CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

NSvc4 Encoded by Rice Stripe Virus Targets Host Chloroplasts to Suppress Chloroplast-Mediated Defense

Our previous research found that NSvc4, the movement protein of rice stripe virus (RSV), could localize to the actin filaments, endoplasmic reticulum, plasmodesmata, and chloroplast, but the roles of NSvc4 played in the chloroplast were opaque. Here, we confirm the accumulation of NSvc4 in the chloroplasts and the N-terminal 1–73 amino acids of NSvc4 are sufficient to localize to chloroplasts. We provide evidence to show that chloroplast-localized NSvc4 can impair the chloroplast-mediated immunity. Expressing NSvc4 in Nicotiana benthamiana leaves results in the decreased expression of defense-related genes NbPR1, NbPR2, and NbWRKY12 and the inhibition of chloroplast-derived ROS production. In addition, generation of an infectious clone of potato virus X (PVX) carrying NSvc4 facilitates PVX infection in N. benthamiana plants. Moreover, we identify two chloroplast-related host factors, named NbGAPDH-A and NbPsbQ1, both of which can interact with NSvc4. Knockdown of NbGAPDH-A or NbPsbQ1 can both promote RSV infection. Our results decipher a detailed function of NSvc4 in the chloroplast.

Sublethal Effects of Imidacloprid on Fecundity, Apoptosis and Virus Transmission in the Small Brown Planthopper Laodelphax striatellus

Laodelphax striatellus damages plants directly through sucking plant sap and indirectly as a vector of rice stripe virus (RSV), resulting in serious losses of rice yield. It is one of the most destructive insects of rice in East Asia. Insecticides are primarily used for pest management, but the sublethal concentrations of insecticides may benefit several insects. The present research attempted to explore the effects of sublethal concentrations of imidacloprid on the fecundity, apoptosis and RSV transmission in the viruliferous SBPH. The results showed that the fecundity of SBPH was significantly increased after treatment with the LC10 dose of imidacloprid, while the LC30 dose of imidacloprid reduced the fecundity compared with the control. To further investigate the underlying mechanism of increased fecundity after exposure to the LC10 dose of imidacloprid, we examined the expression levels of vitellogenin (Vg), Vg receptor (VgR) and caspases in the ovaries of SBPH, and observed the apoptosis by terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL). qRT-PCR results indicated that the expression levels of Vg, VgR and four caspase genes were all significantly increased by the LC10 dose of imidacloprid, and TUNEL assays suggested that the frequency of apoptosis was significantly higher in the SBPH treated by the LC10 dose of imidacloprid, suggesting a potential correlation between the increased fecundity and the apoptosis of SBPH ovarioles. Additionally, the expression levels of RNA3 and capsid protein (CP) were both increased significantly by the LC10 dose of imidacloprid, whereas were decreased by the LC30 dose of imidacloprid compared to the control. Therefore, this study clarifies the mechanisms of sublethal effects of imidacloprid on viruliferous SBPH and could be used to optimize pest control strategies.

Alternative splicing landscape of small brown planthopper and different response of JNK2 isoforms to rice stripe virus infection

Alternative splicing (AS) is a frequent posttranscriptional regulatory event occurring in response to various endogenous and exogenous stimuli in most eukaryotic organisms. However, little is known about the effects of insect-transmitted viruses on AS events in insect vectors. The present study used third-generation sequencing technology and RNA sequencing (RNA-Seq) to evaluate the AS response in the small brown planthopper Laodelphax striatellus to rice stripe virus (RSV). The full-length transcriptome of L. striatellus was obtained using single-molecule real-time sequencing technology (SMRT). Posttranscriptional regulatory events, including AS, alternative polyadenylation, and fusion transcripts, were analyzed. A total of 28,175 nonredundant transcript isoforms included 24,950 transcripts assigned to 8,500 annotated genes of L. striatellus , and 5,000 of these genes (58.8%) had AS events. RNA-Seq of the gut samples of insects infected by RSV for 8 d identified 3,458 differentially expressed transcripts (DETs); 2,185 of these DETs were transcribed from 1,568 genes that had AS events, indicating that 31.4% of alternatively spliced genes responded to RSV infection of the gut. One of the c-Jun N-terminal kinase ( JNK ) genes, JNK2 , experienced exon skipping, resulting in three transcript isoforms. These three isoforms differentially responded to RSV infection during development and in various organs. Injection of double-stranded RNAs targeting all or two isoforms indicated that three or at least two JNK2 isoforms facilitated RSV accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors. Importance Alternative splicing (AS) is a regulatory mechanism that occurs after gene transcription. AS events can enrich protein diversity to promote the reactions of the organisms to various endogenous and exogenous stimulations. It is not known how insect vectors exploit AS events to cope with transmitted viruses. The present study used third-generation sequencing technology to obtain the profile of AS events in the small brown planthopper Laodelphax striatellus , which is an efficient vector for rice stripe virus (RSV). The results indicated that 31.4% of alternatively spliced genes responded to RSV infection in the gut of planthoppers. One of the c-Jun N-terminal kinase ( JNK ) genes, JNK2 , produced three transcript isoforms by AS. These three isoforms showed different responses to RSV infection, and at least two isoforms facilitated viral accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors.

eIF4A, a target of siRNA derived from rice stripe virus, negatively regulates antiviral autophagy by interacting with ATG5 in Nicotiana benthamiana

