The rice stripe virus p2 interacts with Lsm1 and disrupts the Lsm1-Lsm4 complex to facilitate the viral infection

The rice stripe virus p2 interacts with Lsm1 and disrupts the Lsm1-Lsm4 complex to facilitate the viral infection | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 January 2026 V1 Latest version Share on The rice stripe virus p2 interacts with Lsm1 and disrupts the Lsm1-Lsm4 complex to facilitate the viral infection Authors : Anqi Hu 0009-0000-2153-7905 , Zizhou Zhao 0009-0003-0707-9778 , Minjie Shen , Yi Chen , Ying Chen , Miaomiao Li , Lin Lin , … Show All … , Hongying Zheng , Jiejun Peng , Yan Liang , Jianping Chen , Fei Yan 0000-0002-0049-8588 , and Yuwen Lu 0000-0002-5292-960X [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.176828245.52279353/v1 Published Molecular Plant Pathology Version of record Peer review timeline 185 views 128 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Processing bodies (P-bodies) play central roles in RNA metabolism and in the regulation of viral infection. However, whether they function during infection by negative-strand RNA viruses, and how such viruses manipulate P-body activities, remain largely unclear. Here, we showed that the p2 protein encoded by rice stripe virus (RSV), a segmented negative-strand RNA virus, interacted with Lsm1, a core subunit of the Lsm1-7 complex responsible for decapping-dependent 5′-3′ mRNA decay in P-bodies. Knockout of OsLsm1 resulted in severe dwarfism and impaired panicle development, leading to no seeds being harvested. In contrast, its overexpression promoted seedling growth without apparent developmental penalties and exhibited resistance against RSV. Further analysis showed that p2 competitively disrupts the interaction between Lsm1 and Lsm4, impairing Lsm1-7 complex function. Our work suggests that P-bodies constitute a post-transcriptional barrier that restricts RSV infection. RSV overcomes this barrier by deploying p2 to associate with Lsm1 and weaken Lsm1-Lsm4 complex assembly. This counter-defense strategy allows RSV to modulate host RNA decay pathways to its advantage. 1 Introduction RNA metabolism constitutes a fundamental layer of antiviral immunity across eukaryotes, shaping the stability, translation, and fate of both host and viral RNAs. In plant cells, processing bodies (P-bodies) function as dynamic, membrane-less cytoplasmic ribonucleoprotein condensates enriched in mRNA decay factors, including the conserved Lsm1-7 complex, which promotes mRNA decapping and 5′-3′ RNA turnover (Hoffmann et al., 2022; Rahman et al., 2022). Beyond serving as sites of RNA degradation, P-bodies actively regulate mRNA storage, translational repression, and stress-responsive remodeling of gene expression (Arluison et al., 2006; Catalá, Carrasco-López, Perea-Resa, Hernández-Verdeja, & Salinas, 2019; Decker & Parker, 2012; Hubstenberger et al., 2017; Moore, 2005; Parker, 2012; Putnam & Seydoux, 2023; Sharif & Conti, 2013; Sheth & Parker, 2003; Xing, Muhlrad, Parker, & Rosen, 2020; Zuo, Roux, Rodriguez, & Petersen, 2022). Increasing evidence suggests that P-bodies are not merely passive repositories of RNA but function as regulatory hubs that integrate RNA metabolism with antiviral defense, thereby influencing infection outcomes (Beckham & Parker, 2008; Reineke & Lloyd, 2013). Plant viruses have evolved diverse strategies to counteract RNA-based defenses, including suppression of RNA silencing, manipulation of translation initiation, and interference with host RNA decay pathways (Akhter, Nakahara, & Masuta, 2021; Ge, Zhou, & Li, 2024; Voinnet, 2025). Multiple positive-strand RNA viruses have been shown to interact with P-body components, either dismantling P-body integrity to relieve antiviral RNA turnover or selectively hijacking decay-associated factors to promote viral RNA translation and replication (Castello, Álvarez, Kamel, Iselin, & Hennig, 2024; Fan, Xu, Liu, Qin, & Chen, 2021). These findings highlight the functional plasticity of P-bodies during viral infection. However, whether plant negative-strand RNA viruses exploit or reprogram P-body-mediated RNA metabolism, and the underlying molecular mechanisms, remain largely unexplored. Rice stripe virus (RSV) is a segmented negative-strand RNA virus that causes severe yield losses in rice (Muthayya, Sugimoto, Montgomery, & Maberly, 2014; Y. Xu, Fu, Tao, & Zhou, 2021) and represents an important model for studying plant-virus interactions (Cui et al., 2021; Han et al., 2020; Jiang et al., 2021; Shi et al., 2016; X. Zhang et al., 2023; X. Zhang et al., 2021). Among the RSV-encoded proteins, p2 is a multifunctional virulence factor implicated in immune suppression and pathogenicity through its interactions with host regulators involved in RNA silencing, hormone signaling, autophagy, and protein degradation (Z. Du et al., 2011; Li et al., 2021; Yang et al., 2024; H. Zhang et al., 2020; H. Zhang et al., 2023; X. Zhang et al., 2023; Zheng et al., 2015). Despite its central role in RSV infection, whether p2 directly targets host RNA metabolic machineries, particularly those associated with P-bodies, has not been investigated. Here, we identify the P-body core component Lsm1 as a host restriction factor that limits RSV infection. We further show that RSV overcomes this restriction via its p2 protein, which directly associates with Lsm1 and disrupts Lsm1-Lsm4 complex assembly. These findings uncover a previously unappreciated layer of post-transcriptional immune regulation targeted by a plant negative-strand RNA virus. 