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Background: MicroRNAs (miRNAs) play a key role in various biological processes by influencing the translation of target messenger RNAs (mRNAs) through post-transcriptional regulation. The miR-184-3p has been identified as an abundant conserved miRNA in insects. However, less is known about its functions in insect-plant virus interactions., Results: The function of miR-184-3p in regulation of plant viral infection in insects was investigated using a rice black-streaked dwarf virus (RBSDV) and Laodelphax striatellus (Fallén) interaction system. We found that the expression of miR-184-3p increased in L. striatellus after RBSDV infection. Injection of miR-184-3p mimics increased RBSDV accumulation, while treatment with miR-184-3p antagomirs inhibits the viral accumulation in L. striatellus. Ken, a zinc finger protein, was identified as a target of miR-184-3p. Knockdown of Ken increased the virus accumulation and promoted RBSDV transmission by L. striatellus., Conclusion: This study demonstrates that RBSDV infection induces the expression of miR-184-3p in its insect vector L. striatellus. The miR-184-3p targets Ken to promote RBSDV accumulation and transmission. These findings provide a new insight into the function of the miRNAs in regulating plant viral infection in its insect vector. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

MicroRNAs (miRNAs) are non-coding RNAs, which, as members of the RNA interference pathway, play a pivotal role in antiviral infection. Almost 80% of plant viruses are transmitted by insect vectors; however, little is known about the interaction of the miRNAs of insect vectors with plant viruses. Here, we took rice black-streaked dwarf virus (RBSDV), a devastating virus to rice production in eastern Asia, and the small brown planthopper, (SBPH, Laodelphax striatellus ) as a model to investigate the role of microRNA750-3p (miR750-3p) in regulating viral transmission. Our results showed that Ls-miR750-3p was downregulated in RBSDV-infected SBPH and predominately expressed in the midgut of SBPH. Injection with miR750-3p agomir significantly reduced viral accumulation, and the injection with the miR750-3p inhibitor, antagomir-750-3p, dramatically promoted the viral accumulation in SBPH, as detected using RT-qPCR and Western blotting. The processing of precursor 7 ( POP7 ), a subunit of RNase P and RNase MRP, was screened, identified, and verified using a dual luciferase reporter assay as one target of miR750-3p. Knockdown of POP7 notably increased RBSDV viral propagation in SBPH and then increased the viral transmission rate by SBPH. Taken together, our data indicate that miR750-3p targets POP7 to suppress RBSDV infection in its insect vector. These results enriched the role of POP7 in modulating virus infection in host insects and shared new insight into the function of miRNAs in plant virus and insect vector interaction.

Hosts can initiate macroautophagy/autophagy as an antiviral defense response, while viruses have developed multiple ways to evade the host autophagic degradation. However, little is known as to whether viruses can target lipids to subvert autophagic degradation. Here, we show that a low abundant signaling lipid, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ), is required for rice black-streaked dwarf virus (RBSDV) to evade the autophagic degradation in the insect vector Laodelphax striatellus . RBSDV binds to PtdIns(3,5)P 2 and elevates its level through its main capsid protein P10, leading to inhibited autophagy and promoted virus propagation. Furthermore, we show that PtdIns(3,5)P 2 inhibits the autophagy pathway by preventing the fusion of autophagosomes and lysosomes through activation of Trpml (transient receptor potential cation channel, mucolipin), an effector of PtdIns(3,5)P 2 . These findings uncover a strategy whereby a plant virus hijacks PtdIns(3,5)P 2 via its viral capsid protein to evade autophagic degradation and promote its survival in insects.

Rice black-streaked dwarf virus (RBSDV) is an important reovirus that infects both plants and its transmission vector small brown planthopper, causing severe crop loss. High affinity binding between RBSDV P10 and PI(3,5)P 2 lipid layer was measured using biolayer interferometry (BLI). Subcellular co-localization of PI(3,5)P 2 and RBSDV P10 was observed on membranous structures in insect cells with stochastic optical reconstruction microscopy (STORM) imaging. Putative interacting sites of PI(3,5)P 2 lipid on a computational predicted RBSDV P10 structure were mapped to its "C-domain" (250-470 aa), using HDXMS data. The BLI and STORM results showed binding and co-localization of RBSDV P10, and PI(3,5)P 2 on vesicle-like membranous structures were corroborated with the prediction of the binding interface. Understanding the lipid binding sites on viral proteins will lead to developing strategies to block viral-lipid interaction and disrupt viral pathogenesis in insect vectors and to block virus transmission and achieve disease control of crops in the field., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 SIGNIFICANCE: RBSDV 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: 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., Results: 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., Conclusion: 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., (© 2021 Society of Chemical Industry.)

