Rational Design of Phytovirucide that Inhibits the Nucleocapsid Condensates of Tomato Spotted Wilt Virus

Rational Design of Phytovirucide that Inhibits the Nucleocapsid Condensates of Tomato Spotted Wilt Virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Rational Design of Phytovirucide that Inhibits the Nucleocapsid Condensates of Tomato Spotted Wilt Virus Runjiang Song, Zan ning, Jiao Li, Jiahui Yao, Shang Wu, Jianzhuan Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4398439/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Chemical management of crop failures caused by plant viruses poses serious challenges in agricultural chemistry. Recently, phase separation has emerged as a key role in the viral lifecycle, with the discovery of corresponding small molecule inhibitors in the medical field sparking significant interest. However, the applicability of this promising antiviral tactic for plant protection remains largely unknown. Herein, we have demonstrated that the aggregation of the tomato spotted wilt virus (TSWV) nucleocapsid protein (N) is regulated by RNA-induced phase separation, and disclosed a new rationally designed inhibitor Z9 . Z9 is capable of binding to TSWV N at the R 94 and Y 184 sites, preventing the assembly of N and RNA into aggregated ribonucleoproteins through phase separation. Mutations of the binding sites substantially diminish the colocalization between N protein and processing bodies, interfering with the normal interaction of N protein with RNA. This study provides opportunities to phase separation-based anti-plant virus strategies. Biological sciences/Drug discovery/Drug regulation Biological sciences/Drug discovery/Drug screening Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Biomolecular condensates have been related to many cellular processes, including compartmentalization of biochemical reactions, sensing and responding to stress, mechanical regulation and signal transduction 1 , 2 . Mounting evidence suggests that phase separation has emerged as a fundamental principle for the formation of biomolecular condensates 3 – 6 . The assembly components of condensates are typically complex, usually involving proteins and nucleic acids, which lead to phase separation via their multivalent interactions 7 – 10 . As obligate intracellular parasites, viruses rely on cellular functions and processes to enable efficient replication. During infection, condensate-like structures commonly referred to as 'replication compartments' (RCs) or 'inclusion bodies' (IBs) were formed by viral proteins. Importantly, recent studies have implicated phase separation as a driving force for the formation of these biomolecular condensates 11 , 12 . The condensates play key role in viral life cycles, including viral entry 13 , genome synthesis and replication 14 , assembly for new particle 15 , and escape antiviral defense pathways 16 . Condensates formed by phase separation have attracted much attention in pharmaceutical industries and academia as novel targets in drug discovery 17 – 19 . Although it has been found that some small molecules could regulate the aggregation of viral biomolecular condensates in recent years, the discovery of new structures is still relatively rare 20 – 23 . Moreover, such modulators have not been reported in the agricultural field. Replication-related proteins encoded by several plant viruses such as barley yellow mosaic stripe virus (BYSMV), tomato yellow mottle-associated virus (TYMaV), tomato bushy stunt virus (TBSV), and carnation Italian ringspot virus (CIRV) have been continuously found form condensates, which proven to be associated with phase separation in the past two years 24 – 26 . Recently, the phytobacterial type III effectors have been shown to exhibit a propensity for phase separation 27 . Thus, targeting biomolecular condensates may be promising for success in developing novel pesticides for plant diseases that are difficult to control. Tomato spotted wilt virus (TSWV) belongs to the family Bunyaviridae , is one of the most devastating plant viruses worldwide, causing an annual economic loss of over one billion US dollars 28 . The use of antiviral agents is the most direct and effective method, but currently commercialized drugs are few and cannot actually serve purpose of managing TSWV-induced disease 29 . Therefore, developing new and effective anti-TSWV agents is of great significance. The few proteins encoded by the TSWV genome play an important role in virus infection. Among them, nucleocapsid protein (N) is an important component of the core template ribonucleoprotein (RNP) for viral replication 30 , and has multiple biological functions in the viral life cycle. The genomic RNA of virus could be well protected in RNP against RISC-mediated cleavage 31 . Similar to animal-infecting Bunyaviruses 32 , TSWV relies on a 'cap snatching' mechanism to initiate transcription of its genome 33 . The N proteins partially co-localize with processing-bodies (PBs) and stress granules (SGs), which are the sources of 5′ capped RNA 34 . Notably, N protein is capable of forming highly motile cytoplasmic IBs that traffic on the actin filaments 35 . Therefore, N protein can serve as an important molecular target for the creation of anti-TSWV agents. Chromones are an important class of natural active ingredients produced during secondary metabolism in plants. Their structure is easy to modify and is considered a good lead skeleton for drug discovery 36 . Chromone compounds derived from natural products possess potential broad-spectrum antiviral effects against human viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), herpes simplex virus type 1 (HSV1), influenza A virus (IAV), and hepatitis B virus (HBV), etc 37 , 38 , and we have previously synthesized several types of chromone compounds, which possess good inhibitory activity against plant viruses, and found that they had good binding ability to coat proteins (Fig. 1 a) 29 , 39 . Cationic amphiphilic drugs (CADs) are a class of commercialized drugs with a wide range of medical activities 40 . They are characterized by a common structure, namely hydrophobic aromatic side chains containing an amine functional group, which is crucial for its pharmacological activity by increasing the pH in acidic intracellular compartments 41 . We have noticed that the phase separation modulators through high-throughput screening method appear to possess properties of CADs (Fig. 1 b) 42 – 46 . Noteworthy, an increasing number of studies emphasize the crucial role of pH in the formation of condensates 47 – 50 . Inspired by the two points, we propose that designing a compound with properties of CADs may be more successful in discovering a novel biomolecular condensate modulator. Therefore, we designed a series of chromone compounds containing hydrophilic amine groups with piperazine dithiocarbamate as the bond-bridge in the study, and obtained the target compounds with high yields (Fig. 1 c). We next conducted a comprehensive study, including biological assay and exploration of mechanism of action. As a result, the introduction of tertiary amine groups significantly enhances the anti-TSWV activities of compounds. The highly active small molecule Z9 could target N protein to inhibit the replication level of the viral ribonucleoprotein (RNP), thereby affecting virus proliferation in vivo . Furthermore, the addition of Z9 or the loss of binding sites could both lead to a decrease in N protein condensations in plant cells. The characteristics of the N protein condensations driven by phase separation were determined, and RNA plays a crucial role in the process. We proved that the binding sites could affect the normal binding of N protein to nucleic acid, and Z9 could intervene the formation of N protein-RNA complex. Collectively, the study is the first strategic application of novel agrochemicals that disintegrate condensates driven by phase separation in the prevention and treatment of plant diseases, which expands a new direction for pesticide discovery. Results and discussion Anti-TSWV activity . The inhibitory activities of the target compounds Z1 - Z25 against TSWV were assessed using the half-leaf method with Nicotiana glutinosa serving as the model plant. Preliminary screening at a concentration of 500 µg/mL showed that the curative activity range of compounds Z1 - Z25 against TSWV ranged from 43.5–64.4%. The protective activity varied between 43.1% and 65.4%, while the inactivation activity spanned from 41.4–77.3%. Among them, the compounds Z9 (77.3%), Z13 (72.8%), Z14 (70.6%), and Z17 (74.2%) had excellent inactivation activity against TSWV, outperforming the control ningnanmycin (69.3%) and were significantly superior to ribavirin (43.7%). We found the inhibitory activities of compounds ( Z1 - Z7 ) without tertiary amine groups on TSWV were average. The EC 50 values of the compounds were further tested, and the results showed that compound Z9 exhibited the best inactivation activity against TSWV, with an inhibitory activity of 65.3 µg/mL, significantly better than that of ningnanmycin (149.4 µg/mL) and ribavirin (801.5 µg/mL) (Table 1). The above results indicate that the introduction of tertiary amine group substantially enhances the anti-TSWV activities of compounds. In addition, the inhibitory effect of compound Z9 on TSWV was further validated in tomatoes beyond model plants. The tomato plants sprayed with 3% tween-80 exhibited severe symptoms, including curled leaves, dark brown spots, delayed development, and yellowing. However, when treated with compound Z9 , the infection symptoms of TSWV in plants were notably alleviated. Compared with the control group, the Z9 -treated plants displayed better growth, with larger and greener leaves (Fig. 2 a). This indicated that compound Z9 could significantly reduce the proliferation of TSWV in plants. Compound Z9 significantly reduces N protein condensates . TSWV N protein can form motile cytoplasmic IBs to assist the proliferation of virus 33 , 34 . We aimed to observe the impact of compound Z9 on the behavior of N protein condensates in vivo . N-YFP was transiently expressed in Nicotiana benthamiana leaves, and after 36 h, drugs were injected into the infiltrating area and IBs 5 h post-injection were observed (Fig. 3 a). A good advantage is, the superior water solubility of compound Z9 avoids the necessity for surfactants like Tween 80, which may harm plant cells or tissues (Fig. 3 b). Compound Z9 , at a concentration of 100 µg/mL, significantly reduced the number of IBs in the leaf cell infiltration area, while compound Z3 , with less effective anti-TSWV activity, did not markedly decrease the number of IBs. Additionally, compound Z3 treatment led to leaf cells appearing compressed, possibly due to the compound entering the cells and forming solid particles, thereby damaging the tissue (Fig. 3 c, 3 d). A time-course experiment was conducted to further analyze the effects of compound Z9 on the behavior of IBs. It was evident that the mobility of IBs treated with compound Z9 remained unchanged, yet their quantity significantly reduced. Instead, due to the destruction of cellular tissues, IBs treated with Z3 were almost unable to move normally inside the plant cell and formed large particles. (Fig. 3 e and Extended Data Videos S1 - S3 ). The above results reveal that compound Z9 with amine group could effectively inhibit the formation of the aggregates involving N protein. Although several types of anti-TSWV compounds targeting N proteins have been previously reported, their impact on IBs had not been explored 52 . Herein, we further investigate whether other highly active compounds similar to CADs can disrupt the aggregation of N protein. It was found that piperazine derivatives containing ethyl morpholine also indeed inhibited IB formation (Supplementary Fig. 1). Thus, the experimental results expose the potential mechanism by which drugs targeting N proteins may inhibit the assembly of condensates. The absence of binding sites reduces the proliferation of viruses in vivo . We next explored the key binding sites of compound Z9 on the N protein. Molecular docking indicated that the compound Z9 could form conventional hydrogen bonds with the amino acids Arg94 (R 94 ), Gly147 (G 147 ), and Tyr184 (Y 184 ) in the core region of N protein, and hydrophobic interactions with Leu126 (L 126 ), Pro151 (P 151 ), and Ile179 (I 179 ) (Fig. 4 a). To validate the effect of binding sites of compound Z9 on RNP proliferation, reverse PCR amplification and recombination technique were employed to construct SR (+)eGFP R94A , SR (+)eGFP G147A , SR (+)eGFP Y184A , and SR (+)eGFP R94A&Y184A mini-replicons (Fig. 4 b and Supplementary Fig. 2). Nicotiana benthamiana plants were then infected using competent minigenome replication system (RNP eGFP ) and fluorescence was observed on the 5th day. The results showed that the absence of amino acid R 94 marginally reduced fluorescence levels. Conversely, the absence of G 147 did not alter the intensity of fluorescence in leaf cells, while the absence of Y 184 or R 94 &Y 184 could diminish fluorescence levels (Fig. 4 c). Average fluorescence intensity was analyzed according to three independent biological replicates. The results showed that RNP eGFP WT and RNP eGFP G147A in leaf cells had the strongest average fluorescence intensity, RNP eGFP R94A slightly decreased, and RNP eGFP Y184A was the weakest among all single-point mutations. RNP eGFP R94A&Y184A showed a more significant weakening (Fig. 4 d). Additionally, the accumulation levels of N protein were further detected. Compared to RNP eGFP WT , the lack of Y 184 or R 94 &Y 184 significantly reduced the accumulation level of N protein, while the absence of R 94 slightly reduced the accumulation of N protein (Fig. 4 e). To verify the impact of missing binding sites on TSWV pathogenicity in plants, the movement-competent minigenome replication system (M (−)opt ) was added to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs). By the 14th day, noticeable pathogenic symptoms, such as leaf wrinkling and spotting, were evident in the systemic leaves of TSWV WT -infected plants. The severity of symptoms in TSWV-infected plants were divided into three grades (Supplementary Fig. 3). By the 21st day, nearly all plants showed significant wilting and mortality (grade Ⅲ), with a disease susceptibility rate of 100%. In the case of TSWV G147A , over 50% of the plants displayed severe disease symptoms (grade Ⅲ) in systemic leaves, with an overall infection rate surpassing 70%. This suggests that the absence of G 147 somewhat delayed the virus’s systemic infection plants. For TSWV R94A or TSWV Y184A , symptom onset in system leaves was slower and considerably milder compared to TSWV WT , with a