2 Materials and Methods 2.1 Plant Materials and Growth Conditions Rice plants ( Oryza sativa L. ssp. japonica cv. Nipponbare) were used as the wild-type background for generating all transgenic lines. Rice plants were grown in a temperature-controlled greenhouse at 26 °C under a 16-h light/8-h dark photoperiod with 60% relative humidity. Nicotiana benthamiana plants were grown in a growth chamber at 24 °C under a 16-h light/8-h dark photoperiod with 70-75% relative humidity and were used for transient expression assays at 3-4 weeks of age. 2.2 Generation of Transgenic Rice Oryza sativa cv. Nipponbare was used for all transformations. For overexpression, the coding sequence of OsLsm1 was cloned into a CaMV 35S-driven pCAMBIA1300 vector and introduced via Agrobacterium tumefaciens (strain EHA105) by a commercial service (Wuhan Biorun). For knockout, target-specific gRNAs were cloned into a pRGEB32 Cas9 vector. Transgenic plants were generated following standard Agrobacterium-mediated protocols, and homozygous lines were confirmed by western blot, PCR, sequencing, and RT-qPCR for expression analysis. Homozygous T3 plants were used for all subsequent experiments. not-yet-known not-yet-known not-yet-known unknown 2.3 Plasmid Construction The coding sequences (CDS) of OsLsm1 (RAP-DB: Os04g0445800), OsLsm4 (RAP-DB: Os01g0256900), RSV p2 (UniProt: Q89717), OsDCP1 (RAP-DB: Os12g0156400), and OsDCP2 (RAP-DB: Os02g0805900) were amplified by PCR from Nipponbare cDNA or RSV-infected Nipponbare cDNA, respectively, using high-fidelity KOD DNA polymerase (TOYOBO). For bimolecular fluorescence complementation (BiFC) assays, the CDS were cloned into pSPYNE-35S (for nYFP fusions) and pSPYCE-35S (for cYFP fusions) vectors using Gateway cloning (Invitrogen) or restriction enzyme-based ligation. For co-immunoprecipitation (Co-IP) and subcellular localization, CDS were inserted into pCAMBIA1300-based vectors to generate C-terminal GFP, RFP, Flag, or Myc fusion constructs under the control of the CaMV 35S promoter. The P-body marker NbDCP5-RFP was constructed by amplifying the N. benthamiana DCP5 CDS and cloning it into a 35S -driven RFP vector. The OsLsm1Δ17AA mutant was generated by overlapping PCR using primers flanking the 17-amino-acid deletion site. For yeast two-hybrid (Y2H) assays, CDS were cloned into pGADT7 (AD) and pGBKT7 (BD) vectors. For in vitro protein expression, OsLsm1 was cloned into the pGEX-4T-1 vector for GST-fusion expression. All primers used for cloning are listed in Supplemental Table 1. All constructs were verified by Sanger sequencing. not-yet-known not-yet-known not-yet-known unknown 2.4 Agroinfiltration for Transient Expression Agrobacterium tumefaciens strain GV3101 carrying the desired binary vectors was grown in LB medium to an OD600 of 0.1-0.3. Cells were harvested and resuspended in infiltration buffer (10 mM MES pH 5.6, 10 mM MgCl2, 150 µM acetosyringone). For co-expression, cultures were mixed in equal ratios to a final OD600 of 0.5 for each construct. The mixtures were incubated at room temperature for 2-3 h before being infiltrated into the leaves of 4- to 5-week-old N. benthamiana plants using a 1-mL needleless syringe. Infiltrated plants were maintained in the growth chamber for 48-72 h before analysis. For heat shock experiments, infiltrated plants were transferred to a 42 °C incubator for 30 min at 48 hours post-infiltration (hpi) before imaging. Tissues were harvested for microscopy or protein extraction at 48-72 hpi. not-yet-known not-yet-known not-yet-known unknown 2.5 VIGS in N. benthamiana For Virus-Induced Gene Silencing (VIGS), a 300-bp fragment of NbLsm1 was cloned into the Tobacco rattle virus (TRV) vector pTRV2. A. tumefaciens cultures containing pTRV1 and pTRV2 constructs (empty pTRV2, pTRV2-NbLsm1, or pTRV2-PDS) were mixed 1:1 and co-infiltrated into two cotyledons of 2-week-old N. benthamiana seedlings. Plants were used for subsequent experiments 2-3 weeks later, when the photobleaching phenotype was visible on PDS-silenced plants. 2.6 Bimolecular Fluorescence Complementation (BiFC) Assay The CDS of genes of interest were cloned into pCV vectors containing the N-terminal (nYFP) or C-terminal (cYFP) fragments of YFP. Agrobacterium cultures carrying the corresponding nYFP and cYFP fusion constructs were co-infiltrated into N. benthamiana leaves. YFP fluorescence was observed 48 h post-infiltration using a Nikon A1R confocal laser scanning microscope. YFP was excited at 488 nm, and emission was collected between 500 and 550 nm. 2.7 Co-Immunoprecipitation (Co-IP) and Western Blotting N. benthamiana leaf tissue (approximately 100 mg) was harvested 48 h post-infiltration, flash-frozen in liquid nitrogen, and ground to a fine powder. Total proteins were extracted in GTEN buffer (10% glycerol, 25 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl) supplemented with 1% IGEPAL-630, 1 mM DTT, 0.2% PVPP, and 1× cOmplete TM Protease Inhibitor Cocktail (Roche). Lysates were clarified by centrifugation at 12,000 rpm for 10 min at 4 °C. For immunoprecipitation, 10 µL of anti-GFP magnetic beads (Miltenyi Biotec) were added to the supernatant and incubated for 2 h at 4 °C with gentle rotation. The beads were washed five times with IP wash buffer (GTEN with 0.1% IGEPAL-630). Bound proteins were eluted by boiling in 2×SDS loading buffer for 10 min. For western blotting, total proteins or IP eluates were separated by SDS-PAGE (12% gel) and transferred to a PVDF membrane. Membranes were blocked with 5% non-fat milk in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 1 hour and incubated with primary antibodies: anti-GFP (1:10,000, Abcam), anti-Flag (1:10,000, Sigma-Aldrich), anti-Myc (1:10,000, Cell Signaling Technology), anti-GST (1:5,000, Abcam), or custom-generated anti-RSV CP (1:5,000) and anti-RSV p2 (1:5,000). After incubation with HRP-conjugated secondary antibodies (1:10,000), signals were detected using an ECL chemiluminescence kit (Thermo Fisher Scientific). Coomassie brilliant blue (CBB) staining was used to verify protein loading. 