Discovery of aphid-transmitted Rice tiller inhibition virus from native plants through metagenomic sequencing.

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation.

The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication.

An engineered mutant of a host phospholipid synthesis gene inhibits viral replication without compromising host fitness.

Viral infections universally rely on numerous hijacked host factors to be successful. It is therefore possible to control viral infections by manipulating host factors that are critical for viral replication. Given that host genes may play essential roles in certain cellular processes, any successful manipulations for virus control should cause no or mild effects on host fitness. We previously showed that a group of positive-strand RNA viruses enrich phosphatidylcholine (PC) at the sites of viral replication. Specifically, brome mosaic virus (BMV) replication protein 1a interacts with and recruits a PC synthesis enzyme, phosphatidylethanolamine methyltransferase, Cho2p, to the viral replication sites that are assembled on the perinuclear endoplasmic reticulum (ER) membrane. Deletion of the CHO2 gene inhibited BMV replication by 5-fold; however, it slowed down host cell growth as well. Here, we show that an engineered Cho2p mutant supports general PC synthesis and normal cell growth but blocks BMV replication. This mutant interacts and colocalizes with BMV 1a but prevents BMV 1a from localizing to the perinuclear ER membrane. The mislocalized BMV 1a fails to induce the formation of viral replication complexes. Our study demonstrates an effective antiviral strategy in which a host lipid synthesis gene is engineered to control viral replication without comprising host growth.

Cleavage of the Babuvirus Movement Protein B4 into Functional Peptides Capable of Host Factor Conjugation is Required for Virulence.

Banana bunchy top virus (BBTV) poses a serious danger to banana crops worldwide. BBTV-encoded protein B4 is a determinant of pathogenicity. However, the relevant molecular mechanisms underlying its effects remain unknown. In this study, we found that a functional peptide could be liberated from protein B4, likely via proteolytic processing. Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects, including dwarfism and sterility, in plants. The released protein fragment targets host proteins, such as the large subunit of RuBisCO (RbcL) and elongation factor 2 (EF2), involved in protein synthesis. Therefore, the peptide released from B4 (also a precursor) may act as a non-canonical modifier to influence host-pathogen interactions involving BBTV and plants.

Structure Analysis of Effective Chemical Compounds against Dengue Viruses Isolated from Isatis tinctoria.

The history of Chinese herb research can be traced back to thousands of years ago, and the abundant knowledge accumulated for these herbs makes them good candidates for developing new natural drugs. Isatis tinctoria is probably the most well-studied Chinese herb, which has been identified to be effective against dengue fever. However, the underlying biological mechanisms are still unclear. In this study, we adopt combined methods of bioactive trace technology and phytochemical extraction and separation, to guide the isolation and purification of the effective chemical constituents on the water-soluble components of aerial parts of Isatis tinctoria. In addition, we apply polarimetry and 1D or 2D nuclear magnetic resonance (NMR) spectroscopy to identify their structures, which lay a foundation for further study on the biological mechanisms underlying medicinal effects of Isatis tinctoria using in vitro and in vivo experiments. Specifically, we identify and infer the structures of 27 types of chemical compounds named GB-1, GB-2, …, GB-27, respectively, among which GB-7 is a novel compound. Further study of these compounds is critical to reveal the secrets behind the medicinal effects of Isatis tinctoria.

Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis.

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection.

Rice stripe virus NS3 protein regulates primary miRNA processing through association with the miRNA biogenesis factor OsDRB1 and facilitates virus infection in rice.

