Immunoprecipitation mass spectrometry (IP-MS) is a popular method for the identification of protein-protein interactions. This approach is particularly powerful when information is collected without a priori knowledge and has been successively used as a first key step for the elucidation of many complex protein networks. IP-MS consists in the affinity purification of a protein of interest and of its interacting proteins followed by protein identification and quantification by mass spectrometry analysis. We developed an R package, named IPinquiry, dedicated to IP-MS analysis and based on the spectral count quantification method. The main purpose of this package is to provide a simple R pipeline with a limited number of processing steps to facilitate data exploration for biologists. This package allows to perform differential analysis of protein accumulation between two groups of IP experiments, to retrieve protein annotations, to export results, and to create different types of graphics. Here we describe the step-by-step procedure for an interactome analysis using IPinquiry from data loading to result export and plot production.

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.

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).

In the expanding repertoire of small noncoding RNAs (ncRNAs), tRNA-derived RNA fragments (tRFs) have been identified in all domains of life. Their existence in plants has been already proven but no detailed analysis has been performed. Here, short tRFs of 19–26 nucleotides were retrieved from Arabidopsis thaliana small RNA libraries obtained from various tissues, plants submitted to abiotic stress or fractions immunoprecipitated with ARGONAUTE 1 (AGO1). Large differences in the tRF populations of each extract were observed. Depending on the tRNA, either tRF-5D (due to a cleavage in the D region) or tRF-3T (via a cleavage in the T region) were found and hot spots of tRNA cleavages have been identified. Interestingly, up to 25% of the tRFs originate from plastid tRNAs and we provide evidence that mitochondrial tRNAs can also be a source of tRFs. Very specific tRF-5D deriving not only from nucleus-encoded but also from plastid-encoded tRNAs are strongly enriched in AGO1 immunoprecipitates. We demonstrate that the organellar tRFs are not found within chloroplasts or mitochondria but rather accumulate outside the organelles. These observations suggest that some organellar tRFs could play regulatory functions within the plant cell and may be part of a signaling pathway.

In the model plant Arabidopsis thaliana, four Dicer-like proteins (DCL1-4) mediate the production of various classes of small RNAs (sRNAs). Among these four proteins, DCL4 is by far the most versatile RNaseIII-like enzyme, and previously identified dcl4 missense alleles were shown to uncouple the production of the various classes of DCL4-dependent sRNAs. Yet little is known about the molecular mechanism behind this uncoupling. Here, by studying the subcellular localization, interactome and binding to the sRNA precursors of three distinct dcl4 missense alleles, we simultaneously highlight the absolute requirement of a specific residue in the helicase domain for the efficient production of all DCL4-dependent sRNAs, and identify, within the PAZ domain, an important determinant of DCL4 versatility that is mandatory for the efficient processing of intramolecular fold-back double-stranded RNA (dsRNA) precursors, but that is dispensable for the production of small interfering RNAs (siRNAs) from RDR-dependent dsRNA susbtrates. This study not only provides insights into the DCL4 mode of action, but also delineates interesting tools to further study the complexity of RNA silencing pathways in plants, and possibly other organisms.

In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear-encoded and imported into the mitochondria through posttranslational or co-translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome-scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions (UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'-UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria.

As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites.