Sen Lian

Phylogenetic and Recombination Analysis of Tomato Spotted Wilt Virus

Tomato spotted wilt virus (TSWV) severely damages and reduces the yield of many economically important plants worldwide. In this study, we determined the whole-genome sequences of 10 TSWV isolates recently identified from various regions and hosts in Korea. Phylogenetic analysis of these 10 isolates as well as the three previously sequenced isolates indicated that the 13 Korean TSWV isolates could be divided into two groups reflecting either two different origins or divergences of Korean TSWV isolates. In addition, the complete nucleotide sequences for the 13 Korean TSWV isolates along with previously sequenced TSWV RNA segments from Korea and other countries were subjected to phylogenetic and recombination analysis. The phylogenetic analysis indicated that both the RNA L and RNA M segments of most Korean isolates might have originated in Western Europe and North America but that the RNA S segments for all Korean isolates might have originated in China and Japan. Recombination analysis identified a total of 12 recombination events among all isolates and segments and five recombination events among the 13 Korea isolates; among the five recombinants from Korea, three contained the whole RNA L segment, suggesting reassortment rather than recombination. Our analyses provide evidence that both recombination and reassortment have contributed to the molecular diversity of TSWV.

Comparative analysis of chrysanthemum transcriptome in response to three RNA viruses: Cucumber mosaic virus, Tomato spotted wilt virus and Potato virus X

The chrysanthemum is one of popular flowers in the world and a host for several viruses. So far, molecular interaction studies between the chrysanthemum and viruses are limited. In this study, we carried out a transcriptome analysis of chrysanthemum in response to three different viruses including Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Potato virus X (PVX). A chrysanthemum 135K microarray derived from expressed sequence tags was successfully applied for the expression profiles of the chrysanthemum at early stage of virus infection. Finally, we identified a total of 125, 70 and 124 differentially expressed genes (DEGs) for CMV, TSWV and PVX, respectively. Many DEGs were virus specific; however, 33 DEGs were commonly regulated by three viruses. Gene ontology (GO) enrichment analysis identified a total of 132 GO terms, and of them, six GO terms related stress response and MCM complex were commonly identified for three viruses. Several genes functioning in stress response such as chitin response and ethylene mediated signaling pathway were up-regulated indicating their involvement in establishment of host immune system. In particular, TSWV infection significantly down-regulated genes related to DNA metabolic process including DNA replication, chromatin organization, histone modification and cytokinesis, and they are mostly targeted to nucleosome and MCM complex. Taken together, our comparative transcriptome analysis revealed several genes related to hormone mediated viral stress response and DNA modification. The identified chrysanthemums genes could be good candidates for further functional study associated with resistant to various plant viruses.

Current Insights into Research on Rice stripe virus

Rice stripe virus (RSV) is one of the most destructive viruses of rice, and greatly reduces rice production in China, Japan, and Korea, where mostly japonica cultivars of rice are grown. RSV is transmitted by the small brown plant-hopper (SBPH) in a persistent and circulative-propagative manner. Several methods have been developed for detection of RSV, which is composed of four single-stranded RNAs that encode seven proteins. Genome sequence data and comparative phylogenetic analysis have been used to identify the origin and diversity of RSV isolates. Several rice varieties resistant to RSV have been selected and QTL analysis and fine mapping have been intensively performed to map RSV resistance loci or genes. RSV genes have been used to generate several genetically modified transgenic rice plants with RSV resistance. Recently, genome-wide transcriptome analyses and deep sequencing have been used to identify mRNAs and small RNAs involved in RSV infection; several rice host factors that interact with RSV proteins have also been identified. In this article, we review the current statues of RSV research and propose integrated approaches for the study of interactions among RSV, rice, and the SBPH.

Interaction study of rice stripe virus proteins reveals a region of the nucleocapsid protein (NP) required for NP self-interaction and nuclear localization.

Rice stripe virus (RSV), which belongs to the genus Tenuivirus, is an emergent virus problem. The RSV genome is composed of four single-strand RNAs (RNA1-RNA4) and encodes seven proteins. We investigated interactions between six of the RSV proteins by yeast-two hybrid (Y2H) assay in vitro and by bimolecular fluorescence complementation (BiFC) in planta. Y2H identified self-interaction of the nucleocapsid protein (NP) and NS3, while BiFC revealed self-interaction of NP, NS3, and NCP. To identify regions(s) and/or crucial amino acid (aa) residues required for NP self-interaction, we generated various truncated and aa substitution mutants. Y2H assay showed that the N-terminal region of NP (aa 1-56) is necessary for NP self-interaction. Further analysis with substitution mutants demonstrated that additional aa residues located at 42-47 affected their interaction with full-length NP. These results indicate that the N-terminal region (aa 1-36 and 42-47) is required for NP self-interaction. BiFC and co-localization studies showed that the region required for NP self-interaction is also required for NP localization at the nucleus. Overall, our results indicate that the N-terminal region (aa 1-47) of the NP is important for NP self-interaction and that six aa residues (42-47) are essential for both NP self-interaction and nuclear localization.

