Rice Black-streaked Dwarf Virus Research Articles

Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

Dimeric artificial microRNAs mediate high resistance to RSV and RBSDV in transgenic rice plants

Infection of rice with Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) causes a significant loss of grain production. Due to the lack of natural resistance resources against these viruses, it is imperative to discover a biotechnological approach that will provide effective and safe immunity to RSV and RBSDV. In this study, we constructed three dimeric artificial microRNA (amiRNA) precursor expression vectors (pamiR-M, pamiR-3 and pamiR-U) that simultaneously target the CP genes of RSV and RBSDV based on the structure of the rice (Oryza sativa L.) osa-MIR528 precursor. The transgenic plants were obtained by Agrobacterium tumefaciens-mediated transformation and were shown to express amiRNAs successfully. Viral challenge assays revealed that these transgenic plants demonstrated different resistance (26.66–54.17 %) against RSV and RBSDV infection simultaneously. The amiRNA-targeting 3′-UTR region of CP gene (pamiR-U) induced higher virus resistance: 54.17 % against RSV and 45.83 % against RBSDV. A northern blot assay indicated that there was a good correlation between the resistance level and amiRNAs accumulation. The RNA silencing induced by the original amiRNAs could be bilaterally extended by the siRNA pathway. The amiRNAs, together with the secondary siRNAs, mediated the degradation of viral RNAs. A genetic stability assay showed that transgenes and amiRNA-mediated virus resistance could be stably inherited in the transgenic plants.

Screening of rice (Oryza sativa) cultivars for resistance to rice black streaked dwarf virus using quantitative PCR and visual disease assessment

Rice black streaked dwarf virus (RBSDV) causing rice black streaked dwarf disease is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent propagative manner. The disease is considered among the most destructive in rice production in east and southeast Asia. For sustainable control of the disease, planting resistant cultivars is the most economical and efficient method. The virus content in different rice cultivars was quantified using a TaqMan RT‐qPCR assay under greenhouse conditions and the disease was visually assessed in these cultivars in both greenhouse and field conditions. Results revealed significant positive moderate correlation (r = 0.3787; P = 0.0009) between the virus content and visual disease assessment in the greenhouse under forced inoculation. Among 66 rice cultivars, there was no significant difference in RBSDV genome equivalent copies (GEC) in seven cultivars, namely Lian‐dao 9805 (200.2 ± 12), Liangyou 3399 (206.6 ± 28), Ningjing 4 (206.6 ± 28), DaLiang 207 (302.0 ± 61), X 008 (354.0 ± 30), Shengdao 301 (658.4 ± 69) and Liangyou 1129 (679.5 ± 98). These cultivars were also visually assessed as resistant under greenhouse and field conditions. These cultivars could be used in disease management to reduce the likelihood of epidemics.

Reverse transcription loop-mediated isothermal amplification to rapidly detect Rice ragged stunt virus.

Rice ragged stunt virus (RRSV) is a very important virus that infects rice and causes serious yield losses in Asian countries and other major rice planting areas. Thus, it is urgent to establish an efficient and practical approach for identification and diagnosis in the field. Our results indicated that reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions are more efficient and sensitive than RT-PCR for RRSV detection. The optimal LAMP conditions were as follows: 0.4–1.2 μM internal primers, 0.2–0.25 μM external primers, 0.8 μM loop primers, and incubation at 62 °C or 63 °C for 30 min. Furthermore, the RT-LAMP primers specifically targeted RRSV virus and resulted in typical waterfall-like bands by gel electrophoresis and sigmoidal amplification curves. The primers could not be used to amplify other common plant viruses including Papaya ringspot virus (PRSV), Rice yellow stunt virus (RYSV), Sorghum mosaic virus (SrMV), Cactus virus X (CVX), Melon yellow spot virus (MYSV) and Southern rice black-streaked dwarf virus (SRBSDV). Ten-fold serial dilutions of RRSV cDNA indicated that RT-LAMP is much faster and at least ten times more sensitive than RT-PCR in detecting the virus. The waterfall-like product bands could be observed within one hour. In the field study, about 77% samples were identified as RRSV. RT-LAMP has many benefits over RT-PCR such as low cost and high accuracy, sensitivity, and specificity. This technology meets the requirements for rapid diagnosis of plant virus diseases in the field to best guide management practices for growers.