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.

Rice black-streaked dwarf virus (RBSDV) was known to be transmitted by the small brown planthopper (SBPH) in a persistent, circulative and propagative manner in nature. Here, we show that RBSDV major outer capsid protein (also known as P10) suppresses the protein kinase C (PKC) activity of SBPH through interacting with the receptor for activated protein kinase C 1 (LsRACK1). The N terminal of P10 (amino acids (aa) 1-270) and C terminal of LsRACK1 (aa 268-315) were mapped as crucial for the interaction. Confocal microscopy and subcellular fractionation showed that RBSDV P10 fused to enhanced green fluorescent protein formed vesicular structures associated with endoplasmic reticulum (ER) membranes in Spodoptera frugiperda nine cells. Our results also indicated that RBSDV P10 retargeted the initial subcellular localization of LsRACK1 from cytoplasm and cell membrane to ER and affected the function of LsRACKs to activate PKC. Inhibition of RACK1 by double stranded RNA-induced gene silencing significantly promoted the replication of RBSDV in SBPH. In addition, the PKC pathway participates in the antivirus innate immune response of SBPH. This study highlights that RACK1 negatively regulates the accumulation of RBSDV in SBPH through activating the PKC signalling pathway, and RBSDV P10 changes the subcellular localization of LsRACK1 and affects its function to activate PKC. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

RNA interference (RNAi) is a conserved mechanism against viruses in plants and animals. It is thought to inactivate the viral genome by producing virus-derived small interfering RNAs (vsiRNAs). Rice black-streaked dwarf virus (RBSDV) is transmitted to plants by the small brown planthopper (Laodelphax striatellus), and seriously threatens production of rice in East Asia, particularly Oryza sativa japonica subspecies. Through deep sequencing, genome-wide comparisons of RBSDV-derived vsiRNAs were made between the japonica variety Nipponbare, and the indica variety 9311. Four small RNA libraries were constructed from the leaves and shoots of each variety. We found 659,756 unique vsiRNAs in the four samples, and only 43,485 reads were commonly shared. The size distributions of vsiRNAs were mostly 21- and 22-nt long, and A/U bias (66-68%) existed at the first nucleotide of vsiRNAs. Additionally, vsiRNAs were continuously but heterogeneously distributed along S1-S10 segments of the RBSDV genome. Distribution profiles of vsiRNA hotspots were similar in different hosts and tissues, and the 5'- and 3'-terminal regions of S4, S5, and S8 had more hotspots. Distribution and abundance of RBSDV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. Degradome analysis found 25 and 11 host genes appeared to be targeted by vsiRNAs in 9311 and Nipponbare. We report for the first time vsiRNAs derived from RBSDV-infected rice., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) could be produced during the plant-virus compatible interaction. However, the cell responses regulated by the enhanced H2O2 in virus infected plant are largely unknown. To make clear the influence of Rice black-streaked dwarf virus (RBSDV) infection on H2O2 accumulation, we measured the content of H2O2 and found the H2O2 level was increased in rice seedlings inoculated with RBSDV. To reveal the responses initiated by the enhanced H2O2 during plant-virus interaction, the present study investigated the global proteome changes of rice under long-term RBSDV infection. Approximately 1800 protein spots were detected on two-dimensional electrophoresis (2-DE) gels. Among them, 72 spots were found differently expressed, of which 69 spots were successfully identified by MALDI-TOF/TOF-MS. Furthermore, the differentially expressed proteins induced by RBSDV infection were compared to that induced by H2O2. 19 proteins corresponding to 37 spots, which were differentially expressed under RBSDV infection, were observed differentially expressed under H2O2 stress as well. These overlapping responsive proteins are mainly related to photosynthesis, redox homeostasis, metabolism, energy pathway, and cell wall modification. The increased H2O2 in RBSDV infected plant may produce an oxidative stress, impair photosynthesis, disturb the metabolism, and eventually result in abnormal growth. The data provide a new understanding of the pivotal role of H2O2 in rice-RBSDV compatible interaction. [ABSTRACT FROM AUTHOR]