susceptibility rate of around 50% (grade I and grade Ⅱ). In contrast, plants infected with TSWV R94A&Y184A showed fewer spots in the systematic leaves and a lower incidence rate of about 20% (grade I and grade Ⅱ). Therefore, through a comprehensive comparative analysis of symptoms and infection rates, it was revealed that the solely absence of R 94 or Y 184 effectively hindered the virus’s systemic spread, and their joint absence drastically reduced it (Fig. 4 f- 4 h). The order of N protein accumulation level in the infected system leaves was TSWV WT > TSWV G147A > TSWV R94A > TSWV Y184A > TSWV R94A&Y184A (Fig. 4 i). These findings underscore that the binding sites of compound Z9 had a crucial impact on viral proliferation in vivo by affecting RNP replication. The binding sites significantly reduce the aggregation of N protein. To further explore the interaction between compound Z9 and the N protein, mutant proteins (N R94A , N Y184A , and N R94A&Y184A ) were obtained by prokaryotic expression and purification (Supplementary Fig. 4a,4b). Bio-layer interferometry was employed to examine the binding dynamics between compound Z9 and the N protein. Compared to N WT (7.6 µM), the binding ability of compound Z9 for double mutant N R94A&Y184A was significantly diminished, with a K D value exceeding 400 µM (Fig. 5 a). The MST test results also indicated that the binding constants of N R94A&Y184A with compound Z9 were decreased by 180-fold (Fig. 5 b). Similarly, binding constants for the single mutant proteins N R94A and N Y184A with compound Z9 were reduced by approximately 103 and 135 times, respectively (Supplementary Fig. 4c). These results indicate that the amino acids R 94 and Y 184 of N protein were the binding sites for compound Z9 . To investigate the influence of binding sites on the formation of IBs, we engineered a construct that the N R94A&Y184A protein into a yellow fluorescent protein (N R94A&Y184A -YFP) (Supplementary Fig. 5). When transiently expressed in Nicotiana benthamiana leaves, the N WT -YFP formed numerous discrete cytoplasmic inclusions, in stark contrast, both YFP alone and N R94A&Y184A -YFP were predominantly dispersed throughout the cytoplasm, indicating a diffuse distribution pattern (Fig. 5 c). Expanding our observation field, we significantly noted that although small IBs formed by N R94A&Y184A -YFP also migrated, while their quantity was substantially reduced compared to N WT -YFP (Fig. 5 d and Extended Data Video S4 , S5 ). These observations suggest that mutations at binding sites markedly decrease IB formation and is basically consistent with the phenomenon of adding compound Z9 . Like animal-infecting viruses in the family Bunyaviridae 32 , TSWV N protein is known to co-localize with P-bodies 33 , 34 . This was further explored by transient co-expression of N WT -YFP with DCP5-mCherry by agroinfiltration in leaves. It was found that cytoplasmic granules of N WT -YFP co-localized with DCP5-mCherry foci within the cytoplasm. However, the formation of fewer granules by N R94A&Y184A -YFP resulted in a significantly reduced degree of overlap (Fig. 5 e). This reduction points to the impact of missing binding sites on the viral ‘cap snatching’ process. Condensates are formed by phase separation. We further delved into the mechanism behind the reduction in condensate numbers observed upon the addition of compound Z9 or due to the absence of binding sites in N proteins. It is well-established that proteins with IDRs often engage in phase separation to form condensates 3 – 6 . With this premise, the phase separation potential of N protein was assessed using the Predictor of Natural Disordered Regions (PONDR) VL-XT algorithm. This sequence-based prediction honed in on the N protein’s IDR, primarily located within its central core domain—a region notably implicated in nucleic acid interactions (Fig. 6 a) 53 , 54 . Encouraged by these bioinformatic insights, we pursued experimental verification to discern whether N protein could indeed engage in phase-separation activities. To this end, a construct encoding N-YFP was transiently expressed in Nicotiana benthamiana leaves. Time-lapse confocal imaging documented the behavior of small, dynamic inclusions that frequently merged, coalescing into larger formations (Fig. 6 b and Extended Data Video S6 ). Indeed, it was observed that these large inclusions occasionally divided slowly, spawning smaller ones that dispersed (Fig. 6 c and Extended Data Video S7 ). The characteristics of these inclusions, such as their sphericity, motility, and fusion/fission, as seen in N-YFP-infected cells, reveal that the IBs may exhibit properties akin to those of liquid droplets. Fluorescence recovery after photobleaching (FRAP) experiment showed that small droplets achieved up to 50% recovery efficiencies within 2 mins (Fig. 6 d and Extended Data Video S8 ), indicating a rapid dynamic nature of these droplets. Taken together, these results point the propensity of N protein-containing condensates to undergo phase separation in vivo . Compound Z9 inhibits nucleic acid mediate N protein condensation . RNP granules assemble through phase separation of their molecular components, and RNA is thought to be a critical element for the sizing and composition of the condensates 54 – 58 . Notably, the N protein appears to interact with nucleic acids at approximately 25 binding sites (Supplementary Fig. 6a), with residues K 65 , R 94 , R 95 , K 183 , and Y 184 identified as particularly significant 31 , 53 , 54 . Considering that the IDR of N protein mainly resides in the area forming these nucleic acid binding sites, our focus shifted to investigating whether nucleic acids could mediate the formation of condensates. To explore this possibility, we purified the recombinant His-sfGFP-N from Escherichia coli and examined its aggregation investigating under conditions of higher concentration and low salinity (Fig. 7 a). Confocal microscopy analysis showed no significant aggregation of His-sfGFP-N protein under these conditions. Subsequently, total RNA was extracted from TSWV-infected Nicotiana benthamiana leaves. Protein phase separation assay was performed with increasing concentrations of RNA, ranging from 50 to 100 ng/µL. Remarkably, the His-sfGFP-N displayed a phase transition in the presence of RNA, rapidly forming larger, aggregate-like structures at the high concentration of RNA. However, the features of these N-RNA condensates exhibited a solid-like physical state rather than a fluidic one (Supplementary Fig. 6b). This rapid solidification parallels phenomenon observed in amyloid-like fibrils or oskar ribonucleoprotein granules 59 , 60 . Another important reason is that the physiological condition of in vitro assay makes it challenging to achieve consistency with those in the plant body. In conclusion, our results indicate that N protein undergoes RNA-induced condensation in vitro , revealing a critical interplay between nucleic acids and N protein in condensate formation. To verify whether mutations at the binding sites R 94 and Y 184 in N protein significantly affect RNA-induced N protein condensation, we generated mutations of His-sfGFP-N by substituting the residues with alanine residues R 94A /Y 184A (His-sfGFP-N R94A&Y184A ) (Fig. 7 a). Confocal microscopy data showed that both the numbers and sizes of N protein condensates were greatly reduced by the binding site mutations (Fig. 7 b). To further investigate the impact of these specific amino acids on the N protein’s ability to bind RNA, digoxigenin-labeled RNA probe was prepared and electrophoretic mobility shift assay (EMSA) was conducted in the study (Supplementary Fig. 7a). We found that in the presence of the N WT , the rate of RNA migration began to change slightly at a protein dosage of 0.1 µg, with a significant decrease observed at dosages exceeding 0.1875 µg. Conversely, for N R94A&Y184A mutant, a slight slowing in RNA migration was noted at a protein dosage of 1.5 µg, with a substantial decrease only evident at dosages greater than 3.0 µg (Supplementary Fig. 7b). These results indicate that the absence of these binding sites notably impairs the RNA binding capability of the N protein. We next immobilized the biotin-labeled RNA (100 nM) onto the streptavidin (SA) biosensors, and the binding affinities of N WT and N R94A&Y184A with RNA were measured by BLI assay. The results suggested that the affinity constant ( K D) for N WT and N R94A&Y184A binding to RNA were 7.3 nM and 36 nM, respectively (Fig. 7c,7d ). The mutation of the binding sites reduced the affinity between N protein and RNA approximately 5-fold. We observed that N WT binds to RNA more rapidly than N R94A&Y184A at low concentrations, and it dissociates from RNA slower than N R94A&Y184A . When the biosensor was continuously loaded with WT and mutant N protein, and we noticed a significant decrease in the binding curve (Fig. 7e ). Upon reversing the loading sequence, the binding curve increased significantly (Fig. 7f ). These results demonstrate that the absence of binding sites affected the binding ability of N protein to RNA. To evaluate the binding efficiency of compound Z9 with N-RNA complex, we first determined the affinity constant ( K D=150 µM) between compound Z9 and RNA (Fig. 7g ). The interaction between compound Z9 and N protein ( K D=7.6 µM) was significantly stronger than that of Z9 -RNA, suggesting a preference of compound Z9 to bind with N protein. In the competitive binding assay, we discovered that the binding signal value of N protein and RNA (0.26 nm) was remarkably higher than that of the mixture of compound Z9 and N protein (0.17 nm) (Fig. 7h ). From the constants of binding rate and dissociation rate, we found that pre-binding of compound Z9 to N protein significantly blocked RNA binding. Collectively, these results reveal that compound Z9 acts as a novel inhibitor, blocking phase separation of N condensations and inhibiting viral proliferation (Fig. 7i ). Discussion Biomolecular condensate modulators have received great attention in the pharmaceutical field 17 – 23 , however, their application in agriculture has not been reported yet. Consequently, based on the advantages of the privileged scaffold (benzopyrone) in drug discovery, a series of novel benzopyrone compounds were rationally designed and synthesized by analyzing the structures of biomolecular condensates modulators in the pharmaceutical field (Fig. 1 ). We discovered that the introduction of amine groups into the side chain significantly improved the inhibitory activity of the compounds against TSWV (Table. 1). Further investigations into the mechanism of action revealed that the highly active compound Z9 could inhibit the replication of viral RNP (a complex of N protein, nucleic acid, and RdRp) in vivo . Subsequent BLI screening indicated that the introduction of an amine group significantly improved the binding of N protein to the compounds. The binding signal values showed a positive correlation with their activity to some extent, with Z9 displaying the highest signal value (Fig. 2 f). Moreover, the affinity of Z9 for N protein was measured at 7.6 µM, indicating that N protein is a direct target of Z9 . When, N-YFP protein was transiently expressed in plants and treated with the Z9 , a significant reduction in the number of N protein condensates was observed, demonstrating that our designed small molecules effectively regulate N protein condensates (Fig. 3 ). This study supports our initial hypothesis that compounds designed with CADs are potentially more effective in discovering regulators of phase separation. To further elucidate the mechanism of action of Z9 , we employed molecular docking to identify potential binding sites between Z9 and the N protein. Our findings indicate that the absence of these binding sites significantly inhibited the replication of the RNP and alleviated the pathogenic symptoms of TSWV, which was consistent with the mode of action of Z9 . We identified that R 94 and Y 184 as critical binding sites for Z9 by small molecule-protein interaction assays. In vivo experiments further demonstrated that the absence of R 94 and Y 184 led to a diffuse distribution of the N protein within the cell, significantly reducing the number of condensates and consequently impairing TSWV’s cap snatching and infection processes (Fig. 5 ). Phase separation is recognized as a fundamental principle driving the formation of condensates 3 – 6 . The N protein was predicted to contain disordered regions, and our observations showed that N protein condensates underwent a fusion-fission process in their early stages and exhibited a degree of recovery after bleaching (Fig. 6 ). These results suggest that condensate formation in vivo is driven by phase separation, a process resulting from multivalent interactions between proteins or between proteins and nucleic acids 7 – 10 , 55 – 58 , 61 . We speculated that the loss of key amino acids would significantly affect the binding of the N protein to RNA. A series of in vitro experiments revealed a marked reduction in both the quantity and size of the aggregates formed by the N protein and RNA. Furthermore, the absence of key binding sites substantially impacted the N protein’s ability to bind RNA. Notably, the addition of Z9 was found to indeed inhibit the binding of the N protein to RNA (Fig. 7 h). Thus, we have conducted comprehensive research and developed a novel inhibitor targeting condensates by directly blocking protein–nucleic acid interactions (Fig. 7 i). We discovered that the introduction of basic amine groups is critical, enabling classification as as pH-responsive drugs (CADs). Our observations show that most biomolecular condensate inhibitors identified through high-content screening methods in the medical field possess alkalic groups 20 , 42 – 46 . Increasingly, research emphasizes that physiological pH plays a significant role in regulating the formation of certain biomolecular condensates 47 – 50 . This study focuses on elucidating the mechanism through which compound Z9 disrupts the multivalent interaction between N protein and RNA, thereby inhibiting the forces driving phase separation. At present, it appears that there have no instance of research combining drug discovery with the physiological pH of condensates formation. Therefore, a thorough understanding of the formation mechanism of condensates is of great significance for the further development of biomolecular condensate modulators. Given the increasing evidence that many plant viruses and phytopathogenic bacteria rely on the condensation of replication proteins for proliferation 24 – 27 , 62 , it is conceivable that the discovery of novel biomolecular condensate inhibitors for use in agriculture could play a significant role in