Rice Black-streaked Dwarf Virus Research Articles

ITRAQ-based quantitative proteomics suggests mitophagy involvement after Rice black-streaked dwarf virus acquisition in insect vector small brown planthopper Laodelphax striatellus Fallén

Plant viruses trigger numerous responses in their insect vectors. Using iTRAQ-based quantitative proteomics analysis, early responses of the insect vector, the small brown planthopper (Laodelphax striatellus Fallén, SBPH), after acquiring Rice black-streaked dwarf virus (RBSDV) at 3 days and 5 days post first access to diseased plants (padp) were revealed. A total of 582 differentially abundant proteins (DAPs) in SBPH with a fold change >1.500 or <0.667 (p-value < 0.05) were identified. The proteomic analysis in SBPH at 3 days padp revealed 106 highly abundant proteins and 193 of low abundance, while 5 days padp revealed 214 highly abundant proteins and 182 of low abundance. Among them, 51 highly abundant proteins and 42 of low abundance were shown consistently at both 3 days and 5 days padp. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapping and Gene Ontology (GO) term classification suggested impairment of mitochondria in SBPH after RBSDV acquisition, and the 77 out of 582 differentially abundant SBPH proteins analyzed by the STRING program revealed the interaction network of the mitochondrial DAPs, showing an overall down-regulation of mitochondrial proteins including the electron transport chain proteins and mitochondrial ribosome proteins. The high abundance of Parkin at 5 days padp suggests that activation of mitophagy induced degradation of mitochondria occurred. Further verification of autophagy/mitophagy-related genes by reverse-transcription quantitative RT-PCR (RT-qPCR) in SBPH after RBSDV acquisition showed up-regulation of the autophagy receptors Optineurin (OPTN), Sequestosome-1 (SQSTM1, also known as p62) and Tax1-binding protein 1 (TAX1BP1) which targets ubiquitinated damaged mitochondria during mitophagy. The phosphorylation of the three autophagy receptors may be up-regulated through an increase of transcription level TRAF-associated NFκB activator (TANK)-binding kinase 1 (TBK1). As a result, an overall reduction in the abundance of mitochondrial proteins was observed and the selective autophagic degradation was up-regulated through increased transcription level of OPTN, p62/SQSTM1, TAX1BP1 and TBK1. Therefore, acquisition of RBSDV associated with up-regulated autophagy and selective mitochondrial degradation in SBPH suggest prevention of mitochondrial-mediated apoptosis and extension of the vector life span. Biological significanceRBSDV causes severe yield loss in rice plants. RBSDV is transmitted efficiently only through SBPH. It is important to understand how RBSDV infects SBPH in a persistent, circulative and propagative manner. However, there has been no study on the interaction between RBSDV and SBPH at the early acquisition stage using a proteomics approach. In this study, we combined iTRAQ technique and LC-MS/MS to analyze the vector proteomics at both the initial and latent infection stages after RBSDV acquisition and verified the results by RT-qPCR. Our results revealed that significantly low DAPs were involved in various pathways, including biosynthesis of secondary metabolites, ribosomes, carbon metabolism, biosynthesis of amino acids and TCA cycle. Further clustering of the DAPs revealed significant changes in SBPH mitochondria, including decreased proteins in mitochondrial ribosomes and electron transport chain complex I, II and V. On the other hand, there was a high abundance of Parkin, suggesting the occurrence of mitochondria damage and subsequent Parkin-mediated mitophagy for clearance of impaired mitochondria. Moreover, the decreased level of PMPCB in terms of gene expression and protein abundance suggested decreased PINK1 turnover, promoting Parkin/PINK1-mediated mitophagy. Further analysis on autophagy/mitophagy-related gene transcription level indicated up-regulation of OPTN, p62/SQSTM1, TAX1BP1 and TBK1, promoting selective autophagy in SBPH after RBSDV acquisition. These findings provided new insights into the effects of RBSDV on SBPH after early acquisition by selective degradation of mitochondria, especially on reprogramming of energy metabolism and decreased mitochondria biogenesis, to prevent apoptosis and prolong the life span of SBPH post virus acquisition.

