Functional analyses of dopamine receptors involved in virus transmission and reproduction in the small brown planthopper Laodelphax striatellus.

Dopamine (DA) is the most abundant biogenic amine present in the insect central nervous system, and regulates multiple functions in physiology and behaviors through dopamine receptors (DARs). The small brown planthopper Laodelphax striatellus is an important agricultural pest and causes serious damage by transmitting diverse plant viruses, such as rice stripe virus (RSV). However, DARs have not yet been molecularly characterized in planthoppers, and their roles in virus infection and transmission remain largely unknown in insect vectors. In this study, we cloned four LsDARs (LsDOP1, LsDOP2, LsDOP3 and LsDopEcR) from L.striatellus. LsDARs share considerable sequence identity with their orthologous DARs, and cluster nicely with their corresponding receptor groups. The transcript levels of LsDARs varied in different developmental stages and adult tissues, with the highest expressions in the egg stage and in the brain. The expression levels of LsDARs were significantly higher in RSV-viruliferous L.striatellus. Knockdown of LsDOP2 and LsDOP3 significantly downregulated the expressions of viral genes of capsid protein (CP) and RNA3 segment (RNA3), while LsDOP1 knockdown upregulated their expressions. Silencing LsDopEcR upregulated and then downregulated CP and RNA3 expressions. Moreover, LsDOP2 and LsDOP3 knockdown significantly decreased the vertical transmission rates of RSV. Meanwhile, DA injection promoted RSV transmission and accumulation. We further demonstrated that silencing of LsDARs significantly altered the expressions of vitellogenin (LsVg) and Vg receptor (LsVgR). Furthermore, the reproduction performance of L.striatellus was reduced by LsDOP2 and LsDOP3 knockdown, but increased by LsDopEcR knockdown, and not affected by LsDOP1 silencing. These results provide critical information concerning the roles of DARs in virus transmission and reproduction in L.striatellus, and open the way for the development of innovative strategies for planthopper control.

Inhibition of Rice Stripe Virus Accumulation by Polyubiquitin-C in Laodelphax striatellus.

Many hosts utilize the ubiquitin system to defend against viral infection. As a key subunit of the ubiquitin system, the role of polyubiquitin in the viral infection of insects is unclear. Here, we identified the full-length cDNA of the polyubiquitin-C (UBC) gene in Laodelphax striatellus, the small brown planthopper (SBPH). LsUBC was expressed in various tissues and was highly expressed in salivary glands, midgut, and reproductive systems. Furthermore, the LsUBC expression profiles in the developmental stages showed that LsUBC was ubiquitously expressed in seven developmental stages and was highest expressed in female adults with SBPH. qRT-PCR analyses indicated that rice stripe virus (RSV) infection promoted the LsUBC expression. Knockdown of LsUBC mRNA via RNA interference increased RSV accumulation. These findings suggest that LsUBC inhibits RSV accumulation in L. striatellus.

Integrated ATAC-seq and RNA-seq data analysis identifies transcription factors related to rice stripe virus infection in Oryza sativa.

Animal studies have shown that virus infection causes changes in host chromatin accessibility, but little is known about changes in chromatin accessibility of plants infected by viruses and its potential impact. Here, rice infected by rice stripe virus (RSV) was used to investigate virus-induced changes in chromatin accessibility. Our analysis identified a total of 6462 open- and 3587 closed-differentially accessible chromatin regions (DACRs) in rice under RSV infection by ATAC-seq. Additionally, by integrating ATAC-seq and RNA-seq, 349 up-regulated genes in open-DACRs and 126 down-regulated genes in closed-DACRs were identified, of which 34 transcription factors (TFs) were further identified by search of upstream motifs. Transcription levels of eight of these TFs were validated by reverse transcription-PCR. Importantly, four of these TFs (OsWRKY77, OsWRKY28, OsZFP12 and OsERF91) interacted with RSV proteins and are therefore predicted to play important roles in RSV infection. This is the first application of ATAC-seq and RNA-seq techniques to analyse changes in rice chromatin accessibility caused by RSV infection. Integrating ATAC-seq and RNA-seq provides a new approach to select candidate TFs in response to virus infection.

Maintenance of persistent transmission of a plant arbovirus in its insect vector mediated by the Toll-Dorsal immune pathway.

