Rapid and Specific Detection of Apple stem grooving virus by Reverse Transcription-recombinase Polymerase Amplification

Kim, Nam-Yeon,Oh, Jonghee,Lee, Su-Heon,Kim, Hongsup,Moon, Jae Sun,Jeong, Rae-Dong The Korean Society of Plant Pathology 2018 Plant Pathology Journal Vol.34 No.6

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as $4.7ng/{\mu}l$ of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of $42^{\circ}C$. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

Comparison of Fruit Yield and Quality of Virus-infected and Virus-free ‘Hongro’ Apple Trees

Hongsup Kim,Dasom Baek,Jeongran Park,Jinyoung Lee,Misun Kim,Eunsun Chung 한국원예학회 2020 한국원예학회 학술발표요지 Vol.2020 No.11

Survey of Major Viruses in Commercial Nursery Trees of Major Pear Cultivars in Korea

Kim, Nam-Yeon,Lee, Hyo-Jeong,Kim, Na-Kyeong,Oh, Jonghee,Lee, Su-Heon,Kim, Hongsup,Moon, Jae Sun,Jeong, Rae-Dong The Korean Society of Plant Pathology 2019 식물병연구 Vol.25 No.1

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), and Apple scar skin viroid (ASSVd) are economically important viruses that infect pear tree species worldwide. To evaluate the prevalence of these viruses in Korea, we investigated infection degree of three viruses and one viroid for the commercial nursery trees of the pear cultivars, Niitaka, Chuwhang, Wonwhang, and Whasan in 2017 and 2018. The results showed that the infection ratio of ACLSV, ASPV, ASGV, and ASSVd for the scion of pear cultivar Niitaka was 10%, 45%, 77%, and 50%, respectively. From the scion of pear cultivar Chuwhang, infection ratios of ASPV, ASGV, and ASSVd were found to be 70%, 50%, and 60%, respectively. From the scion of pear cultivar Whasan, infection ratios of ACLSV, ASPV, ASGV and ASSVd were found to be 40%, 60%, 93%, and 20%, respectively. From the root stock of pear cultivar Wonwhang, infection ratios of ACLSV, ASPV, ASGV, and ASSVd showed 28%, 57%, 100%, and 14%, respectively. ASGV had the highest recorded infection rate, and ACLSV was characterized by the lowest infection rate. The mixed infection ratio of Niitaka, Chuwhang, Whasan, and Wonwhang was 45%, 60%, 70%, and 85%, respectively.

Biochemical Evidence for ToxR and ToxJ Binding to the tox Operons of Burkholderia glumae and Mutational Analysis of ToxR

Kim, Jinwoo,Oh, Jonghee,Choi, Okhee,Kang, Yongsung,Kim, Hongsup,Goo, Eunhye,Ma, Jun,Nagamatsu, Tomohisa,Moon, Jae Sun,Hwang, Ingyu American Society for Microbiology 2009 Journal of Bacteriology Vol.191 No.15

<B>ABSTRACT</B><P><I>Burkholderia glumae</I> produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the <I>toxABCDE</I> (toxoflavin biosynthesis genes) and <I>toxFGHI</I> (toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for <I>tox</I> gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both <I>tox</I> operons. ToxR and ToxJ both bound to the <I>toxA</I> and <I>toxF</I> regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both <I>tox</I> operons possessed T-N11-A motifs. Following random mutagenesis of <I>toxR</I>, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (<I>toxR</I>′<I>A</I>′::Ω <I>toxF</I>::Tn<I>3</I>-<I>gusA34</I>) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred <I>toxF</I> expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that <I>B. glumae</I> cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR.</P>

Occurrence Pattern of Viral Infection on Pear in Korea and Genetic Characterization of Apple Scar Skin Viroid Isolates

Nam-Yeon Kim,Hyo-Jeong Lee,Na-Kyeong Kim,Jonghee Oh,Su-Heon Lee,Hongsup Kim,JAE SUN MOON,Rae-Dong Jeong 한국원예학회 2019 원예과학기술지 Vol.37 No.6

Apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), and apple scar skin viroid (ASSVd) are economically important viruses infecting pear trees worldwide. A large-scale survey was carried out in five major pear producing area sof Korea in 2017 and 2018 to investigate the occurrence patterns of these viruses in pear leaves and fruit. The virus incidence rate was 13.8% for ACLSV, 31.7% for ASPV, 95.3% for ASGV, and 3.6% for ASSVd. Fruit samples were 2.8% for ACLSV, 28.4% for ASPV, 99.0% for ASGV, and 13.8% for ASSVd. The most common type of mixed infection was ASGV+ASPV (22%). Furthermore, the ASSVd genome was completely sequenced and showed 99% sequence identity at a nucleotide level to other isolates.

Proteomic analysis of the proteins regulated by HrpB from the plant pathogenic bacterium Burkholderia glumae

Kang, Yongsung,Kim, Jinwoo,Kim, Suhyun,Kim, Hongsup,Lim, Jae Yun,Kim, Minkyun,Kwak, Jangyul,Moon, Jae Sun,Hwang, Ingyu WILEY-VCH Verlag 2008 Proteomics Vol.8 No.1

<P>Plant pathogenic bacteria transfer effector proteins into plant cells via the hypersensitive response and pathogenicity (Hrp) type III protein secretion system (T3SS) during infection. The genes encoding the Hrp T3SS are expressed only under plant apoplast-mimicking conditions in an AraC-type transcriptional activator HrpB-dependent manner. To identify the proteins controlled by HrpB in Burkholderia glumae in vitro, we constitutively expressed hrpB and analyzed the proteins showing altered expression using 2-DE and ESI-MS/MS. Among 46 proteins exhibiting consistently altered expression, which were encoded by 34 different genes, 34 were secretory proteins and 12 were cytoplasmic. Twenty-eight of the secreted proteins showed increased accumulation, whereas the other six showed decreased accumulation. None of the HrpB-dependent proteins had significant homology to known T3SS-dependent proteins, except for HrpK from Pseudomonas syringae pv. syringae and two T3SS-associated cytoplasmic proteins from Ralstonia solanacearum. Twenty-one of the 34 genes had putative HrpB-binding sequences in their upstream regulatory regions. Secretion of all 34 extracellular proteins was independent of the Hrp T3SS, and 16 were secreted via a type II protein secretion system (T2SS). Mutants lacking the T2SS or Hrp T3SS produced toxoflavin but were less virulent to rice panicles, indicating the importance of these proteins in pathogenicity.</P>

A novel light‐dependent selection marker system in plants

Koh, Serry,Kim, Hongsup,Kim, Jinwoo,Goo, Eunhye,Kim, Yun‐,Jung,Choi, Okhee,Jwa, Nam‐,Soo,Ma, Jun,Nagamatsu, Tomohisa,Moon, Jae Sun,Hwang, Ingyu Blackwell Publishing Ltd 2011 Plant biotechnology journal Vol.9 No.3

<P><B>Summary</B></P><P>Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by <I>Burkholderia glumae</I>, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H<SUB>2</SUB>O<SUB>2</SUB> during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, <I>tflA</I>, which encodes a toxoflavin‐degrading enzyme in the <I>Paenibacillus polymyxa</I> JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring‐cleavage extradiol dioxygenase in the <I>Exiguobacterium</I> sp. 255‐15; however, unlike other extradiol dioxygenases, Mn<SUP>2+</SUP>and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light‐dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/<I>tflA</I> and hygromycin/hygromycin phosphotransferase (<I>hpt</I>) in both rice and <I>Arabidopsis</I>. The toxoflavin/<I>tflA</I> selection was more remarkable than hygromycin/<I>hpt</I> selection in the high‐density screening of transgenic <I>Arabidopsis</I> seeds. Based on these results, we propose the toxoflavin/<I>tflA</I> selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.</P>

Survey of Major Viruses in Commercial Nursery Trees of Major Pear Cultivars in Korea

