F-box proteins (FBPs), the substrate-recognition subunits of SCF (SKP1-Cullin1-F-box) E3 ubiquitin ligases, are pivotal regulators of protein turnover and play central roles in shaping cellular signaling dynamics. In plants, the repertoire of FBP-encoding genes has undergone remarkable expansion, giving rise to one of the largest and most functionally diverse protein families in the plant kingdom. This diversification underpins an extensive regulatory capacity, enabling FBPs to modulate processes such as hormone perception, developmental patterning, circadian rhythm, and responses to a wide spectrum of biotic and abiotic stresses. Here, we synthesize recent advances that illuminate the molecular mechanisms governing FBP activity, including insights into substrate recognition and their potential applications.

RNA viruses co-opt host endomembranes to form replication complexes, often triggering cellular stress and immune responses. Here, we show that Arabidopsis thaliana activates selective autophagy to respond to viruses targeting mitochondria, chloroplasts, and the endoplasmic reticulum. Rather than degrading viral components, autophagy selectively removes the immune regulator Enhanced Disease Susceptibility 1 (EDS1) to prevent cell death. This targeted mechanism is mediated by oligomeric metabolic enzymes that moonlight as selective autophagy receptors, linking organelle stress to immune homeostasis. Our findings establish selective autophagy as an essential immune rheostat that fine-tunes defense responses and safeguards cellular integrity to promote host survival during viral infections.

F-box proteins of SCF E3 ligases have been documented to control the abundance of numerous critical regulatory proteins. In Arabidopsis, one of them, F-BOX-LIKE17 (FBL17), stands out for playing a key role in DNA replication, DNA damage, and, more recently, for the control of cell size. FBL17 null mutants exhibit severe cellular defects leading to lethality. However, the molecular mechanisms by which FBL17 operate remain poorly understood. Here, we show that FBL17 interacts with different components of the RETINOBLASTOMA-RELATED1/E2F module and is involved in the protein turnover of E2Fa and E2Fb. However, mutations in E2Fa or E2Fb do not alleviate the severe fbl17 phenotype but worsen it. By contrast, it is the accumulation of the transcriptional repressor E2Fc that causes fbl17 mutant lethality. Our results highlight a key role for FBL17 in modulating the transcriptional control of E2F target genes ensuring precise control of cell cycle progression and avoiding uncontrolled DNA damage response.

Plants have evolved sophisticated mechanisms to balance growth and defense. The evolutionarily conserved polymerase-associated factor 1 complex (PAF1C) plays multiple roles in transcription. Here, we show that PAF1C regulates the growth-defense tradeoff by repressing defense genes expression in Arabidopsis. Loss of PAF1C leads to increased expression of defense genes, enhanced disease resistance, but compromised growth. Mechanistically, PAF1C binds to defense genes, where it interacts with histone deacetylases, such as HDA6, to promote histone deacetylation, thereby repressing defense genes expression. The plant immune hormone salicylic acid (SA) promotes the interaction between PAF1 and the SA receptor NPR1, which functions as an E3 ubiquitin ligase to mediate the polyubiquitination and degradation of PAF1. Genetically, loss of PAF1 suppresses the immune defects of the npr1 mutant, supporting the notion that PAF1 functions downstream of NPR1. Collectively, this study identifies the NPR1-PAF1C-HDA6 module that regulates the growth-defense tradeoff.