MicroRNAs (miRNAs) are small regulatory RNAs processed from primary miRNA transcripts, and plant miRNAs play important roles in plant growth, development, and response to infection by microbes. Microbial infections broadly alter miRNA biogenesis, but the underlying mechanisms remain poorly understood. In this study, we report that the Rice stripe virus (RSV)-encoded nonstructural protein 3 (NS3) interacts with OsDRB1, an indispensable component of the rice (Oryza sativa) miRNA-processing complex. Moreover, the NS3-OsDRB1 interaction occurs at the sites required for OsDRB1 self-interaction, which is essential for miRNA biogenesis. Further analysis revealed that NS3 acts as a scaffold between OsDRB1 and pri-miRNAs to regulate their association and aids in vivo processing of pri-miRNAs. Genetic evidence in Arabidopsis showed that NS3 can partially substitute for the function of double-stranded RNA binding domain (dsRBD) of AtDRB1/AtHYL1 during miRNA biogenesis. As a result, NS3 induces the accumulation of several miRNAs, most of which target pivotal genes associated with development or pathogen resistance. In contrast, a mutant version of NS3 (mNS3), which still associated with OsDRB1 but has defects in pri-miRNA binding, reduces accumulation of these miRNAs. Transgenic rice lines expressing NS3 exhibited significantly higher susceptibility to RSV infection compared with non-transgenic wild-type plants, whereas the transgenic lines expressing mNS3 showed a less-sensitive response. Our findings revealed a previously unknown mechanism in which a viral protein hijacks OsDRB1, a key component of the processing complex, for miRNA biogenesis and enhances viral infection and pathogenesis in rice.

Genomic Characterization of Travel-Associated Dengue Viruses Isolated from the Entry-Exit Ports in Fujian Province, China, 2013-2015.

Over the past decade, indigenous dengue outbreaks have occurred occasionally in Fujian province in southeastern China because of sporadic imported dengue viruses (DENV). In this study, 3 DENV-2 and 2 DENV-4 strains were isolated from suspected febrile travelers at 2 ports of entry in Fujian between 2013-2015. Complete viral genome sequences of these new isolates were obtained with Sanger chemistry. Genomic sequence analyses revealed that these strains belonged to genotypes of 2-Cosmopolitan and 4-II. Consistent with the patients' travel information, phylogenetic analyses of the complete coding regions also indicated that most of the new isolates were genetically similar to the circulating strains in Southeast Asia rather than previous Chinese strains that were available. Therefore, phylogenetic analyses of the imported DENV demonstrated that multiple introductions of DENV emerged continuously in Fujian, and highlighted the importance of dengue surveillance at entry-exit ports in the subtropical regions of southern China.

Translation Initiation Factor eIF4E and eIFiso4E Are Both Required for Peanut stripe virus Infection in Peanut (Arachis hypogaea L.).

Peanut stripe virus (PStV) belongs to the genus Potyvirus and is the most important viral pathogen of cultivated peanut (Arachis hypogaea L.). The eukaryotic translation initiation factor, eIF4E, and its isoform, eIF(iso)4E, play key roles during virus infection in plants, particularly Potyvirus. In the present study, we cloned the eIF4E and eIF(iso)4E homologs in peanut and named these as PeaeIF4E and PeaeIF(iso)4E, respectively. Quantitative real-time PCR (qRT-PCR) analysis showed that these two genes were expressed during all growth periods and in all peanut organs, but were especially abundant in young leaves and roots. These also had similar expression levels. Yeast two-hybrid analysis showed that PStV multifunctional helper component proteinase (HC-Pro) and viral protein genome-linked (VPg) both interacted with PeaeIF4E and PeaeIF(iso)4E. Bimolecular fluorescence complementation assay showed that there was an interaction between HC-Pro and PeaeIF4E/PeaeIF(iso)4E in the cytoplasm and between VPg and PeaeIF4E/PeaeIF(iso)4E in the nucleus. Silencing either PeaeIF4E or PeaeIF(iso)4E using a virus-induced gene silencing system did not significantly affect PStV accumulation. However, silencing both PeaeIF4E and PeaeIF(iso)4E genes significantly weakened PStV accumulation. The findings of the present study suggest that PeaeIF4E and PeaeIF(iso)4E play important roles in the PStV infection cycle and may potentially contribute to PStV resistance.

Adaptive evolution and demographic history contribute to the divergent population genetic structure of Potato virus Y between China and Japan.