Nam-Yeon Kim,Hyo-Jeong Lee,Na-Kyeong Kim,Jonghee Oh,Su-Heon Lee,Hongsup Kim,JAE SUN MOON,정래동 한국식물병리학회 2019 식물병연구 Vol.25 No.1

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), and Apple scar skin viroid (ASSVd) are economically important viruses that infect pear tree species worldwide. To evaluate the prevalence of these viruses in Korea, we investigated infection degree of three viruses and one viroid for the commercial nursery trees of the pear cultivars, Niitaka, Chuwhang, Wonwhang, and Whasan in 2017 and 2018. The results showed that the infection ratio of ACLSV, ASPV, ASGV, and ASSVd for the scion of pear cultivar Niitaka was 10%, 45%, 77%, and 50%, respectively. From the scion of pear cultivar Chuwhang, infection ratios of ASPV, ASGV, and ASSVd were found to be 70%, 50%, and 60%, respectively. From the scion of pear cultivar Whasan, infection ratios of ACLSV, ASPV, ASGV and ASSVd were found to be 40%, 60%, 93%, and 20%, respectively. From the root stock of pear cultivar Wonwhang, infection ratios of ACLSV, ASPV, ASGV, and ASSVd showed 28%, 57%, 100%, and 14%, respectively. ASGV had the highest recorded infection rate, and ACLSV was characterized by the lowest infection rate. The mixed infection ratio of Niitaka, Chuwhang, Whasan, and Wonwhang was 45%, 60%, 70%, and 85%, respectively.

The Quorum Sensing-Dependent Gene katG of Burkholderia glumae Is Important for Protection from Visible Light

Chun, Heejin,Choi, Okhee,Goo, Eunhye,Kim, Nayeon,Kim, Hongsup,Kang, Yongsung,Kim, Jinwoo,Moon, Jae Sun,Hwang, Ingyu American Society for Microbiology 2009 Journal of Bacteriology Vol.191 No.13

<B>ABSTRACT</B><P>Quorum sensing (QS) plays important roles in the pathogenicity of <I>Burkholderia glumae</I>, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in <I>B. glumae</I>. The QS-defective mutant of <I>B. glumae</I> exhibited less catalase activity than wild-type <I>B. glumae</I>. A β-glucuronidase assay of a <I>katG</I>::Tn<I>3-gusA78</I> reporter fusion protein revealed that <I>katG</I> expression is under the control of QS. Furthermore, <I>katG</I> expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates <I>katG</I> expression. The <I>katG</I> mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 μmol<SUP>−1</SUP> m<SUP>−2</SUP>, the survival rate of the <I>katG</I> mutant was 10<SUP>5</SUP>-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the <I>katG</I> mutant.</P>

Characterization of Type VI Secretion System in Xanthomonas oryzae pv. oryzae and Its Role in Virulence to Rice

Yeounju Choi,Namgyu Kim,Mohamed Mannaa,Hongsup Kim,Jungwook Park,Hyejung Jung,Gil Han,Hyun-Hee Lee,Young-Su Seo 한국식물병리학회 2020 Plant Pathology Journal Vol.36 No.3

Type VI secretion system (T6SS) is a contact-dependent secretion system, employed by most gram-negative bacteria for translocating effector proteins to target cells. The present study was conducted to investigate T6SS in Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight in rice, and to unveil its functions. Two T6SS clusters were found in the genome of Xoo PXO99A. The deletion mutants, Δhcp1, Δhcp2, and Δhcp12, targeting the hcp gene in each cluster, and a double-deletion mutant targeting both genes were constructed and tested for growth rate, pathogenicity to rice, and inter-bacterial competition ability. The results indicated that hcp in T6SS-2, but not T6SS-1, was involved in bacterial virulence to rice plants. However, neither T6SS-1 nor T6SS-2 had any effect on the ability to compete with Escherichia coli or other bacterial cells. In conclusion, T6SS gene clusters in Xoo have been characterized, and its role in virulence to rice was confirmed.