), SS/pCoYMV:P0-HA WT (CWSS6-3) and SS/pCoYMV-P0-HA LP1 (CLSS6-3) leaves and vasculatures. -5J: Northern blots TIFF images/raw phoshphoimager .gel files for the detection of SUL siRNA, miR160c, miR160*, miR168, miR159 and U6 (PPM89) in leaf and vasculature from SUC-SUL (SS), SS/pCoYMV:P0-HA WT (CWSS6-3) and SS/pCoYMV-P0-HA LP1 (CLSS6-3) plants. Total RNA quantification and input volumes on gel (spreadsheet). -5K: qPCR data for the quantification of CHLI1, CHLI2 and AGO1 RNA in leaves and vasculature of SUC-SUL (SS), SS/pCoYMV:P0-HA WT (CWSS6-3) and SS/pCoYMV-P0-HA LP1 (CLSS6-3) plants. -5L: Raw heatmap of all AGO1 IP libraries (DESeq2). Top 20 most deregulated loci. FIGURE S1: S1A: qPCR data for the quantification of TuYV RNA in whole leaves of TuYVs81 infected Col-0 and different ago single and combination mutants. S1B: Western blot and Coomassie staining raw image files for the detection of AGO1 and AGO2 (W603) from Col-0, ago2-1, ago1-57 and ago2-1/ago1-57 infected with TuYVs81 WT and TRV-PDS S1C: Northern blots raw TIFF image files for the detection of TuYV RNA 3’, TuYV siRNA, s81 insert siRNA, miR403, miR408, miR159 and U6 on membrane PPM60 (AGO1 IP) and PPM62 (AGO2 IP). Total RNA quantification and input volumes on gel (spreadsheet). S1D: Western blot and Coomassie staining raw image files for the detection of AGO1 (W537) and AGO2 (W538) from Col-0, ago1-57, ago1-27 infected with TuYVs81 WT. S1E: Northern blots TIFF images/raw phoshphoimager .gel files for the detection of SUL siRNA, miR408, miR159 and U6 (PPM93) in AGO1 and AGO2 IPs from Col-0, SUC-SUL (SS), SS/ago1-57 rosette leaves. Total RNA quantification and input volumes on gel (spreadsheet). S1F: Western blot and Coomassie staining raw image files for the detection of AGO1 and AGO2 (W766) from Col-0, SUC-SUL (SS), SS/ago1-57 rosette leaves before and after AGO1 and AGO2 IP. FIGURE S2: S2A: Total RNA quantification of TuYVs81 infected Col-0 and ago1-57 plant pools (#1 and #2) used for sRNA deepseq. Powerpoint presentation of all plant pool used, WB results and bioanalyzer profile of the total RNA. Spreadsheet with sequencing and mapping statistic for all 16 JBT libraries. S2B: Western blot and Coomassie staining raw image files for the detection of AGO1 (W398-W402) before and after AGO1 IP for sRNA deepseq samples. S2C: See figure 1K. Original bargraphs for 21-nt, 22-nt and 24-nt mapped reads in all JBT libraries. S2D: Distribution of TuYVs81-derived sRNA reads (20-nt to 25-nt) along the TuYVs81 genome in Col-0 total RNA (libraries JBT5 and JBT6), ago1-57 total RNA (libraries JBT7 and JBT8), Col-0 AGO1 IP (libraries JBT13 and JBT14) and ago1-57 AGO1 IP (libraries JBT15 and JBT16), with MISIS. Output files from MISIS and final .png image of the distribution. S2E: TuYVs81 nucleotide composition % (spreadsheet) and 5’ nucleotide frequency of the vsiRNA reads mapped to the TuYVs81 genome in JBT5, JBT7, JBT13 and JBT15 (.png). 5’ graphs were generated using MISIS. FIGURE S3: S3A: Kinetic of systemic TuYVs81, TuMV-GFP and TRV-PDS infection in Col-0 and ago1-57, represented as the cumulated percentage of infected plants in the inoculated population. Raw spreadsheet with day to day counting. Original graphs. S3B: qPCR data for the quantification of TuYV RNA in inoculated leaves of Col-0, ago1-27 and ago1-57. Biological replicate to the experiment in Figure 2E. FIGURE S4: S4A: Leaf pictures of Col-0, dcl2-1, dcl4-2, dcl2-1/dcl4-2, rdr6-12, sgs3-14 infected with TuYVs81 WT at 17dpi. Leaves are from two different individuals. S4B: Northern blots raw TIFF images and scans files for the detection of TuYVs81 RNA 3’ siRNA and U6 in Col-0, ago1-57, dcl2-1, dcl4-2, dcl2-1/dcl4-2, rdr6-12, sgs3-14 infected with TuYVs81 WT on membrane PPM39. Total RNA