Alternanthera mosaic virus - an alternative ‘model’ potexvirus of broad relevance

John Hammond,Ik-Hyun Kim,Hyoun-Sub Lim 충남대학교 농업과학연구소 2017 Korean Journal of Agricultural Science Vol.44 No.2

Seed Transmission Rates of Bean pod mottle virus and Soybean mosaic virus in Soybean May Be Affected by Mixed Infection or Expression of the Kunitz Trypsin Inhibitor

Nam, Moon,Bae, Hanhong,Hammond, John,Domier, Leslie L.,Youn, Young-Nam,Lee, Bong-Choon,Lim, Hyoun-Sub The Korean Society of Plant Pathology 2013 식물병연구 Vol.19 No.2

To facilitate their spread, plant viruses have developed several methods for dispersal including insect and seed transmission. While insect transmission requires virus stability against insect digestion, seed-transmitted viruses have to overcome barriers to entry into embryos. Bean pod mottle virus (BPMV) is transmitted through seed at levels typically below 0.1%, but co-infection with Soybean mosaic virus (SMV) enhanced the seed transmission rate of BPMV in one experiment. In contrast, the rate of SMV seed transmission was not affected by BPMV co-infection. In a second preliminary study, the rate of SMV transmission was lower in an isoline of Williams 82 that contained a null mutation for the Kunitz trypsin inhibitor gene than in Williams 82. In this preliminary study, we observed that factors such as protease inhibitor expression and dual infection may affect the frequency of seed transmission of BPMV and SMV.

Seed Transmission Rates of Bean pod mottle virus and Soybean mosaic virus in Soybean May Be Affected by Mixed Infection or Expression of the Kunitz Trypsin Inhibitor

남문,임현섭,배한홍,이봉춘,윤영남,John Hammond,LeslieL.Domier 한국식물병리학회 2013 식물병연구 Vol.19 No.2

To facilitate their spread, plant viruses have developed several methods for dispersal including insect and seed transmission. While insect transmission requires virus stability against insect digestion, seed-transmitted viruses have to overcome barriers to entry into embryos. Bean pod mottle virus (BPMV) is transmitted through seed at levels typically below 0.1%, but co-infection with Soybean mosaic virus (SMV) enhanced the seed transmission rate of BPMV in one experiment. In contrast, the rate of SMV seed transmission was not affected by BPMV co-infection. In a second preliminary study, the rate of SMV transmission was lower in an isoline of Williams 82 that contained a null mutation for the Kunitz trypsin inhibitor gene than in Williams 82. In this preliminary study, we observed that factors such as protease inhibitor expression and dual infection may affect the frequency of seed transmission of BPMV and SMV.

Dual infections of Tomato mosaic virus (ToMV) and Tomato yellow leaf curl virus (TYLCV), or Tomato mosaic virus (ToMV) and Tomato chlorosis virus (ToCV), detected in tomato fields located in Chungcheongnam-do in 2017

최고운,김보람,주혜경,조상원,서은영,김정규,박종석,John Hammond,임현섭 충남대학교 농업과학연구소 2018 Korean Journal of Agricultural Science Vol.45 No.1

Demand for tomatoes has been increasing every year as people desire more healthy food. In Korea, tomatoes are mainly grown in the Chungnam, Chunnam and Kyungnam provinces. Recently, reports of whitefly-transmitted viral diseases have increased due to newly emerging whitefly pressures caused by climate change in Korea. Specifically, in 2017, the main tomato growing areas, Buyeo and Nonsan in Chungnam, showed damage typical of viral infection; therefore, we investigated viral diseases in these areas. We collected samples with virus-like symptoms and found that not only whitefly transmitted Tomato yellow leaf curl virus (TYLCV) and Tomato chlorosis virus (ToCV) were detected but also Tomato mosaic virus (ToMV, for which no specific vector is known) and Tomato spotted wilt virus (TSWV, transmitted by thrips). The ToMV-infected samples were mostly co-infected with either TYLCV or ToCV. Mixed infections of different combinations of TYLCV, ToCV and ToMV were detected with the mixed infection of two whitefly-transmitted viruses (TYLCV and ToCV) causing the most severe symptoms. According to the CP sequence of each virus, the 100% identities were shown to be Mexico/ABG73017.1 (TYLCV), Greece/CDG34553.1 (ToCV), China/AKN79752 (TSWV), and Australia/NP078449.1 (ToMV). Based on the sequence data, we presumed that these tomato infecting viruses were transmitted through insects and seeds introduced from neighboring countries.