Time-Course RNA-Seq Analysis Reveals Transcriptional Changes in Rice Plants Triggered by Rice stripe virus Infection

Rice stripe virus (RSV) has become a major pathogen of rice. To determine how the rice transcriptome is modified in response to RSV infection, we used RNA-Seq to perform a genome-wide gene expression analysis of a susceptible rice cultivar. The transcriptomes of RSV-infected samples were compared to those of mock-treated samples at 3, 7, and 15 days post-infection (dpi). From 8 to 11% of the genes were differentially expressed (>2-fold difference in expression) in RSV-infected vs. noninfected rice. Among them, 532 genes were differentially expressed at all three time points. Surprisingly, 37.6% of the 532 genes are related to transposons. Gene ontology enrichment analysis revealed that many chloroplast genes were down-regulated in infected plants at 3 and 15 dpi. Expression of genes associated with cell differentiation and flowering was significantly down-regulated in infected plants at 15 dpi. In contrast, most of the up-regulated genes in infected plants concern the cell wall, plasma membrane, and vacuole and are known to function in various metabolic pathways and stress responses. In addition, transcripts of diverse transcription factors gradually accumulated in infected plants with increasing infection time. We also confirmed that the expression of gene subsets (including NBS-LRR domain-containing genes, receptor-like kinase genes, and genes involving RNA silencing) was changed by RSV infection. Taken together, we demonstrated that down-regulation of genes related to photosynthesis and flowering was strongly associated with disease symptoms caused by RSV and that up-regulation of genes involved in metabolic pathways, stress responses, and transcription was related to host defense mechanisms.

Generation of Antibodies Against Rice stripe virus Proteins Based on Recombinant Proteins and Synthetic Polypeptides

Rice stripe virus (RSV) is one of serious epidemic pathogens for rice species grown in many Asian countries. Therefore, it is necessary to produce a diagnostic detection kit applicable in fields for RSV detection. In this study, RSV proteins that were derived from recombinant proteins and synthetic polypeptides as antigens were generated and were raised in rabbits for antiserum production. Among seven proteins in RSV, genes that code for NCP and NS3 proteins were cloned and subcloned into vector carrying His-tag protein and were expressed in E. coli. Of two recombinant proteins, only anti-NCP displayed stable hybridization signals in western blot analysis. Alternately, synthetic RSV polypeptides for CP, NCP, NS3 and NSvc4 we also generated and only antibodies against CP and NCP were very effective to detect RSV in both RSV infected rice and weed plants. However, antibodies against NS3 and NSvc4 showed weak specific bands as well as strong non-specific background due to the difference of viral proteins produced in the infected leaves. In summary, the antibodies generated against RSV proteins produced in this study will be useful for various assays such as for RSV diagnostic detection, immunoprecipitation, protein purification, and western blot analysis.

Bacterial communities in the phylloplane of Prunus species

Bacterial populations in the phylloplane of four different Prunus species were investigated by 16 S rRNA pyrosequencing. Bioinformatic analysis identified an average of 510 operational taxonomic units belonging to 159 genera in 76 families. The two genera, Sphingomonas and Methylobacterium, were dominant in the phylloplane of four Prunus species. Twenty three genera were commonly identified in the four Prunus species, indicating a high level of bacterial diversity dependent on the plant species. Our study based on 16 S rRNA sequencing reveals the complexity of bacterial diversity in the phylloplane of Prunus species in detail.

Time-Course Small RNA Profiling Reveals Rice miRNAs and Their Target Genes in Response to Rice Stripe Virus Infection.

It has been known that many microRNAs (miRNAs) are involved in the regulation for the plant development and defense mechanism by regulating the expression of the target gene. Several previous studies has demonstrated functional roles of miRNAs in antiviral defense mechanisms. In this study, we employed high-throughput sequencing technology to identify rice miRNAs upon rice stripe virus (RSV) infection at three different time points. Six libraries from mock and RSV-infected samples were subjected for small RNA sequencing. Bioinformatic analyses revealed 374 known miRNAs and 19 novel miRNAs. Expression of most identified miRNAs was not dramatically changed at 3 days post infection (dpi) and 7 dpi by RSV infection. However, many numbers of miRNAs were up-regulated in mock and RSV-infected samples at 15 dpi by RSV infection. Moreover, expression profiles of identified miRNAs revealed that only few numbers of miRNAs were strongly regulated by RSV infection. In addition, 15 resistance genes were targets of six miRNAs suggesting that those identified miRNAs and 15 NBS-LRR resistance genes might be involved in RSV infection. Taken together, our results provide novel insight into the dynamic expression profiles of rice miRNAs upon RSV infection and clues for the understanding of the regulatory roles of miRNAs via time-course.