Throughout evolution, arboviruses have developed various strategies to counteract the host's innate immune defenses to maintain persistent transmission. Recent studies have shown that, in addition to bacteria and fungi, the innate Toll-Dorsal immune system also plays an essential role in preventing viral infections in invertebrates. However, whether the classical Toll immune pathway is involved in maintaining the homeostatic process to ensure the persistent and propagative transmission of arboviruses in insect vectors remain unclear. In this study, we revealed that the transcription factor Dorsal is actively involved in the antiviral defense of an insect vector (Laodelphax striatellus) by regulating the target gene, zinc finger protein 708 (LsZN708), which mediates downstream immune-related effectors against infection with the plant virus (Rice stripe virus, RSV). In contrast, an antidefense strategy involving the use of the nonstructural-protein (NS4) to antagonize host antiviral defense through competitive binding to Dorsal from the MSK2 kinase was employed by RSV; this competitive binding inhibited Dorsal phosphorylation and reduced the antiviral response of the host insect. Our study revealed the molecular mechanism through which Toll-Dorsal-ZN708 mediates the maintenance of an arbovirus homeostasis in insect vectors. Specifically, ZN708 is a newly documented zinc finger protein targeted by Dorsal that mediates the downstream antiviral response. This study will contribute to our understanding of the successful transmission and spread of arboviruses in plant or invertebrate hosts.

N6-methyladenosine modification of the mRNA for a key gene in purine nucleotide metabolism regulates virus proliferation in an insect vector.

The titer of viruses that persist and propagate in their insect vector must be high enough for transmission yet not harm the insect, but the mechanism of this dynamic balance is unclear. Here, expression of inosine monophosphate dehydrogenase (LsIMPDH), a rate-limiting enzyme for guanosine triphosphate (GTP) synthesis, is shown to be downregulated by increased levels of N6-methyladenosine (m6A) on LsIMPDH mRNA in rice stripe virus (RSV)-infected small brown planthoppers (SBPHs; Laodelphax striatellus), the RSV vector, which decreases GTP content, thus limiting viral proliferation. Moreover, planthopper methyltransferase-like protein 3 (LsMETTL3) and m6A reader protein LsYTHDF3 are found to catalyze and recognize the m6A on LsIMPDH mRNA, respectively, and cooperate in destabilizing LsIMPDH transcripts. Co-silencing assays show that negative regulation of viral proliferation by both LsMETTL3 and LsYTHDF3 is partially dependent on LsIMPDH. This distinct mechanism limits virus replication in an insect vector, providing a potential gene target to block viral transmission.

A plant virus manipulates the long-winged morph of insect vectors.

Wing dimorphism of insect vectors is a determining factor for viral long-distance dispersal and large-area epidemics. Although plant viruses affect the wing plasticity of insect vectors, the potential underlying molecular mechanisms have seldom been investigated. Here, we found that a planthopper-vectored rice virus, rice stripe virus (RSV), specifically induces a long-winged morph in male insects. The analysis of field populations demonstrated that the long-winged ratios of male insects are closely associated with RSV infection regardless of viral titers. A planthopper-specific and testis-highly expressed gene, Encounter, was fortuitously found to play a key role in the RSV-induced long-winged morph. Encounter resembles malate dehydrogenase in the sequence, but it does not have corresponding enzymatic activity. Encounter is upregulated to affect male wing dimorphism at early larval stages. Encounter is closely connected with the insulin/insulin-like growth factor signaling pathway as a downstream factor of Akt, of which the transcriptional level is activated in response to RSV infection, resulting in the elevated expression of Encounter. In addition, an RSV-derived small interfering RNA directly targets Encounter to enhance its expression. Our study reveals an unreported mechanism underlying the direct regulation by a plant virus of wing dimorphism in its insect vectors, providing the potential way for interrupting viral dispersal.

Plasmodesmata-associated Flotillin positively regulates broad-spectrum virus cell-to-cell trafficking.

Viral diseases seriously threaten rice production. Plasmodesmata (PD)-associated proteins are deemed to play a key role in viral infection in host plants. However, few PD-associated proteins have been discovered in rice to afford viral infection. Here, inspired by the infection mechanism in insect vectors, we identified a member of the Flotillin family taking part in the cell-to-cell transport of rice stripe virus (RSV) in rice. Flotillin1 interacted with RSV nucleocapsid protein (NP) and was localized on PD. In flotillin1 knockout mutant rice, which displayed normal growth, RSV intercellular movement was retarded, leading to significantly decreased disease incidence. The PD pore sizes of the mutant rice were smaller than those of the wild type due to more callose deposits, which was closely related to the upregulation of two callose synthase genes. RSV infection stimulated flotillin1 expression and enlarged the PD aperture via RSV NP. In addition, flotillin1 knockout decreased disease incidences of southern rice black-streaked dwarf virus (SRBSDV) and rice dwarf virus (RDV) in rice. Overall, our study reveals a new PD-associated protein facilitating virus cell-to-cell trafficking and presents the potential of flotillin1 as a target to produce broad-spectrum antiviral rice resources in the future.