controlling previously untreatable plant diseases. Finally, we believe this study shall establish a cornerstone for the strategic design of innovative virucides targeting phase separation process in the viral lifecycle. Methods Materials and virus sources. The seeds of Nicotiana glutinosa and Nicotiana benthamiana were preserved by our laboratory. All plants were cultured in an artificial climate chamber (25℃ during the day, 50% relative humidity/23℃ at night, 40% relative humidity). The full-length infectious clone vectors (L (+)opt , M (−)opt , and SR (+)eGFP ), expression vector of silencing suppressors (VSRs), vector pCambia2300-N-YFP, and DCP5-mCherry were provided by Professor Xiaorong Tao (Nanjing Agricultural University). TSWV lettuce isolate was maintained in Nicotiana benthamiana and the infected leaves were maintained in a -80℃ refrigerator. Plasmid construction. For generating the mutant plasmids SR (+)eGFP Mut (SR (+)eGFP R94A , SR (+)eGFP Y184A , SR (+)eGFP G147A , and SR (+)eGFP R94A&Y184A ), the insertion fragments and vectors were obtained with SR (+)eGFP WT as a template by reverse polymerase chain reaction. For the construct p2300-N R94A&Y184A -YFP, the cDNA of the N R94A&Y184A gene was amplified from SR (+)eGFP R94A&Y184A , and then inserted into binary vector pCambia2300-YFP to generate. All constructs were verified by DNA sequencing. The primers used are shown in Table S1 . Synthesis. All commercial reagents and solvents were used without further purification. Thin layer chromatography (TLC) was performed on pre-coated silica gel GF254 plates. Visualization of TLC was achieved using UV light (wavelength 254 nm). Column chromatography was performed on silica gel (300–400 mesh) using a proper eluent. NMR was recorded on Bruker DPX 400 MHz (Bruker, Germany). Chemical shifts were reported in parts per million (ppm) referenced to the appropriate solvent peaks ( δ 7.26 ppm for CDCl 3 in proton spectra, 77.0 ppm for CDCl 3 in fully decoupled 13 C spectra). The following abbreviations were used to describe peak splitting patterns: s = singlet, d = doublet, t = triplet, m = multiplet. Coupling constants J were reported in hertz (Hz). High resolution mass spectra were obtained with thermo Scientific Q Exactive (Thermo Scientific, USA). The specific synthesis steps can be found in the supplementary information . Anti-TSWV assay. Nicotiana glutinosa was used to as the model plant, and the inhibitory activities of compounds against TSWV were tested using the half-leaf method. The specific operations were performed as we described recently 29 . The extracted TSWV was inoculated on the lower leaves of tomatoes. The plants were cultured in an artificial climate chamber (25℃ during the day, 50% relative humidity/23℃ at night, 40% relative humidity). After 24 hours, 100 µg/mL agent (2.0 mg compound Z9 , 50 µL DMSO, 20 mL 2% (v/v) tween 80 water) was sprayed onto the tomato leaves, with 20 mL 3% tween 80 water containing DMSO (50 µL) as control. The pathogenic symptom was observed after about 20 days. Agrobacterium infiltration. Agrobacterium tumefaciens strain GV3101 was transfected with the recombinant plasmids and the monoclonal clone was selected and cultured at 28 ℃ for about 24–36 hours. A. tumefaciens cells were resuspended using agroinfiltration buffer (10 mM MES, pH = 5.6, 10 mM MgCl 2 , and 100 µM acetosyringone) and incubated for 2 to 3 h in the dark at room temperature after the optical density at 600 nm (OD 600 ) was adjusted. The cells were used for agroinfiltration of Nicotiana benthamiana leaves. For the competent minigenome replication system (L (+)opt + SR (+)eGFP + VSRs) or reconstituted infectious TSWV (L (+)opt + SR (+)eGFP + M (−)opt + VSRs), equal volumes of Agrobacterium cultures harboring full-length infectious clone vector (final concentration OD 600 = 0.20), were mixed with bacterial mixture (final concentration OD 600 = 0.05) containing the VSRs ( P19 - HcPro - γb). For expression vectors p2300-N-YFP, the cells were resuspended in agroinfiltration buffer adjusted to an optical density OD 600 of 0.50. Leaves of Nicotiana benthamiana plants at the 5-6-leaf stage were infiltrated with Agrobacterium cultures using syringes. The infiltrated plants were grown in an artificial climate chamber. The operation of the mutants is consistent with the above description. Fluorescence microscopy. Agro -infiltrated Nicotiana benthamiana leaves were examined for fluorescence expression using an OLYMPUS model BX53F Fluorescence Microscope (OLYMPUS, Tokyo, Japan) with a green barrier filter. The samples were fixed in water on a microslider under a coverslip to detect eGFP fluorescence. Fluorescence images were processed using ImagePro (OLYMPUS, Tokyo, Japan). Protein extracts and immunoblot assays. 1.0 g healthy, Agrobacterium -infiltrated leaf patches, or TSWV-systemically infected leaves of Nicotiana benthamiana were collected, and total protein was extracted in 1 mL of extraction buffer (25 mM Tris-HCl, pH = 7.5, 150 mM NaCl, 1 mM EDTA, 2% w/v polyvinylpolypyrrolidone, 10 mM dithiothreitol, 0.5% v/v Triton X-100, 10% v/v glycerol, and 1× protease inhibitor cocktail). The supernatants were collected after 10 min centrifugation at 20000 × g at 4°C and directly used for immunoblot assays. The samples were transferred to PVDF membranes after separated by SDS-PAGE. The membranes were blocked with 5% w/v skim milk solution and probed with antibodies against GFP or N. Actin antibody was used as internal control, and then incubated with HRP-conjugated secondary antibody. Finally, the signal was visualized by the ChemiDoc MP Imaging System (Bio-Rad). Protein purification, and microscale thermophoresis (MST) assay. The prokaryotic expression vectors (pET-32a-N WT , pET-32a-N R94A , pET-32a-N Y184A , pET-32a-N R94A&Y184A , pET-32a-sfGFP-N WT , and pET-32a-sfGFP-N R94A&Y184A ) were successfully introduced into Escherichia coli BL21 (DE3), respectively. The expression strains were induced by isopropyl- β -D-thiogalactopyranoside at 28℃ overnight, harvested by centrifugation at 4°C, and the precipitates were lysed (power 35%, 40 min, 4 s ultrasound, and 6 s interval) with buffer (0.869 g NaCl, 3.75 mL PBS, 36 µL mercaptoethanol, 5 mL glycerol, 41.25 mL H 2 O, pH = 7.4). The lysates were clarified by centrifugation at 16000 g for 30 min. The proteins were purified via affinity chromatography using a Trap high-performance column (GE Healthcare, USA). The binding affinities of compound Z9 to the proteins were determined by the Monolith NT.115 instrument (NanoTemper, Munich, Germany). The specific operation was performed according to the reported literature 29 . Electrophoretic mobility shift assay (EMSA). Digoxigenin-labeled RNA probe was prepared following previously described protocols 31 . Briefly, DNA template with the T7 promoter sequence was generated by polymerase chain reaction and purified using the gel recovery kit. RNA was then transcribed in vitro using T7 RNA polymerase (Promega, Madison, USA). 1 µL RNA probe with a concentration of approximately 1200 ng/µL was placed in a PCR tube and diluted to 40 ng/µL using DEPC water. 1 µL RNA probe (40 ng/µL) was added into the PCR tubes and mixed with 9 µL different qualities of the purified N WT or N R94A&Y184A (0.025, 0.05, 0.1, 0.1875, 0.375, 0.75, 1.5, 3.0, and 6.0 µg), respectively. The mixtures were incubated in ice water bath for 10 min. The samples were transferred onto a nylon membrane. The membrane was exposed to UV light and probed with anti-digoxin alkaline phosphatase antibody (Roche, Basel, Switzerland) for 1–2 hours, and then incubated with BCIP-NBT for staining at 37℃. The signal was directly visualized and photographed. Bio-layer interferometry (BLI) assays. The purified His-tagged N protein was loaded onto Ni-NTA biosensors, the binding signal values of different compounds (50 µg/mL) were measured using a bio-layer interferometry method with Octet RED 96 (ForteBio, San Francisco, USA). All compounds were diluted in PBST. The binding affinities ( K D) between compound Z9 and the recombinant wild and mutant proteins were measured using this method. The binding constants of the compound Z9 with proteins were determined by global fit analyses of experimental binding curves using the Octet Red software. To analyze the effect of binding sites on N-RNA interactions, 100 nM of biotinylated RNA (bio-GAGAGCAAUUGUGUCAGAA) was loaded onto the streptavidin (SA) biosensors and balanced with PBS. The biosensors were then exposed to diluted recombinant N WT , or N R94A&Y184A , followed by washing (dissociation) with PBS. The binding constant values ( K D) were calculated using the Octet Red software. For binding inhibition experiments, the binding constant value ( K D) of compound Z9 with RNA was first measured using BLI, and compound Z9 (5 µM) was then combined with N protein (12.5 nM) and incubated for 30 min at room temperature. The biosensors were exposed to recombinant N protein (12.5 nM), incubated mixture (compound Z9 and N protein), and compound Z9 (5 µM), followed by washing (dissociation) with PBST. Association rate constants ( K on ) and dissociation rate constants ( K off ) were obtained using Octet data analysis software. Confocal microscopy and fluorescence recovery after photobleaching assay. Confocal laser scanning micrographs of Agro-infiltrated leaves were captured with a Zeiss LSM900 confocal laser scanning microscope (Zeiss, Oberkochen, Germany). Fluorescent protein was excited at 488 nm and time-lapse images were taken over a 1 or 2 - min period. For drug treatments, when N-YFP was significantly expressed in leaves of Nicotiana benthamiana after 36 h, small molecule (2.0 mg) was dissolved into a 100 µL DMSO solution, and then 20 mL of ddH 2 O was added to prepare a 100 µg/mL agent, rapidly inject it into the infiltration area of leaves. After 5 h, micrographs or time-lapse images were captured by confocal microscopy. For fluorescence recovery after photobleaching (FRAP) experiments, IB droplets formed in infected leaf cells were bleached with a laser at 488 nm (100% intensity) under a confocal microscope with × 20 water immersion and magnify the field of view by × 2.5. After bleaching, at least 40 frames of recovery images were collected by the time-lapse modes. Fluorescence intensity of the bleaching region was measured using Olympus CellSens software. The data was normalized and recovery curves were generated by GraphPad Prism 8. Declarations Data availability Supplementary Figs and full experimental details for the preparation of all new compounds, and their spectroscopic data, can be found in the supplementary materials. Acknowledgements The authors acknowledge financial support from the National Natural Science Foundation of China (Nos. 32330087 and 32302388). The authors thank Dr. Mingfeng Feng and Prof. Dr. Xiaorong Tao (College of Plant Protection, Nanjing Agricultural University, China) for assistance in the plasmid construction. Author contributions N.Z. conducted most of the experiments. J.L., S.W., J.L., and F.C. contributed to some experiments. J.Y. created the pattern diagram. B.S. and R.S. conceptualized and directed the project and drafted the manuscript with assistance from all co-authors. All authors contributed to part of the experiments and/or discussions. Competing interests The authors declare no competing interests. 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Cell 162:1066–1077 Bose M, Lampe M, Mahamid J, Ephrussi A (2022) Liquid-to-solid phase transition of oskar ribonucleoprotein granules is essential for their function in Drosophila embryonic development. Cell 185:1308–1324 Rekhi S et al (2024) Expanding the molecular language of protein liquid-liquid phase separation. Nat Chem. 10.1038/s41557-024-01489-x May JP (2024) Plant viruses and biomolecular condensates: novel perspectives in virus replication strategies. New Phytol. 10.1111/nph.19778 Tables Table 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedDataVideoS1.avi Video S1 The effect of DMSO on the formation of inclusion bodies in Nicotiana benthamiana cells. ExtendedDataVideoS2.avi Video S2 The effect of compound Z9 on the formation of inclusion bodies in Nicotiana benthamiana cells at 100 µg/mL. ExtendedDataVideoS3.avi Video S3 The effect of compound Z3 on the formation of inclusion bodies in Nicotiana benthamiana cells at 100 µg/mL. ExtendedDataVideoS4.avi Video S4 Highly motile cytoplasmic inclusion bodies formed by N-YFP in Nicotiana benthamiana cells. ExtendedDataVideoS5.avi Video S5 Cytoplasmic inclusion bodies formed by NR94A&Y184A-YFP in Nicotiana benthamiana cells. ExtendedDataVideoS6.avi Video S6 Representative time-lapse video showing the fission of inclusion bodies formed by N-YFP in Nicotiana benthamiana leaf cells. ExtendedDataVideoS7.avi Video S7 Representative time-lapse video showing the fusion of inclusion bodies formed by N-YFP in Nicotiana benthamiana leaf cells. ExtendedDataVideoS8.avi Video S8 Fluorescence recovery after photobleaching of inclusion bodies formed by N-YFP in Nicotiana benthamiana leaf cells. supplementaryinformation.docx Table1.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4398439","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":308133070,"identity":"3cbcd4ae-4ab7-45a4-9809-e1a173cc0ddc","order_by":0,"name":"Runjiang Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYNACHhsoTYKWNJK1MBwmQYt8e+/h1xUy5+3mz0hgfPC2jUHenJAWgzPn0izP8NxObpyRwGw4t43BcGcDIS0SOWaGDUAtzBIJbNK8bQwJBgcIOWwGWMu5ZDaJBPbfRGlhuJFj/LCB54AdD9AWZqK0GJw5Y8bYwJOcIMHzsFlyzjkJww0EHdbeY/yxscfOXr49+eCHN2U28oQdxsDAJsHYw5DYwMDYAORIEFYPBMwfGH4w2BOldBSMglEwCkYmAABAkjsmbvR6cwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2502-778X","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Runjiang","middleName":"","lastName":"Song","suffix":""},{"id":308133071,"identity":"2f51f665-7615-4109-b07e-e8828173561f","order_by":1,"name":"Zan ning","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zan","middleName":"","lastName":"ning","suffix":""},{"id":308133072,"identity":"07d3e080-82a6-42dd-85e0-73447a52ce7f","order_by":2,"name":"Jiao Li","email":"","orcid":"https://orcid.org/0000-0003-0751-6846","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Li","suffix":""},{"id":308133073,"identity":"ddfa86e5-036c-477d-9c15-f1f5e185db8c","order_by":3,"name":"Jiahui Yao","email":"","orcid":"https://orcid.org/0009-0008-6626-9658","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiahui","middleName":"","lastName":"Yao","suffix":""},{"id":308133074,"identity":"a0b3a527-4867-49c3-a130-0068e222584b","order_by":4,"name":"Shang Wu","email":"","orcid":"https://orcid.org/0000-0002-2220-7518","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shang","middleName":"","lastName":"Wu","suffix":""},{"id":308133075,"identity":"5c7bf9cf-1617-47be-aba4-1e1e04479972","order_by":5,"name":"Jianzhuan Li","email":"","orcid":"https://orcid.org/0000-0003-0052-8094","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jianzhuan","middleName":"","lastName":"Li","suffix":""},{"id":308133076,"identity":"33b560e8-4ac7-40ff-b0a1-97d46254d756","order_by":6,"name":"Feifei Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Feifei","middleName":"","lastName":"Chen","suffix":""},{"id":308133077,"identity":"5f2ced01-7c8b-45ec-b741-20606824292f","order_by":7,"name":"Bao-An Song","email":"","orcid":"https://orcid.org/0000-0002-4237-6167","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Bao-An","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2024-05-10 06:00:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4398439/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4398439/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58031663,"identity":"58ab1ad0-172c-4ea3-8d7c-7a327147dc01","added_by":"auto","created_at":"2024-06-10 07:56:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":778015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign Strategy for Novel Biomolecular Condensate-inhibiting Phytovirucides. \u003c/strong\u003ea) The structures of chromone and its antiviral analogues. b) The structures of reported aggregation regulators containing tertiary amines. c) Rational design of novel condensates-inhibiting phytovirucides in the study.