Rice stripe virus (RSV) often causes severe rice yield loss in temperate regions of East Asia. Although the correlation of small interfering RNAs (siRNAs) with transgenic virus resistance of plants using RNA interference (RNAi) is known for decades, no systematical research has been done on the profiling of siRNAs from a genomic scale. Our research is aiming to systematically study the RNAi impact in RSV-resistant transgenic rice, which was generated by introducing an inverted repeat construct that targets RSV nucleocapsid protein (NCP) gene. In this paper, three independent RSV-resistant transgenic rice lines were generated, their stable integration of the T-DNA fragment and the expression of siRNAs were confirmed by Southern blotting and Northern blotting analyses, and the majority of siRNAs were in lengths of 21, 22, and 24 nucleotides (nt), which have validated a connection between the presence of the RSV NCP homologous siRNAs and the RSV resistance in those transgenic rice lines. In one of these transgenic lines (T4-B1), the T-DNA fragment was found to have been inserted at chromosome 1 of the rice genome, substituting the rice genome fragment from 32 158 773 to 32 158 787 nt. Bioinformatics analysis of small RNA-Seq data on the T4-B1 line also confirmed the large population of NCP-derived siRNAs in transgenic plants, and the RSV-infected library (T4-B1-V) possessed more siRNAs than its mock inoculated libraries (T4-B1-VF), these results indicating the inverted repeat construct and RSV could introduce abundance of siRNAs in transgenic rice. Moreover, a varied expression level of specific siRNAs was found among different segments of the NCP gene template, about 47% of NCP-derived siRNAs reads aligned with the fragment from 594 to 832 nt (239 nt in length) in NCP gene (969 nt in length) in the T4-B1-V, indicating a potential usage of hotspot regions for RNAi silencing in future research. In conclusion, as the first study to address the siRNA profile in RSV-resistant transgenic plant using next generation sequencing (NGS) technique, we confirmed that the massive abundance of siRNA derived from the inverted repeat of NCP is the major reason for RSV-resistance.

During the last decade, the leafhopper transmitted Wheat dwarf virus (WDV) has become a serious problem both in northwestern China and Hungary. In order to study the molecular diversity and population structure of WDV in these two countries, 39 Chinese isolates and 16 Hungarian isolates were collected from different regions of China and Hungary, and their genomes were sequenced. All isolates belonged to the wheat strain of WDV and showed limited genetic diversity. The highest and lowest nucleotide sequence identities among isolates from China and Hungary were 99.9 and 90%, respectively. In all isolates, the lowest nucleotide sequence identity was 89.5% between MO10-1 and KP10-5, which were collected from Martonvásár and Kompolt, Hungary. Phylogenetic analyses showed the genome sequences of 55 WDV isolates belong to two big clades, but no clear correlation to geographical location. Population difference analyses indicated that the Chinese and Hungarian WDV populations have no significant difference. The regions in WDV genome with relatively low nucleotide diversities represented protein coding regions suggested that these regions evolved under negative selection, and might be one of the causes restricting the number of genetic variants.