Complete Genomic Sequence of Maize Rough Dwarf Virus, a Fijivirus Transmitted by the Small Brown Planthopper.

The nucleotide sequences of the 10 genomic segments of an Italian isolate of maize rough dwarf virus (MRDV) were determined. This first complete genomic sequence of MRDV will help understand the phylogenetic relationships among group 2 fijiviruses and especially the closely related rice black-streaked dwarf virus, which is also found to naturally infect maize.

Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective.

Maize (Zea mays L.) is a host to numerous pathogenic species that impose serious diseases to its ear and foliage, negatively affecting the yield and the quality of the maize crop. A considerable amount of research has been carried out to elucidate mechanisms of maize-pathogen interactions with a major goal to identify defense-associated proteins. In this review, we summarize interactions of maize with its agriculturally important pathogens that were assessed at the proteome level. Employing differential analyses, such as the comparison of pathogen-resistant and susceptible maize varieties, as well as changes in maize proteomes after pathogen challenge, numerous proteins were identified as possible candidates in maize resistance. We describe findings of various research groups that used mainly mass spectrometry-based, high through-put proteomic tools to investigate maize interactions with fungal pathogens Aspergillus flavus, Fusarium spp., and Curvularia lunata, and viral agents Rice Black-streaked Dwarf Virus and Sugarcane Mosaic Virus.

Reference Gene Selection and Evaluation for Gene Expression Studies Using qRT-PCR in the White-Backed Planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

The white-backed planthopper, Sogatella furcifera (Hemiptera, Delphacidae), is one of the most devastating rice pests. For a better control strategy, various genetic studies have been conducted using reverse-transcription quantitative real-time polymerase chain reaction (qRT-PCR). The appropriate application of qRT-PCR requires reliable endogenous controls; however, studies on this aspect of the white-backed planthopper are lacking. In the present study, nine commonly used reference genes, elongation factor 1-α (EF1-α), polyubiquitin (UB), ribosomal protein S18 (RPS18), actin 1 (ACT), α-1 tubulin (TUB), glyceraldehyde-3-phosphate (GAPDH), ribosomal protein L9 (RPL9), ribosomal protein L10 (RPL10), and 18S ribosomal RNA (18S), were evaluated by qRT-PCR for their expression stability under four different experimental conditions (different developmental stages, acquisition of Southern rice black-streaked dwarf virus (SRBSDV), different tissues, and different temperature stress). These results were analyzed using four software programs (geNorm, NormFinder, BestKeeper, and the delta Ct method) and a Web-based comprehensive tool RefFinder to compare and rank candidate reference genes. According to the results of RefFinder analysis, which integrates the abovementioned four software programs, TUB was ranked as the most suitable reference gene at different developmental stages and under different temperature stress, and GAPDH and RPL9 showed the highest stability for acquisition of SRBSDV and different tissues, respectively. These results will provide a solid foundation for future gene expression study on the white-backed planthopper, and also will give aids in establishing a standardized qRT-PCR procedure for other related insects.

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector.

ARGONAUTE SUBFAMILY GENES IN THE SMALL BROWN PLANTHOPPER, Laodelphax striatellus (HEMIPTERA: DELPHACIDAE).

Argonaute (AGO) proteins are essential catalytic components of the RNA-induced silencing complex and play central roles in RNA interference. Using a combination of bioinformatics and rapid amplification of cDNA ends (RACE) methods, putative AGO subfamily members, ls-AGO1 and ls-AGO2, were cloned and characterized from the small brown planthopper, Laodelphax striatellus. The open reading frame (ORF) of ls-AGO1 is 2,820 bp long, encoding a putative protein of 939 amino acid residues, and ls-AGO2 contains an ORF of 2,490 bp, encoding 829 amino acid residues. The expected conserved PAZ and PIWI domains, and the conserved Asp-Asp-His (DDH) catalytic triad motif in the PIWI domain were observed in both ls-AGO1 and ls-AGO2. Reverse transcription-qPCR (RT-qPCR) results showed that both ls-AGO1 and ls-AGO2 were expressed in all developmental stages of L. striatellus with highest mRNA abundance in eggs. Expression of ls-AGO1 and ls-AGO2 was significantly decreased in adult insects in response to acquisition of rice black-streaked dwarf virus by second instar nymphs. mRNA expression of ls-AGO1 was significantly downregulated in response to low and high temperatures, but expression of ls-AGO2 was only affected by low temperature. ls-AGO1 and ls-AGO2 were initially downregulated when insects were transferred from rice to maize and to the wild grass Brachypodium distachyon, but expression showed partial or complete recovery 7 days after transfer. These results document that AGO subfamily members of L. striatellus are ubiquitously expressed at different developmental stages and respond to various stresses. Thus, AGO subfamily may act in regulating the stress-response of L. striatellus by controlling related gene expression.