2.8 Confocal Laser Scanning Microscopy and Image Analysis All imaging was performed on a Nikon A1R HD25 confocal microscope. YFP was excited with a 488 nm laser, and RFP was excited with a 561 nm laser. Emission was collected at 500-550 nm for YFP and 570-620 nm for RFP. Images were acquired using a 20× objective. Bright-field images were captured using a transmitted light detector. Image processing, including channel merging and fluorescence intensity quantification, was performed using NIS-elements software (Nikon) and ImageJ/Fiji. For quantification, Z-stacks were brightness increased and a median filter of 2 pixels applied. Stomata were manually removed from the micrographs, and a mask was generated using thresholding. Foci were counted using the “Analyze Particles” tool. At least 12 independent images from three biological replicates were analyzed for each condition. 2.9 Sequence Analysis Protein sequences of OsLsm1 (RAP-DB: Os04g0445800) and NbLsm1 (Niben101Scf11312g01002) were aligned using the ClustalW algorithm within the Jalview (2.11.1.4) software package. 2.10 Virus Source and Inoculation RSV was maintained in persistently infected rice plants and transmitted by its insect vector, the small brown planthopper ( Laodelphax striatellus ). For RSV inoculation, non-viruliferous small brown planthoppers were allowed to feed on RSV-infected rice plants for 48 h to acquire the virus. After a 10-day latency period, the now-viruliferous planthoppers were used to inoculate 2-week-old rice seedlings. Each seedling was challenged with three viruliferous small brown planthoppers for 3 days. Disease symptoms and viral accumulation were assessed at 20-21 days post-inoculation (dpi). Symptom severity was classified into three categories: N (asymptomatic), Grade I (mild chlorotic stripes), and Grade II (severe chlorosis, stunting, and necrosis). Mechanical inoculation of RSV onto N. benthamiana was performed to establish systemic infection for subsequent experiments. Inoculum was prepared from systemically infected rice leaves collected at 20 days post-inoculation (dpi). Approximately 1 g of leaf tissue was ground to a fine powder in liquid nitrogen and homogenized in 5 ml of inoculation buffer (50 mM sodium phosphate, pH 7.2, containing 0.1% sodium sulfite). Leaves of 4- to 5-week-old N. benthamiana plants were lightly dusted with carborundum powder (600 mesh). The prepared inoculum was then gently rubbed onto the surface of two to three upper leaves of each plant. The inoculated leaves were rinsed with deionized water approximately 10 minutes after inoculation, and the plants were maintained in a controlled growth chamber under standard conditions. Systemic infection in newly emerged upper leaves was confirmed at 20 dpi by immunoblotting with an anti-RSV CP antibody. These systemically infected leaves were then used for subsequent co-expression and protein interaction analyses. 2.11 RNA Extraction and Quantitative Real-Time PCR (RT-qPCR) Total RNA was extracted from rice leaf tissue using TRIzol reagent (Invitrogen) and treated with DNase I. First-strand cDNA was synthesized using the TransScript II All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech). RT-qPCR was performed on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems) using ChamQ SYBR qPCR Master Mix (Vazyme). The relative expression levels of target genes were calculated using the 2 -ΔΔCt method, with OsUBQ5 or NbActin serving as the internal reference gene. Primers are listed in Supplemental Table 1. 2.12 Cordycepin Treatments and mRNA Decay Assay For mRNA decay assays, N. benthamiana leaves were infiltrated with 500 µM cordycepin (3’-deoxyadenosine; Sigma-Aldrich) to inhibit transcription. Leaf samples were collected at 3, 6, 9, 12, and 24 hours post-treatment. Total RNA was extracted at each time point, and the relative abundance of NbEXPL1 and NbSEN1 transcripts was determined by RT-qPCR. 2.13 Recombinant Protein Expression and Purification The pGEX-4T-1-OsLsm1 plasmid was transformed into E. coli strain BL21 (DE3). Protein expression was induced with 0.5 mM IPTG at 16 °C for 16 h. Cells were harvested and lysed by sonication. The GST-OsLsm1 protein was purified from the soluble fraction using ProteinIso GST Resin (TransGen Biotech) according to the manufacturer’s instructions. The pGEX-4T-1 empty vector was used to express and purify GST as a control. 2.14 Microscale Thermophoresis (MST) Assay The binding affinity between OsLsm1-GST and p2-RFP was measured using a Monolith NT.115 instrument (NanoTemper Technologies). p2-RFP was affinity-purified from transiently expressed N. benthamiana leaves. The concentration of fluorescently-labeled p2-RFP was kept constant (50 nM), while the concentration of OsLsm1-GST or GST was serially diluted (from 10 µM to 0.3 nM). Measurements were performed in MST buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) at 25 °C. The dissociation constant ( Kd ) was calculated by fitting the dose-response curve using MO. Affinity Analysis software. 2.15 Protein Structure Modeling The protein structures of OsLsm1, OsLsm4, and RSV p2 were predicted using the AlphaFold server (Jumper et al., 2021). Protein-protein docking and interaction interface prediction for OsLsm1-OsLsm4 and OsLsm1-p2 complexes were performed using the HDOCK server. The final structural models were visualized and rendered using PyMOL. 