DICER-LIKE2 Plays a Crucial Role in Rice Stripe Virus Coat Protein-Mediated Virus Resistance in Arabidopsis.

Virus coat protein (CP)-mediated resistance is considered an effective antiviral defense strategy that has been used to develop robust resistance to viral infection. Rice stripe virus (RSV) causes significant losses in rice production in eastern Asia. We previously showed that the overexpression of RSV CP in Arabidopsis plants results in immunity to RSV infection, using the RSV-Arabidopsis pathosystem, and this CP-mediated viral resistance depends on the function of DCLs and is mostly involved in RNA silencing. However, the special role of DCLs in producing t-siRNAs in CP transgenic Arabidopsis plants is not fully understood. In this study, we show that RSV CP transgenic Arabidopsis plants with the dcl2 mutant background exhibited similar virus susceptibility to non-transgenic plants and were accompanied by the absence of transgene-derived small interfering RNAs (t-siRNAs) from the CP region. The dcl2 mutation eliminated the accumulation of CP-derived t-siRNAs, including those generated by other DCL enzymes. In contrast, we also developed RSV CP transgenic Arabidopsis plants with the dcl4 mutant background, and these CP transgenic plants showed immunity to virus infection and accumulated comparable amounts of CP-derived t-siRNAs to CP transgenic Arabidopsis plants with the wild-type background except for a significant increase in the abundance of 22 nt t-siRNA reads. Overall, our data indicate that DCL2 plays an essential, as opposed to redundant, role in CP-derived t-siRNA production and induces virus resistance in RSV CP transgenic Arabidopsis plants.

Alternative splicing impacts the rice stripe virus response transcriptome.

Alternative splicing (AS) is an important form of post transcriptional modification present in both animals and plants. However, little information was obtained about AS events in response to plant virus infection. In this study, we conducted a genome-wide transcriptome analysis on AS change in rice infected by a devastating virus, Rice stripe virus (RSV). KEGG analysis was performed on the differentially expressed (DE) genes and differentially alternative spliced (DAS) genes. The results showed that DE genes were significantly enriched in the pathway of interaction with plant pathogens. The DAS genes were mainly enriched in basal metabolism and RNA splicing pathways. The heat map clustering showed that DEGs clusters were mainly enriched in regulation of transcription and defense response while differential transcript usage (DTU) clusters were strongly enriched in mRNA splicing and calcium binding. Overall, our results provide a fundamental basis for gene-wide AS changes in rice after RSV infection.

Rice stripe virus nonstructural protein 3 suppresses plant defence responses mediated by the MEL-SHMT1 module.

Our previous study identified an evolutionarily conserved C4HC3-type E3 ligase, named microtubule-associated E3 ligase (MEL), that regulates broad-spectrum plant resistance against viral, fungal and bacterial pathogens in multiple plant species by mediating serine hydroxymethyltransferase (SHMT1) degradation via the 26S proteasome pathway. In the present study, we found that NS3 protein encoded by rice stripe virus could competitively bind to the MEL substrate recognition site, thereby inhibiting MEL interacting with and ubiquitinating SHMT1. This, in turn, leads to the accumulation of SHMT1 and the repression of downstream plant defence responses, including reactive oxygen species accumulation, mitogen-activated protein kinase pathway activation, and the up-regulation of disease-related gene expression. Our findings shed light on the ongoing arms race between pathogens and demonstrate how a plant virus can counteract the plant defence response.

Genetic variability of rice stripe virus after its pandemic in Jiangsu.

