ARGONAUTE 2 increases rice susceptibility to rice black-streaked dwarf virus infection by epigenetically regulating HEXOKINASE 1 expression

ARGONAUTE (AGO) proteins play crucial roles in plant defence against virus invasion. To date, the role of OsAGO2 in rice antiviral defence remains largely unknown. In this study, we determined that the expression of OsAGO2 in rice was induced upon rice black-streaked dwarf virus (RBSDV) infection. Using transgenic rice plants overexpressing OsAGO2 and Osago2 mutants generated through transposon-insertion or CRISPR/Cas9 technology, we found that overexpression of OsAGO2 enhanced rice susceptibility to RBSDV infection. Osago2 mutant lines exhibited strong resistance to RBSDV infection through the elicitation of an early defence response, including reprogramming defence gene expression and production of reactive oxygen species (ROS). Compared to Nipponbare control, the expression level of OsHXK1 (HEXOKINASE 1) increased significantly, and the methylation levels of its promoter decreased in the Osago2 mutant on RBSDV infection. The expression profile of OsHXK1 was the opposite to that of OsAGO2 during RBSDV infection. Overexpression of OsHXK1 in rice also induced ROS production and enhanced rice resistance to RBSDV infection. These results indicate that OsHXK1 controls ROS accumulation and is regulated by OsAGO2 through epigenetic regulation. It is noteworthy that the Osago2 mutant plants are also resistant to southern rice black-streaked dwarf virus infection, another member of the genus Fijivirus. Based on the results presented in this paper, we conclude that OsAGO2 modulates rice susceptibility to fijivirus infection by suppressing OsHXK1 expression, leading to the onset of ROS-mediated resistance. This discovery may benefit future rice breeding programmes for virus resistance.     

Molecular characterization of segments S7 to S10 of a southern rice black-streaked dwarf virus isolate from maize in northern China

Southern rice black-streaked dwarf virus (SRBSDV) is a novel Fijivirus prevalent in rice in southern and central China, and northern Vietnam. Its genome has 10 segments of double-stranded RNA named S1 to S10 according to their size. An isolate of SRBSDV, JNi4, was obtained from naturally infected maize plants from Ji’ning, Shandong province, in the 2008 maize season. Segments S7 to S10 of JNi4 share nucleotide identities of 72.6%–73.1%, 72.3%–73%, 73.9%–74.5% and 77.3%–79%, respectively, with corresponding segments of Rice black-streaked dwarf virus isolates, and identities of 99.7%, 99.1%–99.7%, 98.9%–99.5%, and 98.6%–99.2% with those of SRBSDV isolates HN and GD. JNi4 forms a separate branch with GD and HN in the phylogenetic trees constructed with genomic sequences of S7 to S10. These results confirm the proposed taxonomic status of SRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4 is an isolate of SRBSDV. Shandong is so far the northernmost region where SRBSDV is found in China.     

Suppression of auxin signalling promotes rice susceptibility to Rice black streaked dwarf virus infection

Auxin plays a fundamental role in plant growth and development, and also influences plant defence against various pathogens. Previous studies have examined the different roles of the auxin pathway during infection by biotrophic bacteria and necrotrophic fungi. We now show that the auxin signalling pathway was markedly down-regulated following infection of rice by Rice black streaked dwarf virus (RBSDV), a dsRNA virus. Repression of the auxin receptor TIR1 by a mutant overexpressing miR393 increased rice susceptibility to RBSDV. Mutants overexpressing the auxin signalling repressors OsIAA20 and OsIAA31 were also more susceptible to RBSDV. The induction of jasmonic acid (JA) pathway genes in response to RBSDV was supressed in auxin signalling mutants, suggesting that activation of the JA pathway may be part of the auxin signalling-mediated rice defence against RBSDV. More importantly, our results also revealed that OsRboh-mediated reactive oxygen species levels played important roles in this defence. The results offer novel insights into the regulatory mechanisms of auxin signalling in the rice–RBSDV interaction.     