quantification and input volumes on gel (spreadsheet). S4C: Northern blots raw TIFF images files for the detection of TuYVs81 RNA 3’ in Col-0, ago1-57, dcl2-1, rdr6-12, sgs3-14 infected with TuYVs81 WT on membrane HMW8. Methylene blue stain of the membrane. Total RNA quantification and input volumes on gel (spreadsheet) with figure S4B. S4D: Kinetic of systemic TuYVs81 infection in Col-0, ago1-57, dcl2-1, dcl4-2, dcl2-1/dcl4-2, rdr6-12, sgs3-14 represented as the cumulated percentage of infected plants in the inoculated population. Raw spreadsheet with day to day counting. Original graphs. FIGURE S5: All raw .lif files and Fiji processed TIFF microscopy images of B2-GFP expressing N. benthamiana plants infiltrated with tRFP, DCL2-tRFP and DCL4-tRFP. Note that more images are available that those in the manuscript. FIGURE S6: S6A: qPCR data for the quantification of TuYV RNA in whole leaves and vasculatures of TuYVs81 infected Col-0. S6B: See Figure 4B S6C: qPCR data for the quantification of DCL2 and DCL4 RNA in whole leaves and vasculatures of TuYVs81 infected Col-0 plants. FIGURE S7: S7A: qPCR data for the quantification of TuYV RNA in vasculatures and protoplast cells of TuYVs81 infected Col-0. S7B: Western blot and Coomassie staining raw image files for the detection of AGO1 and RT viral protein (W283), AGO2 and AGO4 (W328) in vasculatures and protoplast cells of TuYVs81 infected Col-0 and ago1-57. S7C: Northern blots raw TIFF images for the detection of miR168 and U6 on membrane PPM47 in vasculatures and protoplast cells of TuYVs81 infected Col-0. Total RNA quantification and input volumes on gel (spreadsheet). S7D: qPCR data for the quantification of TuYV and AGO1 RNA in mock and TuYVs81 infected leaves of Col-0, ago1-57, pSUC:Flag-AGO1 #1 (ASWW1b3) and #2 (ASWW2b1). S7E: qPCR data for the quantification of AGO1 RNA in TuYVs81 WT and P0- infected leaves and vasculature of Col-0 and dcl2-1 plants. S7F: Western blot and Coomassie staining raw image files for the detection of AGO1 and RT viral protein (W708) in whole leaves of TuYVs81 infected Col-0, ago1-57, pSUC:Flag-AGO1 #1 (ASWW1b3) and #2 (ASWW2b1). S7G: qPCR data for the quantification of Flag-AGO1 RNA in mock and TuYVs81 infected leaves of Col-0, pSUC:Flag-AGO1 #1 (ASWW1b3) and #2 (ASWW2b1). FIGURE S8: S8A: All raw .lif files and Fiji processed TIFF microscopy images of B2-GFP expressing N. benthamiana plants infiltrated with tRFP-AGO1. Note that more images are available that those in the manuscript. S8B: All raw .lif files and Fiji processed TIFF microscopy images of B2-GFP expressing N. benthamiana plants infiltrated with P0-tRFP. Note that more images are available that those in the manuscript. FIGURE S9: S9A: Pictures of 11-day old seedlings grown on MS media: Col-0, SUC-SUL (SS), SS/pSuc:P15-FHA, SS/pCoYMV:P0-HA WT (CWSS6-3) and SS/pCoYMV:P0-HA LP1 (CLSS6-3). S9B: qPCR data for the quantification of P0-HA and AGO1 RNA in seedlings of Col-0, SUC-SUL (SS), SS/pSuc:P15-FHA, SS/pCoYMV:P0-HA WT (CWSS6-3) and SS/pCoYMV:P0-HA LP1 (CLSS6-3). FIGURE S10: S10A: Raw heatmap of all AGO1 Total RNA libraries (DESeq2). Top 18 most deregulated loci. S10B: MA plot of AGO1 IP in Col-0 TuYV vs. AGO1 IP in Col-0 mock (DESeq2). MA plot of AGO1 IP in ago1-57 TuYV vs. AGO1 IP in ago1-57 mock (DESeq2). S10C: Northern blots raw TIFF images/raw phoshphoimager .gel files for the detection of SUL3’ siRNA, TuYV 3’ siRNA and U6 on membrane PPM64 (Col-0, ago1-57, dcl2-1, dcl4-2, dcl2/4 infected with TuYVs81 and Col-0 plus ago1-57 infected with aphid transmitted TuYV WT) and PPM68 (Col-0, ago1-57, rdr6-12, sgs3-14, dcl2/4 infected with TuYVs81 as well as Col-0 and ago1-57 infected with TuMV-GFP). Total RNA quantification and input volumes on gel (spreadsheet).