Interplay of Rice Stripe Virus and Rice Black Streaked Dwarf Virus during Their Acquisition and Accumulation in Insect Vector.

Plant viruses transmitted by hemipteran vectors commonly cause losses to crop production. Rice stripe virus (RSV) and rice black streaked dwarf virus (RBSDV) are transmitted to rice plants by the same vector, the small brown planthopper (SBPH), Laodelphax striatellus Fallén, in a persistent propagative manner. However, rarely do the respective diseases they cause occur simultaneously in a field. Here, we determined the acquisition efficiency of RSV and RBSDV when acquired in succession or simultaneously by SBPH. When RBSDV was acquired first, RSV acquisition efficiency was significantly lower than when only acquiring RSV. However, RBSDV acquisition efficiency from insects that acquired RSV first was not significantly different between the insects only acquiring RBSDV. Immunofluorescence assays showed that the acquisition of RBSDV first might inhibit RSV entry into midgut epithelial cells, but RSV did not affect RBSDV entry. SBPHs were more likely to acquire RBSDV when they were feeding on plants coinfected with the two viruses. When RBSDV was acquired before RSV, RBSDV titer was significantly higher and RSV titer first declined, then increased compared to when only acquiring RBSDV or RSV. Only 5% of the SBPHs acquired both viruses when feeding on plants coinfected with RSV and RBSDV. These results provide a better understanding of the interaction between two persistent viruses when present in the same vector insect and explain why RSV and RBSDV occur in intermittent epidemics.

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.

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts.

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

Genome-Wide Analysis of the RAV Transcription Factor Genes in Rice Reveals Their Response Patterns to Hormones and Virus Infection.

The RAV family is part of the B3 superfamily and is one of the most abundant transcription factor families in plants. Members have highly conserved B3 or AP2 DNA binding domains. Although the RAV family genes of several species have been systematically identified from genome-wide studies, there has been no comprehensive study to identify rice RAV family genes. Here, we identified 15 genes of the RAV family in the rice genome and analyzed their phylogenetic relationships, gene structure, conserved domains, and chromosomal distribution. Based on domain similarity and phylogenetic topology, rice RAV transcription factors were phylogenetically clustered into four groups. qRT-PCR analyses showed that expression of these RAV genes was significantly up-regulated or down-regulated by plant hormone treatments, including BL, NAA, IAA, MeJA, and SA. Most of the rice RAV genes were dramatically down-regulated in response to rice stripe virus (RSV) and mostly up-regulated in response to Southern rice black-streaked dwarf virus (SRBSDV). These results suggest that the rice RAV genes are involved in diverse signaling pathways and in varied responses to virus infection.

MicroRNA-315-5p promotes rice black-streaked dwarf virus infection by targeting a melatonin receptor in the small brown planthopper.

MicroRNAs (miRNAs), a class of small non-coding endogenous RNAs, play key roles in various biological processes. Most plant viruses are transmitted by insect vectors. However, little is known about the function of miRNAs on plant virus-insect host interaction. We investigated the role of miR-315-5p in regulation of plant viral infection in insects using a rice black-streaked dwarf virus (RBSDV) and small brown planthopper (SBPH) interaction system. Our results showed that miR-315-5p had the highest expression level in 2nd-instar nymph, and was highly expressed in the salivary gland and midgut in SBPH. miR-315-5p was in response to and regulated RBSDV infection in SBPH. Injection of miR-315-5p mimic, agomir-315, significantly increased the RBSDV accumulation, whereas injection of miR-315-5p inhibitor, antagomir-315, reduced virus accumulation in SBPH. Furthermore, a melatonin receptor was identified as a target gene of miR-315-5p by the dual luciferase reporter assay. Knockdown of the melatonin receptor significantly increased the expression of RBSDV coat protein gene S10 and replication related genes, S5-1, S6, and S9-1. Furthermore, treatment with melatonin receptor antagonist luzindole and activator agomelatine significantly increased and reduced RBSDV accumulation in SBPH, respectively. Compared to the control, miR-315-5p did not affect the efficiency of RBSDV acquisition in SBPH. However, the efficiency of RBSDV transmission was significantly reduced after injecting antagomir-315. Taken together, our data reveal that miR-315-5p is beneficial for RBSDV infection in its insect vector by directly targeting a melatonin receptor. These findings provide a new insight to the function of miRNAs in virus-insect vector interaction. © 2021 Society of Chemical Industry.