BACKGROUND: Rice stripe virus (RSV) caused a serious disease pandemic in rice in East China between 2001 and 2010. The continuous integrated managements reduced virus epidemic year by year until it was non-epidemic. As an RNA virus, its genetic variability after undergoing a long-term non-epidemic period was meaningful to study. While in 2019, the sudden occurrence of RSV in Jiangsu provided an opportunity for the study. METHODS AND RESULTS: The complete genome of JY2019, an RSV isolate from Jiangyan, was determined. A genotype profile of 22 isolates from China, Japan and Korea indicated that the isolates from Yunnan formed the subtype II, and other isolates clustered the subtype I. RNA 1-3 of JY2019 isolate well-clustered in the subtype I clade, and RNA 4 was also in subtype I, but it had a slight separation from other intra-group isolates. After phylogenetic analyses, it was considered NSvc4 gene contributed to the tendency, because it exhibited an obvious trend towards the subtype II (Yunnan) group. High sequence identity (100%) of NSvc4 between JY2019 and barnyardgrass isolate from different regions demonstrated genetic variation of NSvc4 was consistent in RSV natural populations in Jiangsu in the non-epidemic period. In the phylogenetic tree of all 74 NSvc4 genes, JY2019 belonged to a minor subtype Ib, suggesting the subtype Ib isolates might have existed in natural populations before the non-epidemic period, but not a dominant population. CONCLUSIONS: Our results suggested that NSvc4 gene was susceptible to selection pressure, and the subtype Ib might be more adaptable for the interaction between RSV and hosts in the non-epidemic ecological conditions.

Rice stripe virus p2 protein interacts with ATG5 and is targeted for degradation by autophagy.

Autophagy can be induced by viral infection and plays antiviral roles in plants, but the underlying mechanism is not well understood. In our previous reports, we have demonstrated that the plant ATG5 plays an essential role in activating autophagy in rice stripe virus (RSV)-infected plants. We also showed that eIF4A, a negative factor of autophagy, interacts with and inhibits ATG5. We here found that RSV p2 protein interacts with ATG5 and can be targeted by autophagy for degradation. Expression of p2 protein induced autophagy and p2 protein was shown to interfere with the interaction between ATG5 and eIF4A, while eIF4A had no effect on the interaction between ATG5 and p2. These results indicate an additional information on the induction of autophagy in RSV-infected plants.

Transcription start site mapping of geminiviruses using the in vitro cap-snatching of a tenuivirus.

Geminiviruses are a family of single-stranded DNA viruses that cause significant yield losses in crop production worldwide. Transcription start site (TSS) mapping is crucial in understanding the gene expression mechanisms of geminiviruses. However, this often requires costly and laborious experiments. Rice stripe virus (RSV) has a mechanism called cap-snatching, whereby it cleaves cellular mRNAs and uses the 5' cleavage product, a capped-RNA leader (CRL), as primers for transcription. Our previous work demonstrated that RSV snatches CRLs from geminiviral mRNAs in co-infected plants, providing a convenient and powerful approach to map the TSSs of geminiviruses. However, co-infections are not always feasible for all geminiviruses. In this study, we evaluated the use of in vitro cap-snatching of RSV for the same purpose, using tomato yellow leaf curl virus (TYLCV) as an example. We incubated RNA extracted from TYLCV-infected plants with purified RSV ribonucleoproteins in a reaction mixture that supports in vitro cap-snatching of RSV. The RSV mRNAs produced in the reaction were deep sequenced. The CRLs snatched by RSV allowed us to locate 28 TSSs in TYLCV. These results provide support for using RSV's in vitro cap-snatching to map geminiviral TSSs.

A Novel miRNA in Rice Associated with the Low Seed Setting Rate Symptom of Rice Stripe Virus.

MicroRNAs play key regulatory roles in plant development. The changed pattern of miRNA expression is involved in the production of viral symptoms. Here, we showed that a small RNA, Seq119, a putative novel microRNA, is associated with the low seed setting rate, a viral symptom of rice stripe virus (RSV)-infected rice. The expression of Seq 119 was downregulated in RSV-infected rice. The overexpression of Seq119 in transgenic rice plants did not cause any obvious phenotypic changes in plant development. When the expression of Seq119 was suppressed in rice plants either by expressing a mimic target or by CRISPR/Cas editing, seed setting rates were extremely low, similar to the effects of RSV infection. The putative targets of Seq119 were then predicted. The overexpression of the target of Seq119 in rice caused a low seed setting rate, similar to that in Seq119-suppressed or edited rice plants. Consistently, the expression of the target was upregulated in Seq119-suppressed and edited rice plants. These results suggest that downregulated Seq119 is associated with the low seed setting rate symptom of the RSV in rice.

Molecular Hydrogen Confers Resistance to Rice Stripe Virus.