Low Temperature Storage of Southern Rice Black-Streaked Dwarf Virus-Infected Rice Plants Cannot Sustain Virus Transmission by the Vector.

Southern rice black-streaked dwarf virus (SRBSDV) is a novel virus transmitted by white-backed planthopper Sogatella furcifera (Hováth) (Hemiptera: Delphacidae). Due to low virus transmission efficiency by the planthopper, researchers are frequently confronted with shortage of viruliferous vectors or infected rice plants, especially in winter and the following spring. To find new ways to maintain virus-infected materials, viral rice plants were stored at -80°C for 45 or 140 d and evaluated as virus sources in virus transmission by the vector. SRBSDV virions were not degraded during storage at -80°C as indicated by reverse transcription-polymerase chain reaction and reverse transcription real-time PCR detection. The planthopper nymphs fed on the infected thawed plants for 48 h survived at about 40% and showed positive detection of SRBSDV, but they lost the virus after feeding for another 20 d (the circulative transmission period) on noninfected plants. Transmission electron microscope images indicated broken capsid of virions in infected thawed leaves in contrast to integrity capsid of virions in infected fresh leaves. These results show that low temperature storage of SRBSDV-infected rice plants cannot sustain virus transmission by white-backed planthopper.

Rice Responses and Resistance to Planthopper-Borne Viruses at Transcriptomic and Proteomic Levels

Rice (Oryza sativa) is one of the most important cereal crops in the world, especially in Asian areas. Rice virus diseases are considered as the most serious threat to rice yields. Most rice viruses are transmitted by hemipteran insects such as planthoppers and leafhoppers. In Asia five rice viruses are transmitted mainly by three planthopper species in a persistent manner: Rice stripe virus, Rice black-streaked dwarf virus, Rice ragged stunt virus, Rice grassy stunt virus, and Southern rice black-streaked dwarf virus. In rice antivirus studies, several individual genes have been shown to function in rice resistance to viruses. Since plant responses to viral infection are complex, system-level omic studies are required to fully understand the responses. Recently more and more omic studies have appeared in the literatures on relationships between planthoppers and viruses, employing microarray, RNA-Seq, small RNA deep sequencing, degradome sequencing, and proteomic analysis. In this paper, we review the current knowledge and progress of omic studies in rice plant responses and resistance to four planthopper-borned viruses. We also discuss progress in the omic study of the interactions of planthoppers and rice viruses. Future research directions and translational applications of fundamental knowledge of virus-vector-rice interactions are proposed.

NAC transcription factor family genes are differentially expressed in rice during infections with Rice dwarf virus, Rice black-streaked dwarf virus, Rice grassy stunt virus, Rice ragged stunt virus, and Rice transitory yellowing virus.

Expression levels of the NAC gene family were studied in rice infected with Rice dwarf virus (RDV), Rice black-streaked dwarf virus (RBSDV), Rice grassy stunt virus (RGSV), Rice ragged stunt virus (RRSV), and Rice transitory yellowing virus (RTYV). Microarray analysis showed that 75 (68%) OsNAC genes were differentially regulated during infection with RDV, RBSDV, RGSV, and RRSV compared with the control. The number of OsNAC genes up-regulated was highest during RGSV infection, while the lowest number was found during RTYV infection. These phenomena correlate with the severity of the syndromes induced by the virus infections. Most of the genes in the NAC subgroups NAC22, SND, ONAC2, ANAC34, and ONAC3 were down-regulated for all virus infections. These OsNAC genes might be related to the health stage maintenance of the host plants. Interestingly, most of the genes in the subgroups TIP and SNAC were more highly expressed during RBSDV and RGSV infections. These results suggested that OsNAC genes might be related to the responses induced by the virus infection. All of the genes assigned to the TIP subgroups were highly expressed during RGSV infection when compared with the control. For RDV infection, the number of activated genes was greatest during infection with the S-strain, followed by the D84-strain and the O-strain, with seven OsNAC genes up-regulated during infection by all three strains. The Os12g03050 and Os11g05614 genes showed higher expression during infection with four of the five viruses, and Os11g03310, Os11g03370, and Os07g37920 genes showed high expression during at least three viral infections. We identified some duplicate genes that are classified as neofunctional and subfunctional according to their expression levels in different viral infections. A number of putative cis-elements were identified, which may help to clarify the function of these key genes in network pathways.