Molecular phylogenetic analysis shows that causal agent of maize rough dwarf disease in Iran is closer to rice black-streaked dwarf virus

Maize rough dwarf disease (MRDD) is one of the most important viral diseases around the world. Rice black-streaked dwarf virus (RBSDV) and maize rough dwarf virus (MRDV) cause MRDD in East Asia and Mediterranean regions, respectively. RBSDV and MRDV are similar in many features such as host range, disease symptoms, vector, and serological relationships, which make it hard to differentiate them based on serological and biological properties. However, molecular analyses as powerful tools could be used for differentiating species of fijiviruses. In this study, the complete genome sequence, genetic variation of 13 isolates using the nucleotide sequence of segment 10 ORF, and host range of MRDD causal agent in Iran were determined. Whole-genome comparisons and phylogenetic analysis showed that Iranian isolate shared high nucleotide sequence identities ranging from 86.5–91.3% and closest evolutionary relationship to the RBSDV Chinese isolates. The Iranian isolates showed over 99% homology based on amino acid sequence of ORF10 and were grouped in one clade through phylogenetic analysis. The results revealed that ORF10 of MRDD causal agent was under negative selection. Iranian isolate of MRDD was experimentally transmitted to maize, rice, barley, wheat, millet, oat, rye, and some grasses by Loadelphax striatellus with about 32% efficiency. The results of this study revealed that the MRDD causal agent in Iran had the closest genetic similarity to RBSDV demonstrating it is closer to RBSDV than MRDV.

A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions.

In the field, many insect-borne crop viral diseases are more suitable for maintenance and spread in hot-temperature areas, but the mechanism remains poorly understood. The epidemic of a planthopper (Sogatella furcifera)-transmitted rice reovirus (southern rice black-streaked dwarf virus, SRBSDV) is geographically restricted to southern China and northern Vietnam with year-round hot temperatures. Here, we reported that two factors of endoplasmic reticulum-associated degradation (ERAD) machinery, the heat shock protein DnaJB11 and ER membrane protein BAP31, were activated by viral infection to mediate the adaptation of S. furcifera to high temperatures. Infection and transmission efficiencies of SRBSDV by S. furcifera increased with the elevated temperatures. We observed that high temperature (35°C) was beneficial for the assembly of virus-containing tubular structures formed by nonstructural protein P7-1 of SRBSDV, which facilitates efficient viral transmission by S. furcifera. Both DnaJB11 and BAP31 competed to directly bind to the tubule protein P7-1 of SRBSDV; however, DnaJB11 promoted whereas BAP31 inhibited P7-1 tubule assembly at the ER membrane. Furthermore, the binding affinity of DnaJB11 with P7-1 was stronger than that of BAP31 with P7-1. We also revealed that BAP31 negatively regulated DnaJB11 expression through their direct interaction. High temperatures could significantly upregulate DnaJB11 expression but inhibit BAP31 expression, thereby strongly facilitating the assembly of abundant P7-1 tubules. Taken together, we showed that a new temperature-dependent protein quality control pathway in the ERAD machinery has evolved for strong activation of DnaJB11 for benefiting P7-1 tubules assembly to support efficient transmission of SRBSDV in high temperatures. We thus deduced that ERAD machinery has been hitchhiked by insect-borne crop viruses to enhance their transmission in tropical climates.

Detection of Southern Rice Black-Streaked Dwarf Virus Using Western Blotting With P6

The southern rice black-streaked dwarf virus (SRBSDV) is a severe threat to the yield and quality of rice products worldwide. Traditional detection methods for diagnosing SRBSDV infection show several false positives and thus provide inaccurate findings. However, Western blotting (WB) can precisely solve this problem. In this study, P6—a viral RNA-silencing suppressor—was expressed and purified in vitro. Two polyclonal P6 antibodies were obtained and quantified by enzyme-linked immunosorbent assay and WB. Subsequently, WB was performed using the P6 antibodies to identify SRBSDV antigens derived from the suspected rice samples collected from nine districts in Guizhou, China. The assay results showed that Libo, Pingtang, Huishui, Dushan, and Anshun districts had experienced an SRBSDV outbreak. The virus content in the sampled rice tissues was quantified by WB. Our results revealed that SRBSDV mainly accumulated in rice stems rather than rice leaves. Thus, the findings of our study show that the SRBSDV P6 antibody can be used in WB for detecting and monitoring SRBSDV infection in infected rice plants.

Genome-Wide Association Study on Resistance to Rice Black-Streaked Dwarf Disease Caused by Rice black-streaked dwarf virus.

Rice black-streaked dwarf disease caused by Rice black-streaked dwarf virus (RBSDV) is one of the most destructive viral diseases of rice. Thus, it is imperative that resistant rice germplasms are screened for novel RBSDV-resistant genes. RBSDV resistance of a diverse global collection comprising 1,953 rice accessions was evaluated under natural conditions across 3 years. The average disease incidences of the Xian/indica (XI) subgroup were significantly lower than those of the Geng/japonica (GJ) subgroup. Interestingly, most XI-1A accessions in the Xian subgroup were significantly more susceptible than XI-1B accessions, even though XI-1A and XI-1B have a close phylogenetic relationship. Four Xian accessions stably and highly resistant to RBSDV were consistently identified in 2 years. Ten genomic regions (GRs) with 147 single nucleotide polymorphisms associated with RBSDV resistance were detected by a single-locus genome-wide association study (GWAS), of which five were repeatedly identified in a multilocus GWAS. Two previously reported GRs, grRBSDV-6.1 and grRBSDV-6.3, which were repeatedly detected as stably and highly associated with RBSDV resistance, contained 17 and seven genes, respectively, with significant differences of resistance among haplotypes. Haplotype analyses of the candidate genes LOC_Os06g03150 in grRBSDV-6.1 and LOC_Os06g31190 in grRBSDV-6.3 suggested that the former gene is mainly associated with the differentiation of resistance within the Xian subgroup and the latter gene mainly explains the difference in the resistance between Xian and Geng. Another three novel resistance GRs (grRBSDV-1.1, grRBSDV-7.1, and grRBSDV-9.1) were identified. Our findings may enhance the application of disease-resistant rice germplasms for breeding RBSDV-resistant varieties.

RNAi silencing of rice black-streaked dwarf virus P10 and two insect vector genes to reduce virus transmission protects rice plants against RBSDV

ABSTRACT Rice black-streaked dwarf virus (RBSDV) is a virus transmitted by Laodelphax striatellus (L. striatellus), which poses a serious threat to rice production. P10, a major outer capsid protein of RBSDV, which plays a key role in viral infection. In this study, two L. striatellus transmission-related proteins, novel cuticular protein (NCuP) and receptor for activated protein kinase C (RACK), were identified as interacting with P10. Results demonstrated that virus-induced NCuP and RACK were silenced using double-stranded RNA (dsRNA) to reduce the quantity of virus in L. striatellus. The detection results after inoculation of RBSDV by L. striatellus showed that rice plants sprayed with dsP10+NCuP and dsP10+RACK and transgenic rice plants expressing both had higher disease resistance than those expressing dsP10, dsNCuP and dsRACK alone. Therefore, simultaneously interfering with both the expression of viral protein and the proteins that interact with them from insect vectors could enhance the control of viral transmission.

Maize AKINβγ Proteins Interact with P8 of Rice Black Streaked Dwarf Virus and Inhibit Viral Infection.

Rice black streaked dwarf virus (RBSDV) is an important agent causing maize rough dwarf disease, whereas the host factors responding to RBSDV infection are poorly understood. To uncover the molecular interactions between RBSDV and maize, a yeast two-hybrid screen of a maize cDNA library was carried out using the viral P8 protein as a bait. ZmAKINβγ-1 and ZmAKINβγ-2 (βγ subunit of Arabidopsis SNF1 kinase homolog in maize) possessing high sequence similarities (encoded by two gene copies) were identified as interaction partners. Their interactions with P8 were confirmed in both Nicotiana benthamiana cells and maize protoplasts by bimolecular fluorescence complementation assay. The accumulation levels of ZmAKINβγ mRNAs were upregulated at the stage of the viral symptoms beginning to appear and then downregulated. ZmAKINβγs are putative regulatory subunits of the SnRK1 complex, a core regulator for energy homeostasis. Knockdown of ZmAKINβγs in maize regulated the expression levels of the genes involved in sugar synthesis or degradation, and also the contents of both glucose and sucrose. Importantly, downregulation of ZmAKINβγs expressions facilitated the accumulation of RBSDV in maize. These results implicate a role of ZmAKINβγs in the regulation of primary carbohydrate metabolism, and in the defense against RBSDV infection.