2014년 전국 무 재배지의 바이러스 병 발생 조사

정진수,한재영,김정규,주혜경,공준수,서은영,John Hammond,임현섭 한국식물병리학회 2015 식물병연구 Vol.21 No.3

A 2014 nationwide survey in radish fields investigated the distribution of common viruses and possible emerging viruses. Radish leaves with virus-like symptoms were collected and 108 samples assayed by RT-PCR using specific primers for Radish mosaic virus (RaMV), Cucumber mosaic virus (CMV), and Turnip mosaic virus (TuMV); 47 samples were TuMV positive, and RaMV and CMV were detected in 3 and 2 samples, respectively. No samples showed double infection of TuMV/RaMV, or RaMV/CMV, but two double infections of TuMV/ CMV were detected. TuMV isolates were sorted by symptom severity, and three isolates (R007-mild; R041 and R065-severe) selected for BLAST and phylogenetic analysis, which indicated that the coat protein (CP) of these isolates (R007, R041, and R065) have approx. 98-99% homology to a previously reported TuMV isolate. RaMV CP showed approx. 99% homology to a previously reported isolate, and the CMV CP is identical to a previously reported Korean isolate (GenBank : GU327368). Three isolates of TuMV showing different pathogenicity (degree of symptom severity) will be valuable to study determinants of pathogenicity. 2014년 전국 무 포장의 바이러스병 발생양상과 분포조사를실시하였다. 바이러스 병징을 나타내는 108개 시료 중 47개 시료는 TuMV가 진단되었으며 RaMV는 3개, CMV는 2개 시료에서각각 진단되었다. TuMV와 RaMV, RaMV와 CMV의 복합감염은나타나지 않았으나 TuMV와 CMV의 복합감염이 2개 시료에서진단되었다. N. benthamiana에 대한 TuMV 병원성 테스트 결과, 병징의 세기에 따라 세 개의 분리주를 나누었으며 외피단백질의 아미노산 서열 분석을 통하여 약한 병징을 나타내는 분리주R007, 강한 병징을 나타내는 분리주 R041, R065를 선발하여 계통수 분석에 이용하였다. TuMV 계통수분석과 BLAST 검색결과TuMV 분리주 3개와 기 보고된 분리주들 간의 외피단백질 아미노산 서열은 약 98-99%의 상동성을 나타내었으며, RaMV의 외피단백질의 경우 99%의 상동성을 나타내었고, CMV의 외피 단백질은 국내 기 보고된 분리주(GenBank : GU327363)와 동일하였다. TuMV 계통수에서 R007과 R041, R065는 서로 다른 그룹에 속하였으며 그룹간의 차이는 바이러스의 기주 선호도와 관련이 있을 것이라 예상하며 추후 TuMV의 병원성 결정인자 연구에 도움이 될 것으로 사료된다.

AltMV TGB1 Nucleolar Localization Requires Homologous Interaction and Correlates with Cell Wall Localization Associated with Cell-to-Cell Movement

남지륜,임현섭,남문,배한홍,이철호,John Hammond,이봉춘 한국식물병리학회 2013 Plant Pathology Journal Vol.29 No.4

The Potexvirus Alternanthera mosaic virus (AltMV) hasmultifunctional triple gene block (TGB) proteins, amongwhich our studies have focused on the properties of theTGB1 protein. The TGB1 of AltMV has functionsincluding RNA binding, RNA silencing suppression,and cell-to-cell movement, and is known to form homologousinteractions. The helicase domains of AltMVTGB1 were separately mutated to identify which regionsare involved in homologous TGB1 interactions. Theyeast two hybrid system and Bimolecular FluorescenceComplementation (BiFC) in planta were utilized to examinehomologous interactions of the mutants. Helicasemotif I of AltMV TGB1 was found to be critical tomaintain homologous interactions. Mutations in theremaining helicase motifs did not inhibit TGB1 homologousinteractions. In the absence of homologous interactionof TGB1, subcellular localization of helicasedomain I mutants showed distinctively different patternsfrom that of WT TGB1. These results provide importantinformation to study viral movement and replicationof AltMV.

Deep Sequencing Analysis of Apple Infecting Viruses in Korea

조인숙,Davaajargal Igori,임승모,최국선,John Hammond,임현섭,문재선 한국식물병리학회 2016 Plant Pathology Journal Vol.32 No.5

Deep sequencing has generated 52 contigs derivedfrom five viruses; Apple chlorotic leaf spot virus(ACLSV), Apple stem grooving virus (ASGV), Applestem pitting virus (ASPV), Apple green crinkle associatedvirus (AGCaV), and Apricot latent virus (ApLV)were identified from eight apple samples showingsmall leaves and/or growth retardation. Nucleotide (nt)sequence identity of the assembled contigs was from68% to 99% compared to the reference sequences ofthe five respective viral genomes. Sequences of ASPVand ASGV were the most abundantly represented bythe 52 contigs assembled. The presence of the five virusesin the samples was confirmed by RT-PCR usingspecific primers based on the sequences of each assembledcontig. All five viruses were detected in threeof the samples, whereas all samples had mixed infectionswith at least two viruses. The most frequentlydetected virus was ASPV, followed by ASGV, ApLV,ACLSV, and AGCaV which were withal found inmixed infections in the tested samples. AGCaV wasidentified in assembled contigs ID 1012480 and 93549,which showed 82% and 78% nt sequence identity withORF1 of AGCaV isolate Aurora-1. ApLV was identifiedin three assembled contigs, ID 65587, 1802365,and 116777, which showed 77%, 78%, and 76% ntsequence identity respectively with ORF1 of ApLVisolate LA2. Deep sequencing assay was shown to bea valuable and powerful tool for detection and identificationof known and unknown virome in infectedapple trees, here identifying ApLV and AGCaV incommercial orchards in Korea for the first time.