Autophagy is induced by viral infection and has antiviral functions in plants, but the underlying mechanism is poorly understood. We previously identified a viral small interfering RNA (vsiRNA) derived from rice stripe virus (RSV) RNA4 that contributes to the leaf-twisting and stunting symptoms caused by this virus by targeting the host eukaryotic translation initiation factor 4A (eIF4A) mRNA for silencing. In addition, autophagy plays antiviral roles by degrading RSV p3 protein, a suppressor of RNA silencing. Here, we demonstrate that eIF4A acts as a negative regulator of autophagy in Nicotiana benthamiana. Silencing of NbeIF4A activated autophagy and inhibited RSV infection by facilitating autophagic degradation of p3. Further analysis showed that NbeIF4A interacts with NbATG5 and interferes with its interaction with ATG12. Overexpression of NbeIF4A suppressed NbATG5-activated autophagy. Moreover, expression of vsiRNA-4A, which targets NbeIF4A mRNA for cleavage, induced autophagy by silencing NbeIF4A. Finally, we demonstrate that eIF4A from rice, the natural host of RSV, also interacts with OsATG5 and suppresses OsATG5-activated autophagy, pointing to the conserved function of eIF4A as a negative regulator of antiviral autophagy. Taken together, these results reveal that eIF4A negatively regulates antiviral autophagy by interacting with ATG5 and that its mRNA is recognized by a virus-derived siRNA, resulting in its silencing, which induces autophagy against viral infection.

Comparative Transcriptome Analysis of Chemoreception Organs of Laodelphax striatellus in Response to Rice Stripe Virus Infection

Many vector-borne viruses possess the ability to manipulate vector behaviors to facilitate their transmission. There is evidence that the mechanism of this phenomenon has been described in part as direct manipulation through regulating vector chemosensation. Rice stripe virus (RSV) is transmitted by the small brown planthopper, Laodelphax striatellus (Fallen), in a persistent, circulative–propagative manner. The effect of RSV infection on the olfactory system of L. striatellus has not been fully elucidated. Here, we employed transcriptomic sequencing to analyze gene expression profiles in antennae, legs and heads (without antennae) from L. striatellus females and males with/without RSV infection. Comparisons of the differentially expressed genes (DEGs) among antennae, legs and heads indicated that tissue-specific changes in the gene expression profile were greater than sex-specific changes. A total of 17 olfactory related genes were differentially expressed in viruliferous antennae as compared to nonviruliferous antennae, including LstrOBP4/9, LstrCSP1/2/5, LstrGR28a/43a/43a-1, LstrIR1/2/NMDA1, LstrOR67/85e/56a/94 and LstrSNMP2/2-2. There are 23 olfactory related DEGs between viruliferous and nonviruliferous legs, including LstrOBP2/3/4/12/13, LstrCSP13/5/10, LstrIR1/2/Delta2/Delta2-1/kainate2/NMDA2, LstrOR12/21/31/68 and LstrORco. A low number of olfactory related DEGs were found between viruliferous and nonviruliferous heads, including LstrCSP1, LstrOBP2, LstrOR67 and LstrSNMP2-2. Among these DEGs, the expression patterns of LstrOBP2, LstrOBP3 and LstrOBP9 in three tissues was validated by quantitative real-time PCR. The demonstration of overall changes in the genes in L. striatellus’ chemoreception organs in response to RSV infection would not only improve our understanding of the effect of RSV on the olfactory related genes of insect vectors but also provide insights into developing approaches to control the plant virus transmission and spread as well as pest management in the future.

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

In the original publication of the article Fig. 3A and Fig. 33B were incorrectly published. The correct figures are available in this correction.

Regulation of RNA Interference Pathways in the Insect Vector Laodelphax striatellus by Viral Proteins of Rice Stripe Virus

RNA interference (RNAi), especially the small interfering RNA (siRNA) and microRNA (miRNA) pathways, plays an important role in defending against viruses in plants and insects. However, how insect-transmitted phytoviruses regulate the RNAi-mediated antiviral response in vector insects has barely been uncovered. In this study, we explored the interaction between rice stripe virus (RSV) and the miRNA and siRNA pathways of the small brown planthopper, which is a vector insect. The transcript and protein levels of key genes in the two RNAi pathways did not change during the RSV infection process. When the expression of insect Ago1, Ago2, or Translin was silenced by the injection of double-stranded RNAs targeting these genes, viral replication was promoted with Ago2 silencing but inhibited with Translin silencing. Protein-protein binding assays showed that viral NS2 and RNA-dependent RNA polymerase interacted with insect Ago2 and Translin, respectively. When NS2 was knocked down, the transcript level of Ago2 increased and viral replication was inhibited. Therefore, viral NS2 behaved like an siRNA suppressor in vector insects. This protein-binding regulation of insect RNAi systems reflects a complicated and diverse coevolution of viruses with their vector insects.

The dynamics of N6-methyladenine RNA modification in interactions between rice and plant viruses

Background N6-methyladenosine (m6A) is the most common RNA modification in eukaryotes and has been implicated as a novel epigenetic marker that is involved in various biological processes. The pattern and functional dissection of m6A in the regulation of several major human viral diseases have already been reported. However, the patterns and functions of m6A distribution in plant disease bursting remain largely unknown. Results We analyse the high-quality m6A methylomes in rice plants infected with two devastating viruses. We find that the m6A methylation is mainly associated with genes that are not actively expressed in virus-infected rice plants. We also detect different m6A peak distributions on the same gene, which may contribute to different antiviral modes between rice stripe virus or rice black-stripe dwarf virus infection. Interestingly, we observe increased levels of m6A methylation in rice plant response to virus infection. Several antiviral pathway-related genes, such as RNA silencing-, resistance-, and fundamental antiviral phytohormone metabolic-related genes, are also m6A methylated. The level of m6A methylation is tightly associated with its relative expression levels. Conclusions We revealed the dynamics of m6A modification during the interaction between rice and viruses, which may act as a main regulatory strategy in gene expression. Our investigations highlight the significance of m6A modifications in interactions between plant and viruses, especially in regulating the expression of genes involved in key pathways.