Screening of Rice Stripe Virus(RSV)-resistant Germplasm from Landraces of Rice(Oryza sativa L.)

[Objective] The aim of this study was to screen rice strip virus （RSV）-resistant landraces. [Method] The resistance of 119 rice landraces to rice stripe virus was identified in field spontaneously infected with smal plant-hopper. [Result] There were 55 landraces resistant to rice strip disease in 56 indica rice landraces, but on-ly two resistant to rice strip disease in 63 japonica rice landraces. [Conclusion] The results revealed that there were abundant rice landscapes resistant to RSV in Chi-na, and these varieties can be used to develop more genes resistant to RSV.

Screening and Application of SSR Markers of Resistant Gene against Rice Stripe Virus

[Objective] New SSR primers were designed and screened to apply in the backcross breeding for modified resistance against rice stripe virus.[Method] The conventional late japonica rice varieties including 502 with high resistance to stripe virus,Xiushui 09 with high susceptibility to stripe virus and their derived strains were adopted as the test materials,SSR and SAPR markers were used to locate RSV1 gene with high resistance against stripe virus,and three pairs of SSR markers （M-11-1,M-11-2,M-11-3） were further designed.Through screening and analysis,M-11-3 was selected as the RSV1 detection marker gene for tracking RSV1 gene,thus RSV1 gene was successfully introduced to the backcross breeding of late japonica rice varieties such as Xiushui 09,and the resistance expression of different strains was identified.[Result]The resistance of improved strains against stripe virus was significantly higher than Xiushui 09,the resistance of most strains was close to the level of donor,and the expression of resistance among years was stable.Therefore,the resistance effect of RSV1 gene used in the test was very obvious,which was accurate with the assisted selection of RSV1 gene linked markers M-11-3.[Conclusion]The study certified the feasibility of molecular markers application in resistance improvement against rice stripe virus,which also showed that optimization and development of new marker genes could effectively improve the efficiency of marker-assisted selection.

LncRNAs are potentially involved in the immune interaction between small brown planthopper and rice stripe virus

Small brown planthopper(SBPH, Laodelphax striatellus Fallén) is an important vector of major crop pathogen rice stripe virus(RSV). Controlling SBPH population is an efficient approach to control RSV. Long non-coding RNAs(lnc RNA) have been reported to block virus replication in hosts. However, the function of lnc RNAs in RSV infection and replication is still unknown. Here, we aimed to study regulatory mechanisms of lnc RNA in an immune system during RSV infection. First, lnc RNA genes were predicted from SBPH transcriptomes using a bioinformatics pipeline based on characteristics of lnc RNA. We identified 4 786 lnc RNA genes corresponding to 5 790 transcripts in SBPH from an RNA-Seq dataset of 15 transcriptomes. Differential expression analysis indicated that 3, 11, and 25 lnc RNA genes were highly expressed in gut, salivary gland, and ovary, respectively, of viruliferous SBPH(Student’s t-test, P<0.05). We randomly selected eight lnc RNAs for expression validation using quantitative real-time PCR, confirming the differential expression of these lnc RNAs between viruliferous and non-viruliferous SBPH. In summary, we present evidence that the expression of lnc RNA genes was induced by RSV infection, suggesting that RSV might be involved in the antivirus immune system in SBPH and participate in regulating the RSV replication mechanism. These data provide helpful information for future investigations of the interaction between lncRNA and RSV.

