Resistance of TransgenicNicotiana benthamianaPlants to Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses: Evidence of Involvement of the N Protein and N Gene RNA in Resistance

1994 ◽  
Vol 84 (3) ◽  
pp. 243 ◽  
Author(s):  
Sheng- Zhi Pang
Keyword(s):  
N Gene ◽  
Necrotic Spot ◽  
N Protein ◽  
Tomato Spotted Wilt

scholarly journals Tomato Spotted Wilt Virus Resistance in Chrysanthemum Expressing the Viral Nucleocapsid Gene

Plant Disease ◽  
1998 ◽  
Vol 82 (4) ◽  
pp. 407-414 ◽  
Author(s):  
John M. Sherman ◽  
James W. Moyer ◽  
Margaret E. Daub
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Genetically Engineered ◽  
Full Length ◽  
N Gene ◽  
Gene Transcript ◽  
Nucleocapsid Gene ◽  
N Protein ◽  
Tomato Spotted Wilt ◽  
Insertion Sites ◽  
Wilt Virus

Three tomato spotted wilt virus (TSWV) nucleocapsid (N) gene constructs were employed for Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora) cv. Polaris. These constructs contained either a full-length N gene (pTSWVN+), a full-length N gene encoding a truncated N protein (pTSWVNt), or an antisense version of the full-length N gene (pTSWVN-), all derived from a dahlia isolate of TSWV (TSWV-D). Initial resistance screens were conducted on cuttings made from 152 pTSWVN+, 37 pTSWVNt, and 47 pTSWVN- transformed plants employing a highly virulent, heterologous strain of TSWV (TSWV-GB) isolated from chrysanthemum and vectored by thrips. This screening served to eliminate the majority of TSWV-susceptible transgenic lines. More rigorous resistance tests with three rounds of mechanical inoculation with TSWV-GB identified one pTSWVNt and two pTSWVN- transformed lines that exhibited a total lack of systemic symptoms and no virus accumulation. Six other lines, including some pTSWVN+, exhibited a lack of one or more of the destructive necrotic TSWV symptoms (stem canker and apical bud death) and a delay in symptom expression. Both sense and antisense constructs, therefore, were found to be effective at yielding TSWV resistance in chrysanthemum. Molecular analysis revealed that the highly TSWV-resistant pTSWVNt line had no detectable levels of N protein. All three resistant lines had low levels of N gene transcript and at least three transgene insertion sites within their genomes, although susceptible lines often had a similar number of insertion sites. The generation of Polaris lines resistant to TSWV transmitted either mechanically or by thrips represents the first time a major ornamental crop has been genetically engineered for disease resistance.

scholarly journals Partial sequencing of a putative Alstroemeria necrotic streak orthotospovirus isolate detected on lettuce in Colombia

2021 ◽  
Vol 51 ◽  
Author(s):  
Diego Alejandro Jurado-Rincón ◽  
Linda Jeimmy Rincón-Rivera ◽  
Angela María Vargas-Berdugo ◽  
Adriana González-Almario
Keyword(s):  
Viral Infection ◽  
N Gene ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrotic Spot ◽  
Partial Sequencing ◽  
Tomato Spotted Wilt ◽  
Foliar Symptoms ◽  
Necrotic Spots ◽  
Leaf Distortion ◽  
Das Elisa

ABSTRACT Lettuce is the most cultivated leafy salad vegetable in Colombia, being the municipality of Madrid, in the Department of Cundinamarca, the second largest producer. In this region, lettuce plants with foliar symptoms characterized by brown necrotic spots forming an extended necrotic area, chlorosis, leaf distortion and plant stunting have been detected, possibly caused by a viral infection associated with the Orthotospovirus genus. This study aimed to identify the orthotospovirus species associated with those symptoms, contributing to updating the lettuce phytosanitary status in this region. The presence of orthotospovirus was confirmed by the enzyme-linked immunosorbent assay (DAS-ELISA), although the sequence of the nucleocapsid (N) gene confirmed the presence of Alstroemeria necrotic streak orthotospovirus, disregarding the Tomato spotted wilt orthotospovirus and Impatiens necrotic spot orthotospovirus previously reported for this crop, being this its first report in lettuce crops in Colombia.

scholarly journals VIRAL PROTECTION IN TRANSGENIC TOBACCO PLANTS EXPRESSING THE TOMATO SPOTTED WILT VIRUS N GENE

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 621b-621
Author(s):  
Jong Wook Kim ◽  
Thomas L. German ◽  
Samuel S. M. Sun
Keyword(s):  
Transgenic Tobacco ◽  
Blot Analysis ◽  
Tomato Spotted Wilt Virus ◽  
First Generation ◽  
N Gene ◽  
Transgenic Tobacco Plants ◽  
Tobacco Plants ◽  
N Protein ◽  
Tomato Spotted Wilt ◽  
Wilt Virus