Potato virus Y (PVY) is an important plant pathogen causing considerable economic loss to potato production. Knowledge of the population genetic structure and evolutionary biology of the pathogen, particularly at a transnational scale, is limited but vital in developing sustainable management schemes. In this study, the population genetic structure and molecular evolution of PVY were studied using 127 first protein (P1) and 137 coat protein (CP) sequences generated from isolates collected from potato in China and Japan. High genetic differentiation was found between the populations from the two countries, with higher nucleotide diversity in Japan than China in both genes and a KST value of .216 in the concatenated sequences of the two genes. Sequences from the two countries clustered together according to their geographic origin. Further analyses showed that spatial genetic structure in the PVY populations was likely caused by demographic dynamics of the pathogen and natural selection generated by habitat heterogeneity. Purifying selection was detected at the majority of polymorphic sites although some clade-specific codons were under positive selection. In past decades, PVY has undergone a population expansion in China, whereas in Japan, the population size of the pathogen has remained relatively constant.

A DNA Barcoding Based Study to Identify Main Mosquito Species in Taiwan and its Difference from Those in Mainland China.

AIM AND OBJECTIVE: Mosquitoes can transmit many types of viruses such as West Nile virus and Zika virus and are responsible for a number of virus-causing diseases including malaria, dengue fever, yellow fever, lymphatic filariasis, and Japanese B encephalitis. On January 19, 2016, the first case of Zika virus infection was identified in Taiwan, which presents the need for studying the mosquito species in the Taiwan Strait and evaluating the risk of the outbreak of this infection. MATERIALS AND METHOD: In this study, we have collected 144 mosquito specimens from 42 species belonging to nine genera from both sides of the Taiwan Strait during 2013 and 2014. We then applied the COI DNA Barcoding technique to classify the specimens and performed a phylogenetic analysis to infer the evolutionary history of these mosquitoes. Based on the analyses, we found that though the mosquitoes from different sides of the Taiwan Strait share a lot of commonality, they have a few regional specificities. RESULTS: Our results also suggested a very small divergences (1%~9%) between specimens from the same mosquito species and relatively large divergences (8%~25%) between specimens from different mosquito species. Within the same species, the divergence of specimens from the same region is significantly smaller than that between two regions. A few highly divergent species between Fujian and Taiwan (e.g., An.maculatus and Ae.elsiae) might be formed due to the so-called "cryptic evolutionary events", in which the species has differentiation into cryptic species due to geographical differences without changing morphological characteristics. CONCLUSION: In conclusion, the phylogenetic analyses showed a very similar taxonomy to the historical one based on morphological characteristics, validating again the application of COI DNA Barcoding technique in classifying mosquito species. However, there are also some inconsistencies between COI DNA Barcoding and historical taxonomy, which points out the differences between mosquito DNA and morphological characteristics and suggests the possibility to improve mosquito taxonomy based on DNA techniques.

Identification and characterization of Bamboo mosaic virus isolates from a naturally occurring coinfection in Bambusa xiashanensis.

Bamboo mosaic virus (BaMV) is a well-characterized virus and a model of virus-host interaction in plants. Here, we identified naturally occurring BaMV isolates from Fujian Province, China and furthermore describe a naturally occurring BaMV coinfection in bamboo (Bambusa xiashanensis) plants. Two different types of BaMV were identified, represented by isolates BaMV-XSNZHA7 (X7) and BaMV-XSNZHA10 (X10). The phylogenetic relationships between X7- and X10-like isolates and published BaMV isolates were determined based on genomic RNA and amino acid sequences. Three clusters were identified, indicating that BaMV is highly diverse. The in planta viral replication kinetics were determined for X7 and X10 in single infections and in an X7/X10 coinfection. The peak viral load during coinfection was significantly greater than that during single infection with either virus and contained a slightly higher proportion of X10 virus than X7, suggesting that X10-like viruses may have a fitness advantage when compared to X7-like viruses.

ROS accumulation and antiviral defence control by microRNA528 in rice.

MicroRNAs (miRNAs) are key regulators of plant-pathogen interactions. Modulating miRNA function has emerged as a new strategy to produce virus resistance traits1-5. However, the miRNAs involved in antiviral defence and the underlying mechanisms remain largely elusive. We previously demonstrated that sequestration by Argonaute (AGO) proteins plays an important role in regulating miRNA function in antiviral defence pathways6. Here we reveal that cleavage-defective AGO18 complexes sequester microRNA528 (miR528) upon viral infection. We show that miR528 negatively regulates viral resistance in rice by cleaving L-ascorbate oxidase (AO) messenger RNA, thereby reducing AO-mediated accumulation of reactive oxygen species. Upon viral infection, miR528 becomes preferentially associated with AGO18, leading to elevated AO activity, higher basal reactive oxygen species accumulation and enhanced antiviral defence. Our findings reveal a mechanism in which antiviral defence is boosted through suppression of an miRNA that negatively regulates viral resistance. This mechanism could be manipulated to engineer virus-resistant crop plants.