2.16 Yeast Two-Hybrid (Y2H) Assay The Y2H assay was performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech). pGADT7 and pGBKT7-based constructs were co-transformed into Saccharomyces cerevisiae strain AH109. Transformants were selected on SD/-Leu/-Trp medium. To test for interactions, colonies were grown on selective medium SD/-Leu/-Trp/-His/-Ade. Plates were incubated at 30 °C for 2-3 days. not-yet-known not-yet-known not-yet-known unknown 2.17 Quantification and Statistical Analysis All experiments were repeated at least three times with consistent results. Data are presented as mean ± SD. Statistical significance between two groups was determined using Student’s t -test. Statistical analyses were performed using GraphPad Prism 10. For violin plots, the median and quartiles are indicated. P -values < 0.05 were considered statistically significant. In figures, asterisks denote significance levels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 3 Results 3.1 The RSV p2 Interacts with the P-body Component OsLsm1 To identify host factors directly targeted by the RSV virulence protein p2, we performed affinity purification of interaction proteins coupled with IP-MS using transgenic rice stably expressing Myc-tagged p2 (p2-Myc). Comparative analysis of p2-associated proteins relative to wild-type controls revealed a distinct subset of host proteins specifically enriched in the p2 immunoprecipitates (Supplemental Table 2). Among the highest-confidence candidates, OsLsm1 (UniProt: Q0JCW8), a conserved subunit of the Lsm1-7 mRNA decapping activator complex, was selected for further investigation. The interaction between p2 and OsLsm1 was examined in planta using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana ( N. benthamiana ). Strong yellow fluorescent protein (YFP) signals were observed in the cytoplasm and nucleus of cells co-expressing OsLsm1 and p2 fused to complementary YFP fragments (OsLsm1-nYFP/p2-cYFP and OsLsm1-cYFP/p2-nYFP). In contrast, no fluorescence was detected in negative controls where OsLsm1 was replaced with GUS (Figure 1A). Co-immunoprecipitation assays further confirmed the interaction, as OsLsm1-GFP efficiently co-precipitated p2-Flag but not GUS-Flag (Figure 1B). To determine whether this interaction is direct, microscale thermophoresis (MST) assays were conducted using recombinant OsLsm1-GST and fluorescently labeled p2-RFP. OsLsm1-GST bound p2-RFP in a dose-dependent manner, whereas GST alone showed no detectable binding (Figure 1C and Supplemental Figure 1). These results demonstrate that RSV p2 directly interacts with OsLsm1. 3.2 Lsm1 Negatively Regulates RSV Infection To examine the role of OsLsm1 during RSV infection, transgenic rice lines overexpressing OsLsm1 (OE- OsLsm1 ) or carrying loss-of-function mutations were generated. Two independent homozygous overexpression lines (OE-#3 and OE-#13) exhibiting elevated OsLsm1 transcript levels were selected for further experiments (Supplemental Figure 2A and 2B). Overexpression of OsLsm1 did not cause obvious developmental defects, whereas OsLsm1 knockout plants displayed severe dwarfism and failed to produce seeds (Supplemental Figure 2C-2E). Following RSV inoculation, OE- OsLsm1 plants exhibited milder disease symptoms compared with wild-type plants, including reduced chlorotic striping and growth inhibition (Figure 2A), which was supported by quantitative disease scoring (Figure 2B). Viral accumulation was also reduced in OE- OsLsm1 plants, as indicated by lower levels of RSV coat protein (CP) transcripts and decreased accumulation of CP and p2 proteins (Figure 2C and 2D). Given the central role of P-bodies in post-transcriptional regulation, we also examined the transcriptional response of the P-body network during RSV infection. In wild-type plants, OsLsm1 transcript levels were not induced by RSV infection (Supplemental Figure 3A). In addition, RSV infection did not lead to coordinated transcriptional induction of other P-body-associated genes in wild-type plants (Supplemental Figure 3B). In contrast, RSV infection triggered a robust and concerted induction of multiple components of the mRNA decay machinery, including OsLsm2 , OsLsm4 , OsLsm5 , OsLsm6 , OsLsm7 , OsDCP1 , and OsDCP5 , in OE- OsLsm1 plants (Supplemental Figure 3C), suggesting that elevated OsLsm1 levels are associated with enhanced activation of the RNA decay network during infection. To assess whether the antiviral role of Lsm1 is conserved across plant species, we compared Lsm1 sequences from rice and N. benthamiana . Sequence alignment revealed a high degree of conservation within the core Lsm domain, including residues implicated in RNA binding and assembly of the decapping complex (Supplemental Figure 4). Consistent with functional conservation, virus-induced gene silencing (VIGS) of NbLsm1 in N. benthamiana resulted in markedly enhanced susceptibility to RSV. NbLsm1 -silenced plants developed accelerated and severe disease symptoms, accompanied by significantly increased viral RNA and protein accumulation compared with control plants (Figure 2E-2I). Together, these results demonstrate that OsLsm1 positively regulates resistance to RSV in rice and that Lsm1 functions as a conserved host factor restricting RSV accumulation across plant species. 