Won Kyong Cho

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.

Insight into mycoviruses infecting Fusarium species.

Most of the major fungal families including plant-pathogenic fungi, yeasts, and mushrooms are infected by mycoviruses, and many double-stranded RNA (dsRNA) mycoviruses have been recently identified from diverse plant-pathogenic Fusarium species. The frequency of occurrence of dsRNAs is high in Fusarium poae but low in other Fusarium species. Most Fusarium mycoviruses do not cause any morphological changes in the host but some mycoviruses like Fusarium graminearum virus 1 (FgV1) cause hypovirulence. Available genomic data for seven of the dsRNA mycoviruses infecting Fusarium species indicate that these mycoviruses exist as complexes of one to five dsRNAs. According to phylogenetic analysis, the Fusarium mycoviruses identified to date belong to four families: Chrysoviridae, Hypoviridae, Partitiviridae, and Totiviridae. Proteome and transcriptome analysis have revealed that FgV1 infection of Fusarium causes changes in host transcriptional and translational machineries. Successful transmission of FgV1 via protoplast fusion suggests the possibility that, as biological control agents, mycoviruses could be introduced into diverse species of fungal plant pathogens. Research is now needed on the molecular biology of mycovirus life cycles and mycovirus-host interactions. This research will be facilitated by the further development of omics technologies.

Development of RT-PCR Based Method for Detecting Five Non-reported Quarantine Plant Viruses Infecting the Family Cucurbitaceae or Solanaceae

For quarantine purpose, we selected five plant RNA viruses including Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Potato aucuba mosaic virus (PAMV), Potato yellow dwarf virus (PYDV), and Tomato chlorosis virus (ToCV), which are not reported in Korea and cause serious economic losses to the family Cucurbitaceae or Solanaceae. To detect those viruses, we employed RT-PCR technique with specific oligonucleotide primer pairs and tested their detection efficiency for each virus. To design RT-PCR primers, coat protein was used for CVYV, CYSDV, and ToCV whereas RNA polymerase and nucleocapsid regions were used for PAMV and PYDV, respectively. The development of an RT-PCR based method proved a useful tool for rapid detection and identification of quarantine virus infections.

Cis-acting element (SL1) of Potato virus X controls viral movement by interacting with the NbMPB2Cb and viral proteins

A number of candidate tobacco proteins that bind to cis-acting elements (SL1 RNAs) of Potato virus X (PVX) have been identified in previous studies. We further characterized TMV-MP30 binding protein 2C (MPB2C) homologous protein. We isolated NbMPB2Cb from Nicotiana benthamiana and confirmed the interaction of NbMPB2Cb with SL1 RNAs in vitro. The mRNA level of NbMPB2Cb was increased upon infection by PVX and Tobacco mosaic virus. The movement of PVX was reduced by overexpression of NbMPB2Cb and increased by silenced of NbMPB2Cb. In contrast, PVX RNA accumulation was not significantly altered in protoplasts. Protein-protein interaction assays showed that NbMPB2Cb interacts with PVX movement-associated proteins. PVX infection altered the subcellular localization of NbMPB2Cb from microtubules to endoplasmic reticulum. These data suggest that the NbMPB2Cb negatively affects PVX movement by interacting with SL1 RNAs and movement-associated proteins of PVX and by re-localizing in response to PVX infection.

Complete genome sequences of three tomato spotted wilt virus isolates from tomato and pepper plants in Korea and their phylogenetic relationship to other TSWV isolates

Tomato spotted wilt virus (TSWV) infects numerous host plants and has three genome segments, called L, M and S. Here, we report the complete genome sequences of three Korean TSWV isolates (TSWV-1 to -3) infecting tomato and pepper plants. Although the nucleotide sequence of TSWV-1 genome isolated from tomato is very different from those of TSWV-2 and TSWV-3 isolated from pepper, the deduced amino acid sequences of the five TSWV genes are highly conserved among all three TSWV isolates. In phylogenetic analysis, deduced RdRp protein sequences of TSWV-2 and TSWV-3 were clustered together with two previously reported isolates from Japan and Korea, while TSWV-1 grouped together with a Hawaiian isolate. A phylogenetic tree based on N protein sequences, however, revealed four distinct groups of TSWV isolates, and all three Korean isolates belonged to group II, together with many other isolates, mostly from Europe and Asia. Interestingly, most American isolates grouped together as group I. Together, these results suggested that these newly identified TSWV isolates might have originated from an Asian ancestor and undergone divergence upon infecting different host plants.

A comparison of transcriptional patterns and mycological phenotypes following infection of Fusarium graminearum by four mycoviruses.