Although molecular hydrogen (H2) has potential therapeutic effects in animals, whether or how this gas functions in plant disease resistance has not yet been elucidated. Here, after rice stripe virus (RSV) infection, H2 production was pronouncedly stimulated in Zhendao 88, a resistant rice variety, compared to that in a susceptible variety (Wuyujing No.3). External H2 supply remarkably reduced the disease symptoms and RSV coat protein (CP) levels, especially in Wuyujing No.3. The above responses were abolished by the pharmacological inhibition of H2 production. The transgenic Arabidopsis plants overexpressing a hydrogenase gene from Chlamydomonas reinhardtii also improved plant resistance. In the presence of H2, the transcription levels of salicylic acid (SA) synthetic genes were stimulated, and the activity of SA glucosyltransferases was suppressed, thus facilitating SA accumulation. Genetic evidence revealed that two SA synthetic mutants of Arabidopsis (sid2-2 and pad4) were more susceptible to RSV than the wild type (WT). The treatments with H2 failed to improve the resistance to RSV in two SA synthetic mutants. The above results indicated that H2 enhances rice resistance to RSV infection possibly through the SA-dependent pathway. This study might open a new window for applying the H2-based approach to improve plant disease resistance. IMPORTANCE Although molecular hydrogen has potential therapeutic effects in animals, whether or how this gas functions in plant disease resistance has not yet been elucidated. RSV was considered the most devastating plant virus in rice, since it could cause severe losses in field production. This disease was thus selected as a classical model to explore the interrelationship between molecular hydrogen and plant pathogen resistance. In this study, we discovered that both exogenous and endogenous H2 could enhance plant resistance against Rice stripe virus infection by regulating salicylic acid signaling. Compared with some frequently used agrochemicals, H2 is almost nontoxic. We hope that the findings presented here will serve as an opportunity for the scientific community to push hydrogen-based agriculture forward.

The small brown planthopper (Laodelphax striatellus) as a vector of the rice stripe virus.

The small brown planthopper, Laodelphax striatellus, is a destructive pest insect found in rice fields. L. striatellus not only directly feeds on the phloem sap of rice but also transmits various viruses, such as rice stripe virus (RSV) and rice black-streaked dwarf virus, resulting in serious loss of rice production. RSV is a rice-infecting virus that is found mainly in Korea, China, and Japan. To develop novel strategies to control L. striatellus and L. striatellus-transmitted viruses, various studies have been conducted, based on vector biology, interactions between vectors and pathogens, and omics, including transcriptomics, proteomics, and metabolomics. In this review, we discuss the roles of saliva proteins during phloem sap-sucking and virus transmission, the diversity and role of the microbial community in L. striatellus, the profile and molecular mechanisms of insecticide resistance, classification of L. striatellus-transmitted RSV, its host range and symptoms, its genome composition and roles of virus-derived proteins, its distribution, interactions with L. striatellus, and resistance and control, to suggest future directions for integrated pest management to control L. striatellus and L. striatellus-transmitted viruses.

c-Jun regulates flotillin 2 transcription to benefit viral accumulation in insect vectors.

The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in viral infection in host cells. In addition to triggering immune reactions against pathogens, the JNK signaling pathway has also been found to benefit viral infection. Our previous work showed that JNK activation facilitated rice stripe virus (RSV) accumulation in the insect vector small brown planthopper, but the underlying mechanisms remain elusive. Here, we revealed a link between JNK activation and the transcriptional upregulation of the plasma membrane protein flotillin 2, which mediates RSV cell entry. c-Jun, a downstream substrate of JNKs, was identified as a transcription factor that targets the promoter of flotillin 2 at three binding sites. Phosphorylated c-Jun, especially at the serine 63 site, promoted the transcriptional activity of c-Jun on flotillin 2. JNK activation or inhibition affected c-Jun phosphorylation status and flotillin 2 expression. In the midguts of planthoppers, RSV infection significantly increased flotillin 2 expression and the phosphorylation level of JNKs and c-Jun. Manipulation of JNK status impacted viral acquisition in midgut cells. These findings reveal a new regulatory mechanism of the JNK signaling pathway and shed light on the virus-supportive effect of this pathway.

Voltage-dependent anion channel 2 (VDAC2) facilitates the accumulation of rice stripe virus in the vector Laodelphax striatellus.