3.3 The p2-OsLsm1 Interaction Localizes to Cytoplasmic P-bodies and Is Enhanced by Heat Stress To determine the cellular context in which p2 engages OsLsm1, BiFC analysis was performed in the presence of the P-body marker NbDCP5-RFP. Fluorescence signals arising from the interaction between OsLsm1 and p2 were detected exclusively within discrete cytoplasmic puncta labeled by NbDCP5-RFP, indicating that p2 associates with OsLsm1 within cytoplasmic P-bodies (Figure 3A and 3B). Because P-body assembly and activity are dynamically regulated by cellular stresses such as heat, salt, and pathogen challenge (Buchan, 2024; Kearly, Nelson, Skirycz, & Chodasiewicz, 2024), we next examined whether the p2-OsLsm1 association responds to heat stress. Following heat shock treatment at 42 °C, both the number and fluorescence intensity of puncta co-labeled by p2, OsLsm1, and NbDCP5 were significantly increased compared with mock-treated controls (Figure 3C-3E). These results indicate that the association between RSV p2 and OsLsm1 occurs within stress-responsive P-bodies. 3.4 RSV p2 Disrupts OsLsm1-OsLsm4 Complex Assembly Structural and genetic studies in other systems indicate that Lsm1 interacts with Lsm4 to stabilize the Lsm1-7 ring that activates mRNA decapping (Lührmann & Stark, 2009; Maldonado-Bonilla, 2014; Perea-Resa, Hernández-Verdeja, López-Cobollo, Castellano, & Salinas, 2012; Sobti, Cubeddu, Haynes, & Mabbutt, 2010; Tharun et al., 2000). Using bimolecular fluorescence complementation (BiFC), we confirmed that OsLsm1 and OsLsm4 form a complex in plant cells, producing distinct punctate signals that co-localized with the P-body marker NbDCP5 (Supplemental Figure 5A). Although both OsLsm1 and OsLsm4 were enriched in p2-associated complexes identified by IP-MS, direct interaction between p2 and OsLsm4 could not be detected by yeast two-hybrid or Co-IP assays (Supplemental Table 2; Supplemental Figure 5B-5E), indicating that p2 primarily targets OsLsm1. To determine whether p2 affects Lsm1-Lsm4 complex formation, p2 was transiently expressed in N. benthamiana together with OsLsm1 and OsLsm4. Expression of p2 markedly reduced the OsLsm1-OsLsm4 BiFC signal intensity, while immunoblot analysis showed no significant changes in the protein levels of either OsLsm1 or OsLsm4 (Figure 4A-4F). These results indicate that p2 alone is sufficient to interfere with OsLsm1-OsLsm4 complex assembly. We next examined whether disruption of the OsLsm1-OsLsm4 interaction also occurs during RSV infection. In mock-infected cells, OsLsm1-OsLsm4 BiFC signals formed prominent puncta that co-localized with P-bodies. In contrast, systemic RSV infection resulted in a marked reduction in the intensity of these BiFC signals (Figure 4G, 4H, 4K and 4L). Consistent with the p2 expression experiments, immunoblot analyses showed that RSV infection did not significantly alter the abundance of OsLsm1 or OsLsm4 proteins (Figure 4I and 4J). These observations indicate that RSV infection disrupts OsLsm1-OsLsm4 complex assembly without affecting protein stability. To investigate how p2 interferes with OsLsm1-OsLsm4 interaction, structural modeling using AlphaFold was performed. The predicted structures indicated that both OsLsm4 and p2 interact with a shared α-helical surface located in the N-terminal region of OsLsm1 (Figure 5A). Sequence analysis identified a conserved 17-amino acid hydrophobic motif likely forming this shared interface (Figure 5B). Deletion of this motif (OsLsm1 Δ17AA ) significantly weakened both OsLsm1-OsLsm4 and OsLsm1-p2 interactions in BiFC assays (Figure 5C-5F). In addition, competitive co-immunoprecipitation assays showed that increasing p2 expression progressively reduced the association between OsLsm1 and OsLsm4, whereas a control protein had no effect (Figure 5G). not-yet-known not-yet-known not-yet-known unknown 3.5 RSV p2 Impairs Lsm1-Dependent mRNA Decay Because Lsm1 is an essential component of processing bodies (P-bodies), we first examined whether RSV infection affects P-body formation or integrity. Confocal microscopy using the P-body markers NbDCP1 and NbDCP2 showed that RSV infection did not cause detectable changes in P-body number, size, or subcellular distribution compared with mock-treated controls (Supplemental Figure 6). These observations indicate that RSV infection does not disrupt P-body biogenesis or stability. Previous studies have identified Expansin-Like1 (EXPL1 ) and Senescence-related 1 (SEN1 ), as unstable transcripts that are degraded via the P-body-associated 5′-3′ mRNA decay pathway (J. Xu & Chua, 2009; Jun Xu, Yang, Niu, & Chua, 2006). Indeed, in NbLsm1 -silenced plants, both NbEXPL1 and NbSEN1 transcripts accumulated to higher levels and exhibited significantly prolonged decay kinetics compared with control plants (Figure 6A and 6B). As expected, transient expression of p2 protein also led to sustained accumulation of both transcripts (Figure 6E and 6F; Supplemental Figure 7B). Consistently, RSV infection induced a transient increase in NbEXPL1 and NbSEN1 transcript levels (Figure 6C and 6D; Supplemental Figure 7A). The convergence of Lsm1 loss-of-function and p2 expression on the same molecular outcome indicates that RSV, through p2, directly antagonizes the functional activity of Lsm1 in mRNA turnover. not-yet-known not-yet-known not-yet-known unknown 4 Discussion P-bodies are central hubs of cytoplasmic RNA metabolism, coordinating mRNA decapping, turnover, and storage in response to developmental and environmental cues. Although P-bodies have been implicated in antiviral defense against several positive-strand RNA viruses, whether and how plant negative-strand RNA viruses exploit P-body-associated pathways has remained largely unexplored. Here, we demonstrate that RSV actively subverts P-body-mediated mRNA decay through a highly specific mechanism that functionally disables the Lsm1-7 decapping activator complex without disrupting P-body integrity. The roles of Lsm1 and its associated Lsm complexes during viral infection are notably complex and virus-specific. Previous reports have shown that Lsm1 is not only a component of the Lsm1-7 ring complex but can