RNAi-mediated transgenic rice resistance to Rice stripe virus

Rice stripe virus（RSV） often causes severe rice yield loss in temperate regions of East Asia. Although the correlation of small interfering RNAs（si RNAs） with transgenic virus resistance of plants using RNA interference（RNAi） is known for decades, no systematical research has been done on the profiling of si RNAs from a genomic scale. Our research is aiming to systematically study the RNAi impact in RSV-resistant transgenic rice, which was generated by introducing an inverted repeat construct that targets RSV nucleocapsid protein（NCP） gene. In this paper, three independent RSV-retsistant transgenic rice lines were generated, their stable integration of the T-DNA fragment and the expression of si RNAs were confirmed by Southern blotting and Northern blotting analyses, and the majority of si RNAs were in lengths of 21, 22, and 24 nucleotides（nt）, which have validated a connection between the presence of the RSV NCP homologous si RNAs and the RSV resistance in those transgenic rice lines. In one of these transgenic lines（T4-B1）, the T-DNA fragment was found to have been inserted at chromosome 1 of the rice genome, substituting the rice genome fragment from 32 158 773 to 32 158 787 nt. Bioinformatics analysis of small RNA-Seq data on the T4-B1 line also confirmed the large population of NCP-derived si RNAs in transgenic plants, and the RSV-infected library（T4-B1-V） possessed more si RNAs than its mock inoculated libraries（T4-B1-VF）, these results indicating the inverted repeat construct and RSV could introduce abundance of si RNAs in transgenic rice. Moreover, a varied expression level of specific si RNAs was found among different segments of the NCP gene template, about 47% of NCP-derived si RNAs reads aligned with the fragment from 594 to 832 nt（239 nt in length） in NCP gene（969 nt in length） in the T4-B1-V, indicating a potential usage of hotspot regions for RNAi silencing in future research. In conclusion, as the first study to address the si RNA profile in RSV-resistant transgenic plant using next generation sequencing（NGS） technique, we confirmed that the massive abundance of si RNA derived from the inverted repeat of NCP is the major reason for RSV-resistance.

Transcriptome analysis reveals different response of resistant and susceptible rice varieties to rice stripe virus infection

Rice stripe disease,caused by rice stripe virus (RSV) which is transmitted by small brown planthopper (SBPH,Laodelphax striatellus Fallen),resulted in serious losses to rice production during the last 2 decades.Research on the molecular differences between resistant and susceptible rice varieties and the interaction between rice and RSV remains inadequate.In this study,RNA-Seq was used to analyze the transcriptomic differences between the resistant and susceptible rice varieties at different times post RSV infection.Through Gene Ontology (GO) annotation,the differentially expressed genes (DEGs) related to transcription factors,peroxidases,and kinases of 2 varieties at 3 time points were identified.Comparing these 2 varieties,the DEGs associated with these 3 GOs were numerically less in the resistant variety than in the susceptible variety,but the expression showed a significant up-or down-regulation trend under the conditions of|log_2(Fold change)|>0&P_(adj)<0.05 by significance analysis.Then through Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation,DEGs involved in some pathways that have a contribution to disease resistance including plant hormone signal transduction and plant–pathogen interaction were found.The results showed that resistance responses regulated by abscisic acid (ABA) and brassinosteroids (BR) were the same for 2 varieties,but that mediated by salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) were different.The DEGs in resistant and susceptible varieties at the 3 time points were identified in both PAMP-triggered immunity (PTI) and Effector proteintriggered immunity (ETI),with that most of the unigenes of the susceptible variety were involved in PTI,whereas most of the unigenes of the resistant variety were involved in ETI.These results revealed the different responses of resistant and susceptible varieties in the transcription level to RSV infection.

Surface Display of Rice Stripe Virus NSvc2 and Analysis of Its Membrane Fusion Activity

Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper. How RSV enters an insect cell to initiate the infection cycle is poorly understood. Sequence analysis revealed that the RSV NSvc2 protein was similar to the membrane glycoproteins of several members in the family Bunyaviridae and might induce cell membrane fusion. To conveniently study the membrane fusion activity of NSvc2, we constructed cell surface display vectors for expressing Nsvc2 on the insect cell surface as the membrane glycoproteins of the enveloped viruses. Our results showed that NSvc2 was successfully expressed and displayed on the surface of insect Sf9 cells. When induced by low pH, the membrane fusion was not observed in the cells that expressed NSvc2. Additionally, the membrane fusion was also not detected when co-expressing Nsvc2 and the viral capsid protein on insect cell surface. Thus, RSV NSvc2 is probably different from the phlebovirus counterparts, which could suggest different functions. RSV might enter insect cells other than by fusion with plasma or endosome membrane.