Nucleocapsid protein (N) gene was isolated from tomato spotted wilt virus (TSWV) Hawaiian L isolate, and introduced into Nicotiana tabacum cv. Xanthi nc in order to test for “CP-mediated protection”. Agrobacterium tumefaciens-mediated transformation was performed. The integrity and the expression of N gene were verified by Southern blot and Northern blot analysis, and the N protein in the transgenic tobacco plants were determined by ELISA and Western blot analysis. Several first generation of transgenic tobacco were tested for virus resistance. Comparably smaller numbers of the local lesions were developed with several day of delay in the in-frame transformants.

scholarly journals First Report of Tomato spotted wilt virus in Blackberry Lily in North America

Plant Disease ◽  
2003 ◽  
Vol 87 (1) ◽  
pp. 102-102 ◽  
Author(s):  
S. Adkins ◽  
L. Breman ◽  
C. A. Baker ◽  
S. Wilson
Keyword(s):  
North America ◽  
Tomato Spotted Wilt Virus ◽  
Amino Acid Sequences ◽  
South Florida ◽  
N Gene ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Wilt Virus

Blackberry lily (Belamcanda chinensis (L.) DC.) is an herbaceous perennial in the Iridaceae characterized by purple-spotted orange flowers followed by persistent clusters of black fruit. In July 2002, virus-like symptoms including chlorotic ringspots and ring patterns were observed on blackberry lily leaves on 2 of 10 plants in a south Florida ornamental demonstration garden. Inclusion body morphology suggested the presence of a Tospovirus. Tomato spotted wilt virus (TSWV) was specifically identified by serological testing using enzyme-linked immunosorbent assay (Agdia, Elkhart, IN). Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579 base pair region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. Since these 2-year-old plants were grown on-site from seed, they were likely inoculated by thrips from a nearby source. Together with a previous observation of TSWV in north Florida nursery stock (L. Breman, unpublished), this represents, to our knowledge, the first report of TSWV infection of blackberry lily in North America although TSWV was observed in plants of this species in Japan 25 years ago (2). References: (1) S. Adkins, and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) T. Yamamoto and K.-I. Ohata. Bull. Shikoku Agric. Exp. Stn. 30:39, 1977.

scholarly journals Thrips as the Transmission Bottleneck for Mixed Infection of Two Orthotospoviruses

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 509
Author(s):  
Kaixi Zhao ◽  
Cristina Rosa
Keyword(s):  
Genetic Diversity ◽  
Virus Replication ◽  
Mixed Infection ◽  
Viral Fitness ◽  
Mixed Infections ◽  
Virus Species ◽  
Necrotic Spot ◽  
Tomato Spotted Wilt ◽  
Transmission Bottleneck

Mixed infections provide opportunities for viruses to increase genetic diversity by facilitating genomic reassortment or recombination, and they may lead to the emergence of new virus species. Mixed infections of two economically important orthotospoviruses, Tomato spotted wilt orthotospovirus (TSWV) and Impatiens necrotic spot orthotospovirus (INSV), were found in recent years, but no natural reassortants between INSV and TSWV were ever reported. The goal of this study was to establish how vector preferences and the ability to transmit INSV and TSWV influence transmission and establishment of mixed infections. Our results demonstrate that thrips prefer to oviposit on TSWV and INSV mixed-infected plants over singly infected or healthy plants, providing young nymphs with the opportunity to acquire both viruses. Conversely, we observed that thrips served as a bottleneck during transmission and favored transmission of one of the two viruses over the second one, or over transmission of both viruses simultaneously. This constraint was relaxed in plants, when transmission of TSWV and INSV occurred sequentially, demonstrating that plants serve as orthotospovirus permissive hosts, while thrips serve as a bottleneck. Viral fitness, as measured by virus replication, transmission, and competition with other viral strains, is not well studied in mixed infection. Our study looks at the success of transmission during mixed infection of orthotopoviruses, enhancing the understanding of orthotospovirus epidemiology and evolution.