also act independently during viral infection. In turnip mosaic virus (TuMV) infection, Lsm1 modulates the activity of RNA-dependent RNA polymerase 6 (RDR6) to influence antiviral RNA silencing (Zuo et al., 2022). Several positive-strand RNA viruses, including Brome mosaic virus (BMV), hijack the Lsm1-7 complex to promote viral RNA translation and replication (Galão et al., 2010). Conversely, Lsm proteins exhibit antiviral activity in other cases. For example, knockdown of Lsm1 enhances viral RNA accumulation in hepatitis B virus (HBV) infection (Rahman et al., 2022). In animal systems, Lsm1 and other P-body-associated factors have likewise been implicated in viral replication, as observed for hepatitis C virus (HCV) and West Nile virus (WNV), where viral RNAs exploit host mRNA decay machinery to enhance replication efficiency (Chahar, Chen, & Manjunath, 2013; Giménez-Barcons et al., 2013; Scheller et al., 2009). Our results here demonstrate that Lsm1 functions as a conserved antiviral factor restricting RSV accumulation. Overexpression of OsLsm1 in rice significantly enhanced resistance to RSV infection, whereas silencing of NbLsm1 in N. benthamiana led to pronounced hypersusceptibility. Although the essential role of Lsm1 in development prevented the generation of stable knockout rice lines, the consistent phenotypes observed across two plant species provide strong genetic support for a conserved antiviral function of Lsm1. We propose that elevated levels of Lsm1 effectively titrate out RSV p2, ensuring that sufficient Lsm1 remains available to assemble productively with Lsm4 and other Lsm subunits, thereby maintaining the integrity and antiviral capacity of the decapping machinery. Viruses have evolved to encode multifunctional proteins that promote efficient replication by reshaping host cellular environments through interactions with key regulatory nodes. This principle is evident across diverse viral taxa, in which single viral effectors integrate multiple activities to overcome host defenses and optimize infection (Kumar, 2025). In plant viruses, this strategy is exemplified by proteins such as the betasatellite-encoded βC1 and the cucumber mosaic virus (CMV) 2b protein, which integrate immune suppression with transcriptional and post-transcriptional reprogramming to promote infection (Zhiyou Du et al., 2014; Zhao et al., 2025). In this context, RSV p2 represents a highly versatile viral effector. Previous studies have shown that p2 suppresses RNA silencing via SGS3 (Z. Du et al., 2011), facilitates long-distance movement through interaction with fibrillarin (Zheng et al., 2015), modulates hormone signaling through OsARF17 (H. Zhang et al., 2020), and and regulates antiviral immunity via ATG5-associated autophagy pathways (X. Zhang et al., 2023; X. Zhang et al., 2021). Our study expands this functional repertoire by identifying OsLsm1 as a direct host target of p2. Within the canonical Lsm1-7 complex, the interaction between Lsm1 and Lsm4 is essential for maintaining ring integrity and licensing decapping-dependent 5′-3′ mRNA decay (Beckham & Parker, 2008; Zhou et al., 2014). Structural and biochemical studies in yeast and mammals have established that the Lsm1-Lsm4 interface anchors the complex and licenses efficient 5′-3′ mRNA decay, while the C-terminal region of Lsm4 is critical for P-body localization (Catalá et al., 2019; Decker, Teixeira, & Parker, 2007; He & Jacobson, 2015; Reijns, Alexander, Spiller, & Beggs, 2008). Consistent with this conserved architecture, we found that RSV p2 competitively binds to OsLsm1 and disrupts its interaction with OsLsm4, thereby destabilizing the Lsm1-7 complex. Notably, RSV infection did not alter the abundance, size, or subcellular distribution of P-bodies. Instead, p2 selectively interfered with the functional output of these RNA granules while preserving their architecture. This strategy contrasts with those employed by certain animal viruses, such as poliovirus, which actively dismantle P-bodies to evade RNA surveillance (Dougherty, White, & Lloyd, 2011). Our findings support an emerging view of RNA granules as modular reaction platforms whose biochemical activities can be selectively rewired without overt structural remodeling (Eulalio, Behm-Ansmant, & Izaurralde, 2007). By targeting a single, critical protein-protein interaction within the Lsm complex, RSV achieves precise control over mRNA decay while minimizing broader cellular perturbations. RSV relies on cap-snatching to initiate viral transcription, acquiring 5′ m⁷G caps from host mRNAs (Barbier, Takahashi, Nakamura, Toriyama, & Ishihama, 1992; Tian et al., 2025). Results here showed that the decapping-sensitive mRNAs EXPL1 and SEN1 were selectively stabilized upon NbLsm1 silencing, RSV infection, or p2 expression, phenocopying defects observed in core decapping components such as DCP1, DCP2, VCS, and DCP5 (J. Xu & Chua, 2009; Jun Xu et al., 2006). It is reasonable to assume that reduced decapping activity may therefore help maintain a pool of capped host transcripts that are accessible for this process. Additionally, stabilization of EXPL1- and SEN1-associated pathways may also have broader physiological consequences for infected tissues. Expansin-mediated cell wall loosening can facilitate pathogen (e.g., bacteria and fungi) movement through plant tissues, while senescence-associated transcriptional programs may create cellular environments permissive to animal viral replication and spread (Cosgrove, 2024; Seoane, Vidal, Bouzaher, El Motiam, & Rivas, 2020). Although our data do not directly address these outcomes, they raise the possibility that RSV-induced modulation of Lsm1-dependent mRNA decay influences host physiology in ways that could indirectly favor viral spread. Together, our work suggests that P-bodies, through Lsm1-dependent mRNA decapping, constitute a post-transcriptional barrier that restricts RSV infection (Figure 7). RSV overcomes this barrier by deploying p2 to associate with Lsm1 and weaken Lsm1-Lsm4 complex assembly (Figure 7). This counter-defense strategy allows RSV to modulate host RNA decay pathways to its advantage. not-yet-known not-yet-known not-yet-known unknown Acknowledgements We thank Professor M. J. Adams (Minehead, UK) for manuscript correction. This work was financially supported by the National Natural Science Foundation of China (U22A20479, U23A6006, 32270144 and 32170145) and K. C. Wong Magna Fund in Ningbo University. 