抗黑条矮缩病水稻品种资源的筛选与鉴定

近年来,水稻黑条矮缩病严重影响我国水稻生产,培育高抗黑条矮缩病的水稻新品种迫在眉睫。获得抗性好的水稻资源是育种的基础。本研究通过对保存的2000份水稻地方品种连续2年设置2个试验点的黑条矮缩病抗性鉴定,初步筛选出连续2年没有发病的资源38份。对这38份中农艺性状较好的6份粳稻资源进行人工室内接虫鉴定,获得高抗水稻黑条矮缩病的水稻资源1份。与感病对照相比,水稻黑条矮缩病毒（Rice black-streaked dwarf virus, RBSDV）在抗病品种体内的表达量下降71.5倍,表明抗病品种对RBSDV在体内的复制有明显的抑制作用。本研究为水稻黑条矮缩病抗病育种和抗性基因的定位、克隆奠定了材料基础。     

Transmission by Laodelphax striatellus Fallen of Rice black‐streaked dwarf virus from Frozen Infected Rice Leaves to Healthy Plants of Rice and Maize

Rice black‐streaked dwarf virus (RBSDV) is transmitted naturally to important crops such as rice, maize, barley and wheat in a persistent manner by the planthoppers, Laodelphax striatellus, Unkanodes sapporona and Unkanodes albifascia. Insect vector transmission tests are the basis for identifying viral incidence, evaluating the resistance of varieties and selecting resistance sources for rice and maize breeding. A simple, rapid and reliable method is described by which virus‐free small brown planthoppers (L. striatellus) acquired RBSDV from frozen infected rice leaves and transmitted it to healthy rice and maize plants. After feeding on frozen infected rice leaves, the planthoppers were tested by RT‐PCR for the presence of virus after 10, 15, and 22 days, respectively. The percentages of RBSDV‐containing insects were 0, 25 and 71.43% of L. striatellus fed on frozen infected rice leaves compared to 0, 28.25 and 71.43% of L. striatellus fed on fresh infected rice leaves, respectively. In transmission tests, three of eight rice seedlings (37.5%) and four of eight maize seedlings (50%) were inoculated by the planthoppers that had fed previously on frozen leaves and had allowed a 22 days latent period and showed typical disease symptoms. As a positive control, four of eight rice seedlings (50%) and four of six maize seedlings (66.67%) became infected. All rice and maize plants expressing disease symptoms were identified as virus‐positive by RT‐PCR. These results indicated that the planthoppers acquired RBSDV from frozen infected leaves and transmitted the virus to healthy plants.     

Efficient Inoculation of Rice black‐streaked dwarf virus to Maize Using Laodelphax striatellus Fallen

Maize rough dwarf disease caused by Rice black‐streaked dwarf virus (RBSDV) is the most important disease of maize in China. Although deploying disease resistant hybrids would be the most effective way to control the disease, development of resistant hybrids has been limited by virus transmission rates that are too low for effective screening. An efficient inoculation technique for RBSDV was developed using Laodelphax striatellus Fallen, in which a virus‐free planthopper colony was developed and viruliferous planthoppers were obtained by allowing a 3‐ to 4‐day acquisition access period on RBSDV‐infected wheat plants. Planthoppers were then allowed a 25‐ to 28‐day latent period on wheat seedlings followed by a 3‐day inoculation access period on two‐to‐three‐leaf stage maize seedlings. By 35 days postinoculation, susceptible hybrid ‘Zhengdan 958’, inbred lines of ‘Ye 107’ and ‘Ye 478’ plants showed 100% RBSDV infection with symptoms of stunting plants, darkening leaves and white waxy swellings on underside of leaves. At tasseling stage, average disease indices were from 96.4 to 100.0%. Enzyme‐linked immunosorbent assays were correlated with the presence of symptoms. The high efficiency of RBSDV transmission obtained using this technique provides a reliable procedure to screen for RBSDV resistance in maize.     