Characterisation of siRNAs derived from new isolates of bamboo mosaic virus and their associated satellites in infected ma bamboo (Dendrocalamus latiflorus).

We characterised the virus-derived small interfering RNAs (vsiRNA) of bamboo mosaic virus (Ba-vsiRNAs) and its associated satellite RNA (satRNA)-derived siRNAs (satsiRNAs) in a bamboo plant (Dendrocalamus latiflorus) by deep sequencing. Ba-vsiRNAs and satsiRNAs of 21-22 nt in length, with both (+) and (-) polarity, predominated. The 5'-terminal base of Ba-vsiRNA was biased towards A, whereas a bias towards C/U was observed in sense satsiRNAs, and towards A in antisense satsiRNAs. A large set of bamboo genes were identified as potential targets of Ba-vsiRNAs and satsiRNAs, revealing RNA silencing-based virus-host interactions in plants. Moreover, we isolated and characterised new isolates of bamboo mosaic virus (BaMV; 6,350 nt) and BaMV-associated satRNA (satBaMV; 834 nt), designated BaMV-MAZSL1 and satBaMV-MAZSL1, respectively.

Molecular characterization and detection of a recombinant isolate of bamboo mosaic virus from China.

The complete genome sequences of three isolates of bamboo mosaic virus (BaMV) from mainland China were determined and compared to those of BaMV isolates from Taiwan. Sequence analysis showed that isolate BaMV-JXYBZ1 from Fuzhou shares 98 % nucleotide sequence identity with BaMV-YTHSL14 from nucleotides 2586 to 6306, and more than 94 % nucleotide sequence identity with BaMV-MUZHUBZ2 in other regions. Recombination and phylogenetic analyses indicate that BaMV-JXYBZ1 is a recombinant with one recombination breakpoint. To our knowledge, this is the first report of a BaMV recombinant worldwide.

Viruliferous rate of small brown planthopper is a good indicator of rice stripe disease epidemics.

Rice stripe virus (RSV), its vector insect (small brown planthopper, SBPH) and climatic conditions in Jiangsu, China were monitored between 2002 and 2012 to determine key biotic and abiotic factors driving epidemics of the disease. Average disease severity, disease incidence and viruliferous rate of SBPH peaked in 2004 and then gradually decreased. Disease severity of RSV was positively correlated with viruliferous rate of the vector but not with the population density of the insect, suggesting that the proportion of vectors infected by the virus rather than the absolute number of vectors plays an important role in RSV epidemics and could be used for disease forecasting. The finding of a positive correlation of disease severity and viruliferous rate among years suggests that local infection is likely the main source of primary inoculum of RSV. Of the two main climatic factors, temperature plays a more important role than rainfall in RSV epidemics.

The Subcellular Localization and Functional Analysis of Fibrillarin2, a Nucleolar Protein in Nicotiana benthamiana.

Nucleolar proteins play important roles in plant cytology, growth, and development. Fibrillarin2 is a nucleolar protein of Nicotiana benthamiana (N. benthamiana). Its cDNA was amplified by RT-PCR and inserted into expression vector pEarley101 labeled with yellow fluorescent protein (YFP). The fusion protein was localized in the nucleolus and Cajal body of leaf epidermal cells of N. benthamiana. The N. benthamiana fibrillarin2 (NbFib2) protein has three functional domains (i.e., glycine and arginine rich domain, RNA-binding domain, and α-helical domain) and a nuclear localization signal (NLS) in C-terminal. The protein 3D structure analysis predicted that NbFib2 is an α/ß protein. In addition, the virus induced gene silencing (VIGS) approach was used to determine the function of NbFib2. Our results showed that symptoms including growth retardation, organ deformation, chlorosis, and necrosis appeared in NbFib2-silenced N. benthamiana.