Kinetochores are large protein complexes that serve as attachment sites for spindle microtubules, ensuring proper chromosome segregation during cell division. KINETOCHORE NULL2 (αKNL2) is a key kinetochore protein required for the incorporation of the centromeric histone variant CENH3. The precise regulation of αKNL2 levels is crucial, but the molecular mechanisms controlling this process remain largely unexplored. In this study, we demonstrated that the Anaphase-Promoting Complex/Cyclosome (APC/C) mediates the ubiquitin-dependent proteolysis of αKNL2 during mitosis. Our findings revealed that αKNL2 accumulates in the presence of 26S proteasome inhibitors, and our yeast 2-hybrid and proteomic screens showed that proteins from the ubiquitin-proteasome pathway interact with KNL2 in Arabidopsis (Arabidopsis thaliana) and nematode (Caenorhabditis elegans). Arabidopsis αKNL2 directly interacts with Anaphase-Promoting Complex subunit 10 (APC10) and Cell Division Cycle 20.1 (CDC20.1), 2 substrate recognition components of the APC/C. RNAi-mediated depletion of APC/C resulted in the accumulation and mislocalization of endogenous αKNL2. Additionally, mutation or deletion of the D-box1 region, or substitution of residues K336 and K339, impaired αKNL2 degradation. The expression of a proteasome-resistant αKNL2 variant in planta caused severe defects in growth, fertility, and mitotic division. These findings show that APC/CCDC20-mediated degradation of αKNL2 is critical for proper kinetochore function and centromere integrity.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Proteolysis is an essential cellular function mediating the processing and turnover of proteins to remove damaged or inactive proteins, alter protein function (binding or enzymatic activities), and ensure appropriate protein stoichiometries in the cell.The post-translational control of protein stability is also a central feature of cellular signaling in eukaryotes, i.e. regulating the turnover of important regulatory proteins, and is crucial for almost all aspects of plant biology, including vegetative growth, development, reproduction, and stress responses.Proteolysis plays a central role in hormone signaling pathways, plant defense against pests and pathogens, abiotic stress responses, and basic cell functions like the cell cycle, metabolism, organellar biogenesis and maintenance, and senescence.Compared with animals, plant genomes encode a highly expanded number of components related to proteolytic pathways including the ubiquitin-proteasome system, autophagy, programmed cell death, endosomal trafficking, and organelle-associated protein degradation.Recent advances in molecular genetics and cell biology, microscopy and high-resolution imaging, in vivo labeling, proteomics, mass spectrometry, and structural biology have led to new insights and understanding in many areas of plant proteolysis, including autophagy of chloroplasts and other organelles, degradation of membrane proteins, the discovery of plant N-degron pathways, and proteolytic processing involved in plant development and environmental responses (immunity and abiotic stress).Knowledge of plant proteolytic systems is also important for agriculture and plant breeding, given the impact on plant growth and development related to yield, as well as plant resistance to pests and diseases.This focus issue on plant proteolysis includes two letters to the editor, one commentary, nine review articles, and eight original research articles.The two letters address the question of whether the polyubiquitin pathway operates inside intact chloroplasts.The question is posed by van Wijk and Adam (2024), with a Reply from Jarvis et al. (2024).Researchers on both sides of the debate make valid points to be considered.We eagerly await more definitive data to arrive at a complete understanding of protein degradation inside chloroplasts.The commentary by Eckardt et al.

RNA viruses co-opt the host endomembrane system and organelles to build replication complexes for infection. How the host responds to these membrane perturbations is poorly understood. Here, we explore the autophagic response of Arabidopsis thaliana to three viruses that hijack different cellular compartments. Autophagy is significantly induced within systemically infected tissues, its disruption rendering plants highly sensitive to infection. Contrary to being an antiviral defense mechanism as previously suggested, quantitative analyses of the viral loads established autophagy as a tolerance pathway. Further analysis of one of these viruses, the Turnip Crinkle Virus (TCV) that hijack mitochondria, showed that despite perturbing mitochondrial integrity, TCV does not trigger a typical mitophagy response. Instead, TCV and Turnip yellow mosaic virus (TYMV) infection activates a distinct selective autophagy mechanism, where oligomeric metabolic enzymes moonlight as selective autophagy receptors and degrade key executors of defense and cell death such as EDS1. Altogether, our study reveals an autophagy-regulated metabolic rheostat that gauges cellular integrity during viral infection and degrades cell death executors to avoid catastrophic amplification of immune signaling.### Competing Interest StatementThe authors have declared no competing interest.