Specificity of accumulation and transmission of tomato spotted wilt virus (TSWV) in two genera, Frankliniella and Thrips (Thysanoptera: Thripidae)

2004 ◽  
Vol 94 (6) ◽  
pp. 501-507 ◽  
Author(s):  
T. Inoue ◽  
T. Sakurai ◽  
T. Murai ◽  
T. Maeda
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Virus Transmission ◽  
Leaf Disc ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Accumulation ◽  
Accumulation Pattern ◽  
N Protein ◽  
Tomato Spotted Wilt ◽  
Wilt Virus ◽  
Elisa Titre

AbstractThe accumulation and transmission of tomato spotted wilt virus (TSWV) was examined in second instar larvae and adults of two thrips genera, Frankliniella and Thrips. The species tested were F. occidentalis (Pergande), F. intonsa (Trybom), T. tabaciLindeman, T. setosus Moulton, T. palmi Karny and T. hawaiiensis (Morgan). In a standard petunia leaf disc assay, the efficiencies of TSWV transmission by two species of Frankliniella were higher than those of any Thrips species in the adult stage. A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) showed that large amounts of the TSWV-nucleocapsid (N) protein were present in the ELISA-positive larvae of each species, with the exception of T. palmi. The ELISA titre of and the proportion of virus-infected individuals of the two Frankliniella species increased or did not significantly change from the larval to the adult stages, whereas those of the four Thrips species decreased significantly. These results show that the specificity of virus transmission by adult thrips is probably affected by the amount of viral N protein accumulation in the adults and that the accumulation pattern from the larval to the adult stages is in between the two genera tested in the present study.

scholarly journals First Report of Tomato spotted wilt virus on Chrysanthemum in Serbia

Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 150-150 ◽  
Author(s):  
I. Stanković ◽  
A. Bulajić ◽  
A. Vučurović ◽  
D. Ristić ◽  
K. Milojević ◽  
...  
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Disease Incidence ◽  
N Gene ◽  
Impatiens Necrotic Spot Virus ◽  
Maximum Parsimony Tree ◽  
Rt Pcr ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Negative Controls ◽  
Wilt Virus

In July 2011, greenhouse-grown chrysanthemum hybrid plants (Chrysanthemum × morifolium) with symptoms resembling those associated with tospoviruses were observed in the Kupusina locality (West Bačka District, Serbia). Disease incidence was estimated at 40%. Symptomatic plants with chlorotic ring spots and line patterns were sampled and tested by double antibody sandwich (DAS)-ELISA using polyclonal antisera (Bioreba AG, Reinach, Switzerland) against the two of the most devastating tospoviruses in the greenhouse floriculture industry: Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) (2). Commercial positive and negative controls and extracts from healthy chrysanthemum tissue were included in each ELISA. TSWV was detected serologically in 16 of 20 chrysanthemum samples and all tested samples were negative for INSV. The virus was mechanically transmitted from ELISA-positive chrysanthemum samples to five plants each of both Petunia × hybrida and Nicotiana tabacum ‘Samsun’ using chilled 0.01 M phosphate buffer (pH 7) containing 0.1% sodium sulfite. Inoculated plants produced local necrotic spots and systemic chlorotic/necrotic concentric rings, consistent with symptoms caused by TSWV (1). The presence of TSWV in ELISA-positive chrysanthemum plants and N. tabacum‘Samsun’ was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primers TSWVCP-f/TSWVCP-r specific to the nucleocapsid protein (N) gene (4). A Serbian isolate of TSWV from tobacco (GenBank Accession No. GQ373173) and RNA extracted from a healthy chrysanthemum plant were used as positive and negative controls, respectively. An amplicon of the correct predicted size (738-bp) was obtained from each of the plants assayed, and that derived from chrysanthemum isolate 529-11 was purified (QIAqick PCR Purification Kit, Qiagen) and sequenced (JQ692106). Sequence analysis of the partial N gene, conducted with MEGA5 software, revealed the highest nucleotide identity of 99.6% (99% amino acid identity) with 12 TSWV isolates deposited in GenBank originating from different hosts from Italy (HQ830186-87, DQ431237-38, DQ398945), Montenegro (GU355939-40, GU339506, GU339508), France (FR693055-56), and the Czech Republic (AJ296599). The consensus maximum parsimony tree obtained on a 705-bp partial N gene sequence of TSWV isolates available in GenBank revealed that Serbian TSWV isolate 529-11 from chrysanthemum was clustered in the European subpopulation 2, while the Serbian isolates from tomato (GU369723) and tobacco (GQ373172-73 and GQ355467) were clustered in the European subpopulation 1 denoted previously (3). The distribution of TSWV in commercial chrysanthemum crops is wide (2). To our knowledge, this is the first report of TSWV infecting chrysanthemum in Serbia. Since chrysanthemum popularity and returns have been rising rapidly, the presence of TSWV may significantly reduce quality of crops in Serbia. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) Daughtrey et al. Plant Dis. 81:1220, 1997. (3) I. Stanković et al. Acta Virol. 55:337, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012.

scholarly journals Fruit melanotic ringspot (FMRS) – a disease of resistant Capsicum genotypes infected with Tomato spotted wilt virus (TSWV) on the fruits