Disaster Prevention and Mitigation Technologies of Rice Virus Diseases Spread by Small Brown Planthopper (SBPH)

Rice stripe virus disease （RSVD） and rice black-streaked dwarf virus disease （RBSDVD） are two epidemic diseases in Yancheng City, Jiangsu Province in the last decade. The authors investigated the disaster regularity, prevention and control technology of RSVD and RBSDVD systematically. The occurrence and virus transmission of SBPH and disaster regularity of virus diseases were studied; the resistance of some rice varieties was cleared; the effects of physical and agricultural measures such as insect net blocking, appropriate late sowing and plowing on controlling occurrence and virus transmission of SBPH were figured out; a hatch of chemical agents were screened, providing efficient and harmless pesticides for effective control against SBPH and prevention against virus diseases. A set of disaster control and mitigation technologies was proposed in this paper, which was practical, sustainable, and easy to operate for the local planting patterns.

Subcellular localization of the stripe disease-specific protein encoded by rice stripe virus (RSV) in its vector,the small brown planthopper,Laodel-phax striatellus

The stripe disease-specific protein (SP) encoded by the rice stripe virus (RSV) was successfully used as a localization signal of the virus in its vector, the small brown lanthopper, Laodelphax striatellus Fallen. Immunogold particles in large numbers were detected in various parts of the viruliferous females: the ovum, surface of chorion, the mid-gut lumen, and the columnar cells. Whereas there was noneof these particles in the non-viruliferous females and males, and testis of viruliferous males. Endosymbionts (mycetocytes) were abundant, harboring ovaries of both viruliferous and non-viruliferous females, but none in the testis of males. The results provided us with the direct proof that RSV is a ciruculative and propagative plant virus and it was transovarially transmitted alongside with endosymbionts of its vector. Therefore, we deem it a nice lead for future studies on the mechanism of RSV transmission and functioning of its viral proteins.

2013-2014年云南主要稻区病毒病调查及RSV的分子变异

于2013-2014连续两年对云南主要水稻种植区的水稻病毒病进行了调查,发现当前为害云南水稻的病毒病主要有水稻条纹叶枯病、南方水稻黑条矮缩病、水稻矮缩病和水稻齿叶矮缩病。其中,条纹叶枯病是当前为害云南水稻最严重的病毒病,部分田块最高发病率可达15.0%;其次是水稻矮缩病,调查田块最高发病率达10.0%;南方水稻黑条矮缩病的平均发病率达5.3%;水稻齿叶矮缩病只在局部地区发生且发病率都不高。对云南当前发生较普遍的RSV不同分离物RNA3基因间隔区(IR3)及RNA4基因间隔区(IR4)开展了分子变异分析。根据不同RSV分离物IR3、IR4核苷酸序列构建系统进化树,所分析的RSV分离物均可被划分为两个组,部分云南分离物和云南以外的中国、日本、韩国分离物聚在Ⅰ组,其余绝大多数云南分离物单独聚在Ⅱ组。不同RSV分离物的IR3和IR4长度差异较大,按每10个核苷酸的长度差异进行划分,均可划分为A、B、C三种不同的类型。部分RSV云南分离物IR3、IR4中有17-21nt的序列与其水稻、小麦和大麦等寄主的部分序列相同,这可能是病毒在进化过程中与寄主植物发生了重组。云南RSV分离物的IR3、IR4分子变异较大,可能与其特殊的地理、生态条件有一定的相关性。

灰飞虱高带毒(RSV)群体酵母双杂交cDNA文库的构建

为了研究灰飞虱Laodelphax striatellus Fallén与水稻条纹病毒(rice stripe virus,RSV)互作机制,本研究构建了灰飞虱高带毒群体酵母双杂交cDNA文库。以实验室筛选的灰飞虱高带毒群体为材料,分离纯化mRNA,反转录合成双链cDNA,并连接三框型接头,层析柱分级纯化。采用同源重组反应制备三框型cDNA入门文库,再通过同源重组将入门文库转移到Gateway兼容载体pGADT7-DEST上,构建获得酵母双杂交cDNA文库。检测结果表明:文库库容量为3.68×107cfu,扩增文库滴度为2.62×1010cfu/mL;文库重组率大于95%,cDNA插入片段平均长度>1kb,达到了标准cDNA文库的要求。灰飞虱高带毒群体酵母双杂交cDNA文库的构建为开展昆虫介体与水稻条纹病毒互作机制的研究奠定了基础。