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. AGO1 associates to the rough endoplasmic reticulum to conduct miRNA-mediated translational repression, mRNA cleavage, and biogenesis of phased siRNAs. Here, we show that a 37°C heat stress (HS) promotes AGO1 protein accumulation in cytosolic condensates where it colocalizes with components of siRNA bodies and of stress granules. AGO1 contains a prion-like domain in its poorly characterized N-terminal Poly-Q domain, which is sufficient to undergo phase separation independently of the presence of SGS3. HS only moderately affects the small RNA repertoire, the loading of AGO1 by miRNAs, and the signatures of target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting or impairing its activity in reprogramming gene expression during stress. Collectively, our work sheds new light on the impact of high temperature on a main effector of RNA silencing in plants.

Studies in plants were often pioneering in the field of RNA silencing and revealed a broad range of small RNA (sRNA) categories. When associated with ARGONAUTE (AGO) proteins, sRNAs play important functions in development, genome integrity, stress responses, and antiviral immunity. Today, most of the protein factors required for the biogenesis of sRNA classes, their amplification through the production of double-stranded RNA, and their function in transcriptional and post-transcriptional regulation have been identified. Nevertheless, and despite the importance of RNA silencing, we still know very little about their post-translational regulation. This is in stark contrast with studies in metazoans, where different modifications such as prolyl hydroxylation, phosphorylation, sumoylation, ubiquitylation, and others have been reported to alter the activity and stability of key factors, such as AGO proteins. Here, we review current knowledge of how key components of the RNA silencing machinery in plants are regulated during development and by microbial hijacking of endogenous proteases.

SUMMARY In Arabidopsis thaliana , ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. Nuclear AGO1 is loaded with miRNAs and exported to the cytosol where it associates to the rough ER to conduct miRNA-mediated translational repression, mRNA cleavage and biogenesis of phased siRNAs. These latter, as well as other cytosolic siRNAs, are loaded into cytosolic AGO1, but in which compartment this happens is not known. Moreover, the effect of stress on AGO1 localization is still unclear. Here, we show that a 37°C heat stress (HS) promotes AGO1 protein accumulation in cytosolic condensates where it co-localizes with components of siRNA bodies and of stress granules (SGs). AGO1 contains a prion-like domain in its poorly characterized N-terminal Poly-Q domain, which, is sufficient to undergo phase separation, independent of the presence or absence of SGS3. HS only moderately affects the small RNA repertoire, the loading of AGO1 by miRNAs and the signatures of target cleavage, suggesting that its localization in condensates protects AGO1 rather than promotes or impairs its activity in reprograming gene expressing during stress. Collectively, our work shed new light on the impact of high temperature on a main effector of RNA silencing in plants.

Most cellular proteins involved in genome replication are conserved in all eukaryotic lineages including yeast, plants and animals. However, the mechanisms controlling their availability during the cell cycle are less well defined. Here we show that the Arabidopsis genome encodes for two ORC1 proteins highly similar in amino acid sequence and that have partially overlapping expression domains but with distinct functions. The ancestral ORC1b gene, present before the partial duplication of the Arabidopsis genome, has retained the canonical function in DNA replication. ORC1b is expressed in both proliferating and endoreplicating cells, accumulates during G1 and is rapidly degraded upon S-phase entry through the ubiquitin-proteasome pathway. In contrast, the duplicated ORC1a gene has acquired a specialized function in heterochromatin biology. ORC1a is required for efficient deposition of the heterochromatic H3K27me1 mark by the ATXR5/6 histone methyltransferases. The distinct roles of the two ORC1 proteins may be a feature common to other organisms with duplicated ORC1 genes and a major difference with animal cells.

The ubiquitin-proteasome system is vital to hormone-mediated developmental and stress responses in plants. Ubiquitin ligases target hormone-specific transcriptional activators (TAs) for degradation, but how TAs are processed by proteasomes remains unknown. We report that in Arabidopsis , the salicylic acid– and ethylene-responsive TAs, NPR1 and EIN3, are relayed from pathway-specific ubiquitin ligases to proteasome-associated HECT-type UPL3/4 ligases. Activity and stability of NPR1 were regulated by sequential action of three ubiquitin ligases, including UPL3/4, while proteasome processing of EIN3 required physical handover between ethylene-responsive SCF EBF2 and UPL3/4 ligases. Consequently, UPL3/4 controlled extensive hormone-induced developmental and stress-responsive transcriptional programs. Thus, our findings identify unknown ubiquitin ligase relays that terminate with proteasome-associated HECT-type ligases, which may be a universal mechanism for processive degradation of proteasome-targeted TAs and other substrates.