水稻黑条矮缩病传毒昆虫的防治实践与研究

水稻黑条矮缩病 (RBSDV)是由传毒媒介灰飞虱Laodelphaxstriatellus (Fall n)传播所致 ,治虫防病是目前防治水稻黑条矮缩病的重要手段。生产实践证明 ,防治该病应以控制灰飞虱种群数量增长为首选目标 ,把带毒灰飞虱尽可能地扑灭在迁移到水稻秧苗进行传毒侵染之前。因此掌握防治适期 ,选用高效低毒农药 ,切断初次侵染来源 ,才能达到控制该病发生的目的。     

饲食感染SRBSDV的水稻病株对传毒介体白背飞虱相关能源物质含量的影响

南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus, SRBSDV)近年对我国南方稻区水稻生产造成了极大危害。为探讨SRBSDV发生流行的原因,研究了饲食感染SRBSDV水稻病株对其介体昆虫白背飞虱体内能源物质含量的影响。结果表明,饲食感染SRBSDV水稻病株对白背飞虱若虫体内能源物质含量没有明显的影响,但是对雌成虫有明显的影响;饲食感染SRBSDV水稻病株使白背飞虱3日龄和5日龄雌成虫体内可溶性糖及游离氨基酸含量显著升高;此外,饲食感染SRBSDV水稻病株使5日龄雌成虫体内甘油酯含量和可溶性蛋白含量含量明显升高。上述结果证明饲食感染SRBSDV水稻病株会促进白背飞虱雌成虫体内糖类、脂类、氨基酸等物质的积累,有可能会导致其迁飞能力的提升,增加SRBSDV暴发流行的可能性。     

南方水稻黑条矮缩病毒介体昆虫白背飞虱的传毒特性

白背飞虱Sogatella furcifera(Horváth)为传播南方水稻黑条矮缩病病毒(SRBSDV)的媒介昆虫,阐明其传毒特性将有助于了解南方水稻黑条矮缩病的发生流行规律和建立相应的防治方法。本研究通过RT-PCR技术测定了白背飞虱的传毒参数。结果表明,白背飞虱初孵若虫、3龄若虫、5龄若虫、长翅型成虫和短翅型成虫的最短获毒时间分别是11、6、3、2和2min,最长获毒时间分别是19、12、9、8和8min。在26℃时,SRBSDV在不同虫态(初孵若虫、3龄若虫、5龄若虫、长翅型成虫和短翅型成虫)的白背飞虱体内的循回期分别是7～11、5～8、3～7、4～8和3～6d。5龄若虫、长翅型和短翅型成虫在三叶一心稻苗的最短接毒时间为4、5和6min,最长接毒时间为8、10和11min;它们在分蘖初期稻苗上的最短接毒时间分别是5、7和7min,最长接毒时间分别是10、12和12min。白背飞虱获毒后可终身传毒,但不能经白背飞虱卵传毒,单虫最多传毒株数为87株,平均传毒48.3±0.8株。可见,该虫具有较强的获毒能力和传毒能力,秧苗易感染SRBSDV。因此,在防治上应尽量清除田间SRBSDV毒源植物,减少白背飞虱获毒的机会,在秧田期和移栽初期应重点防治飞虱。     

Southern rice black-streaked dwarf virus hijacks SNARE complex of its insect vector for its effective transmission to rice