Changes in Transcriptome and Gene Expression in Sogatella furcifera (Hemiptera: Delphacidae) in Response to Cycloxaprid.

The white-backed planthopper, Sogatella furcifera (Horváth), causes substantial damage to crops by direct feeding or virus transmission, especially southern rice black-streaked dwarf virus, which poses a serious threat to rice production. Cycloxaprid, a novel cis-nitromethylene neonicotinoid insecticide, has high efficacy against rice planthoppers, including imidacloprid-resistant populations. However, information about the influence of cycloxaprid on S. furcifera (Hemiptera: Delphacidae) at the molecular level is limited. Here, by de novo transcriptome sequencing and assembly, we constructed two transcriptomes of S. furcifera and profiled the changes in gene expression in response to cycloxaprid at the transcription level. We identified 157,906,456 nucleotides and 131,601 unigenes using the Illumina technology from cycloxaprid-treated and untreated S. furcifera. In total, 38,534 unigenes matched known proteins in at least one database, accounting for 29.28% of the total unigenes. The number of coding DNA sequences was 28,546 and that of amino acid sequences in the coding region was 22,299. In total, 15,868 simple sequence repeats (SSRs) were identified. The trinucleotide repeats accounted for 45.1% (7,157) of the total SSRs and (AAG/CTT)n were the most frequent motif. There were 359 differentially expressed genes that might have been induced by cycloxaprid. There were 131 upregulated and 228 downregulated genes. Twenty-two unigenes might be involved in resistance against cycloxaprid, such as cytochrome P450, glutathione S-transferase (GST), acid phosphatase (ACP), and cadherin. Our study provides vital information on cycloxaprid-induced resistance mechanisms, which will be useful to analyze the molecular mechanisms of cycloxaprid resistance and may lead to the development of novel strategies to manage S. furcifera.

Selection of DNA Aptamers for Subcellular Localization of RBSDV P10 Protein in the Midgut of Small Brown Planthoppers by Emulsion PCR-Based SELEX.

Rice black-streaked dwarf virus (RBSDV), classified under the Reoviridae, Fijivirus genus, caused an epidemic in the eastern provinces of China and other East Asian countries and resulted in severe yield loss in rice and wheat production. RBSDV is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus Fallén) in a persistent manner. In order to provide a stable and cost-effective detection probe, in this study we selected three DNA aptamers (R3, R5 and R11) by an optimized, standardized and time saving emulsion PCR-based SELEX, for the detection of RBSDV outer-shell P10 protein for in situ localization studies in the midgut of SBPH. The specificity of these three DNA aptamers was tested through detection of the P10 protein using an enzyme-linked oligonucleotide assay (ELONA) and aptamer-based dot-blot ELISA. All three DNA aptamers can be used to detect RBSDV P10 protein by immunofluorescent labeling in the midgut of RBSDV-infected SBPH. These data show that the selected aptamers can be used for the detection of RBSDV P10 protein in vitro and in vivo. This is the first report of aptamers being selected for detection of a rice virus capsid protein.

Comprehensive codon usage analysis of rice black-streaked dwarf virus based on P8 and P10 protein coding sequences.