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/24b3930703585551fee0b7d2.png"},{"id":58032227,"identity":"159e977b-3ecc-49ef-8733-445ca91e33af","added_by":"auto","created_at":"2024-06-10 08:04:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":942973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAction Mode of Compound Z9 Inhibiting TSWV\u003c/strong\u003e. a) Compound \u003cstrong\u003eZ9\u003c/strong\u003e could inhibit the proliferation of TSWV in tomatoes from the appearance of symptoms. b) Schematic diagrams of the constructs SR\u003csub\u003e(+)eGFP\u003c/sub\u003e and L\u003csub\u003e(+)opt\u003c/sub\u003e. L\u003csub\u003e(+)opt\u003c/sub\u003e, a full-length infectious clone of TSWV L genomic RNA fragment with optimized sequence; SR\u003csub\u003e(+)eGFP\u003c/sub\u003e, an infectious clone of TSWV S genomic RNA, in which NSs of TSWV S genome RNA fragment is replaced by eGFP. c) The amounts of fluorescence in the infiltrated leaves were observed at the 5\u003csup\u003eth\u003c/sup\u003e day. Bars, 500 µm. d) Western blot was used to detect the accumulation level of protein N in the infiltrated leaves. Staining of RuBisCO with Ponceau S was used as a sample loading control. e) Pattern diagram of continuous detection of binding signal values between small molecules and N protein using BLI. f) The binding signal values between compounds with different activities and N protein. Compounds \u003cstrong\u003eZ1\u003c/strong\u003e-\u003cstrong\u003eZ6\u003c/strong\u003e are compounds with average activities, while compounds \u003cstrong\u003eZ8\u003c/strong\u003e, \u003cstrong\u003eZ9\u003c/strong\u003e, \u003cstrong\u003eZ12\u003c/strong\u003e, \u003cstrong\u003eZ13\u003c/strong\u003e, \u003cstrong\u003eZ14\u003c/strong\u003e, and \u003cstrong\u003eZ16\u003c/strong\u003e are compounds with good activity. g) The binding ability of compound \u003cstrong\u003eZ9\u003c/strong\u003e with N protein was measured by BLI.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/d214fc892dec9b6853fc04ea.png"},{"id":58031666,"identity":"13d753c2-4d30-4ac3-bad2-efda8f323717","added_by":"auto","created_at":"2024-06-10 07:56:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":851314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCompound Z9 Significantly Disrupt the Formation of IBs\u003c/strong\u003e. a) Schematic method of exploring the effect of compound \u003cstrong\u003eZ9\u003c/strong\u003e on the formation of IBs \u003cem\u003ein vivo\u003c/em\u003e. b) Preparation of agents. Compound\u003cstrong\u003e Z3\u003c/strong\u003eor \u003cstrong\u003eZ9\u003c/strong\u003e (2.0 mg) was dissolved in DMSO (100 µL) and water (20 mL) was added. c) Representative fluorescence images showing compound \u003cstrong\u003eZ9\u003c/strong\u003e could significantly reduce the formation of IBs \u003cem\u003ein vivo\u003c/em\u003e. DMSO and compound \u003cstrong\u003eZ3\u003c/strong\u003ewere used as controls. The experiments were repeated at least three times with similar results. Bars, 20 μm. d) The total number of IBs in confocal images. Data are shown as individual data points and mean ± SD (n=4). Statistical P-values were calculated using the two-tailed Student’s t-test (**, P \u0026lt; 0.01; ***, P \u0026lt; 0.001). e) Fluorescence time lapse confocal images showing the behavior of IBs under different treatments. Bars, 10 μm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/72589034e10bcdf397ecc283.png"},{"id":58031117,"identity":"80c0d998-cab5-41f4-b67a-f4e1f3abfbe5","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":881476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Binding Sites of Compound Z9 and N Protein Significantly Affect the Replication of RNP and the Proliferation of TSWV\u003c/strong\u003e. a) Analysis of binding sites of compound \u003cstrong\u003eZ9\u003c/strong\u003e and N protein by molecular docking. b) Schematic diagram of the constructs SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eMut\u003c/sup\u003e,\u003csub\u003e \u003c/sub\u003eand the sequence encoding amino acids (R\u003csup\u003e94A\u003c/sup\u003e, G\u003csup\u003e147A\u003c/sup\u003e, Y\u003csup\u003e184A\u003c/sup\u003e, or R\u003csup\u003e94A\u003c/sup\u003e\u0026amp;Y\u003csup\u003e184A\u003c/sup\u003e) on the N protein gene were mutated to that of alanine. c) Constructs (L\u003csub\u003e(+)opt\u003c/sub\u003e + SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eWT\u003c/sup\u003e) or its mutant (L\u003csub\u003e(+)opt\u003c/sub\u003e+ SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eMut\u003c/sup\u003e) with three RNA silencing virus inhibitors VSRs (P19, Hc-Pro, and γb) co-expression in plants together with at least three plants in each experiment, the expression of eGFP protein in leaves was observed by fluorescence microscope on the 5\u003csup\u003eth\u003c/sup\u003e day. Bars, 500 μm. d) The average fluorescence intensities in infiltrated leaves on the 5\u003csup\u003eth\u003c/sup\u003e day) was measured by ImageJ. Error bars indicate ± SD of the mean three independent experiments (n = 3). Statistical P-values were calculated using the two-tailed Student’s t-test (*, P \u0026lt; 0.05; ***, P \u0026lt; 0.001; ****, P \u0026lt; 0.0001; ns, P \u0026gt; 0.05 not significant). e) The accumulation level of N protein in the infiltrated leaves was detected by western blot. Staining of RuBisCO with Ponceau S was used as a sample loading control. f) Phenotypes of TSWV\u003csup\u003eWT \u003c/sup\u003eand TSWV\u003csup\u003eMut\u003c/sup\u003e (TSWV\u003csup\u003eR94A\u003c/sup\u003e, TSWV\u003csup\u003eG147A\u003c/sup\u003e, TSWV\u003csup\u003eY184A\u003c/sup\u003e, or TSWV\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e) -infected plants on the 21\u003csup\u003est\u003c/sup\u003e day. Constructs (L\u003csub\u003e(+)opt\u003c/sub\u003e + SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eWT\u003c/sup\u003e + M\u003csub\u003e(-)opt\u003c/sub\u003e) or its mutant (L\u003csub\u003e(+)opt\u003c/sub\u003e+ SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eMut\u003c/sup\u003e + M\u003csub\u003e(-)opt\u003c/sub\u003e) with three RNA silencing virus inhibitors VSRs (P19, Hc-Pro, and γb) co-expression in plants together with at least ten plants in each experiment. g) The infection rates of TSWV\u003csup\u003eWT \u003c/sup\u003eand TSWV\u003csup\u003eMut\u003c/sup\u003e -infected plants on days 14, 17, and 21. Error bars indicate ± SD of the mean three independent experiments (n = 3). h) The percentages of TSWV\u003csup\u003eWT \u003c/sup\u003eand TSWV\u003csup\u003eMut\u003c/sup\u003e -infected plants with different disease symptom grades. Error bars indicate ± SD of the mean three independent experiments (n = 3).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/93968f4982f8769c4d83aa63.png"},{"id":58031119,"identity":"915ef867-2cca-480b-bdf6-052003ec066c","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1038883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Key Binding Sites Significantly Reduce the Aggregation of N protein \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein Vivo\u003c/strong\u003e\u003c/em\u003e. a) The binding ability of compound \u003cstrong\u003eZ9\u003c/strong\u003e with N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e was measured by BLI. b) The binding ability of compound \u003cstrong\u003eZ9\u003c/strong\u003e with N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e was measured by MST. c) Confocal micrographs showing the localization patterns of green fluorescence derived from N\u003csup\u003eWT\u003c/sup\u003e-YFP, N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e-YFP, or YFP infections. Bars, 20 μm. d) Fluorescence time lapse confocal images showing the behavior of IBs formed by N\u003csup\u003eWT\u003c/sup\u003e-YFP or N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e-YFP. Bars, 10 μm.\u003cstrong\u003e \u003c/strong\u003ee) The subcellular localization of N\u003csup\u003eWT\u003c/sup\u003e-YFP or N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e-YFP partially overlaps with processing bodies (PB) component (DCP5-mCherry). The fluorescence after merged was yellow. Confocal images were taken 48 hpi post-infiltration. Bars, 20 μm. c, e, Experiments were repeated at least three times with similar results.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/775e05181348d1fe0ba905ff.png"},{"id":58031114,"identity":"ecd9f631-c69a-4ef9-ad0b-0d239c0e354c","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":817480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhase Separation Drives the Formation of N-protein Condensates\u003c/strong\u003e. a) Prediction of IDRs in N protein by the PONDR VL-XT algorithm. b) Fluorescence time lapse confocal images showing the fission of IBs. The white circles indicated the IBs undergoing fission at 36 hpi. Bars, 10 μm. c) Fluorescence time lapse confocal images showing the fusion of IBs. The white circles indicated the IBs undergoing fusion at 36 hpi. Bars, 10 μm. d) Fluorescence recovery after photobleaching (FRAP) of IBs formed in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e cells. The images were taken every 2.5 s for 120 s to document fluorescence recovery, each time point was normalized to before photobleaching. Bars, 20 μm. e) Quantification of the N-GFP condensates in FRAP assays. Data are mean ± s.d. (n = 4 puncta).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/6da66c2aae71def0393ae234.png"},{"id":58031115,"identity":"b4e9787e-c711-4884-81b0-d6e37799727a","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":682619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCompound Z9 Inhibits Nucleic Acid Mediate N Protein Condensation\u003c/strong\u003e. a) The purified protein (sfGFP-N\u003csup\u003eWT\u003c/sup\u003e or sfGFP-N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e) was identified by SDS-PAGE and showed green fluorescence. b) R\u003csup\u003e94\u003c/sup\u003e and Y\u003csup\u003e184\u003c/sup\u003e mutations disrupt RNA mediate N condensation. The condensates of sfGFP-N\u003csup\u003eWT\u003c/sup\u003e and RNA were larger than that of sfGFP-N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e and RNA under enlarged field of view. 50 ng/µL extracted RNA was incubated with 10 μM sfGFP-N\u003csup\u003eWT\u003c/sup\u003e or sfGFP-N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e protein at 25 °C for 10 min. Bars, 20 μm. The experiments were repeated at least three times with similar results. c) The binding ability of RNA with N\u003csup\u003eWT\u003c/sup\u003e was measured by BLI. d) The binding ability of RNA with N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e was measured by BLI. e) Binding curve of continuous injection of N\u003csup\u003eWT\u003c/sup\u003e and N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e protein. f) Binding curve of continuous injection of N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e and N\u003csup\u003eWT\u003c/sup\u003e protein. g) The binding ability of RNA with \u003cstrong\u003eZ9\u003c/strong\u003e was measured by BLI. h) Competitive binding experiment confirmed that \u003cstrong\u003eZ9\u003c/strong\u003e blocking the binding of RNA and N protein. The experiments were repeated three times with similar results. 5 µM \u003cstrong\u003eZ9\u003c/strong\u003e was first incubated with 12.5 nM N\u003csup\u003eWT\u003c/sup\u003e protein at 25 °C for 30 min, and then detected using SA sensors with fixed RNA. i) Proposed model for \u003cstrong\u003eZ9\u003c/strong\u003e as biomolecular condensate inhibitor.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/58b06423af1bd356576f70cb.png"},{"id":58032228,"identity":"99af8c66-c937-456f-ba04-37f18b564d05","added_by":"auto","created_at":"2024-06-10 08:04:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7084490,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/12bd248f-895a-4f58-97aa-bf1197ff8213.pdf"},{"id":58031118,"identity":"e1a20735-c77b-4b1f-bac1-934ab7756e09","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"avi","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16972288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVideo S1\u003c/strong\u003e The effect of DMSO on the formation of inclusion bodies in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e cells.\u003c/p\u003e","description":"","filename":"ExtendedDataVideoS1.avi","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/ba412ef450e566f9223e5168.avi"},{"id":58031665,"identity":"e811d602-6478-4cde-b909-e265bcf8cf8d","added_by":"auto","created_at":"2024-06-10 07:56:44","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12534272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVideo S2\u003c/strong\u003e The effect of compound\u003cstrong\u003e Z9\u003c/strong\u003e on the formation of inclusion bodies in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e cells at 100 µg/mL.\u003c/p\u003e","description":"","filename":"ExtendedDataVideoS2.avi","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/a7b8b87f70101bf8e13e34c6.avi"},{"id":58031120,"identity":"69de2610-bac3-4393-a9e1-d898a9f26414","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11143168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVideo S3 \u003c/strong\u003eThe effect of compound\u003cstrong\u003e Z3\u003c/strong\u003e on the formation of inclusion bodies in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e cells at 100 µg/mL.