Plant RNA viruses are obligate intracellular parasites that hijack specific cellular membranes to replicate their genomes in what are commonly known as viral replication complexes (VRC). These contain host- and virus-encoded proteins and viral RNA. Double-stranded RNA (dsRNA) is a mandatory intermediate of RNA replication and a hallmark feature of VRCs. We have recently developed a method to isolate viral dsRNA and its associated proteins through pull-down of an ectopically expressed dsRNA-binding protein (B2:GFP) from infected Arabidopsis thaliana plants. After mass spectrometry analysis to identify the dsRNA-associated proteins, resulting candidate proteins of interest are tagged with a red fluorescent protein and their subcellular localization in relation to VRCs is assessed by transient expression within leaves of B2:GFP-transgenic Nicotiana benthamiana plants. In this chapter we describe in detail these experimental procedures to allow investigators to characterize the replication complexes of their plant RNA virus of interest.

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant–microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant–virus interactions in complement to Saccharomyces cerevisiae.

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.

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.

In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles1-3. 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)4, 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.

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 × 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 plants, RNA interference (RNAi) is the main antiviral defense mechanism. It is initiated through the processing of viral RNA into 21-22nt long siRNA by DCL4 and DCL2, respectively. These siRNA can mediate sequence-specific local defense reactions (cell-autonomous RNAi) or move to distant tissues to prime defenses in naive cells (systemic RNAi). Consequently, viruses have evolved proteins (VSRs) to suppress both aspects of RNAi. In this in vivo study, I show that P15, the VSR of Peanut clump virus (PCV), binds and sequesters both 21nt and 22nt siRNA. Importantly, it stops the movement of 22nt siRNA more efficiently than 21nt siRNA. During infection, P15 is shuttled into peroxisomes, and is able to « piggyback » siRNA into these organelles. By confining mobile DCL4-dependent antiviral 21nt siRNA within peroxisomes, P15 is able to shut down systemic RNAi and strongly promote PCV movement. This work describes a novel pathogenic strategy in which an organelle is used to neutralize host defensive molecules.

Plants employ multiple layers of innate immunity to fight pathogens. For both RNA and DNA viruses, RNA silencing plays a critical role in plant resistance. To escape this antiviral silencing-based immune response, viruses have evolved various counterdefense strategies, the most widespread being production of viral suppressors of RNA silencing (VSRs) that target various stages of the silencing mechanisms. Recent findings from in planta analyses have provided new insights into the mode of action of VSRs and revealed that plants react to the perturbation of the silencing pathways brought by viral infection by deploying a battery of counter-counterdefense measures. As well as discussing which experimental approaches have been most effective in delivering clear and unambiguous results, this review provides a detailed account of the surprising variety of offensive and defensive strategies set forth by both viruses and hosts in their struggle for survival.