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus of the family Reoviridae and is an important pathogen that damages rice, maize and wheat worldwide. Previously, several reports have described the genetic variation and population structure of RBSDV. However, the details of the evolutionary changes, synonymous codon usage patterns and host adaptation of the virus are largely unclear. Here, we performed a detailed analysis of the codon usage and host adaptability of RBSDV based on 130 full-length P8 and 234 full-length P10 sequences. Infrequent recombination and frequent segment reassortment influence the genomic evolution of RBSDV. Our phylogenetic analysis found three and four lineages based on the P8 and P10 non-recombinant sequences respectively. We found relatively stable and conserved genomic composition with lower codon usage choice in the RBSDV P8 and P10 protein coding sequences. Both ENC-plot and neutrality-plot analyses showed that natural selection is the key factor that shapes the codon usage pattern of RBSDV. Codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses indicated strong correlation between RBSDV and rice rather than maize, wheat or Laodelphax striatellus. Our study provides deep insight into the evaluation of the codon usage pattern and adaptive evolution of RBSDV based on P8 and P10 sequences and should be taken into consideration for the prevention and control of this virus.

Systematic Identification and Functional Analysis of Circular RNAs During Rice Black-Streaked Dwarf Virus Infection in the Laodelphax striatellus (Fallén) Midgut.

Circular RNAs (circRNAs) are endogenous RNAs that have critical regulatory roles in numerous biological processes. However, it remains largely unknown whether circRNAs are induced in response to plant virus infection in the insect vector of the virus as well as whether the circRNAs regulate virus infection. Rice black-streaked dwarf virus (RBSDV) is transmitted by Laodelphax striatellus (Fallén) in a persistent propagative manner and causes severe losses in East Asian countries. To explore the expression and function of circRNAs in the regulation of virus infection, we determined the circRNA expression profile in RBSDV-free or RBSDV-infected L. striatellus midgut tissues by RNA-Seq. A total of 2,523 circRNAs were identified, of which thirteen circRNAs were differentially expressed after RBSDV infection. The functions of these differentially circRNAs were predicted by GO and KEGG pathway analyses. The expression changes of five differentially expressed circRNAs and eight parental genes were validated by RT-qPCR. The circRNAs-microRNAs (miRNAs) interaction networks were analyzed and two miRNAs, which were predicted to bind circRNAs, were differentially expressed after virus infection. CircRNA2030 was up-regulated after RBSDV infection in L. striatellus midgut. Knockdown of circRNA2030 by RNA interference inhibited the expression of its predicted parental gene phospholipid-transporting ATPase (PTA) and enhanced RBSDV infection in L. striatellus. However, none of the six miRNAs predicting to bind circRNA2030 was up-regulated after circRNA2030 knockdown. The results suggested that circRNA2030 might affect RBSDV infection via regulating PTA. Our results reveal the expression profile of circRNAs in L. striatellus midgut and provide new insight into the roles of circRNAs in virus–insect vector interaction.

Transcriptome Analysis of Rice Reveals the lncRNA-mRNA Regulatory Network in Response to Rice Black-Streaked Dwarf Virus Infection.

The plant genome can produce long non-coding RNAs (lncRNAs), some of which have been identified as important regulators of gene expression. To better understand the response mechanism of rice plants to Rice black-streaked dwarf virus (RBSDV) infection, we performed a comparative transcriptome analysis between the RBSDV-infected and non-infected rice plants. A total of 1342 mRNAs and 22 lncRNAs were identified to be differentially expressed after RBSDV infection. Most differentially expressed transcripts involved in the plant–pathogen interaction pathway were upregulated after RBSDV infection, indicating the activation of rice defense response by RBSDV. A network of differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) was then constructed. In this network, there are 56 plant–pathogen interaction-related DEmRNAs co-expressing with 20 DElncRNAs, suggesting these DElncRNAs and DEmRNAs may play essential roles in rice innate immunity against RBSDV. Moreover, some of the lncRNA–mRNA regulatory relationships were experimentally verified in rice calli by a quick and effective method established in this study. Three DElncRNAs were selected to be tested, and the results indicated that five mRNAs were found to be regulated by them. Together, we give a whole landscape of rice mRNAs and lncRNAs in response to RBSDV infection, and a feasible method to rapidly verify the lncRNA–mRNA regulatory relationship in rice.

Development of marker-free rice with stable and high resistance to rice black-streaked dwarf virus disease through RNA interference.