Identification and expression profiling analysis of calmodulin-binding transcription activator genes in maize (Zea mays L.) under abiotic and biotic stresses.

The calmodulin-binding transcription activators (CAMTA) play critical roles in plant growth and responses to environmental stimuli. However, how CAMTAs function in responses to abiotic and biotic stresses in maize (Zea mays L.) is largely unknown. In this study, we first identified all the CAMTA homologous genes in the whole genome of maize. The results showed that nine ZmCAMTA genes showed highly diversified gene structures and tissue-specific expression patterns. Many ZmCAMTA genes displayed high expression levels in the roots. We then surveyed the distribution of stress-related cis-regulatory elements in the −1.5 kb promoter regions of ZmCAMTA genes. Notably, a large number of stress-related elements present in the promoter regions of some ZmCAMTA genes, indicating a genetic basis of stress expression regulation of these genes. Quantitative real-time PCR was used to test the expression of ZmCAMTA genes under several abiotic stresses (drought, salt, and cold), various stress-related hormones [abscisic acid, auxin, salicylic acid (SA), and jasmonic acid] and biotic stress [rice black-streaked dwarf virus (RBSDV) infection]. Furthermore, the expression pattern of ZmCAMTA genes under RBSDV infection was analyzed to investigate their potential roles in responses of different maize cultivated varieties to RBSDV. The expression of most ZmCAMTA genes responded to both abiotic and biotic stresses. The data will help us to understand the roles of CAMTA-mediated Ca2+ signaling in maize tolerance to environmental stresses.

Genetic analysis and molecular mapping of QTLs for resistance to rice black-streaked dwarf disease in rice.

Rice black-streaked dwarf disease, caused by rice black-streaked dwarf virus (RBSDV), is transmitted by small brown planthoppers (Laodelphax striatellus Fallén, SBPH) and causes severe yield loss in epidemic years in China and other East Asian countries. Breeding for resistance to RBSDV is a promising strategy to control the disease. We identified Tetep that showed resistance to RBSDV using a field test and artificial inoculation test. An evaluation of the resistance mechanism revealed that Tetep was resistant to RBSDV but not to SBPH. Genetic analysis showed that the resistance of Tetep to RBSDV was controlled by quantitative trait loci (QTLs). Three new QTLs for RBSDV resistance were identified in this study, i.e., qRBSDV-3, qRBSDV-10 and qRBSDV-11. The LOD scores of qRBSDV-3, qRBSDV-10 and qRBSDV-11 were 4.07, 2.24 and 2.21, accounting for 17.5%, 0.3% and 12.4% of the total phenotypic variation, respectively. All the resistance loci identified in this study were associated with virus resistance genes. The alleles for enhancing resistance on chromosomes 3 and 11 originated from Tetep, whereas the other allele on chromosome 10 originated from a susceptible parent. The identified new resistance QTLs in this study are useful resources for efficiently breeding resistant rice cultivars to RBSDV.