Vesicular trafficking is an important dynamic process that facilitates intracellular transport of biological macromolecules and their release into the extracellular environment. However, little is known about whether or how plant viruses utilize intracellular vesicles to their advantage. Here, we report that southern rice black-streaked dwarf virus (SRBSDV) enters intracellular vesicles in epithelial cells of its insect vector by engaging VAMP7 and Vti1a proteins in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The major outer capsid protein P10 of SRBSDV was shown to interact with VAMP7 and Vti1a of the white-backed planthopper and promote the fusion of vesicles into a large vesicle, which finally fused with the plasma membrane to release virions from midgut epithelial cells. Downregulation of the expression of either VAMP7 or Vti1a did not affect viral entry and accumulation in the gut, but significantly reduced viral accumulation in the haemolymph. It also did not affect virus acquisition, but significantly reduced the virus transmission efficiency to rice. Our data reveal a critical mechanism by which a plant reovirus hijacks the vesicle transport system to overcome the midgut escape barrier in vector insects and provide new insights into the role of the SNARE complex in viral transmission and the potential for developing novel strategies of viral disease control.     

Molecular Characterization of Southern Rice Blacked‐Streaked Dwarf Virus (SRBSDV) from Vietnam

Southern rice black‐streaked dwarf virus (SRBSDV) is a novel putative member of the genus Fijivirus, family Reoviridae. We report here the genomic sequences of a Vietnamese isolate (SRBSDV‐V). The total genome of SRBSDV‐V has 29 115 nucleotides (nt), nine nt shorter than SRBSDV‐GD or ‐HN, but similar in organization to these two Chinese isolates. Nucleotide diversities among SRBSDV isolates were much lower than those among the corresponding ORFs of the available RBSDV isolates and there was a lower purifying selection pressure on SRBSDV than RBSDV, providing first molecular evidence for the view that SRBSDV is of recent origin. In studies of all available SRBSDV sequences, there was no obvious correlation between geographic distances and phylogenetic distribution. A high frequency of genetic recombination was found among both Chinese and Vietnam SRBSDV isolates, suggesting that recombination may play an important role in the molecular variation and evolution of SRBSDV.     

Heat shock protein 70 is necessary for Rice stripe virus infection in plants

Heat shock proteins 70 (HSP70s) are a highly conserved family of genes in eukaryotes, and are involved in a remarkable variety of cellular processes. In many plant positive‐stranded RNA viruses, HSP70 participates in the construction of a viral replication complex and plays various roles during viral infection. Here, we found increased expression of HSP70 following infection by Rice stripe virus (RSV), a negative‐stranded RNA virus, in both rice (the natural host) and Nicotiana benthamiana (an experimental host). Heat treatment of N. benthamiana (Nb) plants enhanced viral infection, whereas RSV infection was retarded and viral RNAs accumulated at a low level when HSP70 was silenced. In both bimolecular fluorescence complement and in vitro pull‐down assays, the N‐terminus of RSV RNA‐dependent RNA polymerase (RdRp) interacted and co‐localized with the HSP70s of both plants (OsHSP70 and NbHSP70). The localization of the N‐terminus of RdRp when expressed alone was not obviously different from when it was co‐expressed with OsHSP or NbHSP, and vice versa. RSV infection also had no effect on the localization of host HSP70. These results demonstrate that host HSP70 is necessary for RSV infection and probably plays a role in viral replication by interacting with viral RdRp, which provides the first evidence of an interacting host protein related to RSV replication, which has been little studied to date.     

江苏水稻黑条矮缩病毒S10的cDNA克隆序列分析

从来自江苏连云港并在本实验室保存的水稻黑条矮缩病毒接种的病株玉米中提取dsRNA ,采用改进的单引物扩增技术获得了病毒基因组片段S10的cDNA克隆并测定了其全序列。结果表明S10全长 180 1bp ,含有一个ORF ,组织结构与日本报道的RBSDV基本一致 ,核苷酸和推导的氨基酸序列与MRDV的相似性分别为 87.5 %和92 .6 % ,与RBSDV的相似性分别为 93.3%和 96 .4%。该研究也为病毒dsRNA克隆和序列分析奠定了基础     

Expression of rice gall dwarf virus outer coat protein gene (<Emphasis Type="Italic">S8</Emphasis>) in insect cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48–72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.     

