Exploration The Interactions Between Rice Viruses With Their Transmitting Vectors And Functional Analysis Of Rice Stripe Virus-encoded Proteins

Rice is the principal food and is widely cultivated in China. Rice viruses are one of the most destructive pathogens in rice production. It has broken out many times in different provinces in China, causing huge rice yield losses. A lot of rice viruses are transmitted by rice planthoppers. As a result of epidemic outbreak of rice planthoppers, it is really a great threat in rice production. To get better strategies for rice protection, the interactions between rice virus with their transmitting vectors and functional analysis of Rice stripe virus encoded genes were described in this thesis.1Transcriptome and comparative gene expression analysis of Sogatella furcifera (Horva’th) in response to Southern rice black-streaked dwarf virusBy de novo transcriptome assembling and massive parallel pyrosequencing, we constructed two transcriptomes of Sogatella furcifera (the white-backed planthopper, WBPH) and profiled the alternation of gene expression in response to SRBSDV infection in transcriptional level. Over25million reads of high-quality DNA sequences and81388different unigenes were generated using Illumina technology from both viruliferous and non-viruliferous WBPH. WBPH has a very similar gene ontological distribution to other two closely related rice planthoppers, Nilaparvata lugens and Laodelphax striatellus.7291microsatellite loci were also predicted which could be useful for further evolutionary analysis. Furthermore, comparative analysis of the two transcriptomes generated from viruliferous and non-viruliferous WBPH provided a list of candidate transcripts that potentially were elicited as a response to viral infection. Pathway analyses of a subset of these transcripts indicated that SRBSDV infection might perturb primary metabolism and the ubiquitin-proteasome pathways. In addition,5.5%(181out of3315) of the genes in cell cytoskeleton organization pathway showed obvious changes. Our data also demonstrated that SRBSDV infection activated the immunity regulatory systems of WBPH, such as RNA interference, autophagy and antimicrobial peptide production. We employed massively parallel pyrosequencing to collect ESTs from viruliferous and nonviruliferous samples of WBPH.81388different unigenes have been obtained. We for the first time described the direct effects of a Reoviridae family plant virus on global gene expression profiles of its insect vector using high-throughput sequencing. Our study will provide a road map for future investigations of the fascinating interactions between Reoviridae viruses and their insect vectors, and provide new strategies for crop protection.2Rice stripe Tenuivirus nonstructural protein3(NS3) hijack L. striatellus26S proteasome via direct interacting with regulatory-particle non-ATPase subunit3(RPN3)In this research, the cDNA library of L. striatellus was constructed. Using RSV NS3protein as bait, a protein named regulatory-particle non-ATPase subunit3(RPN3) that could interacted with NS3was isolated. Full length of RPN3was cloned by searching transcriptome of L. striatellus and5’-RACE RCR. The NS3protein also interacted with RPN3in vitro by pull down test. NS3protein is a RNA silencing suppressor in a size independent and noncooperative recognition of dsRNA manner. The PRN3expression had no effect on NS3gene silencing suppressor activity by transient expression induced gene silencing system in Nicotiana benthamiana. L. striatellus RPN3could complement the functions of rpn3mutant yeast, indicating a conserved evolutionary function of26S proteasome. Furthermore, when L. striatellus rpn3-expressing vector was co-transformed with NS3plasmid, the complementary yeast lost the activities of protein degradation and caused the accumulation of ubiquitinated protein. An amino acid point mutant at the position of1298of RPN3was found not to interact with NS3. This mutant could also complement the functions of rpn3mutant yeast no matter transformed itself alone or co-transformed with NS3, indicating the direct interaction of RPN3and NS3was responsible for inhibition of L. striatellus RPN3complementary function.DsRNA induced gene silencing technology was carried out to silence RPN3expession level.2,4,6and8days after injection of dsRNA, the RSV titer level was measured by northern blot and western blot. Accumulation of RSV was observed as a result of silencing of RPN3, indicated a defense roles may played by the26S proteasome in RSV infection. Furthermore, RPN3-repressed L. striatellus had a higher virus transmitting activity. That may be caused by higher accumulation of RSV.To better understand the evolutionary relationship between RSV and L. striatellus, we collected at least12places of L. striatellus. We supply the evident that the existence of 1298locus