条纹病毒RSV在灰飞虱与稻株之间传递速率的研究

为进一步深入研究RSV(rice stripe virus)在灰飞虱和稻株之间的传递情况,本研究选用水稻条纹叶枯病易感品种'武育粳7号',让带毒灰飞虱在健康稻株上取食1.5h和3h后,于1.5h、3h、6h、12h、1天、2天和4天采样,采用RT-PCR技术检测稻株被RSV侵染情况。结果表明,当带毒灰飞虱在健株上取食3h后,可在其体内检测出RSV,说明RSV已被有效地传入到稻株内,且带毒灰飞虱取食时间越长,稻株的获毒概率越大;采用斑点免疫吸附法(dot immunobinding assay,DIBA法)检测非带毒灰飞虱在携带RSV稻株上取食0.25h、0.5h、1h、2h、4h和8h后虫体带毒情况。结果表明,当无毒灰飞虱在携毒稻株上取食0.5h后就能顺利获毒,而且随着取食时间的延长,灰飞虱获毒的比率越大。本研究首次从分子水平上对带毒灰飞虱传递RSV到稻株体内的情况进行了检测,同时结合灰飞虱获毒速率的研究结果,可为水稻条纹叶枯病的预测预报提供一种新的途径。

RSV编码的4种蛋白在“AcMNPV-sf9昆虫细胞”体系中的重组表达

应用反转录聚合酶链式反应(RT-PCR)获得水稻条纹病毒(Rice stripe virus,RSV)的4个基因NS2、NS3、CP和SP,并将它们克隆至pMD-18-T载体上.得到的重组质粒pMD-18-T-NS2、pMD-18-T-NS3、pMD-18-T-CP和pMD-18-T-SP经XbaⅠ/HindⅢ双酶切,分别与经相同方法酶切的苜蓿银纹夜蛾核型多角体病毒(Autographa california nuclear polyhedrosis virus,AcM-NPV)转移载体pFastBacHTb相连接,构建重组转移质粒pFastBacHTb-X(pFastBacHTb-NS2、pFastBacHTb-NS3、pFastBacHTb-CP和pFastBacHTb-SP).序列测定表明,目的基因准确地插入到表达载体中.重组质粒pFastBacHTb-X通过转化包含有穿梭载体的大肠杆菌(Escherichia coli)感受态细胞DH10Bac,得到重组穿梭质粒rb-X(rb-NS2、rb-NS3、rb-CP和rb-SP).rb-X侵染草地贪夜蛾(Spodoptera frugiperda)离体细胞(sf9)24-72 h后,在荧光倒置显微镜可见光200倍视野下观察到细胞增大、培养液和细胞内出现颗粒状物质、部分细胞破裂甚至裂解等一系列与正常sf9细胞形态有明显区别的现象.rb-X侵染细胞72h后,从细胞提取蛋白,电泳分析得到4个条带,大小分别为28.2、29.2、40.2和25.2 ku,与预测的4种融合蛋白大小一致.Western blotting分析分别得到4条单一条带,证明了RSV NS2、NS3、CP和SP基因在"AcMNPV-sf9昆虫细胞"真核表达体系中成功表达.

Interaction between Rice stripe virus Disease- Specific Protein and Host PsbP Enhances Virus Symptoms