\u003c/p\u003e","description":"","filename":"ExtendedDataVideoS3.avi","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/d5f6b212cc0ebdd400202afe.avi"},{"id":58031121,"identity":"86be35cb-070e-47e2-b46d-acff653043bd","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"avi","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":6175232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVideo S4\u003c/strong\u003e Highly motile cytoplasmic inclusion bodies formed by N-YFP in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e cells.\u003c/p\u003e","description":"","filename":"ExtendedDataVideoS4.avi","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/0ec70163836bc93da6e2353c.avi"},{"id":58031669,"identity":"0d133fae-a458-4902-a239-5070d760e8cd","added_by":"auto","created_at":"2024-06-10 07:56:44","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":9405440,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVideo S5 \u003c/strong\u003eCytoplasmic inclusion bodies formed by NR94A\u0026amp;Y184A-YFP in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e cells.\u003c/p\u003e","description":"","filename":"ExtendedDataVideoS5.avi","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/c4210c3f9fdc3b1e86e3d6b3.avi"},{"id":58031123,"identity":"2053a74e-183e-4927-8bc7-9c3bf9b6cfee","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"avi","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":7343104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVideo S6 \u003c/strong\u003eRepresentative time-lapse video showing the fission of inclusion bodies formed by N-YFP in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaf cells.\u003c/p\u003e","description":"","filename":"ExtendedDataVideoS6.avi","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/a534120b63a909a1f5eda122.avi"},{"id":58031125,"identity":"bbfff0c9-5cea-437e-98bd-ff757c3e62f3","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"avi","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":7219200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVideo S7 \u003c/strong\u003eRepresentative time-lapse video showing the fusion of inclusion bodies formed by N-YFP in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaf cells.\u003c/p\u003e","description":"","filename":"ExtendedDataVideoS7.avi","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/212f521a877006fdc2d26ea6.avi"},{"id":58031127,"identity":"fe1f4bc9-18dc-4327-b308-0281b8ecd9a5","added_by":"auto","created_at":"2024-06-10 07:48:44","extension":"avi","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14127616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVideo S8\u003c/strong\u003e Fluorescence recovery after photobleaching of inclusion bodies formed by N-YFP in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaf cells.\u003c/p\u003e","description":"","filename":"ExtendedDataVideoS8.avi","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/8fa0f80bbcf5a7af842c75f1.avi"},{"id":58031668,"identity":"9c3a4df4-dee6-4936-adb5-2bc2e63b1662","added_by":"auto","created_at":"2024-06-10 07:56:44","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":11793429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/315b8447f9c50073ccf7a35c.docx"},{"id":58031667,"identity":"e0a6a414-dbb7-4c7e-83f6-fd2d40a3ffd5","added_by":"auto","created_at":"2024-06-10 07:56:44","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":24751,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4398439/v1/eef4d14e957675776b606d88.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Rational Design of Phytovirucide that Inhibits the Nucleocapsid Condensates of Tomato Spotted Wilt Virus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiomolecular condensates have been related to many cellular processes, including compartmentalization of biochemical reactions, sensing and responding to stress, mechanical regulation and signal transduction\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Mounting evidence suggests that phase separation has emerged as a fundamental principle for the formation of biomolecular condensates\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The assembly components of condensates are typically complex, usually involving proteins and nucleic acids, which lead to phase separation via their multivalent interactions\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As obligate intracellular parasites, viruses rely on cellular functions and processes to enable efficient replication. During infection, condensate-like structures commonly referred to as 'replication compartments' (RCs) or 'inclusion bodies' (IBs) were formed by viral proteins. Importantly, recent studies have implicated phase separation as a driving force for the formation of these biomolecular condensates\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The condensates play key role in viral life cycles, including viral entry\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, genome synthesis and replication\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, assembly for new particle\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and escape antiviral defense pathways\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Condensates formed by phase separation have attracted much attention in pharmaceutical industries and academia as novel targets in drug discovery\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although it has been found that some small molecules could regulate the aggregation of viral biomolecular condensates in recent years, the discovery of new structures is still relatively rare\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, such modulators have not been reported in the agricultural field. Replication-related proteins encoded by several plant viruses such as barley yellow mosaic stripe virus (BYSMV), tomato yellow mottle-associated virus (TYMaV), tomato bushy stunt virus (TBSV), and carnation Italian ringspot virus (CIRV) have been continuously found form condensates, which proven to be associated with phase separation in the past two years\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Recently, the phytobacterial type III effectors have been shown to exhibit a propensity for phase separation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Thus, targeting biomolecular condensates may be promising for success in developing novel pesticides for plant diseases that are difficult to control.\u003c/p\u003e \u003cp\u003eTomato spotted wilt virus (TSWV) belongs to the family \u003cem\u003eBunyaviridae\u003c/em\u003e, is one of the most devastating plant viruses worldwide, causing an annual economic loss of over one billion US dollars\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The use of antiviral agents is the most direct and effective method, but currently commercialized drugs are few and cannot actually serve purpose of managing TSWV-induced disease\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, developing new and effective anti-TSWV agents is of great significance. The few proteins encoded by the TSWV genome play an important role in virus infection. Among them, nucleocapsid protein (N) is an important component of the core template ribonucleoprotein (RNP) for viral replication\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and has multiple biological functions in the viral life cycle. The genomic RNA of virus could be well protected in RNP against RISC-mediated cleavage\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Similar to animal-infecting \u003cem\u003eBunyaviruses\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, TSWV relies on a 'cap snatching' mechanism to initiate transcription of its genome\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The N proteins partially co-localize with processing-bodies (PBs) and stress granules (SGs), which are the sources of 5\u0026prime; capped RNA\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Notably, N protein is capable of forming highly motile cytoplasmic IBs that traffic on the actin filaments\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Therefore, N protein can serve as an important molecular target for the creation of anti-TSWV agents.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChromones are an important class of natural active ingredients produced during secondary metabolism in plants. Their structure is easy to modify and is considered a good lead skeleton for drug discovery\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Chromone compounds derived from natural products possess potential broad-spectrum antiviral effects against human viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), herpes simplex virus type 1 (HSV1), influenza A virus (IAV), and hepatitis B virus (HBV), etc\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and we have previously synthesized several types of chromone compounds, which possess good inhibitory activity against plant viruses, and found that they had good binding ability to coat proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Cationic amphiphilic drugs (CADs) are a class of commercialized drugs with a wide range of medical activities\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. They are characterized by a common structure, namely hydrophobic aromatic side chains containing an amine functional group, which is crucial for its pharmacological activity by increasing the pH in acidic intracellular compartments\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. We have noticed that the phase separation modulators through high-throughput screening method appear to possess properties of CADs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb)\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44 CR45\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Noteworthy, an increasing number of studies emphasize the crucial role of pH in the formation of condensates\u003csup\u003e\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Inspired by the two points, we propose that designing a compound with properties of CADs may be more successful in discovering a novel biomolecular condensate modulator. Therefore, we designed a series of chromone compounds containing hydrophilic amine groups with piperazine dithiocarbamate as the bond-bridge in the study, and obtained the target compounds with high yields (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We next conducted a comprehensive study, including biological assay and exploration of mechanism of action. As a result, the introduction of tertiary amine groups significantly enhances the anti-TSWV activities of compounds. The highly active small molecule \u003cb\u003eZ9\u003c/b\u003e could target N protein to inhibit the replication level of the viral ribonucleoprotein (RNP), thereby affecting virus proliferation \u003cem\u003ein vivo\u003c/em\u003e. Furthermore, the addition of \u003cb\u003eZ9\u003c/b\u003e or the loss of binding sites could both lead to a decrease in N protein condensations in plant cells. The characteristics of the N protein condensations driven by phase separation were determined, and RNA plays a crucial role in the process. We proved that the binding sites could affect the normal binding of N protein to nucleic acid, and \u003cb\u003eZ9\u003c/b\u003e could intervene the formation of N protein-RNA complex. Collectively, the study is the first strategic application of novel agrochemicals that disintegrate condensates driven by phase separation in the prevention and treatment of plant diseases, which expands a new direction for pesticide discovery.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eAnti-TSWV activity\u003c/b\u003e. The inhibitory activities of the target compounds \u003cb\u003eZ1\u003c/b\u003e - \u003cb\u003eZ25\u003c/b\u003e against TSWV were assessed using the half-leaf method with \u003cem\u003eNicotiana glutinosa\u003c/em\u003e serving as the model plant. Preliminary screening at a concentration of 500 \u0026micro;g/mL showed that the curative activity range of compounds \u003cb\u003eZ1\u003c/b\u003e - \u003cb\u003eZ25\u003c/b\u003e against TSWV ranged from 43.5\u0026ndash;64.4%. The protective activity varied between 43.1% and 65.4%, while the inactivation activity spanned from 41.4\u0026ndash;77.3%. Among them, the compounds \u003cb\u003eZ9\u003c/b\u003e (77.3%), \u003cb\u003eZ13\u003c/b\u003e (72.8%), \u003cb\u003eZ14\u003c/b\u003e (70.6%), and \u003cb\u003eZ17\u003c/b\u003e (74.2%) had excellent inactivation activity against TSWV, outperforming the control ningnanmycin (69.3%) and were significantly superior to ribavirin (43.7%). We found the inhibitory activities of compounds (\u003cb\u003eZ1\u003c/b\u003e-\u003cb\u003eZ7\u003c/b\u003e) without tertiary amine groups on TSWV were average. The EC\u003csub\u003e50\u003c/sub\u003e values of the compounds were further tested, and the results showed that compound \u003cb\u003eZ9\u003c/b\u003e exhibited the best inactivation activity against TSWV, with an inhibitory activity of 65.3 \u0026micro;g/mL, significantly better than that of ningnanmycin (149.4 \u0026micro;g/mL) and ribavirin (801.5 \u0026micro;g/mL) (Table\u0026nbsp;1). The above results indicate that the introduction of tertiary amine group substantially enhances the anti-TSWV activities of compounds. In addition, the inhibitory effect of compound \u003cb\u003eZ9\u003c/b\u003e on TSWV was further validated in tomatoes beyond model plants. The tomato plants sprayed with 3% tween-80 exhibited severe symptoms, including curled leaves, dark brown spots, delayed development, and yellowing. However, when treated with compound \u003cb\u003eZ9\u003c/b\u003e, the infection symptoms of TSWV in plants were notably alleviated. Compared with the control group, the \u003cb\u003eZ9\u003c/b\u003e-treated plants displayed better growth, with larger and greener leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This indicated that compound \u003cb\u003eZ9\u003c/b\u003e could significantly reduce the proliferation of TSWV in plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCompound Z9 significantly reduces N protein condensates\u003c/b\u003e. TSWV N protein can form motile cytoplasmic IBs to assist the proliferation of virus\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We aimed to observe the impact of compound \u003cb\u003eZ9\u003c/b\u003e on the behavior of N protein condensates \u003cem\u003ein vivo\u003c/em\u003e. N-YFP was transiently expressed in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves, and after 36 h, drugs were injected into the infiltrating area and IBs 5 h post-injection were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). A good advantage is, the superior water solubility of compound \u003cb\u003eZ9\u003c/b\u003e avoids the necessity for surfactants like Tween 80, which may harm plant cells or tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Compound \u003cb\u003eZ9\u003c/b\u003e, at a concentration of 100 \u0026micro;g/mL, significantly reduced the number of IBs in the leaf cell infiltration area, while compound \u003cb\u003eZ3\u003c/b\u003e, with less effective anti-TSWV activity, did not markedly decrease the number of IBs. Additionally, compound \u003cb\u003eZ3\u003c/b\u003e treatment led to leaf cells appearing compressed, possibly due to the compound entering the cells and forming solid particles, thereby damaging the tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). A time-course experiment was conducted to further analyze the effects of compound \u003cb\u003eZ9\u003c/b\u003e on the behavior of IBs. It was evident that the mobility of IBs treated with compound \u003cb\u003eZ9\u003c/b\u003e remained unchanged, yet their quantity significantly reduced. Instead, due to the destruction of cellular tissues, IBs treated with \u003cb\u003eZ3\u003c/b\u003e were almost unable to move normally inside the plant cell and formed large particles. