Analysis of genetic variation and diversity of Rice stripe virus populations through high-throughput sequencing.

Plant RNA viruses often generate diverse populations in their host plants through error-prone replication and recombination. Recent studies on the genetic diversity of plant RNA viruses in various host plants have provided valuable information about RNA virus evolution and emergence of new diseases caused by RNA viruses. We analyzed and compared the genetic diversity of Rice stripe virus (RSV) populations in Oryza sativa (a natural host of RSV) and compared it with that of the RSV populations generated in an infection of Nicotiana benthamiana, an experimental host of RSV, using the high-throughput sequencing technology. From infected O. sativa and N. benthamiana plants, a total of 341 and 1675 site substitutions were identified in the RSV genome, respectively, and the average substitution ratio in these sites was 1.47 and 7.05 %, respectively, indicating that the RSV populations from infected N. benthamiana plant are more diverse than those from infected O. sativa plant. Our result gives a direct evidence that virus might allow higher genetic diversity for host adaptation.

Identification of Himetobi P virus in the small brown planthopper by deep sequencing and assembly of virus-derived small interfering RNAs.

Profiling and assembly of virus-derived small interfering RNAs (siRNAs) using next-generation sequencing technologies have been very useful for identification and diagnosis of a number of plant and invertebrate viruses. In this work, we have conducted high-throughput pyrosequencing and bioinformatic analysis of the small brown planthopper (SBPH, Laodelphax striatellus), and these analyses unexpectedly showed that the Himetobi P virus (HiPV) was present in our laboratory cultures. HiPV was also found to infect our brown planthopper (BPH, Nilaparvata lugens) and the white-backed planthopper (WBPH, Sogatella furcifera) cultures. The majority of the HiPV-derived siRNAs (Hd-siRNAs) were 21 and 22 nucleotides in length and nearly two-thirds of the siRNAs originated from the HiPV genomic RNA strand. The Hd-siRNAs were evenly distributed across the genome and this indicates that the HiPV genome contributes uniformly to production of Hd-siRNAs. Although HiPV infection appeared to be innocuous to the SBPH, alterations of gene expressions involved in reproduction, cytoskeleton structure and defense responses such as RNA interference pathways (RNAi) genes were observed. Furthermore, we demonstrated that silencing Agronaute 2 in L. striatellus enhanced HiPV accumulation, and this observation provides evidence for the existence of RNAi defenses against HiPV in the SBPH.

Genetic variability and evolution of rice stripe virus.

Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV is known to have four segmented, single-stranded RNA molecules and causes rice stripe disease in the rice fields of China, Japan, and Korea. Based on the complete genomic sequences of the determined 6 RSV isolates (from Yunnan, Jiangsu, Zhejiang, and Liaoning Provinces, China) and 27 other RSV isolates (from Yunnan, Jiangsu, Anhui, Henan, and Shandong Provinces of China, also Japan and Korea) downloaded from GenBank, we provided a genotyping profile of RSV field isolates and described the population structure of RSV. All RSV isolates, except isolate CX, could be divided into two subtypes, one including 6 isolates from Yunnan Province, and the other including 26 isolates from different parts of China, Japan, and Korea, which were referred to as subtype II and subtype I, respectively. The amino acid distances between subtypes range from 0.053 to 0.085. RSV isolates in Yunnan Province were genetically differentiated from other parts of China, Japan, and Korea and showed infrequent gene flow. The RSV populations collected from other parts of China, Japan, and Korea were only composed of subtype I and showed very low genetic diversity. We speculated that isolate CX may be the result of recombination of isolates from two subtypes. Two potential recombination events were detected in RNA4 of isolate CX.

Population diversity of rice stripe virus-derived siRNAs in three different hosts and RNAi-based antiviral immunity in Laodelphgax striatellus.

BACKGROUND: Small 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. METHODOLOGY/PRINCIPAL FINDINGS: We examined Rice stripe virus (RSV)-derived small interfering RNAs in three different hosts, Oryza sativa, Nicotiana benthamiana and a natural RSV transmitting vector Laodelphgax 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 RNAs 3 and 4. In contrast, the vsiRNAs identified in L. striatellus distributed uniformly along the whole genome of RSV. We have also shown that silencing Agronaute 2 in L. striatellus enhanced RSV accumulation in this host. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that the core RNA-induced gene silencing (RNAi) machinery is present in L. striatellus. 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.