2011 ◽  
pp. 64-69
Author(s):  
Pál Salamon ◽  
Katalin Nemes ◽  
Katalin Salánki
Keyword(s):  
Frankliniella Occidentalis ◽  
N Gene ◽  
Tomato Spotted Wilt ◽  
Pepper Fruit ◽  
Fruit Samples ◽  
Dna Fragment ◽  
Heterozygous Form ◽  
Systemic Symptoms ◽  
Wilt Virus ◽  
Virus Isolates

Etiology of pepper fruit melanotic ringspot (FMRS) disease (Salamon, 2009) was studied on fruit samples collected in forced pepper populations. It was noticed that in spite of heavy thrips (Frankliniella occidentalis) infestations and of TSWV epidemy detected in the forcing houses, FMRS occurred only in plants having healthy foliage. Symptomatological surveys strongly suggested that FMRS appeared exclusively in specific pepper genotypes. The size of melanotic ringspots has been observed to grow at room temperature during postripening of diseased fruits. A mechanically transmitted plant virus was isolated from symptomatic parts of 9 white pepper fruits affected by FMRS. On test plants each of the virus isolates caused systemic symptoms characteristic to TSWV. Using cDNA/PCR technique and TSWV N-gene specific primers a ca. 300 bp long DNA fragment has been amplified from total nucleic acid extracted from symptomatic tissues but never from asymptomatic parts of the fruits showing FMRS. Plant progenies grown from seeds of FMRS diseased fruits segregated in respect of resistance and/or susceptibility to TSWV infection. TSWV was also detected in and isolated from three fruits showed non-melanotic yellow rings (one of them was infected with a tobamovirus, too). Seedlings derived from these fruits proved to be susceptible to TSWV. Based on the above results we could conclude that the FMRS disease developed on fruits of “cecei” type white peppers that carry a TSWV resistance gene, most likely the Tsw gene in heterozygous form. These fruits were infected with thrips transmitted TSWV and FRMS appeared as a hypersentive reaction (HR) manifested in fruits.

scholarly journals SARS-CoV-2 disinfection in aqueous solution by UV222 from a krypton chlorine excilamp

2021 ◽  
Author(s):  
Richard T. Robinson ◽  
Najmus Mahfooz ◽  
Oscar Rosas-Mejia ◽  
Yijing Liu ◽  
Natalie M. Hull
Keyword(s):  
Aqueous Solution ◽  
Dose Response ◽  
Rate Constants ◽  
Uv Light ◽  
N Gene ◽  
Elisa Assay ◽  
Peak Wavelength ◽  
N Protein ◽  
Excimer Lamps ◽  
Gene Damage

ABSTRACTThere is an urgent need for evidence-based development and implementation of engineering controls to reduce transmission of SARS-CoV-2, the etiological agent of COVID-19. Ultraviolet (UV) light can inactivate coronaviruses, but the practicality of UV light as an engineering control in public spaces is limited by the hazardous nature of conventional UV lamps, which are Mercury (Hg)-based and emit a peak wavelength (254 nm) that penetrates human skin and is carcinogenic. Recent advances in the development and production of Krypton Chlorine (KrCl) excimer lamps hold promise in this regard, as these emit a shorter peak wavelength (222 nm) and are recently being produced to filter out emission above 240 nm. However, the disinfection kinetics of KrCl UV excimer lamps against SARS-CoV-2 are unknown. Here we provide the first dose response report for SARS-CoV-2 exposed to a commercial filtered KrCl excimer light source emitting primarily 222 nm UV light (UV222), using multiple assays of SARS-CoV-2 viability. Plaque infectivity assays demonstrate the pseudo-first order rate constant of SARS-CoV-2 reduction of infectivity to host cells to be 0.64 cm2/mJ (R2 = 0.95), which equates to a D90 (dose for 1 log10 or 90% inactivation) of 1.6 mJ/cm2. Through RT-qPCR assays targeting the nucleocapsid (N) gene with a short (<100 bp) and long (∼1000 bp) amplicon in samples immediately after UV222 exposure, the reduction of ability to amplify indicated an approximately 10% contribution of N gene damage to disinfection kinetics. Through ELISA assay targeting the N protein in samples immediately after UV222 exposure, we found no dose response of the ability to damage the N protein. In both qPCR assays and the ELISA assay of viral outgrowth supernatants collected 3 days after incubation of untreated and UV222 treated SARS-CoV-2, molecular damage rate constants were similar, but lower than disinfection rate constants. These data provide quantitative evidence for UV222 doses required to disinfect SARS-CoV-2 in aqueous solution that can be used to develop further understanding of disinfection in air, and to inform decisions about implementing UV222 for preventing transmission of COVID19.ABSTRACT ART / TOC GRAPHIC

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