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and Extended Data Videos \u003cb\u003eS1\u003c/b\u003e-\u003cb\u003eS3\u003c/b\u003e). The above results reveal that compound \u003cb\u003eZ9\u003c/b\u003e with amine group could effectively inhibit the formation of the aggregates involving N protein. Although several types of anti-TSWV compounds targeting N proteins have been previously reported, their impact on IBs had not been explored\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Herein, we further investigate whether other highly active compounds similar to CADs can disrupt the aggregation of N protein. It was found that piperazine derivatives containing ethyl morpholine also indeed inhibited IB formation (Supplementary Fig.\u0026nbsp;1). Thus, the experimental results expose the potential mechanism by which drugs targeting N proteins may inhibit the assembly of condensates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe absence of binding sites reduces the proliferation of viruses\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e. We next explored the key binding sites of compound \u003cb\u003eZ9\u003c/b\u003e on the N protein. Molecular docking indicated that the compound \u003cb\u003eZ9\u003c/b\u003e could form conventional hydrogen bonds with the amino acids Arg94 (R\u003csup\u003e94\u003c/sup\u003e), Gly147 (G\u003csup\u003e147\u003c/sup\u003e), and Tyr184 (Y\u003csup\u003e184\u003c/sup\u003e) in the core region of N protein, and hydrophobic interactions with Leu126 (L\u003csup\u003e126\u003c/sup\u003e), Pro151 (P\u003csup\u003e151\u003c/sup\u003e), and Ile179 (I\u003csup\u003e179\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). To validate the effect of binding sites of compound \u003cb\u003eZ9\u003c/b\u003e on RNP proliferation, reverse PCR amplification and recombination technique were employed to construct SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eR94A\u003c/sup\u003e, SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eG147A\u003c/sup\u003e, SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eY184A\u003c/sup\u003e, and SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e mini-replicons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;2). \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants were then infected using competent minigenome replication system (RNP\u003csub\u003eeGFP\u003c/sub\u003e) and fluorescence was observed on the 5th day. The results showed that the absence of amino acid R\u003csup\u003e94\u003c/sup\u003e marginally reduced fluorescence levels. Conversely, the absence of G\u003csup\u003e147\u003c/sup\u003e did not alter the intensity of fluorescence in leaf cells, while the absence of Y\u003csup\u003e184\u003c/sup\u003e or R\u003csup\u003e94\u003c/sup\u003e\u0026amp;Y\u003csup\u003e184\u003c/sup\u003e could diminish fluorescence levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Average fluorescence intensity was analyzed according to three independent biological replicates. The results showed that RNP\u003csub\u003eeGFP\u003c/sub\u003e\u003csup\u003eWT\u003c/sup\u003e and RNP\u003csub\u003eeGFP\u003c/sub\u003e\u003csup\u003eG147A\u003c/sup\u003e in leaf cells had the strongest average fluorescence intensity, RNP\u003csub\u003eeGFP\u003c/sub\u003e\u003csup\u003eR94A\u003c/sup\u003e slightly decreased, and RNP\u003csub\u003eeGFP\u003c/sub\u003e\u003csup\u003eY184A\u003c/sup\u003e was the weakest among all single-point mutations. RNP\u003csub\u003eeGFP\u003c/sub\u003e\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e showed a more significant weakening (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Additionally, the accumulation levels of N protein were further detected. Compared to RNP\u003csub\u003eeGFP\u003c/sub\u003e\u003csup\u003eWT\u003c/sup\u003e, the lack of Y\u003csup\u003e184\u003c/sup\u003e or R\u003csup\u003e94\u003c/sup\u003e\u0026amp;Y\u003csup\u003e184\u003c/sup\u003e significantly reduced the accumulation level of N protein, while the absence of R\u003csup\u003e94\u003c/sup\u003e slightly reduced the accumulation of N protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eTo verify the impact of missing binding sites on TSWV pathogenicity in plants, the movement-competent minigenome replication system (M\u003csub\u003e(\u0026minus;)opt\u003c/sub\u003e) was added to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs). By the 14th day, noticeable pathogenic symptoms, such as leaf wrinkling and spotting, were evident in the systemic leaves of TSWV\u003csup\u003eWT\u003c/sup\u003e-infected plants. The severity of symptoms in TSWV-infected plants were divided into three grades (Supplementary Fig.\u0026nbsp;3). By the 21st day, nearly all plants showed significant wilting and mortality (grade Ⅲ), with a disease susceptibility rate of 100%. In the case of TSWV\u003csup\u003eG147A\u003c/sup\u003e, over 50% of the plants displayed severe disease symptoms (grade Ⅲ) in systemic leaves, with an overall infection rate surpassing 70%. This suggests that the absence of G\u003csup\u003e147\u003c/sup\u003e somewhat delayed the virus\u0026rsquo;s systemic infection plants. For TSWV\u003csup\u003eR94A\u003c/sup\u003e or TSWV\u003csup\u003eY184A\u003c/sup\u003e, symptom onset in system leaves was slower and considerably milder compared to TSWV\u003csup\u003eWT\u003c/sup\u003e, with a susceptibility rate of around 50% (grade I and grade Ⅱ). In contrast, plants infected with TSWV\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e showed fewer spots in the systematic leaves and a lower incidence rate of about 20% (grade I and grade Ⅱ). Therefore, through a comprehensive comparative analysis of symptoms and infection rates, it was revealed that the solely absence of R\u003csup\u003e94\u003c/sup\u003e or Y\u003csup\u003e184\u003c/sup\u003e effectively hindered the virus\u0026rsquo;s systemic spread, and their joint absence drastically reduced it (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). The order of N protein accumulation level in the infected system leaves was TSWV\u003csup\u003eWT\u003c/sup\u003e \u0026gt; TSWV\u003csup\u003eG147A\u003c/sup\u003e \u0026gt; TSWV\u003csup\u003eR94A\u003c/sup\u003e \u0026gt; TSWV\u003csup\u003eY184A\u003c/sup\u003e \u0026gt; TSWV\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). These findings underscore that the binding sites of compound \u003cb\u003eZ9\u003c/b\u003e had a crucial impact on viral proliferation \u003cem\u003ein vivo\u003c/em\u003e by affecting RNP replication.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe binding sites significantly reduce the aggregation of N protein.\u003c/b\u003e To further explore the interaction between compound \u003cb\u003eZ9\u003c/b\u003e and the N protein, mutant proteins (N\u003csup\u003eR94A\u003c/sup\u003e, N\u003csup\u003eY184A\u003c/sup\u003e, and N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e) were obtained by prokaryotic expression and purification (Supplementary Fig.\u0026nbsp;4a,4b). Bio-layer interferometry was employed to examine the binding dynamics between compound \u003cb\u003eZ9\u003c/b\u003e and the N protein. Compared to N\u003csup\u003eWT\u003c/sup\u003e (7.6 \u0026micro;M), the binding ability of compound \u003cb\u003eZ9\u003c/b\u003e for double mutant N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e was significantly diminished, with a \u003cem\u003eK\u003c/em\u003eD value exceeding 400 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The MST test results also indicated that the binding constants of N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e with compound \u003cb\u003eZ9\u003c/b\u003e were decreased by 180-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Similarly, binding constants for the single mutant proteins N\u003csup\u003eR94A\u003c/sup\u003e and N\u003csup\u003eY184A\u003c/sup\u003e with compound \u003cb\u003eZ9\u003c/b\u003e were reduced by approximately 103 and 135 times, respectively (Supplementary Fig.\u0026nbsp;4c). These results indicate that the amino acids R\u003csup\u003e94\u003c/sup\u003e and Y\u003csup\u003e184\u003c/sup\u003e of N protein were the binding sites for compound \u003cb\u003eZ9\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the influence of binding sites on the formation of IBs, we engineered a construct that the N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e protein into a yellow fluorescent protein (N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e-YFP) (Supplementary Fig.\u0026nbsp;5). When transiently expressed in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves, the N\u003csup\u003eWT\u003c/sup\u003e-YFP formed numerous discrete cytoplasmic inclusions, in stark contrast, both YFP alone and N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e-YFP were predominantly dispersed throughout the cytoplasm, indicating a diffuse distribution pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Expanding our observation field, we significantly noted that although small IBs formed by N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e-YFP also migrated, while their quantity was substantially reduced compared to N\u003csup\u003eWT\u003c/sup\u003e-YFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and Extended Data Video \u003cb\u003eS4\u003c/b\u003e, \u003cb\u003eS5\u003c/b\u003e). These observations suggest that mutations at binding sites markedly decrease IB formation and is basically consistent with the phenomenon of adding compound \u003cb\u003eZ9\u003c/b\u003e. Like animal-infecting viruses in the family \u003cem\u003eBunyaviridae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, TSWV N protein is known to co-localize with P-bodies\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This was further explored by transient co-expression of N\u003csup\u003eWT\u003c/sup\u003e-YFP with DCP5-mCherry by agroinfiltration in leaves. It was found that cytoplasmic granules of N\u003csup\u003eWT\u003c/sup\u003e-YFP co-localized with DCP5-mCherry foci within the cytoplasm. However, the formation of fewer granules by N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e-YFP resulted in a significantly reduced degree of overlap (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). This reduction points to the impact of missing binding sites on the viral \u0026lsquo;cap snatching\u0026rsquo; process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCondensates are formed by phase separation.\u003c/b\u003e We further delved into the mechanism behind the reduction in condensate numbers observed upon the addition of compound \u003cb\u003eZ9\u003c/b\u003e or due to the absence of binding sites in N proteins. It is well-established that proteins with IDRs often engage in phase separation to form condensates\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. With this premise, the phase separation potential of N protein was assessed using the Predictor of Natural Disordered Regions (PONDR) VL-XT algorithm. This sequence-based prediction honed in on the N protein\u0026rsquo;s IDR, primarily located within its central core domain\u0026mdash;a region notably implicated in nucleic acid interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Encouraged by these bioinformatic insights, we pursued experimental verification to discern whether N protein could indeed engage in phase-separation activities. To this end, a construct encoding N-YFP was transiently expressed in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. Time-lapse confocal imaging documented the behavior of small, dynamic inclusions that frequently merged, coalescing into larger formations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and Extended Data Video \u003cb\u003eS6\u003c/b\u003e). Indeed, it was observed that these large inclusions occasionally divided slowly, spawning smaller ones that dispersed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and Extended Data Video \u003cb\u003eS7\u003c/b\u003e). The characteristics of these inclusions, such as their sphericity, motility, and fusion/fission, as seen in N-YFP-infected cells, reveal that the IBs may exhibit properties akin to those of liquid droplets. Fluorescence recovery after photobleaching (FRAP) experiment showed that small droplets achieved up to 50% recovery efficiencies within 2 mins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and Extended Data Video \u003cb\u003eS8\u003c/b\u003e), indicating a rapid dynamic nature of these droplets. Taken together, these results point the propensity of N protein-containing condensates to undergo phase separation \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCompound Z9 inhibits nucleic acid mediate N protein condensation\u003c/b\u003e. RNP granules assemble through phase separation of their molecular components, and RNA is thought to be a critical element for the sizing and composition of the condensates\u003csup\u003e\u003cspan additionalcitationids=\"CR55 CR56 CR57\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Notably, the N protein appears to interact with nucleic acids at approximately 25 binding sites (Supplementary Fig.