Rice stripe virus （RSV） causes severe diseases in Oryza sativa （rice） in many Eastern Asian countries. Diseasespecific protein （SP） of RSV is a non-structural protein and its accumulation level in rice plant was shown to determine the severity of RSV symptoms. Here, we present evidence that expression of RSV SP alone in rice or Nicotiana benthamiana did not produce visible symptoms. Expression of SP in these two plants, however, enhanced RSV- or Potato virus X （PVX）- induced symptoms. Through yeast two-hybrid screening, GST pull-down, and bimolecular fluorescence complementation assays, we demonstrated that RSV SP interacted with PsbP, a 23-kDa oxygen-evolving complex protein, in both rice and N. benthamiana. Furthermore, our investigation showed that silencing of PsbP expression in both plants increased disease symptom severity and virus accumulation. Confocal microscopy using N, benthamiana protoplast showed that PsbP accu- mulated predominantly in chloroplast in wild-type N. benthamiana cells. In the presence of RSV SP, most PsbP was recruited into cytoplasm of the assayed cells. In addition, accumulation of SP during RSV infection resulted in alterations of chloroplast structure and function. Our findings shed light on the molecular mechanism underlying RSV disease symptom development.

Monoclonal Antibody-Based Serological Detection of Rice Stripe Mosaic Virus Infection in Rice Plants or Leafhoppers

Rice stripe mosaic virus(RSMV) is a rhabdovirus recently found in southern part of China and can cause severe reduction in rice production. To establish serological methods for RSMV epidemiological studies and to establish a control strategy for this virus, we first purified RSMV virions from infected rice plants and then used them as an immunogen to produce four RSMV-specific monoclonal antibodies(MAbs)(i.e.,1D4, 4A8, 8E4 and 11F11). With these MAbs, we have developed a highly specific and sensitive antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA), a Dot-ELISA and a Tissue print-ELISA for rapid detections of RSMV infection in rice plants or in leafhoppers. Our results showed that RSMV can be readily detected in RSMV-infected rice plant tissue crude extracts diluted at 1:20,971,520(w/v, g/m L)through ACP-ELISA or diluted at 1:327,680(w/v, g/m L) through Dot-ELISA. Both ACP-ELISA and Dot-ELISA can also be used to detect RSMV infection in individual RSMV viruliferous leafhopper(Recilia dorsalis) homogenate diluted at 1:307,200 and 1:163,840(individual leafhopper/l L), respectively. Detection of RSMV infection in field-collected rice samples or in RSMV viruliferous leafhoppers indicated that the three serological methods can produce same results with that produced by RT-PCR(19 of the 33 rice samples and 5 of the 16 leafhoppers were RSMV-positive). We consider that the four MAbs produced in this study are very specific and sensitive, and the three new serological methods are very useful for detections of RSMV infection in rice plants or in leafhoppers and the establishment of the disease control strategies.

RSV和HiPV在灰飞虱中传播方式的比较与分析

水稻条纹病毒(Rice stripe virus,RSV)是由介体灰飞虱(Laodelphax striatellus)传播的重要水稻病毒,严重危害我国水稻生产。Himetobi P病毒(Hi PV)是广泛存在于灰飞虱体内的一类昆虫类微小核糖核酸病毒,灰飞虱感染Hi PV后无明显的致病表现。对Hi PV不同分离物及Cripavirus属多个病毒的序列同源性分析表明,Hi PV不同分离物之间相似度非常高,但和同属其他病毒的核苷酸及氨基酸序列均存在明显差异。对两种病毒在水稻中的复制情况进行检测,结果表明,RSV可在水稻中复制,Hi PV可在水稻中稳定存在一段时间(7 d),但无法在水稻中复制。进一步的试验表明,灰飞虱的分泌物中存在Hi PV,但无法检测到RSV。

The nucleocapsid protein of rice stripe virus in cell nuclei of vector insect regulates viral replication

Rice stripe virus(RSV)transmitted by the small brown planthopper causes severe rice yield losses in Asian countries.Although viral nuclear entry promotes viral replication in host cells,whether this phenomenon occurs in vector cells remains unknown.Therefore,in this study,we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells.We observed that the nucleocapsid protein(NP)and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin a nuclear transport system.When blocking NP nuclear localization,cytoplasmic RSV accumulation significantly increased.In the vector cell nuclei,NP bound the transcription factor YY1 and affected its positive regulation to FAIM.Subsequently,decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction.Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells,providing new insights into the balance between viral load and the immunity pressure in vector insects.