The antagonistic effect of Banana bunchy top virus multifunctional protein B4 against Fusarium oxysporum.

The viral-induced banana bunchy top disease and the fungal-induced banana blight are two major causes of concern for industrial scale production of bananas. Banana blight is particularly troublesome, affecting ∼80% of crops worldwide. Strict guidelines and protocols are in place in order to ameliorate the effects of this devastating disease, yet little success has been achieved. From the data presented here, we have found that Banana bunchy top virus (BBTV)-infected bananas are more resistant to Fusarium oxysporum f. sp. cubense (Foc). BBTV appears to be antagonistic towards Foc, thus improving the survivability of plants against blight. The BBTV suppressor of RNA silencing, namely protein B4, displays fungicidal properties in vitro. Furthermore, transgenic tomatoes expressing green fluorescent protein (GFP)-tagged protein B4 demonstrate enhanced resistance to F. oxysporum f. sp. lycopersici (Fol). Differential gene expression analysis indicates that increased numbers of photogenesis-related gene transcripts are present in dark-green leaves of B4-GFP-modified tomato plants relative to those found in WT plants. Conversely, the transcript abundance of immunity-related genes is substantially lower in transgenic tomatoes compared with WT plants, suggesting that plant defences may be influenced by protein B4. This viral-fungal interaction provides new insights into microbial community dynamics within a single host and has potential commercial value for the breeding of transgenic resistance to Fusarium-related blight/wilt.

Analysis of genetic diversity and population structure of peanut cultivars and breeding lines from China, India and the US using simple sequence repeat markers.

Cultivated peanut is grown worldwide as rich-source of oil and protein. A broad genetic base is needed for cultivar improvement. The objectives of this study were to develop highly informative simple sequence repeat (SSR) markers and to assess the genetic diversity and population structure of peanut cultivars and breeding lines from different breeding programs in China, India and the US. A total of 111 SSR markers were selected for this study, resulting in a total of 472 alleles. The mean values of gene diversity and polymorphic information content (PIC) were 0.480 and 0.429, respectively. Country-wise analysis revealed that alleles per locus in three countries were similar. The mean gene diversity in the US, China and India was 0.363, 0.489 and 0.47 with an average PIC of 0.323, 0.43 and 0.412, respectively. Genetic analysis using the STRUCTURE divided these peanut lines into two populations (P1, P2), which was consistent with the dendrogram based on genetic distance (G1, G2) and the clustering of principal component analysis. The groupings were related to peanut market types and the geographic origin with a few admixtures. The results could be used by breeding programs to assess the genetic diversity of breeding materials to broaden the genetic base and for molecular genetics studies.

Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector.

BACKGROUND: Rice dwarf virus (RDV), a plant reovirus, is mainly transmitted by the green rice leafhopper, Nephotettix cincticeps, in a persistent-propagative manner. Plant reoviruses are thought to replicate and assemble within cytoplasmic structures called viroplasms. Nonstructural protein Pns4 of RDV, a phosphoprotein, is localized around the viroplasm matrix and forms minitubules in insect vector cells. However, the functional role of Pns4 minitubules during viral infection in insect vector is still unknown yet. METHODS: RNA interference (RNAi) system targeting Pns4 gene of RDV was conducted. Double-stranded RNA (dsRNA) specific for Pns4 gene was synthesized in vitro, and introduced into cultured leafhopper cells by transfection or into insect body by microinjection. The effects of the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene on viral replication and spread in cultured cells and insect vector were analyzed using immunofluorescence, western blotting or RT-PCR assays. RESULTS: In cultured leafhopper cells, the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene strongly inhibited the formation of minitubules, preventing the accumulation of viroplasms and efficient viral infection in insect vector cells. RNAi induced by microinjection of dsRNA from Pns4 gene significantly reduced the viruliferous rate of N. cincticeps. Furthermore, it also strongly inhibited the formation of minitubules and viroplasms, preventing efficient viral spread from the initially infected site in the filter chamber of intact insect vector. CONCLUSIONS: Pns4 of RDV is essential for viral infection and replication in insect vector. It may directly participate in the functional role of viroplasm for viral replication and assembly of progeny virions during viral infection in leafhopper vector.