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RSV p2 directly and specifically interacts with the P-body component OsLsm1. (A) Bimolecular fluorescence complementation (BiFC) assays in N. benthamiana epidermal cells. OsLsm1 and p2 were fused with the N-terminal (nYFP) or C-terminal (cYFP) fragments of YFP. Co-expression of OsLsm1-nYFP and RSV p2-cYFP (top panel) or OsLsm1-cYFP and RSV p2-nYFP (bottom panel) reconstituted YFP fluorescence. GUS (β-glucuronidase) fusions served as negative controls. Images were taken 48 hours post-infiltration: scale bars, 100 µm. (B) Co-immunoprecipitation (Co-IP) analysis of the p2-OsLsm1 interaction. OsLsm1-GFP was co-expressed with either RSV p2-Flag or GUS-Flag (negative control). Total protein extracts from N. benthamiana leaves expressing the indicated constructs were subjected to immunoprecipitation with anti-GFP magnetic beads (IP: GFP). Co-precipitated p2-Flag was detected by immunoblotting with an anti-Flag antibody. (C) Microscale thermophoresis (MST) analysis. MST assay quantifying the direct binding between OsLsm1 and p2. The change in normalized fluorescence (ΔFnorm) was plotted against the concentration of the ligand (OsLsm1-GST or Mock-GST). A constant concentration of RSV p2-RFP was titrated with serially diluted OsLsm1-GST (red) or GST (green). Data points represent the mean ± SD from three independent biological replicates. Figure 2. Lsm1 negatively regulates RSV infection. (A) Disease symptoms of wild-type (Nip) and OsLsm1 -overexpressing (OE- OsLsm1 ) lines (3# and 13#) following mock or RSV infection at 20 days post-infection (dpi). Scale bars, 5 cm and 1 cm. (B) Quantification of RSV disease severity. The percentage of plants exhibiting different symptom grades (N, asymptomatic; Grade I, mild symptoms; Grade II, severe symptoms) was scored at 20 dpi. Data were collected from at least 40 plants per line. (C) RT-qPCR analysis of RSV coat protein (CP) transcript levels in RSV-infected Nip and OE- OsLsm1 plants at 20 dpi. Data were normalized to OsUBQ5 and presented as mean ± SD (n=3 independent biological replicates), using Student’s t -test (*** P < 0.001). (D) Western blot analysis of viral protein accumulation in mock- and RSV-infected plants using antibodies against CP, p2, and Myc. Coomassie brilliant blue (CBB): loading control. Relative band intensities were quantified using ImageJ and are indicated below each lane. (E) Symptomatic analysis of NbLsm1 -silenced (TRV: NbLsm1 ), control (TRV: 00 ), and phytoene desaturase-silenced (TRV: PDS ) N. benthamiana plants, followed by RSV infection. Representative whole-plant and leaf images are shown at the indicated time points. Scale bars, 5 cm and 2 cm. (F) RT-qPCR analysis of NbLsm1 transcript levels in silenced (TRV: NbLsm1 ) and control (TRV: 00 ) plants. Data were normalized to NbActin and presented as mean ± SD (n=3 independent biological replicates). Student’s t -test (*** P < 0.001). (G) Western blot analysis of RSV CP accumulation in TRV: 00 and TRV: NbLsm1 plants, with CBB staining as a loading control. (H) RT-qPCR analysis of RSV CP transcript levels in silenced and control plants at 20 dpi with RSV. (I) Disease severity index of RSV-infected TRV: NbLsm1 and TRV: 00 plants at the indicated time points. Disease severity was scored on a 0-4 scale and converted to percentage values. Data are presented as mean ± SD (n=30 plants per group) by two-way ANOVA. Figure 3. RSV p2 drives an interaction-dependent and stress-responsive localization to P-bodies. (A) Co-localization of the p2-OsLsm1 complex with the P-body marker NbDCP5-RFP. The YFP signal from the OsLsm1-cYFP/RSV p2-nYFP interaction (green) precisely co-localizes with NbDCP5-RFP (red), appearing as yellow puncta in the merged bright-field (BF) image. Scale bars, 50 µm. (B) Fluorescence intensity line-scan profile along the white arrow in the enlarged image of panel A. The plot shows the relative fluorescence intensity of the reconstituted YFP and RFP channels with peaks coinciding. (C) Heat shock potentiates the recruitment of the p2-OsLsm1 complex to P-bodies. The top row (Mock) shows the basal co-localization, while the bottom row (Heatshock) shows a marked increase in puncta after a 42 °C treatment. Scale bars, 20 µm. (D) Quantification of the relative density of co-localized puncta from panel C. The violin plot displays the distribution of data points from multiple cells (n = 12 cells from three independent experiments). Statistical significance was determined using Student’s t -test. ****, P < 0.0001. (E) Fluorescence intensity profile along the white arrow in the enlarged heat-shock image of panel C. Figure 4. RSV infection and p2 protein disrupt the OsLsm1-OsLsm4 complex in P-bodies. (A-B) The BiFC pairs of OsLsm1-OsLsm4 (green) in N. benthamiana leaves were co-expressed with either RSV p2-Flag or GUS-Flag (control). NbDCP5-RFP (red) as P-body marker. Scale