point mutant in nature. Our results reveal a coevolved adaptation relationship between RSV and its transmiting vector-L. striatellus.3Population diversity of Rice stripe virus-derived siRNAs in three different hosts and RNAi-based antiviral immunity in L. striatellusSmall RNA-mediated gene silencing plays evolutionarily conserved roles in gene regulation and defense against invasive nucleic acids. Virus-derived small interfering RNAs (vsiRNAs) are one of the key elements involved in RNA silencing-based antiviral activities in plant and insect. VsiRNAs produced after viruses infecting hosts from a single kingdom (i.e., plant or animal) are well described. In contrast, vsiRNAs derived from viruses capable of infecting both plants and their insect vectors have not been characterized.We examined Rice stripe virus (RSV)-derived small interfering RNAs in three different hosts, Oiyza sativa, N. benthamiana and a natural RSV transmitting vector L. striatellus, through deep sequencing. Our results show that large amounts of vsiRNAs generated in these hosts after RSV infection. The vsiRNAs from N. benthamiana and L. striatellus mapped equally to the genomic-and antigenomic-strand of RSV RNAs. They showed, however, a significant bias in those from O. sativa. Furthermore, our results demonstrate that the number and size distributions of vsiRNAs in the three hosts were very different. In O. sativa and N. benthamiana, most vsiRNAs were mapped to the discrete regions in the RSV genome sequence, and most of the vsiRNAs from these*two hosts were generated from RSV genomic RNAs3and4. In contrast, the vsiRNAs identified in L. striatellus distributed uniformly along the whole genome. We have also shown that silencing Agronaute2in L. striatellus enhanced RSV accumulation in this host.Our study demonstrates that the core RNA-induced gene silencing (RNAi) machinery is present in L. striatellus, a member of Hemipteran that transmits about55%of the known plant viruses. We also provide evidence that the RNAi-mediated immunity against RSV is present in L. striatellus. We propose that a common small RNA-mediated virus defense mechanism exists in both helipterum insects and plants, but the vsiRNAs are generated differentially in different hosts. 4Role of Rice stripe virus NSvc4in cell-to-cell movement and symptom development in Nicotiana benthamianaOur previous work has demonstrated that the NSvc4protein of Rice stripe virus (RSV) functions as a cell-to-cell movement protein. However, the mechanisms whereby RSV traffics through plasmodesmata (PD) are unknown. Here we provide evidence that the NSvc4moves on the actin filament and endoplasmic reticulum network, but not microtubules, to reach cell wall PD. Disruption of cytoskeleton using different inhibitors altered NSvc4localization to PD, thus impeding RSV infection of N. benthamiana. Sequence analyses and deletion mutagenesis experiment revealed that the N-terminal125amino acids (AAs) of the NSvc4determine PD targeting and that a transmembrane domain spanning AAs106-125is critical for PD localization. We also found that the NSvc4protein could localize to chloroplasts in infected cells. Analyses using deletion mutants revealed that the N-terminal73AAs are essential for chloroplast localization. Furthermore, expression of NSvc4from a Potato virus X (PVX) vector resulted in more severe disease symptoms than PVX alone in systemically infected N. benthamiana leaves. Expression of NSvc4in Spodoptera frugiperda9cells did not elicit tubule formation, but instead resulted in punctate foci at the plasma membrane. These findings shed new light on our understanding of the movement mechanisms whereby RSV infects host plants.5The mechanism of the Rice stripe virus RNA silencing suppressor NS3Nucleic acid-binding proteins frequently use basic arginine and lysine residues to contact negatively charged phosphate groups through electrostatic interactions. To identify the dsRNA-binding residues of NS3, we replaced a few arginine and lysine residues with glycine or positively charged aspartate and glutamate residues.To examine the relationship between the dsRNA binding activity and suppressor activity of NS3, we analyzed these mutants for their suppression of dsRNA-induced GFP silencing using an agrofiltration assay. The suppressor activities of11NS3mutants were examined and found to generally correlate with their dsRNA-binding affinities. Mutants that were normal in dsRNA binding (R94G, K127G, K165G, and R169E) maintained their ability to efficiently inhibit GFP silencing. On the other hand, mutants that were defective in dsRNA binding (R50G, K77G, K112G, R124G, K173GK174G, K173E/K174D/R175E, and R190G) were also defective in the suppression of GFP silencing. We conclude that the in vivo suppressor activity of NS3is correlated to its dsRNA binding ability, providing a mechanistic link between the two activities.