Selective Interaction Between Chloroplast β-ATPase and TGB1L88 Retards Severe Symptoms Caused by Alternanthera mosaic virus Infection

서은영,임현섭,남지륜,김현승,박영환,홍석명,배한홍,Dilip Lakshman,John Hammond 한국식물병리학회 2014 Plant Pathology Journal Vol.30 No.1

The multifunctional triple gene block protein 1 (TGB1)of the Potexvirus Alternanthera mosaic virus (AltMV)has been reported to have silencing suppressor, cellto-cell movement, and helicase functions. Yeasttwo hybrid screening using an Arabidopsis thalianacDNA library with TGB1 as bait, and co-purificationwith TGB1 inclusion bodies identified several hostproteins which interact with AltMV TGB1. Hostprotein interactions with TGB1 were confirmed bybiomolecular fluorescence complementation, whichshowed positive TGB1 interaction with mitochondrialATP synthase delta′ chain subunit (ATP synthasedelta′), light harvesting chlorophyll-protein complex Isubunit A4 (LHCA4), chlorophyll a/b binding protein1 (LHB1B2), chloroplast-localized IscA-like protein(ATCPISCA), and chloroplast β-ATPase. However,chloroplast β-ATPase interacts only with TGB1L88,and not with weak silencing suppressor TGB1P88. Thisselective interaction indicates that chloroplast β-ATPaseis not required for AltMV movement and replication;however, TRV silencing of chloroplast β-ATPase inNicotiana benthamiana induced severe tissue necrosiswhen plants were infected by AltMV TGB1L88 but notAltMV TGB1P88, suggesting that β-ATPase selectivelyresponded to TGB1L88 to induce defense responses.

Generation of an Infectious Clone of a New Korean Isolate of Apple chlorotic leaf spot virus Driven by Dual 35S and T7 Promoters in a Versatile Binary Vector

김익현,임현섭,한재영,조인숙,주혜경,문재선,서은영,김홍기,John Hammond 한국식물병리학회 2017 Plant Pathology Journal Vol.33 No.6

The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacteriophage T7 RNA promoter and the Cauliflower mosaic virus 35S promoter. Chenopodium quinoa was successfully infected using in vitro transcripts synthesized using the T7 promoter, detected at 20 days post inoculation (dpi), but did not produce obvious symptoms. Nicotiana occidentalis and C. quinoa were inoculated through agroinfiltration. At 32 dpi the infection rate was evaluated; no C. quinoa plants were infected by agroinfiltration, but infection of N. occidentalis was obtained.

Pseudomonas oleovorans Strain KBPF-004 Culture Supernatants Reduced Seed Transmission of Cucumber green mottle mosaic virus and Pepper mild mottle virus, and Remodeled Aggregation of 126 kDa and Subcellular Localization of Movement Protein of Pepper mild

김남규,서은영,한상혁,공준수,박철남,박호섭,Leslie L Domier,John Hammond,임현섭 한국식물병리학회 2017 Plant Pathology Journal Vol.33 No.4

Efforts to control viral diseases in crop production include several types of physical or chemical treatments; antiviral extracts of a number of plants have also been examined to inhibit plant viral infection. However, treatments utilizing naturally selected microorganisms with activity against plant viruses are poorly documented. Here we report isolation of a soil inhabiting bacterium, Pseudomonas oleovorans strain KBPF-004 (developmental code KNF2016) which showed antiviral activity against mechanical transmission of tobamoviruses. Antiviral activity was also evaluated in seed transmission of two tobamoviruses, Pepper mild mottle virus (PMMoV) and Cucumber green mottle mosaic virus (CGMMV), by treatment of seed collected from infected pepper and watermelon, respectively. Pepper and watermelon seeds were treated with culture supernatant of P. oleovorans strain KBPF-004 or control strain ATCC 8062 before planting. Seeds germinated after treatment with water or ATCC 8062 yielded about 60% CGMMV or PMMoV positive plants, whereas < 20% of KBPF-004-treated seeds were virusinfected, a significantly reduced seed transmission rate. Furthermore, supernatant of P. oleovorans strain KBPF-004 remodeled aggregation of PMMoV 126 kDa protein and subcellular localization of movement protein in Nicotiana benthamiana, diminishing aggregation of the 126 kDa protein and essentially abolishing association of the movement protein with the microtubule network. In leaves agroinfiltrated with constructs expressing the coat protein (CP) of either PMMoV or CGMMV, less full-size CP was detected in the presence of supernatant of P. oleovorans strain KBPF-004. These changes may contribute to the antiviral effects of P. oleovorans strain KBPF-004.