bars, 20 µm. (C-D) Western blot analysis showing stable expression of OsLsm fusion proteins (detected by anti-GFP) with GUS (control) or p2 proteins. Protein extracts from (A) and (B). (E-F) Quantification of the relative fluorescence intensity of the BiFC signal from images in (A) and (B). All violin plots represent data from at least 12 cells across three independent biological replicates. Statistical significance was determined using Student’s t -test. ****, P < 0.0001. (G-H) BiFC visualization of OsLsm1-OsLsm4 interactions in N. benthamiana leaves under mock or RSV-infected conditions. OsLsm1-nYFP/OsLsm4-cYFP and OsLsm1-cYFP/OsLsm4-nYFP combinations (green) were co-expressed with the P-body marker NbDCP5-RFP (red). Scale bars, 20 µm. (I-J) Western blot analysis showing stable expression of OsLsm fusion proteins under mock and RSV-infected conditions. Protein extracts from the experiments in (G) and (H) were immunoblotted. Anti-GFP antibody detects the individual Lsm fusion proteins. Anti-RSV CP antibody detects the accumulation of RSV. CBB staining serves as a loading control. (K-L) Quantification of the relative fluorescence (FL) intensity of the BiFC signal under mock and RSV-infected conditions from images in (G) and (H), **** P < 0.0001. Figure 5. RSV p2 competes with OsLsm4 for binding to a conserved OsLsm1 interface. (A) Structural models of OsLsm1 (yellow) in complex with OsLsm4 (green) or RSV p2 (cyan). Predicted interaction residues on OsLsm1 are highlighted in red. (B) Sequence alignment of the OsLsm1 interaction interface. Residues predicted to be involved in binding both OsLsm1 with OsLsm4 (OsL1&OsL4) and p2 (OsL1&p2) are highlighted in red. The 17-amino-acid deletion region is marked. (C, E) BiFC assay and quantification showing that the interaction between wild-type (WT) OsLsm1 and OsLsm4 is abolished by the △17AA mutation. (D, F) BiFC assay and quantification demonstrating that the △17AA mutation also abolishes the interaction between OsLsm1 and RSV p2. Scale bars in C, D, 25 µm. For all violin plots, data were collected from at least 12 cells across three independent biological replicates. Statistical significance was determined by Student’s t -test. ****, P < 0.0001. (G) Competitive co-immunoprecipitation assay. OsLsm1 was co-expressed with OsLsm4 in N. benthamiana . Increasing amounts of RSV p2 protein extract were added to the lysate before immunoprecipitation with anti-GFP beads. Relative band intensities for the IP fractions are shown below the blots. Figure 6. RSV p2 impairs Lsm1-dependent mRNA decay. (A) RT-qPCR analysis of unstable genes NbEXPL1 ( Expansin-Like1 ) transcript level in TRV: 00 and TRV: NbLsm1 plants at different time points after cordycepin treatment. (B) RT-qPCR analysis of unstable genes NbSEN1 ( Senescence-related 1 ) transcript level in TRV: 00 and TRV: NbLsm1 plants at different time points after cordycepin treatment. (C, D) RT-qPCR analysis of unstable genes NbEXPL1 and NbSEN1 in mock- or RSV-infected wild-type N. benthamiana at different time points after cordycepin treatment. (E, F) RT-qPCR analysis of NbEXPL1 and NbSEN1 expression in N. benthamiana leaves transiently overexpressing GUS-Myc or RSV p2-Myc at the indicated time points after cordycepin treatment. For all RT-qPCR data, expression was normalized to NbActin . Data are presented as mean ± SD from three biological replicates. Statistical significance was determined by Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant). Figure 7. A working model for RSV p2-mediated subversion of Lsm1-dependent defense. P-bodies, probably through Lsm1-dependent mRNA decapping, constitute a post-transcriptional barrier that restricts RSV infection (left). Meanwhile, RSV overcomes this barrier by deploying p2 to associate with Lsm1 and weaken Lsm1-Lsm4 complex assembly (right). This counter-defense strategy allows RSV to modulate host RNA decay pathways to its advantage. Information & Authors Information Version history V1 Version 1 13 January 2026 Peer review timeline Published Molecular Plant Pathology Version of Record 6 May 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords lsm1 p-body p2 rice stripe virus (rsv) signaling Authors Affiliations Anqi Hu 0009-0000-2153-7905 Zhejiang University College of Agriculture and Biotechnology View all articles by this author Zizhou Zhao 0009-0003-0707-9778 Zhejiang University College of Agriculture and Biotechnology View all articles by this author Minjie Shen Ningbo University View all articles by this author Yi Chen Ningbo University View all articles by this author Ying Chen Ningbo University View all articles by this author Miaomiao Li Ningbo University View all articles by this author Lin Lin Ningbo University View all articles by this author Hongying Zheng Ningbo University View all articles by this author Jiejun Peng Ningbo University View all articles by this author Yan Liang Zhejiang University College of Agriculture and Biotechnology View all articles by this author Jianping Chen Ningbo University View all articles by this author Fei Yan 0000-0002-0049-8588 Ningbo University View all articles by this author Yuwen Lu 0000-0002-5292-960X [email protected] Ningbo University View all articles by this author Metrics & Citations Metrics Article Usage 185 views 128 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Anqi Hu, Zizhou Zhao, Minjie Shen, et al. The rice stripe virus p2 interacts with Lsm1 and disrupts the Lsm1-Lsm4 complex to facilitate the viral infection. Authorea . 13 January 2026. DOI: https://doi.org/10.22541/au.176828245.52279353/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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