Genetic Reassortment of Rice stripe virus RNA Segments Detected by RT-PCR Restriction Enzyme Analysis-based Method

Department of Agricultural Biology, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Korea(Received on March 22, 2011; Accepted on April 11, 2011)Our previous sequence and phylogenetic analyses ofthe Korean Rice stripe virus (RSV) suggested possiblegenetic reassortment of RNA segments, but whether thisRNA variation contributed to the recent RSV outbreaksin Korea is yet unclear. To further clarify these RSV-RNA segment variations, we developed a reverse tran-scription-polymerase reaction/restriction enzyme (RT-PCR/RE) analysis-based method. We identified fiveREs, including DraI, EcoR1, NdeI/AseI, and SpeI, thatcould differentiate RSV RNA 1–4 subtypes, respectively.Our RT-PCR/RE results provided a clear pattern ofRNA reassortment, i.e., different groups of isolates havingtheir RNA segments derived from two to three differentRSV ancestors, such as from Eastern and SouthwesternChinese or Japanese M and T isolates. We also foundthat the migratory small brown planthopper from EasternChina caught by aerial net traps that possesses RSV-RNA3 genotypes corresponds mainly to Eastern China,with a few for Southwestern China based on RT-PCR/RE, sequence and phylogenetic analyses, indicating thatRSV populations in Eastern China may also have strongRNA variation. The development of an RE analysis-based method proved a useful epidemiological tool forrapid genotyping and identification of mixed infectionsby RSV strain and by different subtype.Keywords : genotyping, genetic structure, phylogeneticrelationships, restriction enzyme analysis, Rice stripe virusThe genome of Rice stripe virus (RSV), the prototypemember of the genus Tenuivirus, consists of four ssRNAs,with a total genome size of about 23 kb (Fig. 1; Toriyamaand Tomaru, 1995). RNA1 is the largest segment, havingnegative sense, while the other three segments are ambisense,whereby a single protein is encoded at the 5 proximal endin viral (v) and viral complementary (vc) RNA strands(Hamamatsu et al., 1993; Takahashi et al., 1993; Zhu et al.,1991, 1992). The RSV genome has a total of seven proteinsand most of their functions are known, except for NS2coded in vRNA2. The function of proteins coded in vcRNA1,vcRNA2, vcRNA3, and vRNA4 have long been known asRNA-dependent RNA polymerase (RdRp; Toriyama et al.,1994), glycoprotein-membrane protein (Takahashi et al.,1993), nucleocapsid protein, and noncapsid protein ordisease-specific protein (Kakutani et al., 1990; Zhu et al.,1992), respectively. However, proteins coded in vRNA3and vcRN4 were only recently reported as suppressors ofposttranscriptional gene silencing (PTGS; Hemmes et al.,2007) and movement protein (Xiong et al., 2008), respec-tively. RSV-infected plants usually show chlorotic stripes on theleaves and stunted growth, while severely infected seedl-ings are lethal. The virion morphology of the causal virushas circular filament and is actively and persistently trans-mitted by the small brown planthopper (SBPH) Laodelphaxstriatellus (Koganezawa, 1977). RSV outbreaks have alsooccurred in China, Japan, Taiwan, and the former USSR(Hibino, 1996). For insect transmitted viruses like RSV, its spread is mainlydependent on vector migration. In a previous study on theNilaparvata lugens (Stal), migration processes in Korea usingthe boundary layer atmospheric model and a geographicinformation system showed that the main source of insectsimmigrated into South Korea in the early part of June werefrom the Eastern part of China (Zhu et al., 2000). China andJapan are in close proximity to Korea, and reports show thatChina has a longer history of RSV outbreaks than Japan (Weiet al., 2009). During 2007−2009, RSV had burgeoning incidences thatcaused significant yield losses, posing an enigma to ricegrowers and many rice scientists in South Korea. The cause

De novo transcriptome assembly of a sour cherry cultivar, Schattenmorelle

Sour cherry (Prunus cerasus) in the genus Prunus in the family Rosaceae is one of the most popular stone fruit trees worldwide. Of known sour cherry cultivars, the Schattenmorelle is a famous old sour cherry with a high amount of fruit production. The Schattenmorelle was selected before 1650 and described in the 1800s. This cultivar was named after gardens of the Chateau de Moreille in which the cultivar was initially found. In order to identify new genes and to develop genetic markers for sour cherry, we performed a transcriptome analysis of a sour cherry. We selected the cultivar Schattenmorelle, which is among commercially important cultivars in Europe and North America. We obtained 2.05 GB raw data from the Schattenmorelle (NCBI accession number: SRX1187170). De novo transcriptome assembly using Trinity identified 61,053 transcripts in which N50 was 611 bp. Next, we identified 25,585 protein coding sequences using TransDecoder. The identified proteins were blasted against NCBIs non-redundant database for annotation. Based on blast search, we taxonomically classified the obtained sequences. As a result, we provide the transcriptome of sour cherry cultivar Schattenmorelle using next generation sequencing.