\u0026nbsp;6a), with residues K\u003csup\u003e65\u003c/sup\u003e, R\u003csup\u003e94\u003c/sup\u003e, R\u003csup\u003e95\u003c/sup\u003e, K\u003csup\u003e183\u003c/sup\u003e, and Y\u003csup\u003e184\u003c/sup\u003e identified as particularly significant\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Considering that the IDR of N protein mainly resides in the area forming these nucleic acid binding sites, our focus shifted to investigating whether nucleic acids could mediate the formation of condensates. To explore this possibility, we purified the recombinant His-sfGFP-N from \u003cem\u003eEscherichia coli\u003c/em\u003e and examined its aggregation investigating under conditions of higher concentration and low salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Confocal microscopy analysis showed no significant aggregation of His-sfGFP-N protein under these conditions. Subsequently, total RNA was extracted from TSWV-infected \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. Protein phase separation assay was performed with increasing concentrations of RNA, ranging from 50 to 100 ng/\u0026micro;L. Remarkably, the His-sfGFP-N displayed a phase transition in the presence of RNA, rapidly forming larger, aggregate-like structures at the high concentration of RNA. However, the features of these N-RNA condensates exhibited a solid-like physical state rather than a fluidic one (Supplementary Fig.\u0026nbsp;6b). This rapid solidification parallels phenomenon observed in amyloid-like fibrils or oskar ribonucleoprotein granules\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Another important reason is that the physiological condition of \u003cem\u003ein vitro\u003c/em\u003e assay makes it challenging to achieve consistency with those in the plant body. In conclusion, our results indicate that N protein undergoes RNA-induced condensation \u003cem\u003ein vitro\u003c/em\u003e, revealing a critical interplay between nucleic acids and N protein in condensate formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify whether mutations at the binding sites R\u003csup\u003e94\u003c/sup\u003e and Y\u003csup\u003e184\u003c/sup\u003e in N protein significantly affect RNA-induced N protein condensation, we generated mutations of His-sfGFP-N by substituting the residues with alanine residues R\u003csup\u003e94A\u003c/sup\u003e/Y\u003csup\u003e184A\u003c/sup\u003e (His-sfGFP-N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Confocal microscopy data showed that both the numbers and sizes of N protein condensates were greatly reduced by the binding site mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). To further investigate the impact of these specific amino acids on the N protein\u0026rsquo;s ability to bind RNA, digoxigenin-labeled RNA probe was prepared and electrophoretic mobility shift assay (EMSA) was conducted in the study (Supplementary Fig.\u0026nbsp;7a). We found that in the presence of the N\u003csup\u003eWT\u003c/sup\u003e, the rate of RNA migration began to change slightly at a protein dosage of 0.1 \u0026micro;g, with a significant decrease observed at dosages exceeding 0.1875 \u0026micro;g. Conversely, for N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e mutant, a slight slowing in RNA migration was noted at a protein dosage of 1.5 \u0026micro;g, with a substantial decrease only evident at dosages greater than 3.0 \u0026micro;g (Supplementary Fig.\u0026nbsp;7b). These results indicate that the absence of these binding sites notably impairs the RNA binding capability of the N protein.\u003c/p\u003e \n\u003cp\u003eWe next immobilized the biotin-labeled RNA (100 nM) onto the streptavidin (SA) biosensors, and the binding affinities of N\u003csup\u003eWT\u003c/sup\u003e and N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e with RNA were measured by BLI assay. The results suggested that the affinity constant (\u003cem\u003eK\u003c/em\u003eD) for N\u003csup\u003eWT\u003c/sup\u003e and N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e binding to RNA were 7.3 nM and 36 nM, respectively (Fig.\u003cstrong\u003e\u0026nbsp;7c,7d\u003c/strong\u003e). The mutation of the binding sites reduced the affinity between N protein and RNA approximately 5-fold. We observed that N\u003csup\u003eWT\u003c/sup\u003e binds to RNA more rapidly than N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e at low concentrations, and it dissociates from RNA slower than N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e. When the biosensor was continuously loaded with WT and mutant N protein, and we noticed a significant decrease in the binding curve (Fig.\u003cstrong\u003e\u0026nbsp;7e\u003c/strong\u003e). Upon reversing the loading sequence, the binding curve increased significantly (Fig.\u003cstrong\u003e\u0026nbsp;7f\u003c/strong\u003e). These results demonstrate that the absence of binding sites affected the binding ability of N protein to RNA. To evaluate the binding efficiency of compound \u003cstrong\u003eZ9\u003c/strong\u003e with N-RNA complex, we first determined the affinity constant (\u003cem\u003eK\u003c/em\u003eD=150 \u0026micro;M) between compound \u003cstrong\u003eZ9\u003c/strong\u003e and RNA (Fig.\u003cstrong\u003e\u0026nbsp;7g\u003c/strong\u003e). The interaction between compound \u003cstrong\u003eZ9\u003c/strong\u003e and N protein (\u003cem\u003eK\u003c/em\u003eD=7.6 \u0026micro;M) was significantly stronger than that of \u003cstrong\u003eZ9\u003c/strong\u003e-RNA, suggesting a preference of compound \u003cstrong\u003eZ9\u003c/strong\u003e to bind with N protein. In the competitive binding assay, we discovered that the binding signal value of N protein and RNA (0.26 nm) was remarkably higher than that of the mixture of compound \u003cstrong\u003eZ9\u003c/strong\u003e and N protein (0.17 nm) (Fig.\u003cstrong\u003e\u0026nbsp;7h\u003c/strong\u003e). From the constants of binding rate and dissociation rate, we found that pre-binding of compound \u003cstrong\u003eZ9\u003c/strong\u003e to N protein significantly blocked RNA binding. Collectively, these results reveal that compound \u003cstrong\u003eZ9\u003c/strong\u003e acts as a novel inhibitor, blocking phase separation of N condensations and inhibiting viral proliferation (Fig.\u003cstrong\u003e\u0026nbsp;7i\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBiomolecular condensate modulators have received great attention in the pharmaceutical field\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, however, their application in agriculture has not been reported yet. Consequently, based on the advantages of the privileged scaffold (benzopyrone) in drug discovery, a series of novel benzopyrone compounds were rationally designed and synthesized by analyzing the structures of biomolecular condensates modulators in the pharmaceutical field (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We discovered that the introduction of amine groups into the side chain significantly improved the inhibitory activity of the compounds against TSWV (Table. 1). Further investigations into the mechanism of action revealed that the highly active compound \u003cb\u003eZ9\u003c/b\u003e could inhibit the replication of viral RNP (a complex of N protein, nucleic acid, and RdRp) \u003cem\u003ein vivo\u003c/em\u003e. Subsequent BLI screening indicated that the introduction of an amine group significantly improved the binding of N protein to the compounds. The binding signal values showed a positive correlation with their activity to some extent, with \u003cb\u003eZ9\u003c/b\u003e displaying the highest signal value (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Moreover, the affinity of \u003cb\u003eZ9\u003c/b\u003e for N protein was measured at 7.6 \u0026micro;M, indicating that N protein is a direct target of \u003cb\u003eZ9\u003c/b\u003e. When, N-YFP protein was transiently expressed in plants and treated with the \u003cb\u003eZ9\u003c/b\u003e, a significant reduction in the number of N protein condensates was observed, demonstrating that our designed small molecules effectively regulate N protein condensates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This study supports our initial hypothesis that compounds designed with CADs are potentially more effective in discovering regulators of phase separation.\u003c/p\u003e \u003cp\u003eTo further elucidate the mechanism of action of \u003cb\u003eZ9\u003c/b\u003e, we employed molecular docking to identify potential binding sites between \u003cb\u003eZ9\u003c/b\u003e and the N protein. Our findings indicate that the absence of these binding sites significantly inhibited the replication of the RNP and alleviated the pathogenic symptoms of TSWV, which was consistent with the mode of action of \u003cb\u003eZ9\u003c/b\u003e. We identified that R\u003csup\u003e94\u003c/sup\u003e and Y\u003csup\u003e184\u003c/sup\u003e as critical binding sites for \u003cb\u003eZ9\u003c/b\u003e by small molecule-protein interaction assays. \u003cem\u003eIn vivo\u003c/em\u003e experiments further demonstrated that the absence of R\u003csup\u003e94\u003c/sup\u003e and Y\u003csup\u003e184\u003c/sup\u003e led to a diffuse distribution of the N protein within the cell, significantly reducing the number of condensates and consequently impairing TSWV\u0026rsquo;s cap snatching and infection processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Phase separation is recognized as a fundamental principle driving the formation of condensates\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The N protein was predicted to contain disordered regions, and our observations showed that N protein condensates underwent a fusion-fission process in their early stages and exhibited a degree of recovery after bleaching (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results suggest that condensate formation \u003cem\u003ein vivo\u003c/em\u003e is driven by phase separation, a process resulting from multivalent interactions between proteins or between proteins and nucleic acids \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56 CR57\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. We speculated that the loss of key amino acids would significantly affect the binding of the N protein to RNA. A series of \u003cem\u003ein vitro\u003c/em\u003e experiments revealed a marked reduction in both the quantity and size of the aggregates formed by the N protein and RNA. Furthermore, the absence of key binding sites substantially impacted the N protein\u0026rsquo;s ability to bind RNA. Notably, the addition of \u003cb\u003eZ9\u003c/b\u003e was found to indeed inhibit the binding of the N protein to RNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). Thus, we have conducted comprehensive research and developed a novel inhibitor targeting condensates by directly blocking protein\u0026ndash;nucleic acid interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eWe discovered that the introduction of basic amine groups is critical, enabling classification as as pH-responsive drugs (CADs). Our observations show that most biomolecular condensate inhibitors identified through high-content screening methods in the medical field possess alkalic groups\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR43 CR44 CR45\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Increasingly, research emphasizes that physiological pH plays a significant role in regulating the formation of certain biomolecular condensates\u003csup\u003e\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This study focuses on elucidating the mechanism through which compound \u003cb\u003eZ9\u003c/b\u003e disrupts the multivalent interaction between N protein and RNA, thereby inhibiting the forces driving phase separation. At present, it appears that there have no instance of research combining drug discovery with the physiological pH of condensates formation. Therefore, a thorough understanding of the formation mechanism of condensates is of great significance for the further development of biomolecular condensate modulators.\u003c/p\u003e \u003cp\u003eGiven the increasing evidence that many plant viruses and phytopathogenic bacteria rely on the condensation of replication proteins for proliferation\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, it is conceivable that the discovery of novel biomolecular condensate inhibitors for use in agriculture could play a significant role in controlling previously untreatable plant diseases. Finally, we believe this study shall establish a cornerstone for the strategic design of innovative virucides targeting phase separation process in the viral lifecycle.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eMaterials and virus sources.\u003c/b\u003e The seeds of \u003cem\u003eNicotiana glutinosa\u003c/em\u003e and \u003cem\u003eNicotiana benthamiana\u003c/em\u003e were preserved by our laboratory. All plants were cultured in an artificial climate chamber (25℃ during the day, 50% relative humidity/23℃ at night, 40% relative humidity). The full-length infectious clone vectors (L\u003csub\u003e(+)opt\u003c/sub\u003e, M\u003csub\u003e(\u0026minus;)opt\u003c/sub\u003e, and SR\u003csub\u003e(+)eGFP\u003c/sub\u003e), expression vector of silencing suppressors (VSRs), vector pCambia2300-N-YFP, and DCP5-mCherry were provided by Professor Xiaorong Tao (Nanjing Agricultural University). TSWV lettuce isolate was maintained in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e and the infected leaves were maintained in a -80℃ refrigerator.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmid construction.\u003c/b\u003e For generating the mutant plasmids SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eMut\u003c/sup\u003e (SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eR94A\u003c/sup\u003e, SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eY184A\u003c/sup\u003e, SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eG147A\u003c/sup\u003e, and SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e), the insertion fragments and vectors were obtained with SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eWT\u003c/sup\u003e as a template by reverse polymerase chain reaction. For the construct p2300-N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e-YFP, the cDNA of the N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e gene was amplified from SR\u003csub\u003e(+)eGFP\u003c/sub\u003e\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e, and then inserted into binary vector pCambia2300-YFP to generate. All constructs were verified by DNA sequencing. The primers used are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis.\u003c/b\u003e All commercial reagents and solvents were used without further purification. Thin layer chromatography (TLC) was performed on pre-coated silica gel GF254 plates. Visualization of TLC was achieved using UV light (wavelength 254 nm). Column chromatography was performed on silica gel (300\u0026ndash;400 mesh) using a proper eluent. NMR was recorded on Bruker DPX 400 MHz (Bruker, Germany). Chemical shifts were reported in parts per million (ppm) referenced to the appropriate solvent peaks (\u003cem\u003eδ\u003c/em\u003e 7.26 ppm for CDCl\u003csub\u003e3\u003c/sub\u003e in proton spectra, 77.0 ppm for CDCl\u003csub\u003e3\u003c/sub\u003e in fully decoupled \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC spectra). The following abbreviations were used to describe peak splitting patterns: s\u0026thinsp;=\u0026thinsp;singlet, d\u0026thinsp;=\u0026thinsp;doublet, t\u0026thinsp;=\u0026thinsp;triplet, m\u0026thinsp;=\u0026thinsp;multiplet. Coupling constants \u003cem\u003eJ\u003c/em\u003e were reported in hertz (Hz). High resolution mass spectra were obtained with thermo Scientific Q Exactive (Thermo Scientific, USA). The specific synthesis steps can be found in the \u003cb\u003esupplementary information\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnti-TSWV assay.\u003c/b\u003e \u003cem\u003eNicotiana glutinosa\u003c/em\u003e was used to as the model plant, and the inhibitory activities of compounds against TSWV were tested using the half-leaf method. The specific operations were performed as we described recently\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The extracted TSWV was inoculated on the lower leaves of tomatoes. The plants were cultured in an artificial climate chamber (25℃ during the day, 50% relative humidity/23℃ at night, 40% relative humidity). After 24 hours, 100 \u0026micro;g/mL agent (2.0 mg compound \u003cb\u003eZ9\u003c/b\u003e, 50 \u0026micro;L DMSO, 20 mL 2% (v/v) tween 80 water) was sprayed onto the tomato leaves, with 20 mL 3% tween 80 water containing DMSO (50 \u0026micro;L) as control. The pathogenic symptom was observed after about 20 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAgrobacterium\u003c/b\u003e \u003cb\u003einfiltration.\u003c/b\u003e \u003cem\u003eAgrobacterium\u003c/em\u003e tumefaciens strain GV3101 was transfected with the recombinant plasmids and the monoclonal clone was selected and cultured at 28 ℃ for about 24\u0026ndash;36 hours. A. tumefaciens cells were resuspended using agroinfiltration buffer (10 mM MES, pH\u0026thinsp;=\u0026thinsp;5.6, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 100 \u0026micro;M acetosyringone) and incubated for 2 to 3 h in the dark at room temperature after the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) was adjusted. The cells were used for agroinfiltration of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. For the competent minigenome replication system (L\u003csub\u003e(+)opt\u003c/sub\u003e + SR\u003csub\u003e(+)eGFP\u003c/sub\u003e + VSRs) or reconstituted infectious TSWV (L\u003csub\u003e(+)opt\u003c/sub\u003e + SR\u003csub\u003e(+)eGFP\u003c/sub\u003e + M\u003csub\u003e(\u0026minus;)opt\u003c/sub\u003e + VSRs), equal volumes of \u003cem\u003eAgrobacterium\u003c/em\u003e cultures harboring full-length infectious clone vector (final concentration OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.20), were mixed with bacterial mixture (final concentration OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05) containing the VSRs ( P19 - HcPro - γb). For expression vectors p2300-N-YFP, the cells were resuspended in agroinfiltration buffer adjusted to an optical density OD\u003csub\u003e600\u003c/sub\u003e of 0.50. Leaves of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants at the 5-6-leaf stage were infiltrated with \u003cem\u003eAgrobacterium\u003c/em\u003e cultures using syringes. The infiltrated plants were grown in an artificial climate chamber. The operation of the mutants is consistent with the above description.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence microscopy.\u003c/b\u003e \u003cem\u003eAgro\u003c/em\u003e-infiltrated \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves were examined for fluorescence expression using an OLYMPUS model BX53F Fluorescence Microscope (OLYMPUS, Tokyo, Japan) with a green barrier filter. The samples were fixed in water on a microslider under a coverslip to detect eGFP fluorescence. Fluorescence images were processed using ImagePro (OLYMPUS, Tokyo, Japan).\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein extracts and immunoblot assays.\u003c/b\u003e 1.0 g healthy, \u003cem\u003eAgrobacterium\u003c/em\u003e-infiltrated leaf patches, or TSWV-systemically infected leaves of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e were collected, and total protein was extracted in 1 mL of extraction buffer (25 mM Tris-HCl, pH\u0026thinsp;=\u0026thinsp;7.5, 150 mM NaCl, 1 mM EDTA, 2% w/v polyvinylpolypyrrolidone, 10 mM dithiothreitol, 0.5% v/v Triton X-100, 10% v/v glycerol, and 1\u0026times; protease inhibitor cocktail). The supernatants were collected after 10 min centrifugation at 20000 \u0026times; g at 4\u0026deg;C and directly used for immunoblot assays. The samples were transferred to PVDF membranes after separated by SDS-PAGE. The membranes were blocked with 5% w/v skim milk solution and probed with antibodies against GFP or N. Actin antibody was used as internal control, and then incubated with HRP-conjugated secondary antibody. Finally, the signal was visualized by the ChemiDoc MP Imaging System (Bio-Rad).\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein purification, and microscale thermophoresis (MST) assay.\u003c/b\u003e The prokaryotic expression vectors (pET-32a-N\u003csup\u003eWT\u003c/sup\u003e, pET-32a-N\u003csup\u003eR94A\u003c/sup\u003e, pET-32a-N\u003csup\u003eY184A\u003c/sup\u003e, pET-32a-N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e, pET-32a-sfGFP-N\u003csup\u003eWT\u003c/sup\u003e, and pET-32a-sfGFP-N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e) were successfully introduced into Escherichia coli BL21 (DE3), respectively. The expression strains were induced by isopropyl-\u003cem\u003eβ\u003c/em\u003e-D-thiogalactopyranoside at 28℃ overnight, harvested by centrifugation at 4\u0026deg;C, and the precipitates were lysed (power 35%, 40 min, 4 s ultrasound, and 6 s interval) with buffer (0.869 g NaCl, 3.75 mL PBS, 36 \u0026micro;L mercaptoethanol, 5 mL glycerol, 41.25 mL H\u003csub\u003e2\u003c/sub\u003eO, pH\u0026thinsp;=\u0026thinsp;7.4). The lysates were clarified by centrifugation at 16000 g for 30 min. The proteins were purified via affinity chromatography using a Trap high-performance column (GE Healthcare, USA). The binding affinities of compound \u003cb\u003eZ9\u003c/b\u003e to the proteins were determined by the Monolith NT.115 instrument (NanoTemper, Munich, Germany). The specific operation was performed according to the reported literature\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eElectrophoretic mobility shift assay (EMSA).\u003c/b\u003e Digoxigenin-labeled RNA probe was prepared following previously described protocols\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Briefly, DNA template with the T7 promoter sequence was generated by polymerase chain reaction and purified using the gel recovery kit. RNA was then transcribed \u003cem\u003ein vitro\u003c/em\u003e using T7 RNA polymerase (Promega, Madison, USA). 1 \u0026micro;L RNA probe with a concentration of approximately 1200 ng/\u0026micro;L was placed in a PCR tube and diluted to 40 ng/\u0026micro;L using DEPC water. 1 \u0026micro;L RNA probe (40 ng/\u0026micro;L) was added into the PCR tubes and mixed with 9 \u0026micro;L different qualities of the purified N\u003csup\u003eWT\u003c/sup\u003e or N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e (0.025, 0.05, 0.1, 0.1875, 0.375, 0.75, 1.5, 3.0, and 6.0 \u0026micro;g), respectively. The mixtures were incubated in ice water bath for 10 min. The samples were transferred onto a nylon membrane. The membrane was exposed to UV light and probed with anti-digoxin alkaline phosphatase antibody (Roche, Basel, Switzerland) for 1\u0026ndash;2 hours, and then incubated with BCIP-NBT for staining at 37℃. The signal was directly visualized and photographed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBio-layer interferometry (BLI) assays.\u003c/b\u003e The purified His-tagged N protein was loaded onto Ni-NTA biosensors, the binding signal values of different compounds (50 \u0026micro;g/mL) were measured using a bio-layer interferometry method with Octet RED 96 (ForteBio, San Francisco, USA). All compounds were diluted in PBST. The binding affinities (\u003cem\u003eK\u003c/em\u003eD) between compound \u003cb\u003eZ9\u003c/b\u003e and the recombinant wild and mutant proteins were measured using this method. The binding constants of the compound \u003cb\u003eZ9\u003c/b\u003e with proteins were determined by global fit analyses of experimental binding curves using the Octet Red software. To analyze the effect of binding sites on N-RNA interactions, 100 nM of biotinylated RNA (bio-GAGAGCAAUUGUGUCAGAA) was loaded onto the streptavidin (SA) biosensors and balanced with PBS. The biosensors were then exposed to diluted recombinant N\u003csup\u003eWT\u003c/sup\u003e, or N\u003csup\u003eR94A\u0026amp;Y184A\u003c/sup\u003e, followed by washing (dissociation) with PBS. The binding constant values (\u003cem\u003eK\u003c/em\u003eD) were calculated using the Octet Red software. For binding inhibition experiments, the binding constant value (\u003cem\u003eK\u003c/em\u003eD) of compound \u003cb\u003eZ9\u003c/b\u003e with RNA was first measured using BLI, and compound \u003cb\u003eZ9\u003c/b\u003e (5 \u0026micro;M) was then combined with N protein (12.5 nM) and incubated for 30 min at room temperature. The biosensors were exposed to recombinant N protein (12.5 nM), incubated mixture (compound \u003cb\u003eZ9\u003c/b\u003e and N protein), and compound \u003cb\u003eZ9\u003c/b\u003e (5 \u0026micro;M), followed by washing (dissociation) with PBST. Association rate constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e) and dissociation rate constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e) were obtained using Octet data analysis software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConfocal microscopy and fluorescence recovery after photobleaching assay.\u003c/b\u003e Confocal laser scanning micrographs of Agro-infiltrated leaves were captured with a Zeiss LSM900 confocal laser scanning microscope (Zeiss, Oberkochen, Germany). Fluorescent protein was excited at 488 nm and time-lapse images were taken over a 1 or 2 - min period. For drug treatments, when N-YFP was significantly expressed in leaves of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e after 36 h, small molecule (2.0 mg) was dissolved into a 100 \u0026micro;L DMSO solution, and then 20 mL of ddH\u003csub\u003e2\u003c/sub\u003eO was added to prepare a 100 \u0026micro;g/mL agent, rapidly inject it into the infiltration area of leaves. After 5 h, micrographs or time-lapse images were captured by confocal microscopy. For fluorescence recovery after photobleaching (FRAP) experiments, IB droplets formed in infected leaf cells were bleached with a laser at 488 nm (100% intensity) under a confocal microscope with \u0026times; 20 water immersion and magnify the field of view by \u0026times; 2.5. After bleaching, at least 40 frames of recovery images were collected by the time-lapse modes. Fluorescence intensity of the bleaching region was measured using Olympus CellSens software. The data was normalized and recovery curves were generated by GraphPad Prism 8.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Figs and full experimental details for the preparation of all new compounds, and their spectroscopic data, can be found in the supplementary materials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge financial support from the National Natural Science Foundation of China (Nos. 32330087 and 32302388). The authors thank Dr. Mingfeng Feng and Prof. Dr. Xiaorong Tao (College of Plant Protection, Nanjing Agricultural University, China) for assistance in the plasmid construction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.Z. conducted most of the experiments. J.L., S.W., J.L., and F.C. contributed to some experiments. J.Y. created the\u0026nbsp;pattern diagram. B.S. and R.S. conceptualized and directed the project and drafted the manuscript with assistance from all co-authors. All authors contributed to part of the experiments and/or discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBanani SF, Lee HO, Hyman AA, Rosen MK (2017) Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18:285\u0026ndash;298\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMayr C, Mittag T, Tang T-YD, Wen W, Zhang H, Zhang H (2023) Frontiers in biomolecular condensate research. 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New Phytol. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/nph.19778\u003c/span\u003e\u003cspan address=\"10.1111/nph.19778\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4398439/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4398439/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChemical management of crop failures caused by plant viruses poses serious challenges in agricultural chemistry. Recently, phase separation has emerged as a key role in the viral lifecycle, with the discovery of corresponding small molecule inhibitors in the medical field sparking significant interest. However, the applicability of this promising antiviral tactic for plant protection remains largely unknown. Herein, we have demonstrated that the aggregation of the \u003cem\u003etomato spotted wilt virus\u003c/em\u003e (TSWV) nucleocapsid protein (N) is regulated by RNA-induced phase separation, and disclosed a new rationally designed inhibitor \u003cb\u003eZ9\u003c/b\u003e. \u003cb\u003eZ9\u003c/b\u003e is capable of binding to TSWV N at the R\u003csup\u003e94\u003c/sup\u003e and Y\u003csup\u003e184\u003c/sup\u003e sites, preventing the assembly of N and RNA into aggregated ribonucleoproteins through phase separation. Mutations of the binding sites substantially diminish the colocalization between N protein and processing bodies, interfering with the normal interaction of N protein with RNA. This study provides opportunities to phase separation-based anti-plant virus strategies.\u003c/p\u003e","manuscriptTitle":"Rational Design of Phytovirucide that Inhibits the Nucleocapsid Condensates of Tomato Spotted Wilt Virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 07:48:39","doi":"10.21203/rs.3.rs-4398439/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"21427e35-52bc-425e-88d3-a428d9add8f3","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":32548065,"name":"Biological sciences/Drug discovery/Drug regulation"},{"id":32548066,"name":"Biological sciences/Drug discovery/Drug screening"}],"tags":[],"updatedAt":"2024-06-10T07:48:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-10 07:48:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4398439","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4398439","identity":"rs-4398439","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}