Whole-genome expression analysis of Rice black-streaked dwarf virus in different plant hosts and small brown planthopper

Rice black-streaked dwarf virus (RBSDV) can infect a number of gramineous plants and cause severe crop yield losses in southeast Asian countries. The virus is transmitted by small brown planthopper (SBPH) in a persistent circulative manner. The interactions between RBSDV and its different hosts remain unknown. Besides, how the virus adjusts itself to infect different hosts is unclear. In the present study, the relative RNA levels of the thirteen RBSDV genes in rice, maize, wheat, and SBPH were measured by real-time quantitative PCR. P7-1 and P10 genes were predominantly expressed whereas P8 and P7-2 genes were expressed at low levels in plant hosts. Similar to the expression in rice, P7-1 was the most abundantly expressed gene and P8 was expressed at the lowest level in SBPH, indicating that RBSDV adopts the same strategy to infect distinct hosts. The high expression levels of the P7-1 gene in both plants and insect suggest that it can be used as the target gene for disease diagnostics. However, the expression levels of some genes varied from host to host. P5-1, P6 and P9-1, the components of the RBSDV viroplasm, are differentially expressed in different hosts. Moreover, western blot analysis showed that the quantity of the P9-1 protein was more abundant in SBPH than in plant hosts. These data indicate that the virus may adjust its own gene expression to replicate in different hosts. Analysis of time course of gene expression revealed that P7-1 stands out as the only gene highly expressed at the earliest time point and its expression precedes all others throughout infection from 8 to 24days post-inoculation. The high expression levels of the P7-1 gene suggest that it plays a significant role in RBSDV–host interactions.

Molecular Genetic Analysis and Evolution of Segment 7 in Rice Black-Streaked Dwarf Virus in China.

Rice black-streaked dwarf virus (RBSDV) causes maize rough dwarf disease or rice black-streaked dwarf disease and can lead to severe yield losses in maize and rice. To analyse RBSDV evolution, codon usage bias and genetic structure were investigated in 111 maize and rice RBSDV isolates from eight geographic locations in 2013 and 2014. The linear dsRNA S7 is A+U rich, with overall codon usage biased toward codons ending with A (A3s, S7-1: 32.64%, S7-2: 29.95%) or U (U3s, S7-1: 44.18%, S7-2: 46.06%). Effective number of codons (Nc) values of 45.63 in S7-1 (the first open reading frame of S7) and 39.96 in S7-2 (the second open reading frame of S7) indicate low degrees of RBSDV-S7 codon usage bias, likely driven by mutational bias regardless of year, host, or geographical origin. Twelve optimal codons were detected in S7. The nucleotide diversity (π) of S7 sequences in 2013 isolates (0.0307) was significantly higher than in 2014 isolates (0.0244, P = 0.0226). The nucleotide diversity (π) of S7 sequences in isolates from Jinan (0.0391) was higher than that from the other seven locations (P < 0.01). Only one S7 recombinant was detected in Baoding. RBSDV isolates could be phylogenetically classified into two groups according to S7 sequences, and further classified into two subgroups. S7-1 and S7-2 were under negative and purifying selection, with respective Ka/Ks ratios of 0.0179 and 0.0537. These RBSDV populations were expanding (P < 0.01) as indicated by negative values for Tajima's D, Fu and Li's D, and Fu and Li's F. Genetic differentiation was detected in six RBSDV subpopulations (P < 0.05). Absolute Fst (0.0790) and Nm (65.12) between 2013 and 2014, absolute Fst (0.1720) and Nm (38.49) between maize and rice, and absolute Fst values of 0.0085-0.3069 and Nm values of 0.56-29.61 among these eight geographic locations revealed frequent gene flow between subpopulations. Gene flow between 2013 and 2014 was the most frequent.

Quantification of southern rice black streaked dwarf virus and rice black streaked dwarf virus in the organs of their vector and nonvector insect over time

Southern rice black streaked dwarf virus (SRBSDV) and rice black streaked dwarf virus (RBSDV) are serious rice-infecting reoviruses, which are transmitted by different planthoppers in a persistent propagative manner. In this study, we quantitatively compared the spatial distribution of SRBSDV and RBSDV contents over time in their vector and nonvector insects using real time-PCR. Genome equivalent copies (GEC) were assessed every 2 days from 0 to 14 days after a 3-days acquisition access period (AAP) on infected plants. Results revealed 293.2±21.6 to 404.1±46.4 SRBSDV GEC/ng total RNA in whole body of white-backed planthopper (WBPH, Sogatella furcifera) at day 0 and 12 and 513.5±88.4 to 816.8±110.7 RBSDV GEC/ng total RNA in the whole body of small brown planthopper (SBPH, Laodelphax striatellus) at day 0 and 14, respectively, after 3-days AAP. Highest GEC of both viruses were found in the gut of their respective vectors. Although SRBSDV was detected in the gut of SBPH, it did not spread into the hemolymph or other organs. After an 8-day latent period, the transmission efficiency of SRBSDV and RBSDV by their respective vectors was significantly positively correlated with GEC in the salivary gland (r2=0.7808, P=0.0036 and r2=0.9351, P<0.0001, respectively, at α=0.05). Together, these results confirm that accumulation of >200 SRBSDV or RBSDV GEC/ng total RNA in the gut of vector, indicated threshold for further spread and the virus content in the salivary gland was significantly correlated with transmission efficiency by their respective vectors.