Rice stripe virus (RSV) causes enormous losses in rice production and is transmitted by the small brown planthopper, Laodelphax striatellus, in a persistent-propagative manner. RSV accumulation within the gut lumen of the vector is indispensable for the successful transmission to rice and insects. In this study, we obtained a 1464 bp full-length cDNA of a voltage-dependent anion channel 2 from L. striatellus (LsVDAC2), which encodes a 283 amino acid protein. RSV infection increased the expression of LsVDAC2 in the midguts and ovaries of L. striatellus by 260% and 228%, respectively. Silencing of LsVDAC2 resulted in a 88% reduction of RSV loads at 24 h after RNAi, indicating that LsVDAC2 facilitates RSV accumulation in the vector. Yeast two-hybrid and GST pulldown assays demonstrated that LsVDAC2 interacted with RSV RNA-dependent RNA polymerase, RdRp. Furthermore, experiments in vivo and in vitro showed that LsVDAC2 induced the apoptotic response in RSV-infected insects and tissues. Silencing of LsVDAC2 via RNAi significantly reduced the expression of genes for apoptosis-related caspases 1a and 1c by 62% and 78%, respectively, in RSV-infected vectors. Whether LsVDAC2-induced RSV accumulation is related to RSV RdRp and LsVDAC2-induced cell apoptosis deserves further investigation.

Ammonium protects rice against rice stripe virus by activating HDA703/OsBZR1-mediated BR signaling.

Ammonium (NH4+) is a major inorganic nitrogen source for plants and also as a signal regulates plant growth and defense. Brassinosteroids (BRs) are a class of steroid hormones that control plant developmental and physiological processes through its signaling pathway. Rice is a kind of NH4+-preferring plant which responds to virus infection involving in the regulation of BR biosynthesis and signaling. However, the BR-mediated regulatory mechanisms in rice-virus interactions are not fully understood. In addition, it remains unknown whether there is a direct link between NH4+ and BRs in regulating rice response to virus. HDA703, a histone deacetylase and OsBZR1, a transcription factor, are two positive regulator of BR signaling and interact with each other. In this study, we show that rice plants grown with NH4+ as the sole N source have enhanced resistance to rice stripe virus (RSV), one of the most devastating viruses of rice, than those grown with NO3- as the sole N source. We also show that in contrast to NO3-, NH4+ does not affect BR biosynthesis but promotes BR signaling by upregulating the expression of HDA703 and promoting the accumulation of OsBZR1 in rice shoots. We further show that BR biosynthesis and signaling is required for rice defense against RSV and BR-mediated resistance to RSV attributes to activating HDA703/OsBZR1 module, then decreasing the expression of Ghd7, a direct target of HDA703/OsBZR1. Consistently, increase of the expression of HDA703 or decrease of the expression of Ghd7 enhances rice resistance to RSV. Together, our study reveals that activation of HDA703/OsBZR1-Ghd7 signaling cascade is an undescribed mechanism conferring BR-mediated RSV resistance and NH4+ protects rice against RSV by activating HDA703/OsBZR1-Ghd7-mediated BR signaling in rice.

Antiviral Activity of Ailanthone from Ailanthus altissima on the Rice Stripe Virus.

Rice stripe disease caused by the rice stripe virus (RSV), which infects many Poaceae species in nature, is one of the most devastating plant viruses in rice that causes enormous losses in production. Ailanthone is one of the typical C20 quassinoids synthesized by the secondary metabolism of Ailanthus altissima, which has been proven to be a biologically active natural product with promising prospects and great potential for use as a lead structure for pesticide development. Based on the achievement of the systemic infection and replication of RSV in Nicotiana benthamiana plants and rice protoplasts, the antiviral properties of Ailanthone were investigated by determining its effects on viral-coding RNA gene expression using reverse transcription polymerase chain reaction, and Western blot analysis. Ailanthone exhibited a dose-dependent inhibitory effect on RSV NSvc3 expression in the assay in both virus-infected tobacco plants and rice protoplasts. Further efforts revealed a potent inhibitory effect of Ailanthone on the expression of seven RSV protein-encoding genes, among which NS3, NSvc3, NS4, and NSvc4 are the most affected genes. These facts promoted an extended and greater depth of understanding of the antiviral nature of Ailanthone against plant viruses, in addition to the limited knowledge of its anti-tobacco mosaic virus properties. Moreover, the leaf disc method introduced and developed in the study for the detection of the antiviral activity of Ailanthone facilitates an available and convenient screening method for anti-RSV natural products or synthetic chemicals.