The rice black‐streaked dwarf virus (RBSDV) disease causes severe rice yield losses in Asia. RNA interference (RNAi) has been widely applied to develop antiviral varieties in plants. So far, only a few studies reported the application of RNAi in rice against RBSDV and most of them are lack of enough data to support its breeding potential, which limited the progress on developing RBSDV‐resistant variety. In this study, we generated three RNAi constructs to specifically target three RBSDV genes (S1, S2 and S6), respectively. We confirmed that RNAi targeting RBSDV S6 conferred rice with almost full immunity to RBSDV through phenotyping test in eight consecutive years in both artificial inoculation and field trials, while RNAi of S1 or S2 only leads to partially increased resistance. The S6RNAi was also found conferring strong resistance to southern rice black‐streaked dwarf virus (SRBSDV), a novel species closely related to RBSDV that outbroke recently in Southern China. In particular, no adverse effects on agronomical and developmental traits were found in S6RNAi transgenic lines. The marker‐free transgenic lines with S6RNAi, driven by either maize ubiquitin‐1 promoter or rice rbcS green tissue expression promoter, in elite rice background should have great potential in breeding of resistant varieties to both RBSDV and SRBSDV and provide a basis for further safety evaluation and commercial application.

Comprehensive Identification and Characterization of Long Non-coding RNAs Associated With Rice Black-Streaked Dwarf Virus Infection in Laodelphax striatellus (Fallén) Midgut

Long non-coding RNAs (lncRNAs) are involved in a variety of biological functions through transcriptional and post-transcriptional regulation. However, little is known about their functions in the process of insect mediated virus transmission. In the present study, we analyzed using RNA-Seq, the lncRNAs that were differentially expressed in response to Rice black-streaked dwarf virus (RBSDV) infection in Laodelphax striatellus (Fallén) midgut. A total of 13,927 lncRNAs were identified and over 69% were assigned to intergenic regions. Among them, 176 lncRNAs were differentially expressed and predicted to target 168 trans-regulatory genes. Ten differentially expressed lncRNAs were selected and their expression changes were validated by RT-qPCR. KEGG analysis showed that these target genes were enriched in the essential biological process, such as Purine metabolism, Valine, leucine and isoleucine degradation, and Fatty acid elongation. The expression levels of the differentially expressed lncRNAs and the predicted target genes that were significantly enriched in the Human papillomavirus infection pathway were analyzed by RT-qPCR. The results showed that several lncRNAs were co-expressed with their target genes. One of the lncRNAs called MSTRG15394 and its target gene, encoding a secreted protease inhibitor (PI), were up-regulated at the transcriptional level after RBSDV infection. Knockdown of MSTRG15394 could down-regulate the PI expression at mRNA level. Inhibition of either MSTRG15394 or PI expression by RNA interference promoted RBSDV accumulation in L. striatellus midgut. Our finding provides new insights into the function of lncRNAs in regulating virus infection in an important insect vector.

Alterations of Rice (Oryza sativa L.) DNA Methylation Patterns Associated with Gene Expression in Response to Rice Black Streaked Dwarf Virus.

Rice black-streaked dwarf virus (RBSDV) causes severe yield losses in rice (Oryza sativa L.) in China. Studies have shown that the mechanisms of DNA methylation-mediated plant defense against DNA viruses and RNA viruses are different. However, in rice its function in response to infection of RBSDV, a double-stranded RNA virus, remains unclear. In this study, high-throughput single-base resolution bisulfite sequencing (BS-Seq) was carried out to analyze the distribution pattern and characteristics of cytosine methylation in RBSDV-infected rice. Widespread differences were identified in CG and non-CG contexts between the RBSDV-infected and RBSDV-free rice. We identified a large number of differentially methylated regions (DMRs) along the genome of RBSDV-infected rice. Additionally, the transcriptome sequencing analysis obtained 1119 differentially expressed genes (DEGs). Correlation analysis of DMRs-related genes (DMGs) and DEGs filtered 102 genes with positive correlation and 71 genes with negative correlation between methylation level at promoter regions and gene expression. Key genes associated with maintaining DNA methylation in rice were analyzed by RT-qPCR and indicated that OsDMT702 might be responsible for the global increase of DNA methylation level in rice under RBSDV stress. Our results suggest important roles of rice DNA methylation in response to RBSDV and provide potential target genes for rice antiviral immunity.