Seungmo Lim

Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans

Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)600 of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments.

Development of Multiplex RT-PCR for Simultaneous Detection of Garlic Viruses and the Incidence of Garlic Viral Disease in Garlic Genetic Resources

Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic.

Deep Sequencing Analysis of Apple Infecting Viruses in Korea

Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time.

Characterization of Brugmansia mosaic virus Isolated from Brugmansia spp. in Korea

School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Korea Institute of Plant Medicine, Kyungpook National University, Daegu 702-701, Korea Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, Wanju 565-851, Korea Apple Research Station, National Institute of Horticultural & Herbal Science, Gunwi 716-812, Korea Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305350, Korea Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea

Characteristics of a Lettuce mosaic virus Isolate Infecting Lettuce in Korea.

Lettuce mosaic virus (LMV) causes disease of plants in the family Asteraceae, especially lettuce crops. LMV isolates have previously been clustered in three main groups, LMV-Yar, LMV-Greek and LMVRoW. The first two groups, LMV-Yar and LMV-Greek, have similar characteristics such as no seed-borne transmission and non-resistance-breaking. The latter one, LMV-RoW, comprising a large percentage of the LMV isolates contains two large subgroups, LMV-Common and LMV-Most. To date, however, no Korean LMV isolate has been classified and characterized. In this study, LMV-Muju, the Korean LMV isolate, was isolated from lettuce showing pale green and mottle symptoms, and its complete genome sequence was determined. Classification method of LMV isolates based on nucleotide sequence divergence of the NIb-CP junction showed that LMV-Muju was categorized as LMV-Common. LMV-Muju was more similar to LMV-O (LMV-Common subgroup) than to LMV-E (LMV-RoW group but not LMV-Common subgroup) even in the amino acid domains of HC-Pro associated with pathogenicity, and in the CI and VPg regions related to ability to overcome resistance. Taken together, LMV-Muju belongs to the LMV-Common subgroup, and is expected to be a seed-borne, non-resistance-breaking isolate. According to our analysis, all other LMV isolates not previously assigned to a subgroup were also included in the LMV-RoW group.

A Survey of Viral Diseases of Proso Millet ( Panicum miliaceum L.) and Sorghum ( Sorghum bicolor L.) in South Korea

Throughout year 2015 to 2016, 101 proso millet and 200 sorghum samples were collected from five provinces in South Korea. The samples were subjected to paired-end RNA sequencing and further analyzed by RT-PCR. The results indicated that Rice black-streaked dwarf virus (RBSDV) was detected from sorghum collected in Gyeongsang province. The other four viruses, including RBSDV, Rice stripe virus (RSV), Barley virus G (BVG), and Cereal yellow dwarf virus (CYDV), were detected from proso millet. Among four viruses, both RSV and RBSDV were identified high frequency from proso millet collected from Gyeongsang province. Otherwise, BVG was nearly equally identified from five provinces, suggesting that the virus was supposedly widespread nationwide. RBSDV was first identified from both proso millet and sorghum in South Korea. The other virus annotated CYDV identified proso millet was shown to have relatively low identities compared to CYDV previously reported, suggesting that the virus might be new member of Polerovirus.

First report of Maize yellow mosaic virus infecting Panicum miliaceum and Sorghum bicolor in South Korea

Maize yellow mosaic virus (MaYMV) is a tentative new Polerovirus, which was recently identified from maize (Zea mays) in China (Chen et al. 2016). MaYMV has also been reported to infect sugarcane (Saccharum spp.) and itch grass (Rottboellia cochinchinensis), and it has been reported in Asia, Africa and South America (Goncalves et al. 2017; Palanga et al. 2017; Yahaya et al. 2017). In this study, MaYMV was detected in Panicum miliaceum and Sorghum bicolor using Illumina HiSeq2500 system by Theragen Etex Bio Institute (Suwon, Korea) and SG-VIPdb by SeqGenesis (Daejeon, Korea), as described by Lim et al. (2015). Twenty samples of P. miliaceum and sixty three samples of S. bicolor were collected from July to September and from June to October, 2016, respectively, in South Korea. The high-throughput RNA sequencing of the samples mixed into one pool resulted in a single large contig (5606-nt) with nearly complete MaYMV genome coverage. The contig was assembled from a total of 234,537 reads; the maximum, minimum...

First Report of Soybean mosaic virus and Soybean yellow mottle mosaic virus in Vigna angularis

In 2015 and 2016, 203 samples of adzuki bean (Vigna angularis) exhibiting viral disease-like symptoms, such as mosaic, mottle, yellowing, dwarfing and leaf rolling, were collected from 18 cities/counties in Korea. Total RNA was prepared from a pooled sample using easy-spinTM Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and used for high-throughput paired-end RNA sequencing. Raw data analysis was conducted by Illumina HiSeq 2500 System at the Theragen Etex Bio Institute (Suwon, Korea) and SG-VIPdb at SeqGenesis (Daejeon, Korea), respectively, as previously described (Lim et al. 2015). As expected, 239 and 10 contigs related to Bean common mosaic virus and Cucumber mosaic virus, which have been previously reported in adzuki bean, were obtained from the adzuki bean transcriptome, respectively. However, in addition to this, 143 and 8 contigs were attributed to two soybean viruses, Soybean mosaic virus (SMV) and Soybean yellow mottle mosaic virus (SYMMV), respectively. To confirm the presen...

First Complete Genome Sequence of Barley Virus G Identified from Proso Millet (Panicum miliaceum) in South Korea

ABSTRACT The complete genome sequence of an Uiseong isolate of barley virus G (BVG) on proso millet plants in a field in South Korea was determined by RNA sequencing and Sanger sequencing. To our knowledge, this is the first complete genome sequence report of BVG infecting proso millet in South Korea.