Many fungi-infecting viruses, which are termed mycoviruses, have been identified, and most do not cause any visible symptoms. Some mycoviruses, however, can attenuate the virulence of the infected fungi, a phenomenon referred to as hypovirulence. To study fungus responses to virus infection, we established a model system composed of Fusarium graminearum and four mycoviruses including FgV1 (Fusarium graminearum virus 1), FgV2, FgV3, and FgV4. FgV1 and FgV2 infections caused several phenotypic alterations in F. graminearum including abnormal colony morphology, defects in perithecium development, and reductions in growth rate, conidiation, and virulence. In contrast, FgV3 and FgV4 infections did not cause any phenotypic change. An RNA-Seq-based analysis of the host transcriptome identified four unique Fusarium transcriptomes, one for each of the four mycoviruses. Unexpectedly, the fungal host transcriptome was more affected by FgV1 and FgV4 infections than by FgV2 and FgV3 infections. Gene ontology (GO) enrichment analysis revealed that FgV1 and FgV3 infections resulted in down-regulation of host genes required for cellular transport systems. FgV4 infection reduced the expression of genes involved in RNA processing and ribosome assembly. We also found 12 genes that were differentially expressed in response to all four mycovirus infections. Unfortunately, functions of most of these genes are still unknown. Taken together, our analysis provides further detailed insights into the interactions between mycoviruses and F. graminearum.

Evolution of and Horizontal Gene Transfer in the Endornavirus Genus

The transfer of genetic information between unrelated species is referred to as horizontal gene transfer. Previous studies have demonstrated that both retroviral and non-retroviral sequences have been integrated into eukaryotic genomes. Recently, we identified many non-retroviral sequences in plant genomes. In this study, we investigated the evolutionary origin and gene transfer of domains present in endornaviruses which are double-stranded RNA viruses. Using the available sequences for endornaviruses, we found that Bell pepper endornavirus-like sequences homologous to the glycosyltransferase 28 domain are present in plants, fungi, and bacteria. The phylogenetic analysis revealed the glycosyltransferase 28 domain of Bell pepper endornavirus may have originated from bacteria. In addition, two domains of Oryza sativa endornavirus, a glycosyltransferase sugar-binding domain and a capsular polysaccharide synthesis protein, also exhibited high similarity to those of bacteria. We found evidence that at least four independent horizontal gene transfer events for the glycosyltransferase 28 domain have occurred among plants, fungi, and bacteria. The glycosyltransferase sugar-binding domains of two proteobacteria may have been horizontally transferred to the genome of Thalassiosira pseudonana. Our study is the first to show that three glycome-related viral genes in the genus Endornavirus have been acquired from marine bacteria by horizontal gene transfer.

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP.

Genome-wide expression profiling shows transcriptional reprogramming in Fusarium graminearum by Fusarium graminearum virus 1-DK21 infection

BackgroundFusarium graminearum virus 1 strain-DK21 (FgV1-DK21) is a mycovirus that confers hypovirulence to F. graminearum, which is the primary phytopathogenic fungus that causes Fusarium head blight (FHB) disease in many cereals. Understanding the interaction between mycoviruses and plant pathogenic fungi is necessary for preventing damage caused by F. graminearum. Therefore, we investigated important cellular regulatory processes in a host containing FgV1-DK21 as compared to an uninfected parent using a transcriptional approach.ResultsUsing a 3′-tiling microarray covering all known F. graminearum genes, we carried out genome-wide expression analyses of F. graminearum at two different time points. At the early point of growth of an infected strain as compared to an uninfected strain, genes associated with protein synthesis, including ribosome assembly, nucleolus, and ribosomal RNA processing, were significantly up-regulated. In addition, genes required for transcription and signal transduction, including fungal-specific transcription factors and cAMP signaling, respectively, were actively up-regulated. In contrast, genes involved in various metabolic pathways, particularly in producing carboxylic acids, aromatic amino acids, nitrogen compounds, and polyamines, showed dramatic down-regulation at the early time point. Moreover, genes associated with transport systems localizing to transmembranes were down-regulated at both time points.ConclusionThis is the first report of global change in the prominent cellular pathways in the Fusarium host containing FgV1-DK21. The significant increase in transcripts for transcription and translation machinery in fungal host cells seems to be related to virus replication. In addition, significant down-regulation of genes required for metabolism and transporting systems in a fungal host containing the virus appears to be related to the host defense mechanism and fungal virulence. Taken together, our data aid in the understanding of how FgV1-DK21 regulates the transcriptional reprogramming of F. graminearum.

Identification of natural hybrids in Korean Phragmites using haplotype and genotype analyses

To elucidate natural hybridization of Korean Phragmites, we collected Phragmites plants from 29 regions in South Korea. Haplotypes of the samples, which were determined using two known chloroplast intergenic sequences in this study, were combined with previously known haplotypes. Phylogenetic analysis identified that 30 Korean Phragmites were grouped with two different haplotypes, ‘P’ or ‘W’, respectively, indicating that introduced Phragmites samples from other continents were not present in Korea. The vast majority (26) of the 27 test samples were grouped with the P haplotype, while the E4 sample and the three control Phragmites japonicus samples were grouped with haplotype W. Interestingly, parsimony network analysis revealed that Phragmites australis in Korea might have originated from various regions including Busan (S1), Icheon (M2), and Ansan (W2). Genotype analysis using the PhaHKT1 nuclear gene identified the M3 sample as Phragmites japonicus. For the first time, we found two hybrids (E4 and M3) in the wild by haplotype and genotype analyses, implying that the phenotype of Phragmites australis might be dominant in the hybrids. In summary, we suggest that hybrid speciation might be an important factor in the genetic diversity of Korean Phragmites.