RNA silencing is a conserved mechanism in eukaryotes involved in development and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA- and small interfering RNA-directed silencing, and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 assembles an SCF complex that selectively targets for proteolysis AGO1 when it is unloaded and mutated. Although FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA-silencing activity. Interestingly, under conditions in which small-RNA accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Accordingly, the non-degradable AGO1 protein assembles high-molecular-weight complexes and binds illegitimate small RNA, leading to off-target cleavage. Therefore, control of AGO1 homeostasis by FBW2 plays an important role in quality control of RNA silencing.

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).

Due to their sessile lifestyle, plants are especially exposed to various stresses, including genotoxic stress, which results in altered genome integrity. Upon the detection of DNA damage, distinct cellular responses lead to cell cycle arrest and the induction of DNA repair mechanisms. Interestingly, it has been shown that some cell cycle regulators are not only required for meristem activity and plant development but are also key to cope with the occurrence of DNA lesions. In this review, we first summarize some important regulatory steps of the plant cell cycle and present a brief overview of the DNA damage response (DDR) mechanisms. Then, the role played by some cell cycle regulators at the interface between the cell cycle and DNA damage responses is discussed more specifically.

In Arabidopsis (Arabidopsis thaliana), the F-box protein F-BOX-LIKE17 (FBL17) was previously identified as an important cell-cycle regulatory protein. FBL17 is required for cell division during pollen development and for normal cell-cycle progression and endoreplication during the diploid sporophyte phase. FBL17 was reported to control the stability of the CYCLIN-DEPENDENT KINASE inhibitor KIP-RELATED PROTEIN (KRP), which may underlie the drastic reduction in cell division activity in both shoot and root apical meristems observed in fbl17 loss-of-function mutants. However, whether FBL17 has other substrates and functions besides degrading KRPs remains poorly understood. Here we show that mutation of FBL17 leads not only to misregulation of cell cycle genes, but also to a strong upregulation of genes involved in DNA damage and repair processes. This phenotype is associated with a higher frequency of DNA lesions in fbl17 and increased cell death in the root meristem, even in the absence of genotoxic stress. Notably, the constitutive activation of DNA damage response genes is largely SOG1-independent in fbl17 In addition, through analyses of root elongation, accumulation of cell death, and occurrence of γH2AX foci, we found that fbl17 mutants are hypersensitive to DNA double-strand break-induced genotoxic stress. Notably, we observed that the FBL17 protein is recruited at nuclear foci upon double-strand break induction and colocalizes with γH2AX, but only in the presence of RETINOBLASTOMA RELATED1. Altogether, our results highlight a role for FBL17 in DNA damage response, likely by ubiquitylating proteins involved in DNA-damage signaling or repair.

Protein degradation is essential in plant growth and development. The stability of Cullin3 substrate adaptor protein BPM1 is regulated by multiple environmental cues pointing on manifold control of targeted protein degradation. A small family of six MATH-BTB genes (BPM1-6) is described in Arabidopsis thaliana. BPM proteins are part of the Cullin E3 ubiquitin ligase complexes and are known to bind at least three families of transcription factors: ERF/AP2 class I, homeobox-leucine zipper and R2R3 MYB. By targeting these transcription factors for ubiquitination and subsequent proteasomal degradation, BPMs play an important role in plant flowering, seed development and abiotic stress response. In this study, we generated BPM1-overexpressing plants that showed an early flowering phenotype, resistance to abscisic acid and tolerance to osmotic stress. We analyzed BPM1-GFP protein stability and found that the protein has a high turnover rate and is degraded by the proteasome 26S in a Cullin-dependent manner. Finally, we found that BPM1 protein stability is environmentally conditioned. Darkness and salt stress triggered BPM1 degradation, whereas elevated temperature enhanced BPM1 stability and accumulation in planta.