Suppression of NS3 and MP is important for the stable inheritance of RNAi-mediated rice stripe virus (RSV) resistance obtained by targeting the fully complementary RSV-CP gene

Rice stripe virus (RSV) is a viral disease that seriously impacts rice production in East Asia, most notably in Korea, China, and Japan. Highly RSV-resistant transgenic japonica rice plants were generated using a dsRNAi construct designed to silence the entire sequence region of the RSV-CP gene. Transgenic rice plants were inoculated with a population of viruliferous insects, small brown planthoppers (SBPH), and their resistance was evaluated using ELISA and an infection rate assay. A correlation between the expression of the RSV-CP homologous small RNAs and the RSV resistance of the transgenic rice lines was discovered. These plants were also analyzed by comparing the expression pattern of invading viral genes, small RNA production and the stable transmission of the RSV resistance trait to the T3 generation. Furthermore, the agronomic trait was stably transmitted to the T4 generation of transgenic plants.     

Characterization of Rice Black-Streaked Dwarf Virus- and Rice Stripe Virus-Derived siRNAs in Singly and Doubly Infected Insect Vector Laodelphax striatellus

Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi) which generates viral-derived small interfering RNAs (siRNAs). However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus) was infected by Rice black-streaked dwarf virus (RBSDV) (Reoviridae; Fijivirus), more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV), a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5′- and 3′-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.     

转人工miRNA抗玉米粗缩病毒载体玉米的培育及其抗病能力

玉米是重要的粮食作物,水稻黑条矮缩病毒(RBSDV)是玉米粗缩病的病原,由其引起的玉米粗缩病给玉米生产造成重大损失。利用人工mi RNA构建抗病毒植物的技术已经在多种植物中被证明有效,但是在玉米中的尝试未见报道。实验根据玉米zea-mi R159a的前体序列和RBSDV基因组中编码功能蛋白的基因和基因沉默抑制子的序列信息设计引物,构建了用于沉默RBSDV编码基因和基因沉默抑制子的ami RNA(Artificial mi RNA)基因。构建p CAMBIA3301-121-ami RNA植物表达载体,利用农杆菌介导法转化玉米自交系综31(Z31)。对转基因玉米进行分子检测,选择mi RNA表达量高的纯合体株系进行自然发病实验,按0-4的分级标准调查玉米粗缩病的严重度。结果表明,转抗粗缩病毒人工mi RNA载体玉米纯合体株系的抗病表现好于野生型玉米,其中针对基因组6的S6-mi R159转基因玉米抗病情况较好。研究表明利用人工mi RNA技术构建抗病毒病玉米新品种是可行的。     

Overexpression of Rice Black-Streaked Dwarf Virus P7-1 in Arabidopsis Results in Male Sterility Due to Non-Dehiscent Anthers

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, is propagatively transmitted by the small brown planthopper (Laodelphax striatellus Fallén). RBSDV causes rice black-streaked dwarf and maize rough dwarf diseases, which lead to severe yield losses in crops in China. Although several RBSDV proteins have been studied in detail, the functions of the nonstructural protein P7-1 are still largely unknown. To investigate the role of the P7-1 protein in virus pathogenicity, transgenic Arabidopsis thaliana plants were generated in which the P7-1 gene was expressed under the control of the 35S promoter. The RBSDV P7-1-transgenic Arabidopsis plants (named P7-1-OE) were male sterility. Flowers and pollen from P7-1-transgenic plants were of normal size and shape, and anthers developed to the normal size but failed to dehisce. The non-dehiscent anthers observed in P7-1-OE were attributed to decreased lignin content in the anthers. Furthermore, the reactive oxygen species levels were quite low in the transgenic plants compared with the wild type. These results indicate that ectopic expression of the RBSDV P7-1 protein in A. thaliana causes male sterility, possibly through the disruption of the lignin biosynthesis and H₂O₂-dependent polymerization pathways.     

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2‐YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2‐YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2‐YFP fluorescence were affected in fibrillarin‐silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.