Development of a colloidal gold-based immunochromatographic strip for rapid detection of Rice stripe virus

Rice stripe virus(RSV) causes dramatic losses in rice production worldwide. In this study, two monoclonal antibodies(MAbs) 16E6 and 11 C1 against RSV and a colloidal gold-based immunochromatographic strip were developed for specific, sensitive, and rapid detection of RSV in rice plant and planthopper samples. The MAb 16E6 was conjugated with colloidal gold and the MAb 11C1 was coated on the test line of the nitrocellulose membrane of the test strip. The specificity of the test strip was confirmed by a positive reaction to RSV-infected rice plants and small brown planthopper(SBPH), and negative reactions to five other rice viruses, healthy rice plants, four other vectors of five rice viruses, and non-viruliferous SBPH. Sensitivity analyses showed that the test strip could detect the virus in RSV-infected rice plant tissue crude extracts diluted to 1:20 480(w/v, g/mL), and in individual viruliferous SBPH homogenate diluted to 1:2560(individual SPBH/μL). The validity of the developed strip was further confirmed by tests using field-collected rice and SBPH samples. This newly developed test strip is a low-cost, fast, and easy-to-use tool for on-site detection of RSV infection during field epidemiological studies and paddy field surveys, and thus can benefit decision-making for RSV management in the field.

运用代谢组学研究RSV与水稻的互作初探

水稻条纹叶枯病严重威胁着我国的水稻生产,该病由水稻条纹病毒(Rice stripe virus,RSV)引起。目前,对水稻与RSV互作机制的认识还较少,这是制定有效措施来对RSV进行防控的一大障碍。代谢组学是新近发展起来的一种研究手段,它通过考察生物体系包括细胞、组织或整个生物体在受到刺激或扰动后,在代谢水平上的应答,来研究一系列的生物现象。运用代谢组学对水稻与RSV的互作进行了初步探索。GC-MS分析表明,RSV的侵染能显著影响水稻的代谢谱,且在代谢组水平上,不同抗感性的水稻对RSV的响应存在着差异。与NIST质谱数据库中的信息比较,从武育粳3号健康植株中鉴定到内源性代谢物12种、武育粳3号发病植株中11种、KT95-418健康植株中9种、KT95-418发病植株中14种。

Laodelphax striatellus Atg8 facilitates Rice stripe virus infection in an autophagy-independent manner

Rice stripe virus(RSV)is the causative agent of rice stripe disease and is completely dependent on insect vectors for its plant-to-plant transmission.Laodelphax striatellus is the major insect vector for RSV.In this study,we explored the interactions be-tween RSV infection and L.striatellus autophagy,a potential intrinsic antiviral mechanism in insects.We found that L.striatellus autophagic activity did not affect RSV infection;however,the autophagy related-8(Atg8)gene significantly enhanced virus infection.Dur-ing RSV initial infection within the L.striatellus midgut,silencing of Atg8 expression significantly decreased the phosphorylation of c-Jun N-terminal kinase(p-JNK);however,when RSV infection is absent,silencing of Atg8 did not alter p-JNK levels.Thesc results indicated that Atg8 might activate the JNK machinery by allowing more virus infection into cells.We further revealed that Atg8-deficiency significantly decreased RSV accumu-lation on the surface of the insect midgut epithelial cells,suggesting a receptor trafficking function of the y-aminobutyric acid receptor-associated protein family.Using the RSV ovary entry as a model,in which vitellogenin receptor(V gR)mediates RSV cell entry,we clarified that Atg8-deficiency decreased the abundance of V gR localizing on the cytomem-brane and disturbed the attachment of RSV in the germarium zones.Collectively,these results revealed an autophagy-independent function of L.striatellus Atg8 that enhances RSV initial infection by increasing virus attachment on the infection sites.

THE STUDY OF MOLECULAR BIOLOGY OF RICE STRIPE VIRUS (Ⅰ)——MOLECULAR WEIGHT, AMINO ACID COMPOSITION AND PROPERTY OF COAT PROTEIN

Rice stripe virus (RSV)causes rice stripe disease which is one of the main damages to rice. The virus is transmitted by Laodelphax striatellus and three other plant hoppers. It infects 37 cereals including rice, wheat and maize, and results in a significant reduction of pro-