An RNA interference pathway modulates the persistent infection of &amp;lt;italic&amp;gt;Southern rice black-streaked dwarf virus&amp;lt;/italic&amp;gt; in insect vector, &amp;lt;italic&amp;gt;Sogatella furcifera&amp;lt;/italic&amp;gt;

Southern rice black-streaked dwarf virus (SRBSDV) is transmitted by the white-backed planthopper (WBPH; Sogatella furcifera Horvath) in a persistent-propagative manner. The mechanism for the highly efficient propagation of SRBSDV in the WBPH vector is unknown. RNA interference (RNAi) is generally thought to be an evolutionarily conserved antiviral immunity pathway in insects. To determine whether the persistent propagation of SRBSDV in the WBPH vector is modulated by an RNAi antiviral immunity pathway, we used small-RNA deep sequencing to detect 21- and 22-nt virus-derived small RNAs in cultured cells derived from WBPH infected with SRBSDV; the presence of these small RNAs implied that an RNAi response was trigged by viral infection. In addition, knockdown of the expressions of key RNAi pathway factors Dicer2 and Argonaute2 by treatment with dsRNA synthesized from Dicer2 and Argonaute2 genes strongly increased viral infection and accumulation in the WBPH vector and its cultured cells. Furthermore, the knockdown of Dicer2 and Argonaute2 expression reduced the survival rate and lifespan of the viruliferous insects. Taken together, these results indicate that the RNAi pathway effectively modulates the persistent infection of SRBSDV in the WBPH vector. This pathway thus plays a crucial role by preventing excessive plant virus accumulation in insect vectors, thereby protecting them from the negative impact of these viruses.

Characterization of the importance of terminal residues for southern rice black-streaked dwarf virus P9-1 viroplasm formations

Southern rice black-streaked dwarf virus (SRBSDV) P9-1 is involved in viroplasm formations in the SRBSDV-infected plants and insects. During infection, SRBSDV P9-1 is an important protein. However, the function characterization of P9-1 octamers in vitro and in yeast is still obscure. In this study, the secondary and 3D structure of SRBSDV P9-1 was predicted, then SRBSDV P9-1 was expressed and analyzed in vitro, a size exclusion chromatography assay showed that P9-1 had the ability to form octamers in vitro. Mutational analysis of terminal residues showed that the C-terminal arm (residues 324–347) of P9-1 was importance for the formation of octamers. Furthermore, a yeast two-hybrid assay showed that there were strong interactions between the full-length P9-1s, after deleting the C-terminal arm of P9-1, the interactions between the truncated P9-1s were disappeared in yeast. Collectively, the non-structural protein P9-1 played an essential role in self-interact viroplasm formation in vitro and in yeast, and the C-terminal arm region of P9-1 was important for assembling octamers in vitro.

Studies of binding interactions between Dufulin and southern rice black-streaked dwarf virus P9-1

Southern rice black-streaked dwarf virus (SRBSDV) is an important rice virus that can cause considerable crop losses. During infection, SRBSDV P9-1 accumulates viroplasms in SRBSDV-infected plant and insect cells, which may facilitate the formation of SRBSDV particles. Our field trial results showed that Dufulin inhibited infection activity in SRBSDV-infected rice. However it is not clear how Dufulin inhibits the infection activity. Therefore we studied the interactions between Dufulin and SRBSDV using P9-1 as a potential target, and found that Dufulin inhibited SRBSDV P9-1 gene expression and had a micromole affinity for the arginine 175 residue. Substituting arginine with glycine resulted in a mutant that was significantly less sensitive to Dufulin. Based on these results, we speculate that Dufulin inhibits the virulence of SRBSDV by binding with arginine 175 in the internal pore of P9-1 octameric protein.