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.

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.

Study on the control of rice stripe disease with Bilken virusicide

The paper reviewed the function mechanism of Bilken virusicide against rice stipe disease, and then introduced its control effects in field test as well as its application method.

Effect of Rice Sowing Date on Occurrence of Small Brown Planthopper and Epidemics of Planthopper-Transmitted Rice Stripe Viral Disease

To understand the relationship between rice sowing date and occurrence of the rice small brown planthopper （SBPH） Laodelphax striatellus Fallen and the epidemics of the planthopper-transmitted rice stripe viral （RSV） disease, four sowing dates of rice were evaluated in 2006 and 2007. The results showed that the peak density of SBPH and RSV incidence in the nursery and in the transplanted field decreased with the delay of sowing date in single crop of japonica rice in north Zhejiang Province of China. The relationship between seedling RSV incidence at the end of the nursery trial with sowing date was well described by Weibull equation. The area under the curve of population dynamics （AUCPD or planthopper-day accumulation） or the peak density of the planthopper in the nursery could be summarized by a logistic equation. RSV incidence in the transplanted fields could be characterized quantitatively by a multivariate regression equation, including the variables of sowing date, peak density of the vector, and RSV incidence at the end of the nursery trial. That the descriptive model excluded the AUCPD in transplanted field implies that this variable is not necessary in forecasting disease epidemics in the field. The 2-year experiments sufficiently indicated that suitable sowing of rice could be used as one of the effective measures to control the vector population and therefore the planthopper-transmitted RSV on a larger scale. The optimal sowing date for the single-cropped transplanted japonica rice is recommended from late May to early June in north Zhejiang, China.

Detection,Occurrence,and Survey of Rice Stripe and BlackStreaked Dwarf Diseases in Zhejiang Province,China

The major viral diseases that occur on rice plants in Zhejiang Province, eastern China, are stripe and rice black-streaked dwarf diseases. Rice stripe disease is only caused by rice stripe tenuivirus （RSV）, while rice black-streaked dwarf disease can be caused by rice black-streaked dwarf fijivirus （RBSDV） and/or southem rice black-streaked dwarf fijivirus （SRBSDV）. Here we review the characterization of these viruses, methods for their detection, and extensive surveys showing their occurrence and spread in the province.

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-b＇ and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region （SCAR） marker tightly linked with Stv-b＇ and a cleaved amplified polymorphic sequence （CAPS） marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.

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.

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.

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.

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.

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.

Genetic Analysis of Stripe Disease Resistance in Rice Restorer Line C224 Using Major Gene plus Polygene Mixed Effect Model

The inheritance of stripe disease resistance in a rice restorer line C224 was analyzed using the mixed effect model of major gene plus polygene for quantitative traits.In addition,the resistance was investigated in seven crosses of C224 with maintainer lines.The results showed that the stripe resistance of C224 was controlled by two major genes with additive-dominance-epistasis effects plus polygenes with additive-dominance effects （E-1 model）.These two genes had additive effects of-12.47% and-24.75%,respectively,showing negative dominance effects.There were significant epistasis and interaction effects between the two major genes.The heritability of the two major genes was 92.12%,while that of polygenes was 2.74%,indicating that the stripe resistance had dominant major gene effect.Of the seven crosses,five displayed high or medium resistance to the stripe disease.

Quantitative Trait Loci for Resistance to Stripe Disease in Rice(Oryza sativa)

In order to map the quantitative trait loci for rice stripe resistance, a molecular linkage map was constructed based on the F2 population derived from a cross between Zhaiyeqing 8 and Wuyujing 3. Reactions of the two parents, F1 individual and 129 F2：3 lines to, rice stripe were JnvestJgated by both artificial Jnoculation at laboratory and natural infection in the field, and the ratios of disease rating index were scored. The distribution of the ratios of disease rating index in Zhaiyeqing 8/Wuyujing 3 F2：3 population ranged from 0 to 134,08 and from 6.25 to 133.6 under artificial inoculation at laboratory and natural infection in the field, respectively, and showed a marked bias towards resistant parent （Zhaiyeqing 8）, indicating that the resistance to rice stripe was controlled by quantitative trait loci （QTL）. QTL analysis showed that the QTLs detected by the two inoculation methods were completely different. Only one QTL, qSTVT, was detected under artificial inoculation, at which the Zhaiyeqing 8 allele increased the resistance to rice stripe, while two QTLs, qSTV5 and qSTV1, were detected under natural infection, in which resistant alleles came from Zhaiyeqing 8 and Wuyujing 3, respectively. These results showed that resistant parent Zhaiyeqing 8 carried the alleles associated with the resistance to rice stripe virus and the small brown planthopper, and susceptible parent Wuyujing 3 also carried the resistant allele to rice stripe virus. In comparison with the results previously reported, QTLs detected in the study were new resistant genes to rice stripe disease. This will provide a new resistant resource for avoiding genetic vulnerability for single utilization of the resistant gene Stvb-i.

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.

Genetic variability and evolution of rice stripe virus

Rice stripe virus(RSV) is the type member of the genus Tenuivirus.RSV is known to have four segmented,single-stranded RNA molecules and causes rice stripe disease in the rice fields of China,Japan,and Korea.Based on the complete genomic sequences of the determined 6 RSV isolates(from Yunnan,Jiangsu,Zhejiang,and Liaoning Provinces,China) and 27 other RSV isolates(from Yunnan,Jiangsu,Anhui,Henan,and Shandong Provinces of China,also Japan and Korea) downloaded from GenBank,we provided a genotyping profile of RSV field isolates and described the population structure of RSV.All RSV isolates,except isolate CX,could be divided into two subtypes,one including 6 isolates from Yunnan Province,and the other including 26 isolates from different parts of China,Japan,and Korea,which were referred to as subtype II and subtype I,respectively.The amino acid distances between subtypes range from 0.053 to 0.085.RSV isolates in Yunnan Province were genetically differentiated from other parts of China,Japan,and Korea and showed infrequent gene flow.The RSV populations collected from other parts of China,Japan,and Korea were only composed of subtype I and showed very low genetic diversity.We speculated that isolate CX may be the result of recombination of isolates from two subtypes.Two potential recombination events were detected in RNA4 of isolate CX.

Polyamine-metabolizing enzymes are activated to promote the proper assembly of rice stripe mosaic virus in insect vectors

Both viruses and host cells compete for intracellular polyamines for efficient propagation.Currently,how the key polyamine-metabolizing enzymes,including ornithine decarboxylase 1(ODC1)and its antizyme 1(OAZ1),are activated to co-ordinate viral propagation and polyamine biosynthesis remains unknown.Here,we report that the matrix protein of rice stripe mosaic virus(RSMV),a cytorhabdovirus,directly hijacks OAZ1 to ensure the proper assembly of rigid bacilliform non-enveloped virions in leafhopper vector.Viral matrix protein effectively competes with ODC1 to bind to OAZ1,and thus,the ability of OAZ1 to target and mediate the degradation of ODC1 is significantly inhibited during viral propagation,which finally promotes polyamines production.Thus,OAZ1 and ODC1 are activated to synergistically promote viral persistent propagation and polyamine biosynthesis in viruliferous vectors.Our data suggest that it is a novel mechanism for rhabdovirus to exploit OAZ1 for facilitating viral assembly.

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.

Genetic Improvement of Japonica Rice Variety Wuyujing 3 for Stripe Disease Resistance and Eating Quality by Pyramiding Stv-b^i and Wx-mq

Japonica rice variety Kanto 194 as the donor of resistance gene Stv-b^i and Iow-amylose content gene Wx-mq was used to improve the resistance and eating quality of Wuyujing 3 by the breeding strategy of backcross. In continuous backcross and selfcross generations, the related molecular markers with Stvobi and Wx-mq genes were utilized for genotypic detection by associated with resistance identification of rice stripe disease and agronomic traits selection. Finally, 10 improved lines with homozygous genotype Stv-b^i Stv-b^iWx-mqWx-mq were obtained from BC3F4 generation. The results of comparative analysis indicated that most characters of these lines were consistent with those of recipient parent Wuyujing 3, except for the improved resistance, appearance and eating quality. By evaluation of the comprehensive performance of them, two excellent lines K01 and K04 were selected for further experiments.